UNIVERZITET U BEOGRADU FARMACEUTSKI FAKULTET mr Dragica Kne!evi" Ili" KOMPARATIVNA ANALIZA PROCESA, SISTEMA I REGULATIVA U IZRADI I PRIMENI MATI!NIH !ELIJA I DRUGIH !ELIJSKIH TERAPIJA doktorska disertacija Beograd, 2013. UNIVERSITY OF BELGRADE FACULTY OF PHARMACY Dragica Kne!evi" Ili" BSc MSc MBA COMPARATIVE STUDY OF PROCESSES, SYSTEMS AND REGULATIONS IN MANUFACTURING AND APPLICATION OF STEM CELLS AND OTHER CELL THERAPIES Doctoral Dissertation Belgrade, 2013 KOMISIJA: Mentor ____________________________ Dr Sne!ana Savi", vanredni profesor Univerzitet u Beogradu - Farmaceutski fakultet Mentor ____________________________ Dr Ljiljana Tasi", redovni profesor Univerzitet u Beogradu - Farmaceutski fakultet ____________________ ____________________________ Datum odbrane Dr Bela Balint, nau#ni savetnik Na#elnik Instituta za transfuziologiju i hemobiologiju Vojnomedicinske akademije u Beogradu Ova doktorska disertacija je ura!ena na Katedri za farmaceutsku tehnologiju i Katedri za socijalnu farmaciju i istra"ivanja farmaceutske prakse Farmaceutskog fakulteta u Beogradu, u saradnji sa School of Pharmacy, Pharmaceutical Center of Excellence, PACE - University of Queensland, Brisbane, Australia. Pre svega, "elim posebno da se zahvalim svojim mentorima prof. dr Ljiljani Tasi# i prof. dr Sne"ani Savi#, pod $ijim rukovodstvom je izra!ena ova disertacija, na nesebi$noj pomo#i, razumevanju, savetima i podr%ci koje su mi pru"ile kao i prof. dr Beli Balintu na ukazanom poverenju i stru$noj pomo#i sa klini$kog aspekta. Zahvalnost dugujem i svojim kolegama sa Univerziteta u Kvinslendu me!u kojima se nalaze prof. dr Kerry Atkinson, prof. dr Evan Siegel i mnogi drugi. Posebnu zahvalnost dugujem svim u$esnicima u istra"ivanju $ija imena, na osnovu eti$kih prinicpa istra"ivanja, nisam u mogu#nosti da navedem. Bez njihovog doprinosa ovo istra"ivanje ne bi imalo isti zna$aj i sveobuhvatnost. Zahvaljujem se kolegama na Farmaceutskom fakultetu i Vojnomedicinskoj Akademiji u Beogradu kao i rukovodstvu i kolegama sa Farmaceutskog fakulteta Univerziteta u Kvinslendu (School of Pharmacy, University of Queensland, Brisbane) na saradnji i pru"enoj podr%ci tokom izrade doktorske disertacije. Veliku zahvalnost dugujem i administrativnom osoblju na oba fakulteta, tehni$kom osoblju i kolegama na poslediplomskim studijama. Inspiraciju i motivaciju, nesebi$nu ljubav, kao i sveprisutnu podr%ku i pomo# za izradu ove disertacije su mi pru"ili $lanovi moje porodice na $emu sam im beskrajno zahvalna. IZVOD Terapije mati!nim "elijama i drugim vrstama "elija se mogu uvrstiti u grupu novih bio- terapeutika za koje se smatra da "e u slede"oj dekadi zna!ajno uve"ati mogu"nost izle!enja mnogih oboljenja. Uz to, "elijske terapije ili terapije bazirane na upotrebi mati!nih i drugih "elija obuhvataju novo polje u oblasti medicine, farmacije i razvoja bioterapeutskih sredstava. Ove terapije uklju!uju izradu i primenu razli!itih tipova "elija kao nove kategorije bioloskih lekova. #elijske terapije koje se koriste u terapeutske svrhe moraju da budu okarakterisane, prikupljene, procesovane, !uvane, transportovane i primenjene. Svaki od ovih koraka mora da obuhvati protokole koji "e osigurati njihov integritet i kvalitet. Zbog toga se o!ekuje da se izradi i primeni ovih terapija pridje na isti na!in kao i proizvodnji drugih terapeutskih sredstava u farmaceutskoj industriji - kako uklju!uju"i odgovaraju"e fizi!ko okru$enje, tako i neophodne protokole i procedure obuhva"ene slo$enim sistemima kvaliteta. Ipak, u nizu svojih primena, "elijske terapije koje se koriste u medicini uglavnom su jo% uvek okarakterisane kao eksperimentalne terapije. Transplantacija mati!nih "elija iz periferne krvi (HSC) nakon ablativnih terapija je jedna od retkih ustanovljenih klini!kih procedura koja se ve" du$e vreme koristi sa ciljem da se uspostavi hematopoeza nakon inicijalne faze oporavka krvnih "elija u cirkulaciji i da se uspostavi njihov fiziolo%ki status i broj neophodan za suzbijanje infekcija, krvavljenja i anemije. Broj indikacija za primenu visokih terapijskih doza u hematolo%kim i drugim malignitetima je u porastu i u direktnoj je srazmeri sa potrebom za smanjenjem tro%kova ovakvih terapija, njihovom pristupa!no%"u i pojednostavljenjem rutinskih laboratorijskih procedura koje podrazumevaju zamrzavanje i !uvanje "elija. Adultne mezenhimske mati!ne/stromalne "elije se, za razliku od embrionalnih mati!nih "elija bez ve"ih eti!kih dilema tradicionalno dobijaju iz kostne sr$i i ne formiraju tumore, ali se relativno sporo umno$avaju i sa odredjenim ograni!enjima ulaze u proces diferencijacije. Mezenhimske "elije se relativno lako izoluju i uzgajaju. Zahvaljuju"i svojoj multipotentnosti, parakrinom efektu, imunomodulatornim svojstvima i migracijskim sposobnostima, ove "elije su u stanju da obezbede relativno nove pravce u razvoju "elijskih terapeutika. Dendriti!ne "elije (DC) su antigen prezentuju"e "elije prisutne u gotovo svim tkivima. Ove "elije prezentuju antigene na svojoj povr%ini i mogu indukovati imunski sistem aktiviraju"i CD8+ i CD4+ T "elije u okviru sna$nog anti-tumorskog odgovora. Nove generacije mati!nih "elija, kao %to je uspe%no reprogramiranje razli!itih telesnih tj. somatskih "elija u stanje pluripotentnosti bi moglo da omogu"i stvaranje pacijent- specifi!nih ili oboljenje- specifi!nih mati!nih "elija. Shodno svojim karakteristikama i ogromnom interesovanju istra$iva!a, indukovane pluripotentne mati!ne "elije (iPS) imaju potencijal da dramati!no uti!u na nove pristupe u razvoju terapeutika pre svega u regenerativnoj medicini. Primena mati!nih "elija nudi ogroman potencijal u le!enju kako naslednih oboljenja tako i ste!enih i degenerativnih oboljenja, ali idealan pristup i postupak za dobijanje ove vrste "elija jos uvek nije otkriven. U poredjenju sa nekim standardnim terapeutskim sredstvima, "elijske terapije nose druga!ije rizike, imaju veoma specifi!ne procese proizvodnje, kontrole kvaliteta i upotrebe, kao i mnoga ve" aktuelna ali i anticipirana eti!ka razmatranja. Nakon %to su analizirani zakonsko regulatorni modeli i opseg klini!kih istra$ivanja, prikazane savremene laboratorijske procedure i odr$ivi organizacioni sistemi za njihovu podr%ku – izvr%eno je teorijsko modelovanje, koje mo$e da se koristi u svrhe planiranja i predvidjanja; takodje je izveden prakti!ni model evaluacije klini!ke procedure u oblasti prikupljanja i obrade "elijskih terapija. Ovakav pristup do sad nije zabele$en u literaturi, %to izdvaja ovo istra$ivanje uz njegovu multidisciplinarnost i sveobuhvanost. Razvijena metodologija predstavlja zna!ajan doprinos u daljem razvoju klini!ko laboratorijskih modela za izradu novih terapeutskih sredstava baziranih na izradi i primeni "elijskih terapija, kao i za razvoj novih klini!kih istra$ivanja u istoj oblasti. Modelovanje karakteristika i procesa u okviru izrade i primene "elijskih terapija omogu"uje dalji razvoj teorijske platforme za unapredjenje novih klini!kih pristupa (upotrebe bioterapeutika), prakti!no izvodenje ovih skupih i kompleksnih procesa u strogo kontrolisanom okru$enju, u najkra"em mogu"em roku i uz najmanje mogu"e tro%kove - dovode"i time do njihove %ire primene i mogu"nosti u!e%"a projekata iz na%e zemlje u Evropskim fondovima za razvoj srodnih klini!kih istra$ivanja i inicijativa vezanih za napredne terapije (ATMPs). Klju!ne re!i: mati!ne "elije, "elijske terapije, laboratorijski protokoli, klini!ki pristupi, zakonske regulative. ABSTRACT Cellular therapies, cell and tissue-based therapies incl. tissue engineering encompasses a broad rapidly growing field of medicine, regenerative medicine and (bio)pharmaceutical industry that involves the manipulation and administration of cells for the treatment of disease. This field can be categorized in various ways, based on - the starting cell population, the type of cells generated in the process, the disease or organ targeted, the type of manipulation or the complexity of manipulation (e.g. basic or minimally-manipulated to highly-manipulated). Common features of all cell therapies (used as therapeutics) are requirements that source cells be identified, collected, processed, stored, transported, and administered. Each step must incorporate procedures that ensure that the integrity of the end product is maintained. Hence, approach to the production of a biopharmaceutical drug or advanced therapy medicinal product - including requirements that the entire process occur in appropriate physical environments with protocols and procedures linked to high-level quality systems. In many cases, the manipulation of human cells to produce cellular therapeutic products is, with a few exceptions, still considered an experimental therapy. Supportive reinfusion of haematopoietic stem and progenitor cells following ablative therapy ultimately aims to re-establish haematopoiesis following an initial recovery of end- stage blood circulating cells to a level necessary for reducing the risk of side-effects, such as infections, bleeding or anaemia. The expanding indications of high-dose therapy in hematologic and non-hematologic malignancies are associated with an increasing request for simplification and cost reduction of routine freezing procedures. Adult mesenchymal stem/ stromal cells (MSC) are traditionally derived from bone marrow free of ethical concerns and do not form tumours, but grow more slowly and with limited differentiation potential. MSC are relatively easy to isolate and expand in culture, and due to their multipotency, paracrine effects, immunomodulatory properties and migratory behavior, are likely to provide novel therapeutic alternatives. Dendritic cells (DC) are antigen-presenting cells present in nearly all tissues. Dendritic cells present antigens and can induce immune response by activating both CD8+ and CD4+ T cells to develop a potent antitumor response. Successful reprogramming of differentiated human somatic cells into a pluripotent state may allow creation of patient- and disease-specific stem cells. The data suggest that induced pluripotent stem cells (iPS) should be considered a unique subtype of pluripotent cells. The iPS have potential to revolutionise new therapeutic approaches and regenerative medicine, as they may be derived into all three germ layers in vitro but since derived from adult cells – the research and downstream applications are not encumbered by ethical objections. Stem cells hold great promise to treat a wide range of unmet medical needs in hereditary, acquired and degenerative human disease but the optimal source remains unclear. In comparison with some standard therapeutics (small molecules or protein-based drugs), cell therapies are not entirely characterised products, often manufactured/tested in “real-time”, have costly and labour- intensive laboratory processes, QA and QC methods. Production process of an effective cell therapy treatment is often long, expensive and individualized or tailored for a particular patient (e.g. autologous cell therapies), it cannot be obtained on a large scale, seldom easily characterised or prepared in advance. These caracteristics often bring additional risks and unknowns to the cell therapy application in the clinical area or the clinical research. Following analysis of regulatory framewoks, range and type of current clinical trials, three different laboratory and clinical protocols were analysed in addition to feasible organisational models supporting development of cell therapies projects and applications. Theoretical models were developed for the purpose of planning and forecasting; a practical model/ method was utilised to assess efficiency and reliablility of a clinical procedure used in cell therapy manufacturing. This approach and a range of used methodologies were not previously described in literature, which makes the reserach unique along with its mulidisciplinary and comprehensive features. Methodology used in this research could significantly contribute further advancement of clinical and laboratory protocols for new biotherapeutics through an efficient translational research approach. Proposed theoretical and practical models could be utilized both globally and locally. If implemented, it would enable establishing mechanisms necessary for obtaining EU funding for cell therapy-based and other advanced therapy medicinal products based (ATMPs) projects developed in the country. Key words: stem cells, cell therapies, laboratory protocols, clinical applications, regulatory frameworks. Nau!na oblast: Farmacija U"a nau!na oblast: Farmaceutska tehnologija / Socijalna farmacija i istra!ivanje farmaceutske prakse UDK broj: 611.018.1 : 577.2 : 616-85 (043.3) SADR!AJ Izvod Abstract Sadr"aj I OP#TI DEO 1.0 Uvod 1 1.1 Mati$ne %elije i %elijske terapije: definicija, razvoj, karakterizacija i pregled terminologije (eng. Definitions & Terminology) 1.1.1 Definicija: mati!ne "elije i druge "elijske terapije 4 1.1.2 Istorijski razvoj "elijskih terapija 5 1.1.3 Karakterizacija i tipovi mati!nih "elija i "elijskih terapija 7 1.1.3.1 Totipotentne mati!ne "elije 7 1.1.3.2 Pluripotentne mati!ne "elije 8 1.1.3.3 Multipotentne mati!ne "elije 8 1.1.3.3.1 Mati!ne "elije hematopoeze 9 1.1.3.3.2 Hematopoeza 10 1.1.3.3.3 Mati!ne "elije iz krvi pup!anika 12 1.1.3.3.4 Mezenhimske mati!ne/stromalne "elije 13 1.1.4 Terminologija 15 1.2 Metode u izradi i primeni mati$nih %elija i ostalih %elijskih terapija 17 (eng. Clinical & Lab Science) 1.2.1 Klini!ki pristupi u prikupljanju biolo#kog materijala za izradu mati!nih 17 "elija i "elijskih terapija 1.2.1.1 Separacija "elija krvi i drugih krvnih komponenti: afereza 21 1.2.1.2 Aspiracija "elija kostne sr$i 22 1.2.1.3 Alternativne metode u prikupljanju biolo#kog materijala za izradu 23 "elijskih terapija: upotreba placente 1.2.2 Izrada konvencionalnih terapija mati!nim "elijama uz minimalnu 24 manipulaciju (eng. Peripheral Blood Stem Cells,PBSC/Haematopoietic Stem Cells,HPC) 1.2.3 Slo$ene metode u izradi "elijskih terapija: visoko manipulisane "elije 24 1.2.4 Novi pristupi u izradi mati!nih "elija: indukovane pluripotentne "elije (iPS) 26 1.3 Kontekst i principi u izradi %elijskih terapija: farmacija, biotehnologija 27 i regenerativna medicina (eng. Biotechnology, Cell Therapy & Regenerative Medicine) 1.3.1 Kontekst 27 1.3.2 Problematika izrade "elijskih terapija 30 1.3.3 Osnovni principi kvaliteta u farmaceutskoj industriji i u izradi 33 naprednih terapija: dobra proizvodjacka praksa i sistemi kvaliteta 1.3.4 Principi regulatorne nauke u farmaciji i naprednim terapijama 34 (eng. Regulatory Science and Advanced Therapies) 1.4 Kvalitet, efikasnost i bezbednost: terapije mati!nim "elijama i 36 novi regulatorni principi (eng. Prinicples of Regulatory Science) 1.4.1 Globalni pristup i definicija osnovnih zakonskih regulativa 38 u izradi !elijskih terapija 1.4.1.1 Model zakonskih regulativa u Sjedinjenim Ameri"kim Dr#avama 38 1.4.1.2 Model zakonskih regulativa u Australiji 41 1.4.2 Model zakonskih regulativa u Evropskoj Zajednici 44 1.4.3 Model zakonskih regulativa u Srbiji 48 1.5 Novi pristupi i trendovi u istra#ivanju 50 1.5.1 Najnoviji pristupi razvoju terapija mati"nim !elijama i drugim 50 !elijskim terapijama (eng. Stats & Trends) 1.5.2 Primena kvalitativne metodologije istra#ivanja u analizi globalnog i 52 multidisciplinarnog pristupa razvoju !elijskih terapija (eng. Qualitative research) 1.5.3 Doprinos ovog istra#ivanja 54 2. Ciljevi istra#ivanja 56 II EKSPERIMENTALNI DEO 3. Dizajn i metode 58 3.1 Materijali 58 3.1.1 Materijali za komparativnu analizu 1 58 3.1.1.1 Materijali za analizu zakonsko-regulatornih modela 58 3.2.1.2 Materijali za analizu klini"kih istra#ivanja 60 3.1.2 Materijali za komparativne analize 2 i 3 60 3.1.2.1 Materijali za analizu modela laboratorijsko-klini"kih protokola 60 3.1.2.2 Materijali za analizu laboratorijsko-organizacionih modela 61 3.1.3 Materijali za komparativne analize 4 i 5 62 3.1.3.1 Materijali za analizu percepcije i problematike oblasti 62 3.1.4.1 Materijali za modelovanje pristupa i re$enja 63 3.2 Metode 63 3.2.1 Analiza dokumenata 64 3.2.1.1 Analiza zakonsko regulatornih modela 64 (KOMPARATIVNA ANALIZA 1) 3.2.1.2 Analiza klini"kih istra#ivanja 65 (KOMPARATIVNA ANALIZA 1) 3.2.2 Pojedina!na i uporedna analiza slu!aja 66 3.2.2.1 Pojedina"na analiza laboratorijsko-klini"kih protokola 66 (KOMPARATIVNA ANALIZA 2) 3.2.2.2 Uporedna analiza laboratorijsko-organizacionih modela 66 (KOMPARATIVNA ANALIZA 3) 3.2.3 Tematska i fenomenolo!ka analiza uz analizu izvodjenja teorije 66 bazirane na podacima iz prakse 3.2.3.1 Analiza percepcije i problematike oblasti: prakti!ni pristupi i re"enja 66 (KOMPARATIVNA ANALIZA 4) 3.2.3.2 Teorijsko modelovanje 67 (KOMPARATIVNA ANALIZA 5) 3.2.3.3 Evaluacija klini!ke metode 68 (KOMPARATIVNA ANALIZA 5) 4. Rezultati i diskusija 70 4.1 Komparativna analiza 1: Poredjenje sadr"aja zakonsko regulatornih 70 odredbi i ispitivanje obima i pravca klini#kih istra"ivanja 4.1.1 Sadr#aj reprezentativnih zakonskih regulativa i podzakonskih akata 70 4.1.2 Teme i koncepti reprezentativnih zakonskih regulativa i podzakonskih akata 75 4.1.3 Geografska distribucija klini!kih istra#ivanja 87 4.1.4 Trendovi u klini!kim istra#ivanjima prema tipu $elijskih terapija 90 4.2 Komparativna analiza 2: Karakterizacija laboratorijsko klini#kih protokola 95 4.2.1 Protokol za izradu i primenu mati!nih $elija hematopoeze izolovanih 96 iz periferne krvi 4.2.2 Protokol za izradu i primenu mezenhimskih mati!nih/stromalnih $elija izolovanih 100 iz placente 4.2.3 Protokol za izradu i primenu anti-tumor vakcina baziranih na primeni antigen 104 prezentuju$ih dendriti!nih $elija 4.3 Komparativna analiza 3: Struktura i primena specifi#nih organizacionih modela 108 4.3.1 Karakteristike ne-komercijalnog organizacionog modela 108 4.3.2 Karakteristike komercijalnog organizacionog modela 109 4.3.3 Karakteristike kombinovanog organizacionog modela 111 4.4 Komparativna analiza 4: Procena percepcije i problematike oblasti 114 4.4.1 Segmenti podataka (1): 115 Poslovni i finansijski aspekti uz osnovna razmatranja (eng. Business Models, Funding, Strategic Decision Making & Main Considerations) 4.4.2 Segmenti podataka (2): 118 Uspostavljanje saradnje sa partnerima i druga razvojna problematika (eng. Partnerships, Main Challenges for the Industry, Science, Technology, Tech Transfer & Up Scaling) 4.4.3 Segmenti podataka (3): 121 Primena zakonsko regulatornih odredbi i percepcija njihovog razvoja (eng. Regulatory Compliance & Regulatory Landscape) 4.4.4 Segmenti podataka (4): 122 Osnovne teme i kategorije (eng. Frequent themes and categories) 4.4.5 Segmenti podataka (5): 124 Utemeljenje rezultata u podacima (eng. “Grounding” the data) 4.5 Komparativna analiza 5: Modelovanje pristupa i re!enja u svrhu 131 predvidjanja i planiranja 4.5.1 Teorijski model 1 131 4.5.2 Teorijski model 2 132 4.5.3 Prakti!ni model: evaluacija klini!ke metode 137 4.6 Diskusija 141 5. Zaklju"ci 156 6. Literatura i-xii 7. Prilozi tezi Prilog 1: Lista skra"enica i definicija Prilog 2: Re!nik termina i izraza (englesko- srpski) Prilog 3: Lista tabela i slika Prilog 4: Visoko manipulisane "elije MSC Prilog 5: Visoko manipulisane "elije DC Prilog 6: Regulatorne agencije (FDA, TGA, EMA) Prilog 7: Ostali regulatorni standardi, preporuke i pravilnici Prilog 8: Mogu"nosti primene i kompleksnost "elijskih terapija Prilog 9: Metodolo#ki pregled Prilog 10: Methodology Overview (eng.) Prilog 11: Primer standardne operativne procedure za izradu minimalno manipulisanih "elija Prilog 12: Primer standardne operativne procedure za izradu visoko manipulisanih "elija 1 Prilog 13: Primer standardne operativne procedure za izradu visoko manipulisanih "elija 2 Prilog 14: Pregled najva$nijih GMP elemenata u izradi visoko manipulisanih "elija za primenu u klini!ke svrhe Prilog 15: Primer liste za GMP inspekciju Prilog 16: Rezultati korelacione analize dokumenata Prilog 17: Dodatni rezultati iz analize intervjua Prilog 18: Dodatne mape iz pretrage baze podataka Prilog 19: Biografija autora Prilog 20: Izjava o autorstvu Prilog 21: Izjava o istovetnosti #tampane i elektronske verzije doktorske disertacije Prilog 22: Izjava o kori#"enju 1 I OP!TI DEO 1.0 Uvod !elijske terapije (eng. cell or cellular therapies) i razvoj regenerativne medicine (eng. regenerative medicine) obuhvataju novo polje u oblasti medicinskih, (bio)farmaceutskih i biolo"kih nauka. Bazirane su na upotrebi mati#nih (eng. stem cells) i drugih vrsta $elija u terapeutske svrhe. U budu$nosti se o#ekuje njihova ekspanzija i sve "ira klini#ka primena§. Primena $elijskih terapija uklju#uje prikupljanje, obradu i #uvanje razli#itih tipova $elija kao novih terapeutskih sredstava (Burger, 2003b; Wall, 2003; Prince, 2004) "to ih na odredjeni na#in svrstava ne samo u oblast biofarmaceutskih nauka i izrade bioterapeutika (eng. biotherapeutics or biopharmaceuticals) ve$ i srodnih zakonsko regulatornih nau#nih oblasti* (eng. regulatory science) § (Burger, 2003b; Wall, 2003; Pamphilon, 2004; Dutton, 2007; Marwaha, 2007; Ilic, 2012a; Ilic, 2012b). U naj"irem smislu, mati#ne $elije se mogu razvrstati na embrionalne (eng. embrional stem cells) i adultne* (eng. adult stem cells) dok se $elijske terapije mogu kategorisati na osnovu nekoliko veoma razli#itih pristupa - prema po#etnoj populaciji $elija koju koriste (npr. adipozne $elije ili $elije placente), prema tipu $elija koji $e biti kultivisan tokom procesa njihove izrade (npr. mezenhimske $elije), na osnovu bolesti ili organa na koji se primenjuju (npr. le#enje neurodegenerativnih, autoimunskih oboljenja ili regeneracija sr#anog mi"i$a nakon infarkta miokarda) ili na osnovu nivoa manipulacije koji uklju#uju (od minimalno do visoko manipulisanih). U nizu svojih primena, $elijski bioterapeutici* koji se koriste u medicini su jo" uvek uglavnom okarakterisani kao eksperimentalni deo terapije. Izuzetak predstavljaju transplantacije mati#nih $elija iz kostne sr%i i periferne krvi #iji se __________ § 2020: A New Vision – A Future for Regenerative Medicine, US Department of Health and Human Services: www.dhhs.gov/reference/newfuture.shtml ^ Guidance for industry: PAT – A framework for innovative pharmaceutical development, manufacturing, and quality assurance (2004): http://www.fda.gov.cder/guidance/6419 * Dalja terminolo"ka razmatranja se nalaze u posebnom poglavlju ove doktorske teze. 2 protokoli neprekidno usavr"avaju (Serke, 1997; Balint, 1999; Hubel, 2001; Sartor, 2005; Hubel, 2006; Parkins, 2006; Rowley, 2009) a koje se ve$ vi"e decenija rutinski primenjuju u le#enju bolesnika sa malignim oboljenjima krvi, autoimunskim poreme$ajima i nekim tumorskim oboljenjima (Godlman, 1978; Reiffers, 1988; Eaves, 1992; Perseghin, 1997; Haylock, 1997; Roddie, 2002; Mimeault, 2006). Mati#ne $elije i druge vrste $elija, koje se koriste u terapeutske svrhe ili za klini#ka istra%ivanja, moraju da budu okarakterisane, prikupljene, procesovane, #uvane, transportovane i primenjene u strogo kontrolisanim uslovima (Wall, 2003; Pamphilon, 2004; Dutton, 2007; Marwaha, 2007; Ilic, 2012a; Ilic 2012b). Svaki od ovih koraka trebalo bi da obuhvati protokole koji $e osigurati njihov integritet i kvalitet. Sa zakonsko regulatornog aspekta, o#ekuje da se izradi i primeni $elijskih terapija pristupi na isti na#in kao i proizvodnji drugih terapeutskih sredstava u farmaceutskoj industriji - kako uklju#uju$i odgovaraju$e fizi#ko okru%enje, tako i laboratorijske procedure obuhva$ene slo%enim sistemima kvaliteta u skladu sa propisanim zakonskim regulativama (Burger, 2000; Serke, 2001; Burger, 2003a; FDA, 2004^; Halme, 2006; Areman, 2009; Ilic, 2012). Izrada $elijskih terapija predstavlja kako tehnolo"ki tako organizacioni i zakonsko regulatorni izazov (Wall, 2003; Areman, 2009). Modeli i modaliteti usmereni na zakonske regulative, koji uklju#uju jasno definisane i sveobuhvatne slo%ene sisteme kvaliteta, pokazali su se kao odr%ivi pristup i kao preduslov za uspe"ne projekte u izradi i primeni $elijskih terapija na globalnom nivou. Da li $e ove terapije dopreti do pacijenata, bilo u klini#koj primeni ili u klini#kom istra%ivanju, u krajnjoj instanci zavisi od adekvatno kontrolisanih, organizaciono-finansijski isplativih tehnologija za njihovu izradu i aplikaciju (Mansbridge, 2006; Weber, 2006; Kirouac, 2008; Areman, 2009). Ova doktorska disertacija obuhvata komparativnu studiju procesa, sistema i regulativa u izradi i primeni mati#nih $elija i drugih $elijskih terapija kao novih terapeutskih sredstava namenjenih za primenu u klini#kim istra%ivanjima i za rutinsku klini#ku primenu. Osnovni cilj istra%ivanja je da ponudi kako detaljan i sveobuhvatan protokol izrade i primene vi"e vrsta $elijskih bioterapeutika tako i modelovanje 3 rezultata uporedne i fenomenolo"ke/ tematske analize u svrhu nastanka novih teorijskih pristupa i prakti#nih modela§§. Ovo se obezbedjuje na osnovu #etiri do pet kvalitativnih analiza, koje u okviru istra%ivanja uklju#uju: -dokumentarnu analizu i pregled zakonsko regulatornih okvira u Evropi, Sjedinjenim Ameri#kim Dr%avama (SAD) i Australiji; -pregled baze podataka klini#kih istra%iva#kih projekata baziranih na primeni mati#nih $elija i drugih $elijskih terapija u istom geografskom podru#ju; -pojedina#nu analizu laboratorijsko-klini#kih procesa upotrebom case study metodologije, -uporednu analizu razli#itih laboratorijsko-organizacionih modela (cross-case study) i fenomenolo"ku/ tematsku analizu (thematic analysis) transkripata intervjua sa stru#njacima koji su ili direktno uklju#eni u izradu $elijskih terapija za klini#ku primenu ili se bave oblastima razvoja bazi#nih i klini#ko- primenljivih biolo"kih i medicinskih istra%ivanja, bioetike i strate"kog razvoja $elijskih terapija za klini#ku primenu§§. Obzirom da je izrada efikasnih $elijskih bioterapeutika #esto dug, kompleksan i skup proces, visoko prilagodjen svakom pacijentu tj. individualizovana terapija (eng. individually customized therapy) - nije uvek mogu$e obezbediti adekvatne uslove za njihovu pripremu (Tran, 2007; Mooney, 2008; Parson, 2008; Areman, 2009). Pored toga, u procesu izrade $elijskih terapija uglavnom je te"ko u potpunosti okarakterisati finalni proizvod ili pripremiti ga unapred za specifi#nu svrhu npr. za odredjeno oboljenje ili odredjenog pacijenta (Yamanaka, 2007). Slo%enost zakonsko regulatornih pristupa i modaliteta na globalnom nivou (Ilic, 2013a, 2013b), raznovrsnost nau#no-tehnolo"kih pristupa i rizici u klini#koj primeni $elijskih terapija diktiraju visoko definisane laboratorijske procedure za njihovu izradu, obezbedjivanje zna#ajnih nov#anih sredstava i anga%ovanje tima stru#njaka u okviru svakog pojedina#nog projekta. Medjutim, ovakva slo%enost i izvanredno visoka finansijska ulaganja, kao i brojni rizici u klini#koj upotrebi $elijskih bioterapeutika, mogli bi se zna#ajno umanjiti primenom standardizovanih procesa i sistema, od kojih su neki ponudjeni u ovoj doktorskoj disertaciji. __________ §§ Dalja metodolo"ka razmatranja se nalaze u posebnom poglavlju ove doktorske teze. 4 1.1 Mati"ne #elije i #elijske terapije: definicija, razvoj, karakterizacija i pregled terminologije (eng. Definitions & Terminology) 1.1.1 Definicija: mati"ne #elije i druge #elijske terapije Mati#ne $elije imaju potencijal da se razviju u vi"e $elijskih tipova u organzimu tokom procesa ranog rasta i razvoja. Uz to, slu%e kao unutra"nji sistem za obnovu jer su sposobne za prakti#no neograni#eni broj deoba pri tome obnavljaju$i druge $elije u organizmu tokom zivota. Kada se mati#na $elija podeli, svaka nova $elija ima potencijal da ostane mati#na $elija ili da se diferencira u neki drugi $elijski tip (uz ostvarivanje specijalizovane funkcije). Mati#ne $elije se razlikuju od ostalih $elijskih tipova na osnovu dve osnovne karakteristike. Prvo, one su nespecijalizovane $elije sposobne da se samoobnavljaju kroz deobu, ponekad i nakon vrlo dugog perioda neaktivnosti. Drugo, u odredjenim fiziolo"kim ili eksperimentalnim uslovima, mati#ne $elije mogu biti usmerene ka razvoju $elija nekog specijalizovanog tkiva ili organa. U nekim organima, kao "to je na primer epitel creva ili kostna sr%, mati#ne $elije se neprestano dele i obnavljaju razli#ite $elijske tipove u tim tkivima ili organima. U drugim organima, kao "to je na primer srce ili pankreas, mati#ne $elije se dele samo u specifi#nim situacijama, uglavnom nakon povrede ili o"te$enja. Do nedavno nau#nici su se uglavnom bavili istra%ivanjem dve vrste mati#nih $elija kod ljudi i na %ivotinjskim modelima – primenjuju$i tako osnovnu podelu na embrionalne mati#ne $elije (eng. embryonic stem cells, ESC) i mati#ne $elije ne- embrionalnog porekla ili adultne mati#ne $elije (eng. somatic/ adult stem cells, ASC). Tehnika izolovanja embrionalnih mati#nih $elija iz embriona mi"a otkrivena je jo" 1981. godine od strane dve istraziva#ke grupe. Martin Evans i Matthew Kaufman, (eng. Department of Genetics, University of Cambridge), objavili su u julu 1981. godine novu tehniku za kultivisanje $elija izolovanih iz uterusa mi"a pri tome uzgajaju$i embrionalne $elije izolovane iz embriona. Zatim, u decembru iste godine Gail Martin, (eng. Department of Anatomy, University of California, San Francisco), objavljuje svoj rad sa sli#nim rezultatima i po prvi put koristi termin embrionalne mati#ne $elije (eng. Embryonic Stem Cell, EMS) (Martin, 1981). Nakon iscrpnih istra%ivanja, grupa istra%ivaca pod rukovodstvom James Thomson-a, (eng. University of Wisconsin-Madison) 1998. godine uspostavila je prvu tehniku izolacije i kultivacije embrionalnih mati#nih $elija iz ljudskog embriona (Thomson, 1998). 5 U unutra"njem sloju embriona starog 3 do 5 dana, koji se naziva blastocist (eng. blastocyst), nalaze se $elije koje svojom deobom grade sve slojeve specijalizovanih $elija u organima kao "to su srce, plu$a, ko%a, reproduktivni organi i sva druga tkiva. Smatra se da su ove, ili veoma sli#ne $elije, prisutne u kostnoj sr%i, mi"i$ima, mozgu i drugim delovima odraslog organizma kao izvestan broj adultnih mati#nih $elija koje tokom #itavog %ivota nadoknadjuju razli#ite $elijske tipove u datim organima – usled normalnog obnavljanja $elijskih populacija ili zbog nastalih povreda i oboljenja. Embrionalne mati#ne $elije ljudi su u proteklom periodu intenzivno kori"$ene u procesima vantelesne oplodnje i u istra%iva#ke svrhe. Pri tome su izazvale ogromnu pa%nju nau#ne i op"te javnosti zbog potencijalnih dilema u okviru eti#kih i zakonskih na#ela njihove primene. Od 2006. godine pa%nja nau#nika i javnosti je ponovo usmerena na adultne mati#ne $elije jer je otkrivena tehnika genetskog “reprogramiranja” diferenciranih $elija odraslog organizma (npr. fibroblasta ko%e) pri #emu se ve$ diferencirane $elije vra$aju u svoje “mati#no” stanje pri tome izra%avaju$i osobine pluripotentnih mati#nih $elija embriona. Ovaj tip $elija je nazvan indukovanim pluripotentnim mati#nim $elijama (eng. induced pluripotent stem cells, iPS/ human-induced pluripotent stem cells, hiPSC) (Patterson et al., 2012). Danas se iPS $elije koriste u nizu istra%ivanja pri tome, do odredjene mere, zamenjujuci kontraverznu upotrebu ljudskih embrionalnih mati#nih $elija. 1.1.2 Istorijski razvoj #elijskih terapija !elijska teorija predstavlja fundamentalni apsekt moderne biologije, obavezni i implicitni deo na"eg razumevanja strukture organizama, mehanizma nasledjivanja, razvoja i diferencijacije, jedinice %ivota od najednostavnijih do slo%enih organizama i teorije evolucije. Re$i da je $elija osnovna jedinica %ivog sveta predstvalja istovremeno izuzetnu generalizaciju koja objedinjuje istra%ivanje: strukture i funkcije, reprodukcije i nasledjivanja, rasta i diferencijacije. Ipak, predstavljaju$i osnovu telesne organizacije pomo$u $elija i $elijskih produkata - $elijska teorija obezbedjuje samo jedan od mogu$ih odgovora na pitanje - !ta je organizam? (Magner, 2002). 6 Tridestih godina 19. veka Theodor Schwann (1810-1882) primenjuje $elijsku teoriju (do tada prihva$enu samo na biljnim organizmima) na %ivotinjski organizam. Schwann sumira svoj rad i generalizaciju $elijske teorije kao univerzalnog principa svih delova tela. Osim toga on opisuje plasti"nost kao mogu$nost molekula da formiraju $eliju, kao i hemijske promene komponenti $elije (ili njenog okru%enja) koji se naziva metabolizam (od gr#ke re#i koja opisuje “ono "to zavisi od okru%enja ili ono "to je podlo%no promeni”) (Magner, 2002). Moderna $elijska teorija $e kasnije biti izvedena od strane niza nau#nika kao "to su Karl Nägeli, Hugo Von Mohl i Rudolf Virchow. 1855. godine Virchow objavljuje svoj rad “!elijska patologija” u kome ne samo navodi $eliju kao osnovnu kariku u slo%enom lancu hijerarhije izmedju tkiva, organa, sistema organa i kompletnog organizma, ve$ postavlja zna#ajno pitanje: gde se u organizmu, na $elijskom nivou, nalazi bolest? (Magner, 2002). Pri tome Virchow dovodi u vezu osnovne procese patologije i osnovne procese fiziologije kao neodvojive – na primer rad na polju leukemije pokazuje da se kancerske $elije ne razlikuju od normalnih $elija toliko po svojoj strukturi koliko po svom pona"anju. Nema#ki (pruski) nau#nici su jo" sredinom 19. veka, posmatraju$i preseke kostne sr%i ispod mikroskopa, primetili nekoliko razli#itih tipova $elija. Medju njima je, bez obzira na njihovu o#iglednu razli#itost, postojala evidentna 'familijarnost' odnosno srodnost. Izgledalo je ne samo da svaka $elija postaje od iste takve $elije, ve$ i da grupa $elija postaje tj. proisti#e od jedne 'izvorne' ili osnovne (mati#ne) $elije (eng. 'one cell had stemmed form another'). Jedan od najistaknutijih pruskih nau#nika ovog perioda, Rudolf Ludwig Karl Virchow (1821-1902) postavio je veoma zna#ajnu konstataciju da svaka $elija proizilazi iz iste takve $elije (lat. Omnis cellula e cellula; eng. all cells derive from other cells) koju je objavio 1858. godine (Silver, 1987). Smatra se da termin 'stem' ili mati#na $elija vodi poreklo iz ovih istra%ivanja u 19. veku (Magner, 2002). Naziv 'adultne' mati#ne ili stem $elije nije u potpunosti adekvatan. Adultne mati#ne $elije postoje kako u organizmu odrasle osobe tako i u fetusu i u organizmu novorodjen#eta. Ove $elije obnavljaju sve $elijske populacije u __________ http://stemcells.nih.gov/ National institute of Health, USA. Patterson M, Chan DN, Ha I et al. Defining the nature of human pluripotent stem cell progeny. Cell Res. 2012 January; 22(1): 178–193. 7 organizmu, u svim stupnjevima njegovog razvoja i postoje u svakom organu (Bjornson, 1999; Mezey, 2003; Mitalipov & Wolf, 2009; Witkowska-Zimny & Wrobel, 2011). Prvobitno se smatralo da su organ-specifi#ne ali krajem 20. i pocetkom 21. veka u nizu eksperimenata se pokazalo da populacija mati#nih $elija iz jednog organa mo%e da se razvije u funkcionalne $elije nekog drugog organa u organizmu (Bjornson, 1999; Mezey, 2003). Danas se smatra da ovde ipak postoje odredjeni ograni#avaju$i faktori (Fox, 2007; Finkel, 2005) i da su deoba i diferenciranje mati#nih $elija iz jednog organa manje uspe"ni (ili nepotpuni, uklju#uju$i termin fuzija $elija) pri diferencijaciji u tkiva drugih organa (Mitalipov & Wolf, 2009; Witkowska-Zimny & Wrobel, 2011); ovo pitanje jo" uvek ostaje delimi#no nerazja"njeno#. 1.1.3 Karakterizacija i tipovi mati"nih #elija i #elijskih terapija Mati#ne $elije su, pre svega, definisane svojom sposobno"$u da se samoobnavljaju i da se diferenciraju u progenitorske $elije (eng. progeny/daughter cells) iz jednog ili vi"e germinalnih slojeva (eng. germ layers). Mati#ne $elije se mogu klasifikovati kao a) totipotentne (eng. totipotent), b) pluripotentne (eng. pluripotent) ili c) multipotentne (eng. multipotent). 1.1.3.1 Totipotentne mati"ne #elije Najprimitivnije mati#ne $elije koje poseduju najve$i ili prakti#no neograni#eni kapacitet za diferencijaciju, su totipotentne $elije zigota ili prve blastomere (Mitalipov & Wolf, 2009). Ove $elije nastaju u prvoj deobi zigota (oplodjene jajne $elije) i imaju sposobnost da formiraju #itav organizam jedinke. Totipotentne $elije zigota, na osnovu svoje deobe, formiraju embrion i placentu. Embrion je na nivou od 32 $elije (morula) sagradjen od $elija koje su izgubile svoju totipotentnost i koje se __________ Analiza bilo kog nau#nog problema automatski vodi ka izu#avanju njegove istorije (eng. an analysis of almost any scientific problem leads automatically to a study of its history)*. Nau#no saznanje samo po sebi, van svog dru"tvenog i kulturolo"kog konteksta, mo%e u pojedinom slu#aju da izgleda pojednostavljeno i banalno. Ukoliko se nau#no saznanje posmatra kao ljudski izum i dinami#ki koncept koji obuhvata niz saznanja, ono postaje na#in da se sagleda univerzum (Magner, 2002). * Ernst Walter Mayr (1904 - 2005), jedan od vode$ih evolutivnih biologa 20. veka koji je u#estvovao u nau#noj sintezi moderne teorije evolucije. # 'No consensus on stem cells' editorial in Nature 2004, 428: 587. 8 smatraju pluripotentnim (Mitalipov & Wolf, 2009, Witkowska-Zimny & Wrobel, 2011). Ovde prisutne pluripotentne $elije izgradjuju tri germinalna sloja embriona: endoderm, mezoderm i ektoderm (eng. endoderm, mesoderm & ectoderm). 1.1.3.2 Pluripotentne mati"ne #elije Pluripotentnim $elijama se smatraju embrionalne mati#ne $elije (eng. embryonic stem cells, ESC) koje se razvijaju iz unutra"njeg sloja zigota u ranom embrionalnom razvoju (eng. inner cell mass). Ove pluripotentne $elije su sposobne da izgrade sva tri germinalna sloja (endoderm, mezoderm i ektoderm) i da svojom deobom obezbede $elije za razvoj svih tkiva i organa u organizmu jedinke. Indukovane pluripotentne $elije (eng. induced pluripotent stem cells, iPS) imaju mnogo sli#nih karakteristika sa embrionalnim mati#nim $elijama ali su nastale re- programiranjem adultnih $elija kao "to su fibroblasti ili $elije placente (Mitalipov & Wolf, 2009). Pokazano je da indukovane pluripotentne $elije imaju mogu$nost da se dele u neograni#enom broju i da formiraju benigna teratomska tkiva sva tri germinalna sloja, "to ih svrstava u pluripotentne $elije. Diferencirane progenitorske $elije koje poti#u iz embrionalnih mati#nih $elija i indukovanih pluripotentnih $elija (na primer, neuroni, kardiomiociti i hepatociti) mogu biti odba#ene od strane imunskog sistema alogenog primaoca i zahtevaju primenu imunosupresivnih lekova da bi se izbegla ova ne%eljena reakcija. Uz to postoji niz eti#kih dilema koje prate upotrebu embrionalnih mati#nih $elija u klini#ke istra%iva#ke svrhe; ovo nije slu#aj sa iPS $elijama koje nastaju re-programiranjem adultnih $elija. Ipak, klini#ka istra%ivanja su ve$ neko vreme u toku uz upotrebu neurona koji su nastali manipulacijom embrionalnih mati#nih $elija, kao i drugih vrsta $elija^. 1.1.3.3 Multipotentne mati"ne #elije Mati#ne $elije sa ograni#enom mogu$no"$u diferencijacije se nazivaju multipotentnim mati#nim $elijama. Smatra se da ove mati#ne $elije mogu da se diferenciraju samo u odredjeni broj tkiva koja poti#u iz istog germinalnog sloja. Multipotentne mati#ne $elije su usmerene ka razvoju odredjenog organa ili tkiva i u tom smislu predstavljaju najzreliji tip mati#nih $elija (Witkowska-Zimny & Wrobel, 2011). __________ ^ www.geron.com; www.clintrials.gov 9 1.1.3.3.1 Mati"ne #elije hematopoeze Najpoznatiji primer multipotentnih mati#nih $elija su mati#ne $elije hematopoeze (eng. Hematopoietic Stem Cells, HSC) koje se mogu izolovati iz kostne sr%i, krvi pup#anika ili iz periferne krvi. Ne"to manje poznata #injenica je da je placenta takodje zna#ajan izvor mati#nih $elija hematopoeze, "to je do sada dokazano na nekim %ivotinjskim modelima (samo u odredjenom stupnju embrionalnog razvoja) (Gekas et al., 2008, Gekas et al., 2010). Mati#ne $elije hematopoeze predstavljaju izvor svih $elija krvi i konstantno obnavljaju krvni i imunski sistem tokom %ivota jedinke. Ovo su istovremeno najbolje okarakterisane adultne mati#ne $elije i jedine mati#ne ili progenitorske $elije koje se danas koriste u rutinskoj klini#koj praksi (Appelbaum, 2007; Areman, 2009). Transplantacija mati#nih $elija hematopoeze mo%e da se koristi kao tretman za niz imunodeficijentnih oboljenja, malignih oboljenja krvi (leukemije, mijelodisplazije i mijeloma) i za neka od oboljenja kostne sr%i (aplasticne anemije). Pionirski rad u ovoj oblasti zapo#eo je jos pedesetih godina pro"log veka da bi se prva uspe"na alogena transplantacija kostne sr%i izvr"ila 1968. godine. Danas se putem tehnika afereze (separacijom $elija krvi) obezbedjuju mati#ne $elije hematopoeze iz periferne krvi koje su naj#e"$i izvor za transplantaciju. Sve ove tehnike transplantacije iziskuju testiranje na HLA markere (eng. human leukocyte antigen, HLA) da bi se izbegle ne%eljene reakcije kao "to je potencijalno fatalna reakcija graft- versus-host disease, GVHD, koja nastaje usled imunske reakcije od strane leukocita davaoca koji ‘odbacuju’ odnosno ‘napadaju’ tkiva primaoca (Vander Lugt et al., 2013). &ezdesetih godina 20. veka, nakon iskustava sa o"te$enjima koje ljudskom organizmu nanosi nuklearno zra#enje, nau#nici su u nastojanjima da le#e radijacijska oboljenja spoznali da odredjene $elije krvi ne samo mogu svojom deobom da obnove razli#ite populacije krvnih $elija, ve$ i da se 'samoobnavljaju' tj. obnavljaju svoju sopstvenu $elijsku populaciju (Becker, 1963). Neke to #ine samo u ograni#enom, kra$em vremenskom periodu (kasnije nazvane progenitorne krvne $elije) a neke u neograni#eno dugom vremenskom periodu (danas okarakterisane kao mati#ne $elije krvi) (Becker, 1963; Bradford, 1997; Areman, 2009). Iako se svi nau#nici ne sla%u da je uop"te mogu$e izolovati 'apsolutnu mati#nu' $eliju krvi, za sada je op"teprihva$eno stanovi"te da se mati#na $elija krvi deli u proseku jednom u toku mesec dana daju$i dve nove $elije. Jedna od njih ostaje kao mati#na $elija krvi a druga se diferencira u 10 progenitornu krvnu $eliju koja se u toku svog %ivota deli jo" nekih nekih dvadesetak puta obnavljaju$i pritom sve populacije zrelih diferenciranih krvnih $elija (Bradford, 1997; Areman, 2009). 1.1.3.3.2 Hematopoeza Hematopoeza je izuzetno kompleksan proces stvaranja uobli#enih elemenata krvi, koji bez obzira na njihovu morfolosku i funkcionalnu raznolikost i specijalizovanost, vode poreklo od zajedni#ke pluripotentne mati#ne $elije hematopoeze (Petakov, Bela, Bugarski, 2004; Areman, 2009). Heterogena $elijska populacija mati#nih $elija hematopoeze predstavlja 'razvojni kontinuum' $elija razli#itog nivoa zrelosti; opisuje se se kao populacija piramidalne organizacije koju #ine tri glavna odeljka: pluripotentne mati#ne $elije, opredeljene mati#ne $elije i morfolo"ki prepoznatljive, ali jos uvek nezrele $elije (slika 1.1). Legenda: HSC, Hematopoietic Stem Cell; CMP, Common Myeloid Progenitor; LMPP, Lymphoid-primed Multipotential Progenitor; MEP, Megakaryocyte-Erythroid Progenitor; GMP, Granulocyte-Macrophage Progenitor; CLP, Common Lymphoid Progenitor; Other commited cells: EP, MkP, GP, MacP, Pro-B, Pro-T, Pro-NK. Slika 1.1 Razvoj i hijerarhija mati#nih $elija hematopoeze (preuzeto iz: Nature Review Immunology Vol.6 Feb 2006). 11 Razvoj brojnih sofisticiranih tehnika izdvajanja $elija omogu$io je neposredan eksperimentalni pristup pojedinim kategorijama ovih $elija a samim tim i veliki napredak u izu#avanju (i terapijskoj primeni) mati#nih $elija hematopoeze. Ove tehnike su zasnovane na razli#itim pristupima kao sto su fizi#ko/hemijska izolacija mati#nih $elija, imunocitohemijska identifikacija povr"inskih markera mati#nih $elija i odredjivanje njihove metaboli#ke aktivnosti (Petakov, Bela, Bugarski, 2004; Areman, 2009). Plasti#nost je jedna od osnovnih karakteristika mati#nih $elija hematopoeze. Ovo podrazumeva da put njihove diferencijacije nije striktno odredjen i da samim tim one ispoljavaju neo#ekivani potencijal transformacije u druga tkiva. Najmanje tri svojstva mati#nih $elija hematopoeze karakteri"u njihovu plasti#nost: svojstvo odr%avanja ravnote%e izmedju procesa samoobnove i procesa diferencijacije, svojstvo da mogu da se diferenciraju u najmanje deset razli#itih zrelih hematopoeznih $elijskih tipova, i svojstvo regulacije sopstvene proliferativne aktivnosti (Lemischka, 2002). Smatra se da hematopoetske $elije ispoljavaju dve vrste plasti#nosti – intrahematopoeznu (unutra"nju ili linijsku plasti#nost) i intersistemsku plasti#nost (sposobnost da se diferenciraju u $elije miokarda, $elije jetre, epitelne i nervne $elije) (Lagasse et al., 2000; Orlic et al., 2001). Ova karakteristika je zapa%ena nakon transplantacije $elija kostne sr%i tako da se ne mo%e sa sigurno"$u re$i da li diferencijacija zapo#inje od hematopoeznih mati#nih $elija ili od mezenhimskih mati#nih $elija (ili od angiogenih prethodnika) prisutnih u kostnoj sr%i. Gotovo sve stromalne $elije hematopoezne mikrosredine (osim makrofaga) su embrionalno-mezenhimalnog porekla (kao i endotelske $elije, fibroblasti, adipociti, osteoblasti, miociti i adventicijske $elije retikuluma). Sli#no hematopoeznim $elijama, stromalne $elije hematopoezne mikrosredine nastaju od jedne zajedni#ke pluripotentne mezenhimske mati#ne $elije (eng. Mesenchymal Stem/Stromal Cell, MSC) (Gamiche et al., 1993). Ovo $e biti razmatrano u slede$em poglavlju. 12 1.1.3.3.3 Mati"ne #elije iz krvi pup"anika Mati#ne $elije iz krvi pup#anika (eng. Umbilical cord blood stem cells, UCBSC) se danas sve vi"e koriste kao izvor mati#nih $elija hematopoeze. Izolacija i klini#ka primena mati#nih $elija hematopoeze iz pup#anika je posebno zanimljiva sa klini#kog stanovi"ta. Ove mati#ne $elije su relativno lako dostupne, prikupljaju se ne- invazivnom metodom i danas se privatne i javne banke krvi iz pup#anika (eng. cord blood banks) nalaze u celom svetu (Bradley & Cairo, 2005). Ono "to je najva%nije sa klini#kog stanovi"ta je da mogu biti kori"$ene sa manje optimalnom kompatibilno"$u HLA-markera, u poredjenju sa mati#nim $elijama iz kostne sr%i i periferne krvi. Ovo je mogu$e usled imunolo"ke 'naivnosti' mati#nih $elija hematopoeze izolovanih iz pup#anika i njihove takozvane ontogenetske primitivnosti u poredjenju sa drugim tipovima mati#nih $elija hematopoeze (Haylock & Nilsson, 2007). Sve mati#ne $elije hematopoeze, uklju#uju$i i one izolovane iz krvi pup#anika, mogu biti okarakterisane prisustvom povr"inskog glikoproteinskog markera CD34 (Andrews et al., 1992, Okuno et al., 2002, Osawa et al., 1996). Poznato je da se populacija CD34+ $elija sastoji od ve$eg dela ve$ delimi#no diferenciranih progenitorskih $elija (eng. committed progenitor cells) uz koje se nalazi manje od 1% istinski primitivnih $elija (Wognum et al., 2003). Zbog toga se u laboratotijskim i transplantacionim esejima CD34 marker #esto koristi zajedno sa drugim markerima kao "to su Lin-, CD38- i CD90+ (Park et al., 2008). Uz upotrebu povr"inskih markera razvijene su i druge (funkcionalne) tehnike da bi se identifikovala populacija $elija bogata mati#nim $elijama hematopoeze, kao "to je primena fluorescentnih boja (Hoechst-33342, Ho i Rhodamine-123, Rho) (Bertoncello et al., 1988; Bertoncello and Williams, 2004; Goodell et al., 1996; Li & Johnson, 1995; Schroeder, 2010; Wognum et al., 2003). Najpouzdaniji testovi za utvrdjivanje kvaliteta mati#nih $elija hematopoeze su ipak oni koji se obavljaju in vivo, pri #emu se na %ivotinjskom modelu npr. dugoro#no letalno ozra#enih mi"eva (eng. lethally irradiated, >3 months) testira sposobnost transplantiranih mati#nih $elija da uspostave razvoj $elijske populacije hematopoeze. Jedan od ograni#avaju$ih faktora u upotrebi mati#nih $elija hematopoeze izolovanih iz krvi pup#anika predstavlja njihov ukupan broj, u odnosu na broj $elija neophodnih za uspe"an transplant (broj $elija/ kg telesne mase primaoca) kao i u 13 odnosu na prinos $elija koji se dobija aspiracijom iz kostne sr%i ili iz periferne krvi. Zbog toga je jedna doza mati#nih $elija hematopoeze izolovane iz krvi jednog pup#anika uglavnom nedovoljna za klini#ke potrebe (naro#ito za odraslog primaoca). U tom smislu se razvijaju tehnike umno%avnja i kultivisaja ovih $elija u procesu njihove pripreme za #uvanje ili transplant (Areman, 2009). Prva uspe"na transplantacija mati#nih $elija hematopoeze izolovane iz krvi pup#anika izvr"ena je 1989. godine na "estogodi"njem pacijentu sa Fankonijevom anemijom (Gluckman et al., 1989) i od tada se ova klini#ka procedura intenzivno razvija i primenjuje kao alternativni izvor mati#nih $elija hematopoeze. 1.1.3.3.4 Mezenhimske mati"ne/stromalne #elije Jo" jedan tip multipotentnih mati#nih $elija je otkriven u kostnoj sr%i 1976. godine (Friedenstein et al., 1976). Danas su ove $elije poznate kao mezenhimske mati#ne/stromalne $elije (eng. Mesenchymal Stem /Stromal Cells) i one u#estvuju u formiranju tkiva mezodermalnog porekla kao "to su kost, hrskavica, mi"i$i, tetive i masno tkivo. U smislu izolovanja, manipulacije i potenicjalne primene u terapeutske svrhe (kao jednog od tipova $elijskih terapija) mezenhimske mati#ne/stromalne $elije se odlikuju relativno lakom izolacijom, potencijalom za deobu in vitro, stabilnim fenotipskim karakteristikama u procesu deobe kao i mogu$no"$u transplantacije tj. infuzije primaocu na vi"e razli#itih na#ina i u razli#itim kombinacijama (lokalno, sistemski, u infuzionom rastvoru, uz transplant drugih $elijskih terapija ili samostalno, kao deo medicinskih pomagala ili u okviru primene scaffold tehnika) (Brooke et al., 2007; Ilic et al., 2011; Steiner et al., 2009; Jing et al., 2010; Song et al., 2010; Wagner et al., 2008; Wagner et al., 2007; Wein et al., 2010; Zhang et al.; 2006; Heazlewood et al., 2012). Razumevanje i nau#na saznanja u oblasti mikrosredine neophodne za razvoj i funkciju mati#nih $elija hematopoeze napredovali su zna#ajno od prvobitne ideje o specijalizovanom okru%enja u kojoj se mati#ne $elije nalaze in vivo (Schofield, 1978). Ovakvo specijalizovano okru%enje obezbedjuje homeostazu mati#nim $elijama hematopoeze pri tome osiguravaju$i optimalne uslove za njihovu funkciju u zdravom 14 organizmu kao i u procesima njihove deobe usled gubitka razli#itih $elija koje nadoknadjuju i obnavljaju. Poznato je da u ovom procesu u#estvuju brojni citokini, faktori rasta, proteini ekstracelularnog matriksa, adhezioni molekuli kao i same medju-$elijske interakcije (eng. cell-cell interactions). Mnogi od ovih signala obezbedjeni su od strane $elija u neposrednom okru%enju mati#nih $elija hematopoeze, a najve$im brojem su to $elije mezenhimskog porekla. Stoga se mezenhimske mati#ne/stromalne $elije koriste kao deo ex vivo sistema za kultivaciju mati#nih $elija hematopoeze. Izolacija mezenhimskih mati#nih/stromalnih $elija, koje se upotrebljavaju kao dodatak i pomo$ u ekspanziji mati#nih $elija hematopoeze ex vivo, se naj#e"$e vr"i iz kostne sr%i ali je pokazano da su podjednako efektne mezenhimske $elije izolovane iz drugih tkiva kao sto su placenta (Zhang et al., 2004; Heazlewood et al., 2012), krv pupcanika (Bakhshi et al., 2008; Huang et al., 2007; Wang et al., 2004) i masno tkivo (Nakao et al., 2010). U proteklih nekoliko godina pokazano je da postoje mnoge specifi#ne medju- $elijske interakcije izmedju mati#nih $elija hematopoeze i mezenhimskih mati#nih/stromalnih $elija koje su zna#ajne za regulaciju razvoja mati#nih $elija hematopoeze in vivo (Steiner et al., 2009) i in vitro (Jing et al., 2010; Song et al., 2010; Wagner et al., 2008; Wagner et al., 2007; Wein et al., 2010), kao i da postoji neosporna interakcija veoma primitivnih mati#nih $elija hematopoeze sa mezenhimskim $elijama u njihovom neposrednom okru%enju (Zhang et al., 2006; Song et al., 2010). Ostale karakteristike mezenhimskih mati#nih/stromalnih $elija, metode za njihovu izolaciju i kultivaciju ex vivo su razmatrani u jednom od slede$ih poglavlja. 15 1.1.4 Terminologija Termin #elijske terapije (u smislu aktivnosti* i dela biofarmaceutske industrije kojoj pripada^) koji se koristi u ovoj doktorskoj disertaciji, uklju#uje "iroki spektar: tehnolo"kih pristupa, klini#kih projekata i instituta/organizacija uklju#enih u bazi#na istra%ivanja i klini#ke istra%iva#ke aktivnosti (eng. research & development, R&D) – baziranih na nizu nau#nih otkri$a u oblasti biologije, medicine, biohemije, imunologije, transfuziologije i regenerativne medicine (eng. biology, medicine, biochemistry, immunology, transfusiology & regenerative medicine) §§. Sveobuhvatan pristup* koji je usvojen u okviru ovog istra%ivanja obezbedjuje neophodnu multidisciplinarnost⌘ i integraciju nau#nih saznanja^ u okviru pomenutih nau#nih disciplina, kao i novih nau#nih disciplina u koje spada oblast regulatornih nauka (eng. regulatory science). Neophodno je naglasiti da se u terminolo"kom smislu ova doktorska disertacija bavi jednim domenom $elijske biologije i naprednim terapijama koje njihova primena donosi u oblasti medicine, ali da se pri tome ne bavi njihovom primenom u drugim oblastima (kao "to su neke "ire industrijske primene). U skladu sa nau#nim disciplinama i nau#nim metodama koje koriste (Kirouac & Zandstra, 2008; Halme & Kessler, 2006; Burger, 2003) projekti i organizacije zasnovani na radu sa #elijskim terapijama mogu se svrstati u "iru oblast biotehnologije¶ (Rader, 2008) kao i relativno nove oblasti biofarmaceutske industrije^. __________ * To thwart disease, apply now (Editorial). Nature, 453 7197 (2008). ^^ http://dictionary.cambridge.org ^ FDA Guidance for industry: PAT - A framework for innovative pharmaceutical development, manufacturing, and quality assurance, 2004. ⌘Advancing Regulatory Science at FDA, Strategic Plan: August 2011. Food and Drug Administration U.S. Department of Health and Human Services. & Implementing the European Medicines Agency’s Road map to 2015: The Agency’s contribution to Science, Medicine, Health – From Vision to Reality. 6 October 2011 EMA/MB/550544/2011. §§ Dalja terminoloska razmatranja se nalaze u drugim poglavljima ove doktorske teze kao i u Prilogu 1 i Prilogu 2. ¶The financing of biopharmaceutical product development in Europe, Final Report. European Commission, Enterprise and Industry, October 2009. ** http://www.infoport.ca/life/bins/content_page.asp?cid=4364 ## http://www.merriam-webster.com/dictionary 16 Termin biotehnologija se ipak ovde ne primenjuje u zna#ajnoj meri jer se u literaturi vrlo #esto koristi za komercijalne organizacije i na taj na#in ozna#ava skup komercijalnih aktivnosti u svrhu kreiranja profita¶ ili nekog dugoro#nog oblika intelektualnog vlasni"tva koje obezbedjuje profit (eng. intellectual property, IP based income). Isti termin se ovde koristi uglavnom u svojoj primarnoj definiciji “primene biolo"kih tehnika u istra%ivanju i razvoju produkata koji koriste %ive organizme ili njihove delove” (eng. “use of biological techniques in research and product development using living organisms or their parts”)** ili “primene %ivih organizama, posebno $elija i bakterija u industrijske svrhe” (eng. “the use of living things, especially cells and bacteria, in industrial processes”)^^. Lista skra$enica i definicija nalazi se u Prilogu 1, dok se englesko-srpski re#nik termina i izraza nalazi u Prilogu 2. __________ * To thwart disease, apply now (Editorial). Nature, 453 7197 (2008). ^^ http://dictionary.cambridge.org ^ FDA Guidance for industry: PAT - A framework for innovative pharmaceutical development, manufacturing, and quality assurance, 2004. ⌘Advancing Regulatory Science at FDA, Strategic Plan: August 2011. Food and Drug Administration U.S. Department of Health and Human Services. & Implementing the European Medicines Agency’s Road map to 2015: The Agency’s contribution to Science, Medicine, Health – From Vision to Reality. 6 October 2011 EMA/MB/550544/2011. §§ Dalja terminoloska razmatranja se nalaze u drugim poglavljima ove doktorske teze kao i u Prilogu 1 i Prilogu 2. ¶The financing of biopharmaceutical product development in Europe, Final Report. European Commission, Enterprise and Industry, October 2009. ** http://www.infoport.ca/life/bins/content_page.asp?cid=4364 ## http://www.merriam-webster.com/dictionary 17 1.2 Metode u izradi i primeni mati"nih #elija i ostalih #elijskih terapija (eng. Clinical & Lab Science) 1.2.1 Klini"ki pristupi u prikupljanju biolo$kog materijala za izradu mati"nih #elija i #elijskih terapija Kao "to je vec izlo%eno u prethodnom poglavlju, hematopoeza (eng. haematopoiesis) je slo%en proces nastanka krvnih $elija putem $elijske proliferacije (umno%avanja) i diferencijacije (sazrevanja) koji obezbedjuje grupa samoobnavljaju$ih pluripotentnih mati#nih $elija (eng. haematopoietic stem cells, HSC) pod slo%enim sistemom kontrole citokina. HSC su, kao pluripotentne $elije (sa ‘nezrelim’ antigenskim fenotipom CD34+, HLA-DR-, CD33- i CD38-), kompletno sposobne za samoobnavljanje (deobu) i dalju diferencijaciju (sazrevanje) u mijeloidne i limfoidne grupe krvnih $elija (Balint B., Hemomodulatorni mehanizmi i terapijski pristupi 1999, str. 20-25; Areman E.M., Cellular Therapy: Principles, Methods, and Regulations, 2009). Tabela 1.1: Tipovi transplantacije, bilo da su u pitanju mati#ne $elije iz kostne sr%i, mati#ne $elije izolovane iz pup#anika ili iz periferne krvi. Tip transplantacije &ta predstavlja Svrha Alogena ·Upotreba hematopoetskih progenitornih $elija (graft) od neke druge osobe /kao "to je #lan porodice ili dobrovoljni davalac/ ·Uspostavlja normalnu hematopoezu i/ili spre#ava imunodeficijenciju ·Potpoma%e primenu hemoterapije i/ili radijacije /u svrhu le#enja malignog oboljenja/ Autologna ·Upotreba hematopoetskih progenitornih $elija (graft) od iste osobe ·Potpoma%e primenu hemoterapije i/ili radijacije /u svrhu le#enja malignog oboljenja/ Singena ·Upotreba hematopoetskih progenitornih $elija (graft) od genetski identi#nog blizanca ·Potpoma%e primenu hemoterapije i/ili radijacije /u svrhu le#enja malignog oboljenja/ Adaptirano iz: Hematopoietic Stem Cell Transplantation – A Handbook for Clinicians, 2009 AABB (USA). 18 Tabela 1.2: Osnovne komponente transplantacije mati#nih $elija hematopoeze. Elementi/ komponente Svrha Autologna transplantacija Alogena transplantacija Anti-tumorski tretman (hemoterapija i/ili zra#enje) ·Uklanjanje tumorskih $elija ·Uklanjanje tumorskih $elija ·Supresovanje imuniteta primaoca Mati#ne $elije (graft) ·Uspostavljanje hematopoeze i normalizovanje imuniteta ·Uspostavljanje hematopoeze i normalizovanje imuniteta ·Uklanjanje imunodeficijencije ili metaboli#ke abnormalnosti /koju izazivaju obolele krvne $elije usled naslednog poremecaja imunskog sistema ili imunodeficijencije/ ·Obezbedjivanje ‘novog’ imuniteta da bi se uklonile tumorske $elije Terapija (uklju#uju$i imunosupresivnu terapiju) ·Antiobiotici (da bi se spre#ile ili le#ile infekcije) ·Transfuzija (da bi se kompenzovala aplazija) ·Imunosupresivno dejstvo da bi se izbegla imunolo"ka reakcija primaoca (i odbacivanje transplantiranih $elija) ·Imunosupresivno dejstvo da bi se izbegla GVHD (eng. graft-vs- host disease) ·Antiobiotici (da bi se spre#ile ili le#ile infekcije) ·Transfuzija /da bi se kompenzovala aplazija/ Adaptirano iz: Hematopoietic Stem Cell Transplantation – A Handbook for Clinicians, 2009 AABB (USA). GVHD = graft-vs-host disease. Pri tome, uz odredjeni citokinski stimulus, HSC mogu da se diferenciraju u HPC $elije (eng. haematopoietic progenitor cells) koje imaju veoma ograni#enu ili nikakvu sposobnost samoobnavljanja (dalje deobe). U kostnoj sr%i HPC $elije nastavljaju svoj proces diferencijacije pod kontrolom citokina, pri tome obezbedjuju$i nastanak grupa $elija koje daju razli#ite vrste zrelih, funkcionalno sposobnih krvnih $elija (eritrocita, trombocita i leukocita) (Balint B., Hemomodulatorni mehanizmi i terapijski pristupi 1999, str. 20-25; E.M. Areman, Cellular Therapy: Principles, Methods, and Regulations, 2009; Wingard et al., 2009). Postoje tri tipa transplantacije mati#nih $elija hematopoeze (eng. hematopoietic stem cell transplantation, HSCT) (Tabela 1.1) i tri osnovne komponente procesa transplantacije (Tabela 1.2). 19 Diferencijacija pluripotentnih mati#nih $elija (HSC) u progenitorne (HPC) $elijske linije manifestuje se kroz promenu antigenskog fenotipa i gubljenje CD34+ antigenskog profila. In vitro studije obezbedile su kvantifikaciju obe vrste $elija u normalnoj kostnoj sr%i (koncentracija HSC je priblizno 0.005%) i u perifernoj krvi zdrave osobe (100 puta ni%a HSC koncentracija, pribli%no 0.00005%) (Balint B., Transfuziologija 2004; Areman E.M., Cellular Therapy: Principles, Methods, and Regulations, 2009). Nakon kultivacije progenitornih (HPC) $elija u in vitro $elijskoj kulturi (14 dana, u specifi#noj citokinski pripremljenoj polu#vrstoj podlozi) dobijaju se kolonije $elija (eng. colony forming units, CFU ili eng. bursting forming units, BFU) iz kojih se razvijaju zrele, funkcionalno sposobne krvne $elije. Istra%ivanjima na %ivotinjskim modelima, a kasnije i u okviru klini#kih istra%ivanja, pokazano je da se primenom agenasa kao sto je G-CSF (eng. granulocyte-colony stimulating factor) mogu pove$ati koncentracije obe $elijske grupe (HSC i HPC) u perifernoj krvi (Balint B., Od hemoterapije do hemomodulacije 2001, str. 259; Areman, 2009). Ovo se koristi kao proces ‘mobilizacije’ mati#nih (HSC) i progenitornih (HPC) $elija u perifernoj krvi, kao priprema za proces odvajanja (filtracije) krvnih $elija u postupku afereze (eng. apheresis) (Balint B., Od hemoterapije do hemomodulacije 2001, str. 255-274; Areman, 2009; Wingard et al., 2009). Osamdestih godina dvadestog veka razvoj procedura za procesovanje mati#nih $elija dovodi do mogu$nosti za njihovo zamrzavanje i #uvanje u dugoro#nom periodu. Ovo je omogu$ilo da mati#ne $elije mogu biti prikupljene unapred, procesovane i zamrznute a zatim transplantovane nakon visokih doza hemoterapije ili terapeutskog zra#enja (Wingard J.R. et al. (Ed.), 2009, Hematopoietic Stem Cell Transplantation – A Handbook for Clinicians. AABB, USA). Iako je svrha visokih doza hemoterapije da uklone sve kancerske $elije (ablativne terapije) pri tome dolazi i do o"te$enja normalnih $elija kostne sr%i. Transplantacijom mati#nih $elija (ili ta#nije, progenitornih $elija) uspostavlja se normalna hematopoeza. !elije namenjene za transplantaciju su se inicijalno prikupljale iz kostne sr%i, danas sve #e"$e iz periferne krvi (eng. peripheral blood progenitor cells, PBPC) zbog manje invazivnosti klini#ke procedure kao i ve$eg broja $elija koje se mogu prikuptiti u toku jedne procedure (Tabela 1.3). 20 Tabela 1.3: Izvor mati#nih $elija za klini#ku upotrebu u svrhe transplantacije. Izvor mati#nih $elija Prednosti Nedostaci Komentar Kostna sr% ·Bogat izvor mati#nih $elija ·Manji broj limfocita nego iz periferne krvi /manje GVHD/ ·Potrebna je generalna anestezija davaoca ·Manje progenitorskih $elija nego iz periferne krvi ·Tradicionalno se koristi kao izvor mati#nih $elija, najve$e klini#ko iskustvo ·Ostaje kao naj#e"$i izbor za alogene transplantacije i mnoge transplantacije medju srodnim davaocem i primaocem Periferna krv ·Visok broj mati#nih $elija ·Ve$i broj limfoidnih $elija /ve$i anti- tumorski (GVT) efekat/ ·Davalac mora da prima G-CSF i potrebno je du%e vreme za pripremu ·Ostaje kao naj#e"$i izbor za autologne transplantacije ·Ostaje kao naj#e"$i izbor za: -autologne transplantacije sa manjim intenzitetom pripremnih terapija/re%ima (eng. conditioning regimens) /jer obezbedjuje vi"e mati#nih $elija i vi"e limfoidnih $elija koje suzbijaju imunski odgovor primaoca/; -leukemije u odmaklom stadijumu /zbog ve$eg anti- tumorskog (GVT) efekta/ Krv iz pup#anika ·Prikupljanje $elija se vr"i bez rizika za majku ili novorodjen#e ·Manji su rizici preno"enja infektivnih agenasa ·Prakti#no je stalno dostupan izvor $elija /u poredjenju sa prikupljanjem $elija od odraslog davaoca/ ·limfoidne $elije su imunoloski naivne ·Limfoidne $elije su nezrele tako da je potrebno du%e vreme da ispolje svoj anti- tumorski efekat (GVT) ·Obi#no je dovoljan broj $elija za pedijatrijske pacijente ali ne i za ve$inu odraslih primaoca ·U poslednje vreme postaje sve #e"$i izbor za alogene transplantacije kod pedijatrijskih pacijenata Adaptirano iz: Hematopoietic Stem Cell Transplantation – A Handbook for Clinicians, 2009 AABB (USA). GVHD = graft-vs-host disease; GVT = graft-vs-tumor; G-CSF = granulocyte colony-stimulating factor. 21 Tabela 1.4: Prednosti i nedostaci razli#itih tipova transplantacije mati$nih $elija. Tip transplantacije Prednosti Nedostaci Alogena ·Obezbedjuje anti-tumorski (GVT) efekat ·Progenitorske $elije su zdrave ·Dozvoljava upotrebu manje agresivnih citotoksi#nih re%ima u procesu pripreme (eng. conditioning regimen) ·Moze biti te%e da se identifikuje odgovaraju$i davalac ·GVHD ·Rizik od odbacivanja Autologna ·Ne zahteva tra%enje adekvatnog davaoca ·Nema rizika od GVHD ·Transplantirane mati#ne $elije mogu biti kontaminirane tumorskim $elijama ·Transplantirane mati#ne $elije mogu biti o"te$ene prethodnim terapijama, proces prikupljanja odgovaraju$eg broja $elija mo%e biti ote%an, ili mo%e da doprinese riziku od nastanka mijelodisplazije nakon transplantacije ·Nema anti-tumorskog (GVT) efekta Singena ·Nema rizika od GVHD ·Nema anti-tumorskog (GVT) efekta Adaptirano iz: Hematopoietic Stem Cell Transplantation – A Handbook for Clinicians, 2009 AABB (USA). GVHD = graft-vs-host disease; GVT = graft-vs-tumor. Svi tipovi transplantacije imaju odredjene prednosti i nedostatke (Tabela 1.4). Primena mati#nih $elija u ovom trenutku nudi ogroman potencijal u le#enju kako naslednih oboljenja tako i ste#enih i degenerativnih oboljenja, ali idealan pristup i postupak za dobijanje ove vrste $elija jos uvek nije otkriven (Wingard et al. (Ed.), 2009, Hematopoietic Stem Cell Transplantation – A Handbook for Clinicians. AABB USA). 1.2.1.1 Separacija #elija krvi i drugih krvnih komponenti - afereza Afereza predstavlja skup postupaka vezanih za eksfuziju davao#eve ili bolesnikove krvi, njeno razdvajanje na komponente, uz zadr%avanje – prikupljanje ili otklanjanje – nekog sastojka i reinfuziju preostalog ili “rekombinovanog” dela krvi (Balint B., Transfuziologija 2010, str. 455; Areman, 2009; Wingard, 2009). Pri 22 izvodjenju nekih afereznih procedura bolesniku se vra$a autologna plazma oslobodjena odredjenih komponenti, sopstvene imunokompetentne krvne $elije nakon ex vivo modulacije njihove aktivnosti ili sopstvene multipotentne mati#ne $elije hematopoeze (nakon njihove krioprezervacije i #uvanja). Sa aspekta terapijske primene, afereze mogu biti primenjene u svrhu pripremanja pojedinih krvnih konstituenata ili u cilju postizanja odredjenog terapijskog efekta kao "to je uklanjanje odredjene koli#ine nekog krvnog sastojka (bilo da je u pitanju “patolo"ki supstrat” ili ekscesna koli#ina normalnog ili izmenjenog krvnog sastojka) (Balint, 2010). Savremena afereza se primenjuje uz upotrebu $elijskih separatora (eng. apheresis machine/cell processor) koje se programiraju za odredjenu funkciju na osnovu veli#ine pora na filterima, koli#ine krvi koja $e biti filtrirana, te%ine pacijenta, tipa $elija i drugih parametera. Izvodjenje savremenih afereznih postupaka nije mogu$e bez adekvatne osposobljenosti osoblja i savremene opreme. Aferezno prikupljanje krvnih komponenata #ine donorske afereze, plazmafereze i citafereze, medju kojima se naj#e"$e sre$u trombocitafereza i razli#iti tipovi leukafereza. U zavisnosti od vrste prikupljenih leukocita, mo%e se govoriti o granulocitaferezi i aferezi mononuklearnih $elija – uglavnom u svrhu transplantacije mati#nih $elija hematopoeze (Balint, 2010). Pri postupku prikupljanja i transplantacije mati#nih $elija hematopoeze izolovanih iz periferne krvi, izbegava se invazivni postupak aspiracije $elija iz kostne sr%i. Pri tome je omogu$eno prikupljanje ve$eg broja $elija, koje danas mogu da se koriste u razli#itm rutinskim klini#kim protokolima ili u svrhu klini#kih istra%ivanja (npr. eksperimentalne procedure uz primenu genskih terapija ili za primenu nepromenjenih $elija ali u novim klini#kim indikacijama transplantacije i ko-transplantacije). 1.2.1.2 Aspiracija #elija kostne sr%i Osim $elijske separacije afereznim tehnikama, mati#ne $elije hematopoeze se prikupljaju vi"estrukim aspiracijama iz spongioznih delova kostiju, pri #emu $elije mogu biti transplantirane neposredno nakon pripreme, posle kratkotrajnog #uvanja ili nakon zamrzavanja (Balint B., Transfuziologija 2004; Areman, 2009; Wingard, 2009). !elije kostne sr%i se prikupljaju aspiracijom iz zadnjeg i prednjeg grebena 23 bedrene kosti ili iz grudne kosti. Procedura je invazivne prirode, izvodi se u operacionoj sali, u asepti#nim uslovima i uklju#uje primenu lokalne ili op"te anestezije (Balint B., Transfuziologija 2004; Areman, 2009). Ovom metodom se dobija relativno mali broj $elija po ubodu/aspiratu (volumen jednog aspirata nije ve$i od 5ml a broj aspirata se obi#no kre$e od 3 do 5 po jednoj lokaciji/kosti) tako da se vremenom razvijaju i druge tehnike manje invazivne prirode – a uz obezbedjivanje ve$eg broja $elija i bolje selektivnosti tehnike (Balint B., Transfuziologija 2004; Areman, 2009; Wingard, 2009). U cilju obezbedjivanja dovoljnog broja $elija sa jedrom, medju kojima su i mati#ne $elije hematopoeze, smatra se da je neophodno prikupiti 3x108 celija po kg telesne mase primaoca – bilo da je u pitanju autologna ili alogena primena. Nakon aspiracije, $elije se filtriraju u cilju eliminacije eritrocita, limfocita T i/ili malignih $elija u aspiratu; a zatim su pripremljene u antiokoagulatnom rastvoru uz dodatak hranljivih sastojaka u cilju o#uvanja $elijske vijabilnosti i bolje efikasnosti terapije (Balint B., Transfuziologija 2004; Areman, 2009). Tehnike u prikupljanju i primeni $elija kostne sr%i su se intenzivno razvile u toku "ezdesetih i sedamdesetih godina 20. veka, naro#ito radom E. D. Thomas-a (1920-2012) koji je jos 1981. godine bio jedan od osniva#a prvog nau#nog #asopisa (eng. Stem Cell) u oblasti mati#nih $elija i regenerativne medicine (Areman, 2009; Stem Cells Translational Medicine, Editorial, 2013; 2:81-82). 1.2.1.3 Alternativne metode u prikupljanju biolo$kog materijala za izradu #elijskih terapija: upotreba placente Ljudska placenta je organ sagradjen iz tkiva fetusa i majke (eng. fetomaternal organ) koji se sastoji iz fetalnog tkiva amniona i horiona (eng. amnion, chorion) uz deciduu (eng. decidua) koja je poreklom samo iz tkiva majke (Parolini et al., 2008). Ovaj kompleksni organ po#inje da se razvija u roku od nekoliko dana nakon fertilizacije jajne $elije i od presudnog je zna#aja za razvoj i opstanak fetusa. Placenta "titi fetus od infekcije tokom trudno$e ali istovremeno vr"i druge brojne funkcije neophodne za razvoj fetusa preuzimaju$i ulogu razli#itih organa kao "to su plu$a, bubrezi i digestivni trakt (Parolini et al., 2008). Placenta je relativno lako dostupno tkivo bogato mati#nim $elijama i progenitorskim $elijama tako da njena upotreba predstavlja alternativu konvencionalnim metodama za prikupljanje mati#nih $elija. 24 1.2.2 Izrada konvencionalnih terapija mati"nim #elijama uz minimalnu manipulaciju Tranplantacija mati#nih i progenitorskih $elija iz periferne krvi koja se koristi nakon ablativnih terapija predstavlja primer minimalno manipulisanih $elija za homogenu upotrebu (eng. homogenous use), uglavnom ili samo za autologne transplantacije i bez upotrebe kombinovanih produkata, bez sistemskih efekata (zabele%enih do sada). Transplantacija mati#nih $elija iz periferne krvi (eng. Peripheral Blood Stem Cells, PBSC ili Haematopoietic Stem Cells, HPC) ve$ du%e vreme se koristi kao terapija sa ciljem da se uspostavi hematopoeza nakon inicijalne faze oporavka krvnih $elija u cirkulaciji i da se uspostavi njihov fiziolo"ki status i broj neophodan za suzbijanje infekcija, krvavljenja i anemija (Serke, 2001; Balint B., Transfuziologija 2004, str. 530-542; E.M. Areman, Cellular Therapy: Principles, Methods, and Regulations, 2009). Broj indikacija za primenu visokih terapijskih doza (eng. high- dose therapy, HDT) u hematolo"kim i drugim malignitetima je u porastu i u direktnoj je srazmeri sa potrebom za smanjenjem tro"kova ovakvih terapija, njihovom pristupa#no"$u i pojednostavljenjem rutinskih laboratorijskih procedura koje podrazumevaju zamrzavanje i #uvanje $elija (Montanari, 2003). Uspeh zabele%en u pripremi i transplantaciji mati#nih $elija iz periferne krvi u proteklih 20 godina pra$en je usavr"avanjem laboratorijskih procedura kao i zapa%enim pobolj"anjem klini#kih pokazatelja (npr. smanjenjem broja neuspe"nih transplantacija). Ovo se pripisuje uvodjenju efikasnih metoda obezbedjenja kvaliteta (eng. Quality Assurance, QA) i kontrole kvaliteta (eng. Quality Control, QC) uz upotrebu novih tehnologija, a postignuto je uglavnom velikim naporima na usavr"avanju veoma jednostavne tehnologije. Pri tome je omogu$eno i sticanje novih znanja u ovoj oblasti i transfer tih znanja na proizvodnju slo%enijih, visoko manipulisanih $elijskih terapija i njihovu primenu (Montanari, 2003). 1.2.3 Slo%ene metode u izradi #elijskih terapija: visoko manipulisane #elije Mezenhimske mati#ne/stromalne $elije (eng. mesenchymal stem cells, MSC) kao grupa adultnih mati#nih $elija se, za razliku od embrionalnih mati#nih $elija (eng. embryonic stem cells, ESC) bez ve$ih eti#kih dilema tradicionalno dobijaju iz kostne 25 sr%i i ne formiraju tumore, ali se relativno sporo umno%avaju i sa odredjenim ograni#enjima ulaze u proces diferencijacije. Mezenhimske $elije se relativno lako izoluju i uzgajaju. Zahvaljuju$i svojoj multipotentnosti, parakrinom efektu, imunomodulatornim svojstvima i migracijskim sposobnostima, ove $elije su u stanju da obezbede relativno nove pravce u razvoju $elijskih terapeutika (Brooke et al., 2008; Ilic et al., 2011). Izolovane mezenhimske $elije (MSC) koje su umno%ene ex vivo zadr%avaju svoju pluripotentnost i mogu$nost da se diferenciraju u tkiva mezodermalnog porekla. Osim sposobnosti da obnavljaju odredjene vrste tkiva, mezenhimske $elije (MSC) (Prilog 4) imaju imunosupresivni efekat na T $elije i antigen-prezentuju$e dendriti#ne $elije in vitro (Prilog 5). Osim toga, #ini se da su mezenhimske $elije (MSC) imunolo"ki privilegovane i mogu biti unakrsno kotransplantirane izmedju dva ili vi"e %ivotinjskih organizama bez potrebe za imunosupresovanjem istih jedinki (pokazano i na 'outbred' %ivotinjskim modelima) (Brooke et al., 2009; Ilic et al., 2011). Ova osobina ima izuzetne implikacije na potencijalnu terapijsku primenu mezenhimskih $elija (MSC) (Brooke et al., 2009; Ilic et al., 2011; Ilic et al., 2013). Slika 1.2: Morfologija mezenhimskih $elija izolovanih iz placente obojenih fluoroscentnom bojom Phalloidin-AF546 (Brooke G, Rossetti T, Pelekanos R, Ilic N et al., 2008). 26 Mezenhimske $elije (MSC) mogu biti izolovane is razli#itih tkiva kao "to su krv iz pup#ane vrpce i fetalna krv, iz fetalne jetre i kostne sr%i, ali takodje i iz placente (Slika 1.2). U poredjenju sa humanim mezenhimskim $elijama (MSC) izolovanim iz kostne sr%i, fetalne mezenhimske $elije (MSC) (iz prvog trimestra) i mezenhimske $elije (MSC) izolovane iz placente (nakon porodjaja) pokazuju razliku u markerima pluripotentnosti i br%e se razvijaju (Brooke et al., 2009; Ilic et al., 2011). Dalja razmatranja i pregled procesa koji se koriste u svrhu ex vivo umno%avanja mezenhimskih mati#nih/stromalnih $elija nalaze se u Prilogu 4. 1.2.4 Novi pristupi u izradi mati"nih #elija: indukovane pluripotentne #elije (iPS) Uspe"no reprogramiranje razli#itih telesnih tj. somatskih $elija u stanje pluripotentnosti bi moglo da omogu$i stvaranje pacijent- specifi#nih ili oboljenje- specifi#nih mati#nih $elija. U ovom trenutku, indukovane pluripotentne mati#ne $elije (eng. induced pluripotent stem cells, iPS) mogu nastati iz humanih fibroblasta, kao i nekih drugih somatskih $elija. Inicijalno, prva generacija indukovanih pluripotentnih mati#nih $elija, sposobnih za “germline transmission“, nastala je iz somatskih $elija mi"a upotrebom #etiri transkripciona faktora (Oct3/4, Sox2, Klf4, and c-Myc) (Takahashi, 2007). Ista tehnika je kasnije kori"$ena za nastanak indukovanih pluripotentnih mati#nih $elija iz adultnih humanih fibroblasta uz upotrebu istih faktora (Takahashi, 2007). Smatra se da su humane indukovane pluripotentne mati#ne $elije (iPS) sli#ne embrionalnim mati#nim $elijama (eng. embryonic stem cells, ESC) po mnogim svojim karakteristikama – morfologiji, $elijskoj deobi, povr"inskim antigenima, ekspresiji gena, epigenetskom statusu pluripotentnih $elija-specifi#nih gena i telomeraznoj aktivnosti. Osim toga, indukovane pluripotentne mati#ne $elije (iPS) mogu se in vitro diferencirati u $elijske tipove sva tri germinalna sloja (endodermalnog, mezodermalnog i ektodermalnog porekla) (Takahashi, 2007). Daljim istra%ivanjima je naknadno pokazano da se indukovane pluripotentne mati#ne $elije (iPS) ipak razlikuju od embrionalnih mati#nih $elija (ESC) u ekspresiji svog genoma (Chin, 2005). Uradjena su mnoga istra%ivanje profila ekspresije gena u embrionalnim (ESC) i indukovanim mati#nim $elijama (iPS) kod mi"a i #oveka. Pokazano je da, iako 27 sli#an ekspresiji gena u embrionalnim mati#nim $elijama, postoji tipi#an profil iPS ekspresije gena – bez obzira na njihov izvor (tip tkiva) ili metod kojim su iPS $elije indukovane u pluripotentno stanje. Ovi podaci sugeri"u da bi indukovane pluripotentne mati#ne $elije (iPS) trebalo da budu svrstane u jedinstveni podtip pluripotentnih $elija (Chin, 2005). Shodno svojim karakteristikama i ogromnom interesovanju istra%iva#a, indukovane pluripotentne mati#ne $elije (iPS) imaju potencijal da dramati#no uti#u na nove pristupe u razvoju terapeutika pre svega u regenerativnoj medicini; ove $elije mogu da se diferenciraju u $elijske tipove sva tri germinalna sloja, ali po"to su nastale manipulacijom somatskih $elija, njihovo istra%ivanje, izrada i primena nemaju u toj meri izra%ene eti#ke dileme (kao u slu#aju upotrebe embrionalnih mati#nih $elija) (Loh, 2009). 1.3 Kontekst i principi u izradi #elijskih terapija: farmacija, biotehnologija i regenerativna medicina (eng. Biotechnology, Cell Therapy & Regenerative Medicine) 1.3.1 Kontekst !elijske terapije potrebno je razmatrati u specifi#nom kontekstu savremenog razvoja biotehnologije, regenerativne medicine i drugih srodnih oblasti nauke (eng. life science). Pojednostavljene definicije regenerativne medicine i $elijskih terapija predstavljaju $elijske terapije kao deo regenerativne medicine (Buckler, 2009) u funkciji obnove normalne funkcije i/ili zamene $elija, tkiva ili organa (eng. “cell therapy is always about the replacement or regeneration of human cells, tissue, or organs, to restore or establish normal function”) (Mason & Dunnill, 2008). Na osnovu iste definicije, regenerativna medicina mo%e ali ne mora da koristi $elije u svrhu zamene tkiva ili organa. Za ovaj proces mogu da se koriste i drugi pristupi, kao "to su upotreba malih molekula kao aktivatora, genske terapije (koje nisu bazirane na $elijskoj manipulaciji) ili drugi biomaterijali (Mason & Dunnill, 2008; Buckler, 2009) (Tabela 1.5). Ne"to "iri kontekst koji je zna#ajan za $elijske 28 terapije, predstavlja polje biotehnologije i razvoja biofarmaceutskih sredstava (Tabela 1.6). Tabela 1.5: Definicije koje se odnose na zna#enje pojmova “regenerativna medicina” i “$elijske terapije” u kontekstu regenerativne medicine. eng. Regenerative Medicine is typically described as having four primary pillars: cells, devices, biomaterials, and bioactive agents (reagents, drugs, and biologics). Their common goal is to replace, repair, or regenerate human cells, tissues, or organs in such a way as to restore or establish normal functions. eng. If cell therapy is about delivering cells as therapeutics, [it is often referred] to Regenerative Medicine as being about affecting cells (including whole tissues or organs) whether through their delivery (cell therapy) or their in vivo recruitment and/or manipulation through molecular or other means. Cell therapy fits within the large and even more diverse Regenerative Medicine industry. eng. Regenerative medicine is already commercially available and entrenched into several types of American and Western European clinical practice: tissue engineering (e.g., skin repair/wound healing), orthopedics (e.g., spinal disc and joint cartilage repair), diabetes (e.g., islet cell transplantation), oncology/hematology (e.g., stem cell transplants), ophthalmics (e.g., limbal stem cell deficiency, corneal disease, and so on), and cosmetic/aesthetic (e.g. body sculpting). Adaptirano iz: Buckler, L (2009), Cell Therapies, Opportunities in Regenerative Medicine, BioProcess International, Vol. 9, No. S1, March 2011. Biofarmaceutska industrija, sli#no kao i biotehnologija, se uglavnon defini"e na osnovu definicije svojih proizvoda ili na osnovu poslovnih aktivnosti koje su vezane za razvoj biofarmaceutskih (ili biotehnolo"kih) produkata (Buckler, 2009) (Tabela 1.6). Moderna biotehnologija je od strane OECD-a definisana kao “application of living organisms, as well as parts, products and models thereof, to alter living or nonliving materials for the production of knowledge, goods and services” (eng. Organisation for Economic Cooperation and Development, OECD 2005)*. __________ *The financing of biopharmaceutical product development in Europe, Final Report. European Commission, Enterprise and Industry, October 2009. ISBN 978-92-79-14055-6 29 Tabela 1.6: Definicije koje se odnose na zna#enje pojmova “biotehnologija”, “nova biotehnologija” i “biofarmaceutska industrija”. eng. Broad Biotechnology - Biopharmaceuticals are defined as pharmaceuticals manufactured by biotechnology methods, with the products obviously having biological sources, usually involving live organisms or their active components (bioprocessing; also usually very obvious; or directly involving surrogates, e.g., protein/gene sequences). This broad view has been adopted by Biopharmaceuticals in the U.S. and European Markets, which includes all recombinant proteins, (monoclonal) antibodies, vaccines, blood/plasma-derived products, nonrecombinant culture-derived proteins, and cultured cells and tissues. This is the view/definition most used/favored by those within the U.S. biopharmaceutical industry, and probably best understood by the public. eng. New Biotechnology - This view or definition only includes those products based on platform technologies developed in recent decades, usually only including recombinant proteins and monoclonal antibodies as being biopharmaceuticals, leaving out nonrecombinant cultured proteins, blood/plasma proteins, vaccines and other classes of products. This view/definition is more predominant in Europe, e.g., European Union formal definitions, or is used by those whose primary/sole interest is in recombinant and monoclonal antibody products. Many obvious biopharmaceutical products are classed as as 'old' or simply ignored, including many products developed and approved recently and incorporating more recent/newer technology than many recombinant proteins and monoclonal antibodies (which are based on now 'old' technologies, invented in the 1970s and commercialized in the 1980s). eng. Biotechnology Business - This company-centric view or definition simply includes all/everything from biotech-like (smaller, entrepreneurial, R&D- intensive) pharmaceutical and life science companies as being biopharmaceutical; plus obvious biopharmaceuticals from large companies (Big Pharma). All/every non-Big Pharma pharmaceutical or life sciences company is viewed as a biopharmaceutical company. By this view/definition, products, technologies and companies need not have any involvement or use of biotechnology to be called biopharmaceutical. Those using this definition/view include many in the press, stock analysts and, regrettably, the Biotechnology Industry Organization (BIO). Many small drug R&D and service companies also describe themselves as biopharmaceutical companies, including those designing or developing small molecule drugs, even when their products, technologies and companies have absolutely no use or involvement with biotechnology. eng. Pharma Business - This view simply includes all pharmaceuticals as biopharmaceuticals, i.e., biopharmaceuticals is used as a synonym for pharmaceuticals; and the pharmaceutical industry is now the biopharmaceutical industry. Biopharmaceuticals are no longer a subset of pharmaceuticals, with biopharmaceuticals now taking in all pharmaceutical and biotechnology companies (with a health care orientation). Various industry analytical reports, including those funded and issued by the Pharmaceutical Research and Manufacturers Association (PhRMA), assert that the pharmaceutical industry (dominated by Big Pharma) has just recently morphed or transformed itself into the biopharmaceutical industry as a result of the "convergence" of biotechnology and pharmaceutical industries/technologies; that the pharmaceutical industry, through adoption of new technologies and close relationships (primarily outsourcing, contracting and licensing) with biotech companies, has undergone a fundamental change; and that 'new' and 'revolutionary' technologies, e.g., high-throughput screening and drug design methodologies, are biotechnologies and have fundamentally changed the industry. [Thus, everything in the pharmaceutical universe is now biopharmaceutical]. Adaptirano iz: Ronald A. Rader, Biopharmaceuticals in the U.S. and European Markets, BioExecutive, March 2005 & May 2005. 30 1.3.2 Problematika izrade #elijskih terapija !elijskim terapijama mogu da se bave klini#ki projekti, instituti/organizacije uklju#eni u bazi#na istra%ivanja i klini#ke istra%iva#ke aktivnosti, komercijalne kompanije (eng. start-up & spin-off companies) i srednje ili velike korporacije. Pri tome su uklju#eni u razvoj nau#no-tehnolo"kih istra%ivanja, klini#ku primenu, komercijalizaciju ili usluge vezane za razvoj $elijskih terapija (npr. testiranje i logistiku). Mnogi projekti i kompanije/organizacije zapo#inju svoje poslovanje kao univerzitetski projekti, bolni#ke ili istra%iva#ke laboratorije (Armstrong, 2001). U ovoj oblasti, kao i mnogim drugim “life-science” oblastima razvoja, ubrzani proces globalizacije, slo%eni procesi izrade (Slika 1.3) i sve stro%ije regulatorne zakonske odredbe (Areman, 2009), kao i razli#itosti u veku trajanja ovih proizvoda (eng. life- cycle of products) – projektuju se, i uz ostale faktore, #ine tr%i"te sve vi"e medjuzavisnim, dinami#nim i kompetitivnim na lokalnom i globalnom nivou. Stoga su se povezivanje adekvatnih poslovnih principa i nau#nih projekata uz regulatorni fokus i sveobuhvatni dizajn kvaliteta – pokazali kao odr%ivi pristup za uspe"an razvoj projekata i organizacija koje se bave $elijskim terapijama (kao razvojnom obla"$u biotehnolo"ke ili biofarmaceutske industrije, Tabela 1.6) (Armstrong, 2001; Pisano, 2006). Tabela 1.7: Prakti#ni aspekti izrade $elijskih terapija koji #esto ograni#avaju proces razvoja novih biolo"kih lekova u toj oblasti. Po!etni materijali i reagensi (eng. Key materials) Proces izrade (eng. Manufacturing) Parametri kvaliteta i stadardizovane analiti!ke metode (eng. Product quality/ analytical methods) Klini!ke metode i logistika za primenu novih terapija (eng. Clinical site logistics) Transport i primo-predaja (eng. Delivery) Ostali klini!ki aspekti (eng. Other clinical considerations) U nekim industrijama broj novih proizvoda prisutnih na tr%i"tu mo%e se smatrati adekvatnim pokazateljem poslovnog rasta i razvoja. U oblasti razvoja $elijskih terapija, mo%da vi"e nego u bilo kojoj drugoj grani industrije, osnovni 31 parametar uspe"nosti le%i u uravnote%enom pristupu nau#no-istra%iva#kom, poslovnom (Armstrong, 2001; Pisano, 2006) i regulatornom (Burger, 2003; Areman, 2009) aspektu – u mnogo ve$oj meri nego prosto obezbedjivanje novih nau#nih ili tehnolo"kih pristupa (eng. “know-how”). Procesi izrade efikasnih $elijskih terapija #esto su dugi, skupi i bazirani na individualnom pristupu odredjenom pacijentu (eng. individualised/ autologous cell therapies). Za razliku od principa izrade vakcina, monoklonalnih antitela ili hormonskih preparata, ovi proizvodi ne mogu biti izradjeni u velikoj koli#ini, retko su u potpunosti okarakterisani (zbog visoke biolo"ke promenljivosti) ili unapred pripremljeni. (injenica da su u pitanju nepotpuno okarakterisani proizvodi, ponekad izradjeni i testirani ta#no u trenutku kada je neophodno ili neposredno pred upotrebu (eng. in real-time) kao i da se izradjuju u dugim i skupim procedurama (Tabela 1.7) – izdvaja $elijske terapije od svih drugih kategorija biolo"kih lekova ili biotehnolo"kih proizvoda (Burger, 2003; Areman, 2009). Osim toga, svi biolo"ki lekovi (medju kojima i $elijske terapije) moraju biti osmi"ljeni i izradjeni u strogo kontrolisanim uslovima**. Ovo se zahteva i kada nije mogu$e pridr%avati se svih tradicionalnih principa farmaceutske tehnologije (iako je to krajnji cilj u ovom procesu), posebno za urgentne terapije (eng. life-saving therapies) i nove modalitete le#enja koji se primenjuju u klini#kim istra%ivanjima!. Stoga se na ove procese izrade $elijskih terapija primenjuju strategije procene rizika u datom kontekstu (eng. risk-benefit ratios) kao i transparetni mehanizmi za finalno odobrenje upotrebe proizvoda (eng. transparent and accountable release of products) (Burger, 2003; Areman, 2009). Smatra se da je tzv. science-business, posmatrano na globalnom nivou i od svog nastanka, do sada izgubio vi"e sredstava nego "to je zaradio (eng. failing to deliver return on investment, ROI) kao i da nije obezbedio dovoljno novih terapeutskih sredstava (na nivou koji je bio o#ekivan ili koji je obe$avao). Takodje je poznato da, u celini gledano, ovaj projekat (eng. science-business) nije zadovoljio o#ekivanja i da se jedan od glavnih razloga nalazi u kontradiktornim ciljevima i zahtevima nau#no-tehnolo"kih i poslovnih aspekata (eng. conflicting objectives and requirements between science and business) (Pisano, 2006). Deo problema, smatra se, mo%e se na$i i u nedostatku mehanizama za obezbedjivanje postojanosti kvaliteta, adekvatnih ispitivanja da bi se obezbedila bezbednost korisnicima (pacijentima), 32 uklju#uju$i testiranje in vitro i in vivo (Pisano, 2006). Osim toga, znacajan broj projekata i kompanija u ovoj oblasti zapo#inje svoje poslovanje pre nego "to imaju oformljene nau#no-tehnolo"ke procedure, ulaze u partnerstva bez odgovaraju$e podr"ke a zatim su suo#eni sa nekim od konfliktnih prioriteta koje postavljaju nau#ni i poslovni aspekt njihovog poslovanja^ (Armstrong, 2001; Pisano, 2006). Neki autori smatraju da “investment and R&D decisions you make today are not easily quantifiable and not coming to fruition for fifteen to twenty years, special intuition and judgment are required”^. Oni takodje predla%u da je neophodan razvoj nove generacije stru#njaka koji $e da vode ovakve projekte i organizacije – posebno kada se uzme u obzir dugoro#nost ovih projekata. Tako se dolazi do zaklju#ka da su u industriji razvoja $elijskih terapija neophodne nove poslovne sposobnosti upravljanja, kao i novi poslovni sistemi bazirani na po"tovanju regulative i strogih zakonskih odredbi^. Grafi#ki prikaz slo%enosti i mogu$nosti za primene $elijskih terapija nalazi se u Prilogu 8. Slika 1.3: Prakti#ni aspekti izrade $elijskih terapija koji #esto ograni#avaju proces razvoja novih biolo"kih lekova u toj oblasti / Adaptirano iz: ISCT presentations Sept 2008, Biologics Consulting Group, by D Weber /. __________ **Business Process Management: Global Trends to 2013 (Strategic Focus), September 2008, Datamonitor !BIO. (2001) Editor’s and Reporter’s Guide to Cell therapy, A report by the Cell Therapy Industry Organization, USA. 33 1.3.3 Osnovni principi kvaliteta u farmaceutskoj industriji i u izradi naprednih terapija: dobra proizvodja"ka praksa i sistemi kvaliteta Koncept kvaliteta u farmaciji kroz implementaciju osnovnih na#ela Dobre proizvodja#ke prakse, DPP (eng. Good Manufacturing Practice, GMP) prvo je za%iveo u farmaceutskoj industriji. Kvalitet proizvedenih lekova je posebno zna#ajan za bezbednost korisnika lekova tako da je oblast proizvodnje i prometa farmaceutskih proizvoda, u najve$em broju zemalja, najuredjenija oblast privrede koju #ine brojne i veoma stroge zakonske odredbe, tehni#ki propisi, podzakonska akta, uredbe, pravilnici i preporuke kao i slo%eni, visoko uredjeni poslovni sistemi (Pisano G., Science Business-The Promise, The Reality and The Future of Biotech 2006; Tasi$ Lj., Farmaceutski menadzment i marketing, 2007; Handfield R., Biopharmaceutical Supply Chains 2012; Pharmaceutical Biotechnology-Drug Discovery and Clinical Applications 2012; Tasi$ Lj., Marinkovi$ V., Kvalitet u farmaciji 2012). U ovom procesu primenjuju su razli#ite “Dobre prakse” koje obuhvataju op"te i "iroko prihva$ene principe. Bilo da su upitanju razvoj, proizvodnja ili plasiranje na tr%i"te (eng. marketing) u integrisanom polju privrede i zdravstva, naro#ito farmaceutske industrije – izrazito je prisutna filozofija “Dobre prakse”. Dobre prakse se zasnivaju na filozofiji sistema upravljanja kvalitetom (eng. Quality Management System, QMS), principima obezbedjivanja kvaliteta (eng. Quality Assurance, QA), kontrole kvaliteta (eng. Quality Control, QC) a danas sve vi"e na principima upravljanja “totalnog kvaliteta” (eng. Total Quality Management, TQM) (Tasi$, 2007; Handfield, 2012; Tasi$ & Marinkovi$, 2012) i sve "ire primene najnovijih, regulatornih nauka, koje se razmatraju u nekoliko slede$ih poglavlja. Globalizacija tr%i"ta i nastojanje farmaceutskih kompanija da ostvare "to ve$i profit plasiraju$i svoje proizvode na "to ve$i broj tr%i"ta – doveli su do inicijativa za harmonizaciju i uskladjivanje razli#itih aspekata razvoja, proizvodnje, ispitivanja i prodaje lekova. Na ovaj na#in se osigurava uniformnost kvaliteta proizvoda i bezbednost korisnika lekova, ali se u isto vreme ulazi u kompleksne procese harmonizacije i uskladjivanja zakonskih regulativa u farmaceutskoj industriji na globanom nivou (Pisano, 2006; Tasi$, 2007; Handfield, 2012; Tasi$ & Marinkovi$, 2012). Osim standradnih i op"te prihva$enih principa “Dobre prakse” i primene slo%enih sistema kvaliteta u konvencionalnoj farmaceutskoj industriji i njoj bliskim oblastima zdravstva, sli#ni principi i procesi harmonizacije i uskladjivanja razvili su 34 se u oblasti izrade biofarmaceutskih proizvoda (eng. biopharmaceuticals) ili farmaceutske biotehnologije (eng. pharmaceutical biotechnology) (Kayser O. & Warzecha H., Pharmaceutical Biotechnology - Drug Discovery and Clinical Applications 2012; Ilic et al., 2012a; Ilic et al., 2012b). Medju biofarmacetskim proizvodima se, u okviru "irokog dijapazona biotehnolo"kih produkata (eng. biotech products), nalaze: monoklonalna antitela (eng. monoclonal antibodies), insulin/ insulin analogni lekovi, eritropoetin i drugi lekovi na bazi proteina – a od nedavno i $elijske terapije (eng. cell therapies) koje vrlo #esto koriste neke od tipova mati#nih $elija (eng. stem cells) kao osnovu za nove biofarmacetske lekove ili bioterapeutike (eng. biotherapeutics) (Kayser & Warzecha, 2012; Ilic et al., 2012a; Ilic et al., 2012b). Smatra se da danas postoji preko 400 biofarmaceutskih proizvoda (lekova i vakcina) koji su u procesu klini#kih ispitivanja a namenjeni su primeni u terapiji preko 200 razli#itih oboljenja (Kayser & Warzecha, 2012). 1.3.4 Principi regulatorne nauke u farmaciji i naprednim terapijama (eng. Regulatory Science and Advanced Therapies) U poredjenju sa nekim standardnim terapeutskim sredstvima, kao "to su na primer proteini i peptidi koji su bili od ogromnog interesa za farmaceutsku industriju (zbog svoje bioreaktivnosti, specifi#nosti, bezbedne upotrebe i uspe"nosti na tr%istu), $elijske terapije nose druga#ije rizike, imaju veoma specifi#ne procese proizvodnje, kontrole kvaliteta i upotrebe, kao i mnoga ve$ aktuelna ali i anticipirana eti#ka razmatranja#. Za razliku od proteina i peptida koji se koriste u terapeutske svrhe (posebno visoko-specifi#nih antitela sa relativno niskom toksi#no"$u) – $elijske terapije nisu dovoljno okarakterisane kao gotov terapijski sistem. Rezultati biomedicinskih istra%ivanja mogu biti preneti u klini#ku praksu uz dugotrajan, kompleksan i #esto veoma skup proces. Ovo je definisano kao translatorno istra%ivanje (eng. translational research) (Lowes, 2010; Sukkar, 2011, Ilic et al., 2012a; Ilic et al., 2012b). Na sli#nom principu se razvijaju napredne terapije (eng. advanced therapies) u obliku biofarmaceutskih lekova, koje se jo" nazivaju biolo"kim lekovima (eng. biologic drugs/ biologics/ biologicals). __________ ^ Harvard Business School: http://hbswk.hbs.edu/cgi-bin/print?id=5987 35 Regulatorne agencije "irom sveta suo#ene su sa novim, visoko dinami#nim poljem nau#nih otkri$a koji poku"avaju, manje ili vi"e uspe"no, da prate i da pri tome garantuju bezbednost pacijentima kao krajnjim korisnicima. Nezavisnost regulatornih agencija i specifi#ni regionalni/ nacionalni uslovi koji su uticali na njihov razvoj doveli su do razlika u pristupu koji su danas vidljivi u radu tih agencija, na primer u zemljama kao sto su SAD, Australija i zemlje Evropske zajednice. Globalnost tr%i"ta i procesi uskladjivnaja postoje$ih i novih regulativa u farmaceutskoj industriji i srodnim granama – kao "to je razvoj naprednih terapija (eng. advanced therapies) ili biolo"kih lekova (eng. biologic drugs) – doveli su do harmonizacije razli#itih zakonskih okvira izmedju regulatornih agencija. Ovo je posebno vidljivo u razvijenim zemljama kao "to su SAD, Australija i zemlje Evropske zajednice, #ije regulatorne agencije (Ameri#ka agencija za hranu i lekove - FDA, Australijska agencija za terapijska sredstva - TGA, Evropska agencija za lekove - EMA)* aktivno u#estvuju u tom procesu a #iji su regulatorni principi i zakonske regulative veoma razvijene u oblasti naprednih terapija. U procesu razvoja i harmonizacije^ izmedju tri pomenute agencije postoje odredjene sli#nosti i zajedni#ki principi koji mogu dalje da se koriste i usavr"avaju od strane regulatornih eksperata kao izvor znanja i ve"tina u okviru domena regulatornih nauka# (eng. regulatory science), izmedju ostalog, omogu$uju$i translatornim istra%ivanjima da se br%e i uspe"nije odvijaju (Lowes, 2010; Sukkar, 2011, Ilic, 2012a: Ilic, 2012b). Ameri#ka agencija FDA je pre nekoliko godina izdala saop"tenje* u kome je nagla"eno da je “nedovoljno investiranje u regulatorne nauke jedan od osnovnih razloga "to manji broj novih terapeutika dospeva na tr%i"te”. Tako se FDA agencija 2010. godine obavezala da uvede svoje regulatorne nauke u 21. vek, a sredinom 2011. godine ista agencija je izdala svoj strate"ki plan* za razvoj regulatornih nauka nazvan ‘Advancing Regulatory Science in FDA’. Osim toga, ubrzo je objavljeno partnerstvo/ program** “Common Fund’s Regulatory Science” izmedju Nacionalnog Instituta za Zdravlje SAD-a (eng. USA National Institute oh Health, NIH) i agencije FDA. __________ * Food and Drug Administration, FDA: http://www.fda.gov ; Therapeutic Goods Administration, TGA: http://www.tga.gov.au European Medicine Agency, EMA: http://www.ema.europa.eu ^ The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH), Available from URL: http://www.ich.org # What is Regulatory Science? The Institute for Regulatory Science, USA: www.nars.org/whatis 36 Krajem 2010. godine evropska regulatorna agencija EMA takodje je objavila^^ da je ona “glavni lider u razvoju i primeni regulatornih nauka”. Kao jedan od dokaza njihove inicijative u tom smislu, navedeni su rezultati konferencije koja je odrzana pod pokroviteljstvom EMA agencije u to vreme i nakon koje je objavljen izve"taj## pod nazivom “Regulatory science: are regulators leaders or followers?”. Svi ovi programi i inicijative se odnose kako na primenu novog domena regulatornih nauka# (eng. regulatory science) u farmaceutskoj industriji tako i na primenu u translatornim istrazivanjima u oblasti naprednih terapija. 1.4 Kvalitet, efikasnost i bezbednost: terapije mati"nim #elijama i novi regulatorni principi (eng. Prinicples of Regulatory Science) Uz razmatranje regulativa vezanih za medicinska sredstva (npr. lekove) koja su proizvedena u tradicionalnim okvirima farmaceutske industrije, uspostavljanje i primena novih regulativa vezanih za $elijske terapije (uop"te, napredne terapije) je neizbe%no vezano za oblasti za"tite javnog zdravlja i obezbedjivanja fondova za njihov razvoj – kao i razvoj novih disciplina ("to je diskutovano u prethodnom poglavlju). Ovo postavlja regulatorno telo ili regulatornu agenciju u veoma delikatnu poziciju i name$e joj neke nove uloge. Pri tome se o#ekuje da se u okviru zakonsko regulatornih okvira ‘pomire’ uloge agencije kao nezavisnog ekspertskog tela za ispitivanje bioterapeutika sa ulogom iste agencije u za"titi javnog zdravlja i formiranju percepcije o bezbednosti i upotrebnoj vrednosti naprednih terapija u stru#noj i op"toj javnosti. Poreklo termina ‘regulatorne nauke’ nije sasvim jasno ali se smatra da je nastao jos sedamdesetih godina 20. veka kada je jedna od ameri#kih agencija za za"titu %ivotne sredine (eng. USA Environmental Protection Agency) morala da donese odredjene odluke bez dovoljno adekvatnih informacija, pri tome uzimaju$i u obzir niz kompleksnosti. Definicija regulatornih nauka koju trenutno koristi agencija FDA je: “nauka koja razvija nove metode, standarde i pristupe za ispitivanje bezbednosti, efikasnosti, kvaliteta i uspe"nosti svih produkata (regulisanih od strane agencije)” (eng. “the science of developing new tools, standards, and approaches to assess safety, efficacy, quality, and performance of all (FDA) regulated products”)*. 37 Jo" uvek postoji neslaganje sa tim terminom jer mnogi stru#njaci smatraju da “je nauka ve$ nauka bez obzira kako se primenjuje”#. Usled pomenute slo%enosti, postoji mno"tvo dokumenata i raznovrsnih informacija u okviru regulatornih propisa svake od pomenutih agencija – a njihov broj svakodnevno raste u skladu sa razvojem nau#nih metoda i propratnih regulativa. Za istra%iva#e, proizvodja#e biofarmaceutskih sredstava, lekare, pacijente i njihove zakonske predstavnike, kao i za investitore, vladine agencije i op"tu javnost, neki od osnovnih problema u primeni i razumevanju regulativa u ovom obilju informacija predstavljaju: - upotreba razli#itih termina kao "to su biolo"ki lekovi (eng. biologic drugs), napredne terapije (eng. advanced tehrapies) i drugi (eng. engineered tissues and somatic cells vs. biologics vs. biologicals), - razli#iti nivoi na kojima su regulative zastupljene (eng. different levels of scrutiny, i.e. manufacturing facility vs. manufacturing process), - razlike u pristupu agencija koje mogu da se kre$u od veoma detaljnih do veoma uop"tenih u smislu preporuka i zahteva koje propisuju (eng. differences in availability of specific formal written procedures: prescriptive vs. overly generic), - veoma razli#iti kriterijumi kvaliteta u odnosu na propisane testove koje zahtevaju (eng. different testing requirements: all inclusive demonstrated biologic effects vs. infectious disease markers). U slede$im poglavljima bi$e, u glavnim crtama, razmatrani principi regulatornih nauka odnosno zakonsko regulatorni okviri koje u oblasti naprednih terapija i srodnih disciplina primenjuju agencije u SAD-u, Australiji, u zemljama Evropske zajednice i u na"oj zemlji. __________ * FDA News Release: Regulatory science plan positions agency to foster innovation through better science, 2011: www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm268293 ** The Common Fund’s Regulatory Science program between NIH and FDA: www.commonfund.nih.gov/regulatoryscience/overview ^^ European Medicines Agency, Special Topics, Regulatory science: www.ema.europa.eu/ema ## EMA Report. Regulatory science: are regulators leaders or followers? www.ema.europa.eu/ema # What is Regulatory Science? The Institute for Regulatory Science, USA: www.nars.org/whatis 38 1.4.1 Globalni pristup i definicija osnovnih zakonskih regulativa u izradi #elijskih terapija Iako su neki od biolo"kih lekova (npr. monoklonalna antitela) bili prethodno regulisani od strane regulatornih agencija, biolo"ki produkti koji se baziraju na transplantaciji $elija i tkiva bili su, istorijski gledano i iz vi"e razloga, isklju#eni iz okvira ovih regulativa (Burger, 2003b; Halme, 2006; Ilic et al., 2012a; Ilic et al., 2012b). Danas se, ipak, smatra, da je neophodno da se grupa biolo"kih terapeutika baziranih na transplantaciji $elija i tkiva takodje reguli"e. Istovremeno, poznato je da tradicionalni sistemi kvaliteta usvojeni iz farmaceutske industrije nisu najpogodniji za ovu svrhu; budu$i da su ovi bioterapeutici slo%eniji i te%i za identifikaciju kao i da podle%u specifi#nim, veoma slo%enim procesima prikupljanja, izrade i primene (Burger, 2003a; Halme, 2006; Kirouac & Zandstra, 2008; Ilic et al., 2012a; Ilic et al., 2012b). Ova nova razmatranja uslovljena rapidnim razvojem nau#nih istra%ivanja, kao i vi"ezna#na uloga regulatornih agencija (razmatrana u prethodnom poglavlju), doveli su do formiranja novih, slo%enih regulatornih okvira koji se primenjuju u sferi razvoja biolo"kih lekova baziranih na transplantaciji $elija i tkiva. Pri tome se formira inovativni sektor biofarmaceutskih proizvoda ‘koji postaje jedan od najintenzivnijih istra%iva#kih sektora sa ogromnim potencijalom da obezbedi nove lekove i napredak medicine u budu$nosti’ (eng. “has become one of the most research-intensive sectors with a great potential for delivering innovative human medicines in the future”) §. 1.4.1.1 Model* zakonskih regulativa u Sjedinjenim Ameri"kim Dr%avama (SAD) Nakon svog osnivanja 1930. godine, kome je prethodio prvi zakon u SAD-u o hrani i lekovima ustanovljen jo" 1906. godine (eng. Food and Drug Act) kao i niz drugih mnogobrojnih zakonskih regulativa i pravilnika u toj oblasti – ameri#ka agencija FDA je 1993. godine objavila svoj prvi interim pravilnik koji je zahtevao adekvatno testiranje tkiva na infektivne bolesti (pre svega HIV, hepatitis B i C), prikupljanje informacije o davaocu tkiva i #uvanje podataka vezanih za transplantaciju (nakon incidenta i upotrebe HIV zara%enog tkiva za transplantaciju zabele%enog u SAD-u u ranim devedesetim). Zbog sveobuhvatnosti svog pristupa koji se bazira na proceni rizika (eng. risk-based approach) kao i zbog ekspertize koju danas poseduju stru#njaci u ovoj agenciji, FDA se smatra jednom od najzna#ajnijih __________ *Model podrazumeva principe i infrastrukturu regulatornih tela. 39 svetskih regulatornih agencija u ovoj oblasti. Agencija FDA je 1997. godine objavila dva bitna dokumenta§§ time ozna#avaju$i planove za dalje regulisanje primene $elija, $elijskih produkata i produkata baziranih na primeni humanih tkiva. Zatim je 2001. godine formiran pristup za registraciju laboratorija koje se bave ovom delatno"$u, a u maju 2004. godine FDA je zapo#ela aktivnu kampanju baziranu na regulativama koje su zahtevale da svi davaoci $elija i tkiva budu podvrgnuti testovima na infektivne bolesti (eng. donor-eligibility final rule, 69 FR 29786). Agencija je krajem 2004. godine donela novu zakonsku regulativu kojom se zahteva da sve laboratorije koje se bave izradom produkata baziranih na ljudskim $elijama i tkivima (eng. human cell and tissue based products, HCT/P) moraju da se pridr%avaju Dobre prakse u toj oblasti (eng. Current Good Tissue Practices, CGTP) koja, izmedju ostalog, zahteva primenu procedura za adekvatno rukovanje, procesovanje, obele%avanje i #uvanje podataka (eng. handling, processing, labelling, and record-keeping procedure). Va%an datum u pristupu i primeni novih zakonskih regulativa agencije FDA u ovoj oblasti je 25. maj 2005. godine kada je objavljeno novo interim pravilo (eng. interim final rule) da se izvr"i revizija do tada postoje$ih regulativa za testiranje i odabir HCT/P donora kao i pravila za njihovo obele%avanje (eng. 70 FR29949). Agencija je ovim odgovorila na zna#ajne komentare eksperata u ovoj oblasti koji su se odnosili na neke neprakti#nosti HCT/P regulativa koje su prethodno bile na snazi. Danas su najzna#ajnije CGTP i druge HCT/P regulative koje primenjuje agencija FDA sadr%ane u federalnom zakonu SAD-a tzv. kodu/ dokumentu 21 CFR Part 1271 (eng. 21 Code of Federal Regulations Part 1271) koji obuhvata i odredbe za registraciju laboratorija koje se bave ovom delatno"$u. Uz to postoji mno"tvo podzakonskih akata, odredbi i preporuka koje agencija FDA primenjuje u ovoj oblasti. Federalni zakonski dokument 21 CFR Part 1271 ima "est osnovnih delova (eng. General provisions pertaining to the scope and purpose of Part 1271, as well as definitions; Registration and listing procedures; Provisions for the screening and testing of donors to determine their eligibility; Current Good Tissue Practice (CGTP) requirements; Certain labelling and reporting requirements; and Inspection and enforcement provisions). __________ *FDA News Release: Regulatory science plan positions agency to foster innovation through better science, 2011: www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm268293 What is Regulatory Science? The Institute for Regulatory Science, USA: www.nars.org/whatis §The financing of biopharmaceutical product development in Europe, Final Report. European Commission, Enterprise and Industry, October 2009. ISBN 978-92-79-14055-6. 40 Tabela 1.8: Kategorije HCT/P proizvoda koje ne zadovoljavaju sve kriterijume propisane u regulativi 21 CFR 1271.10(a). Krv i krvni produkti: eng. Blood and Blood Products are covered under CP 7342.001 "Inspection of Licensed and Unlicensed Blood Banks, Brokers, Reference Laboratories, and Contractors"; and CP 7342.002 "Inspection of Source Plasma Establishments" Medicinske naprave: eng. HCT/Ps that do not meet all 21 CFR 1271.10(a) criteria, and are regulated as Medical Devices are covered under CP 7382.845 "Inspection of Medical Device Manufacturers" Visoko manipulisane "elijske i genske terapije: eng. HCT/Ps that do not meet all 21 CFR 1271.10(a) criteria, i.e. Autologous, Allogeneic, or Xenogeneic Cells whose biological characteristics have been altered (propagate, pharmacologically treated, etc.); Ex Vivo and Gene Therapy products are regulated as biological drugs and are covered under CP 7345.848 "Inspection of Biological Drug Products" Proizvodi bazirani na "elijama i tkivu koje je prikupljeno pre 25. maja 2005. godine: eng. HCT/Ps recovered before May 25, 2005 and regulated under 21 CFR 1270 and subparts A and B of Part 1271 are covered under CP 7341.002A "Inspection of Tissue Establishments". Uz to postoje njegove specifi#ne odredbe (eng. 21 CFR Part 1271 subparts A, B, C, F, 21 CFR 1271.150(c), and 21 CFR 1271.155 of subpart D apply to reproductive HCT/Ps) od kojih se neke odnose na reproduktivna tkiva. Ovaj dokument se smatra “novim” i sve njegove zakonske odredbe se odnose na HCT/P produkte prikupljene nakon 25. maja 2005. godine. HCT/P produkti prikupljeni i/ili izradjeni pre tog datuma su regulisani primenom prethodnog federalnog zakonskog koda/dokumenta 21 CFR 1270 kao i delova A i B “novih” dokumenata u specifi#nim domenima (eng. subparts A and B of Part 1271, as appropriate). Oni HCT/P proizvodi koji ne zadovoljavaju sve kriterijume propisane u regulativi 21 CFR 1271.10(a) regulisani su kao lekovi (eng. drugs), medicinske naprave (eng. devices) ili biolo"ki proizvodi (eng. biological products), a samim tim se uklapaju u neke druge zakonsko regulatorne FDA okvire (Tabela 1.8). Dalja razmatranja o regulatornom okviru koji primenjuje agencija FDA kao i pregled nekih drugih FDA regulativa nalaze se u Prilogu 6. __________ *Food and Drug Administration (FDA) http://www.fda.gov The Center for Biologics Evaluation and Research (CBER) http://www.fda.gov/BiologicsBloodVaccines/DevelopmentApprovalProcess/default.htm The Center for Drugs Evaluation and Research (CDER) http://www.fda.gov/cder/guidance/959fnl.pdf The Center for Biologics Evaluation and Research (CBER) http://www.fda.gov/BiologicsBloodVaccines/DevelopmentApprovalProcess/NewDrugApplicationNDAProcess/default.htm §§ "A Proposed Approach to the Regulation of Cellular and Tissue-Based Products" (62 FR 9721, March 4, 1997) and "Reinventing the Regulation of Human Tissue". 41 1.4.1.2 Model* zakonskih regulativa u Australiji Regulatorna agencija za terapeutska sredstva u Australiji (eng. Therapeutic Goods Administration, TGA) osnovana je 1990. godine kao deo federalne vladine agencije za zdravlje (eng. Commonwealth Department of Health and Aged Care). Njen rad se zasniva na najvi"em zakonskom aktu uspostavljenom od strane federalne vlade (eng. Therapeutic Goods Act of 1989) ^. Agencija TGA kontroli"e promet terapeutskih sredstava u Australiji na tri razli#ita na#ina (eng. pre-market evaluation and approval, licensing of manufacturers and post market surveillance). Lekovita sredstva koja su uvezena ili proizvedena u Australiji moraju biti registrovana u okviru necionalnog registra lekova (eng. Australian Register of Therapeutic Goods, ARTG), ukoliko nisu izuzeta. Pristup lekovitim sredstvima koja su izuzeta obezbedjuje se kroz nekoliko mehanizama/ shema (eng. Special Access Scheme Cat. A and B, Clinical Trials - CTN and CTX Schemes, authorized prescriber/s, and importation for personal use)^. U smislu naprednih terapija, koje se u novom regulatornom okviru agencije TGA# nazivaju biolo"kim produktima (eng. biologicals), zakonske regulative izmedju ostalog, uklju#uje kontrolu $elija i tkiva namenjenih za transplantaciju koji su: proizvedeni, uvezeni u Australiju ili namenjeni izvozu iz zemlje#. Pre nego "to je novi regulatorni okvir# (eng. Biologicals Regulatory Framework) stupio na snagu 31. maja 2011. godine, $elije i tkiva namenjeni za transplantaciju nisu bili regulisani kroz visoke zakonske odredbe u Australiji (eng. Therapeutic Goods [Excluded Goods] Order). Ovi proizvodi su bili uglavnom regulisani kao medicinska sredstva ili kao medicinske naprave (eng. medicines or medical devices) i bili su izuzeti iz primene odgovaraju$e zakonske odredbe kojom se uredjivao ulazak terapeutskih sredstava u registar (eng. specific parts of the Therapeutic Goods Act 1989). Pri tome su se na ove produkte primenjivali neki drugi delovi istog zakonskog akta kao "to je npr. obavezno po"tovanje principa Dobre proizvodja#ke prakse (eng. compliance with Good Manufacturing Principles) #ija je najnovija verzija u ovoj oblasti (eng. Australian Code of Good Manufacturing Practice for human blood and blood components, human tissues and human cellular therapy products) stupila na snagu u avgustu 2013. godine^^. __________ *Model podrazumeva principe i infrastrukturu regulatornih tela. 42 Konferencija ministara zdravlja u Australiji (eng. Australian Health Ministers’ Conference) je 2006. godine preporu#ila da se $elijske terapije i terapije na bazi transplantacije tkiva, kao i nove generacije biolo"kih lekova (uz izuzetak organa i reproduktivnih tkiva) reguli"u kao deo terapeutskih sredstava pod ingerencijom TGA. U tom smislu je novi zakonski okvir, koji se bazira na proceni rizika i shodno tome primenjuje na razli#itim nivoima (tzv. klase proizvoda), stupio na snagu 31. maja 2011 godine; isti zakonski okvir je predvidjen da obezbedi podr"ku razvoju inovativnih terapija koje bi tek trebalo da budu razvijene u budu$nosti. Postavljen je trogodi"nji period tranzicije za ve$ registrovane produkte a svi novi proizvodi u datoj oblasti odmah podle%u zakonskim regulativama pri #emu ulaze u nacionalni registar terapeutskih sredstava (eng. ARTG). Na osnovu najvi"eg zakonskog akta (eng. Therapeutic Goods Act 1989) i njegovih amandmana, agencija TGA u biolo"ke produkte (eng. biologicals) ubraja “sve "to sadr%i ili je naravljeno od ljudskih $elija ili tkiva i "to se koristi za navedene funkcije” (Tabela 1.9). Tabela 1.9: Biolo"ki proizvodi koji zadovoljavaju ili ne zadovoljavaju kriterijume propisane u novom zakonskom okviru (eng. Biologicals Regulatory Framework) #. Proizvodi koji zadovoljavaju kriterijume novog zakonskog okvira Izuzeti proizvodi koji ne zadovoljavaju kriterijume novog zakonskog okvira eng. -treat or prevent disease, ailment, defect or injury, -diagnose a condition of a person, -alter the physiological processes of a person, -test the susceptibility of a person to disease, -replace or modify a person’s body parts. eng. -assisted reproductive technologies (in vitro fertilisation), -fresh viable organs, -fresh haematopoietic progenitor cells (bone marrow transplants), -cells and tissues made by a medical practitioner for a single patient under the care of that medical practitioner. Proizvodi koji se smatraju biolo#kim proizvodima (eng. biologics) Proizvodi koji se ne smatraju biolo#kim proizvodima (eng. non-biologics) eng. -human stem cells, -tissue-based products (skin, bone, ocular, cardiovascular), -cell-based products (genetically modified, in vitro cell expansion or depletion), -combined cell and tissue products (collagen matrices for localised cell delivery). eng. -biological prescription medicines (vaccines, plasma derivatives, recombinant products), -labile blood and blood components, -haematopoietic progenitor cells used for haematopoietic reconstitution (non-fresh transplants). 43 Biolo"ki proizvodi u okviru “kombinovanih produkata” npr. u kombinaciji sa medicinskim napravama/pomagalima (eng. integrated with the medical device), kao "to su metalni stentovi oblo%eni matriksom i endotelijalnim $elijama (eng. metal stent coated with a matrix and endothelial cells) – regulisani su od strane novog zakonskog okvira i moraju biti registrovani kao biolo"ki produkt (eng. biological); dok je sâm metalni stent nezavisno naveden u registru medicinskih naprava/pomagala. Sredstva koja su izuzeta iz regulative kao terapeutska sredstva, nalaze se u okviru posebnog zakonskog akta (eng. Therapeutic Goods /Excluded/ Order) i nisu regulisana od strane agencije TGA. Postoje proizvodi koji su poreklom iz ljudskog tkiva, koji spadaju u terapeutska sredstva iako nisu svrstani u biolo"ke produkte (eng. things that are not biologicals, Determination No.1 of 2011), tako da su oni regulisani od strane TGA agencije (Tabela 1.9), ali u okviru drugih zakonskih regulativa. U svakom slu#aju, zakonski okvir obezbedjuje diskreciono pravo zakonodavcu da proglasi da li postoje$i ili novi terapeutski proizvodi koji su $elijski- bazirani (eng. cell-based therapeutic goods) spadaju u kategoriju “biologicals” ili ne (Tabela 1.9). Dalja razmatranja i detaljan pregled TGA zakonskih regulativa nalaze se u Prilogu 6. Pregled ostalih regulatornih standarda, preporuka i pravilnika koji se koriste na globalnom nivou kao i u Australiji predstavljen je u Prilogu 7. __________ Therapeutic Goods Administration: ^http://www.tga.gov.au/docs/html/clintrials.htm ; http://www.tga.gov.au/docs/pdf/euguide/ich/ich13595.pdf #http://www.tga.gov.au/industry/biologicals-argb.htm ; http://www.tga.gov.au/industry/biologicals-standards.htm ^^http://www.tga.gov.au/industry/manuf-cgmp-human-blood-tissues-revised.htm Therapeutic Goods Administration: #http://www.tga.gov.au/industry/biologicals-argb.htm ; http://www.tga.gov.au/industry/biologicals-standards.htm ^^http://www.tga.gov.au/industry/manuf-cgmp-human-blood-tissues-revised.htm 44 1.4.2 Model* zakonskih regulativa u Evropskoj Zajednici Osnove moderne regulative o oblasti kontrole lekova u Evropi postavljene su 1968. godine kada je uspostavljena regulatorna agencija u Velikoj Britaniji, (eng. Medicines Control Authority, MCA) koja se danas skra$eno naziva MHRA (eng. Medicines and Healthcare products Regulatory Agency). Evropska agencija za kontrolu medicinskih sredstava, kao decentralizovano telo Evropske zajednice (eng. decentralised body of the European Union, EU) sa sedi"tem u Londonu, zapo#ela je svoju aktivnost 1995. godine, prvo kao EMEA a potom kao EMA (eng. European Medicines Agency) §. Prevashodna uloga evropske regulatorne agencije EMA je da obezbedi autorizaciju medicinskih proizvoda "to obezbedjuje centralizovanu proceduru i uzajamno prihvatanje ove procedure u evropskim zemljama (eng. a centralised and a mutual recognition procedure). Agencija EMA je podeljena u pet osnovnih delova, svaki sastavljen od vi"e sektora; ima Upravni odbor i "est komiteta a u svom radu obuhvata ekspertsko znanje iz 30 zemalja (eng. EU and EEA-EFTA) countries. Od kraja 2012. godine se planira restruktuiranje nekih delova agencije koje $e verovatno biti obavljeno u toku ove ili slede$e godine§§. Osnovna odgovornost agencije je za"tita i unapredjenje javnog zdravlja u regionu a time pokriva lekove i druga terapeutska sredstva koji se koriste u medicini i veterinarskoj nauci. Istorijski gledano, nedostatak centralizovanog zakonskog okvira u Evropskoj zajednici je, u odredjenoj meri, onemogu$io da se prevazidju razlike izmedju nacionalnih zakonskih regulativa u oblasti razvoja novih medicinskih sredstava. U svom nastojanju da obezbedi adekvatan okvir za novonastala nau#na dostignu$a u oblasti biofarmaceutskih lekova i novih terapijskih pristupa, agencija EMA je uspostavila zakonske odredbe za napredne terapije (eng. regulatory framework for ATMPs). Regulativa 1394 (eng. Regulation (EC) No 1394/2007) zvani#no je usvojena od strane Ministarskog saveta Evropske zajednice (eng. Council of Ministers, EU) u oktobru 2007. godine. Napredne terapije (eng. Advanced-therapy medicinal products, ATMP) su u okviru nove regulative tada definisane kao lekovi bazirani na genskim __________ *Model podrazumeva principe i infrastrukturu regulatornih tela. 45 terapijama, adultnim mati#nim $elijama ili in%enjeringu tkiva (eng. medicines for human use that are based on gene therapy, somatic-cell therapy or tissue engineering, TE). Period tranzicije za proizvode bazirane na $elijskim terapijama bio je do 30. decembra 2011. godine a za TE proizvode do 30. decembra 2012. godine. Ovo se odnosilo na sve medicinske proizvode koji su bili na tr%i"tu zahvaljujuci nacionalnim regulativama u pojedinim zemljama Evropske zajednice – ili usled nedostatka istih ("to se ponekad baziralo samo na sporadi#nim istra%ivanjima ili minimalnoj koli#ini podataka). U toku i nakon perioda tranzicije, centralna procedura agencije EMA zahteva da svaki ATMP proizvod bude pojedina#no odobren i u potpunosti okarakterisan na osnovu novih zakonskih odredbi kao "to je ne samo Reg.1394/2007 ve$ i druge regulative (eng. Regulations) i direktive (eng. Directives) i njihovi brojni amandmani. Kompleksnost zakonskog pristupa Evropske zajednice koji se odnosi na napredne terapije (ATMP) proizilazi u odredjenoj meri od mno"tva drugih direktiva (eng. a directive is the minimum legal standard issued by the European Parliament requiring adoption by the member states)¶ i regulativa (eng. a regulation completely by passes all member state laws and takes precedence)¶ (Tabela 1.11). Slika 1.4: Definicija naprednih terapija prema Reg. 1394/2007 Evropskog parlamenta. eng. The European Parliament Regulation 1394/2007 (amending directive 2001/83/EC and Regulation 726/2004) identifies an advanced therapy medicinal product (ATMP) as: “! a medicinal product as defined in Directive 2001/83/EC of the European Parliament and of the Council of 6 November 2001 on the Community code relating to medicinal products for human use (the Directive), as amended to reflect new innovative therapeutic products. Specifically, an ATMP is a biological medicinal product which is either: - a gene therapy medicinal product as defined in Part IV of Annex I to Directive 2001/83/EC; - a somatic cell therapy medicinal product as defined in Part IV of Annex I to Directive 2001/83/EC; or - a tissue engineered product as defined in Article 2 1 (b) of the ATMP Regulation (1394/2007/EC)” _________ §European Medicines Agency: http://www.ema.europa.eu/ema/ §European Medicines Agency: http://www.ema.europa.eu/ema/ §§http://www.ema.europa.eu/ema/index.jsp?curl=pages/news_and_events/news/2013/08/news_detail_001868.jsp &mid=WC0b01ac058004d5c1 ¶ The European Medicines Agency (EMA) http://www.ema.europa.eu/ema/index.jsp?curl=pages/about_us/general/general_content_000235.jsp&murl=menus/about_us/abo ut_us.jsp&mid 46 Tabela 1.10: Podr"ka koju agencija EMA nudi individualnim i organizacionim projektima za napredne terapije u ranom stadijumu razvoja kao dodatak inicijativi eng. Innovation Task Force, ITF i uloga Komiteta za napredne terapije (eng. Committee for Advanced Therapies, CAT). Informacije Dokumenta eng. -the classification of ATMP marketing- authorisation applications; -certification of the quality of ATMPs; -non-clinical data submitted by small and medium- sized enterprises (SMEs) developing ATMPs. eng. -European Union regulation on advanced therapies; -ATMP classification; -Certification procedure for small- and medium- sized enterprises; -Marketing-authorisation application submission; -Scientific guidelines; -Risk management; -Interested parties; and -Guidance. Proces za sticanje odobrenja od strane CAT i EMA Ostale uloge CAT eng. The Agency's Committee for Advanced Therapies (CAT) plays a central role in the scientific assessment of advanced-therapy medicines. It provides the expertise that is needed to evaluate advanced-therapy medicines. During the assessment procedure, the CAT prepares a draft opinion on the quality, safety and efficacy of an advanced-therapy medicine. It sends this to the Committee for Medicinal Products for Human Use (CHMP). Based on the CAT opinion, the CHMP adopts a recommendation for the European Commission. The European Commission may grant or refuse a marketing authorisation on the basis of the Agency's recommendation. eng. -gives recommendations on the classification of advanced-therapy medicines; -reviews data on the manufacture and testing of medicines developed by small companies; -contributes towards giving scientific advice on advanced-therapy medicines; -contributes towards an environment that encourages the development of advanced-therapy medicines; -provides scientific expertise and advice for any initiatives related to the development of innovative medicines and therapies, at the request of the European Commission. -engages with its stakeholders through regular interaction with its interested parties. Tabela 1.11: Kompleksnost zakonskih propisa u Evropskoj zajednici koji se odnose na napredne terapije (eng. ATMP), a sastoje se iz vise regulativa i direktiva. Directive 2001/83 Regulation 726 Reg. 668 (Certification) Regulation 1394/2007 Directive 2003/63 Directive 2003/94 Directive 2005/28 Regulativa 726 je takozvana centralna procedura (#iji su amandmani odslikali promene nastale usled dono"enja novog zakonskog okvira vezanog za napredne terapije) i ona defini"e koji produkti moraju da budu autorizovani kroz centralnu proceduru jer ne mogu vi"e biti odobreni kroz pojedina#ne nacionalne regulative. Medjutim, promene regulative ne obavezuju zemlje #lanice da menjaju svoje zakone 47 na isti na#in kao kada se uskladjuju sa novim direktivama!. Regulativa 1394/2007 (eng. Regulation 1394/2007) koju je doneo Evropski parlament, je dopunila direktivu 2001/83/EC i regulativu 726/2004 (eng. directive 2001/83/EC & Regulation 726/2004) novom definicijom naprednih terapija (eng. advanced therapy medicinal product, ATMP) (Slika 1.4). Aneks I direktive 2001/83 (eng. Annex I, Directive 2001/83/EC) je u junu 2003. godine bio zamenjen aneksom I direktivi 2003/63 (eng. Annex I, Directive 2003/63/EC). Ovaj aneks opisuje nau#notehni#ke kriterijume koji su neophodni u dozieru (eng. dosier) za marketin"ku autorizaciju svih klasa medicinskih produkata* i na svim nivoima (eng. national, decentralised & centralised procedure) a prema novoobjavljenoj verziji Zajedni#kog tehni#kog dokumenta (eng. Common Technical Document, CTD), koji propisuje novi, harmonizovan format i terminologiju. Nova direktiva 2003/63 sa aneksom I je stupila na snagu 1. jula 2003. godine a zemlje #lanice su bile u obavezi da usklade svoje neophodne zakonske regulative najkasnije do 31. oktobra 2003. godine. Shodno slo%enosti zakonskih okvira Evropske zajednice, mnogobrojni zakonski propisi (medju kojima je grupa pravilnika i zakonskih propisa za regulisanje medicinskih produkata i naprednih terapija) nalaze se na Eudralex-u (eng. EU Legislation/ Eudralex¶¶) kao i na web sajtu Direktorata evropske komisije (eng. European Commission's Directorate)^^. Dalja razmatranja regulativa EMA agencije u oblasti naprednih terapija nalaze se u Prilogu 6. __________ !There are three bodies in Europe: elected European Parliament (meets at Strasburg); they vote and discuss/ negotiate laws but cannot propose a law; non-elected European Commission in Brussels decides what draft laws should be put before the Parliament; after the Parliament has decided something, it has to be adopted by the Council of Minsters. If we talk about the Health, all of the Ministers of Health of member states, as a Council, has to endorse the new law. ¶ The European Medicines Agency (EMA) http://www.ema.europa.eu/ema/index.jsp?curl=pages/about_us/general/general_content_000235.jsp&murl=menus/about_us/abo ut_us.jsp&mid * Neki odredjeni medicinski produkti moraju da ispune i dodatne zahteve koji su doneti drugim zakonskim propisima, ukoliko tome podlezu. ¶¶http://ec.europa.eu/health/documents/eudralex/index_en.htm ^^http://ec.europa.eu/health/human-use/index_en.htm 48 1.4.3 Model* zakonskih regulativa u Srbiji Zakon o transplantaciji !elija i tkiva stupio je na snagu prvi put u Republici Srbiji nakon objavljivanja u Slu%benom Glasniku broj 72 iz 2009. godine a primenjen je od 1. januara 2010. godine. Danom po#etka primene ovog zakona prestala je da va%i odredba #lana 48. stav 3. Zakona o zdravstvenoj za!titi (Sl. glasnik RS br. 107 iz 2005. godine). Zakonom o transplantaciji !elija i tkiva (u daljem tekstu: Zakon) uredjeno je “dobijanje, doniranje, testiranje, obrada, o#uvanje, skladi!tenje i distribucija ljudskih !elija i tkiva namenjenih za primenu kod ljudi; osnivanje banaka !elija i tkiva; nadzor nad sprovodjenjem ovog zakona i obavljanje odredjenih poslova dr%avne uprave u oblasti transplantacije !elija i tkiva, kao i druga pitanja od zna#aja za organizaciju i sprovodjenje transplantacije !elija i tkiva.” (Zakon o transplantaciji !elija i tkiva, Sl. glasnik RS br. 72/2009). Primenom Zakona je uredjen “postupak dobijanja, doniranja, testiranja, obrade, o#uvanja, skladi!tenja i distribuciju krvotvornih !elija i mati#nih !elija hematopoeze iz periferne krvi, krvi iz placente i ko"tane sr%i, reproduktivnih !elija, tkiva i !elija fetusa i mati#nih !elija odraslih organizama i embriona primenjuju se odredbe ovog zakona, osim ako ovim zakonom nije druk#ije uredjeno. Upotreba !elija i tkiva kao autolognih transplantata u istom hirur"kom postupku, kao i krvi i komponenata krvi, obavlja se u skladu sa zakonom.” (Zakon o transplantaciji !elija i tkiva, Sl. glasnik RS br. 72/2009). U okviru Zakona nagla"eno je da se, za potrebe transplantacije $elija, u zdravstvenim ustanovama organizuju timovi za transplantaciju. Takodje, uredjeno je da se postupak akreditacije zdravstvenih ustanova, za potrebe transplantacije $elija, obavlja u skladu sa zakonom kojim se uredjuje zdravstvena za!tita, a na osnovu standarda za akreditaciju zdravstvenih ustanova koje obavljaju poslove transplantacije !elija, odnosno tkiva, koje propisuje Ministar zdravlja Republike Srbije. Novi zakonski principi odredili su da zdravstvene ustanove mogu podneti zahtev za osnivanje banke !elija i tkiva u roku od 12 meseci od dana po#etka rada Uprave za biomedicinu Ministarstva zdravlja Republike Srbije; a zdravstvene ustanove, odnosno delovi zdravstvenih ustanova koji su ve$ obavljali odredjene poslove banaka !elija i tkiva u Republici Srbiji bili su du%ni da usklade svoj rad sa odredbama ovog zakona u roku od 12 meseci od dana po#etka rada Uprave za biomedicinu. 49 Tabela 1.12: Domeni u procesu transplantacije !elija i tkiva koji su regulisani Zakonom (Zakon o transplantaciji !elija i tkiva, Sl. glasnik RS br. 72/2009). I OSNOVNE ODREDBE II NACELA POSTUPKA TRANSPLANTACIJE !ELIJA I TKIVA o Na!elo solidarnosti, Na!elo medicinske opravdanosti transplantacije; Na!elo za!tite interesa i dostojanstva; Na!elo dostupnosti i zabrane diskriminacije; Na!elo bezbednosti. III ORGANIZACIJA OBAVLJANJA POSLOVA TRANSPLANTACIJE !ELIJA I TKIVA o Zdravstvena ustanova koja obavlja poslove transplantacije !elija, odnosno tkiva; Izdavanje, obnova i oduzimanje dozvole; Uloga koordinatora i eti!kog odbora u ovla!"enoj zdravstvenoj ustanovi. IV POSTUPAK TRANSPLANTACIJE !ELIJA I TKIVA o Dobrovoljnost davanja !elija i tkiva; Medicinska opravdanost postupka transplantacije; Pismeni pristanak punoletnog primaoca; Pismeni pristanak maloletnog primaoca, odnosno primaoca kome je oduzeta poslovna sposobnost; Republi!ka lista !ekanja za transplantaciju; Jedinstveni republi!ki registar davalaca !elija, odnosno tkiva; Razmena !elija i tkiva radi le!enja; Sledivost !elija i tkiva; Postupak transplantacije; Spre!avanje rizika preno!enja bolesti, kao i o!uvanje kvaliteta !elija, odnosno tkiva; Finansiranje postupka transplantacije; Pra!enje ozbiljnih ne!eljenih reakcija i ozbiljnih ne!eljenih pojava; Mogu"nost kori#"enja podataka; Prikupljanje, obrada, vodjenje i kori!"enje podataka o davaocu, odnosno primaocu i medicinska dokumentacija; Obave!tavanje Uprave za biomedicinu; Standardne operativne procedure i vodi!i dobre prakse. V UZIMANJE !ELIJA I TKIVA OD $IVOG DAVAOCA o Uslovi za uzimanje !elija i tkiva od "ivog davaoca; Procena rizika po zdravlje davaoca; Pismeni pristanak i povla!enje pristanka davaoca; Lica koja mogu biti davaoci !elija i tkiva; Pravo na nepristrasno informisanje davaoca !elija, odnosno tkiva radi davanja pismenog pristanka. VI UZIMANJE TKIVA OD UMRLOG LICA VII BANKE !ELIJA I TKIVA o Osnivanje banke !elija i tkiva; Izdavanje, obnova i oduzimanje dozvole za banku !elija i tkiva; Inspekcijski nadzor nad bankom !elija i tkiva; Evidencije koje vodi banka !elija i tkiva i obaveza izve!tavanja; Registar banki, "elija i tkiva; Upravljanje sistemom kvaliteta; Obrada i uslovi skladistenja !elija i tkiva; Obele"avanje, dokumentovanje i ambala"a; Identifikacija !elija, odnosno tkiva; Distribucija; Odnos izmedju banke !elija i tkiva i tre!ih lica. VIII NAU%NOISTRA$IVA%KI RAD o Dozvola za sprovodjenje nau!noistra!iva!kog rada; Dozvola za uvodjenje novih zdravstvenih tehnologija u postupku le!enja primenom !elija, odnosno tkiva. IX NADLE$NOST UPRAVE ZA BIOMEDICINU o Poslovi Uprave za biomedicinu koji se odnose na transplantaciju !elija i tkiva; Nadzor koji sprovodi Uprava za biomedicinu u vezi sa poslovima transplantacije !elija i tkiva. X NADZOR XI OBAVLJANJE POSLOVA TRANSPLANTACIJE !ELIJA I TKIVA U USTANOVAMA KOJE NISU U PLANU MRE$E XII KAZNENE ODREDBE XIII PRELAZNE I ZAVR!NE ODREDBE Istim zakonskim pristupom, od 1. januara 2010. godine regulisan je uvoz i izvoz !elija i tkiva koji vr!i ovla!"ena zdravstvena ustanova ili banka !elija i tkiva preko ovla!"enog distributera (koji mora da ispunjava uslove propisane Zakonom i drugim propisima donetim za sprovodjenje ovog zakona). Na predlog Uprave za biomedicinu, Ministar daje dozvolu za svaki pojedina#ni slu#aj uvoza, odnosno izvoza !elija i tkiva pri tome ocenjuju!i opravdanost izvoza ili uvoza. Domeni u procesu transplantacije !elija i tkiva koji su regulisani Zakonom obuhvataju niz osnovnih i specifi#nih odredbi (Tabela 1.12). Smatra se da $e dalje regulisanje ovog zakonskog pristupa biti uspostavljeno kroz niz prate!ih propisa i podzakonskih akata. 50 1.5 Novi pristupi i trendovi u istra%ivanju 1.5.1 Najnoviji pristupi razvoju terapija mati"nim #elijama i drugim #elijskim terapijama (eng. Stats & Trends) Regenerativna medicina, kao i njen deo koji se zasniva na $elijskim terapijama, se smatra novim poglavljem u medicinskim naukama jer u prakti#nom smislu objedinjuje znanja iz skoro svih nau#nih disciplina do sada ¶. Osim o#iglednih razloga u medicinskom smislu, regenerativna medicina ima ogroman ekonomski zna#aj. Na primer, u proteklih nekoliko godina SAD na godi"njem nivou tro"i oko 13% ukupnog doma$eg proizvoda (eng. Gross Domestic Product, GDP) na zdravstvo ¶. Do 2040. godine se o#ekuje da $e populacija osoba preko 65 godina %ivota u SAD-u dosti$i 70 miliona ¶. Na taj na#in $e, smatra se, potro"nja na zdravstvo u SAD-u dosti$i 25% ukupnog doma$eg proizvoda ¶. U proteklih nekoliko godina zemlje kao Japan, Evropska zajednica, Kina i Australija su zapo#ele razli#ite zasebne inicijative za unapredjenje razvoja regenerativne medicine ¶. Ovo je postalo jedno od osnovnih postavki strate"kog razvoja u mnogim dr%avama koje dugoro#no planiraju svoja ulaganja i time obezbedju budu$i razvoj ove va%ne oblasti ¶. Globalno tr%i"te za farmaceutska sredstva je poraslo sa $693,7 milijardi USD u 2007. godini na o#ekivanih blizu $1 trilion USD ¶¶. Ipak, velike farmaceutske kompanije trenutno imaju problem sa razvojem novih terapeutskih sredstava "irom sveta ¶¶. U ovom trenutku 50% svih novih medicinskih sredstava koja su u procesu razvoja zasniva se na biotehnologiji ¶¶. U Evropskoj zajednici se ovaj deo biotehnolo"kog razvoja zasniva na malim kompanijama koje se suo#avaju sa veoma slo%enim regulatornim i finansijskim sistemima (70% ovih kompanija u Evropskoj zajednici ima ispod 50 zaposlenih) ¶¶. Japan ula%e u private kompanije i uspostavlja izvoz $elijskih terapija na tr%i"te Bliskog istoka ve$ od 2015. godine ¶¶. U smislu novih pravaca tehnolo"kog razvoja, dominira razvoj kombinovanih produkata baziranih na $elijskim terapijama uz primenu nosa#a (eng. scaffolds) (Saif et al., 2010) i medicinskih naprava/pomagala (eng. medical devices) kao i primena biorobotike, automatizacije i 3-D bioreaktora (Dos Santos et al., 2013). Takodje se razvijaju nove tehnologije u istra%ivanju i proizvodnji (eng. upstream processing)^ uz 51 tehnologije za propratne sisteme i testiranja, analiti#ke metode, karakterizaciju i kvantifikaciju u procesu proizvodnje biolo"kih lekova (eng. downstream processing)^. Tabela 1.13: Realnost i o#ekivanja u razvoju $elijskih terapeutika: Najnoviji trendovi u primeni neuralnih mati#nih $elija (eng. Neural Stem Cells, NSC) u klini#kim istra%ivanjima u SAD-u, Velikoj Britaniji i &vajcarskoj i pluripotentnih mati#nih $elija (eng. pluripotent stem cells) u SAD-u. Adaptirano iz: Trounson et al., 2011. Veliki deo populacije u razvijenim zemljama, medju kojima su i zdrave osobe, u poslednjih nekoliko godina po#inje da ispituje mogu$nost prikupljanja i #uvanja mati#nih $elija za sopstvenu upotrebu ili za budu$e primene u alogenoj transplantaciji (za potrebe porodice). __________ ¶ www.dhhs.gov/reference/newfuture.shtml ¶¶ www.ey.com ^ www.biopharminternational.com tissue repair [28]. !ey consist of adipose derived stem cells (ASCs) (CD31-/CD34+/CD45-/CD90+/CD105-/ CD146-), endothelial progenitor cells and pericytes. Autologous ASCs and the stromal vascular fractions are being used for soft tissue engineering with a range of scaffolds, particularly for breast augmentation, fistulas in Crohn’s disease and tissue damaged by radiation [28]. In addition to soft tissue repair, ASCs are also in clinical trial for myocardial infarction and graft versus host disease, with outcomes equivalent to MSCs [29]. !ey have also been used in clinical trials for tracheomediastinal fistula, Calvarial bone defect, skin ulcer and stress induced urinary incontinence. !e relative advantage of ASCs over MSCs remains to be determined for the variety of applications envisaged and further studies may demonstrate the merits of ASCs. Meanwhile, soft tissue repair and fistula repair will remain a primary application of ASCs for the immediate future. Endothelial stem cells Endothelial progenitor cells (CD34+/CD133+/KDR+ or VEGFRII+) may be sourced from several sources inclu- ding bone marrow, umbilical cord blood and adipose tissue. !ey are effective in the stimulation of angio- genesis and in clinical studies requiring revascularization and remodeling of collaterals in atherosclerotic cardio- vascular disease. !e result desired is the regeneration of damaged tissues, preventing amputation of ischemic limbs and other areas, and recovery after myocardial infarction. While efficacy in preclinical trials and safety in Phase I studies has been demonstrated, unequivocal evidence for patient benefit in placebo-controlled trials has not been obtained [30]. !e role of endothelial progenitor cells (EPCs) in neoangiogenesis of plexiform lesions remains uncertain and there is continuing debate about the function of EPCs in the regenerative processes that are the target of EPC therapy. !ese matters require careful consideration in future clinical trials [31]. Pancreatic β islet cells Transplantation of pancreatic β Islet cells has been recently reviewed by Matsumoto [32]. Approximately 70% of Type I diabetes patients can achieve insulin independence but may have difficulty in maintaining this. !ey also have problems due to immunosuppression and generally there is a shortage of donors (patients need multiple donors). Xenotransplants of pig islets using encapsulation to address immune rejection is moving towards the clinic but concerns still exist for transmission of porcine endogenous retrovirus. !e use of embryonic stem cell derived β Islets in special subcutaneous capsules that induce minimal fibrosis may evolve into clinical trials shortly [33]. Neural stem cells Neural stem cells (NSCs) may be sourced from the fetal, neonatal or adult brain. !ey self renew and differentiate to neurons, astrocytes and oligodendrocytes and are used in a variety of indications (Table 1). Clinical trials have been undertaken for the use of fetal neural stem cells for lysosomal storage diseases. Children with advanced stage Batten’s disease (neuronal ceroid lipofuscinosis) tolerated high doses of NSCs in multiple sites in the brain in  Phase  I studies. !e transplanted cells provide widespread global replacement enzyme, renewal for cell replacement and bystander neuroprotection [34]. !e Californian company StemCells Inc. embarked on a second safety and efficacy study in children with less advanced Batten’s disease using CD133+ cell culture expanded NSCs, but discontinued the study because of failure to enroll patients meeting study criteria. !e company is also carrying out a Phase 1 clinical trial using fetal neural stem cell brain transplantation for Pelizaeus- Merzbacher disease (PMD), a mylination disorder that affects male children. Preclinical trials showed NSCs produce oligodendrocytes that remylinate neurons affected by the mutated gene for PMD. Fetal NSCs are also being used for treatment of disabled ischemic stroke patients by the company ReNeuron in the UK. !e NSCs have a conditional form of the oncogene encoding c-Myc under the control of an estradiol receptor that activates propogation for manufacture. Patients are transplanted with these NSCs 6 to 24 months after stroke using a straight-forward neurosurgical implantation into the brain. !e NSCs express several trophic and pro-angiogenic factors that Table 1. Neural stem cell (NSC) clinical trials underway NSC Clinical Trials Regenerative, Cell Replacement StemCells Inc., CA HuCNS-SC® (fetal derived human NSCs) Phase I – completed Batten’s Disease (NCL) USA Phase Ib Discontinued for lack of enrollment Phase I Pelizaeus-Merzbacher Disease (PMD) USA Phase I/II Chronic Spinal Cord Injury Switzerland NeuroGeneration, CA Autologous NSC-derived Neurons Phase I – completed Advanced Parkinson’s Disease USA Phase II – clinical hold Neuralstem Inc., MD Fetal derived hu spinal cord SCs Phase I ALS (Lou Gehrig’s Disease) USA ReNeuron, UK ReN001 Immortalized huNSCs Phase I Stroke UK Targeted Delivery of Therapeutics City of Hope, CA HB1.F3.CD Immortalized hu NSCS Phase I Recurrent High Grade Glioma USA Page 4 of 7Trounson et al. BMC Medicine 2011, 9:52 http://www.biomedcentral.com/1741-7015/9/52 !e studies were originally placed on hold while the company addressed the occurrence of micro-cysts in animal transplants and screened cell line products for freedom of this characteristic. !e company Advanced Cell Technology (ACT, CA and MA) has Phase I/II approval for clinical trials on Stargardt’s Macular Dystrophy, which is a condition of blindness that arises through a photoreceptor cell protein anomaly that causes degeneration of the underlying retinal epithelium monolayer and subsequent loss of the photoreceptor cells. !ey have derived pigmented epithelial progenitor cells that may be injected under the photoreceptor cells to redevelop the polarized retinal epithelium monolayer. Since the primary defect is in the photoreceptor cells, it is possible that the new retinal epithelium will be lost i time and require repeat grafting. Short-term immunosuppression is to be used for grafts of allogenic retinal progenitors to the eye, even though this is considered an immune privileged site. ACT also has approval for use of the same cells for a Phase I/II study of dry macular degeneration. !is a leading cause of loss of central vision in persons older than 55 years of age. !e retinal progenitors should distribute to areas of retinal degeneration and potentially correct the loss of vision. Conclusions Clinical trials on the use of stem cells are underway for a wide variety of conditions and there is an emphasis on the use of bone marrow, hematopoietic (mobilized and recovered in blood and umbilical cord blood) and mesenchymal stem cells. While safety has been consistently demonstrated, particularly with autologous transplants, sustained curative benefit has not been consistently obtained. Allogenic transplants generally have major issues for continual immunosuppression to prevent rejection of grafted cells. In some cases, the benefit of cell therapy is through unidentified trophic effects of transient grafted cells. Nevertheless, progress for therapeutic benefit for patients is increasing and there is clear merit for using stem cells as delivery vehicles for correcting genetic mutations that cause severe disease phenotypes. Increasingly, new stem cell types are being explored and both neural and pluripotent stem cells (embryonic stem cells) are under study in early Phase I/II trials. It is too early to predict the outcome of these trials at present but early observations of patients indicate that they do appear to be safe. Recent studies using induced pluripotent stem cells (iPSCs) have shown sizeable genetic and epigenetic abnormalities in these cells and there is now a clear need to determine the biological significance of those changes before iPSCs are taken to clinical trials [41, 42]. A strong indication of the confidence in the cell therapy field is the increasing participation of the large pharmaceutical companies in stem cell therapies [43]. Strong funding from organizations such as the Californian Institute for Regenerative Medicine and their collaborating partners worldwide is likely to rapidly expand new clinical trials in the next few years. Abbreviations ALS, amyotrophic lateral sclerosis; ASCs, adipose derived stem cells; EPCs, endothelial progenitor cells; G-CSF, granulocyte-colony stimulating factor; GVHD, graft versus host disease; hESCs, human embryonic stem cells; HSCs, hematopoietic stem cells; iPSCs, induced pluripotent stem cells; LDL, low- density lipoprotein; MSCs, mesenchymal stem cells; NSCs, neural stem cells; PMD, Pelizaeus-Merzbacher disease. Competing interests CIRM’s governing board recently voted to provide $25 million in loans to Geron to support the spinal cord injury trial noted in this review. Author contributions AT was lead writer and editor with editorial assistance from DG and data gathering from GL and RGT. All authors read and approved the !nal manuscript. Acknowledgements Thanks to Pat Beaupre Becker for her assistance. Published: 10 May 2011 References 1. Trounson A: New perspectives in human stem cell therapeutic research. BMC Med 2009, 7:29. 2. Yang WZ, Zhang Y, Wu F, Min WP, Minev B, Zhang M, Luo XL, Ramos F, Ichim TE, Riordan NH et al: Safety evaluation of allogeneic umbilical cord blood mononuclear cell therapy for degenerative conditions. J Transl Med 2010, 8:75. 3. Prasad VK, Kurtzberg J: Umbilical cord blood transplantation for non- malignant diseases. Bone Marrow Transplant 2009, 44(10):643-651. 4. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop D, Horwitz E: Minimal criteria for de!ning Table 2. Pluripotent stem cell clinical trials (USA) Trial sponsor Disease target Cell therapy Geron Inc. Complete subacute thoracic spinal cord injuries. Human embryonic stem cell derived Phase I: 10 patients enrolled 2010-12 T3 to T10 segments between seven and 14 days after injury Oligodendrocyte progenitor cells (GRNOPC1) Advanced Cell Technologies (ACT) Stargardt’s Macular Dystrophy (juvenile macular degeneration) Retinal Pigment Epithelium derived from human Phase I/II: 12 patients enrolled 2011 embryonic stem cells Advanced Cell Technologies (ACT) Age-related Macular Degeneration Retinal Pigment Epithelium derived from human Phase I/II: 12 patients enrolled 2011-12 embryonic stem cells California Stem Cell (CSC) Spinal muscular atrophy (SMA) Type 1 Human motor neuron progenitor cells derived Phase I: Currently on hold 2011 from human embryonic stem cells Page 6 of 7Trounson et al. BMC Medicine 2011, 9:52 http://www.biomedcentral.com/1741-7015/9/52 52 Svi oni 'investiraju' o#ekuju$i da $e u bli%oj budu$nosti potencijal razvoja ovih tehnologija (i klini#kih primena) da dovede do realnih mogu$nosti za upotrebu $elija koje su skladi"tene (ukoliko im se za to uka%e potreba). Sektor #uvanja i skladi"tenja mati#nih $elija je u porastu (eng. cell banking) u javnim i privatnim bankama $elija. Ovo prati adekvatan razvoj tehnologije i servisa koje druge delatnosti pru%aju $elijskim bankama (oprema, reagenti, IT podr"ka). Uz to se razvijaju biorobotika, nove tehnologije za $elijsko sortiranje, skeniranje (eng. high speed throughput array), mikroskopija – "to sve zajedno postaje de rigueur infrastruktura za istra%iva#ke laboratorije, razvojne projekte i njihovu primenu u bankama $elija (eng. reserach and development, R&D). Medjutim, jo" uvek ostaje otvoreno pitanje za nau#nu i "iru javnost, za medije i regulatorno zakonska tela - koliko su realna o#ekivanja vezana za sada"nji i budu$i razvoj $elijskih terapeutika i u kojim oblastima (Tabela 1.13). 1.5.2 Primena kvalitativne metodologije istra%ivanja u analizi globalnog i multidisciplinarnog pristupa razvoju #elijskih terapija (eng. Qualitative research) Kvalitativne metodologije istra%ivanja uglavnom omogu$uju iscrpnu, preciznu i detaljnu analizu uz adekvatno prepoznavanje kompleksnosti analiziranog fenomena na vi"e nivoa (Miles & Huberman, 1994; Patton, 2002), izmedju ostalog i na nivou terminologije (Weber, 1990). Analiza dokumenata je jedna od kvalitativnih metodologija istra%ivanja koja omogu$uje sveobuhvatan pristup u prikupljanju informacija kako u smislu konecptualne i tematske analize, tako i terminolo"ke analize (Cabré Castellví, 2003; Meyer, et al., 1997; Felber, 1984; Kageura, 2002; Thomas, 2008). Proces #itanja, razumevanja, izbora i poredjenja dokumenata dodaje novu dimenziju kroz analizu ‘konstrui"u$i’ i reflektuju$i tekst kroz poimanje #itaoca odnosno istra%iva#a (eng. “…Textual analysis involves the mediation between the frames of reference of the researcher and those who produced the text. … The researcher’s own frame of reference becomes the springboard from which the circle is entered, and so the circles reaches back to encompass the dialogue between the researcher and the text” (Scott, 1990). 53 Pojedina"na analiza (eng. Case Study Analysis) i uporedna analiza (eng. Cross-Case Study Analysis) kao metode analize projekata, programa, organizacija ili klini#kih zapa%anja zahtevaju od istra%iva#a da oformi adekvatne teorijske koncepte ili osnovni konceptualni okvir (eng. conceptual framework) (Anderson & Aydin, 1994; Miles & Huberman, 1994; Yin, 1999; Eisenhardt; 1989; Van de Ven & Poole, 1995). Primarno, ovo omogu$ava da se pojedina#an slu#aj ili vi"e njih pa%ljivo pozicioniraju i da istra%ivanje ovom metodom bude fokusirano na odgovaraju$u tematiku ili cilj (Yin, 1994). Osim toga, time se jasno defini"e jedinica analize (eng. unit of analysis; what is the case?) kao i odgovaraju$i parametri i kriterijumi zna#ajni za datu analizu (Anderson & Aydin, 1994; Miles & Huberman, 1994; Yin, 1999; Eisenhardt; 1989; Van de Ven & Poole, 1995). U obe analize - pojedina#noj i uporednoj, selekcija “slu#aja” je od izuzetnog zna#aja (Eisenhardt, 1989; Lee, 1989; Yin, 1994). Smatra se da bi u uporednoj analizi selekcija trebalo da se zasniva na sli#nostima (eng. literal replication) ili na kontrastnom pristupu tj. razli#itosti koja se o#ekuje (eng. theoretical replication) (Eisenhardt, 1989; Lee, 1989; Yin, 1994). Ukoliko se radi o eksploratornom istra%ivanju (pojedina#nom ili uporednom), va%no je da se defini"e hipoteza ("ta se istra%uje) (eng. what is being explored?) ili da se unapred formiraju postupci analize (kako se vr"i analiza) (Eisenhardt, 1989; Miles & Huberman, 1994). Eksploratorno istra%ivanje takodje mo%e da posmatra uzro#no-posledi#ne veze i odnose uz to obja"njavaju$i kako se ne"to odigralo, dok deskriptivne studije nude opis dogadjaja, procesa ili fenomena na zadatom nivou. U svakoj od pomenutih analiza je zna#ajno da se ponudi generalizacija i/ili ekstrapolacija na "iri koncept odnosno na sli#ne fenomene, situacije, dogadjaje, pojave ili procese. Tematska analiza i analiza sadr$aja (eng. Thematic and Content Analysis) uz izvodjenja teorije bazirane na podacima iz prakse (eng. Grounded Theory) nude obilje podataka i iscrpnu analizu kompleksnih fenomena, pojava ili teksta. Metodologija izvodjenja teorije bazirane na podacima iz prakse (eng. Grounded Theory) je ustanovljena jo" "ezdesetih godina pro"log veka (Glaser & Strauss, 1967) ali se jo" uvek uspe"no koristi od strane istra%iva#a (Strauss & Corbin, 1998; Glaser, 2002). Ova metoda identifikuje i obja"njava (procesom indukcije) analiti#ke kategorije koje se izdvajaju iz prikupljenih podataka - razvijaju$i tako teoriju iz bazi#nog tj. 54 prakti#nog dela istra%ivanja (eng. from the ground research) – pre nego je defini"u$i unapred. U ovom procesu se iz prikupljenih podataka (sistemati#nom analizom) identifikuju teme, kategorije ili pod-kategorije koje mogu da se zasnivaju na razli#itim osnovama – na frazama, dogadjajima, pojavama, incidentima, tipu pona"anja, odredjenoj shemi, pravilu ili principu (Pope, 2000). Kvalitativna istra%ivanja u osnovi produkuju velike koli#ine podataka koje je te"ko analizirati, kodirati, razvrstati ili redukovati uz pomo$ automatskih ili jednostavnih metoda (Yin, 1994). U tom smislu, osnovni cilj kvalitativne analize je razumevanje tj. potraga za uspostavljanjem odredjene zakonitosti ili reda (eng. coherence and order) (Kaplan & Maxwell, 1994). U ve$ini kvalitativnih analiza podaci su sa#uvani u svojoj tekstualnoj formi ili su obele%eni u procesu kategorizacije (eng. indexed) da bi se tako organizovali u analiti#ke kategorije i teoretska obja"njenja (Pope, 2000). Analiza ovakvih podataka nije jednostavan ili brz postupak. Ukoliko se obavlja na sistemati#an, rigorozan na#in – predstavlja naporan i dugotrajan proces (Pope, 2000). Razli#ite metodologije kvalitativnih istra%ivanja se u poslednjih petnaest do dvadest godina zna#ajno koriste u dru"tvenim naukama, u medicinskim istra%ivanjima, istra%ivanjima u zdravstvu i ostalim srodnim disciplinama (Green & Britten, 1998; Fairhurst & Huby, 1998; Jain & Ogden, 1999, Marshall, 1999; Maxwell, Streetly & Bevan, 1999; Salmon, Peters & Stanley, 1999; Tomlin, Humphrey & Rogers, 1999; Paré, 2002; Dixon-Woods, 2007; Baxter & Jack, 2008; Hughes at al., 2009). 1.5.3 Dorpinos ovog istra%ivanja Metodologija nau#nog istra%ivanja u ovoj disertaciji se zasniva na primeni kvalitativnih metoda. Pregled metodologije na srpskom jeziku nalazi se u Prilogu 9 a pregled na engleskom jeziku u Prilogu 10. Istra%ivanje je eksploratorno (eng. explorative), istra%uje veze izmedju elemenata i detaljno analizira mali broj uzoraka, sa namerom da doprinese nastanku novih teorijskih pristupa (eng. theory generating). U okviru istra%ivanja je prikupljena velika koli#ina podataka (upotrebom metode “desk analysis”) koji se nalaze u tekstu kao i u okviru 18 priloga ovoj doktorskoj disertaciji. 55 Ekspanzija klini#kih istra%ivanja na nova geografska podru#ja (uglavnom manje razvijenih zemalja ili zemalja sa nepotpuno definisanim zakonskim regulativama u ovoj oblasti) omogu$uje velikim biofarmaceutskim kompanijama da uz manje tro"kove obave inicijalna klini#ka istra%ivanja (faze I i II) u oblasti $elijskih terapija‡. Ukupan broj svih klini#kih istra%ivanja obavljenih u severnoj Americi i zapadnoj Evropi opao je za 50% u korist Azije, Bliskog istoka, srednje i isto#ne Evrope (McDonnell, 2008). Pri tome je veoma zna#ajno unapred obezbediti teorijske i prakti#ne pristupe primenljive za razvoj ovakvih istra%ivanja, kao i centralizovanu bazu podataka za na"u zemlju i usvojiti neki od postoje$ih akreditacijskih sistema priznatih u svetu. Na ovaj na#in bi se u izvesnoj meri za"titili interesi istra%iva#a i medicinskih istra%iva#kih institucija u Srbiji, pacijenata koji u#estvuju u ovim istra%ivanjima, i pacijenata kojima su ovakve eksperimentalne terapije neophodne (eng. life-saving therapies). Postoji mogu$nost da bi se, istovremeno, na ovaj na#in otvorio put za: - ulazak nov#anih sredstava od stranih partnera i evropskih fondova za razvoj naprednih terapija – kao neophodni deo budu$eg razvoja terapeutika u ovoj oblasti. - protok informacija o broju, ishodima i finansiranju klini#kih i bazi#nih istra%ivanja u Srbiji koja se bave primenom $elijskih terapija. - adekvatnu zakonsko regulatornu i eti#ku kontrolu. - mogu$nost publikacija u svetski renomiranim nau#nim #asopisima – koji ne prihvataju rezultate klini#kih istra%ivanja ako nisu sprovedena na svetski priznatim eti#kim principima ili nisu ostvarila formalno odobrenje eti#kih komiteta ili nisu u skladu sa principima propisanim od strane Medjunarodne Konferencije za Harmonizaciju (eng. ICH) i Evropske Medicinske Agencije (eng. European Medicines Agency, EMA) u svrhu sprovodjenja Dobre klini#ke prakse (eng. Good Clinical Practice, GCP) i Helsinki deklaracije o pravima #oveka (eng. Declaration of Helsinki)††. __________ ‡ Business Process Management: Global Trends to 2013 (Strategic Focus), September 2008, Datamonitor. † BIO. (2001) Editor’s and Reporter’s Guide to Cell therapy, A report by the Cell Therapy Industry Organization, USA. †† ICH Topic E6 (R1), Guideline for Clinical Practice, Note for Guidance on Good Clinical Practice CPMP/ICH/135/95 version July 2002. 56 2. Ciljevi istra%ivanja Uz sveobuhvatnu analizu procesa, sistema i zakonskih regulativa vezanih za izradu $elijskih terapija kao novih biofarmaceutskih preparata, cilj ove doktorske disertacije je karakterizacija teorijskih i prakti#nih modela u laboratorijskoj izradi i klini#koj primeni mati#nih $elija i drugih vrsta $elija u terapeutske svrhe. Ciljevi prvog dela istra%ivanja (Komparativna analiza 1) su analiza dokumenata i uporedni pregled (eng. documentary analysis): ! zakonsko regulatornih modela* koji se odnose na primenu mati#nih $elija i drugih vrsta $elija u terapeutske svrhe: -u Evropi propisane od strane Evropske Medicinske agencije EMA (eng. European Medicines Agency), -u Sjedinjenim Ameri#kim Dr%avama (SAD) propisane od strane FDA (eng. Food and Drug Administration), -u Australiji propisane od strane TGA (eng. Therapeutic Goods Administration). ! globalne baze podataka klini"ko istra$iva"kih projekata baziranih na primeni mati#nih $elija i drugih vrsta $elija u terapeutske svrhe, uklju#uju$i pregled podataka dostupnih na internetu, tip i analizu istih podataka i pregled projekata prema kategorijama, geografskom regionu i tipu $elijskih terapija. Cilj drugog dela istra%ivanja (Komparativna analiza 2) je pojedina"na analiza (eng. case study) postoje$ih laboratorijsko klini"kih projekata u rasponu od veoma jednostavnih do veoma slo%enih procedura: -za rutinsku klini"ku primenu - izrada i primena minimalno manipulisanih $elija: umno%avanje i klini#ka primena hematopoetskih $elija iz periferne krvi; __________ *Model podrazumeva principe i infrastrukturu regulatornih tela. 57 -za istra$iva"ke svrhe i za !iru klini"ku primenu (eng. 'off-the-shelf') - izrada i primena visoko manipulisanih $elija: umno%avanje i primena mezenhimskih $elija za razli#ite klini#ke primene i klini#ka istra%ivanja; -za primenu u istra$iva"ke svrhe - izrada i primena visoko manipulisanih $elija: umno%avanje i klini#ka primena antigen-prezentuju$ih dendriti#nih $elija u anti-tumor terapiji. Cilj tre$eg dela istra%ivanja (Komparativna analiza 3) je uporedna analiza (eng. cross-case study) postoje$ih laboratorijsko-organizacionih modela koji su finansijski i zakonski prihvatljivi, a koji se odnose na izradu mati#nih $elija i drugih vrsta $elija u terapeutske svrhe. Cilj #etvrtog dela istra%ivanja (Komparativna analiza 4) je tematska i fenomenolo!ka analiza transkripta intervjua sa stru#njacima u datoj oblasti (eng. thematic analysis/ phenomenology) uz izvodjenje teorije bazirane na podacima iz prakse (eng. grounded theory). Cilj petog dela istra%ivanja (Komparativna analiza 5) je teorijsko i prakti"no modelovanje pristupa i re!enja. 58 II EKSPERIMENTALNI DEO 3. Dizajn i metode Ova doktorska disertacija je dizajnirana kao skup pet komparativnih studija koje koriste slede$e materijale i metode. 3.1 Materijali 3.1.1 Materijali za komparativnu analizu 1 3.1.1.1 Materijali za analizu zakonsko-regulatornih modela* Dokumenti i tekst zakonsko regulatornih propisa analizirani u ovoj studiji nalaze se na zvani#nom web sajtu (eng. web site) svake od regulatornih agencija (FDA, TGA, EMA) kao kompletan, dobro struktuiran i kvalitetno prezentovan materijal. Tabela 3.1: Kriterijumi za izbor analiziranih dokumenata. Preuzeto iz: Ilic et al., 2012a. Parametri Kriterijumi za ukljucenje dokumenata u proces analize Kriterijumi za iskljucenje dokumenata iz procesa analize Parameters Inclusion criteria Exclusion criteria Location EU Regions other than EU, USA, and Australia USA Australia Language English Documents not written in English Type of the document High-tier legal and regulatory domain Documents other than high-tier legal and regulatory domain Purpose of the document Legal requirements for manufacture and use of biologic drugs for human use, including definitions and scope Documents concerned with issues other than legal requirements for manufacture, GMP and GCP applicable to medicinal products and biologic drugs for human use Good Manufacturing Practice (GMP) for manufacture of medicinal products and biologic drugs for human use Good Clinical Practice (GCP) for use of medicinal products and biologic drugs in clinical trials involving humans Scope of the document Medicinal Products, Biologics, Biologicals and Advanced Therapy Medicinal Products (ATMP) for human use Documents concerned with other than medicinal products, Biologics, Biologicals and Advanced Therapy Medicinal Products (ATMP) for human use Time frame Most recent documents published from 2000 (inclusive) Documents published prior to 2000^ ^Only one document was exempt as it was originally published in 1996. 59 Tabela 3.2: Lista analiziranih dokumenata. Preuzeto iz: Ilic et al., 2012a. *Subsequently amended. **In process of being amended. ^Document was exempt from the timeframe. #Pharmaceutical Inspection Convention / Cooperation Scheme. n/a - not applicable. ⌘Disclaimer: Major, specific, and updated FDA guidances relating to key subsets of biologics were not included in Cohort 1 and 2 documents. Although the basic FDA regulations have not been changed much since the expansion of biologic drugs, including vaccines, the guidances have really become more like regulations in the interim. Due to the research sampling criteria, guidances (including the key manufacturing guidances for biologics that the FDA really does enforce in a way similar to that for regulations) have not been analyzed here. These documents can be found on the following link: http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/default.htm. __________ *Model podrazumeva principe i infrastrukturu regulatornih tela. Grupa dokumenata Naslov (ime dokumenta) Verzija Godina izdanja/ revizije Da li je dokument vazeci Group of documents Title Version Published Current Cohort 1 European Medicines Agency (EMA) Document Code EMA DOC1 Directive 2001/83/EC n/a 2001 Yes* EMA DOC2 Regulation 726 /2004 n/a 2004 Yes* EMA DOC3 Regulation 1394/2007 n/a 2007 Yes Food and Drug Administration (FDA)⌘ Document Code FDA DOC1 21CFR312 n/a 2011 Yes FDA DOC2 21CFR600 Biological Products: General n/a 2011 Yes FDA DOC3 21CFR1271 Human Cells, Tissues, and Cellular and Tissue-based Products n/a 2011 Yes Therapeutic Goods Administration (TGA) Document Code TGA DOC1 Australian Regulatory Guidelines for Biologicals Part 1 1.0 2011 Yes TGA DOC2 Australian Regulatory Guidelines for Biologicals Part 2 1.0 2011 Yes TGA DOC3 Australian Regulatory Guidelines for Biologicals Part 3 1.0 2011 Yes Cohort 2 Good Manufacturing Practice (1) Document Code GMP DOC1 Directive 2003/94/EC (EMA) n/a 2003 Yes GMP DOC2 Australian Code of Good Manufacturing Practice – Human Blood and Tissues (TGA) n/a 2000 Yes** GMP DOC3 Australian Code of Good Manufacturing Practice for Medicinal Products (TGA) n/a 2002 Yes Good Manufacturing Practice (2) Document Code GMP DOC4 21CFR211 Current Good Manufacturing Practice for Finished Pharmaceuticals n/a 2011 Yes GMP DOC5 Guide to Good Manufacturing Practice for medicinal Products -Part I (PIC/S#) n/a 2009 Yes GMP DOC6 Guide to Good Manufacturing Practice for medicinal Products - Part II (PIC/S#) n/a 2009 Yes Good Clinical Practice Document Code: GCP DOC1 Directive 2003/20/EC (EMA) n/a 2001 Yes GCP DOC2 Guidance for Industry E6 Good Clinical Practice (FDA) n/a 1996^ Yes GCP DOC3 Note for Guidance on Good Clinical Practice (CPMP/ICH/135/95) Annotated (TGA) n/a 2000 Yes 60 Odabrani materijal obuhvata preko 700 strana teksta. Kori"$en je teorijski ili takozvani svrsishodan uzorak zakonsko-regulatornih propisa na najvi"em nivou, koji je uzet kao adekvatna prezentacija zakonsko-regulatornih modela* iz kojih poti#e. Pristup analizi i kriterijumi za uzorkovanje predstavljeni su u Tabeli 3.1, dok je iscrpna lista analiziranih dokumenata prikazana u Tabeli 3.2. Osnovni cilje ove analize je, pre svega, da se u procesu selekcije od ogromne koli#ine dostupnog materijala izvr"i odabir adekvatnih, reprezentativnih i srodnih zakonsko regulatornih propisa; a zatim da se izvrsi analiza konceptualnog pristupa i terminologije. 3.1.1.2 Materijali za analizu klini#kih istra%ivanja Pretra%ivanjem globalne baze podataka www.clinicaltrials.gov u januaru 2011. i u avgustu 2013. godine izvr"ena je analiza tipa, broja i opsega klini#ko istra%iva#kih projekata baziranih na izradi i primeni mati#nih $elija i drugih $elijskih terapija na svetskom nivou. Pretra%ivanje globalne baze podataka izvr"eno je prema geografskom regionu i prema tipu $elijskih terapija. Dodatna razmatranja koja se odnose na jedinicu analize (eng. unit of analysis) nalaze se u Metodolo"kom pregledu (Prilozi 9 i 10). 3.1.2 Materijali za komparativne analize 2 i 3 3.1.2.1 Materijali za analizu modela laboratorijsko-klini#kih protokola U ovoj analizi kori"$eni su protokoli i laboratorijski modeli izrade $elijskih terapija za klini#ku upotrebu i klini#ka istra%ivanja (Tabela 3.3). Tri specifi#na modela protokola laboratorijske izrade (P1, P2, P3) su odabrana na osnovu nivoa manipulacije koji koriste, od minimalno manipulisanih do visoko manipulisanih procedura. Modeli protokola analizirani u ovoj komparativnoj studiji obuhvataju laboratorijsku izradu i klini#ke parametre; izbor je baziran na teorijskom odabiru (ne prema statisti#kom) da bi se opisale njihove sli#nosti i razlike. __________ *Model podrazumeva principe i infrastrukturu regulatornih tela. 61 Pregled op"tih proceduralnih razmatranja predstavljen je u Prilozima 9 i 10. Primeri standardnih operativnih procedura za izradu $elijskih terapija, razmatranih u okviru ove analize, nalaze se u Prilozima 11, 12 i 13. Tabela 3.3: Odabir analiziranih protokola i laboratorijskih modela izrade $elijskih terapija za klini#ku upotrebu i klini#ka istra%ivanja. P1: LABORATORIJSKO-KLINI!KI PROTOKOL 1 P2: LABORATORIJSKO-KLINI!KI PROTOKOL 2 P3: LABORATORIJSKO-KLINI!KI PROTOKOL 3 ANALIZA KLINI%KOG I LABORATORIJSKOG MODELA IZRADE &ELIJSKIH TERAPIJA ZA RUTINSKU KLINI"KU PRIMENU ANALIZA KLINI%KOG I LABORATORIJSKOG MODELA IZRADE &ELIJSKIH TERAPIJA ZA ISTRA#IVA"KE SVRHE I ZA $IRU KLINI"KU PRIMENU (eng. 'OFF-THE-SHELF') ANALIZA KLINI%KOG I LABORATORIJSKOG MODELA IZRADE &ELIJSKIH TERAPIJA ZA PRIMENU U ISTRA#IVA"KE SVRHE Izrada i primena minimalno manipulisanih %elija: Krioprezervacija i klini!ka primena hematopoetskih "elija iz periferne krvi (eng. Haematopoietic Stem Cells Processing) Izrada i primena visoko manipulisanih %elija: Umno'avanje i klini!ka primena mezenhimskih mati!nih/stromalnih "elija za razli!ite klini!ke primene (eng. Mesenchymal Stem Cell Processing by ex vivo expansion) Izrada i primena visoko manipulisanih %elija: Umno'avanje i klini!ka primena antigen-prezentuju"ih dendriti!nih "elija u anti-tumor terapiji (eng. Dendritic Cells Processing by ex vivo expansion and cell loading) 3.1.2.2 Materijali za analizu laboratorijsko-organizacionih modela U svrhu sagledavanja osnovnih karakteristika postoje$ih laboratorijsko- organizacionih modela u izradi $elijskih terapija izvr"ena je uporedna analiza prakti#nih, finansijski i zakonski prihvatljivih, modela M1, M2 i M3 (Tabela 3.4). Prema svojoj strukturi i izvoru finansiranja izabrane su tri razli#ite organizacione jedinice. Dodatna razmatranja koja se odnose na jedinicu analize (eng. unit of analysis) nalaze se u Metodolo"kom pregledu (Prilozi 9 i 10). 62 Tabela 3.4: Odabir analiziranih laboratorijsko-organizacionih modela. M1: LABORATORIJSKO- ORGANIZACIONI MODEL 1 M2: LABORATORIJSKO- ORGANIZACIONI MODEL 2 M3: LABORATORIJSKO- ORGANIZACIONI MODEL 3 NE-KOMERCIJALNI MODEL (eng. Non-for-profit model) KOMERCIJALNI MODEL (eng. Commercial model) KOMBINOVANI MODEL (eng. Hybrid model) ISTRA$IVA%KA GRUPA ZA RAZVOJ &ELIJSKIH TERAPIJA U OKVIRU NE-KOMERCIJALNE KLINI%KO-BOLNI%KE INSTITUCIJE I/ILI ISTRA$IVA%KOG INSTITUTA NEZAVISNA KOMERCIJALNA KOMPANIJA ZA IZRADU ILI SERVIS NAMENJEN PROCESU IZRADE &ELIJSKIH TERAPIJA KOMPANIJA BAZIRANA NA KOMERCIJALNIM PRINCIPIMA - FORMIRANA U OKVIRU SLO$ENOG SPECIJALISTI%KOG KLINI%KO-BOLNI%KOG KOMPLEKSA/ INSTITUCIJE Izvor: http://www.qimr.edu.au/page/Lab/Translational_ Leukaemia_Research/ http://www.griffith.edu.au/science-aviation/ eskitis-institute/research/st-john-group Izvor: http://www.dendreon.com/ http://www.mesoblast.com/ Izvor: http://www.celltherapies.com.au/ 3.1.3 Materijali za komparativne analize 4 i 5 3.1.3.1 Materijali za analizu percepcije i problematike oblasti Tematska i fenomenolo"ka analiza percepcije i problematike oblasti izvr"ena je uz upotrebu transkripta 24 intervjua sa stru#njacima (#etiri %ene i 20 mu"karaca) od #ega je "est u#esnika bilo iz Evrope (25%), "est iz SAD-a (25%) i 12 iz Australije (50%). Kriterijumi za izbor u#esnika u ovoj studiji, osim stru#no-nau#nog zvanja i rukovode$e pozicije u organizaciji/ kompaniji, bili su: - direktno u#e"$e u projektu izrade $elijskih terapija za klini#ku primenu (eng. adult stem cells, embryonic stem cells and other cell types used for various research and clinical applications), ili - direktno u#e"$e u oblasti razvoja bazi#nih i klini#ko-primenljivih biolo"kih i medicinskih istra%ivanja, bioetike i strate"kog razvoja $elijskih terapija za klini#ku primenu (eng. immunotherapy for cancer patients, treatment of hematological and solid tumors, orthopedic, use, cardiovascular applications, and cell banking). 63 Svi u#esnici u ovoj studiji bili su preporu#eni od strane kolega koji su ve$ prethodno bili uklju#eni u studiju (eng. snow-ball sampling method). Svako od u#esnika potpisao je obrazac za dobrovoljni pristanak za u#e"$e u studiji. Obavljena je serija delimi#no struktuiranih (eng. semi-structured) intervjua u trajanju od 60 minuta koji su snimljeni. Preko 18 sati (1090 minuta) snimljenog materijala je inicijalno obradjeno pri #emu su uklonjeni uvodni i zaklju#ni delovi snimaka. U tom procesu je snimljeni materijal redukovan na 15 sati i pripremljeni su transkripti snimaka (obele%eni kodovima od CTE01 do CTE24). 3.1.3.2 Materijali za modelovanje pristupa i re"enja U teorijskom modelovanju pristupa i re"enja kori"$eni su rezultati iz prethodnih komparativnih studija (1- 4) kao i empirijski podaci. U evaluaciji klini#ke procedure, kao primeru prakti#nog modelovanja u svrhu predvidjanja i planiranja, kori"$eni su istorijski empirijski podaci prikupljeni u datoj klini#koj proceduri za period od dve godine. Dodatna razmatranja koja se odnose na jedinicu analize (eng. unit of analysis) nalaze se u Metodolo"kom pregledu (Prilozi 9 i 10). 3.2 Metode Konceptualni okvir istra%ivanja (eng. conceptual framework) slu%i da identifikuje "ta (ili ko) $e biti ili ne$e biti deo analize/ istra%iva#ke studije, opisuje pristup i logiku istra%iva#kog procesa, teorijski pristup (metode i aparate) kao i prakti#ne aspekte istra%ivanja (Bailey, 1982; Scott, 1990; Weber, 1990; Miles & Huberman, 1994; Mason, 2002; Patton, 2002; Robson, 2002; Maxwell, 2005; Creswell, 2007; Thomas & Harden, 2008). Osim toga, konceptualni okvir istra%ivanja daje priliku istra%iva#u da prikupi i organizuje svoje misaone postavke (eng. constructs) u odgovaraju$e celine i grupi"e ih (eng. intellectual “bins”). Takodje, slu%i kao osnova istra%iva#kog procesa obezbedjuju$i izvor informacija tokom #itave 64 studije, posebno u procesu analize prikupljenih podataka. Ukoliko je vizuelno predstavljen npr. kao shema ili dijagram, konceptualni okvir istra%ivanja ne mora da prika%e sve odnose izmedju postavki u okviru tog istra%ivanja. Trebalo bi da se razvija tokom istra%iva#kog procesa i tada $e se verovatno oformiti i definisati odnosi izmedju postavki, pri #emu $e se finalna verzija konceptualnog okvira oformiti – do kraja prikazuju$i teme i/ili koncepte nastale iz podataka. Vra$aju$i se na polazne postavke istra%ivanja, konceptualni okvir istra%ivanja $e obezbediti da se analiza obavlja u zadatim okvirima iako u nekim situacijama mo%e, u odredjenoj meri, da ograni#i istra%ivanje. Konceptualni okvir ovog istra%ivanja, koji obuhvata metode i aparate opisane u ovom poglavlju, predstavljen je u vidu metodolo"kog pregleda svih studija u Prilozima 9 i 10. 3.2.1 Analiza dokumenata Upotreba dokumenata ili teksta kao izvora podataka zahteva da se njihova analiza izvr"i uz sposobnost kontekstualizacije, procene i interpretacije teksta (Bailey, 1982; Robson, 2002). 3.2.1.1 Analiza zakonsko regulatornih modela* (KOMPARATIVNA ANALIZA 1) Analiza reprezentativnih zakonskih regulativa i podzakonskih akata izvr"ena je manuelnom pretragom dokumenata i uz pomo$ softverskog paketa. Pre toga, izvr"ena je kontekstualna analiza bazirana na kompleksnosti i istorijskoj perspektivi za svaku grupu dokumenata; izvr"ena je priprema za analizu i selekciju dokumenata; zatim su odabrani dokumenti analizirani u procesu koji posmatra tekst u doslovnom smislu i terminologiju kao reprezentaciju fokusa i namene dokumenta, putem manuelne metode (Tabela 3.5). Manuelna analiza dokumenata je izvr"ena na osnovu op"te terminologije koju koriste kao i frekvencije tj. u#estalosti termina i fraza u njima. Rezultati su zabele%eni u tabelarnom i grafi#kom prikazu. 65 Analiza dokumenata uz pomoc softvera je bila slede$i korak u ovom procesu, pri #emu je kori"$en softverski paket za tekstualnu analizu Leximancer v3.1 (eng. Leximancer Pty Ltd, Jindalee, Queensland, Australia) (Smith, 2010). Identifikovani su koncepti i teme i u odredjenoj meri je izvr"ena njihova korelaciona analiza. Tabela 3.5: Proces manuelne analize dokumenata. Manuelna analiza dokumenata Razmatranja Osnovno pitanje Rezultat ove faze Manual Document Analysis Considerations Question Raised Stage Result Stage 1: Selection of Documents (incl. Comparison with Other Frameworks/ Areas) - Regulatory frameworks (by region) - Areas of regulatory frameworks What to analyze - List of documents to be analyzed Stage 2: Reading and ‘Interpretation’ - Purpose and aim of documents - Understanding of the content How to analyze - Thorough understanding of aim, scope and depth of documents - List of terms and phrases to search Stage 3: Search by Key Word and/or by Phrase - Generalizability of terms - Terminology specific to a regional regulations Common (shared) features - Frequencies of specific terminology and/or phrases Stage 4: Recording and Reconciliation of Findings (incl. Tabulation of Results) - Consistency and quality assurance - Presenting results in an adequate manner Reliability of results - Tables and diagrams outlining results of the manual analysis 3.2.1.2 Analiza klini#kih istra%ivanja (KOMPARATIVNA ANALIZA 1) Analiza tipa, broja i opsega klini#kih istra%ivanja u oblasti $elijskih terapija izvr"ena je nizom upita u globalnu bazu podataka #iji su rezultati zabele%eni u tabelarnom i grafi#kom prikazu. __________ *Model podrazumeva principe i infrastrukturu regulatornih tela. 66 3.2.2 Pojedina!na i uporedna analiza slu!aja 3.2.2.1 Pojedina#na analiza laboratorijsko-klini#kih protokola (KOMPARATIVNA ANALIZA 2) U ovoj analizi su odabrana tri reprezentativna modela laboratorijskih protokola koji se baziraju na razli#itom nivou manipulacije $elija (P1, P2, P3) pri #emu je svaki zasebno opisan i okarakterisan. Primeri odgovaraju$ih standardnih operativnih procedura za izradu $elijskih terapija #iji su protokoli analizirani, nalaze se u Prilozima 11, 12 i 13. 3.2.2.2 Uporedna analiza laboratorijsko-organizacionih modela (KOMPARATIVNA ANALIZA 3) U procesu uporedne analize odabrana su tri reprezentativna organizaciona modela (M1, M2, M3) pri #emu su uporedno opisani i okarakterisani u cilju da se identifikuju njihove sli#nosti i razlike. 3.2.3 Tematska i fenomenolo"ka analiza uz analizu izvodjenja teorije bazirane na podacima iz prakse 3.2.3.1 Analiza percepcije i problematike oblasti: prakti#ni pristupi i re"enja (KOMPARATIVNA ANALIZA 4) Intervjui koji su obavljeni kao deo ove analize bili su bazirani na seriji ustanovljenih pitanja (eng. standardized interview protocol). Nakon dobijanja pismenog pristanka u#esnika, intervjui su snimljeni. Svaki snimak je bio u trajanju od 22 do 64 minuta, za koje je izvr"ena transkripcija. Transkripti intervjua su dalje obradjeni tematskom analizom uz detaljnu analizu sadr%aja (eng. thematic & content analysed), a nakon toga su identifikovani reprezentativni delovi za fenomenolo"ku analizu - koji su bili bazirani na doslovnim citatima/ izvodima iz intervjua (eng. narrative analysis). 67 Dva segmenta podataka koja su identifikovana u ovoj analizi bila su uglavnom bazirana oko – poznavanja oblasti, ste#enog kao deo ekspertskih znanja, prakse i iskustva (eng. knowledge, practice & experience); ili mi"ljenja oformljenog kroz posmatranje i percepciju oblasti (eng. view, perception & opinion) (Tabela 3.6). Svaki segment podataka je bio tematski i fenomenolo"ki analiziran, pri #emu su ustanovljene teme i kategorije (eng. themes & categories) koje su nakon toga grupisane u osnovne teme i kategorije (eng. principal themes & categories). Za svaku od osnovnih tema je ustanovljena serija citata (eng. narratives) iz transkripta intervjua pri #emu je izvr"eno utemeljenje svake od glavnih tema u samom tekstu (eng. grounding in the data). Tabela 3.6: Pristup analizi podataka prikupljenih kroz intervjue. Segmenti podataka Poznavanje oblasti kroz znanje, praksu i iskustvo Mi"ljenje formirano kroz posmatranje i percepciju oblasti Segment of data Knowledge, practice & experience View, perception & opinion Main questions What is your approach to … ? *** How do you attain … ? What do you think about … ? *** How do you perceive … ? Topics discussed Business Model, Funding & Strategic Decision Making; Partnerships; Science, Technology, Tech Transfer & Up Scaling. *** Regulatory Compliance; Learning & Access to new people, new technologies and new ideas; Risk Management. Main challenges posed to an organisation, a project or an entity; Main challenges posed to the industry/field. *** Industry/ Field, Regulatory Landscape, Important Lessons Learned; Other Considerations. 3.2.3.2 Teorijsko modelovanje (KOMPARATIVNA ANALIZA 5) Nakon obavljenih analiza u okviru #etiri komparativne studije (1- 4), na osnovu dobijenih rezultata i empirijskih saznanja su oformljena dva teorijska modela koja su predstavljena u vidu shema i dijagrama. Obja"njene su njihove karkteristike, ocenjena je njihova primenljiva vrednost u aproksimaciji proizvodnih procesa bioterapeutskih sredstava a zatim su izvr"ena dodatna razmatranja bazirana na principima dinami#kih sistema. 68 3.2.3.3 Evaluacija klini#ke metode (KOMPARATIVNA ANALIZA 5) Evaluacija klini#ke metode prikupljanja mati#nih/progenitorskih $elija hematopoeze aferezom (eng. HPC-A) uz pomo$ $elijskog separatora (eng. COBE Spectra Mononuclear Cell (MNC)/AutoPBSC) je izvr"ena za na osnovu prakti#nog modelovanja. Kriterijumi kori"$eni u ovom procesu obuhvataju nekoliko parametara (Tabela 3.7). Tabela 3.7. Kriterijumi za evaluaciju klini#ke metode prikupljanja mati#nih/progenitorskih $elija hematopoeze aferezom. Broj prikupljenih CD34+ "elija/ kg Efikasnost procedure Slaganje sa publikovanim podacima > 2 x 106 CD34+ "elija/ kg prikupljeno u vi#e od 50% klini!kih procedura Broj prikupljenih CD34+ "elija/ kg (eng. actual number) stoji u odredjenoj statisti!koj korelaciji sa brojem "elija koje su o!ekivane (eng. predicted number) Srednja aritmeti!ka vrednost efikasnosti klini!ke procedure > 50% Rezultati evaluacije su u skladu sa publikovanim podacima (po izboru) U procesu evaluacije procedure (modelovanja) izvr"eno je prikupljanje podataka a zatim njihova statisti#ka obrada: - te%ina pacijenta/donora (kg), - broj CD34+ $elija u krvi pacijenta/davaoca 24 sata pre procedure, - zapremina krvi koja je procesovana u toku procedure (l), - broj CD34+ $elija/kg prikupljenih u toku procedure. Dobijeni rezultati uporedjeni su sa podacima iz literature po izboru. 69 Slika 3.1: Statisti#ka metoda obrade podataka (eng. Spearman rank correlation) za uspostavljanje korelacije izmedju broja prikupljenih $elija i o#ekivanog broja $elija. O!ekivani broj CD34+ #elija (x 106/kg) 100 Spearman r = 0.95 p < 0.0001 10 1 0.1 1 10 100 Broj prikupljenih CD34+ #elija (x106/ kg) Izra"unavanje broja o"ekivanih #elija i efikasnosti metode O#ekivani broj* CD34+= Broj CD34+ $elija u krvi (/$l) x Zapremina krvi koja je procesovana (l) Te%ina (kg) Efikasnost procedure (%) = Broj prikupljenih* CD34+ $elija x 100 O#ekivani broj* CD34+ $elija *(x106) Statisti"ka obrada podataka Izvr"en je grafi#ki prikaz broja prikupljenih CD34+ $elija (x osa) u odnosu na o#ekivani broj $elija (y osa) i utvrdjena je korelacija na osnovu statisti#ke metode (eng. Spearman rank correlation) (Slika 3.1). 70 4. Rezultati i diskusija 4.1 Komparativna analiza 1: Poredjenje sadr%aja zakonsko regulatornih odredbi i ispitivanje obima i pravca klini"kih istra%ivanja 4.1.1 Sadr%aj reprezentativnih zakonskih regulativa i podzakonskih akata Pregled regionalnih zakonsko-regulatornih principa koji se odnose na primenu mati#nih $elija i drugih $elijskih terapija u Evropi, SAD-u i Australiji obuhvata slede$e rezultate dobijene analizom reprezentativnih zakonskih regulativa i podzakonskih akata. Rezultati dobijeni manuelnom analizom dokumenata iz grupe 1 (eng. cohort 1) predstavljeni su u Tabeli 3.2, uklju#uju vrstu i frekvenciju 10 termina (Tabela 4.1) i 16 fraza (Tabela 4.2). Frekvencije termina predstavljenih u Tabeli 4.1 pokazuju relativnu uskladjenost u upotrebi ‘manufacturing’ (termin koji se pojavljuje 58, 71 i 113 puta u EMA, FDA i TGA dokumentima) kao i ‘human’ (sa relativno visokom u#estalo"$u od 137, 55 i 89 puta u istim dokumentima) (Slika 4.1). Tabela 4.1: Rezultat manuelne analize dokumenata iz grupe 1: u#estalost termina. Pretraga termina (eng.) U"estalost termina u dokumentima agencije EMA U"estalost termina u dokumentima agencije FDA U"estalost termina u dokumentima agencije TGA Frequency in the EMA Documents Frequency in the FDA Documents Frequency in the TGA Documents DOC1 DOC2 DOC3 DOC1 DOC2 DOC3 DOC1 DOC2 DOC3 2001/83 726/2004 1394/ 2007 21CFR 312 21CFR 600 21CFR 1271 ARGB P1 ARGB P2 ARGB P3 approve(d)/ approval(s) 16 5 0 14 9 15 24* 30 55* biologic/ biological(s) 0 2 2 6 89 17 377 219 67 cell(s) 1 2 46 1 5 78 100 6 1 drug(s) 1 0 0 84 34 36 1 3 0 human 0 84 53 11 6 38 83 1 5 manufacturing 41 8 9 2 48 21 56 57 0 medicinal 615 329 148 0 0 0 2 0 0 product(s) 727 350 227 14 197 49 144 21 6 pharmaceutical 54 8 4 1 1 0 3 0 1 therapy(ies) 4 7 141 0 0 0 13 0 0 *Uklju#uje termin (eng.): ‘unapproved’. Preuzeto iz: Ilic et al., 2012a. 71 Tabela 4.2: Rezultat manuelne analize dokumenata iz grupe 1: u#estalost fraza. Pretraga fraza (eng.) U"estalost fraza u dokumentima agencije EMA U"estalost fraza u dokumentima agencije FDA U"estalost fraza u dokumentima agencije TGA Frequency in the EMA Documents Frequency in the FDADocuments Frequency in the TGA Documents DOC1 DOC2 DOC3 DOC1 DOC2 DOC3 DOC1 DOC2 DOC3 2001/ 83 726/ 2004 1394/ 2007 21CFR 312 21CFR 600 21CFR 1271 ARGB P1 ARGB P2 ARGB P3 access to … 4 7 0 5 0 1 8 0 6 clinical investigation 0 0 0 13 1 0 0 0 0 clinical trial 21 8 4 10 2 0 11 2 41 class/ classification of … 7 0 0 0 0 0 5 16 0 human cell(s) and tissue(s) 0 0 1 0 0 1 0 0 0 human cells 0 0 8 0 0 11 12 0 0 investigational new drug(s) 0 0 0 16 2 0 0 0 0 investigational drug(s) 0 0 0 14 0 0 0 0 0 marketing authorisation(s) 110 115 32 0 0 0 0 0 0 marketing approval(s) 0 0 0 5 0 0 0 0 1 medicinal product(s) 599 329 148 0 0 0 2 0 0 product deviation(s) 0 0 0 0 11 0 0 0 0 regulatory process 0 0 0 0 0 0 0 5 0 quality of … 8 1 0 0 1 0 1 4(7 *) 0 quality, safety and efficacy 7 10 4 0 0 0 2 2 2 risk(s) management 1 0 7 0 2 0 23 2 2 *Frekvencija termina (eng.): ‘manufacturing quality’. Preuzeto iz: Ilic et al., 2012a. Termini kao "to su ‘drug(s)’, ‘cell(s)’ i ‘biologic/biological(s)’ se do odredjene mere koriste u svim analiziranim zakonskim regulativama. Ne"to je u#estalija upotreba ‘medicinal’ u regulatornim dokumentima agencije EMA (upotrebljen je ukupno 1092 puta u tri dokumenta) dok je termin ‘pharmaceutical’ samo sporadi#no u upotrebi u svim EMA dokumentima osim EMA DOC1 (gde se koristi 54 puta). Termin ‘therapy(ies)’ nije pomenut ni u jednom od regulatornih dokumenata agencije FDA a vrlo retko je upotrebljen u TGA regulativama (samo 13 puta), dok se pominje 141 put u EMA DOC3. Dok je naj#e"$e upotrebljen termin u EMA 72 regulativama ‘product(s)’ zajedno sa ‘medicinal’, ‘drug(s)’ se konstantno pominje u svim regulatornim dokumentima agencije FDA (84, 34 i 36 puta) ali se prakti#no jedva pojavljuje u dokumentima iz druga dva regulatorna okvira (samo na 5 mesta). Regulative agencije TGA koriste uglavnom ‘biologic/biological(s)’ dok je termin ‘cell(s)’ najvi"e upotrebljen u regulatornom okviru agencije FDA (78 puta u FDA DOC 3) kao i u TGA regulativama (100 puta samo u TGA DOC1). Rezultati pretrage fraza u Tabeli 4.2 pokazuju da je ‘access to’ bila najuniformnije upotreblljena fraza u svim dokumentima iz tri razli#ita regulatorna okvira iako, ukupno gledano, nije bila jedna od u#estalijih fraza (upotrebljena je ukupno 31 put u okviru svih analiziranih dokumenata) (Slika 4.2). Fraze ‘clinical investigation’ i ‘clinical trial’ su se smenjivale u FDA dokumentima dok su EMA i TGA regulative koristile samo ‘clinical trial’. ‘Human cell(s) and tissue(s)’ i ‘human cells’ su pomenuti samo u jednom dokumentu u svakom od analiziranih regulatornih okvira (9 puta u EMA DOC3, 12 puta u FDA DOC3 i 12 puta u TGA DOC1) dok se ‘investigational new drug’ pominje samo u dva FDA dokumenta (ukupno 32 puta u FDA DOC 1 i FDA DOC 2). ‘Marketing authorisation’ je fraza koju u svojim regulatornim odredbama ekskluzivno koristi samo agencija EMA kao i ‘medicinal products’. Ni jedna od ove dve fraze nije upotrebljena u bilo kom dokumentu agencija FDA ili TGA (Slika 4.2). ‘Quality, safety and efficacy’ se sporadi#no pojavljuje u EMA i TGA regulativama a prakti#no se ne pominje u bilo kom od FDA dokumenata. Ovo se odnosi i na ‘quality of’ ili ‘manufacturing quality’. ‘Class’ ili ‘classification of’ nisu upotrebljeni ni u jednom od FDA dokumenata, minimalno se koriste u dokumentima agencije EMA (samo 7 puta) a ne"to u#estalije u TGA dokumentima (ukupno 54 puta). Posmatrano sa generalnog stanovi"ta, regulative agencije EMA koriste u svom razmatranju uglavnom ‘medicinal products’ i ‘marketing authorisation(s)’ – dok FDA regulative razmatraju ‘drug(s)’ ili ‘biologic(s)’ a TGA uglavnom ‘biological(s)’. Regulatorna dokumenta agencije TGA #esto koriste ‘manufacturing’ za razliku od druge dve agencije kao i ‘approved/unapproved’, dok pominju termin ‘cell(s)’ preko 100 puta u samo jednom od dokumenata (TGA DOC1). Osim toga, agencija TGA se u svojim regulativama oslanja na upotrebu termina ‘product(s)’ (koji je upotrebljen 171 put) i ‘risk management’ (koristi se na 27 mesta). ‘Risk management' se pominje 73 vrlo sporadi#no u EMA dokumentima a samo dva puta od strane agencije FDA (Slika 4.2). Preuzeto iz: Ilic et al., 2012a. Slika 4.1: Manuelna analiza dokumenata: frekvencija upotrebe termina u dokumentima iz grupe 1 (eng. cohort 1). 74 Preuzeto iz: Ilic et al., 2012a. Slika 4.2: Manuelna analiza dokumenata: frekvencija upotrebe fraza u dokumentima iz grupe 1 (eng. cohort 1). Cilj ove analize bio je da se uspostavi korelacija izmedju dokumenata i da se doprinese generalizaciji postavki zakonsko regulatornih odredbi, ne samo u smislu razvoja projekata baziranih na primeni $elijskih terapija i drugih bioterapeutika – ve$ i u daljem razvoju nau#ne discipline regulatornih principa (eng. regulatory science). Anticipirano je da se sagledavanjem sli#nosti i razlika u ovoj analizi, mo%e u odredjenoj meri unaprediti proces harmonizacije zakonskih regulativa, da se mo%e doprineti njegovom razvoju u zemljama koje takav zakonski okvir tek formiraju kao i da se mo%e pro"iriti teorijski skup saznanja u novonastaloj disciplini regulatornih nauka. U toku analize posebna pa%nja je posve$ena selektivnosti uzorka i fokusu celokupnog istra%ivanja. Zbog toga su oformljeni kriterijumi za odabir dokumenata kao i precizna metodologija za postupak analize, "to se nadovezuje na osnovna pitanja ontolo"ke (pojednostavljeno – "ta se istra%uje?) i epistemiolo"ke prirode (pojednostavljeno – kako se istra%uje?). U nastojanju da se odgovori na ova pitanja, o#uvana je selektivnost u procesu odabira i analize dokumenata. 75 4.1.2 Teme i koncepti reprezentativnih zakonskih regulativa i podzakonskih akata Analiza uz pomo$ softverskog programa je uklju#ila dokumenta iz grupe 1 i 2 (eng. cohort 1 & cohort 2) predstavljenih u Tabeli 3.2 koji su procesovani uz pomo$ Leximancer v3.1 programa za analizu teksta (Smith, 2005; Smith, 2010). Rezultati analize prikazani su u Tabeli 4.3, Tabeli 4.4, na Slikama 4.3, 4.4, 4.6 kao i na Slici 4.5 koja se nalazi u Prilogu 16. Za potrebe softverske analize koncepti su definisani kao grupe re#i koje se u kontinuitetu zajedno pojavljuju u tekstu (eng. collections of words that generally travel together throughout the text) (Smith, 2005; Smith, 2010). Softverski program obavlja konceptualnu i korelacionu analizu (Smith, 2005; Smith, 2010) pri tome uzimaju$i u obzir koji koncepti su prisutni u tekstu i kako su povezani tj. u kakvoj su korelaciji (Slika 4.3 i 4.4). Pored grafi#kog prikaza rezultata, obezbedjeni su podaci o u#estalosti, kao "to je i)lista naju#estalijih koncepata prikazana u Tabeli 4.3 i ii)frekvencija u#estalosti koncepata koji se zajedno pojavljuju kroz tekst (eng. frequency of concept co-occurrence) koja je predstavljena u obliku 3D grafi#kih prikaza na Slici 4.5 koja se nalazi u Prilogu 16. Koncepti Nakon procesovanja dokumenata, program generi"e primarne konceptualne mape (eng. concept maps) (Slika 4.3) i konceptualne oblake (eng. concept clouds) (Slika 4.4). Konceptualne mape imaju razli#itu kompleksnost i strukturu (eng. complexity, number of concepts or size of circles around each concept) pri tome prikazuju$i neke od osnovnih informacija o tekstu (Smith, 2005; Smith, 2010): • Osnovne tj. naju#estalije koncepte koji se pojavljuju u analiziranom tekstu (dokumentu) ili grupi dokumenata (eng. document set) (Tabela 4.3). • Centralnost tj. zna#aj svakog koncepta (Slika 4.4 i Slika 4.6). • Frekvenciju tj. u#estalost koncepata koji se zajedno pojavljuju kroz tekst (Slika 4.5, Prilog 16). • Sli#nost u kontekstu gde se konceptualne grupe pojavljuju zajedno kao teme (eng. themes) i relativnu u#estalost koncepata (Tabela 4.4). Kao primer funkcionalnosti softverske analize, prikazana je konceptualna mapa dokumenata agencije TGA koji se bave veoma razli#itim aspektima regulative – "to se manifestuje velikim brojem koncepata koji se medjusobno preklapaju (Slika 4.3C). 76 Preuzeto iz: Ilic et al., 2012b. Slika 4.3: Konceptualne mape i koncepti identifikovani u slede$im dokumentima: dokumenta agencije EMA (A), dokumenta agencije FDA (B), dokumenta agencije TGA (C). GMP(1) dokumenta (D), GMP(2) dokumenta (E), GCP dokumenta (F). Lista svih dokumenata je prikazana u Tabeli 3.2 u poglavlju Materijali. E F C D A B 77 Konceptualni oblaci (eng. concept clouds) (Slika 4.4) po svojoj strukturi i funkciji slu%e kao ilustracija razlika izmedju dokumenata u smislu sadr%aja (Smith, 2005; Smith, 2010). Na primer, sadr%aj dokumenata agencija EMA (Slika 4.4D) i TGA (Slika 4.4E) mogu se uporediti vizuelno na osnovu konfiguracije njihovih konceptualnih oblaka. Konceptualni oblak dokumenata agencije EMA ima druga#iju strukturu (uzak, izdu%en skup koncepata) u odnosu na druge konceptualne oblake npr. agencije TGA (Slika 4.4E) – sa centralno postavljenim primarnim konceptima ‘medicinal’ i ‘products’ kao i sa nekoliko sekundarnih konceptualnih postavki kao "to su ‘marketing’, ‘authorisation’, ‘holder’ and ‘referred’ (Slika 4.4D). Konceptualni oblak agencije TGA prikazuje centralnost dva koncepta, ‘biological’ i ‘class’, okru%enih sa jedne strane konceptima ‘human’ i ‘products’, a sa druge strane ‘information’ i ‘application’ (Slika 4.4E). Takodje, ukoliko se uporede konceptualni oblaci dokumenata koji pripadaju razli#itim grupama regulatornih principa GMP i GCP – mogu se zapaziti sli#nosti i razlike koje odlsikavaju njihovu strukturu i sadr%aj. Konceptualni oblak grupe dokumenata GMP(1) (grupa koja obuhvata raznorodna dokumenta agencija EMA i TGA) (Slika 4.4A) je "iri, sa vi"e razli#itih koncepata koji okru%uju centralno postavljen koncept ‘product’ ukazuju$i na "iroki spektar ne naro#ito srodnih koncpetualnih postavki. Konceptualni oblak grupe dokumenata GMP(2) (Slika 4.4B) je, za razliku od prethodnog primera, znatno uredjeniji sa konceptima ‘procedures’, ‘processing’, ‘quality’ i ‘control’ koji na manjoj razdaljini okru%uju centralno postavljen koncept ‘appropriate’; dok se u "irem okru%enju na jednoj strani nalaze ‘intended’ i ‘use’ a na drugoj strani ‘batch’ i ‘records’. Ovakav oblik i struktura konceptualnog oblaka ukazuje na ve$u koherentnost dokumenata koji su zajedno analizirani, u ovom slu#aju radi se o dve GMP regulative koje pokrivaju proizvodnju medicinskih produkata i jednom GMP dokumentu koji se odnosi na dovr"ene farmaceutske proizvode. Ipak, najizra%eniji po svojoj koherentnosti je konceptualni oblak GCP dokumenata (Slika 4.4C) koji obuhvata relativno uzak konceptualni opseg oko centralno postavljenih ‘sponsor’ i ‘trial’, time ukazuju$i na fokus i nameru ovih dokumenata da uka%u na jednu osnovnu konceptualnu postavku. Nasuprot tome, konceptualni oblaci agencije EMA (Slika 4.4D) i TGA (Slika 4.4E) su orijentisani oko svoje vertikalne ili horizontalne ose – ukazuju$i na raznolikost i razudjenost konceptualnih postavki kojima se bave. 78 Preuzeto iz: Ilic et al., 2012b. Slika 4.4 A-C: Konceptualni oblaci i teme (konceptualne grupe) identifikovani u dokumentima softverskom analizom. Lista svih dokumenata je prikazana u Tabeli 3.2 u poglavlju Materijali. U daljoj analizi softver je identifikovao najosnovnije (naj#e"$e upotrebljene) koncepte kao "to je prikazano u Tabeli 4.3 – za agenciju TGA to je bio koncept ‘biological’, za FDA ‘product’ a za agenciju EMA, ‘medicinal’. Ovo je bilo u skladu sa rezultatima manuelne analize dokumenata (Tabele 4.1 i 4.2) ali "to je jo" zna#ajnije, takodje ukazuje na #injenicu da sve tri regulatorne agencije kao osnovnu konceptualnu postavku svojih zakonskih regulativa u ovoj oblasti imaju – produkt, odnosno terapeutsko sredstvo koje se dobija u procesu izrade $elijskih tj. naprednih terapija (iako koriste razli#itu terminologiju: biological vs. product vs. medicinal). Ovo je pronadjeno kao jedna od osnovnih, ali retkih sli#nosti medju agencijama FDA, TGA i EMA u datoj regulatornoj oblasti. Konceptualni oblaci identifikovani u GMP(1) dokumentima (A), GMP(2) dokumentima (B), GCP dokumentima (C). A B C 79 Preuzeto iz: Ilic et al., 2012b. Slika 4.4 D-F: Konceptualni oblaci i teme (konceptualne grupe) identifikovani u dokumentima softverskom analizom. Lista svih dokumenata je prikazana u Tabeli 3.2 u poglavlju Materijali. U grupama dokumenata iz istih regulatornih principa, za GMP(1) grupu osnovni koncept je bio ‘quality’, za GMP(2) ‘appropriate’ a za GCP grupu dokumenata, koncept ‘clinical’ koga slede ‘trial’, ‘subjects’, ‘sponsor’ i ‘data’. Ovo je takodje bilo u skladu sa rezultatima manuelne analize. Konceptualni oblaci identifikovani u regulatornim okvirima (D, E), i preklapanje tema (F) identifikovani od strane softvera u slede#im dokumentima: agencije EMA (D), agencije TGA (E), agencije GCP (preklapanje konceptualnih okvira predstavlja njihovu blisku povezanost) (F). D E F 80 Teme (tematski klasteri) Razli#ite boje kru%nica na grafi#kim prikazima koje generi"e softver, ozna#avaju razli#ite teme tj. tematske grupe (Smith, 2005; Smith, 2010). Krugovi se formiraju oko povezanijih tj. bliskijih koncepata na taj na#in formiraju$i tematske klastere (eng. thematic clusters). Rezultat tematske analize predstavlja i tematski pregled za grupu dokumenata 1 i 2 koji je prikazan u Tabeli 4.4. Konceptualna mapa (Slika 4.4F) prikazuje analizu GCP dokumenata iz razli#itih regulatornih okvira, koji ipak imaju sli#nu udaljenost konceptualnih postavki kao i stepen njihove konektivnosti – predstavljen ne samo bojom ve$ i razmakom na mapi. U ovoj analizi su GCP dokumenta iz razli#itih regulatornih okvira pokazala vi"i stepen koherentnosti i slaganja u svom sadr%aju od drugih (npr. GMP). Centralna tema ‘trial’ je bila okru%ena bliskim temama: ‘sponsor’, ‘subjects’, ‘data’, ‘clinical’, ‘accordance’ i ‘adverse’ (Slika 4.4F). Teme kao "to su ‘written’, ‘including’ i ‘days’ bile su u odredjenoj meri udaljenije ali jo" uvek dovoljno blizu centralne teme ‘trial’. Tema ‘sponsor’ je u sebi sadr%ala koncepte ‘sponsor’, ‘regulatory’, ‘protocol’ i ‘opinion’, dok je tema ‘subjects’ sadr%ala koncepte kao "to su ‘provided’, ‘information’ i ‘consent’ veoma blizu teme ‘data’ (kao i koncepta ‘data’) (Slika 4.4F). Teme ‘clinical’ and ‘products’ su takodje predstavljene na maloj udaljenosti, "to predstavlja njihovu povezanost u tekstu, ali ne"to vi"e udaljene od tema ‘sponsor’, ‘written’ ili ‘data’, koje se nisu zajedno sa njima pojavljivale u tekstu. U"estalost koncepata koji se zajedno pojavljuju U#estalost koncepata koji se zajedno pojavljuju (eng. co-occurrence between concepts) je va%an aspekt analize kojim se utvrdjuje bliskost tj. povezanost konceptualnih postavki u tekstu (Smith, 2005; Smith, 2010). Grafi#ki prikaz (3D) na Slici 4.5 u Prilogu 16 ilustruje rezultate dobijene analizom dokumenata u grupi 1 i 2, uzimaju$i u obzir bliskost konceptualnih postavki koje se pojavljuju zajedno kroz tekst (eng. a window, based on a length of words or sentences, is specified and moved sequentially through the text, taking note of the concepts that occurred together). U dokumentima agencije EMA, maksimalna u#estalost parova koncepata koji se zajedno pojavljuju je u opsegu od 7000 do 8000 (Slika 4.5C, Prilog 16) dok je ovaj 81 opseg za dokumenta druge dve agencije najvi"e do 1000 (Slika 4.5A,B, Prilog 16). Ova mera ne zavisi samo od broja ponavljanja parova koncepata ve$ i od njihove povezanosti u smislu konteksta (Smith, 2005; Smith, 2010). Individualna u#estalost termina ‘medicinal’ je ne"to preko 1000 u tri manuelno analizirana EMA dokumenta. Ipak, zbog sli#nosti konteksta u kojem se pojavljuje sa nekim drugim konceptima (eng. co-occurrence) frekvencija njihovog zajedni#kog pojavljivanja je od strane softvera rangirana u opsegu od 7000 do 8000 (Slika 4.5C, Prilog 16). U ovoj analizi nije bilo dovoljno podataka da bi se zabele%ilo bilo kakvo zna#ajno zajedni#ko pojavljivanje (eng. co-occurrence) kao osnova da se uspostave zajedni#ke ili sli#ne konceptualne postavke izmedju razli#itih regulatornih okvira ili izmedju GMP dokumenata koje razli#ite agencije propisuju u ovoj oblasti. Nedostatak sli#nosti koji je o#igledan u manuelnoj i softverskoj analizi (npr. razli#ita terminologija, upotreba razli#itih fraza i razlike u u#estalosti njihove upotrebe) – potvrdjuje deo hipoteze da se regulatorne agencije razlikuju u svojim konceptualnim postavkama. Takodje, pokazano je da razlike u formalnom smislu postoje kroz njihove najvi"e zakonsko regulatorne odredbe koje u ovoj analizi reprezentuju dati zakonski okvir za svaku agenciju. Zavr"ni deo informacije o tekstu, koji je vidljiv na mapi, predstavlja kontekstualna sli#nost izmedju konceptualnih postavki (eng. contextual similarity between concepts). U procesu formiranja mape, koncepti su slu#ajnim redosledom postavljeni na ivice mape. U slede$oj fazi analize koncepti medjusobno demonstriraju svoj uticaj jedan na drugi koji se bazira na prisustvu ili odsustvu zajedni#kog konteksta kao i na njegovu u#estalost (eng. exert a pull on each other concept with a strength related to their co-occurrence value). Ovo mo%e figurativno da se predstavi kao fizi#ka veza medju konceptima (npr. opruga) koja ‘povla#i’ koncepte jedan ka drugom ili obrnuto (Smith, 2005; Smith, 2010). Naravno, zbog mnogo medjusobnih veza medju konceptima, kao i ‘sila’ privla#enja/odbijanja baziranih na meri njihovog zajedni#kog konteksta i frekvencije, te"ko je o#ekivati da se ovo prika%e u apsolutnoj meri. Tako se koncepti u stvari grupi"u na osnovu svih ovih korelacija. Grupe konceptualnih postavki koje su bliske u tekstu, bi$e identifikovane kao deo iste teme. Najcentralnije teme i njihovi koncepti predstavljeni su na Slici 4.6. Primer kontekstualne sli#nosti nalazi se na Slici 4.6C gde su prikazani rezultati 82 analize TGA dokumenata koji pripadaju istoj grupaciji (eng. Biologicals Framework). Iako koncepti ‘application’ i ‘information’ nemaju direktnu vezu u tekstu (ne pojavljuju se zajedno), oni izgledaju bliski na mapi zbog sli#nosti konteksta (teme) u kojem se pojavljuju. Osim toga, ovi koncepti se pojavljuju zajedno u grupi sa nekim va%nim konceptima kao "to su ‘dossier’, ‘processing’ i ‘goods’ koji se nalaze u istim takvim kontekstima u dokumentu. Bazirano na sli#nosti u kontekstu, na istoj slici se izdvaja i grupa koju #ine koncepti ‘blood’, ‘human’, ‘cells’ i ‘product’/‘products’ koja ne pokazuje direktnu vezu sa ve$ pomenutim konceptima ‘application’ i ‘information’, ‘sponsor’ ili ‘trial’ u drugom delu mape. Preuzeto iz: Ilic et al., 2012b. Slika 4.6: Naju#estalije teme i koncepti (A, B). A. Naju#estalije teme identifikovane u dokumentima agencije FDA. Preklapanje konceptualnih okvira reprezentuje njihovu blisku povezanost. B. Naju#estalije teme identifikovane u dokumentima agencije EMA. 83 Tabela 4.3: Rezultat softverske analize dokumenata iz grupe 1 i 2: naju#estaliji koncepti u pojedina#nim regulatornim okvirima i u okviru regulatornih principa (GMP(1), GMP(2), GCP). Preuzeto iz: Ilic et al., 2012b. Preuzeto iz: Ilic et al., 2012b. Slika 4.6: Naju#estalije teme i koncepti (C). Naju"estaliji koncepti 1 2 3 4 5 Concepts in Order of Frequency U okviru pojedina#nih regulatornih okvira (eng. in individual regulatory frameworks) TGA Biological Human Section Trial - FDA Product Section Used HCT CFR EMA Medicinal Marketing Commission Directive Public U okviru regulatornih principa (eng. across regulatory frameworks) GMP(1) Quality Materials Product Contamination Manufacture GMP(2) Appropriate Drug Records API - GCP Clinical Trial Subjects Sponsor Data C. Naju#estalije teme identifikovane u dokumentima agencije TGA. Primer veoma izra%ene kontekstualne sli#nosti izmedju dokumenata (identifikovane od strane softvera). 84 Tabela 4.4: Rezultati softverske analize dokumenata iz grupe 1 i 2: Tematski pregled u pojedina#nim regulatornim okvirima i u okviru regulatornih principa (GMP(1), GMP(2), GCP). ^Stepen konektivnosti (eng. connectivity) je mera korelacije izmedju koncepata i mera stepena njihove srodnosti/ medjuzavisnosti. Lista u#estalosti za svaki pojedina#ni koncept je deo rezultata koji su izlo%eni ispod njihovog grafi#kog prikaza/mape za dati tekst. Preuzeto iz: Ilic et al., 2012b. Re#enice za koje se smatra da sadr%e koncept su obele%ene od strane softverskog programa samo ako je akumulirano dovoljno podataka za to (npr. u vidu ponavljanja odredjenih grupa re#i) (Smith, 2005; Smith, 2010). Prisustvo svake obele%ene re#i donosi odredjenu vrednost tako da $e grupa re#i ili re#enica u tekstu biti klasifikovana od strane softvera kao concept samo ako se akumulira dovoljno re#i koje nose odredjenu vrednost tj. ako vrednost svih obele%enih re#i zajedno doprinese prelasku u opseg koncepta (Smith, 2005; Smith, 2010). Konceptualna analiza kao deo funkcionalnosti programa, omogu$ava da se tekst jednog ili vise dokumenata Tema Stepen konektivnosti^ Tema Stepen konektivnosti^ Tema Stepen konektivnosti^ Theme Connectivity Theme Connectivity Theme Connectivity TGA FDA EMA biological 100% section 100% medicinal 100% Class 46% donor 82% marketing 88% Use 44% product 53% referred 59% biologicals 42% HCT 49% Community 29% ARTG 34% including 46% Directive 28% Human 20% manufacture 39% market 15% medical 15% products 21% Agency 12% Section 10% requirements 19% appropriate 10% Trial 02% FDA 11% human 10% Page 01% drug 05% therapy 09% Commission 09% GMP(1) GMP(2) GCP Quality 100% appropriate 100% sponsor 100% Product 91% drug 50% subjects 66% Materials 73% use 45% trial 56% Products 63% batch 40% written 34% Used 46% used 39% clinical 28% Manufacture 36% manufacture 28% products 18% Validation 26% written 25% product 15% System 26% established 23% adverse 03% Contamination 19% API 22% Area 18% maintained 09% Containers 08% Apis 08% Filling 06% personnel 04% MANUFACTURE 03% data 03% 85 podeli u celine #ije medjusobne relacije mogu da se kvantifikuju i analiziraju (Smith, 2005; Smith, 2010). U konceptualnoj analizi tekst dokumenata se ispituje na prisustvo i frekvenciju konceptualnih postavki/ koncepata. Nasuprot tome, u korelacionoj analizi meri se kako su tako identifikovani koncepti medjusobno povezani u tekstu (Smith, 2005; Smith, 2010). Tabela 4.5: Frekvencije (eng. high, medium & low) i stepen konektivnosti (%) izmedju tema koje su identifikovane u dokumentima grupe 1 i 2 (eng. cohorts 1 and 2). THEMES CONNECTIVITY^ THEMES IDENTIFIED BY THE SOFTWARE THEMES OVERALL FREQUENCY MIN ( % ) MAX ( % ) TGA Documents FDA Documents EMA Documents GMP(1) Documents GMP(2) Documents GCP Documents 91 100 biological section medicinal quality product appropriate sponsor HIGH ( 9/65 ) 81 90 - donor marketing - - - 51 80 - product referred materials products - subjects trial MEDIUM ( 41/65 ) 11 50 class use biologicals ARTG human medical HCT including manufacture products requirements FDA community directive market agency used manufacture validation system contamination area drug use batch used manufacture written established API written clinical products product adverse !10 - section trial page drug appropriate human therapy commission containers filling manufacture maintained Apis personnel data - LOW ( 15/65 ) ^Stepen konektivnosti (eng. connectivity) je mera korelacije izmedju koncepata i mera stepena njihove srodnosti/ medjuzavisnosti. Lista u#estalosti za svaki pojedina#ni koncept je deo rezultata koji su izlo%eni ispod njihovog grafi#kog prikaza/mape za dati tekst. Preuzeto iz: Ilic et al., 2012b. Ova analiza je zna#ajan doprinos uporednoj kvalitativnoj analizi zakonsko regulatornih odredbi, koja do sada nije bila zastupljena u literaturi. Takodje doprinosi harmonizaciji i razumevanju sli#nosti i razlika izmedju osnovnih postavki razli#itih zakonsko regulatornih okvira formiranih u ovoj oblasti. Kao "to je napomenuo jedan od stru#njaka u svom intervjuu – nije u pitanju razlika u terminologiji ve$ razlika u onome "to se tom terminologijom podrazumeva (CTE022, Tabela 4.25). 86 Primenjena metodologija koristi prednosti kvalitativne analize – omogu$uju$i kontekstualizaciju podataka i detaljnu analizu uzorka. Neka od zna#ajnih metodolo"kih razmatranja u ovom tipu analize uklju#uju nereaktivnost metodologije (eng. non-reactivity) kao "to navode neki autori “the data selection method itself generally does not change the data being collected” (Bailey, 1982) i dug proces pripreme koji prethodi analizi (zbog toga "to je dokument u tekstualnoj formi) (Mason, 2002). U obzir se takodje uzimaju odredjena merila kao "to su: ponovljivost (eng. stability of a measure) (Weber, 1990; Creswell, 2007), pouzdanost i preciznost metodologije (Creswell, 2007; Maxwell, 2005; Scott, 1990; Weber, 1990; Maxwell, 2005), adekvatnost i reprezentativnost uzorka (Robson, 2002); kao i validnost interpretacije (Mason, 2002), mogu$nost generalizacije rezultata (Robson, 2002) (empirijska, koja predstavlja primenljivost u odnosu na "iri ‘univerzum’ i teoretska) (Mason, 2002), transparentnost odnosno standardizacija pristupa (Maxwell, 2005) i validnost konstrukcije odnosno teorijske postavke (eng. construct validity) (Thomas & Harden, 2008). Budu$i da su prvenstveno analizirane sli#nosti i razlike izmedju zakonsko regulatornih pristupa, bilo je sasvim uputno koristiti dokumenta koja sadr%e regulatorne odredbe na najvi"em nivou – za koje se smatra da adekvatno reprezentuju osnovne karakteristike tih pristupa. Dok je manuelna analiza dokumenata obezbedila terminolo"ki osvrt na dati tekstualni materijal, upotreba softverskog programa obezbedila je da se, uz primenu grafi#kih prikaza, izvedu odredjeni koncepti, sagleda njihov kontekst i korelacijske odrednice medju njima (Scott, 1990). Pri tome je usvojen stav da se o#ekuju odredjene razlike u zakonsko regulatornim postavkama, koje su bazirane na kulturolo"kim, istorijskim i drugim faktorima kao i regionalnoj dinamici razvoja biolo"kih lekova. Ipak je, kao "to se moglo o#ekivati, fokus svih analiziranih zakonskih okvira bio na za"titi javnog zdravlja, zdravlja pojedinca i obezbedjenju kvaliteta terapeutskog produkta baziranog na savremenim nau#nim saznanjima. 87 4.1.3 Geografska distribucija klini!kih istra%ivanja Nau#na oblast koja se bavi mati#nim $elijama i drugim $elijskim terapijama, ranije je bila deo bazi#nih istra%ivanja a danas sazreva kao novo polje u domenu translatornih klini#kih istra%ivanja (Atala, 2013). Ove terapije, kao deo regenerativne medicine, imaju prakti#no neograni#en potencijal (Atala, 2013). Regenerativna medicina koristi nekoliko mehanizama kao "to su mati#ne $elije, in%enjering tkiva, biomaterijali i sli#ni prsistupi u zameni obolelih tkiva i organa (Quick Facts on Regenerative Medicine, Canada, 2011). Globalno tr%i"te regenerativne medicine trenutno se procenjuje na $3,6 milijarde dolara i o#ekuje se da prema"i $11 milijardi dolara do 2020. godine, ukoliko se nastavi predvidjeni razvoj po godi"njoj stopi rasta od 30% (Quick Facts on Regenerative Medicine, Canada, 2011). U periodu izmedju 2000. i 2005. godine broj klini#kih istra%ivanja sa primenom ‘biolo"ke intervencije’ (eng. ‘biological intervention’) bio je oko 1200 da bi ubrzo dostigao nivo odo oko 6000 istra%iva#kih projekata (Bourgoin, 2011). Ipak, samo jedan mali procenat ovih projekata $e da nastavi svoj put kroz istra%iva#ki proces/ translaciju u kasnije faze klini#kih istra%ivanja a jo" manji broj $e biti odobren od strane regulatornih agencija za svoju dalju primenu. Zaklju#no sa novembrom 2010. godine, samo je 3% klini#kih istra%ivanja u tre$oj fazi (eng. Phase III clinical trials) bilo registrovano sa primenom ‘biolo"ke intervencije’ iako je 15% istra%ivanja u prvoj fazi (eng. Phase I) bilo registrovano u istoj kategoriji (Bourgoin, 2011). Da bi postao isplativ terapeutski proizvod u regenerativnoj medicini, kompleksni biolo"ki lek (bilo da je baziran na primeni $elija ili tkiva) mora da bude razvijen, ispitan i primenjen u strogo kontrolisanim uslovima. U ovom procesu postoje brojna nau#na, tehnolo"ka, regulatorna, eti#ka, finansijska i druga razmatranja i izazovi (Burger, 2003; Dutton, 2007; Prince et al., 2004; Nature Editorial, 2008; Kirouac & Zandstra, 2008; Atala, 2012). Uprkos tome, nastojanja da se to ostvari mogu da budu uspe"na kao "to je pokazano na primeru najprodavanijih 12 biolo"kih lekova u SAD-u koji su u 2010. godini u svom kumulativnom prihodu dostigli $30 milijardi dolara (Bourgoin, 2011). Shodno tome, klini#ka istra%ivanja u ovoj obasti se nastavljaju i pored ogromne neizvesnosti i koli#ine ulaganja koja iziskuju. Pregled globalne baze podataka koja sadr%i klini#ke istra%iva#ke projekte (eng. clinical trials) na svetskom nivou www.clinicaltrials.gov prikazao je slede$u geografsku distribuciju ukupnih klini#kih istra%ivanja kojih je u januaru 2011. godine bilo preko 100.000 a 88 preko 150.000 u avgustu 2013. godine (Tabela 4.6). U istom periodu, bilo je registrovano preko 18.000 klini#kih istra%ivanja (u januaru 2011. godine) odnosno blizu 25.000 (u avgustu 2013. godine) koja su dobijena pretragom uz kju#ne re#i ‘cell therapies’(Tabela 4.7). Tabela 4.6: Broj klini#kih istra%ivanja na svetskom nivou u januaru 2011. i u avgustu 2013. godine. Celokupna klini"ka istra$ivanja* u januaru 2011. godine Geografski region Broj klini#kih istra%ivanja^ World 101792 Africa 2201 Central America 1451 East Asia 7314 Japan 1726 Europe 25543 Middle East 3883 North America 56557 Canada 7842 Mexico 1304 United States 51967 North Asia 1751 Pacifica* (incl. Australia) 2929 Australia 2764 South America 3178 South Asia 1729 Southeast Asia 1857 *Klini#ka istra%ivanja koja nemaju preciziranu lokaciju ili imaju vi"e nazna#enih lokacija nisu u"la u ovaj broj. ^Prema globalnoj bazi podataka www.clinicaltrials.gov Prema globalnoj bazi podataka www.clinicaltrials.gov Slika 4.7: Mapa klini#kih istra%ivanja na svetskom nivou u januaru 2011. godine. Celokupna klini"ka istra$ivanja* u avgustu 2013. godine Geografski region Broj klini#kih istra%ivanja^ World 150905 Africa 3428 Central America 1884 East Asia 13477 Japan 2844 Europe 41103 Middle East 6237 North America 78019 Canada 11282 Mexico 1936 United States 71010 North Asia 2820 Pacifica* (incl. Australia) 4099 Australia 5139 South America 2655 South Asia 3050 Southeast Asia 2820 Od najmanjeg broja ka najve$em 89 Prema globalnoj bazi podataka www.clinicaltrials.gov Slika 4.8: Mapa klini#kih istra%ivanja u Evropi u januaru 2011. godine. Prema globalnoj bazi podataka www.clinicaltrials.gov Slika 4.9: Mapa klini#kih istra%ivanja u Evropi u avgustu 2013. godine. Od najmanjeg broja ka najve$em Od najmanjeg broja ka najve$em 90 4.1.4 Trendovi u klini!kim istra%ivanjima prema tipu #elijskih terapija Rezultati pretrage na osnovu klju!ne re!i "cell therapies” (Tabela 4.7) pokazali su da je opseg klini!kih istra"ivanja u toj kategoriji porastao za tre#inu u Tabela 4.7: Broj klini#kih istra%ivanja na svetskom nivou u januaru 2011. i u avgustu 2013. godine na osnovu klju#nih re#i ‘cell therapies’. Broj klini"kih istra$ivanja na osnovu klju"nih re"i: ‘cell therapies’ u januaru 2011. godine Geografski region Broj klini#kih istra%ivanja^ World 18833 Africa 295 Central America 363 East Asia 990 Japan 214 Europe 4123 Middle East 499 North America 13037 Canada 1581 Mexico 138 United States 12512 North Asia 247 Pacifica 683 South America 376 South Asia 208 Southeast Asia 254 *Klini#ka istra%ivanja koja nemaju preciziranu lokaciju ili imaju vi"e nazna#enih lokacija nisu u"la u ovaj broj. ^Prema globalnoj bazi podataka www.clinicaltrials.gov periodu od januara 2011. do avgusta 2013. godine. Porast broja ovih istra"ivanja je zabele"en u svim svetskim regionima (Tabela 4.7). Pretragom baze podataka na osnovu klju#nih re#i “cell therapies AND stem cell” dobijeni su rezultati predstavljeni u Tabeli 4.8 i na mapi (Slika 4.10) gde je prikazano da je na svetskom nivou po#etkom 2011. godine bilo registrovano 3.076 klini#kih istra%ivanja u ovoj oblasti – 668 u Evropi, 2.015 u SAD-u i 106 u Australiji. Pribli%no jedna tre$ina ovih istra%ivanja bila je finansirana od strane Nacionalnog instituta za zdravlje u SAD-u (National Institutes of Health, NIH), oko 17% od strane industrije (privatnih kompanija) a iz fondova univerziteta oko 40%. Broj klini"kih istra$ivanja na osnovu klju"nih re"i: ‘cell therapies’ u avgustu 2013. godine Geografski region Broj klini#kih istra%ivanja^ World 24909 Africa 445 Central America 406 East Asia 1808 Japan 346 Europe 5968 Middle East 763 North America 16367 Canada 1952 Mexico 232 United States 15648 North Asia 437 Pacifica 893 South America 589 South Asia 309 Southeast Asia 403 91 Tabela 4.8: Broj klini#kih istra%ivanja na svetskom nivou u januaru 2011. i u avgustu 2013. godine na osnovu klju#nih re#i ‘cell therapies AND stem cell’. Broj klini"kih istra$ivanja* na osnovu kljucnih reci: ‘cell therapies AND stem cell’ u januaru 2011. godine Geografski region Broj klini#kih istra%ivanja^ World 3076 Africa 9 Central America 41 East Asia 164 Japan 21 Europe 668 Middle East 111 North America 2071 Canada 216 Mexico 10 United States 2015 North Asia 14 Pacifica 106 South America 32 South Asia 19 Southeast Asia 23 *Klini#ka istra%ivanja koja nemaju preciziranu lokaciju ili imaju vi"e nazna#enih lokacija nisu u"la u ovaj broj. ^Prema globalnoj bazi podataka www.clinicaltrials.gov Prema globalnoj bazi podataka www.clinicaltrials.gov Slika 4.10: Mapa klini#kih istra%ivanja na osnovu pretrage klju#nih re#i ‘cell therapies AND adult stem cell’ u januaru 2011. godine. Broj klini"kih istra$ivanja* na osnovu kljucnih reci: ‘cell therapies AND stem cell’ u avgustu 2013. godine Geografski region Broj klini#kih istra%ivanja^ World 4311 Africa 16 Central America 41 East Asia 344 Japan 35 Europe 975 Middle East 162 North America 2694 Canada 252 Mexico 24 United States 2606 North Asia 31 Pacifica 126 South America 54 South Asia 52 Southeast Asia 38 Od najmanjeg broja ka najve$em 92 Od ukupno 41.103 registrovanih klini#kih istra%ivanja Evropi (Slika 4.9) 9.450 je registrovano u Francuskoj, 10.567 u Nema#koj, 6.122 u Italiji, 8.052 u Velikoj Britaniji, a ne"to vi"e od 500 klini#kih istra%ivanja je registrovano u na"oj zemlji, sli#no kao u Litvaniji i Latviji. Na osnovu pretrage klju#nih re#i “cell therapies AND adult stem cell” mapa (Slika 4.10) prikazuje ukupno 2.972 klini#kih istra%ivanja, od #ega je 636 klini#kih projekata registrovano u Evropi, 1.951 u SAD-u i 91 u Australiji. Zna#ajno manji broj klini#kih istrazivanja, ukupno 140, registrovan je na svetskom nivou u kategoriji mezenhimskih mati#nih $elija (pretraga uz klju#ne re#i “cell therapies AND mesenchymal stem cells”) sa po pribli%no 40 registrovanih klini#kih projekata u SAD-u i Evropi i 2 u Australiji (Slika 4.11 i 4.12). Mora se uzeti u obzir #injenica da sli#na istra%ivanja mogu biti registrovana pod nekim od drugih kategorija. U isto vreme pretraga bazirana na klju#nim re#ima “cell therapies AND dendritic cell therapy” (Slika 18.3 u Prilogu 18) oznacila je 319 klini#kih istra%ivanja od kojih je 229 registrovano u SAD-u, 59 u Evropi i 2 u Australiji. Mapa 1.496 klini#kih istra%ivanja koja navode upotrebu autolognih $elijskih terapija, prikazana je na Slici 4.13, od kojih su 903 projekta registrovana u SAD-u, 338 u Evropi i 19 u Australiji (pretraga na osnovu klju#nih re#i “cell therapies AND autologous” u januaru 2011. godine). Prema globalnoj bazi podataka www.clinicaltrials.gov Slika 4.11: Mapa klini#kih istra%ivanja uz upotrebu mezenhimskih $elija na svetskom nivou (pretraga uz klju#ne re#i ‘cell therapies AND mesenchymal stem cells’) u januaru 2011. godine. 93 Prema globalnoj bazi podataka www.clinicaltrials.gov Slika 4.12: Mapa klini#kih istra%ivanja uz upotrebu mezenhimskih $elija na svetskom nivou (pretraga uz klju#ne re#i ‘cell therapies AND mesenchymal stem cells’) u avgustu 2013. godine. Prema globalnoj bazi podataka www.clinicaltrials.gov Slika 4.13: Mapa klini#kih istra%ivanja uz upotrebu autolognih terapija (903 u SAD-u, 338 u Evropi i 19 u Australiji) koja su izlistana na osnovu klju#nih re#i ‘cell therapies AND autologous’ u januaru 2011. godine. Od najmanjeg broja ka najve$em Od najmanjeg broja ka najve$em 94 Cilj ove analize nije bio da se daju detaljni broj#ani podaci o klini#kim istra%ivanjima od interesa, niti iscrpni upiti u analiziranu bazu podataka. Prikazani rezultati dati su samo kao ilustracija odnosno kao pokazatelj trendova u opsegu i tipu klini#kih istra%iva#kih projekata vezanih za $elijske terapije. Danas je prepoznata vrednost i sna%an, ali donekle provokativan, potencijal regenerativne medicine i nove generacije terapija baziranih na primeni $elija i tkiva (Atala, 2012). U hiljadama istra%iva#kih laboratorija "irom sveta ula%e se ogroman napor u ovoj oblasti – koji relativno sporo ulazi u proces translatornih klini#kih istra%ivanja, pri tome nude$i veliki broj mogu$nosti ali postavljaju$i i niz prepreka (Atala, 2012). Na osnovu ove analize o#igledno je da se celokupan proces translatornog istra%ivanja ipak obavlja na zavidnom nivou kao i da se njegov obim uve$ava shodno nastanku novih nau#nih i tehnolo"kih saznanja u ovoj oblasti. Ipak postoji #itav niz bazi#nih istra%iva#kih metoda i njihovih primena kojima je potreban dalji rad i unapredjenje; tu spadaju: adekvatni pre-klini#ki modeli, upotreba malih molekula, upotreba novih disciplina kao "to je genomics, sagledavanje dugoro#nih efekata eksperimentalnih terapija na ljudima i profili bezbedne upotrebe novih terapeutika, mnogobrojne klini#ke potrebe za novim terapeutskim pristupima, zakonsko regulatorni tokovi, dizajn klini#kih istra%ivanja, modaliteti klini#ke primene i ostali mnogobrojni aspekti (Atala, 2012). Uspeh u klini#kim translatornim istra%ivanjima zahteva ne samo visoko obu#en kadar i kompleksan set nau#nih znanja i ve"tina, ve$ i razumevanje kompleksnih skupova podataka iz klini#kih primena, njihov zna#aj u zakonsko regulatornom smislu, standardizovane laboratorijske protokole i odr$ive organizacione modele (od kojih $e neki biti analizirani u slede$im poglavljima), adekvatne partnerske sporazume, stabilne poslovne sisteme i druge vrste efikasne podr"ke (Burger, 2003; Dutton, 2007; Prince et al., 2004; Nature Editorial, 2008; Kirouac & Zandstra, 2008; Atala, 2012). 95 4.2 Komparativna analiza 2: Karakterizacija laboratorijsko klini"kih protokola Primena adekvatnih laboratorijskih i klini#kih protokola je osnova u translatornim istra%ivanjima. Klini"ke primene iziskuju neka od slede$ih razmatranja u sprovodjenju protokola klini#kih istra%ivanja: preparativne re%ime (npr. vrste primenjene hemoterapije), alternativne tretmane (autologne vs. transplantacije alogenih $elija), primenu kombinovanih protokola za transplantaciju, redovno posmatranje mnogobrojnih klini#kih parametara i preventivu ne%eljenih reakcija kao i mogu$ih fatalnih ishoda (engraftment, GVHD, stopa mortaliteta i drugi parametri). Sa druge strane, laboratorijski protokoli se sve vi"e uslo%njavaju. Metodologijom pojedina#ne analize slu#aja (eng. case study) okarakterisani su modeli protokola za izradu (laboratorijski aspekt) i primenu (klini#ki pristup) mati#nih $elija i drugih $elijskih terapija, na slede$im nivoima: • Minimalno manipulisane $elije, - #iji proces proizvodnje uklju#uje samo zamrzavanje i #uvanje istih, namenjene za transplantaciju istom pacijentu (eng. autologous use); bez upotrebe kombinovanih proizvoda (npr. oboga$ivanja bilo kakvim proteinskim ili drugim agensima), bez dokazanog sistemskog efekta tj. sa visoko- specifi#nim terapeutskim efektom (eng. homogenous use), kao "to je proizvodnja i transplantacija mati#nih $elija izolovanih iz periferne krvi ili kostne sr%i. • Visoko-manipulisane $elije, - #iji proces proizvodnje uklju#uje kulturu $elija (eng. ex vivo expansion), kao "to je proizvodnja i transplantacija adultnih mezenhimskih mati#nih / stromalnih $elija (eng. mesenchymal stem cells, MSC) izolovanih iz placente, namenjenih za upotrebu u razli#itim klini#kim indikacijama. • Visoko-manipulisane $elije, - #iji proces proizvodnje uklju#uje oboga$ivanje (eng. loading) razli#itim proteinskim ili drugim agensima, kao "to je proizvodnja i transplantacija antigen-prezentuju$ih dendriti#nih $elija (eng. dendritic cells, DC) izolovanih iz krvi pacijenta upotrebom $elijske separacije aferezom (eng. 96 leukapheresis) i oboga$enih rekombinovanim proteinskim produktima, uglavnom namenjenih za antitumorske terapije. 4.2.1 Protokol za izradu i primenu mati!nih #elija hematopoeze izolovanih iz periferne krvi Karakteristike koje izdvajaju ovaj model protokola su: koristi se rutinski u klini#kim uslovima, uklju#uje minimalnu koli#inu manipulacije (uslovno re#eno, osnovnu manipulaciju) i izrada $elijskog produkta obavlja se u klini#ko bolni#kim laboratorijama. Ve$ina zakonsko regulatornih standarda, koji se odnose na minimalno manipulisane $elije nalazi se u nivou GTP (eng. Good Tissue Practice). U tom smislu zahteva se da njihove procedure i protokoli sadr%e odredjene elemente i da se u celokupnom procesu primeni adekvatno prikupljanje i #uvanje podataka (Tabela 4.9). Tabela 4.9: Va%ni elementi u procedurama, protokolima i #uvanju podataka za potrebe laboratorijske izrade i klini#ke primene minimalno manipulisanih mati#nih/ progenitorskih $elija izolovanih iz periferne krvi uz primenu afereze (prema brojnim standardima i regulatornim odredbama). Elementi neophodni u procedurama, protokolima I procesu #uvanja podataka: - donor selection, - assessment, - consent, - microbiological testing, - collection, - labelling, - system of numbering, - processing, - quality management and improvement, - proficiency testing, - storage, including alternative storage strategies if the primary storage device fails, - transportation, - expiry dates, - outcome analysis, - audits, - emergency and safety procedures, - equipment and supplies, - maintenance and monitoring, - cleaning procedures, - personnel training, - disposal of medical and biohazard waste, - release procedures, including criteria for exceptional release, - references, - tolerance limits, - corrective actions, - recall, returns and discard policy. Za izdvajanje mati#nih $elija periferne krvi (eng. Haematopoietic Stem/Progenitor Cells, HSC/HPC) koriste se vi"enamenski $elijski separatori sa kontinuiranim ili diskontinuiranim protokom krvi i softverskim programom za svaki tip aferezne procedure. Princip prikupljanja je zasnovan na centrifugalnom razdvajanju krvnih $elija i izdvajanju mononuklearnog sloja leukocita iz 97 antikoagulisane krvi bolesnika. Faktori koji uti#u na efikasnost ove klini#ke procedure obja"njeni su u delu teze koji se bavi teorijskim i prakti#nim modelovanjem (Komparativna analiza 5) dok su karakteristike samih $elija, procesa hematopoeze i afereznih metoda obja"njeni u uvodnom delu teze. Ve$ina procedura za pripreme mati#nih $elija hematopoeze izolovanih iz periferne krvi, uklju#uje procesovanje (pripremu $elija), njihovo zamrzavanje i #uvanje dok se samo ponekad koristi sve% proizvod. U toku pripreme $elija, koje prethodi zamrzavanju, mo%e da se obavi smanjenje volumena plazme ili da se uklone eritrociti (eng. plasma depletion & red cell depletion). Cilj je da se redukuje ukupna zapremina proizvoda ili da se uklone ne%eljene komponente (npr. antitela ili eritrociti). Ovo se smatra osnovnim procesnim modelom u izradi $elijskih terapija, ne toliko zbog njegove jednostavnosti ve$ zbog u#estale upotrebe u rutinske klini#ke svrhe. Manuelne metode smanjenja zapremine plazme i uklanjanja eritrocita uklju#uju centrifugiranje proizvoda pri #emu se mehani#ki uklanja sloj plazme ili istalo%eni eritrociti. Pri ovoj metodi se gubi odredjeni deo osnovnog #elijskog proizvoda tako da se sve u#estalije koriste automatske metode za odvajanje $elija. U procesu kriokonzervacije mati#ne $elije se postepeno zamrzavaju (eng. control rate freezing) u rastvoru koji #ine: plazma ili albuminski rastvor, krioprotektant (npr. dimetilsulfoksid, DMSO) i citrat dekstroza, obi#no u odnosu 65:20:10:5. Tako pripremljen, $elijski produkt se #uva u te#nom azotu (ako je mogu$e u gasnoj fazi) na temperaturi izmedju -160°C i -190°C. Osnovni elementi laboratorijske izrade i klini#ke primene minimalno manipulisanih mati#nih/ progenitorskih $elija izolovanih iz periferne krvi uz primenu afereze, prikazani su u Tabeli 4.10. Primer standradne operativne procedure za manipulaciju i kriokonzervaciju nalazi se u Prilogu 11. Tri osnovna elementa klini#ke metode transplantacije mati#nih $elija hematopoeze su: i)pripremni tretman (eng. conditioning regimen) #ija svrha je da ukloni tumorske $elije; ii)transplantacija $elijskog grafta (eng. stem cell graft), da bi se ponovo uspostavila hematopoeza; iii)post-transplantacioni tretmani (eng. 98 supportive care, including immunosuppressive regimen) uz primenu antibiotika, transfuzije krvi i/ili imunosupresivnih lekova (kod autolognih transplantacija). Tabela 4.10: Osnovni elementi laboratorijske izrade i klini#ke primene minimalno manipulisanih mati#nih/ progenitorskih $elija izolovanih iz periferne krvi uz primenu afereze. Prikupljanje $elija aferezom Kriokonzervacija i #uvanje Transplantacija Collection of PBSC by apheresis Cryopreservation & storage Reinfusion -Use of PBSC mobilization techniques and a variety of chemotherapy and/or cytokine regimens. -For patients treated with chemotherapy plus a hematopoietic cytokine, apheresis is generally initiated when the peripheral blood white cell count exceeded 1 109/l. -For some patients (as prescribed by the individual patient's physician), apheresis is initiated based on the level of CD34+ cells in the peripheral blood. -Apheresis is continued daily until the target goal of CD34+ cells is achieved (generally, >5 106/kg patient weight, although the physician could prescribe collection of higher numbers) or mobilization is deemed a failure. -Patients with limited numbers of cells collected after one or two apheresis procedures are managed with large volume apheresis procedures or with a temporary postponement of apheresis until adequate numbers of CD34+ cells are present in the peripheral blood. -Two or more attempts at mobilization and collection of PBSC are possible and all cells are frozen using the same technique to which the patient is originally assigned. -The peripheral circuit is anticoagulated with a mixture of 5000 units of heparin in 500 ml ACD-A infused at a blood to anticoagulant ratio of 30 : 1. An additional 40 ml of this anticoagulant solution is placed in the collect bag at the start of the procedure. For most patients, 12-15 l of blood is processed during each apheresis procedure. -The volume of the PBSC component was reduced by centrifugation at 2300g. -Excess supernatant was expressed, the volume of the residual cell pellet adjusted with autologous plasma as necessary, and the cell pellet diluted with an equal volume of the designated 2 cryoprotectant solution. -The cells were frozen in 30–70 ml aliquots in 250 ml freeze bags. -The first collection for each patient was frozen in at least two bags. -Subsequent collections could be frozen in single bags without regard to the concentration of nucleated cells or platelets. -Cells were cooled at -1°C/min using a rate-controlled cooling device to -40°C with compensation for the heat of fusion, and then at -10°C/min to -80°C before storage in the vapor phase of nitrogen at -186°C or colder. -Most components were cryopreserved within 4 h of collection; cells arriving late in the laboratory could be held at 4°C overnight in a monitored refrigerator before concentration and cryopreservation. -Cryopreserved cells were infused at least 24 h after the administration of the last dose of chemotherapy. -All patients were hydrated for 2 h before and after infusion (total, 4 h) with D5W1/2NS containing 20 meq/l KCl at a rate of 125 ml/m2/h. -Patients were medicated before infusion with diphenhydramine (or other antihistamine) and hydrocortisone. -Administration of antiemetics before or during the cell infusion was left to the discretion of the staff caring for the patient. -Cells were transported to the patient's bedside in liquid nitrogen carriers and thawed by immersion in a water bath at 37°C until complete dissolution of ice crystals. -The cells were then infused at a rate of 10–20 ml/min through a platelet product administration set with an inline 170–210 µm filter into a patent central venous catheter. Adaptirano iz: Chepovetsky et al., 2013. Pripremni tretman koji prethodi transplantaciji mati#nih $elija sastoji se od intenzivne primene citostatskih lekova koji imaju ulogu da uklone tumorske $elije, uz 99 manje ili vi"e o#uvan imunitet pacijenta (u zavisnosti od oboljenja i primenjenog terapijskog pristupa/tipa protokola u ovoj fazi). U autolognoj transplantaciji, primena transplantiranih $elija uklanja efekte primenjenog citostatskog tretmana i uspostavlja ponovo hematopoezu tako da antitumorski efekat po#iva pre svega na delovanju primenjenih citostatika. U alogenoj transplantaciji, transplantirane mati#ne $elije ne samo da obnavljaju hematopoezu ve$ ispoljavaju i svoje antitumorsko dejstvo. U rutinskoj klini#koj praksi postoje tri tipi#na izvora mati#nih $elija hematopoeze: kostna sr%, periferna krv i krv iz pup#anika, #ije su prednosti i ograni#enja prethodno prikazani u uvodnom delu teze. Transplantacija mati#nih $elija hematopoeze tj. $elijskog graft-a uklju#uje ne samo pluripotentne $elije ve$ i druge primitivne progenitorske $elije, uklju#uju$i prekursore is razli#itih razvojnih $elijskih linija kao i mnoge zrele krvne $elije. Ova klini#ka metoda se danas koristi u terapiji velikog broja hematolo"kih oboljenja, maligniteta (leukemija, limfoma i solidnih tumora) i oboljenja kao "to su neke bolesti metabolizma, te"ke kongenitalne imunodeficijencije, talasemija i druga. Primene ovakvog terapijskog pristupa su u porastu usled pristupa#nosti metode (izvodi se u klini#kim uslovima i uz podr"ku klini#kih laboratorija bez potrebe za ve$im dodatnim ulaganjima) kao i zbog stalnog usavr"avanja metode, u smislu laboratorijskih pristupa i klini#kih protokola koji slu%e kao podr"ka u ovom terapeutskom pristupu. Sa laboratorijskog aspekta, cilj je da se oporavak $elija odr%i u optimalnom opsegu nakon njihovog odmrzavanja (70-90%), da im se o#uva vijabilnost (ne manje od optimalnih 70-90% u odnosu na vrednost pre zamrzavanja, a meri se pomo$u broja kolonija koje su $elije sposobne da ostvare u ex vivo uslovima; eng. CFU-GM). Sa klini#kog stanovi"ta, cilj je, uglavnom, da se procedure prikupljanja i transplantacije, preparativnih i post-transplantacionih tretmana izvedu u adekvatnom vremenskom intervalu da bi se dobio najoptimalniji terapijski efekat. 100 4.2.2 Protokol za izradu i primenu mezenhimskih mati!nih/stromalnih #elija izolovanih iz placente Karakteristike koje izdvajaju ovaj tip protokola su: koristi se u klini#ko istra%iva#kim uslovima, uklju#uje zna#ajnu koli#inu manipulacije, ex vivo unmo%avanje $elija, "to nosi daleko ve$e rizike od minimalne manipulacije. Celokupan proces laboratorijske izrade i #uvanja mora da uklju#i odredjene GMP (eng. Good Manufactruing Practice) elemente (kao "to je prikazano u Prilogu 14). Obavlja se u za tu svrhu namenjenim GMP laboratorijama, koje moraju da zadovolje visoke standarde proizvodnje a "to se utvrdjuje redovnim periodi#nim inspekcijama (primer liste za inspekciju nalazi se u Prilogu 15). !elijski produkt ove vrste mo%e biti skladi"ten i #uvan (eng. cell banked), u najve$em broju slu#ajeva namenjen je ve$em broju pacijenata, ima nespecifi#nu namenu odnosno mo%e biti upotrebljen za razli#ite klini#ke indikacije (eng. off-the-shelf) "to ga #ini pogodnim za niz klini#kih istra%ivanja (usled specifi#nosti $elijskog tipa koji je prikazan u uvodnom delu teze). Ukoliko se koristi vi"e doza ovakvih $elijskih produkata, preporu#uje se da se za istog pacijenta uvek koriste produkti iz iste serije (eng. batch). Tipi#an primer ovakvog protokola je izrada i klini#ka primena mezenhimskih mati#nih/stromalnih $elija (eng. mesenchymal stem/ stromal cells, MSC) za razli#ite klini#ke primene. Karakteristike mezenhimskih mati#nih/stromalnih $elija su prethodno razmatrane u uvodnom delu teze, a obim klini#kih istra%ivanja koja uklju#uju upotrebu MSC je u stalnom porastu, kao "to je pokazano u Komparativnoj analizi 1. U dosada"njim istra%ivanjima MSC su bile izolovane iz razli#itih tkiva i organa, medju kojima su svi delovi placente – amnion, horion i decidua. Medjutim, za njihovu izolaciju se sve u#estalije koristi cela placenta prikupljena nakon porodjaja uz primenu carskog reza (eng. elective Caesarean sections) da bi se redukovao rizik od mikrobiolo"ke kontaminacije. Usitnjavanje tkiva placente mehani#kim putem pra$eno je enzimatskom obradom (eng. collagenase type 1 & DNase I). Nakon ispiranja, centrifugiranja i pripreme $elija (eng. ficoll density gradient/ cell lysis) za in vitro kultivaciju, vr"i se zasejavanje, inkubacija i uzgajanje u vi"e serija/ pasa%a (Slika 4.14). 101 Adaptirano iz: Ilic et al., 2013. Slika 4.14: Dijagram procesa prikupljanja i obrade visoko manipulisanih mezenhimskih mati#nih/ stromalnih $elija, MSC (dobijenih metodom izolovanja iz placente i ex vivo umno%avanja). U slo%enom procesu izrade mezenhimskih mati#nih/stromalnih $elija postoji niz neophodnih razmatranja i faktora koji moraju da budu zadovoljeni (Tabela 4.11). 102 Primer standardne operativne procedure za njihovu izolaciju iz placente i ex vivo ekspanziju nalazi se u Prilogu 12. Tabela 4.11: Elementi laboratorijske izrade visoko manipulisanih mezenhimskih mati#nih/ stromalnih $elija, MSC (dobijenih metodom izolovanja iz placente i ex vivo umno%avanja). Osnovne karakteristike $elijske terapije Primeri publikovanih protokola za laboratorijsku izradu i klini#ku primenu Problematika translatornog istra%iva#kog procesa Characteristics Examples of published Laboratory & Clinical Protocols Translational Research Process (‘bench-to-bedside’) Highly manipulated, heterogenous use, autologous or allogeneic use (even unmatched); may be combination product; may potentially have systemic effects; G. Brooke, T. Rossetti, R. Pelekanos, N. Ilic, P. Murray, S. Hancock, V. Antonenas, H. Gillian, D. Gottlieb, K. Bradstock, K. Atkinson, Manufacturing of human placenta-derived mesenchymal stem cells for clinical trials. British Journal of Haematology, 2009; vol 144, 4: 571-579. N. Ilic at al. (2011) Manufacturing of clinical grade human placenta- derived mesenchymal stem cells (MSC), Mesenchymal Stem Cell Assays and Applications, Methods in Molecular Biology, Humana Press USA, Vol. 698; Vemuri, Mohan; Chase, Lucas G.; Rao, Mahendra S. (Eds.). From basic science concept – Discovery Phase Through Engineering Phase with some of the following steps: Process development -Tissue processing and cell selection and isolation techniques (yield, loss of cells, purity, feasibility of the process eg. time/ staff) -Basic cell isolating techniques or highly sophisticated cell isolating techniques; pros (high purity of cell population vs. high number of cells); cons (single use disposables, time, cost of equipment, cost per procedure, regulatory issues eg. reagents, instruments and their compliance with device regulatory requirements, vendor-dependability, no back up option) -Use of any animal derived reagents (enzymes, media) -Flasks vs. bags, type of plastic/ materials used in culturing technique Scale Up/ Automation -Cost and feasibility studies (open vs. closed system; consistency of cell growth parameters) Validation of Manufacturing -Use of any animal derived reagents (enzymes, media) -Use of antibiotics, enzymes (clinical grade reagents availability) -Lot release characterisation (how to validate when no standard to compare) -Validation of route of administration (how to validate with limited or no clinical data) -Maintaining sterility through the process, monitoring sterility Custom GMP reagents QC Assays Release criteria/ potency assays -Characterisation of cells, -Viability, -Gram stain, - Sterility 14-day culture, -Endotoxin QA requirements and support Regulatory submission development/ maintenance (eg. IND) 103 Patents/ Intellectual Property (IP) and Commercialisation Multiple contracts and multiple supplier-network arrangements Academic support Regulatory support Funding Zbog svog porekla (placenta je izgradjena iz tkiva majke i fetusa), $elijskih karakteristika i mnogostrukih primena, mezenhimske mati#ne/stromalne $elije izolovane iz placente mogu da zauzimaju specifi#no mesto u zakonsko regulatornim propisima. Ako se uzmu u obzir samo evropske regulative koje primenjuje agencija EMA, postoji najmanje devet regulatornih i podzakonskih akata koji su primenljivi na izradu i klini#ku primenu MSC (Prilog 4, Slika P4.1). Australijski propisi u ovom procesu name$u dve kategorije zakonskih odredbi, one koje su primenljive na mati#ne $elije pup#anika, jer su takodje fetalnog porekla, kao "to su AusCord Guidelines (Prilog 7) kao i GMP propise namenjene za krv i tkivne produkte (eng. Human Blood and Tissues GMP). Zato su testovi za kontrolu kvaliteta ove grupe $elijskih terapeutika opse%ni, i uz standardne testove na infektivne bolesti kao "to su hepatitis B, hepatitis C, HIV-1,2, sifilis i HTLV-1, uklju#uju proveru zdravlja majke i bebe, upitnik pre donacije placente, upitnik 180 dana nakon donacije placente, kariotipske analize i druge testove (Prilog 4, Tabela P4.1). MSC izolovane iz razli#itih tkiva, koriste se kao jedan od $elijskih terapijskih produkata u veoma raznolikim klini#kim aplikacijama: u tretmanu GVHD, le#enju infarkta miokarda, u ortopedskim oboljenjima/nakon povreda, kao i kod autoimunskih oboljenja. Ove terapije mogu biti kombinovane sa drugim $elijskim produktima (npr. ko-transplantirane sa mati#nim $elijama hematopoeze), kombinovane sa medicinskim pomagalima (npr. oblaganje stentova), a mogu da se apliciraju lokalizovano ili sistemski. Raznoliki modaliteti primene MSC name$u dodatni nivo slo%enosti kada su u pitanju zakonsko regulatorne odredbe koje se na njih primenjuju. 104 4.2.3 Protokol za izradu i primenu anti-tumor vakcina baziranih na primeni antigen prezentuju#ih dendriti!nih #elija Karakteristike koje izdvajaju ovu vrstu protokola su: koristi se u klini#ko istra%iva#kim uslovima, uklju#uje zna#ajnu koli#inu manipulacije uz ex vivo umno%avanje $elija i njihovo kombinovanje sa drugim komponentama (eng. loading), koja nosi daleko ve$e rizike od svih prethodno izlo%enih protokola. Celokupan proces izrade mora da zadovolji uslove prema strogim zakonsko regulatornim odredbama (eng. GMP), kao i slo%enoj logistici jer se #esto obavlja u vi"e razli#itih laboratorijskih jedinica (eng. multicentre manufacturing) i primenjuje u vi"e razli#itih klini#kih centara (eng. multicentre clinical trials). !elijski produkt ove vrste mo%e biti pripremljen za jednog ili vi"e pacijenata, u zavisnosti od toga da li se primenjuje kao autologna ili alogena terapija. Priprema se kao visoko specifi#na terapija za odredjeni tip oboljenja. Primer ovakvog protokola je izrada i klini#ka primena antigen prezentuju$ih dendriti#nih $elija koje se, izmedju ostalog, koriste za pripremu antitumor vakcina. Prva istra%ivanja ovog tipa zapo#ela su 1996. godine ali se danas jo" uvek traga za adekvatnom antitumorskom strategijom, sa ili bez upotrebe dendriti#nih $elija. Ove $elije se ipak intenzivno koriste i u drugim vrstama klini#kih istra%ivanja (npr. supresije autoimunskih i alergijskih oboljenja ili aloimunske reakcije odbacivanja presadjenih tkiva) zbog svojih izuzetno zna#ajnih regulatornih i posredni#kih uloga u imunskoj reakciji. Dendriti#ne $elije se aktiviraju preuzimanjem antigena i zauzimaju specifi#no mesto u imunskom sistemu jer istovremeno predstavljaju osnovu urodjenog imunskog sistema kao i vezu sa adaptivnim imunskim odgovorom – imaju veliki imunogeni, migratorni i tolerogeni potencijal. Kao direktni u#esnici u ovim procesima, dendriti#ne $elije igraju va%nu ulogu u genezi razli#itih patolo"kih stanja – od alergijskih i autoimunskih poreme$aja do nastanka malignih tumora. Ove antigen prezentuju$e $elije su prisutne u skoro svim tkivima organizma, imaju receptore za prepoznavanje i vezivanje antigena, kao i sposobnost da obrade antigene i migriraju u periferne limfne organe (gde se nalaze nezreli T-limfociti koje imaju sposobnost da 105 Adaptirano iz: Active cellular immunotherapy monograph (www.dendreon.com). Slika 4.15: Dijagram procesa prikupljanja, obrade, primene i efekta visoko manipulisanih antigen prezentuju$ih dendritskih $elija, DC (dobijenih metodom umno%avanja i oboga$ivanja rekombinantnim antigenom) Infuzija Sipuleucel-T: Patient-Specific Product 7 Day 1 Leukapheresis Day 2-3 sipuleucel-T is manufactured Day 3-4 Patient is infused Apheresis Center Dendreon 'RFWRU¶V2IILFH COMPLETE COURSE OF THERAPY: Weeks 0, 2, 4 Dan (1) Leukafereza Sipuleucel-T: Patient-Specific Product 7 Day 1 Leukapheresis Day 2-3 sipuleucel-T is manufactured Day 3-4 Patient is infused Apheresis Center Dendreon 'RFWRU¶V2IILFH COMPLETE COURSE OF THERAPY: Weeks 0, 2, 4 Dan (2-3) Procesovanje "elija Sipuleucel-T: Patient-Specific Product 7 Day 1 Leukapheresis Day 2-3 sipuleucel-T is manufactured Day 3-4 Patient is infused Apheresis Center Dendreon 'RFWRU¶V2IILFH COMPLETE COURSE OF THERAPY: Weeks 0, 2, 4 Dan (3-4) 6 ACIs Help Immune System Target Tumor Cells and Mount a Strong Anti-Cancer Response APC takes up the antigen Recombinant Prostatic Acid Phosphatase (PAP) antigen combines with resting antigen presenting cell (APC) Fully activated, the APC is now sipuleucel-T The precise mechanism of sipuleucel-T in prostate cancer has not been established. Antigen is processed and presented on surface of the APC !"#$%&'()*!&"*' T-cells proliferate and attack cancer cells Sipuleucel-T activates T-cells in the body Active T-cell Inactive T-cell Procesovan i prezentiran antigen na povr#ini antigen- prezentuju"e "elije (DC). U potpunosti aktivirana antigen-prezentuju"a "elija (DC) postaje terapeutsko sredstvo. Rekombinantni antigen se ‘kombinuje’ sa miruju"om antigen- prezentuju"om "elijom (DC). Antig n- prezentuju"a "elija (DC) ‘uzima’ antigen. 6 ACIs Help Immune System Target Tumor Cells and Mount a Strong Anti-Cancer Response APC takes up the antigen Recombinant Prostatic Acid Phosphatase (PAP) antigen combines with resting antigen presenting cell (APC) Fully activated, the APC is now sipuleucel-T The precise mechanism of sipuleucel-T in prostate cancer has not been established. Antigen is processed and presented on surface of the APC !"#$%&'()*!&"*' T-cells proliferate and attack cancer cells Sipuleucel-T activates T-cells in the body Active T-cell Inactive T-cell 6 A Is Help Immune System Target Tumor Cells and Mount a Strong Anti-Cancer Response APC takes up the antigen Recombinant Prostatic Acid Phosphatase (PAP) antigen combines with resting antigen presenting cell (APC) Fully activated, the APC is now sipuleucel-T The precise mechanism of sipuleucel-T in prostate cancer has not been established. Antigen is processed and presented on surface of the APC !"#$%&'()*!&"*' T-cells proliferate and attack cancer cells Sipuleucel-T activates T-cells in the body Active T-cell Inactive T-cell 6 ACIs Help Immune System Target Tumor Cells and Mount a Strong Anti-Cancer Response APC takes up the antigen Recombinant Prostatic Acid Phosphatase (PAP) antigen combines with resting antigen presenting cell (APC) Fully activated, the APC is now sipuleucel-T The precise mechanism of sipuleucel-T in prostate cancer has not been established. Antigen is processed and presented on surface of the APC !"#$%&'()*!&"*' T-cells proliferate and attack cancer cells Sipuleucel-T activates T-cells in the body Active T-cell Inactive T-cell Table 1. Peripheral Blood Mononuclear Cells and Their Functions15-18 • An essential component of the antigen-specific immune response • There are 2 types of T cells—killer and helper - Killer T cells are the effector cells responsible for cell-mediated immunity - Helper T cells help B cells differentiate into plasma cells, which secrete antibodies • A subset of T cells kills tumor cells or cells infected by pathogens • Can perpetuate an initiated immune response once activated • The effector cells for antigen-specific humoral immunity • Play a supporting rol in antitumor activity by secreting antibodies, which bind to a specific a tigen, thereby marking cells expressing that antigen for destruction by a macrophage • Nonspecific lymphocytes that recognize foreign cells of many different types • Possess lytic enzymes that are an important first-line defense against newly malignant cells • One of the cell types that destroys cells that have been “tagged” by an antibody via the mechanism called antibody-dependent cell-mediated cytotoxicity (ADCC) • Antigen-presenting cells (APCs), including dendritic cells and macrophages • Function as effector cells and confer innate immunity (surround and kill microorganisms, ingest foreign material, and remove dead cells) as well as adaptive immunity (boost immune responses) • APCs can destroy cells that have been tagged by an antibody T ce lls B c el ls N at ur al k ill er c el ls M on oc yt es Monociti uklju!uju makrofage i antigen- prezentuju"e "elije (medju kojima se nalaze i dendriti!ne "elije, DC). Antigen- prezentuju"e "elije u!estvuju u uklanjanju infektivnih agenasa, stranih !estica i mrtvih "elija u organizmu. Uz to stimuli#u adaptivni imunski odgovor aktiviraju"ii T- "elije. Pod uticajem aktivirane antigen- prezentuju"e "elije (DC) vr#i se aktivacija T- "elija. T-"elije se proliferi#u i napadaju tumorske "elije. 106 Tabela 4.12: Elementi laboratorijske izrade visoko manipulisanih $elija (metodom ex vivo & loading umno%avanja i oboga$ivanja $elija proteinima/rekombinantnim antigen proteinima). Osnovne karakteristike $elijske terapije Primeri publikovanih protokola za laboratorijsku izradu i klini#ku primenu Problematika translatornog istra%iva#kog procesa Characteristics Examples of published Laboratory & Clinical Protocols Translational Research Process (‘bench-to-bedside’) Highly manipulated, heterogenous use, autologous or allogeneic use; may be combination product; systemic effects unknown; E.J. Small et al, Immunotherapy of Hormone-Refractory Prostate Cancer With Antigen-Loaded Dendritic Cells. Journal of Clinical Oncology 2000; 18(23): 3894 - 3903. J. Baggers, G. Ratzinger, J. W. Young, Dendritic Cells as Immunologic Adjuvants for the Treatment of Cancer. Journal of Clinical Oncology 2000, 18(23): 3879 – 3882. Laboratory process/ pre-clinical studies -Mobilised Leukapheresis product from patient/ donor; QC issues CD34+ or other marker enrichment using automated techniques (e.g. Isolex Device); validation parameters -Cell staining and sorting (e.g. using MoFlo sorter) ; validation parameters -Formulation and cryopreservation; QC issues Translational research process -From basic science concept – Discovery Phase -Through Engineering Phase with some of the following steps: Process development -Cell depletion techniques (yield, loss of cells, purity, feasibility of the process eg. time/ staff) -Basic cell sorting techniques or high-speed cell sorting techniques -High-speed cell sorting pros (high purity of cell population, enrichment of a subpopulation eg. gene modified cells or tumour cells for vaccine production) -High-speed cell sorting cons (single use disposables, time, cost of equipment, cost per procedure, regulatory issues eg. reagents, instruments and their compliance with device regulatory requirements, vendor-dependability, no back up option) -Use of any animal derived reagents (antibodies, peptides, proteins, media) Scale Up/ Automation -Cost and feasibility studies -Validation of Manufacturing Use of any animal derived reagents (antibodies, peptides, proteins, media) Use of antibiotics, enzymes (clinical grade reagents availability) Lot release characterisation (how to validate when no standard to compare) Validation of route of administration (how to validate with limited or no clinical data) Maintaining sterility through the process, monitoring sterility Custom GMP reagents QC Assays Release criteria/ potency assays -Characterisation of cells, -Viability, -Gram stain, - Sterility 14-day culture, -Endotoxin QA requirements and support Regulatory submission development/ maintenance 107 (eg. IND) Patents/ Intellectual Property (IP) and Commercialisation Multiple contracts and multiple supplier-network arrangements Academic support Regulatory support Funding stimuli"u). Ipak, najva%niji aspekt istra%ivanja dendriti#nih $elija le%i u procesu diskriminacije izmedju indukcije imuniteta i indukcije tolerancije. Uspostavljanje kontrole imunskog sistema nad patolo"kim procesima koji prethode razvoju oboljenja predstavlja zna#ajan cilj mnogih istra%ivanja. Maligna oboljenja predstavljaju poseban izazov jer su tumorske $elije slabi inicijatori reakcije imunskog sistema a tumorska mikrosredina mo%e direktno da inhibira imunolo"ku reaktivnost. Obzirom da su dendriti#ne $elije anga%ovane u prepoznavanju i obradi stranih i sopstvenih (eng. self) antigena, kao i da u#estvuju u balansiranju izmedju autodestrukcije i za"tite tkiva, ne iznenadjuje #injenica da se intenzivno istra%uju kao mogu$i nosioci ili medijatori antitumorskih efekata. Autologne dendriti#ne $elije mogu da se uzgajaju in vitro i da se pri tome izvr"i njihova tarnsfekcija odgovaraju$im antigenom – pre nego "to se infuzijom vrate istom pacijentu da bi indukovale odredjeni imunski odgovor u organizmu (Slika 4.15). U ovom procesu dendriti#ne $elije su prikupljene u procesu afereze (eng. leukapheresis), oboga$ene (eng. loaded) rekombinantnim proteinskim antigenom specifi#ne strukture i namene. Od tog trenutka ove $elije postaju novi biolo"ki lek koji zapo#inje svoj antitumorski efekat (Slika 4.15). Postoji niz neophodnih razmatranja i faktora koji moraju da budu zadovoljeni u izradi i primeni antigen-prezentuju$ih dendriti#nih $elija (Tabela 4.12). Primer standardne operativne procedure za njihovu izradu nalazi se u Prilogu 13. 108 4.3 Komparativna analiza 3: Struktura i primena specifi"nih organizacionih modela Organizacioni modeli koji daju podr"ku izradi i primeni $elijskih terapija/ bioterapeutika predstavljaju zna#ajan preduslov za razvoj ove vrste naprednih terapija. U ovoj analizi, rezultati su izlo%eni kroz karakteristike modela koje uklju#uju – njihova osnovna obele%ja, izvor finansiranja, prednosti i ograni#enja, dinamiku razvoja, rizike koje nose, va%ne aspekte ili preduslove za njihov dalji razvoj, poziciju u odnosu na zakonske regulative, i da li odgovaraju samo odredjenom tipu projekata za razvoj $elijskih i drugih naprednih terapija. 4.3.1 Karakteristike ne-komercijalnog organizacionog modela Ne-komercijalni model (eng. non-for-profit model) istra%iva#ke grupe za razvoj $elijskih terapija u okviru ne-komercijalne klini#ko bolni#ke institucije i/ili istra%iva#kog instituta je naju#estaliji razvojni model u datoj oblasti. On nudi mnoge prednosti kao "to je relativno malo ulaganje nov#anih sredstava (jer se oslanja na postoje$e institucijske mehanizme i kadrove) i specifi#nost pristupa za dati projekat (jer se odvija u bliskom kontaktu sa razvojnim procesom tj. originlanim bazi#nim istra%ivanjem iz kojeg proisti#e). Ipak, ovaj model nosi mnoga ograni#enja i te"ko je na osnovu njegove primene zapo#eti neke od narednih razvojnih faza projekta (npr. klini#ka istra%ivanja druge ili tre$e faze). Istovremeno, zbog nedostatka specifi#nih znanja u okviru zakonsko regulatornih odredbi #esto zanemaruje odredjene va%ne aspekte ove rane faze translatornih istra%ivanja i na taj na#in ote%ava ili #ak onemogu$ava dalji razvoj projekta kroz kasnije faze. Primena ovog modela zapo#inje/ zavr"ava se u skladu sa razvojem projekta koji podr%ava – uglavnom na nivou uvodjenja rezultata bazi#nih istra%ivanja u rane faze klini#kih istra%ivanja (translatorni proces). Pri tome ne postoji adekvatno finanisranje, projekti se oslanjaju na postoje$e organizacione strukture dok samo povremeno ostvaruju priliv finansijskih sredstava za svoj razvoj (uglavnom iz vladinih fondova za razvoj nauke) (Tabela 4.13). Ovaj organizacioni model karakteri"e relativno spora dinamika, obi#no nema ve$u dozu fleksibilnosti, bazira se 109 na kratkoro#nim potrebama jednog ili vi"e projekata i retko je visoko efikasan (Tabela 4.14). U isto vreme, mo%e da pru%i podr"ku ranim fazama klini#kih istra%iva#kih projekata i da preraste u bolje organizovan i adekvatno finansiran model (kao "to je npr. kombinovani model) – ukoliko postoji interes institucije, adekvatno dugoro#no planiranje i pravilan odabir kadrova (Tabela 4.13 i 4.14). 4.3.2 Karakteristike komercijalnog organizacionog modela Nezavisni komercijalni model, specifi#an za novonastale kompanije u privatnom sektoru (eng. biotech start-up), nalazi se na suprotnom kraju organizacionog spektra u odnosu na prethodni model. Pri tome, on ispoljava svoje Tabela 4.13: Karakterizacija organizacionih modela: uporedna analiza (1). Model Ne-komercijalni model (eng. Non-for-profit model) Komercijalni model (eng. Commercial model) Kombinovani model (eng. Hybrid/ partnership) Osnovna obele%ja Highly dependant. Non-for-profit. Mostly project-based. Starts and finishes on “as required” basis so there is no clearly defined hierarchy/ structure. Independent entity. Commercial. Defined hierarchy/ structure and reporting lines. Requires rather high start-up funding. Relatively independant entity. Proft-orientated but not profit-dependant. Rather “fluid” structure and not fully defined elements/ areas and stakeholders’ responsibilities. Izvor finansiranja Grants, fellowships, government (public) funded. Full commercial ownership and funding. Mix of private and public funding. Commercial entity start-up in the hospital/ institute only if/ when one of the products or technology becomes financially viable. Profit-orientated only to certain extent (as uses other financial resources eg research grants, donations, fundraising). Prednosti koje nudi Ideal for early stage R & D projects, pilot projects or clinical trial son a small scale. Use of existing supporting structures, so minimal funding required. Fast decision making. Reactive. Efficient. Offers great range of possibilities and keeps number of options “open” for further development. Promotes number of small projects that may be become viable (or not). Using the best of both worlds – science/ clinical expertise from the hospital/ institute and business knowledge from the commercial partner. 110 osnovne karakteristike kao "to su, pre svega, da je orijentisan ka ostvarivanju profita, finansiran od strane privatnih lica ili korporacija, #esto visoko efikasan, fleksibilan i nudi adekvatnu ekspertizu u svojoj oblasti poslovanja (eng. use of ‘know-how’). Inicijalno zahteva zna#ajna finansijska ulaganja, visoko je podlo%an ekonomskim uticajima (Tabela 4.13). (esto ima centralizovanu, hijerarhijsku strukturu i jak zakonsko regulatorni fokus u svom pristupu (Table 4.14). Tabela 4.14: Karakterizacija organizacionih modela: uporedna analiza (2). Model Ne-komercijalni model (eng. Non-for-profit model) Komercijalni model (eng. Commercial model) Kombinovani model (eng. Hybrid/ partnership) Ograni#enja koja ima Slow decision making. Non-reactive. Rarely efficient. Sub-contractual agreements required (i.e. “know-how” & expertise in area of quality design and production management) 50-50 partnership of a business unit/ entity with the institute/ hospital. Relying on existing hospital/ institute mechanisms and expertise in both science/ clinical, business domain. Dinamika Requires low start-up funding. Slow develoment. Requires medium to high start-up funding. Fast-paced development. Requires low to medium to start-up funding. Keeping number of ongoing projects on lower side due to limited resources and in order to keep projects’ feasibility. Projekti Mostly clincial, clinical research or early stage R&D projects. All for-profit/ comercial projects. Versatile projects, from early stage to comercial. Rizici koje nosi Easy to lose funding. Priority-driven from the executive level. Usually not very supported by the institution (hospital or institute). Can be complex and unreliable. Profitability depends on many factors. Financial viabilty. High-risk for venture capitalists to invest. Sometimes can be very profit-orientated (as the business partner entirely depends on the financial success). Certain level of ambiguity and uncertainty present until the “take-off” (if sucessfull). Complexity. Va%ni aspekti ili preduslovi za dalji razvoj Executive support. Fundng. New contracts. Keeping efficency & profitability. Partnerships. Networking & partnerships (lat. quid pro quo). Open approach for partnerships and service based on the sound financial impact (“fee for service”). Pozicija u odnosu na zakonske regulative i njihovu primenu Not very strong regulatory compliance / quality design focus. Centralised decision making & strong regulatory compliance / quality design focus. Centralised decision making & strong regulatory compliance / quality design focus. Da li odgovara samo odredjenom tipu projekata za razvoj $elijskih i drugih naprednih terapija? Yes, only internal ‘in-house’ projects. No. No. 111 4.3.3 Karakteristike kombinovanog organizacionog modela Kombinovani model (eng. hybrid model) je baziran na komercijalnim principima, a formiran je u okviru slo%enog specijalisti#kog klini#ko bolni#kog kompleksa ili klini#ke institucije. Uklju#uje, na primer, formiranje nezavisnog centra za izradu $elijskih terapija (eng. fee for service) u okviru tradicionalne klini#ke institucije koja zadr%ava deo vlasni"tva nad novonastalom organizacijom (Tabela 4.13). Pri tome se formira odredjena vrsta partnerskog modela ili pridru%enog modela (koji se jednim imenom mo%e nazvati kombinovanim modelom) – izmedju klini#kog centra i privatnih investitora/ kompanije uz u#e"$e partnerskog finansiranja. Bez obzira "to je finansijski kapital me"ovit, postoji strogo definisana organizaciona struktura, centralizovano dono"enje odluka i visoka efikasnost. Akcenat se stavlja na dobro osmi"ljen poslovni model, jak zakonsko regulatorni fokus i dugoro#no planiranje uz ostvarivanje adekvatnih partnerskih sporazuma za saradnju. Model se zasniva na profitabilnosti, na visokom nivou profesionalizma, strate"kom planiranju i dugoro#nim projektima. Ovakav organizacioni model obi#no nastaje iz ne-komercijalnog modela koji je postao dovoljno oformljen i/ili je ostvario ve$u finansijsku dobit na nekom od svojih uspe"nih projekata (Tabela 4.14). Nudi veliki broj mogu$nosti za dalji razvoj, #esto podr%ava ve$i broj manjih projekata (posebno u ranim fazama svog razvoja) i mo%e da preraste u ozbiljan komercijalni entitet. Pored osnovnih prednosti koje nude komercijalni ili kombinovani model, usled nedostatka sredstava u ranim fazama razvoja ve$ina projekata polazi iz ne- komercijalnih modela da bi zatim pro"la kroz neke od tipi#nih razvojnih faza koje su obja"njene u daljem tekstu (Quinn & Cameron, 1983; Maslow, 1998; Weick & Quinn, 1999). U ovom procesu ‘sazrevanja’ organizacionih modela #esto dolazi do promene (eng. shift) iz prve razvojne faze tzv. dobrovoljnog udru$ivanja znanja i ve!tina u okviru projekta (eng. volunteer-based project), koji su usmereni na samo jedan odredjeni projekat i #ija glavna obele%ja predstavljaju osnivanje (eng. foundation) i saradnja medju u#esnicima (eng. collaboration) – u drugu fazu koju karakteri"e partnerski odnos. Partnerski odnos karakteri"u jedinstvo pristupa (eng. synergy) i tendencija specijalizacije (gde svaki partner, pojedinac ili organizaciona jedinica, 112 obavlja svoj deo projekta). Pri tome se ne razmenjuju nov#ana sredstva medju partnerima, a tro"kovi se uglavnom ravnopravno dele. U tre$oj fazi, koja sledi partnerski odnos, formira se polje zajedni#kog upravljanja projektom (eng. shared governance), pri #emu se ostvaruje odredjeno pozicioniranje (eng. positioning) svakog od u#esnika (bilo da su u pitanju pojedinci, organizacione jedinice ili #itave organizacije/institucije) i zapo#inje rast (eng. growth), odnosno po#inje da se ostvaruje manja finansijska dobit (naju#estaliji primer su projekti finansirani od strane vladinih agencija ili drugih eksternih tela kao "to su regionalni fondovi razvoja). U zavr"noj, #etvrtoj fazi, formira se nezavisna organizaciona ili institucionalna jedinica (eng. independent entity) koja mo%e biti u vidu nove organizacione jedinice u okviru iste institucije (eng. department) ili potpuno nezavisne komercijalne kompanije (eng. biotech start-up). Ovu fazu karakteri"u tendencija ka ostvarivanju sopstvenog identiteta (eng. recognition) i ka ostvarivanju zarade (eng. profit). Osnovno obele%je celokupnog razvojnog procesa je 'pomeraj' (eng. shift) – iz stanja bez strukture (eng. volunteer-based stage) u stanje bez inovacije (eng. institutional stage). Izmedju njih se nalazi #itav spektar prelaznih 'stanja' u zavisnosti od razvojne faze i specifi#nih uslova za svaki projekat. Va%no je napomenuti da faze mogu da se preklapaju, da se odredjeni projekti kre$u izmedju njih, pri tome se kra$e ili du%e zadr%avaju$i u odredjenoj fazi. Neki od ovih projekata nikada ne predju u narednu fazu razvoja ve$ ostanu na istom nivou neko vreme, ugase se ili ih nadomeste novi, ve$i projekti. Takav poredak uti#e na ishode mnogih po#etnih translatornih istra%ivanja u oblasti $elijskih odnosno naprednih terapija, pri #emu se najte%i zadatak nalazi u njihovoj selekciji – "ta podr%ati kroz organizaciono finansiranje ili kroz razvojne fondove? – i u modalitetima njihovog razvoja – kako pru%iti adekvatnu podr"ku? Ovo je danas prepoznato kao najve$a prepreka izmedju translacije rezultata bazi#nih istra%ivanja u klini#ke istra%iva#ke projekte, koja se u literaturi naziva eng. translational research gap (Woolf, 2008; Marincola, 2003; Kong & Segre, 2010). Odgovor na gore navedena pitanja nije jednostavan niti jednozna#an. Verovatno se nalazi u sistemskom pristupu, odabiru projekata od interesa i ustanovljavanju jednog strate"kog centra u zemlji ili regionu, koji bi procenjivao i podr%avao projekte od interesa. Na taj na#in bi se omogu$io translatorni razvoj i br%a 113 klini#ka primena novih terapijskih pristupa, posebno u oblasti novih naprednih terapija. 114 4.4 Komparativna analiza 4: Procena percepcije i problematike oblasti Kvalitativna metodologija intervjua obezbedjuje obilje podataka. Zbog toga je ovakvim pristupom ostvaren uvid u prakti#nu problematiku oblasti razvoja $elijskih terapija/bioterapeutika – uz iskustva stru#njaka koji u tom procesu u#estvuju, njihova mi"ljenja i percepciju celokupne oblasti. Pri tome je izvr"ena analiza sadr%aja i tematska analiza transkripta 24 intervjua, a zatim su identifikovani pojmovi utemeljeni u podacima/ izvodima iz transkripata intervjua. Rezultati tematske i fenomenolo"ke analize uklju#uju pet segmenata (1 -5) koji se baziraju na 24 intervjua obavljena sa stru#njacima iz Evrope, SAD-a i Australije. Rezultati su prezentovani na slede$i na#in: ! Segmenti podataka (1): Poslovni i finansijski aspekti uz osnovna razmatranja. ! Segmenti podataka (2): Uspostavljanje saradnje sa partnerima i druga razvojna problematika. ! Segmenti podataka (3): Primena zakonsko regulatornih odredbi i percepcija njihovog razvoja. ! Segmenti podataka (4): Osnovne teme i kategorije. ! Segmenti podataka (5): Utemeljenje rezultata u podacima. Na osnovu pristupa analizi podataka prikupljenih kroz intervjue (Tabela 3.6) rezultati su uvek izlo%eni u okviru dve celine koje manifestuju podatke sakupljene na osnovu – poznavanja oblasti kroz znanje, praksu i iskustvo (eng. knowledge, practice & experience) ili mi!ljenja u"esnika formirana kroz posmatranje i percepciju oblasti (eng. view, perception & opinion). Na osnovu brojnih utisaka i zapa%anja u#esnika, preovladavalo je mi"ljenje da je celokupna oblast jo" uvek u veoma ranim fazama razvoja, da postoji izuzetan stepen neizvesnosti o daljim razvojnim pravcima ("ta i kako razvijati? gde usmeriti najvi"e sredstava i napora?) i da su sve dosada"nje procene vezane za razvoj oblasti uglavnom bile pogre"ne. 115 4.4.1 Segmenti podataka (1): Poslovni i finansijski aspekti uz osnovna razmatranja (eng. Business Models, Funding, Strategic Decision Making & Main Considerations) Analiza sadr%aja mo%e da se obavi na kvalitativnom nivou, kao "to je obavljeno kategorizacijom u segmente podataka, ili kvantitativno. Kvantitativna analiza sadr%aja identifikovala je u#estalost odredjenih termina i fraza kao indikaciju percepcije oblasti. Na osnovu analize transkripta intervjua, najve$i broj u#esnika u studiji pomenuo je ‘quality’ u razli#itim kontekstima (n=19) pri tome nagla"avaju$i zna#aj kvaliteta u razmatranju celokupne oblasti. Termin ‘regulatory’ ili ‘regulations’ pomenulo je ne"to manje u#esnika (n=15) dok je pribli%no 50% njih u nekom delu svog intervjua upotrebilo ‘challenge’ (n=13) i ‘funds’ ili ‘funding’ (n=12). Ovo ukazuje na zna#aj koji su finansijkom aspektu i primeni regulatornih standarda dali intervjuisani stru#njaci. U istoj meri, u pribli%no 50% intervjua, se pojavljuju u razli#itim kontekstima: ‘potential’ (n=11) i ‘cost’, ‘expensive’ ili ‘costly’ (n=10). Opseg i u#estalost termina ‘potential’, koji je upotrebljen u okviru slede$ih fraza: ‘potential applications’, ‘potential of therapies’, ‘potential collaboration’ or ‘potential development’ – u odredjenoj meri ukazuje na percepciju neizvesnosti koja okru%uje celokupnu oblast razvoja $elijskih terapija. Sli#no tome, "irok spektar upotrebljenih fraza koje nose finansijsku konotaciju, ‘cost’, ‘costly’, ‘expensive’, ‘cost of manufacturing’, ‘costly exercise’, ‘regulatory compliance cost’, ‘expensive technology’ i ‘cost of goods’ – ukazuje na finansijsku neizvesnost i zna#aj koji je od strane stru#njaka pridodat finansijskim ulaganjima u ovu oblast. Za razliku od toga, samo manji broj u#esnika je pomenuo termine kao "to su ‘promise’ (n=4), ‘uncertainty’ ili ‘unknown’ (n=3). Rezultati dalje analize sadr%aja nalaze se u okviru tema i kategorija (Tabela 4.21) dok su specifi#ni rezultati vezani za poslovne i finansijske aspekte (uz osnovna razmatranja i izazove identifikovane od strane u#esnika) prikazani u Tabeli 4.15 i 4.16. 116 Tabela 4.15: Rezultati vezani za poslovne i finansijske aspekte uz osnovna razmatranja i izazove identifikovane od strane u#esnika (eng. Business Models & Funding; Strategic Decision Making). B us in es s M od el & F un di ng , St ra te gi c D ec is io n M ak in g KNOWLEDGE, PRACTICE & EXPERIENCE: - “staff switch between projects, portability and flexibility of personnel; process development, consulting & facility design” [CTE01]. - “must do’s; black and white delineation; small adjustments as needed; multidisciplinary skill; strategically ( at the very top of organisation ( an inherent understanding of the environment we work in and therefore our structures need to be built up around that” [CTE03]. - “business model based on the development of new interventions through basic science, prototype development, high quality clinical data and using good clinical need and cost economic data to provide an attractive platform for potential license and/or trade sale or interested parties” [CTE05]. - “where [you have] lots of external funding and almost commercially orientated group which is seeking is fund itself through exploitation of its own capabilities, you may get a group that is set up and has built a facility, it has capabilities and it offers these to the sector on a for-profit basis. So if you’re a group in a university or somewhere you could take your new little cell line and you could, if you had the money, get it expanded and you could get clinical trials going and all that because the centre itself had the full capability of doing it. ( Others may well be more like not-for-profit university based systems, which tend to hold out the olive branch to similar not-for-profit groups of researchers and try to do things collaboratively and collectively and share the costs. So a not-for-profit versus a for-profit [business] model is essentially [here at the moment] and there’re obviously little permutations in between” [CTE14]. - “there’s the most popular ‘let’s just wing it and hope for the best’ strategy ( there’s a real level of over confidence. People who are in senior management tend to be extremely confident and not generally particularly detailed orientated ( let’s use slogans and say things that sound good and have absolutely no substance. I have seen situations where you have these very over confident people who don’t want to talk about risk management because they truly believe it won’t happen to them, hey truly believe the clinical trial is going to be successful, it will enrol early, everyone will be great and of course the regulator will say, sure you can market this early, it is the most popular approach” [CTE23]. Oznake za u#esnike u istra%ivanju: CTE01 - CTE24. Poslovni sistemi i izvori finansiranja koji su diskutovani od strane u#esnika studije, bili su raznoliki – od malih projekata do ogromnih korporacijskih struktura. Ipak, kada su razmatrani organizacioni modeli koji se naj#e"$e pojavljuju u oblasti, ve$ina u#esnika je nagla"avala neophodnost strukturnih, organizacionih i infrastrukturnih resursa koji su neophodni u oblasti. Takodje je nagla"ena #esta pojava da bazi#ni istra%iva#ki projekti prerano zapo#inju svoju komercijalnu aktivnost odnosno da ve$ina projekata prerano ulazi u komercijalizaciju, a da ne postoje adekvatni sistemi za podr"ku (na nivou dr%avnih struktura ili u okviru institucija u kojima se ovo de"ava). Kao osnovni faktor uspeha u ovim modelima, pored dovoljno finansijskih sredstava, naveden je regulatorni fokus i znanja u ovoj oblasti. Zajedni#ki stav koji je dominirao medju u#esnicima je bio nedostatak finansijskih sredstava za translatorna istra%ivanja, dizajn i sprovodjenje klini#kih istra%ivanja (posebno rane 117 faze) kao i transfer tehnologije izmedju istra%iva#kih i komercijalnih laboratorija (tipa ‘fee for service’). Tabela 4.16: Rezultati vezani za poslovne i finansijske aspekte uz osnovna razmatranja i izazove identifikovane od strane u#esnika (eng. Main Considerations in Relation to Project or Organisation). M ai n C on si de ra tio ns (o rg an is at io n/ p ro je ct ) VIEW, PERCEPTION & OPINION: - “we have no single product that's the most important. ( one, which is reimbursed, I believe not only provides the strong commercial value but imposes a manufacturing discipline on the facility. It instills and reinforces high levels of professionalism and actually puts appropriate ‘stress’ and pressure on the facility and the personnel” [CTE01]. - “a lot of people would try and set up a classic commercial organisation and then( find that the commercial organisation runs at odds to the [regulatory] compliance requirement” [CTE03]. - “one is regulatory hurdle, second is the commercial interaction, third is the market needs( you’ve got to understand these three right at the beginning [when developing] cellular product or device - you need to know these three components before you actually [start] development process” [CTE04]. - “there is always longer way to the overall exploitation than expected( from [the] idea to the common use, you need [for example] 15 years and you still don't know which of these products will be ‘the product’ ” [CTE11]. - “finances are the biggest [issue] for this sector, closely followed by changing the culture from a clinical service to a manufacturing service and ( changing the structure from having the doctor who's looking after the patient versus the doctor who signs off the release” [CTE12]. - “some of the gains from these sorts of therapies are going to be much longer term than we can imagine now” [CTE19]. - “the field is definitely growing and it's taking on maturity ( when you begin to see pharma companies move into stem cells, it's a realisation that there is an acceptance that, hey there's something here ( still a very, very, very nascent technology and industry” [CTE21]. - “there’s so much going on in this field and there’s been so many things put in place now that new therapies that are just coming in now will have a much more accelerated pace through those painful early first steps. Simply because there’s systems that they [industry] can use to get going” [CTE23]. - “determining governance structure in [an overarching] organisation; developing enterprise & commercial ‘know-how’ “ [CTE01]. - “resources; competition for peer-reviewed grants is extremely intense; in human clinical trials [to prove] whether there are any therapeutic benefits” [CTE02]. - “establishing a reputation, identifying those organisations, individuals or researchers who were prepared to embrace the concept of outsourcing” [CTE03]. - “funding” [CTE04]. - “the ongoing financial development of the company” [CTE05]. - “the management spends a disproportional amount of time raising money instead of managing day-to-day operation of the company. So raising money is the biggest one. The other challenge is [finding] the right models” [CTE06]. - “you have fixed costs that will run you high, it could be about 60 or 65 per cent fixed cost... have adequate, sufficient amount of volume to compensate, to leverage of those fixed costs” [CTE08]. - “education in a very broad sense ( there needs to be education of all the different participants in the sector. That being the manufacturing, the researchers and investigators, the clinicians, the regulators, and investors because they want to see their money come back quickly” [CTE19]. - “they [companies] might make a mistake but they’re only going to make it once, the next time around things will be done correctly ( we have people who are getting much more savvy about what happens after the product leaves phase I and how do you submit an IND to the FDA and move forward with that process; things have been slow, there’s been a lot of bumps in the road but things will accelerate very rapidly over the next ten years” [CTE23]. Oznake za u#esnike u istra%ivanju: CTE01 - CTE24. 118 4.4.2 Segmenti podataka (2): Uspostavljanje saradnje sa partnerima i druga razvojna problematika (eng. Partnerships, Main Challenges for the Industry, Science, Technology, Tech Transfer & Up Scaling) Ve$i deo u#esnika naglasio je zna#aj partnerstva i uspostavljanja dugoro#nih partnerskih sporazuma radi: upotrebe infrastrukturue (oprema, tehnolologija), pristupa novim nau#nim saznanjima (kroz saradnju sa istra%iva#kim institutima), ili pristupa pacijentima (kroz saradnju sa klini#kim centrima). Komercijalne kompanije su iz takvih sporazuma dobijale pristup novim nau#no tehnolo"kim saznanjima, dok su manji projekti koji se odvijaju u okviru istra%iva#kih institucija, na taj na#in ostvarivali vi"estruku korist (u ostvarivanju novih finansijskih sporazuma ili razmeni iskustava, kadrova, za potrebe publikovanja i sli#no). Od svih vrsta partnerskih sporazuma, najmanje zna#aja je bilo pridodato dr%avnim strukturama, a zna#aj saradnje sa regulatornim agencijama je izdvojen u svetlu stalnog razvoja i uslo%njavanja regulatornih odredbi i potrebe za njihovom implementacijom. Medju najve$im izazovima navedeni su: nedostatak finansijskih sredstava, dugoro#no strate"ko planiranje, slo%enost tehnologija i regulatornih odredbi koje se na njih odnose, bezbedna i efikasna translacija iz bazi#ne nauke u klini#ku primenu, nedovoljno sredstava za komercijalizaciju. Osim toga, nedovoljno mehanizama koji bi ubrzali proces ostvarivanja prihoda od novih biolo"kih lekova na nivou zdravstvene za"tite (eng. reimbursement) i kompetitivnost u projektima finansiranim od strane dr%ave (eng. grants). Neki od u#esnika su pomenuli uspostavljanje odgovaraju$eg profila i reputacije za novonastale kompanije, razvoj novih mehanizama u postoje$im istra%iva#kim institucijama ili klini#kim centrima (logistika neophodna za razvoj novih terapijskih pristupa), obrazovanje kadra, "ire javnosti i prate$e eti#ke dileme u oblasti. Ostali rezultati vezani za uspostavljanje saradnje sa partnerima, tehnolo"ka i druga razmatranja vezana za razvoj proizvodnje bioterapeutika/ $elijskih terapija prikazani su u Tabeli 4.17 i 4.18, kao i u Prilogu 17. 119 Tabela 4.17: Rezultati vezani za uspostavljanje saradnje sa partnerima i druga razmatranja vezana za razvoj proizvodnje bioterapeutika/ $elijskih terapija (eng. Partnerships & Main Challenges in Relation to Industry/ Field). Pa rt ne rs hi ps & M ai n C ha lle ng es (in du st ry / f ie ld ) KNOWLEDGE, PRACTICE & EXPERIENCE: - “we don't want the relationship [with clients] to be over when the commercial contract phases over; we stay in contact and we encourage them to do the same and that results in a lot of repeat work and a strength in a deepening relationship” [CTE01]. -“ partners and alliances in manufacturing and in scientific collaborations; co- investigators on grants, shared and co-supervised PhD students, data sharing, data analysis, discussion and agreement on experimental design” [CTE02]. - “constant dialog, openness” [CTE03] -“lectures, meetings, networking” [CTE04] - “many groups may not appreciate how important it is to them but in time they will reflect on the fact that they would never have got as far without those collaborations. It may well be that we have to form a new type of organisation [e.g. virtual organisations] to keep this stuff going and to keep those innovative ideas flowing and new products coming on stream in a setting where resources are constrained, both human resources and financial resources... we can try and shed some of that rugged individualism ego stuff in favour of a more collaborative approach” [CTE14]. - “it's about those two groups [researchers and clinicians] acting as one to help to influence the regulators in terms of what's realistic e.g. if we are able to provide therapies for patients, then there needs to be consideration about the difficulties of developing these therapies[CTE19]. -this is a field that is very high profile and very intriguing to people so people from all disciplines are coming into cell therapy [industry] ( we have process engineers who maybe last year were working on something for the auto industry or the airline industry but now they are applying it to cell therapy or material engineers who are figuring out how to make new scaffolding that we can stick in joints” [CTE23]. VIEW, PERCEPTION & OPINION: - “competition for peer-reviewed grants is extremely intense; in human clinical trials [to prove] whether there are any therapeutic benefits” [CTE02]. - “the speed to market is an issue because the trials take so long; meeting the regulatory inherent safety requirements” [CTE03]. - “time to deliver their product, not enough financial support from the commercial side” [CTE04]. - “some revenue strands and early commercialisation [is present] but the ongoing funding of the companies to a breakeven point or to a point where they can last until the trade sales of their technology; trying to get reimbursement for cell therapy( the second biggest challenge following the regulatory pathways is the reimbursement pathways” [CTE05]. - “the biggest challenges have to do with both the technology and the regulatory environment. ( there is not sufficient, or adequate, equipment to do the work. ( The second [challenge] is the regulatory environment, where the regulations are very strict. ( use the pharmaceutical standards in order to align with cell therapy and it does not work” [CTE08]. - “the biggest challenge is, let's say, to profile themselves enough” [CTE11]. - “the challenge is the safe and effective translation - so the delivery on the promise, that's the challenge; we're making a great deal of progress using stem cells to understand basic biology, to understand the enormous power of these cells( but we're yet to be able to harness that potential” [CTE20]. - “you might have a regulation that on paper sounds crystal clear but when you’re actually the sponsor and you’re trying to comply with that regulation you run into a lot of things that just don’t make any sense whatsoever ( you have to go back to the regulator and start to negotiate what you would like to do, give your scientific and clinical rationale and then get agreement. Eventually that feeds back into the high-level executive decision making. But it’s not there routinely yet” [CTE23]. Oznake za u#esnike u istra%ivanju: CTE01 - CTE24. 120 Tabela 4.18: Rezultati vezani za tehnolo"ka, nau#na i druga razvojna razmatranja vezana za proizvodnju bioterapeutika/ $elijskih terapija (eng. Science, Technology, Tech Transfer & Up Scaling) uz generalna zapa%anja (eng. About Industry). Sc ie nc e, T ec hn ol og y, Te ch T ra ns fe r, U p Sc al in g & A bo ut In du st ry KNOWLEDGE, PRACTICE & EXPERIENCE: - “cell culture extension incl. basic techniques, automation, purification, in vivo human imaging (labeling, determining presence and distribution of cells); initial transfer to demonstrate comparable outcomes, a process development plan [with] appropriate milestones, full scale engineering runs where we then typically write comparability studies to show that we've got a comparable product at the end of the process” [CTE01]. - “cell culture, plans for bioreactor; safety driven technology; use of same reagents in pre-clinical and clinical production” [CTE02]. - “we work with device and the automation manufacturers; our staff maintain a detailed working knowledge; we work closely with the regulators; we work closely with the manufacturers, such that we can understand the integration; that's part of our value-add [and] we deliver that to our clients” [CTE03]. - “the technology is really about cells in defined conditions” [CTE06]. - “technology transfer, also similar to new product development, is something that only recently dawned on people that it is a process, tech transfer. Of course the commercial sector could have helped here really early on if people had been aware that there is this thing called tech transfer. I mean it’s only relatively - probably five years’ old in the language. People stumbled upon it” [CTE14]. - “typically transfers tend to be small scale to slightly less small scale” [CTE16]. - “cell therapy, whether it’s autologous or allogeneic, involves a service component which small molecules do not whatsoever [and] pharma is not comfortable with that. They are not equipped to do it and over the past 15 to 20 years there’s been increasingly high barriers put between the companies [e.g. drug manufacturers] and patients, also between [manufacturing] companies and practising physicians ( that service component (interaction with the actual human being whether it’s the donor of the source tissue or the recipient of the final product) is very difficult for big pharma to manage” [CTE23]. VIEW, PERCEPTION & OPINION: - “tremendous promise [but] unrealized until 3-5 years ago; rapid growth, improved revenue and returns, more profitable companies; therapies not reimbursable” [CTE01]. - “enormous stem cell potential” [CTE02]. - “the market for cellular therapies and tissue replacement is getting more optimistic every year” [CTE03]. - “there's still an enormous amount of opportunities” [CTE05]. - “cell therapy application will be developed within the next decade [and] we will have meaningful treatments [with] stem cell therapy (derived from embryonic stem cells)” [CTE06]. - “it’s still in its infancy stage, despite the fact that we have been working at this for [over] 16 years” [CTE08]. - “high expectations, not developed yet; it's just starting to profile itself” [CTE11]. - “not too many cell products in the market( more academy sponsors doing early studies, without thinking through the study design (many do not make beyond this stage). Industry sponsors are not interested in phase I or II, only phase III and have business models tailored accordingly; there is a disconnect from pre-phase I to phase III” [CTE17]. - “the reality of cell therapy is that you have a technology that allows you to actually repair diseased tissues rather than just treating the symptoms of the disease with drugs. You actually can undo some of the damage ( the technology and the potential rewards are huge. But it's just going to take time and we just have to be careful and cautious but work very diligently to make it a reality” [CTE21]. - “it still has a long way to go( in terms of the level of support and investment. The pharmaceutical industry and drug development is a massive machine, there’s a huge amount of money and human effort put into it. The cell therapy industry so far is off in a corner struggling, very poorly funded cousin” [CTE023]. Oznake za u#esnike u istra%ivanju: CTE01 - CTE24. 121 4.4.3 Segmenti podataka (3): Primena zakonsko regulatornih odredbi i percepcija njihovog razvoja (eng. Regulatory Compliance & Regulatory Landscape) Zna#ajni rezultati analize transkripta intervjua koji su vezani za primenu zakonsko regulatornih odredbi (eng. Regulatory Compliance) prikazani su u Tabeli 4.19, a za njihov razvoj u "irem smislu (eng. Regulatory Landscape) u Tabeli 4.20. Razvojni pristupi kao "to su obuka osoblja, usvajanje novih tehnologija ili novih ideja (eng. Staff Training & Access to New People, New Technology, New Ideas) kao i zna#ajne pouke nastale u dosada"njem razvoju (Important Lessons Learned) predstavljene su u Prilogu 17. Tabela 4.19: Rezultati vezani za primenu zakonsko regulatornih odredbi (eng. Regulatory Compliance). KNOWLEDGE, PRACTICE & EXPERIENCE: On Regulatory Compliance - “multiple markets [that require] thorough understanding [of regulations] nationally and internationally, trusted partners & local presence, local regulatory knowledge; at a strategic level , discuss the threats and opportunities of various proposed changes to the regulatory framework; work with all clients , get some very clear messages across to the regulator” [CTE01]. - “strategically we've made the decision to accept the structure that's needed to be compliant, and then we work the operational things around that structure” [CTE03]. - “[strategically ] we will always look at the regulatory environment first ( one of the first things we looked at was not just the regulatory environment here (but the regulatory environment across the globe” [CTE05]. - “basically every group comes to grips with [the] GMP. Some try and do it on their own (through trial and error, struggle and at great cost eventually get there) [while] others recognise early on that they need some assistance and they buy in the help. And the whole intention is to try and get them on their learning curve and evolving rapidly into basically a system that is well versed in basically documenting, authoring, validating, capturing data, analysing it, reporting back, doing whatever changes are necessary, auditing and so on that makes up the viable quality system( Over the last five years it’s suddenly become apparent to everybody that this as a parallel organisation - if you’re going to be successful in the space of cell therapies and they’re inexplicably tied up together (understanding of what GMP’s about) and the need for it” [CTE14]. - “incorporating regulatory requirements at the strategic decision making level tends to be hard; industry tends to ‘reinvent the wheel’ and FDA struggles with the new concepts; combination products (e.g. cells on scaffolds) are particularly challenging” [CTE17]. - “one of the major differences [between regulations] is the barrier to entry. To get into a very early stage clinical trial in Australia it’s much lower than in the US ( traditionally in Australia if a physician one and two run a protocol in cell therapy it was a very simple straightforward process of getting local IRB approval and moving forward with it. In the US you still needed to get that federal approval from the FDA in order to implement that kind of trial – that traditionally has been one of the biggest differences[CTE23]. Oznake za u#esnike u istra%ivanju: CTE01 - CTE24. 122 Tabela 4.20: Rezultati vezani za primenu zakonsko regulatornih odredbi i njihov razvoj u "irem smislu (eng. Regulatory Landscape). VIEW, PERCEPTION & OPINION: On Regulatory Landscape -regulatory uncertainty[CTE01]. -regulatory requirements are ( rigorous and expensive but safety is paramount[CTE02]. -it's an absolute challenge and it's a major problem; for example, the Europeans have determined that cells and tissues are a medicinal( but it is what it is and we're going to have to live with it. In Australia they couldn't work out whether it was a medicinal or a device so they've formed a new division of biologics[CTE03]. -cellular therapies have to separate from the medical devices[CT04]. -in a micro sense some of them [regulatory requirements] just don't make any sense at all; and frankly they're not based on strong scientific evidence. What we're asked to continually base our approach on is strong scientific evidence, yet the regulatory environment is often not based on strong scientific evidence[CTE05]. -in the regulatory environment, regulations are increasing, getting more difficult, and that will continue to happen[CTE08]. -they [regulators] worked kind of hard to evolve a regulatory framework that can address the protection of the public health( they tried to engage the sector in the evolution and it’s been a joint learning exercise from a regulator point of view and from the sector’s point of view about what the regulator is there to do[CTE14]. -they [regulatory bodies] have come a long way, they have more clarity around the expectations in what you need to do for most part, what you don’t need to do, more importantly( from the early days of cell therapy, the regulation has caught up with cell therapy. It took FDA several years to pick up and give exemption, so it’s come a long way. The flipside is about maturity – you still don’t get the kind of the guidance you might need on more mature products just because of the variation of product and lack of precedent[CTE16]. -EMA is trying to make a lot of people happy, it never works. The member states, the way things are divided up between the EMA versus the members state is very difficult to negotiate if you’re not within Europe; people can deal with the TGA and FDA externally I think if somebody wants to do a development programme anywhere in one of the European member states they need to have a location there or they can interact with the local regulator. The reason for that is that the rules are quite different in very fundamental ways from country to country and I think coming form Australia or coming from the US we like to think that there’s this one thing called Europe and you coming from Europe know that that is not true[CTE23]. Oznake za u#esnike u istra%ivanju: CTE01 - CTE24. 4.4.4 Segmenti podataka (4): Osnovne teme i kategorije (eng. Frequent themes and categories) Tematskom analizom je identifikovano 16 tema i 19 kategorija koje su dominirale u intervjuima, a koje se mogu svrstati u tri osnovne teme (Tabela 4.21). Osnovne teme uklju#uju: dinamiku nau"no tehnolo!kog razvoja; poslovne modele uz operativna i finansijska razmatranja; kao i stalno usavr!avanje i uslo$njavanje zakonskih regulativa u oblasti. 123 Tabela 4.21: Osnovne teme i kategorije dobijene analizom intervjua. U okviru prve osnovne teme koja se odnosi na dinamiku nau"no tehnolo!kog razvoja, u#esnici studije su se uglavnom fokusirali na teme kao "to su: tipovi $elijskih produkata, modeli njihovog razvoja (usmereni na odredjeno oboljenje/ primenu ili na tip $elijske terapije i/ili tehnologije koju koriste), razudjenost istra%iva#kih napora i ulaganja (u smislu broja i vrste projekata) i izvesna nepovezanost koja dominira u tome (da li se zaista mo%e propratiti toliki broj terapeutskih indikacija na adekvatnom nivou?), novi materijali/ biomaterijali i upotreba automatizacije (npr. primena bioreaktora, biorobotike i koliko smo danas jo" uvek daleko od takvih re"enja) (Tabela 4.21). PRINCIPAL THEMES: ( n=3 ) DYNAMICS OF SCIENCE & TECHNOLOGY DEVELOPMENT REGULATORY IMPACT & CONTINUAL PROGRESS OF REGULATORY REQUIREMENTS BUSINESS MODELS, OPERATIONAL & FUNDING CONSIDERATIONS Themes ( n=16 ) Type of product Disease- centered Hospital- based Contract Manufacturing Segregated focus Technology- centered Focus on a global regulatory landscape University- based Pharmaceutical Companies New materials/ biomaterials Automation (e.g. robotics, bioreactors) Attain local knowledge of regulations New Biotech Startup Global Economic Uncertainty Cell Banking Size of the Market Categories ( n=19 ) Somatic cells vs. Induced pluripotent cells (iPS) Autologous vs. Allogeneic Meet current regulations vs. Establish dialogue with a regulatory agency Private vs. Public Develop in-house vs. mergers & acquisitions Virtual Models vs. Real Partnership s Public engagement High throughput screening Streamline and simplify requirements Preclinical Studies & Testing services Skilled workforce Forecasting Cell expansion technologies Embryonic cells Process vs. Facility Project-based compliance vs. Systemic Compliance Segregated focus GxP compliance & capabilities Infrastructur e 124 Jo" specifi#nije oblasti interesa (kategorije) ovde su uklju#ivale: izbor $elijske populacije od interesa (tzv. somati#ne/ adultne mati#ne $elije vs. indukovane pluripotentne mati#ne $elije, iPS), tip produkata od interesa (autologne vs. alogene terapije), novi pristupi i tehnologije za ‘high throughput screening’ i ‘imaging’, kao i u odredjenoj meri, interes za razumevanje problematike ove oblasti u "iroj javnosti – gde se uticajem medija (i usled drugih faktora) kreiraju nerealna o#ekivanja ili pru%aju mogu$nosti za ne-eti#ke pristupe (Tabela 4.21). U drugoj i tre$oj osnovnoj temi (poslovni pristupi i usavr!avanje/uslo$njavanje zakonskih regulativa), u#esnici su svoju diskusiju uglavnom usmerili na slede$e trendove: uslo%njavanje i neprekidan razvoj regulatornih aspekata u oblasti/ regionu; uspostavljanje biotehnolo"kih inicijativa koje se pojavljuju kao novi poslovni sistemi, uticaj globalne ekonomske krize i globalne ekonomske nesigurnosti. Dok su specifi#nije kategorije u njima bile grupisane oko sve ve$e potrebe za: prihvatljivim servisom (za pre-klini#ka istra%ivanja i testiranje produkata), adkevatno obu#enim kadrovima, ‘virtuelnim’ modelima partnerstva, ali i stvarnim partnerskim modelima, kao i za metodama adekvatnog predvidjanja i planiranja (Tabela 4.21). Iako su izdvojene brojne teme i kategorije, svaki intervju bi, po obilju informacija koje nudi, mogao potencijalno da se iskoristi za individualnu analizu (eng. case study) i da ponudi mno"tvo novih podataka za dalje analize. 4.4.5 Segmenti podataka (5): Utemeljenje rezultata u podacima (eng. “Grounding” the data) Zavr"ni korak u analizi bilo je utemeljenje rezultata u podacima pri #emu su za to kori"$eni citati iz transkripta intervjua (eng. narratives) koji u sebi sadr%e izjave ili opise date od strane u#esnika, a koje doprinose nekoj od identifikovanih tema i kategorija (eng. substantiated by the material or ‘well-grounded’). Utemeljenje rezultata predstavljeno je u Tabeli 4.22 do 4.25, prema prethodno identifikovanim kategorijama i temama. 125 Tabela 4.22: Utemeljenje rezultata u podacima: O industriji (eng. About Industry). ABOUT INDUSTRY: ! “It's an industry that had tremendous promise which was largely unrealised till maybe two or three years ago when we began seeing significant growth, improved revenue and returns and more and more profitable companies. So my view is that it's now moved from a fairly slow growth phase till now, a very rapid growth phase across the world.” [CTE01] ! “So we've shifted from, can we make it and does it do anything, to how do we make it repetitively and reliably and deliver it into the marketplace to thousands of patients? That's where cell therapies is right now.” [CTE03] ! “I'd like to see some more budgets provided to those that have got personnel - enough personnel to be able to service the industry.” [CTE03] ! “We’re five years from curing cancer, but of course we’ve been five years from curing cancer for the last 50 years. The same kind of thing is true of cell therapy.” [CTE07] ! “Differentiation between them [organisations] is mostly based on the funding source, whether they're public or private… with change of models and approaches they use, all money-driven change.” [CTE12] ! “If we looked at Australia, [they] don’t have much in the way of investment, significant investment and ... there’s not a huge amount of money going into this space and the most recent funds are going to more bricks and mortar rather than operating funds. This is where the problem lies - building the capacity of these operations to actually run. In the States [USA] and Europe, venture capitalists are much more involved [and] it would appear raising money through venture capitalists seems to be the nature of the game. However, there’s a very high mortality rate for bio-techs and … the fact that the big pharma are muscling in on the territory and causing grief for start-up bio-techs. It’s really an interesting phase for cellular therapies right now.” [CTE14] ! “Stem cell science is seen as an answer to a lot of medical problems that at the moment have no solutions. So I'm very mindful that while it's fantastic to see progress in our field, we're quite a long way away from being able to routinely apply these developments to help people. I'm concerned that there's a bit of a void between - a gap between the community expectation and the reality of where we're at with stem cell research, [in other words] an expectations vacuum.” [CTE20] ! “There are just issues across everything and there just aren't any easy solutions to any of them. It's going to be a lot of work and I think people were very optimistic early in the days of stem cells [research] that we were going to be in the clinic in a couple of years, that was just grossly naive. People have got be patient, there aren't going to be any quick solutions.” [CTE21] Oznake za u#esnike u istra%ivanju: CTE01 - CTE24. Iz rezultata analize vidi se da su u#esnici studije uspeli da prenesu osnovu problematike u oblasti – kompleksnost. Neki od eksperata su to pripisali ranom stadijumu razvoja a mnogi su upotrebili termin ‘infancy’. Postoji koherentan stav u#esnika studije u tom smislu kao i u stavu da je neophodan dalji razvoj, ne samo u nau#no tehnolo"kom smislu ve$ i u prate$im oblastima koje obezbedjuju podr"ku #itavom sektoru razvoja $elijskih terapija odnosno biolo"kih lekova kao kategorije naprednih terapija. Uz to, ve$ina stru#njaka je izrazila svoje mi"ljenje o zakonsko regulatornim okvirima, njihovom neprestanom razvoju i promenama u pristupu nekih od regulatornih agencija. Pomenuti su, u zna#ajnoj meri, upravljanje rizicima (eng. risk management) na vi"e nivoa i neophodnost po"tovanja specifi#nih standarda koji se primenjuju u proizvodnji novih terapija (eng. GMP). Medjutim, ve$ina stru#njaka 126 se slo%ila da iako su neophodni strogi regulatorni okviri u ovoj oblasti, neophodno je da postoji odredjeni nivo fleksibilnosti u njima, su"tinsko razumevanje razlika izmedju nau#nih pristupa, razli#itih produkata i ograni#enja koja oni nose (Tabela 4.23 i 4.24). Tabela 4.23: Utemeljenje rezultata u podacima: Tehnologija i razvoj (eng. Technology & Development). TECHNOLOGY: ! “So we've changed from first grade media through to improved formulations to animal free formulations. We've moved from enzymes, which were animal derived to recombinant and now to recombinant/animal free. So there's been constant change.“ [CTE01] ! “[One] can argue that bone marrow cell transplantation is a cell therapy, blood transfusion is a stem cell therapy and we have been doing it for many, many years; what we haven’t been [doing] is extracting the cells, expanding in vitro, differentiating into different cell populations and transplanting it.” [CTE06] ! “Make distinction between the use of cells as either tools or for the therapy.” [CTE21] ! “Looking into the future, for example 2D and 3D bioreactors.” [CTE24] DEVELOPMENT: ! “The products are so different. It relates very much to the regulatory status of the product. Whether it's a standard of care or whether it's a clinical trial product. So for a standard care product it's typically a three year cycle, I think, before there's major technology changes. For new products and clinical research products there's typically an annual cycle.” [CTE01] ! “We've seen an evolution from - certainly from the US regulators and some of the Europeans. They are now beginning to give approval for phase I/II cellular therapy trials because there's a body of knowledge that's accumulating that says, cell therapies appear to be either inherently safe - they either do some something interesting and useful or nothing. They don't appear to follow the classical pharma model [where] you need a phase I to determine the level of toxicity. Cell therapies don't appear to be toxic. … Whereas we're working with a living cell and we have difficulty defining the cell letting alone doing it.“ [CTE03] ! “There is a philosophical change, where the time span between research, GMP manufacturing and an attempt of commercial applications is now expected to be so fast (e.g. only a couple of years apart for each stage) while previously, in a small molecule development cycle, it took at least five to ten years for the development cycle between the research, pre-clinical and pre-marketing approval prior to commercial application of a drug.” [CTE15] ! “There are several challenges with that model [from research to manufacturing]. First one is, I think, that a lot of research people are not trained to do development and they don’t understand the vigour of [the] development process.” [CTE16] ! “With the way that society is now … we're really going much more in the direction of expectations of instant gratification, there's a perception that things shouldn't take so long, that it should be able to happen quickly. The techniques in pharmaceutical development that allow to review a huge number of compounds and pick one or two to take forward, and then to find by computational means other molecules that are very similar or can be used in a similar way … with bio-therapeutics, because of the way that they're developed clinically as a clinical trial programme, your whole trial programme is designed with the potential product label for one product and one indication only.” [CTE19] ! “What I've learned both in my own personal interactions with regulators as well as being a regulator, is that you have to work with them, not against them. If you come in with a very antagonistic mindset, as in you're right and they're not; you're not going to get very far. It really is a field that requires mutual understanding and mutual working together because we're all moving into a space that's completely uncharted territory. So it's very important to work with them in a very tight collegial way that will get you further than any other behaviours.”[CTE21] ! “The [cell] therapies have got incredible clinical potential and incredible financial potential. But it’s a complex field and a complicated programme to move forward and needs to be more effort put into it and a more systematic effort. It’s that word ‘systematic’ that has been missing and part of it is that so many of these programmes are being operated by a very small team.” [CTE23] Oznake za u#esnike u istra%ivanju: CTE01 - CTE24. 127 Tabela 4.24: Utemeljenje rezultata u podacima: Zakonske regulative, upravljanje rizicima, Dobra proizvodja#ka praksa (eng. Regulatory compliance, risk management, GMP). REGULATORY/RISK MANAGEMENT/GMP: ! “Where we've had the most risk has been when there's been undisclosed and where there's been no stakeholder discussion. For instance when [regulator] abruptly implemented harmonisation treaty … that caused enormous disruption.“ [CTE01] ! “The change I would apply is I would actually move forward the biologicals framework ... the biological framework that TGA is proposing is a very sophisticated to provide an expedited approval mechanism for biological therapies which is more supportive and tolerant of the special requirements of cell therapy compared to the European and US frameworks. … we have a particular framework that's free of many of the requirements of the medicinal framework like in North America, which has a reduced pre-clinical requirement compared to the European framework. So my push would be to expedite that more quickly because the greatest impediment to product development in Australia is the regulatory uncertainty.” [CTE01] ! “The whole of our business is risk management, because that - we have to essentially leave a paper trail that says we have assessed and put in place a mechanism whereby we've assessed the risks to be delivering an inherently safe product. So the whole of our GMP is based on a risk management strategy…. All or nothing approach; that's what we do for a living.“ [CTE03] ! “Our whole ethos is that we are not against the heavy regulation of the industry because we think it's absolutely mandatory to be inherently safe, and we've followed that … while our operators bitch and moan about having another audit and going through the pain and suffering of the audit, etcetera, we understand it. That's what it needs to play in this space. That's what you need. In terms of [change] … we're actively attempting to facilitate more dialogue [with regulators].“ [CTE03] ! “[If one] cannot achieve the maximum requirement because of [the lack of] staff [try to meet a] minimum requirement from the bottom up, never top down… Use flow-chart of the procedures [and identify] what the potential risks are.” [CTE04] ! A very multi-layered risk management approach from a basic science perspective right through to a formalised risk management, risk assessment approach… everything we do is risk assessed, as a group and as individuals.“ [CTE05] ! “Need to ... increase the funding, increase the regulatory requirements, remove the potential conflicts that exist [on the operational level], separate collection and manufacture versus transplantation, so that the confidentiality of the donor versus the recipient is always maintained.“ [CTE12] ! “I can see significant impacts in terms of them [sector] certainly understanding the stringency with which some of the things need to be done. A deep understanding that suddenly starts to emerge about what validation really is… this is basically moving from a research paradigm into a production steady state paradigm - you need to know everything about what it is you do when you’re in a manufacturing paradigm whereas research you’re pushing the edge of [the] knowledge, different mindset entirely. The impact is big because, depending on the maturity of the group and how long ago it left its research roots on its way to becoming a mature production entity, will depend on the impact of the change.“ [CTE14] ! “For the larger companies with multiple products, more often than not I have seen the ‘focused’ quality systems. They bring in corporate quality policies as such but they tend to find all the GMP standards as too much of specialisation… it is a bit of implementation challenge in a large company. It’s hard to take a risk in a more specialised approach and adapt within the large corporate system.” [CTE16] ! “There are technical risks, there are commercial risks, there are manufacturing risks, there are business risks; there are revenue risks. We navel-gaze in quite considerable detail all the time, we have risk litigation as part of what we do but we are in an inherently risky business.” [CTE21] ! “We need to have levels of multi-skilling, cross-functional training and an attitude within the group that allows them to quickly adopt other roles without feeling threatened by any changes that they may bring upon them in a short period of time. So our development process is really about keeping the skill set that we have and developing a cohesiveness between the group.” [CTE22] ! “Building up GMP facilities, building up solid regulations that industry actually had quite a bit of input to. Now I feel like the foundations are in place so we haven’t seen a huge amount of progress the past five to 10 years. But I think there has been a lot of progress, it’s just not particularly visible because it’s not making the headlines. You don’t get a big headline because the FDA has finally issued the draft guidance on knee cartilage.” [CTE23] Oznake za u#esnike u istra%ivanju: CTE01 - CTE24. 128 Tabela 4.25: Utemeljenje rezultata u podacima: Promene u zakonsko regulatornim odredbama (eng. Regulatory Shift). REGULATORY/SHIFT OF ! “The TGA talks to the FDA on a regular basis, they share information, and the EMEA is similarly moving in that field; the problem with the EMA is the fact that, notwithstanding the common regulatory EU approval across the top, when you've got that common approval then you've got to each of the countries anyway. It's better, but … Europe's still going to be a fragmented market. But it's getting there and they are talking on a regular basis and groups like us are helping keeping that dialogue going so there's some commonality. It's a pity the Europeans chose to regulate cell[s] [and] tissues as a medicinal.” [CTE03] ! ”We're seeing some interesting shifts in terms of the ability of a regulator to send you back when you go in and ask for a phase I, and they said, well why don't you bring it back as a phase I/II. That to me is the most fantastic outcome… so more of that … where the regulator is able to disseminate their knowledge of what's inherently safe and what isn't, and to save you time and dollars.” [CTE03] ! “The regulatory requirements are acceptable and reasonable. The new four-tier process has been put into place … I think is probably fair and reflective.“ [CTE05] ! “Their focus, for years, has been very different, in that the EU regulations were focussed on your design of the facility, on your process and your documentation. The FDA, who is never so focussed on designer facility, or focussed on the process, but they would focus more to the science. What we’re now seeing is an evolution where, Europe is beginning to get into the science and the FDA is beginning to get into the design of the facility.“ [CTE08] ! “Australia has fascinated me. First, you’d never talk to the TGA about anything the FDA does. They [TGA] observe and follow European regulation, but then they’ll also take European regulation and put a twist on it. In other words, they’ll make their own changes to that. Typically, their changes are more of a conservative approach.” [CTE08] ! “FDA has proved core guidance to the extent they care; they got more in writing to dictate how to do the cell therapy.“ [CTE16] ! “Engage with the regulatory bodies as much as we can as early as possible. In many cases, even almost in an informal way - this is what we're thinking. What do you think about this? They give it a very informal but non-binding interpretation and then from that we will build a case to them.“ [CTE21] ! “The difference is not in the actual words of the regulations, but it's the interpretation by the regulator of the countries. In particular, in Europe the interpretation by the regulator is actually very, very lenient in respect to what we would expect to see for example in Australia and also, to some extent, in the US; over time the interpretation tends to come together and move apart over the period of years. We've seen differences in regulations even over the course of the last 10 years where the regulations were upheld by the FDA in the States were supposed to be the gold standard. Then it was Health Canada who had a stronger and more rigid interpretation at that time the TGA was very lenient. Then the TGA went through a period in time where they changed their attitude towards the regulations and they became more strict. So it goes through various periods and it probably tends to follow either what's happening within the market – if there's been a scare within the market or whether or not the people who are actually looking after it are that way inclined.” [CTE22] ! “If they want to do a trial in Europe, US and Australia they really need to plan on speaking with five regulators and treat them separately. The US is one thing, Australia is one thing and then for Europe you have the EMA, the CAT and whatever member state your main principle investigator or your manufacturing site is located in.[CTE23] Oznake za u#esnike u istra%ivanju: CTE01 - CTE24. Rizici i strategije za upravljanje rizicima (eng. risk management), po mi"ljenju svih stru#njaka, su neizbe%an element razvoja oblasti. Rizici su prepoznati na svim nivoima aktivnosti – klini#ki, poslovni, finansijski, operativni, strate"ki i regulatorni (Tabela 4.24). Pored toga, u#esnici studije su se slo%ili da je u toku zna#ajan pomeraj u okviru zakonsko regulatornih tokova na globalnom i lokalnom nivou (Tabela 4.25). Zna#ajno je napomenuti da je ve$ina stru#njaka u odredjenoj meri zabrinuta zbog 129 uticaja koji regulativne odredbe mogu imati na budu$i razvoj sektora/industrije i razvoj novih generacija biolo"kih lekova. Neke od najva%nijih pouka izdvojenih od strane u#esnika u istra%ivanju su – “Thrilling experience.”[CTE02]; “Things are always more complicated than you would think they would be.”[CTE06]; “Communicating and not over-selling the promise.”[CTE19]; “There is no single cell that does everything.”[CTE21]; kao i “Not to underestimate the regulator.”[CTE22]. Ve$ina stru#njaka je naglasila potrebu da postoji otvorenost u komunikaciji i razmeni saznanja u oblasti, iako se od nedavno zapa%a odredjena promena u tome. Navedeni faktori bili su, ulazak velikih farmaceutskih kompanija u ovu oblast (koje poku"avaju da za"tite ili ostvare profit za"titom intelektualne svojine) kao i razlike izmedju komercijalnih i akademskih centara (Tabela 4.26). Tabela 4.26: Utemeljenje rezultata u podacima: Otvorenost i saradnja u industriji (eng. Industry openess). INDUSTRY OPENNESS: ! “It was [there] but it's gradually closing, because it's now serious. There's now real dollars at the other end [for example] cut a $1.7 billion deal. That's deadly serious. There will be more and more tightening of security. Researchers will be asked, more and more, not to talk and not to publish. It's a nature of a new, evolving industry. It's been wide open and lots of the academics will continue and attempt to keep it wide open. But because of the mega dollars involved there will be more and more tighter restraints and I expect less and less information to be available.” [CTE03] ! “In Europe and the US, we would call it a clinical study, we would call it research, maybe a clinical trial, but definitely not a stem cell therapy… in Europe and the US, we would not call it a stem cell therapy.” [CTE10] ! “Sharing information in the field…I think by those who are responsible, yes. I think there's a big difference between the groups that use peer review publications and standard mechanisms to communicate their results, sharing their information at a conference et cetera. Compared to these groups that operate under the radar that don't go to the international meetings, that don't publish their results. That it's not really clear how many patients they're treating, what they're treating them with and doing appropriate pre and post screening on the patients to really see if there's a result.” [CTE20] ! “It’s such a global community that people are very much still in the mode of sharing what they know and what they’ve learned and collaborating; that’s actually been one very positive side of not having much investment and much involvement from big groups, is that people had to collaborate. They had to get out there and find somebody on the other side of the planet that they could work with and they were free to do so because they weren’t hemmed in by IP clauses with their pharmaceutical partner – it’s just been very collaborative and very rapidly growing because of that.“ [CTE23] Oznake za u#esnike u istra%ivanju: CTE01 - CTE24. 130 U procesu prikupljanja i analize podataka bila je interesantna tranzicija izdvojenih tema i kategorija, koje u procesu analize nastaju od uop"tenih mi"ljenja o industriji i njenom trenutnom stanju do visoko specifi#nih i usko stru#nih detalja vezanih za dati projekat. Intervjui su se uglavnom odvijali od op"tih primedbi o razvoju, produkciji i upotrebi novih terapeutika baziranih na primeni $elija i tkiva (biolo"kih lekova), razli#ite terminologije i razli#itih namena (uz detaljne preglede projekata, organizacija, starte"kih pristupa ili detaljnih struktura koje u njima u#estvuju). Zatim bi se diskusija ponovo preusmerila na op"ta razmatranja, komentare ili mi"ljenja o stanju u datoj oblasti – kao "to je ve$ prezentovano u mno"tvu izlo%enih rezultata. Ova komparativna analiza, iako sa relativno malim brojem u#esnika, predstavlja doprinos u sagledavanju problematike razvoja $elijskih terapija kao oblasti (neki od u#esnika se nisu slo%ili sa upotrebom termina ‘industrija’). Ona omogu$uje odredjene generalizacije, ali uglavnom otvara niz pitanja na koja bi tek trebalo da se nadju odgovori. Te"ko je izvr"iti poredjenje sa postoje$im studijama jer do sada nije bilo sli#nih podataka u literaturi. Dok bi budu$a istra%ivanja mo%da mogla da ponude jasniju perspektivu i specifi#niji fokus - ova analiza ve$ zapo#inje taj proces postavljaju$i pitanja, identifikuju$i problematiku i nude$i odredjene prakti#ne modalitete. Ipak, mo%da najzna#ajniji presek dosada"njeg i budu$eg razvoja oblasti daje slede$a izjava jednog od svetskih stru#njaka: “There are just issues across everything and there just aren't any easy solutions to any of them. It's going to be a lot of work and I think people were very optimistic early in the days of stem cells [research] that we were going to be in the clinic in a couple of years, that was just grossly naive. People have got to be patient, there aren't going to be any quick solutions.” [CTE21] 131 4.5 Komparativna analiza 5: Modelovanje pristupa i re$enja u svrhu predvidjanja i planiranja Rezultati prethodne analize u odredjenoj meri prezentuju prakti#na re"enja ali ne i sistemska re"enja u datoj oblasti. U tom smislu je mogu$e ponuditi neke pojednostavljene teorijske modele kao "to su horizontalni i hijerarhijski pristup razvoju procesa izrade $elijskih terapija. Fokus razmatranja prakti#nih i teorijskih re"enja jeste na PROCESU jer bioterapeutska sredstva (uklju#uju$i $elijske terapije) neizbe%no podle%u klasi#noj postavci DIZAJN PRODUKT-PROCES-SPECIFIKACIJA (eng. design product, process, specification) – koja se #esto svodi na jednostavniji oblik PROCES JESTE PRODUKT (eng. process equals product) (Copmann, 2001; Webster, 2003; Rader, 2008). Ova paradigma razvojnih postupaka bioprocesovanja odgovara osnovnim CMC aspektima (eng. chemistry, manufacturing, control, CMC) na kojima po#ivaju osnove Dobre proizvodja#ke prakse (eng. Good Manufacturing Practice, GMP) u biofarmaceutskoj industriji. 4.5.1 Teorijski model 1 Horizontalni model (Slika 4.16) se danas primenjuje u ve$ini projekata, gde u svojoj lineranoj postavci, ne mo%e da obezbedi adekvatno predvidjanje, planiranje i upotrebu resursa. U horizontalnom modelu nema zajedni#ke osnove izmedju faza, kao "to je na primer slu#aj u hijerarhijskom modelu (Slika 4.17). Horizontalni model odlikuje diskretna raspodela resursa koja onemogu$ava standardizaciju procesa kao i dugoro#no planiranje. Ovakav model omogu$ava da se projekti u svakoj, manje ili vi"e nezavisnoj fazi, vode kao fragmentisan niz dogadjaja (od A do B) pri tome zapo#inju$i proces iz po#etka za svaku individualnu fazu (npr. od A do B, od E do F) ili njen deo (od A1 do A2). Ograni#ena finansijska sredstva #esto uslovljavaju ovakav razvoj projekata, "to posredno dovodi do ve$eg utro"ka sredstava i manje efikasnosti. 132 A 1 A 2 B A B C D E F Elementi Faze 2: Elementi Faze 3: Elementi Pre-klinii!ke faze: Faza 1 Faza 2 Faza 3 Pre-klini!ko istra'ivanje Klini!ko istra'ivanje Slika 4.16: Horizontalni model izrade bio-terapeutika sa ‘standardnim’ pristupom (elementi i faze). Na primer, ako su odredjeni elementi obezbedjeni u jednoj od prethodnih faza razvoja, isti ili sli#ni elementi $e morati da se obezbede nezavisno u nekoj od kasnijih faza (pri tome iziskuju$i dodatna finansijska sredstva i vreme za optimizaciju, validaciju ili re-validaciju). Horizontalni model nije pogodan za implementaciju i/ili po"tovanje strogih zakonsko regulatornih odredbi u procesu izrade bioterapeutskih sredstava. Ne obezbedjuje jasnu strate"ku odrednicu, nema zakonsko regulatorni tim koji $e u kontinuitetu da proprati razvojne faze kao ni mogu$nost da vi"e projekata koristi zajedni#ke usluge ili istu komponentu u procesu izrade (npr. logistiku/ transport i skladi"tenje u kontrolisanim uslovima ili testiranje u akreditovanoj laboratoriji). 4.5.2 Teorijski model 2 Hijerarhijski model, za razliku od horizontalnog, ima strukturu koja omogu$uje kontinuitet, fokus i efikasniju upotrebu resursa. U ovakvom modelu izrade bioterapeutika mogu$e je izvoditi nekoliko projekata u razli#itim razvojnim fazama uz optimalnu potro"nju sredstava i u optimalnom vremenskom intervalu. Medjutim, ni ovaj teorijski model nije u stanju da omogu$i adekvatno modelovanje visoko slo%enog procesa razvoja $elijskih terapija bioterapeutika (Slika 4.18). 133 Projekat 1 Projekat 2 Projekat 3 Pre-marketing )))) Faze Elementi Slika 4.17: Hijerarhijski model organizacije i izrade (elementi i faze). Odredjena multidimenizonalnost i slo%enost procesa prikazana je na modelu iz literature (Slika 4.19). Ipak, svi prikazani modeli su drasti#no pojednostavljena reprezentacija realnosti ovakvih projekata, kompleksnih nelinearnih procesa koji ih #ine (Slika 4.18), multidimenzionalnih optimizacija ovih procesa (Slika 4.19) kao i njihove osetljivosti na dinamiku unutra"njih faktora (Slika 4.20) i spolja"nje uticaje. U tu svrhu su u daljem tekstu navedena neka dodatna razmatranja koja bi mogla da doprinesu smernicama u budu$im istra%ivanjima. Zakonsko regulatorni fokus Infrastruktura sa zajedni!kim elementima Pre-klini!ko istra'ivanje Faza 3 klini!kog istra'ivanja Faza 2 klini!kog istra'ivanja Faza 1 klini!kog istra'ivanja Pre-klini!ko istra'ivanje Faza 3 klini!kog istra'ivanja Faza 2 klini!kog istra'ivanja Faza 1 klini!kog istra'ivanja Pre-klini!ko istra'ivanje Faza 3 klini!kog istra'ivanja Faza 2 klini!kog istra'ivanja Faza 1 klini!kog istra'ivanja 134 Faze Problematika Strategije koje koriste Adaptirano iz: Kirouac & Zandstra, 2008. Slika 4.18: Dijagram procesa razvoja $elijskih terapija/bioterapeutskih sredstava i osnovne faze: otkri$e, optimizacija, produkcija i isporuka. Faze mogu medjusobno da se preklapaju i nisu u potpunosti jednosmerne (npr. u toku procesa optimizacije mogu nastati nova otkri$a). Slika 4.19: Multidimenzionalna optimizacija procesa izrade $elijskih terapija/bioterapeutskih sredstava sa prikazom vi"ezna#nih (ponekad kontradiktornih) ‘izlaza’ koji se o#ekuju (npr. smanjenje tro"kova uz maksimalni kvalitet ili koli#inu $elijskih terapija kao produkta) a koji su uslovljeni ne-linearnim ‘ulaznim’ parametrima (eng. ‘design space’). Pri tome se u teorijskom smislu defini"e ‘response surface’ koji je predstavljen obojenom zakrivljenom povr"inom. Adaptirano iz: Kirouac & Zandstra, 2008. Teorija haosa (eng. chaos theory) za #ijeg osniva#a se smatra matemati#ar Benoît B. Mandelbrot, 1924–2010 (Mandelbrot, 1977) nalazi svoje mesto u nizu teorijskih razmatranja i nekim prakti#nim primenama u matematici, fizici, biolo"kim i drugim naukama, u nastojanju da otkrije strukturu i zakonitosti u aperiodi#nim, nepredvidivim dinami#kim sistemima – kao "to su kretanja #estica, formiranje oblaka ili oscilacije u broju i strukturi biolo"kih populacija. Iako se ovi ‘haoti#ni’ sistemi cells and the monitoring and control aspects of the preclinical and clinical production systems are particularly relevant. The extensive roliferative potential that makes stem and progenitor cells an attractive cell source also confers ri k associated with tumorgenesis, particularly since there is evidence that some cancers arise from aberrant tissue-resident stem cells (Lapidot et al., 1994). Indeed, studies suggest that the proliferative burden placed on hematopoietic stem cells (HSC) following bone marrow transplantation plays a role in the increased risk of secondary hematopoietic malignancies and myeloproliferative disorders (Rocci et al., 2007). Even in the absence of culture- induced transformation, pluripotent stem cells form mix d cell tumors when transplanted in vivo (Blum and Benvenisty, 2008), necessitating multistep procedures to remove undifferentiated cells and enrich for target cells (Hentze et al., 2007). We need to face the fact that growing stem cells and their derivatives involves balancing cell growth and proliferation (product yield) with culture-induced genetic or epigenetic changes (product quality). Thus, strategies to produce cells for cell therapies will need to address a multidimensional optimization problem that maximizes target cell output while reducing production costs (necessary for any relevant clinical translation) and regulating cell-culture-associated selective pressure (Figure 2). Such se- lective pressure likely underlies many observations emerging from the in vitro culture of primary cells. Embryonic stem cells (ESCs), the in vitro correlate of the inner cell mass (ICM), can clearly emerge (in the right culture conditions) at high probabili- ties. Likewise, induced Pluripotent stem (iPS) cells reprogram with increased probabilities upon transduction with stem-cell- associated transgenes and conditions. Perhaps other pluripo- tent cells such as Multipotent Adult Progenitor Cells (MAPCs) (Jiang et al., 2002) also fall upon this spectrum, albeit with m ch lower probabilities. Overlaying measurements or ou p ts for selective pressure on bioprocess optimization protocols will b important to determine how long to culture cells and what conditions could yield equivalent outputs with lower probabilities of adverse events. Cell Therapy Bioprocess Optimization and Development The goal of cell therapy bioprocess optimization is to define the conditions that will be brought forward for preclinical validation and clinical approval. Not only does the right cell type have to be produced, strategies for its production in a manner that can yield sufficient numbers of cells, under conditions that are appro- priate for regulatory approval and are cost effective, need to be Figure 1. Roadmap for the Development of Cell Therapies Cell therapy development can be divided into four phases: discovery, process optimization, produc- tion, and therapeutic delivery. In the discovery phase, the main issue is product characterization. Functional assays and cellular and molecular pro- filing are useful tools in this phase. The process optimization phase involves quantifying the rela- tionship between culture parameters and cell output. The rational design of experiments and high-throughput screening using microculture platforms can be used to generate empirical cell- based models, which may be integrated with mo- lecular profiling technologies to develop more mechanistic, molecular-based models. Consider- ations in the production phase include the scale- up strategy and quality control. The final phase is the therapeutic delivery of the cell product. It should be noted that the development phases feed back on one another—during process optimi- zation, biological discoveries may be made, and during production, the design space will become better defined. Figure 2. Multidimensional Optimization of Cell Production Processes Multidimensional optimization problems are an intrinsic part of process design engineering wherein multiple (often conflicting) outputs must be simulta- neously maximized/minimized as a function of multiple (nonlinear) input parameters (the ‘‘design space’’). For cell therapy production processes, one desires to minimize cost (reagents, time, and operator requirements) while maximizing product yield (target cells/day) and quality (cell purity, functionality, sterility, etc.). Depicted is the response surface of a theoretical objective func- tion to be minimized (cost) as a function of two closely linked process param- eters, selective pressure (in this case the rate at which undesired genetic changes occur as a function of the culture conditions) and cell output (the rate t which the target cells are generated). 370 Cel Stem Cell 3, October 9, 2008 ª2008 Elsevier Inc. Cell Stem Cell Review cells and the monitoring and control aspects of the preclinical and clinical production systems are particularly relevant. The extensive proliferative potential that makes stem and progenitor cells an attractive cell source also confers risk associated with tumorgenesis, particularly since there is evidence that some cancers arise from aberrant tissue-resident stem cells (Lapidot et al., 1994). Indeed, studies suggest that the proliferative burden placed on hematopoietic stem cells (HSC) following bone marrow transplantation plays a role in the increased risk of secondary hematopoietic malignancies and myeloproliferative disorders (Rocci et al., 2007). Even in the absence of culture- induced transformation, pluripotent stem cells form mixed cell tumors when transplanted in vivo (Blum and Benvenisty, 2008), necessitating multistep procedures to remove undifferentiated cells and enrich for target cells (Hentze et al., 2007). We need to face the fact that growing stem cells and their derivatives involves balancing cell growth and proliferation (product yield) with culture-induced genetic or epigenetic changes (product quality). Thus, strategies to produce cells for cell therapies will need to address a multidimensional optimization problem that maximizes target cell output while reducing production costs (necessary for any relevant clinical translation) and regulating cell-culture-associated selective pressure (Figure 2). Such se- lective pressure likely underlies many observations emerging from the in vitro culture of primary cells. Embryonic stem cells (ESCs), the in vitro correlate of the inner cell mass (ICM), can clearly emerge (in the right culture conditions) at high probabili- ties. Likewise, induced Pluripotent stem (iPS) cells reprogram with increased probabilities upon transduction with stem-cell- associated transgenes and conditions. Perhaps other pluripo- tent cells such as Multipotent Adult Progenitor Cells (MAPCs) (Jiang et al., 2002) also fall upon this spectrum, albeit with much lower probabilities. Overlaying measurements or outputs for selective pressure on bioprocess optimization protocols will be important to determine how long to culture cells and what conditions could yield equivalent outputs with lower probabilities of adverse events. Cell Th rapy Bioprocess Optimization and Development The goal of cell therapy bioprocess optimization is to define the conditions that will be brought forward for pr clinical validation and clinical approval. Not only does the right cell type have to be produced, strategies for its production in a manner that can yield sufficient numbers of cells, under conditions that are appro- priate for regulatory a proval and re cost effective, need to be Figure 1. Roadmap for the Development of Cell Therapies Cell therapy development can be divided into four phases: discovery, process optimization, produc- tion, and therapeutic delivery. In the discovery phase, the main issue is product characterization. Functional assays and cellular and molecular pro- filing are useful tools in this phase. The process optimization phase involves quantifying the rela- tionship between culture parameters and cell output. The rational design of experiments and high-throughput screening using microculture platforms can be used to generate empirical cell- based models, which may be integrated with mo- lecular profiling technologies to develop more mechanistic, molecular-based models. Consider- ations in the production phase include the scale- up strategy and quality control. The final phase is the therapeutic delivery of the cell product. It should be noted that the development phases feed back on one another—during process optimi- zation, biological discoveries may be made, and during production, the design space will become better defined. Figure 2. Multidimensional Optimization of Cell Production Processes Multidimensional optimization problems are an intrinsic part of process design engineering wherein multiple (often conflicting) outputs must be simulta- neously maximized/minimized as a function of multiple (nonlin ar) input parameters (the ‘‘design space’’). For cell therapy production processes, one desires to minimize cost (reagents, time, and operator requirements) while maximizing product yield (target cells/day) and quality (cell purity, functionality, sterility, etc.). Depicted is the response surface of a theoretical objective func- tion to be minimized (cost) as a function of two closely linked process param- eters, selective pressure (in this c se the rate at which und sired genetic changes occur as a function of the culture conditions) and cell output (the rate at which the target cells are generated). 370 Cell Stem Cell 3, October 9, 2008 ª2008 Elsevier Inc. Cell Stem Cell Review 135 donekle mogu aproksimirati odredjenim matemati#kim funkcijama, teorija haosa ilustruje za"to je te"ko predvideti njihovo dugoro#no pona"anje (Mandelbrot, 1977; Hofstadter, 1985; Strang, 1986; Gleick, 1987; Devaney & Keen, 1989; Stewart, 1997). Linearna analiza koja se primenjuje u klasi#nim matemati#kim modelima uzima u obzir odredjeni stepen uredjenosti koji se retko mo%e na$i u prirodi – tako da je u ovom nastojanju da se otkriju regularnosti, neuredjenost sistema bila dugo zanemarena. Adaptirano iz: http://www.duke.edu/~mjd/chaos.html Slika 4.20: Ilustracija nepredvidivosti dugoro#nog predvidjanja u neuredjenim sistemima usled fenomena nazvanog 'sensitive dependance’ – gde male razlike u polaznom stanju mogu da dovedu do zna#ajnih kvalitativnih razlika u ishodima pri tome onemogu$uju$i adekvatno predvidjanje u dugoro#nom smislu. &to je sistem slo%eniji, postoji ve$a "ansa za neuredjenost odnosno ‘haos’. To je zato "to postoji vi"e "ansi za poreme$aj uredjenosti koje prete stabilnosti sistema (Mandelbrot, 1977; Hofstadter, 1985; Strang, 1986; Gleick, 1987; Devaney & Keen, 1989; Stewart, 1997). (ak ni elementi koji #ine ovakav sistem nisu stati#ni i ‘kona#ni’. Jedno od teorijskih razmatranja blisko teoriji haosa, geometrija fraktala (eng. geometry of fractals), obja"njava prirodu i strukturu fraktala (sastavnih elemenata sistema); pri tome navodi da i najmanje promene u njihovoj inicijalnoj vrednosti mogu u velikoj meri da uti#u na krajnje ishode/ stanja u sistemu (Mandelbrot, 1977; Hofstadter, 1985; Strang, 1986; Gleick, 1987; Devaney & Keen, 1989; Stewart, 1997). Slo%enost fraktala je uporedno proporcionalna rezoluciji njihove analize. Dimenzija fraktala se meri stepenom pove$anja njihove strukturalne slo%enosti koja 136 prati porast skale ili rezolucije njihove analize (Slika 4.21). Dakle, fraktalna dimenzija slu%i i kao mera kompleksnosti (Mandelbrot, 1977; Hofstadter, 1985; Strang, 1986; Gleick, 1987; Devaney & Keen, 1989; Stewart, 1997). Adaptirano iz: Kantz & Schreiber, 2001. Slika 4.21: Primer fraktalne dimenzije (eng. Sierpinski gasket). Zna#ajna definicija u primeni teorije haosa ovde je navedena kao ilustracija kompleksnosti koju aproksimira: ‘haos je neprekidna i neuredjena evolucija koja zadovoljava odredjene matemati#ke zakonitosti i koja se odvija u deterministi#kim nelienarnim sistemima’ (Mandelbrot, 1977; Hofstadter, 1985; Strang, 1986; Gleick, 1987; Devaney & Keen, 1989; Stewart, 1997). U svrhu modelovanja neuredjenih sistema, konstruisani su brojni nelinearni deterministi#ki dinami#ki modeli, od strane autora kao "to su M. Feigenbaum, J. Yorke, E. Lorenz, G.W. Flake; u novije vreme autori kao "to su J. Gleick, I. Stewart, A.A. Tsonis, D.N. Chorafas i drugi, koji nastoje da otkriju i prika%u nepredvidiva pona"anja ili objasne razli#ite primene postoje$ih modela baziranih na teoriji haosa (Mandelbrot, 1977; Hofstadter, 1985; Strang, 1986; Gleick, 1987; Devaney & Keen, 1989; Stewart, 1997). Budu$i da pomenuti procesi u bioprocesovanju tj. izradi $elijskih terapija/ bioterapuetika, nisu linearni po svojoj prirodi, da su kompleksni (gde male razlike u njihovoj osnovnoj konfiguraciji i/ili vrednosti mogu dovesti do kvalitativno razli#itih ishoda) i da su ovi procesi osetljivi na oscilacije u okolini, kao i da po#ivaju na jo" slo%enijim nelinearnim biolo"kim sistemima – dalja razmatranja njihovog razvoja, planiranja i predvidjanja bi, u nekom od budu$ih istra%ivanja, mogla da primene teoriju haosa i matemati#ke modele koji se baziraju na njoj. Quantifying Chaos! INTRODUCTION TO CHAOS THEORY! !!Dimension of a strange attractor: several definitions! Example of fractal dimension: Sierpinski gasket!!! H. Kantz and T. Schreiber, ‘‘Nonlinear Time Series Analysis,’’ Cambridge University Press, (2001)! 137 4.5.3 Prakti"ni model: evaluacija klini"ke metode Za razliku od teorijskog modelovanja koje uglavnom potvrdjuje nepredvidivost sistema u kome se odvija bioprocesovanje, prakti#no modelovanje procesa izrade $elijskih terapija (ili jednog dela tog procesa) mo%e da obezbedi zna#ajan aspekt za njihov razvoj i kvalitet, posebno u procesu evaluacija/ validacije tehnika i pristupa. U ovom poglavlju je prikazano prakti#no modelovanje, kroz statisti#ku obradu podataka, klini#kog procesa prikupljanja mobilizovanih mati#nih/progenitorskih $elija iz periferne krvi aferezom. Mati#ne/progenitorske $elije iz periferne krvi mogu biti prikupljene aferezom od alogenih ili autolognih davalaca. Cilj procedure je da se sakupi odgovaraju$i broj CD34+ $elija po kilogramu telesne te%ine davaoca. Faktori koji uti#u na prinos $elija u proceduri uklju#uju telesnu te%inu davaoca, koncentraciju CD34+ $elija u perifernoj krvi, zapreminu procesovane krvi i efikasnost klini#ke procedure afereze (Trickett et al., 2001; Chepovetsky et al., 2013). Prakti#no modelovanje i evaluacija klini#ke procedure je uklju#ilo uzorak od 111 klini#kih procedura obavljenih u periodu od dve godine (Tabela 4.27). Cilj evaluacije je bio da se utvrdi efikasnost klini#ke procedure afereze i da se uspostavi korelacija sa podacima u literaturi (Trickett et al., 2001; Chepovetsky et al., 2013). Rezultati analize vezani za efikasnost klini#ke procedure afereze pokazali su da je broj prikupljenih CD34+ $elija bio preko 2 x 106 $elija u 58 od 111 procedura (>50%). Korelacija izmedju o#ekivanog i prikupljenog broja $elija je grafi#ki predstavljena na Slici 4.22, 4.23 i 4.24 (u okviru tri razli#ite serije analiziranih podataka) dok se ostali rezultati evaluacije nalaze u Tabeli 2.28. Takodje je pokazano da su rezultati evaluacije u skladu sa literaturnim podacima (Trickett et al., 2001; Chepovetsky et al., 2013). 138 Tabela 4.27: Podaci za uspostavljanje korelacije izmedju broja prikupljenih $elija (x) i o#ekivanog broja $elija (y) u 111 klini#kih procedura afereze obavljenih u periodu od dve godine. Series 1 (Graph 1) Series 1 (Graph 2) Series 1 (Graph 3) Actual (x) Collected CD34/kg Predicted (y) Score CD34 Actual (x) Collected CD34/kg Predicted (y) Score CD34 Actual (x) Collected CD34/kg Predicted (y) Score CD34 3.3 5.411764706 16.43 9.236923077 10.78 33.3 2.67 3.764705882 1.06 2.269090909 7.76 33.3 1.75 2.882352941 0.65 1.701818182 0.73 2.368536585 2.57 7.764705882 0.7 1.985454545 0.41 1.052682927 1.27 7.341176471 0.92 1.134545455 0.39 1.052682927 1.05 7.647058824 0.82 2.269090909 2.5 7.71875 1.15 6.988235294 0.6 2.836363636 2.89 5.566819277 0.5 1.090836364 5.16 24.40120482 0.62 3.363738462 0.7 1.636363636 9.39 11.34939759 4.04 11.874 0.48 1.090909091 5.46 13 5.3 12.696 1.57 3.783928571 7.12 18.72 5.99 13.01821667 4.57 17.83285714 3.92 9.88 8.09 6.015737705 0.71 2.514285714 0.78 1.3 14.4 20.65022951 3.4 7.182333333 0.63 1.56 3.67 3.747890625 7.73 15.99296296 0.37 0.78 26.92 9.852272727 1.16 2.536363636 2.83 6 0.74 1.74890625 1.84 4.363636364 3.72 8.7 0.72 2.257734375 1.97 5.454545455 5.94 16.2 0.68 1.314912281 1.06 2.727272727 1.23 3.502333333 0.79 1.856842105 0.85 2.469272727 3.43 7.004666667 1.08 3.096491228 0.6 1.799552239 7.59 13.509 2.8 5.526285714 0.3 0.899776119 8.8 14.4 5.4 9.016571429 0.26 1.3512 1.04 2.538823529 4.7 9.7 6.89 22.70185185 1.7 3.554352941 10.09 19.27395349 5.39 11.62777778 1.74 4.569882353 10.31 20.28837209 3.81 10.52037037 1.8 3.554352941 20.94 36.63204819 0.29 2.946428571 2.34 21.17142857 12.44 31.07875862 5.98 6.24 4.98 13.2 7.2 9.359714286 0.8 1.69 2.75 9.6 4.75 8.722658537 0.81 2.001333333 0.39 1.04 20.8 40.37142857 0.51 1.000666667 1.9 3.831578947 3.7 6.146341463 0.3 2.2515 0.9 3.284210526 0.81 1.501 0.3 1.642105263 3.64 7.8 2.06 4.740740741 9.8 14.04 2.06 4.148148148 6.44 18.72 1.48 4.148148148 7.5 12.87785714 6.15 11.4 0.91 1.418181818 2.5 2.371621622 0.56 1.134545455 4.9 8.959459459 11.48 24.75 4.85 10.54054054 139 Slika 4.22: Rezultat statisti#ke obrade podataka (eng. Spearman rank correlation) za uspostavljanje korelacije izmedju broja prikupljenih $elija i o#ekivanog broja $elija (Graph 1). Slika 4.23: Rezultat statisti#ke obrade podataka (eng. Spearman rank correlation) za uspostavljanje korelacije izmedju broja prikupljenih $elija i o#ekivanog broja $elija (Graph 2). Slika 4.24: Rezultat statisti#ke obrade podataka (eng. Spearman rank correlation) za uspostavljanje korelacije izmedju broja prikupljenih $elija i o#ekivanog broja $elija (Graph 3). 0.1 1 10 100 0.1 1 10 100 PB SC P re di ct ed PBSC Actual/ Collected Actual vs Predicted (Graph 1) Series1 0.1 1 10 100 0.1 1 10 100 PBSC Actual/ Collected Actual vs Predicted (Graph 2) Series1 1 10 100 0.1 1 10 100 PB SC P re di ct ed PBSC Actual/ Collected Actual vs Predicted (Graph 3) Series1 p < 0.0001 p < 0.0001 p < 0.0001 140 Tabela 4.28: Rezultati statisti#ke obrade podataka i korelacije izmedju broja prikupljenih $elija (x) i o#ekivanog broja $elija (y) u 111 klini#kih procedura afereze obavljenih u periodu od dve godine. Rezultat tri serije analiziranih podataka Srednja efikasnost procedure (n=111) Graph 1 (Slika 4.22) r = 0.8937 p < 0.0001 57% Graph 2 (Slika 4.23) r = 0.8989 p < 0.0001 Graph 3 (Slika 4.24) r = 0.9084 p < 0.0001 Ovde je zna#ajno napomenuti da su u okviru prethodnih teorijskih razmatranja, osnovni postulati bili geometrija sistema, mera njegove neuredjenosti i oscilacije tih istih parametara – dok se u prakti#nom modelu kao osnovni parametar razmatra broj okarakterisanih $elija odnosno numeri"ka vrednost. Dakle, u oba pristupa, teorijskom i prakti#nom, posmatra se dinamika sistema kao osnova njegove karakterizacije. Medjutim, tehnike i aparati koji se koriste u svakoj analizi (modelovanju) se kvalitativno razlikuju – geometrija i mera neuredjenosti sistema vs. numeri#ka vrednost biolo"ke promenljive tj. broja okarakterisanih $elija. Takodje, ako se uzmu u obzir glavne odrednice u teorijskom razmatranju kao "to je, na primer, geometrija fraktala (kao sastavnih elemenata sistema), one mogu ali ne moraju da budu indikator dinamike sistema – iako se smatra da uti#u na nju. Istinski pokazatelj stanja sistema su oscilacije razli"itih parametara u njemu. Sli#no tome, u prakti#nom modelu koji u osnovi razmatra jedan kompleksan i relativno neuredjen biolo"ki sistem, broj okarakterisanih $elija (koje su sastavni deo sistema i imaju svoju specificnu geometriju, "to ih posredno dovodi u vezu sa geometrijom fraktala), posmatrana numeri#ka vrednost istih okarakterisanih $elija konvergira ka svom biolo"kom optimumu i oscilira u datom fiziolo"kom opsegu usled uticaja razli#itih unutra"njih i spolja"njih faktora – upravo kao kod drugih nelinearnih dinami#kih sistema koje razmatra teorija haosa. 141 4.6 Diskusija Zapo#ela je nova era u razvoju nauke (Atala, 2013; Atala, 2012; Klein 2012; Daley et al., 2003) koja nudi mogu$nost da obnavlja i regeneri"e ljudske $elije i tkiva (Trounson, 2009). Odvija se ve$ neko vreme, nudi ogromne mogu$nosti ali postavlja mnoga pitanja i dileme (The Quest Resumes, Time, Feb 9 2009, A. Park; Finkel, 2005). Nau#no tehnolo"ki razvoj koji je prethodio ovome, i koji se jo" uvek odvija nesmanjenim tempom, omogu$ava da se pomeraju granice ljudskih saznanja u istra%iva#kim laboratorijama "irom sveta (Atala, 2012; Trounson et al., 2011). Prate$e tehnologije na sli#an na#in i uz isto toliko novih saznanja nude svoju podr"ku i ubiraju plodove ove tehnolo"ke ekspanzije (Godlman, 1978; Reiffers, 1988; Eaves, 1992; Perseghin, 1997; Haylock, 1997; Roddie, 2002; Mimeault, 2006). Klini#ko bolni#ki centri i istra%iva#ki instituti su mesta gde se odvija tiho i nezaustavljivo stremljenje ka izle#enju mnogih nedostataka, oboljenja i deformiteta (Serke, 1997; Balint, 1999; Hubel, 2001; Sartor, 2005; Hubel, 2006; Parkins, 2006; Rowley, 2009; McDonnell et al., 2008a; McDonnell et al., 2008b). Odvijaju se brojna klini#ka istra%ivanja (Trounson et al., 2011; Daley et al., 2003; Daley, 2008). Nastaju novi zakonski propisi koji poku"avaju da prate, pomognu ali i kontroli"u na#in, ne samo primene novih znanja, ve$ i sprovodjenje istra%ivanja koja vode do njih (Burger, 2000; Serke, 2001; Burger, 2003a; FDA, 2004^; Halme, 2006; Areman, 2009; Ilic, 2012). Nau#na i "ira javnost, uz aktivno u#e"$e medija, svakodnevno govori o zna#ajnim otkri$ima i time se njihove nade i o#ekivanja od ovog novog polja nauke svakim danom uve$avaju. __________ ^ FDA Guidance for industry: PAT - A framework for innovative pharmaceutical development, manufacturing, and quality assurance, 2004. 142 Ulo%ena su ogromna finansijska sredstva od strane javnih fondova za razvoj nauke, kao i privatnih investitora i korporacija "irom sveta. Sve razvijene dr%ave u svetu danas odvajaju jedan zna#ajan deo svog nacionalnog dohotka da bi stekle ili sa#uvale svoje mesto u ovom razvojnom procesu. Jasno je da se ovde, u osnovi, radi o primeni mati"nih i drugih #elija koje se proteklih nekoliko decenija, u manjoj ili ve$oj meri, sve vi"e istra%uju – tako da danas ve$ mo%emo da govorimo o sektoru ili industriji #elijskih terapija. Ipak, #ini se da rezultati tolikog rada i ulaganja jo" uvek ne mogu da se mere sa nastojanjima i o#ekivanjima. Pojednostavljeno re#eno, primena $elijskih terapija uklju#uje prikupljanje, obradu i #uvanje razli#itih tipova $elija kao novih terapeutskih sredstava (Burger, 2003b; Wall, 2003; Prince, 2004). Ovo svrstava $elijske terapije, ne samo u oblast biofarmaceutskih¶ nauka i izrade bioterapeutika^ (eng. biotherapeutics or biopharmaceuticals) (Parson, 2008; Kayser & Warzecha, 2012; Handfield, 2012) – ve$ i srodnih zakonsko regulatornih nau#nih oblasti! (eng. regulatory science) (Burger, 2003b; Wall, 2003; Pamphilon, 2004; Dutton, 2007; Marwaha, 2007; Ilic, 2012a; Ilic 2012b). Za sektor $elijskih terapija (eng. cell or cellular therapies) se jo" uvek smatra da je u svojoj ranoj razvojnoj fazi (eng. 'infancy') – posebno u smislu "ire klini#ke primene* i komercijalizacije (Mansbridge, 2006; Dutton & Scharer, 2007; Santos et al., 2013; Kirouac & Zandstra, 2008; Locke at al., 2011). __________ ^ FDA Guidance for industry: PAT - A framework for innovative pharmaceutical development, manufacturing, and quality assurance, 2004. * To thwart disease, apply now (Editorial). Nature, 453 7197 (2008). ! Advancing Regulatory Science at FDA, Strategic Plan: August 2011. Food and Drug Administration U.S. Department of Health and Human Services. & Implementing the European Medicines Agency’s Road map to 2015: The Agency’s contribution to Science, Medicine, Health – From Vision to Reality. 6 October 2011 EMA/MB/550544/2011. ¶ The financing of biopharmaceutical product development in Europe, Final Report. European Commission, Enterprise and Industry, October 2009. 143 Za ovo se navode mnogi nau"no tehnolo!ki, zakonsko regulatorni, organizacioni, strate!ki razlozi – koji su svi delimi#no razmatrani u ovom istra%ivanju, kao i slo%eni ekonomski i eti#ki razlozi kojima se ova doktorska disertacija nije bavila. Iz tog razloga je, u svrhu sagledavanja dela problematike razvoja $elijskih terapija, ovo istra%ivanje usmereno na vi"e ciljeva. Od njega se o#ekuje da svojim rezultatima posredno ili neposredno doprinese razvoju novih terapijskih pristupa u klini"koj praksi. U odredjenoj meri, ovo je mogu$e ostvariti kroz odgovaraju$e protokole za laboratorijsku izradu i klini"ku primenu $elijskih terapija (Mason & Dunnill, 2008; Mooney, 2008), uz primenu procesa i sistema koji uzimaju u obzir slo$ene zakonske regulative i primenjuju ih na transfer tehnologija i znanja (Slaper- Cortenbach, 2008), uz analizu i sintezu novih organizacionih modela (Rader, 2008) kao i adekvatnu upotrebu teorijskih i prakti"nih modela u svrhu planiranja i predvidjanja (Trounson et al., 2012). Smatra se da je sve ovo neophodno za dalji razvoj $elijskih terapija kako na lokalnom tako i na globalom nivou (Preti, 2005; Buckler, 2009; Bradley & Cairo, 2005). Ovo istra%ivanje tako)e doprinosi razvoju nau#ne misli u oblasti. O#ekuje se da u odre)enoj meri nadomesti nedostatak literature u okviru istra%ivanja procesa i dinamike nove biomedicinske i biofarmaceutske discipline sa svim njenim specifi#nostima¶ (Atala, 2012; Rader, 2008). Do sada kori"$eni modeli izrade i primene $elijskih terapija bili su uglavnom bazirani na biotehnolo"koj i farmaceutskoj industriji (Parson, 2008; Handfield, 2012). __________ ¶ The financing of biopharmaceutical product development in Europe, Final Report. European Commission, Enterprise and Industry, October 2009. 144 Nau"no tehnolo!ki izazovi Postoji niz nau"no tehnolo!kih izazova koji, za sada, ostaju bez odgovora (Joshi, 2003; Burra, 2011; Jing et al., 2010; Song et al., 2010; Wagner et al., 2008; Wagner et al., 2007; Wein et al., 2010). Na primer, stabilnost i isplativost dugoro#nih $elijskih linija, kontrola i primena ekspanzije $elija in vitro, na#in primene i pra$enje efekata i pona"anja $elija in vivo, dugoro#ni efekti ovih terapija, odbacivanje ili gubitak $elija u organizmu nakon terapijskih primena – da pomenemo samo neke (Bertoncello et al., 1988; Bertoncello and Williams, 2004; Goodell et al., 1996; Li & Johnson, 1995; Schroeder, 2010; Wognum et al., 2003). Bazi"na istra$ivanja nude deo odgovora kroz mno"tvo svojih pristupa, dok se klini"ka istra$ivanja, koja su rizi#na, skupa i slo%ena, sporo odvijaju ili daju samo deo neophodnih podataka za dalji razvoj. Uprkos sprovodjenju velikog broja klini#kih projekata (eng. clinical trials) u proteklih nekoliko godina, jo" uvek se smatra da smo izmedju pet i deset godina 'daleko' od neophodnih podataka (pre svega u smislu efikasnosti i klini#ke opravdanosti) (Tournson, 2009; Finkel, 2005). Danas se sve vi"e smatra da $e biti potrebno veoma mnogo vremena da se zaista doka$e efikasnost novih naprednih terapija (mati#nim i drugim $elijama) u poredjenju sa drugim terapijskim pristupima (Blau, 2013; Tournson, 2009; Tournson et al., 2012; Fox, 2007). U smislu novih pravaca tehnolo"kog razvoja, dominira razvoj kombinovanih produkata baziranih na $elijskim terapijama uz primenu nosa#a (eng. scaffolds) (Saif et al., 2010) i medicinskih naprava/pomagala (eng. medical devices) kao i primena biorobotike, automatizacije i 3-D bioreaktora (Dos Santos et al., 2013; Naing & Williams, 2011). Takodje se razvijaju nove tehnologije u istra%ivanju i proizvodnji (eng. upstream processing)^ uz tehnologije za propratne sisteme i testiranja, analiti#ke metode, karakterizaciju i kvantifikaciju u procesu proizvodnje biolo"kih lekova (eng. downstream processing)^. __________ ¶ www.dhhs.gov/reference/newfuture.shtml ¶¶ www.ey.com ^ www.biopharminternational.com 145 Zakonsko regulatorni izazovi Iako su neki od biolo"kih lekova (npr. monoklonalna antitela) bili prethodno regulisani od strane regulatornih agencija, biolo"ki produkti koji se baziraju na transplantaciji $elija i tkiva bili su, istorijski gledano i iz vi"e razloga, isklju#eni iz okvira ovih regulativa (Burger, 2003b; Halme, 2006; Ilic et al., 2012a; Ilic et al., 2012b). Uprkos tome, danas se smatra da je neophodno da se grupa biolo"kih terapeutika baziranih na transplantaciji $elija i tkiva takodje reguli"e. Istovremeno, poznato je da tradicionalni sistemi kvaliteta usvojeni iz farmaceutske industrije nisu najpogodniji za ovu svrhu; budu$i da su ovi bioterapeutici slo%eniji i te%i za identifikaciju kao i da podle%u specifi#nim, veoma slo%enim procesima prikupljanja, izrade i primene (Burger, 2003a; Halme, 2006; Kirouac & Zandstra, 2008; Ilic et al., 2012a; Ilic et al., 2012b). Ova nova razmatranja uslovljena rapidnim razvojem nau#nih istra%ivanja kao i vi"ezna#na uloga regulatornih agencija (razmatrana u prethodnom poglavlju) doveli su do formiranja novih, slo%enih regulatornih okvira koji se primenjuju u sferi razvoja biolo"kih lekova baziranih na transplantaciji $elija i tkiva. Pri tome se formira inovativni sektor biofarmaceutskih proizvoda ‘koji postaje jedan od najintenzivnijih istra%iva#kih sektora sa ogromnim potencijalom da obezbedi nove lekove i napredak medicine u budu$nosti’ (eng. “has become one of the most research-intensive sectors with a great potential for delivering innovative human medicines in the future”) §. Modeli i modaliteti usmereni na razvoj zakonskih regulativa, koji uklju#uju jasno definisane i sveobuhvatne slo%ene sisteme kvaliteta, pokazali su se kao odr%ivi pristup i kao preduslov za uspe"ne projekte u izradi i primeni $elijskih terapija na globalnom nivou (Lowes, 2010; Sukkar, 2011, Ilic et al., 2012a: Ilic et al., 2012b). Regulatorna i zakonodavna tela "irom sveta jo" uvek nisu u mogu$nosti da formiraju jasan stav u odnosu na mnoge od novih terapija, "to kreira ose$aj nesigurnosti u industriji u smislu poslovnih rizika i o#ekivanja. __________ §The financing of biopharmaceutical product development in Europe, Final Report. European Commission, Enterprise and Industry, October 2009. ISBN 978-92-79-14055-6. 146 Sa druge strane, sa zakonsko regulatornog aspekta, o#ekuje se da se izradi i primeni $elijskih terapija pristupi na isti na#in kao i proizvodnji drugih terapeutskih sredstava u farmaceutskoj industriji - kako uklju#uju$i odgovaraju$e fizi#ko okru%enje, tako i laboratorijske procedure obuhva$ene slo%enim sistemima kvaliteta u skladu sa propisanim zakonskim regulativama. Dakle, ovde se radi o dijametralno suprotnom polazi"tu izmedju dva glavna u#esnika u procesu – novog nau#nog sektora tj. industrije i zakonsko regulatornog tela. Organizaciono finansijski izazovi Ve$ina autora se sla%e da $e ishodi razvoja novih terapijskih pristupa u ovoj oblasti u krajnjoj instanci zavisiti od adekvatno kontrolisanih, organizaciono finansijski odr$ivih modela (Mansbridge, 2006; Weber, 2006; Kirouac, 2008; Areman, 2009). U ovom procesu ‘sazrevanja’ organizacionih modela #esto dolazi do promene (eng. shift) iz prve razvojne faze tzv. dobrovoljnog udru%ivanja znanja i ve"tina u okviru projekta (eng. volunteer-based project), koji su usmereni na samo jedan odredjeni projekat i #ija glavna obele%ja predstavljaju osnivanje (eng. foundation) i saradnja medju u#esnicima (eng. collaboration) – u drugu fazu koju karakteri"e partnerski odnos (Quinn & Cameron, 1983; Maslow, 1998; Weick & Quinn, 1999). Partnerski odnos odlikuju jedinstvo pristupa (eng. synergy) i tendencija specijalizacije (gde svaki partner, pojedinac ili organizaciona jedinica, obavlja svoj deo projekta). __________ * Food and Drug Administration, FDA: http://www.fda.gov ; Therapeutic Goods Administration, TGA: http://www.tga.gov.au European Medicine Agency, EMA: http://www.ema.europa.eu ^ The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH), Available from URL: http://www.ich.org # What is Regulatory Science? The Institute for Regulatory Science, USA: www.nars.org/whatis 147 Pri tome se ne razmenjuju nov#ana sredstva medju partnerima, a tro"kovi se uglavnom ravnopravno dele. U tre$oj fazi, koja sledi partnerski odnos, formira se polje zajedni#kog upravljanja projektom (eng. shared governance), pri #emu se ostvaruje odredjeno pozicioniranje (eng. positioning) svakog od u#esnika (bilo da su u pitanju pojedinci, organizacione jedinice ili #itave organizacije/institucije) i zapo#inje rast (eng. growth), odnosno po#inje da se ostvaruje manja finansijska dobit (naju#estaliji primer su projekti finansirani od strane vladinih agencija ili drugih eksternih tela kao "to su regionalni fondovi razvoja). U zavr"noj, #etvrtoj fazi, formira se nezavisna organizaciona ili institucionalna jedinica (eng. independent entity) koja mo%e biti u vidu nove organizacione jedinice u okviru iste institucije (eng. department) ili potpuno nezavisne komercijalne kompanije (eng. biotech start-up). Ovu fazu karakteri"u tendencija ka ostvarivanju sopstvenog identiteta (eng. recognition) i ka ostvarivanju zarade (eng. profit) (Quinn & Cameron, 1983; Maslow, 1998; Weick & Quinn, 1999). Osnovno obele%je celokupnog razvojnog procesa je 'pomeraj' (eng. shift) – iz stanja bez strukture (eng. volunteer-based stage) u stanje bez inovacije (eng. institutional stage). Izmedju njih se nalazi #itav spektar prelaznih 'stanja' u zavisnosti od razvojne faze i specifi#nih uslova za svaki projekat. Pomenute faze mogu da se preklapaju, da se odredjeni projekti kre$u izmedju njih, pri tome se kra$e ili du%e zadr%avaju$i u odredjenoj fazi (Quinn & Cameron, 1983; Maslow, 1998; Weick & Quinn, 1999). Neki od ovih projekata nikada ne predju u narednu fazu razvoja ve$ ostanu na istom nivou neko vreme, ugase se ili ih nadomeste novi, ve$i projekti. Takav poredak uti#e na ishode mnogih po#etnih translatornih istra%ivanja u oblasti $elijskih odnosno naprednih terapija, pri #emu se najte%i zadatak nalazi u njihovoj selekciji – "ta podr%ati kroz organizaciono finansiranje ili kroz razvojne fondove? – i u modalitetima njihovog razvoja – kako pru%iti adekvatnu podr"ku? Ovo je danas prepoznato kao najve$a prepreka izmedju translacije rezultata bazi#nih istra%ivanja u klini#ke istra%iva#ke projekte koja se u literaturi naziva eng. translational research gap (Woolf, 2008; Marincola, 2003; Kong & Segre, 2010). 148 Strate!ko planiranje i predvidjanje (modelovanje) Deo strate"kog planiranja i predvidjanja u sektoru $elijskih terapija trebalo bi da se odvija uz primenu teorijskog i prakti"nog modelovanja. Nekim od ranijih studija ustanovljeno je, na primeru jednostavnih modela, kako se odvijaju faze razvoja i bioprocesi izrade $elijskih bioterapeutika (Kirouac & Zandstra, 2008) ili njihova primena u (linearnom sistemu) translacionih istra%ivanja (Preti, 2005). Prikazana je multidimenzionalna optimizacija procesa izrade $elijskih terapija/bioterapeutskih sredstava sa prikazom vi"ezna#nih (ponekad kontradiktornih) ‘izlaza’ koji se o#ekuju (npr. smanjenje tro"kova uz maksimalni kvalitet ili koli#inu $elijskih terapija kao produkta) a koji su uslovljeni ne-linearnim ‘ulaznim’ parametrima (eng. ‘design space’) (Kirouac & Zandstra, 2008). Pri tome se u teorijskom smislu defini"e eng. response surface koji je predstavljen obojenom zakrivljenom povr"inom. Me)utim, uglavnom je nedostajao adekvatan pristup da se sagleda nivo kompleksnosti ovih procesa (koji je vi"ekomponentan i slojevit). Rezultati ove doktorske disertacije delimi#no su rasvetlili ta pitanja dovode$i u vezu teorije za razmatranje visoko slo%enih dinami#kih sistema i njihove geometrije tj. strukture. U smislu modelovanja dinami#kih sistema, trebalo bi planirati nastavak istra%ivanja, uzimaju$i u obzir sve specifi#nosti procesa izrade $elijskih terapija (kao grupe bioterapeutskih sredstava). Zna#ajno je da su u okviru teorijskih razmatranja u ovom istra%ivanju osnovni postulati bili geometrija sistema, mera njegove neuredjenosti i oscilacije tih istih parametara – dok se u prakti#nom modelu kao osnovni parametar razmatrao broj okarakterisanih $elija odnosno numeri#ka vrednost. Dakle, u oba pristupa, teorijskom i prakti#nom, posmatrana je dinamika sistema kao osnova njegove karakterizacije. Medjutim, tehnike i aparati koji se koriste u svakoj analizi (modelovanju) se kvalitativno razlikuju – geometrija i mera neuredjenosti sistema vs. numeri#ka vrednost biolo"ke promenljive tj. broja okarakterisanih $elija. Takodje, ako se uzmu u obzir glavne odrednice u teorijskom razmatranju kao "to je na primer, geometrija fraktala (kao sastavnih elemenata sistema), one mogu ali ne moraju da budu indikator dinamike sistema – iako se smatra da uti#u na nju. Istinski pokazatelj stanja dinami#kog sistema su oscilacije razli#itih parametara u njemu. Shodno tome, budu$a strate"ka razmatranja i planiranja u ovoj 149 obalsti trebalo bi jednim delom da se baziraju na primeni modelovanja dinami#kih sistema. Finansijski izazovi Finansijski aspekt celokupnog razvoja sektora $elijskih terapija se ne mo%e zanemariti iz vi"e razloga. Pre svega, zna#ajna finansijska sredstva su neophodna za sam razvoj (usled skupih tehnologija), neophodan je visoko stru#an kadar ("to takodje iziskuje zna#ajna sredstva), prate$e tehnologije u dijagnostici i pra$enju klini#kih primena su skupe, a u trenutku kada dodje do njihove "ire primene, o#ekuje se da $e tro"kovi ovakvih terapija biti veoma visoki sa aspekta zdravstvene za"tite (Klein, 2012). Regenerativna medicina, kao i njen deo koji se zasniva na $elijskim terapijama, se smatra novim poglavljem u medicinskim naukama jer u prakti#nom smislu objedinjuje znanja iz skoro svih nau#nih disciplina do sada ¶. Osim o#iglednih razloga u medicinskom smislu, regenerativna medicina ima ogroman ekonomski zna#aj. Na primer, u proteklih nekoliko godina SAD, na godi"njem nivou, tro"i oko 13% ukupnog doma$eg proizvoda (eng. Gross Domestic Product, GDP) na zdravstvo¶. Do 2040. godine se o#ekuje da $e populacija osoba preko 65 godina %ivota u SAD-u dosti$i 70 miliona ¶. Na taj na#in $e, smatra se, potro"nja na zdravstvo u SAD-u dosti$i 25% ukupnog doma$eg proizvoda ¶. Japan, Evropska zajednica, Kina i Australija nedavno su zapo#ele razli#ite zasebne inicijative za unapredjenje razvoja regenerativne medicine i $elijskih terapija ¶. Ovo je postala jedna od osnovnih postavki strate"kog razvoja u mnogim dr%avama koje dugoro#no planiraju svoja ulaganja i time obezbedjuju budu$i razvoj ove va%ne oblasti ¶. __________ ¶ www.dhhs.gov/reference/newfuture.shtml ¶¶ www.ey.com ^ www.biopharminternational.com 150 Globalno tr%i"te farmaceutskih sredstava je poraslo sa $693.7 milijardi USD u 2007. godini na o#ekivanih blizu $1 trilion USD ¶¶. Ipak, velike farmaceutske kompanije trenutno imaju problem sa razvojem novih terapeutskih sredstava "irom sveta ¶¶. Njihov interes za datu oblast bioterapeutika je zna#ajno porastao jer se u ovom trenutku 50% svih novih medicinskih sredstava u procesu razvoja – zasniva na biotehnolo"kim pristupima ¶¶. Dakle, "ira primena $elijskih terapija kao na#ina le#enja $e umnogome zavisiti od izvora finansiranja za zdravstvenu za"titu, bilo da su u pitanju javna finansiranja ili privatno zdravstveno osiguranje (Klein, 2012; Trounson et al., 2012; Parson, 2008; Pisano, 2006). Da bi se ovakav (novi) model zdravstvene za"tite uop"te uspostavio (uzimaju$i pri tom u obzir nove napredne terapije), potrebno je da se demonstrira njihova efikasnost, bezbednost upotrebe ali i finansijska isplativost u odnosu na postoje$e tzv. standardne terapijske pristupe (Chapman & Sonnenberg, 2000). Osim toga, potrebno je da se obezbede op"ti ekonomski uslovi za to. Skoro je izvesno da $e se ovakva promena prvo desiti u oblastima gde uop"te ne postoje adekvatni terapijski modaliteti, kao "to su neka neurodegenerativna oboljenja (na primer demencija ili Parkinsonova bolest) ili u le#enju akutnih povreda. Primena novih terapija $e biti bazirana na finansijkoj isplativosti dugoro#nih modela i modaliteta u zdravstvenoj za"titi, uzimaju$i u obzir trend starenja populacije u razvijenim zemljama kao i tendencije koje pokazuju grupe dominantnih oboljenja u njima (npr. kardiovaskularna oboljenja, bolesti metabolizma ili neurodegenerativne bolesti). __________ ¶ www.dhhs.gov/reference/newfuture.shtml ¶¶ www.ey.com ^ www.biopharminternational.com 151 Posredni rezultati koji su ve$ prepoznatljivi ili se o#ekuju u bli%oj budu$nosti, izmedju ostalog uklju#uju: smanjen potencijal za "iru primenu i komercijalizaciju $elijskih terapija, relativno malo znanja i informisanosti o ovome u "iroj javnosti kao i nizak 'rating' celokupnog sektora u smislu privatnih finansijskih ulaganja na globalnom nivou. Ako se tome doda da intelektualna svojina ovog sektora le%i uglavnom u istra%iva#kim univerzitetskim centrima i klini#kim institucijama, jasno je da model za isplativu primenu ovih terapija mora biti perfektno prezentovan da bi se privukao kapital privatnih investitora. Uz rizike i neizvesnost koje ova oblast nosi u finansijskom smislu, postoji ceo niz drugih problemati#nih aspekata kao "to su nedostatak odgovaraju$e infrastrukture (ili pristupa postoje$oj infrastrukturi), nespremnost istra%iva#a i lekara da udju u privatne kompanije, nedostatak adekvatno obu#enih rukovode$ih kadrova sa poznavanjem problematike oblasti i, jo" uvek nedovoljno podataka iz kontrolisanih rigorozno sprovedenih klini#kih istra%ivanja. Dok univerziteti do odredjene mere razvijaju svoje komercijalne pristupe u ovoj oblasti, bolni#ki centri i istra%iva#ki instituti jo" uvek nemaju mehanizme za to. U ovom trenutku izgleda da se kapital uliva iz velikih farmaceutskih kompanija koje postepeno pokazuju interes za uspostavljanje novih terapijskih pristupa baziranih na primeni $elija i drugih naprednih terapija kao grupe biolo"kih lekova (Kayser & Warzecha, 2012). Model se zasniva na uspostavljanju novih centara za ispitivanje lekova pri specijalizovanim grupama koje se bave istra%ivanjem $elijskih terapija. Na primer GlaxoSmithKline je ulo%io US$25 miliona dolara u Harvardski centar za mati#ne $elije jo" 2008. godine dok je u isto vreme Pfizer ulo%io US$100 miliona u novi Centar za regenerativnu medicinu u Kembrid%u (www.pfizer.com). Najprodavaniji biolo"ki lekovi su u proteklih dve do tri godine napravili ogroman finansijski uspeh tako da se o#ekuju dalja ulaganja velikih farmaceutskih korporacija u ovu vrstu istra%ivanja (Kayser & Warzecha, 2012). Nedostatak adekvatno obu#enih kadrova i infrastrukture, uz nau#no tehnolo"ke izazove i nedostatak podataka i klini#kih parametara (ili diskutabilan 152 kvalitet postoje$ih klini#kih podataka) ostaju kao najve$i problemi u razvoju sektora (Klein, 2012). Neposredni rezultati vi"ezna#ne – nau"no tehnolo!ke, zakonsko regulatorne, organizacione, strate!ke, ekonomske i eti#ke, slo%enosti su u ovom trenutku prepoznati na svim nivoima. Manje ili vi"e se razmatraju od nivoa malih i srednjih projekata, preko korporacijskih sistema do dr%avnih i regionalnih inicijativa (Klein, 2012; Trounson, 2009; Daley & Scadden, 2008; Kirouac & Zandstra, 2008). Usled svih kompleksnosti i prepreka u sada"njem i anticipiranom razvoju oblasti, logi#no je postaviti dva pitanja – koji su razvojni pravci i kako ih ostvariti. Postoji vi"e zna#ajnih primera, od kojih $e ovde biti pomenuti samo nekoliko – eng. Disease Teams i Alpha Stem Cell klinike, virtuelni GMP modeli, planiranje 'unazad' i personalizovana medicina uz 4Ps pristup. Nova re!enja ‘Disease Team’ i ‘The Alpha Stem Cell Clinic’ Modeli kao "to su eng. Disease Team (Klein, 2012) i eng. The Alpha Stem Cell Clinic (Trounson et al., 2012) proiza"li su iz CIRM (eng. California Institute for Regenerative Medicine) inicijativa u SAD-u i njihovih modela za finansiranje translatornih istra%ivanja. Za model nazvan Disease Teams je predvidjeno da integri"e sve neophodne aspekte u translaciji terapijskog kandidata (specifi#nog $elijskog proizvoda) da bi se taj proizvod na"ao u fazi klini#kih istra%ivanja u periodu od #etiri godine. To zna#i da bi u tom periodu, proizvod od interesa trebalo da bude u prvoj ili drugoj fazi klini#kih istra%ivanja (Klein, 2012). Za ovu svrhu su predvidjena sredstva i do $20USD miliona dolara po projektu, a od istra%iva#a se zahteva da obezbede svoj tim koji $e raditi na projektu. Timski pristup je najva%niji aspekt ovog modela a finansiranje je obezbedjeno od strane privatnih investitora (Klein, 2012). U okviru plasiranja ovog 153 modela i njegove prezentacije nau#noj javnosti, nagla"ava se zna#aj: edukacije "ire javnosti, uloge medija u ovom procesu kao i #injenice da se u svetu trenutno de"ava ‘revolucija mati#nih $elija’ (Klein, 2012). Predvidja se da $e model koji se naziva The Alpha Stem Cell Clinic ispuniti osnovne tri funkcije u procesu razvoja $elijskih terapija. U to spadaju – sprovodjenje klini#kih istra%ivanja, procena novog $elijskog proizvoda (pre svega se misli na bezbednost i efikasnost terapije), kao i obezbedjenje pristupa ovim terapijama za pacijente (Trounson et al., 2012). Pacijenti koji bi imali pristup ovim klinikama bili bi grupisani prema kategoriji u odnosu na nivo razvijenosti terapija koje im se nude. Od pacijenata koji nemaju bilo kakve druge terapijske opcije (stoga im se nudi eksperimentalna terapija) do pacijenata kojima su potrebni standardni terapijski pristupi (eng. standard of care treatment) koje bi platilo njihovo zdravstveno osiguranje. Klini#ka istra%ivanja bi u okviru ovog modela bila nezavisno finansirana, tako da mo%e da se posmatra kao umre%en system centara za klini#ka istra%ivanja (Trounson et al., 2012). Ova inicijativa bi trebalo da bude finansirana iz javnih fondova, uz mogu$nost da se deo tro"kova povrati kroz privatno zdravstveno osiguranje. Poslovni pristup u osnovi ove inicijative po svojoj strukturi podse$a na primenjeni model za centre koji obezbedjuju vantelesnu oplodnju (eng. IVF clinics). Pri tome mo%e da ponudi podr"ku projektima u dizajnu klini#kih istra%ivanja, kao i u prikupljanju dugoro#nih podataka o pacijentima i ishodima njihovih terapija ("to je do sada bio jedan od osnovnih nedostataka klini#kih istra%ivanja – nedovoljan broj pacijenata uz nedovoljno podataka vezanih za dugoro#ne efekte terapija) (Trounson et al., 2012). Smatra se da $e jedna od osnovnih uloga ove inicijative biti edukacija pacijenata, njihovih porodica i "ire javnosti. Virtuelni GMP model Za razliku od prethodnih modela, virtual GMP model se ne odnosi na dizajn i izvodjenje kilini#kih istra%ivanja ve$ na izradu #elijskih proizvoda. Jednostavnost ovog modela le%i u #injenici da se tim obu#enih stru#njaka (operativnih i regulatornih kadrova) anga%uje na projektu prema potrebi. Ovakav tim je obi#no iz ve$e institucije 154 ili iz klini#ko bolni#ke ustanove koja se bavi izradom $elijskih proizvoda. Iako je model dosta zastupljen na manjim projektima ne smatra se da je dugoro#no isplativ niti u skladu sa GMP zahtevima. Osim toga, nije prethodno bio zastupljen u farmaceutskoj industriji ili u biotehnolo"kom sektoru (Preti, 2005). Ipak, uz ograni#ena finansijska sredstva mo%e da pru%i podr"ku razvoju eksperimentalnih terapija. Planiranje ‘unazad’ Jedna od inicijativa Nacionalnog instituta za zdravlje (eng. National Institutes of Health, NIH), agencije FDA i istra%iva#a koji se bave $elijskim terapijama uklju#uje seriju edukativnih seminara koja se upravo odvija. Medju njima se u okviru razmatranja vezanih za translatorna istra%ivanja pluripotentnih $elija, pojavila inicijativa tzv. planiranja ‘unazad’ (eng. planning backward) – pri #emu se, pre nego "to osnovno istra%ivanje uop"te otpo#ne, ono planira uzimaju$i u obzir osnovne postulate koji $e biti zna#ajni tek u njegovim kasnijim translatornim fazama (Kleitman et al., 2013). Posebna pa%nja je posve$ena parametrima testiranja (materijala i reagenasa koji se koriste, %ivotinjskih modela, dizajna samog $elijskog proizvoda, dizajna procesa izrade, vrste kontrola u tom procesu). Akcenat je na dizajnu klini#kih istra%ivanja putem dizajna osnovnih (bazi#nih) i pre-klini#kih istra%ivanja koja im prethode. Personalizovana medicina i 4Ps pristup Novi pristupi u medicinskim naukama uklju#uju model nazvan 4Ps (eng. Personalized, Predictive, Preventive, Participatory) (Stanford Center for Genomics & Personalized Medicine www.stanford.edu; Sadee & Dai, 2005; Hood, 2006; Martinez de Lecea & Rossbach, 2012; An integrated map of genetic variation from 1,092 human genomes). Smatra se da je ova promena uslovljena pojavom nove nauke 155 (Hood, 2002) kojom se naziva molekularna tehnologija uz druge integrisane nauke (genomics, IT, matemati#ko modelovanje, integracija podataka, epigenetics). O#ekuje se da novi trendovi u pristupu definicijama bolesti i zdravlja, upotrebi genomics-a i njegovoj translaciji u klini#ku praksu – uspostave nove postulate biomedicinskih istra%ivanja, bioterapeutskih sredstava, kao i da promene fokus sa le#enja na predvidjanje i prevenciju (Stanford Center for Genomics & Personalized Medicine www.stanford.edu; Sadee & Dai, 2005; Hood, 2006; Martinez de Lecea & Rossbach, 2012; An integrated map of genetic variation from 1,092 human genomes). Smatra se da je osim nau#no tehnolo"ke zansovanosti ovog pristupa najva%nija edukacija i multidisciplinarnost u budu$im istra%ivanjima. U literaturi se nagla"ava da su tendencije ka usko stru#noj specijalizaciji u visokom "kolstvu danas u svetu prepoznate kao mogu$i problem za obrazovanje budu$ih stru#njaka koji bi u svom obrazovanju dobili najvi"e od sinteze i integracije saznanja iz vi"e razli#itih oblasti. Ovo se ve$ prepoznaje u okviru novih nau#nih disciplina kao "to su, izmedju ostalog, razvoj novih terapeutskih pristupa, primena genomics-a, proteomics-a i personalizovane/ individualizovane medicine (eng. genomics, proteomics & personalised medicine). U nove multidisciplinarne oblasti spadaju jo": hemijski bioin%enjering, epigenetska istra%ivanja, razvoj medicinskih naprava i pomagala uz primenu bioin%enjerstva, integracija informacione tehnologije, kao i procesni in%enjering, regulatorni in%enjering i druge discipline koje su trenutno u svojim ranim fazama razvoja. Ovo istra%ivanje je obavljeno nau#nim metodologijama koje se uobi#ajeno koriste u okviru medicine, sociolo"kih razmatranja, istra%ivanja u psihologiji, pravnim i politi#kim naukama. Medjutim, kontekst i konceptualna kompleksnost koju ono donosi razvoju egzaktnih nauka u oblasti bioterapeutskih sredstava, svrstava ga u red modernih multidisciplinarnih istra%ivanja #ija ekspanzija se o#ekuje u 21. veku. 156 5. Zaklju"ci Na osnovu sveobuhvatne kvalitativne studije procesa, sistema i regulativa u nizu klini#kih projekata, ovaj rad nudi detaljan protokol izrade i primene vi"e vrsta $elijskih terapija kao nove grupe biofarmaceutskih sredstava (eng. biopharmaceuticals) tj. novonastale grupe biolo"kih lekova (eng. biologic drugs). Uz pretragu literature u datoj oblasti razvoja i primene $elijskih terapija u Evropi, Sjedinjenim Ameri#kim Dr%avama i Australiji, u disertaciji je izvr"ena sveobuhvatna karakterizacija zakonsko regulatornih modela, savremenih tokova u klini#kim istra%ivanjima u ovoj oblasti, kao i teorijskih i prakti#nih modela u laboratorijskoj izradi i klini#koj primeni mati#nih $elija i drugih vrsta $elija u terapeutske svrhe. I U prvom delu istra%ivanja izvr"ena je, manuelna i automatizovana (uz pomo$ softverskog paketa Leximancer) analiza dokumenata i detaljan pregled zakonsko regulatornih modela u Evropi, Sjedinjenim Ameri#kim Dr%avama i Australiji – uklju#uju$i zakonske regulative za dobru proizvodja#ku praksu, dobru klini#ku praksu i druga zakonska dokumenta na najvi"em nivou – koji se odnose na primenu mati#nih $elija i drugih vrsta $elija kao nove generacije bioterapeutika tj. biolo"kih lekova. ! Odredjene su sli#nosti i razlike medju zakonsko regulatornim modelima koji se odnose na primenu mati#nih $elija i drugih vrsta $elija u terapeutske svrhe. ! Prikazana je i detaljno analizirana terminologija, centralne teme i koncepti zakonsko-regulatornih dokumenata na najvi"em nivou. ! Pokazano je da, iako svaki regulatorni model ima sopstvenu terminologiju i koncepte, postoji zajedni#ki fokus na kvalitet, bezbednost i efikasnost krajnjeg 157 proizvoda koji predstavlja bioterapeutsko sredstvo nove generacije tj. biolo"ki lek. ! Takodje je pokazano da razlike medju regulatornim agencijama, u formalnom smislu, postoje kroz najvi"e zakonsko regulatorne odredbe koje u ovoj analizi reprezentuju dati zakonski okvir za svaku od njih. II Analizirana je globalna baza podataka klini#ko istra%iva#kih projekata baziranih na primeni mati#nih $elija i drugih vrsta $elija u terapeutske svrhe u regionima Evrope, Sjedinjenih Ameri#kih Dr%ava, Australije i drugih delova sveta, prema kategorijama, geografskom regionu i tipu $elijskih terapija koje se primenjuju. Na osnovu dobijenih rezultata: ! Utvrdjen je broj ukupnih klini#kih istra%ivanja u datom periodu. ! Prikazani su statisti#ki podaci prema tipu $elijskih terapija od interesa, prema geografskom regionu i klju#nim terminima. ! Ukazano je na zna#ajan porast broja i opsega klini#kih istra%ivanja u oblasti $elijskih terapija. ! Pokazano je da postoje odredjeni trendovi u klini#ko istra%iva#kim projektima – uglavnom zasnovani na odredjenom tipu $elija koje se u tom trenutku koriste kao nova bioterapeutska sredstva tj. biolo"ki lekovi od interesa. III Metodologijom komparativne analize serije uzoraka (eng. case study) u tre$oj komparativnoj studiji prikazani su: a) Detaljni laboratorijski protokoli izrade mati#nih $elija i drugih $elijskih terapija, 158 ! Minimalno manipulisane $elije koje se koriste kao terapeutska sredstva i #iji proces proizvodnje uklju#uje samo zamrzavanje i #uvanje istih, namenjene za transplantaciju istom pacijentu (eng. autologous use); bez upotrebe kombinovanih proizvoda (npr. oboga$ivanja bilo kakvim proteinskim ili drugim agensima), bez dokazanog sistemskog efekta tj. sa visoko-specifi#nim terapeutskim efektom (eng. homogenous use), kao "to je proizvodnja i transplantacija mati#nih $elija hematopoeze izolovanih iz periferne krvi a namenjenih obnavljanju hematopoeze. ! Visoko-manipulisane $elije koje se koriste kao terapeutska sredstva, a #iji proces proizvodnje uklju#uje: • kulturu $elija (eng. ex vivo expansion), kao "to je proizvodnja i transplantacija adultnih mezenhimskih mati#nih/stromalnih $elija (eng. mesenchymal stem cells, MSC) izolovanih iz placente, namenjenih za upotrebu u razli#itim klini#kim indikacijama. • oboga$ivanje (eng. loading) razli#itim proteinskim ili drugim agensima, kao "to je proizvodnja i transplantacija antigen- prezentuju$ih dendriti#nih $elija (eng. dendritic cells, DC) izolovanih iz krvi pacijenta upotrebom $elijske separacije aferezom (eng. leukapheresis) i oboga$enih rekombinovanim proteinskim produktima, namenjenih za anti-tumorske terapije. b) Primena mati#nih $elija i drugih $elijskih terapija (klini#ki pristup) za: ! Rutinsku klini#ku primenu - izrada i primena minimalno manipulisanih $elija: umno%avanje i klini#ka primena hematopoetskih $elija iz periferne krvi; ! Primenu u istra%iva#ke svrhe - izrada i primena visoko manipulisanih $elija: umno%avanje i klini#ka primena dendriti#nih $elija u anti-tumor terapiji; ! Istra%iva#ke svrhe i za "iru klini#ku primenu (eng. 'off-the-shelf') - izrada i primena visoko manipulisanih $elija: umno%avanje i primena mezenhimskih mati#nih/stromalnih $elija za razli#ite klini#ke primene i klini#ka istra%ivanja. 159 IV U #etvrtoj komparativnoj studiji uporednom analizom postoje$ih laboratorijsko- organizacionih modela: ! Analizirana su tri odr%iva modela, koji su se do sada pokazali kao finansijski i zakonski prihvatljivi, a koji se odnose na izradu mati#nih $elija i drugih vrsta $elija kao nove generacije bioterapeutskih sredstava. ! Predlo%eni modeli mogu biti primenjeni kako u okviru akademskih institucija, tako i u okviru komercijalnih jedinica pri velikim akademskim ili klini#kim institucijama, ili u nezavisnim komercijalnim kompanijama (eng. biotech). U okviru iste studije izvr"ena je fenomenolo"ka/tematska analiza i analiza sadr%aja transkripta intervjua sa 24 svetska stru#njaka koji su ili direktno uklju#eni u izradu $elijskih terapija za klini#ku primenu, ili u#estvuju u drugim oblastima razvoja bazi#nih i klini#ko-primenljivih biolo"kih i medicinskih istra%ivanja, bioetike i strate"kog razvoja $elijskih terapija za klini#ku primenu. Procenjeni su zna#ajni aspekti i date su preporuke za prakti#na re"enja i uspe"an razvoj projekata u relativno novoj i veoma kompleksnoj oblasti izrade novih generacija biolo"kih lekova. Na osnovu dobijenih rezultata mo%e se zaklju#iti da ne postoji jedan univerzalan model koji bi u potpunosti zadovoljio potrebe izrade $elijskih terapija ali su opisana tri prakti#na modela od zna#aja za dalji razvoj i primenu ove grupe bioterapeutskih sredstava. 160 V Uz primenu teorijskog i prakti#nog modelovanja razvijena su dva teorijska modela koji, u zavr"nom delu istra%ivanja, nude mogu$nost teorijskih razmatranja, planiranja, predvidjanja (eng. forecasting) i projektovanja vezanog za budu$i razvoj ove nau#ne oblasti; takodje je razvijen jedan prakti#ni model evaluacije klini#ke metode koji je dostupan za primenu u novim projektima. Analizom teorija modelovanja i analizom prakti#nih primera date su osnove za dalja istra%ivanja modelovanja biosistema i mo%e se zaklju#iti da se u oba pristupa, teorijskom i prakti#nom, posmatra dinamika sistema kao osnova njegove karakterizacije. Takodje, ako se uzmu u obzir glavne odrednice u teorijskom razmatranju kao "to je na primer, geometrija fraktala (kao sastavnih elemenata sistema), one mogu ali ne moraju da budu indikator dinamike sistema – iako se smatra da uti#u na nju. Istinski pokazatelj stanja sistema su oscilacije razli"itih parametara u njemu. Sli#no tome, u prakti#nom modelu koji u osnovi razmatra jedan kompleksan i relativno neuredjen biolo"ki sistem, broj okarakterisanih $elija (koje su sastavni deo sistema i imaju svoju specificnu geometriju, "to ih posredno dovodi u vezu sa geometrijom fraktala), posmatrana numeri#ka vrednost istih okarakterisanih $elija konvergira ka svom biolo"kom optimumu i oscilira u datom fiziolo"kom opsegu usled uticaja razli#itih unutra"njih i spolja"njih faktora – upravo kao kod drugih nelinearnih dinami#kih sistema koje razmatra teorija haosa. i 6. Literatura Atala A. The Journal of Tomorrow is a Reality Today (Editorial). Stem Cell Translational Medicine 2013;2:1-2. Atala A. 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Prilozi tezi Prilog 1: Lista skra!enica i definicija Abbreviations (eng.) EMA European Medicines Agency FACT Foundation for the Accreditation of Cell Therapies FDA Food and Drug Administration NPAAC National Pathology Accreditation Advisory Council TGA Therapeutic Goods Administration PBSC Peripheral Blood Stem Cells (also referred to as: PBPC) a.k.a. also known as PBPC Peripheral Blood Progenitor Cells e.g. for example HPC Haematopoietic Progenitor Cells vs. versus HPC-A Haematopoietic Progenitor Cells (harvested by peripheral blood apheresis) R&D Research and Development MSC Mesenchymal Stem Cells (non embryonic stem cells) IP Intellectual Property DC Dendritic Cells (from blood) APC Antigen-presenting cells QMS Quality Management System HDT High-dose therapy SACTP “Standardised approach to cell therapy production” HR Hematologic reconstitution GMP Good Manufacturing Practice ROI Return on investment GCP Good Clinical Practice ESC Embryonic Stem Cells iPS Induced pluripotent stem cells HLA Human Leukocyte Antigen vs. versus ATMP Advanced Therapy Medicinal Products. HCT/P Human Cell and Tissue Products ARTG Australian Register of Therapeutic Goods. ICH International Conference on Harmonisation CBER Center for Biologics Evaluation and Research. IND Investigational New Drug Application CDER Center for Drugs Evaluation and Research IRB Institutional Review Board CFR Code of Federal Regulation MHRA MHRA, Medicines and Healthcare Products Regulatory Agency cGTP Current Good Tissue Practices OECD OECD, Organisation for Economic Cooperation and Development EEA- EFTA European Economic Area- European Free Trade Association PIC/S Pharmaceutical Inspection Convention (PIC) and Pharmaceutical Inspection Co-operation Scheme EMA European Medicines Agency PMDA Pharmaceutical and Medical Devices Agency EU European Union TGA Therapeutics Goods Administration FDA Food and Drug Administration Definitions (eng.) Life science A branch of science (as biology, medicine, and sometimes anthropology or sociology) that deals with living organisms and life processes – usually used in plural. (Merriam-Webster Online Dictionary http://www.merriam-webster.com/dictionary/life+science ) Life science Any of several branches of science, such as biology, medicine, anthropology, or ecology, that deal with living organisms and their organization, life processes, and relationships to each other and their environment. Also called bioscience. (Free Dictionary Online http://www.thefreedictionary.com/life+science ) Biotechnology The manipulation (as through genetic engineering) of living organisms or their components to produce useful usually commercial products (as pest resistant crops, new bacterial strains, or novel pharmaceuticals); also : any of various applications of biological science used in such manipulation. (Merriam-Webster Online Dictionary http://www.merriam-webster.com/dictionary/biotechnology ) Biotechnology The use of living things, especially cells and bacteria, in industrial processes. (Cambridge International Dictionary of English http://dictionary.cambridge.org ) Minimal manipulation Includes centrifugation, refrigeration, freezing, trimming, flushing, washing; processing steps related to preserving function or minimising contamination, including using additives such as cryopreservatives, anticoagulants, antimicrobial agents and irradiation; and freeze drying (of structural tissues only). (Australian Regulatory Guidelines for Biologicals http://www.tga.gov.au/pdf/biologicals-argb-p1.pdf ) Homologous use The repair, reconstruction, replacement or supplementation of a recipient’s cells or tissues with a biological that performs the same basic function in the recipient as in the donor. This definition is internationally consistent and relates to the use of the product independent of whether the recipient is the same as the donor (autologous) or different from the donor (allogeneic). (Australian Regulatory Guidelines for Biologicals http://www.tga.gov.au/pdf/biologicals-argb-p1.pdf ) Complex methods of modification Include processes that are not listed under ‘minimal manipulation’, including such processes as demineralisation or enzymatic dissociation. Genetic modification is also a complex method. (Australian Regulatory Guidelines for Biologicals http://www.tga.gov.au/pdf/biologicals-argb-p1.pdf ) Definicije Davalac Davalac po!etne populacije "elija namenjenih za transplantaciju. Recipijent/ Primalac Primalac zavr#nog "elijskog produkta ("elija pripremljenih za transplantaciju). Singena transplantacija (i/ili "elijska terapija) Transplantacija izmedju genetski podudarnih (identi!nih) osoba kao sto su jednojajni blizanci. 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B5465! 4;! 3K;>>@!8458>51;31045!4C! 17>!3810A01?!<>05K!9>3/2;>@D! *1!/742=@!<>!3//3?>@!6017!>387! K;42:!4C! 1>/1/!14!>/13<=0/7!17>!/>5/010A01?!4;!83=0<;31045!4C!17>!25B5465!1>/1/!05!17>!K;42:D! "'*%+( -&'%*'"*2( %:@E6( ?@3?@43(9:86(><(E<@3I(&@?34643(C6(>D695(A@5?5(4<>4:D6(=6(F3(><( 5>?:>46D3B6(:><4B83D:>4(3B3(96B3F@67386(;4:D6(5(4CDI(F64<@383(4<>4:D6I( ( Prilog 3: Lista tabela i slika Lista tabela: Tabela 1.1: Tipovi transplantacije, bilo da su u pitanju mati!ne "elije iz kostne sr#i, mati!ne "elije izolovane iz pup!anika ili iz periferne krvi. Tabela 1.2: Osnovne komponente transplantacije mati!nih "elija hematopoeze. Tabela 1.3: Izvor mati!nih "elija za klini!ku upotrebu u svrhe transplantacije. Tabela 1.4: Prednosti i nedostaci razli!itih tipova transplantacije mati"nih "elija. Tabela 1.5: Definicije koje se odnose na zna!enje pojmova “regenerativna medicina” i “"elijske terapije” u kontekstu regenerativne medicine. Tabela 1.6: Definicije koje se odnose na zna!enje pojmova “biotehnologija”, “nova biotehnologija” i “biofarmaceutska industrija”. Tabela 1.7: Prakti!ni aspekti izrade "elijskih terapija koji !esto ograni!avaju proces razvoja novih biolo$kih lekova u toj oblasti. Tabela 1.8: Kategorije HCT/P proizvoda koje ne zadovoljavaju sve kriterijume propisane u regulativi 21 CFR 1271.10(a). Tabela 1.9: Biolo$ki proizvodi koji zadovoljavaju ili ne zadovoljavaju kriterijume propisane u novom zakonskom okviru (eng. Biologicals Regulatory Framework). Tabela 1.10: Podr$ka koju agencija EMA nudi individualnim i organizacionim projektima za napredne terapije u ranom stadijumu razvoja kao dodatak inicijativi eng. Innovation Task Force, ITF i uloga Komiteta za napredne terapije (eng. Committee for Advanced Therapies, CAT). Tabela 1.11: Kompleksnost zakonskih propisa u Evropskoj zajednici koji se odnose na napredne terapije (eng. ATMP) a sastoje se iz vise regulativa i direktiva. Tabela 1.12: Domeni u procesu transplantacije !elija i tkiva koji su regulisani Zakonom (Zakon o transplantaciji !elija i tkiva, Sl. glasnik RS br. 72/2009). Tabela 1.13: Realnost i o!ekivanja u razvoju "elijskih terapeutika. Tabela 3.1: Kriterijumi za izbor analiziranih dokumenata. Tabela 3.2: Lista analiziranih dokumenata. Tabela 3.3: Odabir analiziranih protokola i laboratorijskih modela izrade "elijskih terapija za klini!ku upotrebu i klini!ka istra#ivanja. Tabela 3.4: Odabir analiziranih laboratorijsko-organizacionih modela. Tabela 3.5: Proces manuelne analize dokumenata. Tabela 3.6: Pristup analizi podataka prikupljenih kroz intervjue. Tabela 3.7. Kriterijumi za evaluaciju klini!ke metode prikupljanja mati!nih/progenitorskih "elija hematopoeze aferezom. Tabela 4.1: Rezultat manuelne analize dokumenata iz grupe 1: u!estalost termina. Tabela 4.2: Rezultat manuelne analize dokumenata iz grupe 1: u!estalost fraza. Tabela 4.3: Rezultat softverske analize dokumenata iz grupe 1 i 2: naju!estaliji koncepti u pojedina!nim regulatornim okvirima i u okviru regulatornih principa (GMP(1), GMP(2), GCP). Tabela 4.4: Rezultati softverske analize dokumenata iz grupe 1 i 2: Tematski pregled u pojedina!nim regulatornim okvirima i u okviru regulatornih principa (GMP(1), GMP(2), GCP). Tabela 4.5: Frekvencije (eng. high, medium & low) i stepen konektivnosti (%) izmedju tema koje su identifikovane u dokumentima grupe 1 i 2 (eng. cohorts 1 and 2). Tabela 4.6: Broj klini!kih istra"ivanja na svetskom nivou u januaru 2011. i u avgustu 2013. godine. Tabela 4.7: Broj klini!kih istra"ivanja na svetskom nivou u januaru 2011. i u avgustu 2013. godine na osnovu klju!nih re!i “cell therapies”. Tabela 4.8: Broj klini!kih istra"ivanja na svetskom nivou u januaru 2011. i u avgustu 2013. godine na osnovu klju!nih re!i “cell therapies AND stem cell”. Tabela 4.9: Va"ni elementi u procedurama, protokolima i !uvanju podataka za potrebe laboratorijske izrade i klini!ke primene minimalno manipulisanih mati!nih/ progenitorskih #elija izolovanih iz periferne krvi uz primenu afereze (prema brojnim standardima i regulatornim odredbama). Tabela 4.10: Osnovni elementi laboratorijske izrade i klini!ke primene minimalno manipulisanih mati!nih/ progenitorskih #elija izolovanih iz periferne krvi uz primenu afereze. Tabela 4.11: Elementi laboratorijske izrade visoko manipulisanih mezenhimskih mati!nih/ stromalnih #elija, MSC (dobijenih metodom izolovanja iz placente i ex vivo umno"avanja). Tabela 4.12: Elementi laboratorijske izrade visoko manipulisanih #elija (metodom ex vivo & loading umno"avanja i oboga#ivanja #elija proteinima/rekombinantnim antigen proteinima). Tabela 4.13: Karakterizacija organizacionih modela: uporedna analiza (1). Tabela 4.14: Karakterizacija organizacionih modela: uporedna analiza (2). Tabela 4.15: Rezultati vezani za poslovne i finansijske aspekte uz osnovna razmatranja i izazove identifikovane od strane u!esnika (eng. Business Models & Funding; Strategic Decision Making). Tabela 4.16: Rezultati vezani za poslovne i finansijske aspekte uz osnovna razmatranja i izazove identifikovane od strane u!esnika (eng. Main Considerations in Relation to Project or Organisation). Tabela 4.17: Rezultati vezani za uspostavljanje saradnje sa partnerima i druga razmatranja vezana za razvoj proizvodnje bioterapeutika/ #elijskih terapija (eng. Partnerships & Main Challenges in Relation to Industry/ Field). Tabela 4.18: Rezultati vezani za tehnolo$ka, nau!na i druga razvojna razmatranja vezana za proizvodnju bioterapeutika/ #elijskih terapija (eng. Science, Technology, Tech Transfer & Up Scaling) uz generalna zapa"anja (eng. About Industry). Tabela 4.19: Rezultati vezani za primenu zakonsko regulatornih odredbi (eng. Regulatory Compliance). Tabela 4.20: Rezultati vezani za primenu zakonsko regulatornih odredbi i njihov razvoj u $irem smislu (eng. Regulatory Landscape). Tabela 4.21: Osnovne teme i kategorije dobijene analizom intervjua. Tabela 4.22: Utemeljenje rezultata u podacima: O industriji (eng. About Industry). Tabela 4.23: Utemeljenje rezultata u podacima: Tehnologija i razvoj (eng. Technology & Development). Tabela 4.24: Utemeljenje rezultata u podacima: Zakonske regulative, upravljanje rizicima, Dobra proizvodja!ka praksa (eng. Regulatory compliance, risk management, GMP). Tabela 4.25: Utemeljenje rezultata u podacima: Promene u zakonsko regulatornim odredbama (eng. Regulatory Shift). Tabela 4.26: Utemeljenje rezultata u podacima: Otvorenost i saradnja u industriji (eng. Industry openess). Tabela 4.27: Podaci za uspostavljanje korelacije izmedju broja prikupljenih "elija (x) i o!ekivanog broja "elija (y) u 111 klini!kih procedura afereze obavljenih u periodu od dve godine. Tabela 4.28: Rezultati statisti!ke obrade podataka i korelacije izmedju broja prikupljenih "elija (x) i o!ekivanog broja "elija (y) u 111 klini!kih procedura afereze obavljenih u periodu od dve godine. Lista slika: Slika 1.1 Razvoj i hijerarhija mati!nih "elija hematopoeze. Slika 1.2: Morfologija mezenhimskih "elija izolovanih iz placente obojenih fluoroscentnom bojom Phalloidin-AF546. Slika 1.3: Prakti!ni aspekti izrade "elijskih terapija koji !esto ograni!avaju proces razvoja novih biolo#kih lekova u toj oblasti Slika 1.4: Definicija naprednih terapija prema Reg. 1394/2007 Evropskog parlamenta. Slika 3.1: Statisti!ka metoda obrade podataka (eng. Spearman rank correlation) za uspostavljanje korelacije izmedju broja prikupljenih "elija i o!ekivanog broja "elija. Slika 4.1: Manuelna analiza dokumenata: frekvencija upotrebe termina u dokumentima iz grupe 1 (eng. cohort 1). Slika 4.2: Manuelna analiza dokumenata: frekvencija upotrebe fraza u dokumentima iz grupe 1 (eng. cohort 1). Slika 4.3: Konceptualne mape i koncepti identifikovani u slede"im dokumentima: dokumenta agencije EMA (A), dokumenta agencije FDA (B), dokumenta agencije TGA (C). GMP(1) dokumenta (D), GMP(2) dokumenta (E), GCP dokumenta (F). Slika 4.4 A-C: Konceptualni oblaci i teme (konceptualne grupe) identifikovani u dokumentima softverskom analizom. Slika 4.4 D-F: Konceptualni oblaci i teme (konceptualne grupe) identifikovani u dokumentima softverskom analizom. Slika 4.6: Naju!estalije teme i koncepti (A, B). Slika 4.6: Naju!estalije teme i koncepti (C). Slika 4.7: Mapa klini!kih istra$ivanja na svetskom nivou u januaru 2011. godine. Slika 4.8: Mapa klini!kih istra$ivanja u Evropi u januaru 2011. godine. Slika 4.9: Mapa klini!kih istra"ivanja u Evropi u avgustu 2013. godine. Slika 4.10: Mapa klini!kih istra"ivanja na osnovu pretrage klju!nih re!i “cell therapies AND adult stem cell” u januaru 2011. godine. Slika 4.11: Mapa klini!kih istra"ivanja uz upotrebu mezenhimskih #elija na svetskom nivou (pretraga uz klju!ne re!i “cell therapies AND mesenchymal stem cells”) u januaru 2011. godine. Slika 4.12: Mapa klini!kih istra"ivanja uz upotrebu mezenhimskih #elija na svetskom nivou (pretraga uz klju!ne re!i “cell therapies AND mesenchymal stem cells”) u avgustu 2013. godine. Slika 4.13: Mapa klini!kih istra"ivanja uz upotrebu autolognih terapija (903 u SAD-u, 338 u Evropi i 19 u Australiji) koja su izlistana na osnovu klju!nih re!i “cell therapies AND autologous” u januaru 2011. godine. Slika 4.14: Dijagram procesa prikupljanja i obrade visoko manipulisanih mezenhimskih mati!nih/ stromalnih #elija, MSC (dobijenih metodom izolovanja iz placente i ex vivo umno"avanja). Slika 4.15: Dijagram procesa prikupljanja, obrade, primene i efekta visoko manipulisanih antigen prezentuju#ih dendritskih #elija, DC (dobijenih metodom umno"avanja i oboga#ivanja rekombinantnim antigenom). Slika 4.16: Horizontalni model izrade bio-terapeutika sa ‘standardnim’ pristupom (elementi i faze). Slika 4.17: Hijerarhijski model organizacije i izrade (elementi i faze). Slika 4.18: Dijagram procesa razvoja #elijskih terapija/bioterapeutskih sredstava i osnovne faze: otkri#e, optimizacija, produkcija i isporuka. Slika 4.19: Multidimenzionalna optimizacija procesa izrade #elijskih terapija/bioterapeutskih sredstava sa prikazom vi$ezna!nih (ponekad kontradiktornih) ‘izlaza’ koji se o!ekuju. Slika 4.20: Ilustarcija nepredvidivosti dugoro!nog predvidjanja u neuredjenim sistemima usled fenomena nazvanog 'sensitive dependance’. Slika 4.21: Primer fraktalne dimenzije (eng. Sierpinski gasket). Slika 4.22: Rezultat statisti!ke obrade podataka (eng. Spearman rank correlation) za uspostavljanje korelacije izmedju broja prikupljenih #elija i o!ekivanog broja #elija (Graph 1). Slika 4.23: Rezultat statisti!ke obrade podataka (eng. Spearman rank correlation) za uspostavljanje korelacije izmedju broja prikupljenih #elija i o!ekivanog broja #elija (Graph 2). Slika 4.24: Rezultat statisti!ke obrade podataka (eng. Spearman rank correlation) za uspostavljanje korelacije izmedju broja prikupljenih #elija i o!ekivanog broja #elija (Graph 3). Prilog 4: Visoko manipulisane !elije (MSC) Adaptirano iz Slaper-Cortenbach, 2008 Slika P4.1: Polo!aj visoko manipulisanih mezenhimskih mati"nih/stromalnih #elija (MSC) pripremljenih za klini"ku primenu u razmatranjima evropskih regulativa. Transfus Med Hemother 2008;35:295–298Current Regulations for the Production of Multipotent Mesenchymal Stromal Cells for Clinical Application 297 The biological testing must be carried out by a qualified labo- ratory, authorized as a testing center by the CA in the Mem- ber States using EC-marked testing kits. Moreover, even if MSCs are cultured from cells of autologous donors, the same tests need to be done. This is not aiming at the prevention of donation of the cells, but it is included because it is essential for minimization of the risk of cross-contamination during the expansion of MSCs. Adverse events and reactions must also be reported to the CA within the Member States. Clear definitions of the adverse events and reactions which may be caused by MSCs do not yet exist. This may in part be due to the status of the product (still being used in smaller clinical trials only) and the fact that they are being applied in many different diseases with various routes of application (graft versus host disease, inborn errors, autoimmune disease, cardiac failure, as part of a tissue engi- neered product in combination with all sorts of matrices etc.). This, however, should not block the reporting of adverse events and reactions. In my view, it is up to the scientific com- munity to start the discussion on the adverse events and reac- tions very soon, to assist CA and the CAT of EMEA in their evaluation of the quality and safety of the MSCs. European Directive on Clinical Trials and Good Manufacturing Practice In the Clinical Trials Directive 2001/20/EC [8] which came into force in April 2001, the implementation of good clinical prac- tice (GCP) is described. GCP is a set of internationally recog- nized ethical and scientific quality measurements, which must be observed for designing, conducting, recording and report- ing clinical trials that involve the participation of humans. In article 13.3(a) of this EUD, it is stated that each batch of MPs needs to be manufactured according to the good manufactur- ing practice (GMP) for medicinal products for human use (Di- rective 2003/94/EC) [9]. The guidelines for GMP are also pub- lished and can be retrieved from the website of the European Commission as part of the EU Pharmaceutical legislation [10]: volume 4 of ‘The rules governing medicinal products in the European Union’. It contains guidance for the interpretation of the principles and guidelines of GMPs for medicinal prod- ucts for human and veterinary use. So, MSCs as part of a clini- cal trial need to be manufactured under GMP. In addition, it describes the documentation to be sent to the Ethics Commit- tee as well as the Investigational Medicinal Product Dossier (IMPD). For all cellular products, including MSCs, it is still very difficult to complete an IMPD, and input of scientific committees in restructuring this pharmaceutical document for MP is, in my view, essential. EUD on Medicinal Products for Human Use The EU Directive 2001/83/EC [11] regulates products that are classified as MPs for human use and which are being industri- ally produced to be placed on the market. This EUD includes 2 types of products: somatic cell therapy MPs and gene thera- py MPs. The Directive defines a MP as any substance or com- bination of substances presented for treating or preventing disease in human beings. 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Intro/ Background In the early 1990's, the Centers for Disease Control and Prevention (CDC) reported that human immunodeficiency virus (HIV) had been transmitted through transplantation of human tissue. Information was also reported which suggested that potentially unsafe tissue was being imported into the United States for transplantation into humans. Prompted by reports that potentially unsafe bone was being imported, the Commissioner of Food and Drugs ordered an immediate investigation. Information resulting from this investigation identified an immediate need to protect the public health from the transmission of HIV and hepatitis B and C through transplantation of unsuitable tissue. Concerns that disease transmission could occur, coupled with information derived from these investigations, prompted the Food and Drug Administration (FDA, the Agency) to publish an interim rule in December 1993 that specifically required certain communicable disease testing, donor screening, and record-keeping for human tissue intended for transplantation. A final rule was issued in July 1997. FDA chose to regulate tissues under the legal authority of Section 361 (Sec. 361) of the Public Health Service Act (hereafter, PHS Act) [42 USC 264]. This section authorizes the Surgeon General, with the approval of the Secretary, Department of Health and Human Services, to make and enforce such regulations as judged necessary to prevent the introduction, transmission, or spread of communicable diseases from foreign countries into the United States or from State to State. Section 361 of the PHS Act focuses on preventing the introduction, transmission, and spread of communicable diseases. In 1997, the agency announced its plans for human cells, tissues, and cellular and tissue-based products (HCT/Ps) in two documents: "A Proposed Approach to the Regulation of Cellular and Tissue- Based Products" (62 FR 9721, March 4, 1997) and "Reinventing the Regulation of Human Tissue". FDA requested written comments on its proposed approach and, on March 17, 1997, held a public meeting to solicit information and views from the interested public. Since that time, the Agency has published three final rules and one interim final rule to implement aspects of the proposed approach. On January 19, 2001, FDA issued regulations to create a new unified system for registering HCT/P establishments and for listing their HCT/P's (registration final rule, 66 FR 5447). The registration rule became effective in two stages. The first effective date, April 4, 2001 was applicable to establishments that were already regulated under 21 CFR Part 1270. The second effective date was originally January 21, 2003, and was applicable to establishments that manufacture HCT/Ps currently regulated as biological products, drugs, or devices, hematopoietic stem cells from peripheral and cord blood, and reproductive cells and tissues. On January 21, 2003, FDA announced that the registration requirements for these establishments would be further delayed until January 21, 2004. On January 27, 2004, FDA issued an interim final rule to except human dura mater and human heart valve allografts from the scope of that definition until all of the tissue rules became final. On May 25, 2004, FDA promulgated regulations requiring most cell and tissue donors to be tested and screened for relevant communicable diseases (donor-eligibility final rule, 69 FR 29786). On November 18, 2004, FDA issued regulations that require establishments that manufacture HCT/Ps to comply with Current Good Tissue Practices (CGTP), which would include, among other things, proper handling, processing, labeling, and record-keeping procedures. The regulations require each establishment to maintain a quality program to ensure compliance with CGTP. In addition, with the implementation of CGTPs, human dura mater and human heart valve allografts are now included in the scope of HCT/Ps regulated under 21 CFR 1271. On May 25, 2005 FDA published an interim final rule to revise certain regulations regarding the screening and testing of HCT/P donors and related labeling (interim final rule, 70 FR29949). FDA took this action in response to comments from interested persons regarding the impracticability of complying with certain regulations as they affect particular HCT/Ps. ! ! 4 21 CFR Part 1271 The CGTP and other regulations are contained in 21 CFR Part 1271, along with provisions relating to establishment registration. These regulations will apply to HCT/Ps recovered on or after the rule's effective date, May 25, 2005. HCT/Ps that were recovered before the effective date of the new rules are subject to 21 CFR 1270, and subparts A and B of Part 1271, as appropriate. In addition, 21 CFR Part 1271 subparts A, B, C, F, 21 CFR 1271.150(c), and 21 CFR 1271.155 of subpart D apply to reproductive HCT/Ps. The new Part 1271 is made up of six subparts: 1. General provisions pertaining to the scope and purpose of Part 1271, as well as definitions. 2. Registration and listing procedures. 3. Provisions for the screening and testing of donors to determine their eligibility. 4. Current Good Tissue Practice (CGTP) requirements. 5. Certain labeling and reporting requirements. 6. Inspection and enforcement provisions. 21 CFR 1271.10(a) sets out the criteria that form the foundation of our tiered, risk-based approach to regulating HCT/Ps. HCT/Ps that meet all of these criteria are subject only to regulation under section 361 of the PHS Act. These HCT/Ps are subject to the regulations in Part 1271, and no pre-market approval is required. HCT/Ps that do not meet all of the criteria in 21 CFR 1271.10(a) are regulated as drugs, devices, and/or biological products. These HCT/Ps are subject to the regulations specific to drugs, biological products, or medical devices, in addition to applicable sections of Part 1271. HCT/Ps covered by this program include: Bone (including demineralized bone), Ligaments, Tendons, Fascia, Cartilage, Ocular Tissue (Corneas and Sclera), Skin, Arteries and Veins (except umbilical cord veins), Pericardium, Amniotic membrane (when used alone, without added cells for ocular repair), Dura mater, Heart valve allografts, Semen, Oocytes, Embryos and Hematopoietic stem/progenitor cells derived from peripheral and cord blood. 361 HCT/Ps – Definition The above HCT/Ps are regulated solely under section 361of the PHS Act and the regulations in 21 CFR Part 1271 if they meet all of the following criteria: -Minimally manipulated; -Intended for a homologous use only as reflected by the labeling, advertising, or other indications of the manufacturer's objective intent; -Not combined with another article, (except for water, crystalloids, or a sterilizing, preserving, or storage agent, if the addition of the agent does not raise new clinical safety concerns with respect to the HCT/P); AND Either: -Do not have a systemic effect and are not dependent upon the metabolic activity of living cells for the primary function; OR -Have a systemic effect or are dependent upon the metabolic activity of the other cells for the primary function, AND: -Are for autologous use; ! 5 -Are for allogeneic use in a first or second-degree relative; OR -Are for reproductive use. ! HCT/Ps not regulated under section 361of the PHS Act and the regulations in 21 CFR Part 1271 and Products covered by other Compliance Programs Those HCT/Ps that do not meet all 21 CFR 1271.10(a) criteria and are regulated as drugs, devices, or biological products are covered under separate compliance programs, such as: - Blood and Blood Products are covered under CP 7342.001 "Inspection of Licensed and Unlicensed Blood Banks, Brokers, Reference Laboratories, and Contractors"; and CP 7342.002 "Inspection of Source Plasma Establishments" - HCT/Ps that do not meet all 21 CFR 1271.10(a) criteria, and are regulated as Medical Devices are covered under CP 7382.845 "Inspection of Medical Device Manufacturers" - HCT/Ps that do not meet all 21 CFR 1271.10(a) criteria, i.e. Autologous, Allogeneic, or Xenogeneic Cells whose biological characteristics have been altered (propagate, pharmacologically treated, etc.); Ex Vivo and Gene Therapy products are regulated as biological drugs and are covered under CP 7345.848 "Inspection of Biological Drug Products" - HCT/Ps recovered before May 25, 2005 and regulated under 21 CFR 1270 and subparts A and B of Part 1271 are covered under CP 7341.002A "Inspection of Tissue Establishments". Current Good Tissue Practices (CGTPs): HCT/P establishments must follow CGTP requirements to prevent the introduction, transmission, or spread of communicable diseases by ensuring that the HCT/Ps do not contain communicable disease agents, that they are not contaminated, and that they do not become contaminated during manufacturing. The following are Core CGTP requirements as referenced in 21 CFR 1271.150(b): -Requirements relating to facilities (21 CFR 1271.190(a) and (b)) -Requirements relating to environmental controls (21 CFR 1271.195(a)) -Requirements relating to equipment (21 CFR 1271.200(a)) -Requirements relating to supplies and reagents (21 CFR 1271.210(a) and (b)) -Requirements relating to recovery (21 CFR 1271.215) -Requirements relating to processing and process controls (21 CFR 1271.220) -Requirements relating to labeling controls (21 CFR 1271.250(a) and (b)) -Requirements relating to storage (21 CFR 1271.260(a) - (d)) -Requirements relating to receipt, pre-distribution shipment, and distribution of an HCT/P (21 CFR 1271.265(a) - (d)). -Requirements relating to donor eligibility determinations, donor screening, and donor testing (sections 1271.50, 1271.75, 1271.80 and 1271.85). ! 6 Establishment Registration, Listing, and Inspection status: - All establishments engaged in manufacture (as defined in 21 CFR 1271.3(e)) of an HCT/P must register with and submit to FDA, a list of each human tissue product manufactured unless excepted by 21 CFR 1271.15. - New establishments must register and list within 5 days of beginning operations. - CBER maintains an alphabetic listing of currently registered HCT/P establishments that is accessible on the CBER internet web site at http://www.fda.gov/cber/tissue/hctregestabl.htm. Donor Eligibility Determination: - HCT/P establishments must screen and test HCT/P donors for risk factors for, and clinical evidence of, relevant communicable disease agents and diseases and communicable disease risks associated with xenotransplantation. - Procedures for all steps that the HCT/P establishment performs in testing, screening, and determining donor eligibility must be established and maintained. These procedures must be designed to ensure compliance with the requirements of subpart C, 21 CFR Part 1271. - Donor eligibility determination must be based upon the results of donor screening in accordance with 21 CFR 1271.75 and donor testing in accordance with 21 CFR 1271.80 and 1271.85. - Certain records must accompany the HCT/P at all times once a donor eligibility determination has been made (21 CFR 1271.55). 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(Source: official websites). Name of the Standard/ Regulation or Guidelines and Regulatory Agency Abbreviated Name of Standard/ Guidelines or Issuing Authority Purpose and Context Scope Standards for Haemopoietic Progenitor Cell Collection, processing and Transplantation Foundation for the Accreditation of Cellular Therapy, FACT FACT FACT cont’d Founded in 1996, FACT establishes standards for high quality medical and laboratory practice in cellular therapies. FACT is a non- profit corporation co-founded by the International Society for Cellular Therapy (ISCT) and the American Society of Blood and Marrow Transplantation (ABMTR) for the purposes of voluntary inspection and accreditation in the field of cellular therapy. In 2000 and 2006 FACT partnered with NetCord and Joint Accreditation Committee- ISCT & EBMT (JACIE) respectively, and developed international standards in the field of cellular therapy. FACT accredits facilities under the two sets of international standards: -FACT-JACIE International Standards for Cellular Therapy Product Collection, Processing and Administration. These apply to hematopoietic progenitor cells isolated from bone marrow or peripheral blood and to all phases of collection, processing and administration of these cells. - NetCord-FACT International Standards for Cord Blood Collection, Processing, Testing, Banking, Selection and Release. The Joint Accreditation Committee-ISCT & EBMT http://www.jacie.org/portal/jacie /welcome JACIE The Joint Accreditation Committee-ISCT & EBMT is a non-profit body established in 1998 for the purposes of assessment and accreditation in the field of haematopoietic stem cell (HSC) transplantation. The Committee was founded by the European Group for Blood and Marrow Transplantation (EBMT) and the International Society for Cellular Therapy (ISCT), the two leading scientific organisations involved with HSC transplantation in Europe. JACIE's primary aim is to promote high quality patient care and laboratory performance in haematopoietic stem cell collection, processing and transplantation centres through an internationally recognised system of accreditation. AABB, The association has grown to represent much more than the "American Association of Blood Banks." In 2005, the association instituted a name change to reflect its expanding interests and diverse membership, and is now known only by its acronym, AABB. AABB AABB is an international association, based in USA, representing individuals and institutions involved in activities related to transfusion and cellular therapies, including transplantation medicine. AABB member facilities are responsible for collecting virtually all of the nation’s blood supply and transfusing more than 80 percent of all blood and blood components used in the United States. Since its inception in 1947, AABB — formerly known as the American Association of Blood Banks — has continued to support the highest standards of medical, technical and Nearly 2,000 institutions, including community and hospital blood banks, hospital transfusion services and laboratories, and about 8,000 individuals, including physicians, nurses and other health care providers; scientists; administrators; medical technologists; blood donor recruiters; and public relations practitioners, are members of AABB. AABB’s active membership — located in all 50 states and 80 2 administrative performance; scientific investigation; and clinical application through standard setting, accreditation, education, advocacy and other activities. The association also is dedicated to increasing public awareness of the importance of voluntary blood donation. countries — provides direction to the association through its board of directors and more than 30 committees of volunteer professionals. Approved by the ISBT Council in 1994, the ISBT 128 standard has gained international acceptance* and is now officially endorsed by the American Association of Blood Banks, European Plasma Fractionators Association, European Blood Alliance and the American Red Cross. Its use for Cellular Therapy products is supported by the boards of the AABB, ASBMT§, EBMT§, FACT, ISBT, ISCT, ISCT Europe, JACIE, NMDP§ and WMDA§. ISBT ISBT 128 sets a global standard for the identification, labeling and information processing of human blood, tissue and organ products across international borders and disparate health care systems. The standard has been designed and perfected over a period of almost two decades to ensure the highest levels of accuracy, safety and efficiency for the benefit of donors, patients and official ISBT 128 licensed facilities worldwide. *Countries which have facilities that have implemented ISBT 128: Austria, Belgium, Brazil, Canada, China, Denmark, Egypt, Estonia, Finland, Greece, Iceland, Israel, Kuwait, Latvia, Lithuania, Netherlands, Norway, Oman, Portugal, Russia, Singapore, South Korea, Spain, Sweden, Switzerland, Taiwan, Turkey, United Arab Emirates, United Kingdom, United States. The standard has been designed to ensure the highest levels of accuracy, safety and efficiency for the benefit of donors, patients and official ISBT 128 licensed facilities worldwide. Featuring a unique, highly flexible and comprehensive coding method for every collected product, ISBT 128 provides international consistency to support the transfer, transfusion or transplantation of blood and tissue products. A truly global standard, ISBT 128 has been adopted worldwide in numerous countries*. Its adoption has spanned the spectrum from major health care facilities, governmental agencies, and emergency response organizations to medical technology hardware and software developers, laboratories and research organizations. International Cellular Therapy Coding and Labeling Advisory Group **The Boards of AABB, ASBMT, ASFA, EBMT, FACT, ICCBBA Inc, ISBT, ISCT, ISCT Europe, JACIE, NMDP and WMDA: ICCBBA The Boards of relevant agencies** are: recognizing the significant benefits of international standardization of coding and labelling in clinical practice; taking into account the requirements of new regulation, in particular the European Tissue and Cells Directive (2004/23/EC) and the FDA interest in bar coding of biological products [Federal Register: February 26, 2004 (Volume 69, Number 38)]. It is to acknowledge the widespread use of the international information standard ISBT 128 in the fields of blood transfusion and transplantation; recognise the need for international management and technical co- operation for the successful maintenance and development of such standards, and confirm their support for the international use of ISBT 128 in the coding of hematopoietic progenitor cell and other therapeutic cell products and announce the establishment of a co-sponsored International Cellular Therapy Coding and Labeling Advisory Group. The British Pharmacopoeia http://www.pharmacopoeia.co.u k/ BP The British Pharmacopoeia (BP) is the official collection of standards for UK medicinal products and pharmaceutical substances. Produced by the British Pharmacopoeia Commission Secretariat, part of the Medicines and Healthcare products Regulatory Agency, the BP makes an important contribution to public health by setting publicly available standards for the quality of medicines. 3 The United States Pharmacopeia http://www.usp.org/ USP The United States Pharmacopeia (USP) is a non–governmental, official public standards– setting authority for prescription and over–the– counter medicines and other healthcare products manufactured or sold in the United States. USP sets widely recognized standards for food ingredients and dietary supplements. USP sets standards for the quality, purity, strength, and consistency of these products. It is recognized and used in more than 130 countries around the globe. §Abbreviations: EBMT, European Group for Blood and Marrow Transplantation; ASBMT, American Society for Blood and Marrow Transplantation; NMDP, National Marrow Donor Program (USA); WMDA, World Marrow Donor Association. Tabela P7.2: Pregled standarda i regulativa u Australiji koji se odnose na izradu i klini!ku primenu "elijskih terapija (Regulatory Requirements, Standards and Guidelines currently applicable to Cell Therapies used in Clinical and Clinical Research Purposes in Australia). (Source: official websites). Name of the Standard/ Regulation or Guidelines and Regulatory Agency Abbreviated Name of Standard/ Guidelines or Issuing Authority Purpose and Context Scope Requirements for Procedures Related to the Collection, Processing, Storage and Issue of Human Haemopoietic Progenitor Cells This NPAAC Standard is currently administered by the National Association of Testing Authorities, NATA NPAAC The National Pathology Accreditation Advisory Council (NPAAC) was established in 1979 to consider and make recommendations to the Australian, state and territory governments on matters related to the accreditation of pathology laboratories and the introduction and maintenance of uniform standards of practice in pathology laboratories throughout Australia. A function of NPAAC is to formulate standards and initiate and promote guidelines and education programs about pathology tests. Publications produced by NPAAC are issued as accreditation material to provide guidance to laboratories and accrediting agencies about minimum standards considered to be acceptable for good laboratory practice. Failure to meet these minimum standards may pose a risk to public health and patient safety. The standard describes the minimum requirements for competence and quality to be met by facilities and individuals preparing HPCs (PBSC) and/or lymphocytes for infusion, and for providing assured safety and quality of the product. Any manufacturing and use of products will still be subject to regulatory oversight by the Therapeutic Goods Administration (TGA) or its successor body. This document covers Class 1 products only. For further info on HPC see Appendix 2, Table 3. Current: Australian Code of Good Manufacturing Practice – Human Blood and Tissues, cGMP cGMP To meet the requirements of the Therapeutic Goods Act 1989, blood and tissue banks must meet the requirements of the Manufacturing Principles, which reference the Australian Code of Good Manufacturing Practice - Human Blood and Tissues. The current code continues to include the elements of quality systems form the ISO 9000 series of standards and applies these principles to blood and tissue banking. Human tissue for implantation in the human body that is obtained, stored and supplied without any deliberate alteration to its biological and 4 Therapeutic Goods Administration (TGA) Under the present revision of the Therapeutic Goods Act 1989 (amended June 2003), human organs, tissue and cellular products, and tissue and cell based derivatives are regulated by several different routes within the legislation. mechanical properties must comply with the Australian Code of Good Manufacturing Practice, Human Blood and Tissues but are exempt from the requirement for entry on the ARTG. This includes most banked tissue, such as dura mater, heart valves, skin, corneas and bone. Proposed: Australian Code of Good Manufacturing Practice human blood and blood components, human tissues and human cellular therapies - Draft http://www.tga.gov.au/bt/consul t/drbloodstandards-cgmp.pdf Therapeutic Goods Administration (TGA) cGMP In July 2002 the Australian Health Ministers' Conference recommended that the Therapeutic Goods Administration (TGA) develop a new regulatory framework for human cell and tissue therapies and other emerging biological therapies. A framework to regulate these products was proposed by the TGA during the development of the now postponed joint Australia New Zealand Therapeutic Products Agency (ANZTPA). During this time significant consultation was undertaken on the development of standards. Following the postponement of ANZTPA in 2007, the Government agreed in 2008 to move forward with a number of improvements identified during the development of ANZTPA in an Australia-only context. The TGA is inviting stakeholders to comment on five newly drafted Therapeutic Goods Orders (TGOs) and an amended Code of GMP for blood and blood components, human tissues and human cellular therapies. The draft Code of GMP is an amended version of the current Code of GMP for human blood and tissues (2000) which has applied to manufacturers and has been in place for almost 10 years. The five new TGOs are proposed to mandate a standard for minimising the risk of transmission of infectious diseases and four tissue-specific standards for banked cardiovascular tissue, musculoskeletal tissue, ocular tissue and skin. The new TGOs clarify best practice requirements, increase the degree of international harmonisation and ensure ongoing flexibility to respond to new technologies. The draft Code of GMP and the infectious diseases standard are intended to apply to all human blood and blood components, human tissues and human cellular therapy products. Some of these products will be regulated under a new regulatory framework for Biologicals. Products regulated under the new Biologicals framework will also have to comply with the relevant tissue specific standards i.e. standards for banked cardiovascular, musculoskeletal and ocular tissue and skin. Definitions and Exclusions: -Organs used in direct transplant are excluded therapeutic goods (Therapeutic Goods (Excluded Goods) Order No. 1 of 2008. The solutions used to flush the organs or to maintain them during transport must be listed or registered on the Australian Register of Therapeutic Goods (ARTG). -Human tissue and cell extracts, whose principle therapeutic purposes are achieved through chemical, pharmacological, or metabolic actions that are generally able to be batch released, are regulated as medicines. -Human derived tissue and cell products that are not regulated as medicines; and are produced by deliberate alteration of tissue or cells in defined manufacturing processes are being regulated as "therapeutic devices" and declared other therapeutic goods (OTGs) by a subsection 41BD(3) Order, Therapeutic Goods (Articles that are not Medical Devices . Such products are to be manufactured in compliance with the Australian Code of Good Manufacturing Practice for Human Blood and Tissues (Part 3-3 of the Act), and to be registered on the ARTG (Part 3-2 of the Act). -Medicines and other therapeutic goods (OTGs) of human origin produced or prepared for a particular person are regulated differently. 5 Cont’d. Name of the Standard/ Regulation or Guidelines and Regulatory Agency Abbreviated Name of Standard/ Guidelines or Issuing Authority Purpose and Context Scope Access to Unapproved Therapeutic Goods - Clinical Trials in Australia http://www.tga.gov.au/docs/htm l/clintrials.htm Therapeutic Goods Administration (TGA) TGA This document describes the regulations for allowing patients access to unapproved medicines or medical devices by participation in a clinical trial. It is primarily directed at sponsors and investigators, but will also provide useful guidance to Human Research Ethics Committees (HRECs). HRECs are also directed to the TGA publication Human Research Ethics Committees and the Therapeutic Goods Legislation . This document provides: Legislative basis for clinical trials, The Clinical Trial Notification (CTN) Scheme, The Clinical Trial Exemption (CTX) Scheme, Reporting of adverse reactions during a clinical trial of medicines, Reporting of adverse reactions during clinical trials of medical devices, Glossary, CTN form, Clinical trial completion advice - CTN and CTX schemes, Supply of unapproved therapeutic goods under the CTX Scheme, CTX scheme - particulars of the product and the trial, CTX scheme documents for ethics committees, ADRAC blue card format, Medical device incident report form. Good Clinical Practice – Internationally accepted*** standard for the designing, conducting, recording and reporting of clinical trials http://www.tga.gov.au/docs/htm l/ich13595.htm Therapeutic Goods Administration (TGA) GCP The Note for Guidance on Good Clinical Practice (CPMP/ICH/135/95) is an internationally accepted standard for the designing, conducting, recording and reporting of clinical trials. The TGA has adopted CPMP/ICH/135/95 in principle but has recognised that some elements are, by necessity, overridden by the National Statement (and therefore not adopted) and that others require explanation in terms of 'local regulatory requirements'. ***Adopted as a National Standard with TGA Annotations National Statement on Ethical Conduct in Human Research http://www.nhmrc.gov.au/public ations/synopses/e72syn.htm The National Health and Medical Research Council NHRMC This Statement entitled the National Statement on Ethical Conduct in Human Research ('the Statement') consists of a series of Guidelines made in accordance with the National Health and Medical Research Council Act 1992 ('the Act'). This document outlines the values and principles of ethical conduct, themes in research ethics: risk and benefit, consent, ethical considerations specific to research methods or fields, ethical considerations specific to participants, processes of research governance and ethical review. Human Research Ethics Committees and the Therapeutic Goods Legislation Therapeutic Goods Administration (TGA) TGA This document describes the role of Human Research Ethics Committees (HRECs) in relation to the supply of unapproved therapeutic goods. It is in connection with the operation of the Clinical Trial Notification Scheme (CTN), the Clinical Trial Exemption Scheme (CTX), the Special Access Scheme, and in the approval of Authorised Prescribers. 6 Australian Red Cross Blood Services (ARCBS) and National Transplantation Service (NTS) http://www.donateblood.com.au /index.aspx ARCBS- NTS Before the Blood Service came into formal existence in 1996, the collection, processing and distribution of blood products throughout the country's health system was managed by individual State and Territory Red Cross Transfusion Services. The establishment of a national blood service has facilitated new levels of national and international co- operation, resulting in improved consistency, quality and safety across Australia. ARCBS Guidelines for Selection of Blood Donors, to assist in determining the appropriateness of collecting blood from potential donors and the purpose(s) for which their collection should be used. Australian Bone Marrow Donor Registry Guidelines Australian Bone Marrow Donor Registry http://www.abmdr.org.au/ ABMDR The Australian Bone Marrow Donor Registry is the tenth largest registry in the world. The registry is guided by people committed to help any person in need of a haemopoietic stem cell transplant. The ABMDR Board provides oversight and guidance to the ABMDR and its executives. The registry network is comprised of state donor centres, tissue typing centres, marrow collection centres and transplant centres. It receives advice and support from the following advisory committees: Scientific advisory, Donor centre advisory, Ethics and Cord blood national management. AusCord Guidelines http://www.abmdr.org.au/dyna mic_menus.php?id=4&menuid =17&mainid=4 AusCord AusCord is the Australian national network of umbilical cord blood banks and cord blood collection centres. AusCord aims to provide greater opportunities to patients who need a life-saving procedure through cord blood transplantation. AusCord Guide to Selection of Mothers and Donors used by Cord Blood Banks staff in Australia to establish selection criteria. Prilog 8: Mogu!nosti primene i kompleksnost !elijskih terapija Preuzeto iz: Trounson, 2009 Slika P8.1: Mogu!nosti za primenu i slo"enost !elijskih terapija. Razli#ite primene: !elija kostne sr"i, alogenih !elijskih terapija (iz placente), mezenhimskih mati#nih !elija, neuralnih mati#nih !elija, embrionalnih pluripotentnih mati#nih !elija i indukovanih pluripotentnih !elija. !"#$"%&'(')%!"##$%!!&"$ '(()&**+++,-./012314(567,3/0*89:8;9#8<*9*"$ =6>1!"!/?!< *+,-%$)./0%1$)23$421$('3,3'2)$+.1+25%56 These autologous stem cell therapies are in widespread clinical trials with varying reported clinical benefits to patients. Reports on the use of granulocyte-colony stimu- lating factor mobilization of bone marrow stem cells and the intracoronary administration of leukapheretically iso- lated HSCs, show a few subjective benefits but overall no real objective benefit in refractory ischemic heart disease [1]. Some consistency in benefit in left ventricular func- tion and remodeling has been observed in studies of intra- coronary administration of the patient's own bone marrow preparations for severe myocardial infarction [2]. There is also evidence in the few comparative clinical stud- ies available that the endothelial (CD34+) fraction of mononuclear bone marrow cells is the potent cell type responsible for vascular growth and regeneration and the clinical improvements observed in cardiac repair [3]. Aside from autologous adult stem transplants that are based mainly on the use of bone marrow, where is the field moving (Figure 1)? There is an increasing interest in allogenic stem cell therapies and advantage is being taken of cell therapies using mesenchymal (bone marrow stro- mal) stem cells (MSCs), which have both multipotential (by definition are capable of forming bone, adipose, and cartilage tissue), and immunosuppressive properties. The latter enables their grafting without the need for severe immunosupression and selection for histocompatibilty. These clinical trials are being led by the biotechnology companies, Osiris (Columbia, MD, USA) and Angioblast (New York, USA)/Mesoblast (Melbourne, Australia) for primary applications in preventing graft versus host dis- ease (GvH) and treatment of cardiovascular and bone dis- eases. Cartilage repair using MSCs is still in preclinical and phase I studies with no detailed comparative data in humans [4]. Other very accessible sources of MSCs with Human stem cell transplantsFigure 1 Human stem cell transplants. Bone marrow cells are mobilized and recovered for autologos transplantation for a number of applications, including cancer, blood diseases, stroke and multiple sclerosis. Allogenic stem cells are frequently recovered from the placenta and umbilical cord. Mesenchymal stem cells are used for immune suppression and inflammatory conditions. Neural stem cells are of fetal origin or differentiated from embryonic stem (ES) cells and induced pluripotential stem (iPS) cells. Both ES and iPS cells are poised for clinical trials for repair of spinal injury, macular degeneration, diabetes and cancer. Prilog 9: Metodolo!ki prikaz KOMPARATIVNA ANALIZA PROCESA, SISTEMA I REGULATIVA U IZRADI I PRIMENI MATI!NIH "ELIJA I DRUGIH "ELIJSKIH TERAPIJA Metodolo!ki prikaz komparativne studije Elementi i faze Fokus i o!ekivani rezultati Svrha ove faze analize Jedinica analize i materijal (izvor informacija) Kvalitativna metodoogija koja se koristi u ovoj fazi Izvodi novu teoriju i/ili novi model Teorija/ Model (novonastali teorijski pristup, model i/ili modalitet) Teorijski modeli/ re!enja Prakti"ni model/ evaluacija N ova teorijska re!enja i novi m odaliteti. Prakti"na prim ena m odelovanja. Komp. studija 5 Materijal: rezultati prethodnih analiza i empirijski rezultati Analizira zna!enje ljudskih iskustava, percepcije i znanja/ fenomene Fenomen (postoje!i problemi i re"enja) Materijal: transkripti intervjua CTE01 - CTE24 Izvodjenje teorije iz prakse (engl. Grounded theory) " Tematska/ fenomenolo!ka analiza (engl. Phenomenology/ Thematic analysis) Postoje#i problem i i postoje#a re!enja iz prakse. Komp. studija 4 Jedinica analize- intervju (zvu!ni zapis i transkript zvu!nog zapisa) Demonstrira sli!nosti i razlike izmedju modela izrade Model (postoje!i lab-organizacioni model) M-1 # M-2 # M-3 Uporedna analiza modela (engl. Cross-case study) K arakteristike postoje#ih organizacioinh m odela. Materijal: dokumenta/ zvani!ni web sajt Jedinica analize- organizacioni model (koji se bavi izradom $elijskih terapija nezavisno od njihoveog tipa i primene) Komp. studija 3 Ilustruje slo%enost procesa izrade i primene Projekat (postoje!i lab-klini#ki proces) P-1 P-2 P-3 Pojedina"na analiza individualnog projekta (engl. Case study) K arakteristike procesa laboratorijske izrade i klini"ke prim ene u postoje#im projektim a. Materijal: dokumenta/ publikacije Jedinica analize- projekat (proces laboratorijske izrade i klini!ke primene odredjenog tipa $elija) Komp. studija 2 Ilustruje stepen raznovrsnosti primena u klini!ke svrhe Baza podataka klini#kih istra$iva#kih projekata (klini#kih istra$ivanja) Analiza dokumenata (engl. Documentary analysis) (Pregled klini!kih primena/ klini!kih istra%iva!kih projekata) R aznovrsnost, tip i broj klini"kih istra$iva"kih projekata. Komp. studija 1 Materijal: baza podataka/ zvani!ni web sajt Jedinica analize- klini!ki istra%iva!ki projekat (klini!ko istra%ivanje) Ilustruje slo%enost zakonsko regulatornih modela Zakonsko regulatorni model (engl. Regulatory Framework) Analiza dokumenata (engl. Documentary analysis) (Pregled zakonsko regulatornih propisa) K om pleksnost zakonsko regulatornih m odela i okru$enja. Komp. studija 1 Materijal: dokumenta/ zvani!ni web sajt Jedinica analize- zakonsko regulatorni model Prilog 10: Methodology Overview (eng.) COMPARATIVE STUDY OF PROCESSES, SYSTEMS AND REGULATIONS IN MANUFACTURING AND APPLICATION OF STEM CELLS AND OTHER CELL THERAPIES Methodology Overview of the Comparative Study Elements and phases Focus & expected results Purpose of the analysis Unit of analysis & Material (source of information) Qualitative methodology Generates a new theory/ model Theory/ Model (new theoretical approach/ models) Material: results of previous analyses and empirical results New theory/ New models Practical model/ evaluation N ew theories, theoretical & practical m odels. Comp. Study 5 Looks into human experiences / type of phenomenon Phenomenon (existing problems and solutions) Material: interview transcripts CTE01 - CTE24 Grounded theory ! Phenomenology/ Thematic analysis Identifying existing problem s and practical solutions Comp. Study 4 Unit of analysis- interview (recordings and interview transcripts) Demonstrates similarities and differences between manufacturing models Model (existing manufacturing/ organisational model) M-1 " M-2 " M-3 Cross-case study C haracteristics of existing organisational m odels in m anufacturing/ services. Material: documents/ official web site Unit of analysis- organisational model in cell therapies manufacturing/service, irrespective of applications Comp. Study 3 Demonstrates complexity of manufacturing process and clinical appl. Project (existing lab-clinical process) P-1 P-2 P-3 Case study Process characteristics: an existing laboratory m anufacturing & clinical application. Material: documents/ publications Unit of analysis- project (process of laboratory manufacturing & clinical application of a specific cell type) Comp. Study 2 Presents the broad spectrum of clinical applications Clinical Trials Database Documentary analysis (Overview of clinical applications/ clinical trials) V ariety of clinical applications and num ber of clinical trials. Comp. Study 1 Material: data-base/ official web site Unit of analysis- clinical trials Presents the complexity of regulatory environment Regulatory Framework(s) Documentary analysis (Overview of regulatory frameworks) C om plexity of reg. fram ew orks/ global regulatory environm ent. Comp. Study 1 Material: documents/ official web site Unit of analysis- regulatory framework 1 Prilog 11: Primer standardne operativne procedure za izradu minimalno manipulisanih !elija Generic elements: PURPOSE AND SCOPE This method will describe the standard operating procedure for ! REFERENCES Add: standards, guidelines, publications and other external documents. DOCUMENTS Add: manufacturing standard procedures and other internal documents. For example:!Cleaning of Biological Safety Cabinet in Laboratory, Product Release, Product Transfer and Disposal from the Lab, Product Transport and Receipt to the Lab, Cryopreservation, Reinfusion, Collection and Training for Collection, Records Overview, Detailed Records, Records Validation and Reporting, CFU-GM Quantitation - Fresh Assay, CFU-GM Quantitation - Thawed Vial Assay, Storage of Cryopreserved Products, Thawed Vial CD34 Assay, Donor Evaluation and Selection, Quality Control of Cryopreserved Product, DEFINITIONS Add: list of definitions. RESPONSIBILITIES Add: list of staff responsibilities. Product-specific elements: PROCEDURE Stem/progenitor cells (CELLS PRODUCT) harvested by peripheral blood apheresis are to be cryopreserved in a solution comprising autologous plasma or albumex - 4 at 20%, dimethyl sulphoxide (DMSO) 10%, and acid citrate dextrose – A 5%, of the final volume (i.e. a ratio of 65:20:10:5). The cells are then control rate frozen to -160oC and stored in a vapour phase liquid nitrogen cryostorage vessel (tank). DMSO is the primary cellular cryopreservative. It prevents cellular hypo-osmotic stress by minimizing intracellular ice crystal formation, which may rupture cells on thawing. Critical Control Points and Assays Critical Control Points The Critical Control points i.e. points during the cryopreservation process where error may result in product loss or patient harm are listed below: • Time and temperature of storage of CELLS PRODUCT collection prior to completion of cryopreservation should be less than 24 hours at 4oC. • Identification checks of CELLS PRODUCT collection and related labels/records • Review of Infectious serology • Weighing CELLS PRODUCT and Plasma collection bags • Calculation of cryoprotectant volumes • Labelling of Cryobags, Cryovials and Blood Culture Bottles • Sterile connection of CELLS PRODUCT collection bag to linked cryobag, plasma bag and transfer set • Aseptic preparation of cryoprotectant mix – correct volume of DMSO i.e. 10% of final volume • Setup of Rate freezer – correct cryopreservation program • Aliquoting and Heat sealing cryobags • Aliquoting Blood cultures and cryovials • Excel record data entry (Bag Weights, Nucleated Cell Count, CD34+ cell count) • Correct storage and documentation of cryopreserved CELLS PRODUCT in inventory with reference to quarantine status. Critical Assays Assays / testing associated with critical control points are listed below: • Blood Cultures – Apheresis end of procedure and cryobag culture. • CFU-GM Assay • CD34 Assay • CELLS PRODUCT bag white cell count Specimen • Autologous CELLS PRODUCT collection supplied in apheresis harvest bag or equivalent. 2 • Autologous plasma collection supplied in bag or equivalent. • Additional samples are taken off the collected cells by apheresis staff, at the completion of the harvest. These include: • EDTA specimen for bag white cell count. • EDTA specimen for molecular analysis if required. • Lithium Heparin specimen for CD34 analysis. • Blood cultures - to check sterility of apheresis procedure and cells collection. Materials Required • Metal tray with frozen cold packs and freezer rack for cryotubes • Benchcoat sheet • Gown - long sleeve • Sterile gloves - Ansell • Cryocyte freeze containers - size & no. is patient dependent • Connector adaptor set. 1 per apheresis product • Syringes - 50ml, 30ml, and 10ml • 18g needles • 23g needles • Blunt fill needles • Airway needles • Sterile IVA bottle seals • Alcowipes • Cryotubes for aliquots of cryopreserved cells • Sterile plain 5ml screw top tube - for cryobag cell count • Blood culture bottles - aerobic and anaerobic - 1 pair per cells collection • Blood transfer device for blood cultures eg. Angelwings • Sterile disposable scissors • Transfer bags 600ml size with coupler, sterile, may be required if autologous plasma not supplied. Reagents Required • Acid Citrate Dextrose - Solution A, 500ml for injection • Dimethyl sulphoxide –DMSO, 70ml multidose vial - Sterile endotoxin free. • Albumex 4 - 50ml sterile vials, use as replacement for autologous plasma • 70% alcohol solution surface spray disinfectant - for swabbing out laminar flow cabinet before and after use. • Viraclean Disinfectant solution for swabbing out laminar flow cabinet before and after use. Equipment Required • Computer (records) • Biological Class II Safety Cabinet (Laminar Flow Cabinet) • Weighing balance • Calibration weight – 500g • Stainless steel tray • Freezer blocks & bench coat • Sterile Coupling Device & disposable wafers • Tubing Heat Sealer • Freezing Cassettes • Rack for cryovials • Control Rate Freezer • Liquid nitrogen reservoir • Cryostorage Tank with liquid nitrogen (vapour phase) Method Unique ID of sample generation Cryopreservation process Prepare Class II Biological Safety Cabinet for use. Briefly: • Spray Viraclean liberally on all surfaces. Leave for 10 minutes. Soak up and wipe off Viraclean with a disposable cloth. • Spray all surfaces in the cabinet with 70% Isopropyl alcohol. Wipe all surfaces using a clean disposable cloth using a consistent motion from left to right and back to front. • Turn on BSC and allow to equilibrate air flow and pressure by running for at least 5 minutes. • Note any warnings or alarms and use alternate cabinet if fault detected. • Check Identification on cells and plasma bag labels with the accompanying records. • Document identification check performed. • Document transportation and product storage details. Weigh plasma collection bag (if available) and determine collected plasma volume. Collected plasma volume = plasma bag weight – empty bag weight (i.e. tare bag weight) ! NOTE: compare plasma bag against laboratory examples of empty bags for tare weight ! Document weights. 3 Weigh cell produc product and determine collected CELLS PRODUCT volume. Collected cells volume = .cells product bag weight minus empty bag weight (i.e. tare weight) ! NOTE: compare CELLS PRODUCT bag against laboratory examples of empty bags for tare weight) ! Document weights. Compare collected volume of plasma with calculated volume of plasma determined in next step below. Differences of greater than approximately 3% (or 5 mls plasma volume) of the final cryopreserved volume, should be adjusted for by aseptically removing plasma or adding albumex-4 to the plasma, to make up to correct volume. Autoplasma, which will automatically instruct if steps need to be taken to adjust plasma volume. Calculate cryoprotectant volumes and final cryopreservation volume Relative amounts of each component are given in table 1. Table 1. Relative amounts of cells product to cryoprotectant Determine number and type of cryocyte cryopreservation bags required for cryopreservation. ! Check calculated Volume. /Cryobag and based on data in the Table for cryobags, determine appropriate cryobag size to be used. ! If the volume / cryobag is greater than the maximum for the anticipated cryobag size then readjust the number of cryobags eg. from 2 to 4, to give a volume / cryobag compatible with the minimum and maximum cryobag volumes listed in the table below. Generate LABELS for Cryobags, Cryovials, CFU-GM vial and Blood Cultures Prepare laminar flow cabinet with the following: • Benchcoat • Metal tray with frozen cold packs covered by benchcoat above and then with the cells and Plasma collection bags (or empty transfer bag if no plasma bag) • Labelled cryobags of appropriate type (size) & number • Multiflow connector adaptor set • Syringes - 50ml, 20ml, and 10ml • 23g needles • 18g needles • Blunt fill needle • Airway needles • IVA seals • Alcowipes • Chilled cryotubes • Sterile plain 5ml screw top tube • Blood culture bottles • Blood transfer device for blood cultures eg. Angelwings • Acid Citrate Dextrose - Solution A • Dimethyl sulphoxide • Sterile disposable scissors • Albumex 4 - 50ml sterile vials - if no autologous plasma collected. • Sterile transfer bag - if no autologous plasma collected Glove (sterile) and gown up for CELLS PRODUCT processing. When documentation for processing is complete (ie to the stage of processing volumes for cells product, Plasma, DMSO & ACD-A being determined) don disposable gown then sterile gloves in preparation for aseptic processing. Needle + syringe preparation. • ACD-A syringe preparation: In laminar flow cabinet, aseptically prepare a 20ml syringe and 18 g blunt fill needle or similar. • DMSO syringe preparation: In laminar flow cabinet, aseptically prepare a syringe eg. 50ml + 21 g needle. • If required due to insufficient autologous plasma, Albumex 4 syringe/s are prepared as above using 50ml syringe and 18 g needle. • Aseptically prepared needle + syringes are placed aside in laminar flow cabinet until needed. Aseptically open multiflow connector set in laminar flow cabinet and inspect for sterile integrity i.e. all luer connector covers in place and no tubing compromised. If necessary tighten all luer lock covers. Close all clamps and turn 3-way tap away from luer lock port. Lay set out on cabinet bench. Prepare cryocyte/cryomacs bags. In laminar flow cabinet lay out labelled cryobags on cabinet bench. Close all roller clamps. Heat seal off the two female luer lock connectors leaving only the male luer lock connector attached to the bags. Attach cryocyte bag to connector set using aseptic technique. Connection is made using cryocyte bag male connector connected to set cryobag (1 of 4) female connector. Repeat for additional cryocyte bags (maximum of 4 cyrocyte bags per set). COMPONENT % OF FINAL VOLUME CELLS PRODUCT collection 65 Plasma (Albumex 4) 20 ACD-A 5 DMSO 10 4 Attach Plasma bag to connector set using aseptic technique. Connection is via connector set bag spike with adjacent remaining female luer lock fitting close and then piercing using a quarter turn only the plasma bag port. Place Plasma bag on covered ice tray. If no autologous plasma has been collected then prepare albumex -4 substitute bag. Aseptically connect empty transfer bag (or similar) to plasma bag spike /coupling site of connector set using ! turn only or alternately via sterile tubing welder. Swab Albumex 4 vials with alcohol wipe and allow to dry for 10-15 seconds before aseptically withdrawing with prepared 50ml syringe + 18 g needle and airway needle the required "plasma" volume from albumex-4 vial/s. Remove needle/s from syringe/s and aseptically add albumex-4 to 600ml transfer bag via Luer lock fitting of connector set. Leave empty syringe attached to female luer lock fitting to maintain aseptic pathway. Attach cells product bag to connector set using aseptic technique. Connection is via remaining bag spike on connector set and piercing cells bag port with a maximum of a ! turn. Place bag with cells product on covered ice tray. Remove CFU-GM aliquot sample. Attach 10ml syringe to Plasma bag associated female luer lock fitting. Clamp off plasma bag using grips. Open clamps to gently mix bag with cells product. Withdraw 1ml of cells product via syringe. Leave syringe attached to fitting until ready to replace it with ACD-A syringe. Close clamps. Add CFU-GM sample to prelabelled sterile plain tube. DMSO - Swab DMSO bottle with alcowipe and allow to dry for 10-15 seconds. Pierce bung with airway needle. Swab bung again with second alcowipe and allow to dry for 10-15 seconds then pierce with needle+syringe. Aseptically draw up required volume of DMSO. Remove needle+ syringe from DMSO bottle. Aseptically remove needle from syringe and discard needle safely. Displace syringe attached to Plasma bag associated luer lock fitting and attach the DMSO syringe. Open clamps and slowly add DMSO to plasma (or albumex-4) - ACD-A solution mixing well but avoid foaming of the solution. Leave empty syringe attached to luer lock fitting. Close clamps and allow the cryoprotectant solution to cool between covered (benchcoat) frozen cold packs in metal tray. Cooling of the cryoprotectant solution occurs during set up of control rate freezer Start control rate freezer. Add cooled cryoprotectant to bag with cells product. Distribute CELLS PRODUCT/cryoprotectant into cryobags. Remove air from cryobags. In turn open clamps between each cryocyte bag and the CELLS PRODUCT bag. Gently squeeze all the air from cryobag. Close cryobag clamp. Repeat for each cryobag. Ensure all clamps are closed on completion. Heat seal (x3) tubing adjacent to the cryobag. Ensure one seal is close to the tubing attachment site on the cryo bag and is sited beneath the yellow D ring port covers. Detach cryobags - by cutting with sterile disposable scissors across the middle of the third seal distal from the cryobag, i.e. leaving 2.5 seals attached to the cryobag. Repeat for additional cryobags. Store cryobags on covered ice cold blocks. Collect sample of cells for blood cultures and cryovials. Aseptically attach a 20ml syringe to the 3-way tap of the connector set and open this tap and CELLS PRODUCT bag clamp. Aseptically draw up cells/cryomix from CELLS PRODUCT harvest bag into the syringe. Draw all cells/cryoprotectant in bag into the syringe (approx 10mls). Inoculate blood cultures. Exercise care and maintain aseptic technique. Aseptically aliquot 0.5 – 1.0 ml of cells+Cryoprotectant into each of 3 cryovials. Ensure cryovials are securely capped and place in cold rack. Cryobags to control rate freezer. Take sealed cryobags on covered iceblock tray and cryovials in cold rack to the control rate freezer. Using a clean cloth wipe chilled freeze cassettes and cryocyte bag free of any moisture (prevents ice forming on outside of cryocyte bag during freezing which may make it difficult to remove the bag from the freezing cassette / damage cryobags). Place cryobag evenly and with no folds in freeze cassette and close cassette clamp. Place on shelf in Cryomed freeze chamber. Repeat for additional cryobags. Place cryovials in rack on top shelf in the freeze chamber. Close and seal door. Start cryopreservation run, as per the freezing profile. Review freeze curve for any cryopreservation program anomalies. Record run/patient demographics on printout and file in patient file Complete records with the required data including cryobag and vial storage location and sign where appropriate. Deliver cryobag cultures for testing. Record any adverse cryopreservation events, if occur. Endpoints Patient cells cryopreserved in cryoprotectant solution (20% v/v autoplasma or equivalent; 10% DMSO; 5% ACD-A) using standard control rate freeze program, within 24 hours of collection. Sterile cryopreserved cells product. Viability and engraftment potential of cryopreserved cells maintained. END OF DOCUMENT Document Review Record Date Reviewed Reviewed By Reason for Review Appendix X: How to generate a Unique Number/ Product ID Appendix Y: How to operate Control Rate Freezer Appendix Z: Training Guide for the Procedure 1 Prilog 12: Primer standardne operativne procedure za izradu visoko manipulisanih !elija 1 Generic elements: PURPOSE AND SCOPE This method will describe the standard operating procedure for ! REFERENCES Add: standards, guidelines, publications and other external documents. DOCUMENTS Add: manufacturing and related standard operating procedures and other internal documents. DEFINITIONS Add: list of definitions. RESPONSIBILITIES Add: list of staff responsibilities. Product-specific elements: Example of standard operating procedure for manipulation and cryopreservation of human placenta mesenchymal stem cells (hpMSC) Procedure This procedure involves a biohazard. Laboratory scientists must exercise caution when working with biological specimens. Method • Asceptic technique must be used throughout this method • Sterile gloves must be changed EACH TIME Operator 1 enters the biohazard cabinet or every 2 hours. Sterile gloves must be sprayed with 70% IPA for Operator 2 and changed every 2 hours. • All reagents, disposables and tools must be sprayed with sterile 70% IPA each time they enter the biohazard cabinet • A sterile drape must be removed when there is a spill in the biohazard cabinet • Packages and bags containing materiel (listed above) must be opened inside the biohazard cabinet • Used disposables must be discarded in pathological waste bin • Liquid waste must be discarded in waste container • Scalpel blades must be discarded in Sharps container Preparation Day before production DOCUMENTS Print all documents required for the production day.. LABELS Print all labels for this production day Label items throughout this process. CLEANING Check that all equipment required and Production Laboratory have been cleaned according to relevant SOPs and sign the Q-Gen Clean room Cleaning Form (QF-HK-1) located on the door of the clean room. Fill in Batch Record Form. MATERIEL Place FCS, DMEM-LG, gentamicin, Collagenase I and Pulmozyme (DNase I) in the refrigerator in the Production Laboratory. Place cleaning and sterilising, environmental monitoring and stationary materiel on a bench in the Production Laboratory. Place all other reagents, disposables and tools on the trolleys and move them into the Production Laboratory. Fill in Batch Record Form. DOCUMENTS Place all documents printed in Steps 1-2 on a bench in the Production Laboratory. Fill in FRM 2-094, Batch Record Form. Day of production 2 Clean biohazard cabinet with viraclean.. Place 10 x sterile drapes evenly at least 3 layers deep across the biohazard cabinet. Spray the packaged, sterilised waste containers with 70% IPA and place in biohazard cabinet. Remove and discard bags in the pathological waste bin. Label waste containers. Fill in Batch Record Form. Preparation of Digest Medium Place one bottle of 500 ml DMEM-LG in biohazard cabinet. Use Collagenase NB6 and Neutral protease as outlined in the Batch Record. Using 10 ml syringe and hypodermic needle take up 10 ml from fresh 500 ml bottle of DMEM-LG and add to the lyophilised Collagenase NB6 bottle. Using 10 ml syringe and hypodermic needle take up a further 10 ml DMEM-LG from the 500 ml bottle of DMEM-LG and add to the lyophilised Neutral Protease bottle. Swirl bottles to dissolve contents. Using fresh syringes and needles remove dissolved enzymes and transfer back to the 500 ml bottle of DMEM-LG. Add 0.6 ml gentamicin to the bottle of DMEM-LG/Collagenase/Neutral Protease. Add 2.0 ml Pulmozyme (DNase I) to bottle of DMEM-LG/Collagenase/Neutral Protease/gentamicin and mix well. Over-lay previous label on this bottle with “Digest Medium” label. Preparation of Tissue Culture Medium In biohazard cabinet, spray 1x bottle of DMEM-LG and 1x bottle of FCS with 70% IPA. Allow bottles to air dry. Remove lid from DMEM-LG and FCS bottles. Using a 50 ml pipette, add 125 ml FCS to DMEM-LG. In biohazard cabinet, spray 1 vial of gentamicin (2ml @ 40 mg/ml) with 70% IPA. Allow vial to air dry. Break top off a gentamicin vial by twisting until it detaches. Using a 1 ml syringe transfer 0.8 ml of gentamicin to bottle of DMEM-LG/20% FCS. Label the medium bottle with “TCM/gent-01” label. Transfer to 4°C fridge in production laboratory. Discard remaining gentamicin. Fill in Batch Record Form. Isolation of hpMSC from placenta OBTAINING PLACENTA Obtain a placenta with patient consent and ethics approval. Fill in Batch Record Form. Spray 70 % IPA onto gloved hands and the placenta’s outer bag and move into biohazard cabinet DISSECTING PLACENTA Open the two sterile bags which contain the placenta, but keep the placenta in the bags. Label three (x3) 30 cm petri-dishes; Petri Dish A Using tweezers and scissors or a scalpel, remove the umbilical cord and keep in bag. Cut the placenta into cubes of approximately 5 cm x 2 cm x 1 cm (approx. 10g) and distribute them evenly into “Petri dishes A”. Leave any unused placenta and umbilical cord in its own bag and discard into pathological waste bin. WASHING PLACENTA Label four (x4) 600 ml wash containers; ”placenta wash-01” to “placenta wash-04”. Place placenta pieces into placenta wash-01 beaker using tweezers. Add HBSS up to 500 ml mark in placenta wash-01 beaker. Stir the contents of the placenta wash-01 beaker with a 5 ml serological pipette for approximately 20 seconds. Using the pipette, gently squeeze the placental pieces against the sides of the beaker to aid process. With care, using 5 ml pipette to help retain placental pieces in wash beaker, pour HBSS into placenta wash-02 beaker. . Repeat these steps twice more, except this time pouring waste into placenta wash-03 beaker and placenta wash-04 beaker respectively. At least 2x 1 litre bottles of HBSS will be needed to accomplish this. COLLAGENASE I AND DNASE I DIGESTION OF PLACENTA: Label 32x50 ml tubes: Tubes-placental digest (numbered 1-32). Transfer Tubes-placental digest (1-32) in 4 racks into biohazard cabinet and remove lids. Using tweezers transfer all placenta pieces from placenta wash-01 beaker to 4 fresh Petri Dishes. Cut each piece of placenta into approximately 5-10 mm pieces with scissors/scalpel. Transfer placenta pieces up to 10 ml mark in 32 x Tubes-placental digest (1-32) using tweezers. Using a 25 ml pipette, add 15 ml of Digest Media to each of Tubes-placental digest (1-32). Reattach lids to tubes. Tighten firmly. Transfer tubes to incubated shaker and incubate tubes at 250 rpm, 37ºC for 1-1.5 hours (but no more than 2.5 hours) – fix tube racks onto shaker at an angle to aid digestion. During this incubation, label 32 tubes “Tubes-cell suspension (numbered 1-32)” and place in 4 racks (8 per rack). CELL ISOLATION (PART 1) Place 16x Tubes-cell suspension (1-16) in 2 racks in biohazard cabinet. (note: this is only half of the tubes!). Place 16x 100 !m nylon cell strainers in biohazard cabinet. Remove lids from Tubes-cell suspension (1-16) and place a 100 !m nylon cell strainer on top of each tube, positioning cell strainers on an angle so there is a small air gap between the bottom of each cell strainer and the top of each tube. 3 When incubation/tissue digestion step is complete, transfer Tubes-placental digest (1-16) into centrifuge. Set centrifuge to a speed of 540g (rcf). Set acceleration and deceleration to their maximum settings. Set temperature to 200C. As this is a pulse spin, time setting is not critical. It can be set for 5 min, but this will not be used. Start centrifuge, allow to attain 540g, then immediately press stop. Do not allow to run for more than 5 seconds. Transfer tubes back to biohazard cabinet in racks. In biohazard cabinet, being careful not to dislodge solid tissue, pipette as much cell suspension as possible from each Tubes-placental digest (1-16) into Tubes-cell suspension (1-16 , through the cell strainer. The tubes may need to be tipped at a slight angle to achieve this. Using a 25 ml pipette, add 20 ml HBSS to remaining tissue in each Tubes-cell suspension (1-16). Attach lids to Tubes-cell suspension (1-16 , tighten firmly and shake vigorously for 5 seconds. Repeat steps 69-71 Discard all Tubes-placental digest (1-16) and cell strainers into pathological waste bin. Using a 25 ml pipette, adjust volume of Tubes-cell suspension (1-16) to 40 ml with HBSS. Firmly attach lids and transfer Tubes-cell suspension (1-16) to bench beside centrifuge. CELL ISOLATION (PART 2) Repeat steps 62-75 with Tubes-placental digest (17-32), transferring to the tubes labelled Tubes-cell suspension (17-32). Place Tubes-cell suspension (1-16) in centrifuge and spin at 540 g for 5 minutes, 40C. Transfer Tubes-cell suspension (1-16) back to biohazard cabinet. Carefully discard the supernatant using a 50 ml pipette. Swirl/flick tubes to resuspend pellet. Add 5 ml HBSS to each tube Tubes-cell suspension (1-16) and put these tubes to one side. Place Tubes-cell suspension (17-32) in centrifuge and spin at 540 g for 5 minutes, 40C. Transfer Tubes-cell suspension (17-32) back to biohazard cabinet. Carefully discard the supernatant using a 50 ml pipette. Swirl/flick tubes to resuspend pellet. Add 5 ml HBSS to each tube Tubes-cell suspension (17-32). CELL ISOLATION (PART 3) Pool contents of Tubes-cell suspension (1-16) into two of the existing tubes (Tube-cell suspension-1 and Tube-cell suspension-2). Place Tube-cell suspension-1 and Tube-cell suspension-2 to one side Pool contents of Tubes-cell suspension (17-32) into two of the existing tubes (Tube-cell suspension-17 and Tube-cell suspension-18). Transfer Tubes-cell suspension (1217 and 18) to centrifuge and spin 540g, 5 min, 40C. Transfer Tubes-cell suspension (1217 and 18) back to biohazard cabinet. Carefully discard the supernatant using a 50 ml pipette. Swirl/flick tubes to resuspend pellet. Add 9 ml sterile water to each of Tubes-cell suspension (1217 and 18), leave for 5 seconds (10 seconds MAXIMUM), then add 1 ml 10x HBSS to each tube. Re-attach lids and mix well. . Transfer Tubes-cell suspension (1217 and 18) to centrifuge and spin 540g, 5 min, 40C. Transfer Tubes-cell suspension (1217 and 18) back to biohazard cabinet. Carefully remove the supernatant from each tube using a 50 ml pipette, ensuring that the cell pellet is not disturbed. Swirl/flick tubes to resuspend pellet. Transfer TCM+gent-01 bottle from 4°C fridge in production laboratory to Biohazard Cabinet, spray with 70% IPA and allow to air dry. Label a fresh 50 ml tube “Tube p0 cells” and transfer to a rack in Biohazard Cabinet. Using a 5 ml pipette, add 5 ml TCM+gent-01 to each of Tubes-cell suspension (1, 2, 17 & 18). Transfer contents of all Tubes-cell suspension (1, 2, 17 & 18) to Tube p0 cells. Remove 10 µl from Tube p0 cells and place in one well of a 96 well plate. Add 10 µl of trypan blue to this and mix well. Assess cell yield and viability with a trypan blue count and haemocytometer. CULTURING PLACENTA CELLS: Move 8 x 175cm2 flasks into biohazard cabinet. Label these flasks: flask p0-1 to flask p0-8. Add 22 ml medium from TCM+gent-01 bottle to each flask using 25 ml pipette. Add 3.25 ml of cell suspension from Tube-p0 cells to each flask p0-1 to flask p0-8. Keeping flasks horizontal swirl contents gently to disperse cells evenly and ensure base is covered with media. Transfer flask p0-1 to flask p0-8 to TC-incubator. CONCLUSION: Complete Batch Record Form. Transfer unused TCM back to 4°C fridge in production laboratory. From this point use SOP for Culture of hpMSC, to culture placenta-derived cells. Cleaning TOOLS Wipe scissors, tweezers and scalpel holders clean. Add biodyne to liquid waste containers and double bag in a biohazard bag. Take to sterilization room for autoclaving. Send scissors, tweezers and scalpel holders to sterilization room for washing and sterilising. EQUIPMENT Perform a Minor Clean of Biohazard Cabinet and switch off. Switch off centrifuge, incubating shaker and microscope. Clean equipment and Production Laboratory as described in relevant SOPs. Update Q-Gen clean room cleaning form (QF-HK-1). 4 Storage MATERIEL Store gentamicin, DMEM-LG, DMEM-LG + 20% (v/v) FCS (not heat-inactivated) + 50 !g/ml gentamicin, FCS, Collagenase NB6 solution and powder and clinical-grade water in refrigerator at 4OC. Store cleaning and sterilising, environmental monitoring and stationary materiel, 1 x HBSS, unopened disposables and tools that don’t require sterilising at room temperature. Next step: Preparation for cryopreservation hpMSC Before production DOCUMENTS Print the batch record. Print all documents required for the production day, as outlined in the batch record. LABELS Print all labels for this production day. Label items throughout this process. CLEANING Clean equipment required (see the batch record) and Production Laboratory. Fill in the batch record. MATERIEL Place Plasma-Lyte and Albumex-20 in the refrigerator in the Production Laboratory. Place stationary materiel on a bench in the Production Laboratory. Place all other reagents, disposables and tools on the trolleys and move them into the Production Laboratory. Fill in the batch record. DOCUMENTS Place all documents in the Production Laboratory. Fill in the batch record. BIOHAZARD CABINET Clean biohazard cabinet. Fill in the batch record. Place 5 x sterile drapes on the base of the biohazard cabinet. Spray the packaged, sterilised waste containers with 70 % ethanol and place in biohazard cabinet. Remove and discard bags in the pathological waste bin. Fill in The batch record. REAGENTS Place 8x50 ml tubes in the biohazard cabinet and label tubes “Freezing Mix-01” to “Freezing Mix-08” Place 1 litre bag of plasmaLyte and Albumex-20 in biohazard cabinet. Using a 50 ml syringe and sterile needle, transfer 32.5 ml of plasmaLyte into each of 8x50 ml Tubes. Add 12.5 ml of Albumex-20 to each of 8x50 ml tubes. Add 5 ml of Cryo-Sure DMSO to each of 8x50 ml tubes and mix well. Fill in the batch record. Cryopreservation of hpMSC Obtaining the hpMSC: hpMSC at passage 2-5 in media obtained as outlined in SOP 2-93 Passage 2 of hpMSC. Cryopreserving Cells: OVERVIEW Two cryopreserved hpMSC doses; A and B are required, as listed in Appendix 1. Appendix 1 is an example of the number of cryopreserved hpMSC bags (at Doses A and B) that would need to be administered (in full and part) to patients weighing 20 kg, 50 kg and 100 kg, at the three hpMSC administration doses (i.e. 1 x 106 cells/kg, 3.3 x 106 cells/kg and 10 x 106 cells/kg). CALCULATIONS FOR CRYOPRESERVATION DOSES The number of cells to be cryopreserved will depend on the passage number, calculations are explained below: For Passages 2-4 Overview Cells will be used for testing, continuing culture and the remaining cells will be cryopreserved. Testing: 4 aliquots of cells in culture medium after counting will be kept (1x106) and 14 vials will be cryopreserved for testing along with the cryo-bags (Dose A 1.2x108/bag and Dose B 4x107/bag) and clinical dose cryovials (1x106, 2x106 and 8x106). Continuing culture: 47.7x106 cells are required for continuing culture of the cells in 15xT1272 flasks. Cells are plated at 2500 cells/cm2, 3.18x106 cells per flask. Cells will be separated and aliquoted after counting and kept in media in the fridge until after cells have been cryopreserved. Calculation Cells for cryopreservation = Total # of Cells -Cells for aliquoting prior to cryopreservation and continuing culture (49.7x106) For Passage 5 Overview Cells will be used for testing, continuing culture and the remaining cells will be cryopreserved. 5 Testing: 4 aliquots of cells in culture medium after counting will be kept (1x106) and 14 vials will be cryopreserved for testing along with the cryo-bags (Dose A 1.2x108/bag and Dose B 4x107/bag) and clinical dose cryovials (1x106, 2x106 and 8x106). See Appendix 2. No cells are needed for continuing culture. Calculations Cells for cryopreservation = Total # of Cells - Cells for aliquoting prior to cryopreservation (2x106) Volumes of Cryopreservation Reagents in Each Mix Cryobag Cell Dose Size Total Volume Volume of Plasma-Lyte (65%) Volume of Albumex-20 (25%) Volume of DMSO (10%) A - 1.2 x 108 cells/bag 30 ml 19.5 ml 7.5 ml 3 ml B - 4 x 107 cells/bag 10 ml 6.5 ml 2.5 ml 1 ml Cryovials 1ml 0.65 ml 0.25ml 0.10 ml Add cryopreservation mixtures to appropriate tubes Dose A or Dose Busing serological pipettes. Label the correct number of cryocyte freezing bags according to SOP 2-13, Labelling for Manufacture and Sampling during Open Label Trials. There must be the same number of cryocyte bags as there are 50ml tubes Dose A and Dose Bcontaining cell suspensions. Inside the biohazard cabinet, determine which tube (out of the three input tubes) on the cryocyte freezing bags fits a mixing canula in it. Mark the other two tubes with black permanent marker. In the biohazard cabinet, use the heat sealer to seal each tube on every cryocyte bag which has black permanent marker three times. Cut through each third seal (i.e. the seal that is furthest from the cryocyte bag) with scissors. In biohazard cabinet, aseptically attach a mixing canula to a 60 ml syringe. Place the mixing canula at the bottom of one 50ml tube Dose A or Dose B and suck up all of the tube’s contents into the syringe. Insert the mixing canula to the one remaining input tube ON THE CORRECT CRYOCYTE BAG (look at the label). Have a second Operator check that the correct cell dose is added to the appropriate cryocyte freezing bag. Add the cells and cryopreservation mixture to the bag. Use heat-sealing machine to seal each cryocyte bag’s open tube three times, as close to the bag as possible. Cut THROUGH the third seal (the seal furthest from the cryocyte bag) with scissors. Repeat Steps for every tube Dose A and Dose B. Place one cryocyte bag per cryocyte bag cassette. Repeat Step for every cryocyte bag. Place all cryocyte bag cassettes in the controlled-rate freezer and press START. Controlled-rate freezer program: Hold at 4OC for 10 mins. Drop 1OC/min to -90OC. Transfer cryobags to liquid N2. CONCLUSION Discard any remaining hpMSC. Complete the batch record. Cells will be transported to the receiving hospitals. Cleaning TOOLS Soak tube racks in a bucket of warm water with Pyroneg for at least 10 minutes. Send tube racks, pipette boys and power pipettes to Central Sterilizing Supply Department (CSSD) for washing and sterilising. EQUIPMENT Clean Biohazard Cabinet. . Clean equipment and Production Laboratory. Storage MATERIEL Store gentamicin, DMEM-LG + 20% (v/v) FCS (not heat-inactivated) + 50 !g/ml gentamicin, TrypLE™ Select, FCS and in refrigerator at 4OC. 6 Store cleaning and sterilising, environmental monitoring and stationary materiel, 1 x HBSS, unopened disposables and tools that don’t require sterilising, at room temperature. END OF DOCUMENT Document Review Record Date Reviewed Reviewed By Reason for Review Appendix X: Example of how the hpMSC Cryopreservation Doses (Doses A and B) The following could be used for the three hpMSC administration doses (i.e. 1 x 106 cells/kg, 3.3 x 106 cells/kg and 10 x 106 cells/kg) in patients weighing 20kg, 50kg and 100kg: MSC Dose 20 kg person 50 kg person 100 kg person 1.0 x 106 cells/kg 2 x 107 cells 1 x cryobag B 5 x 107 cells 2 x cryobag B 1 x 108 cells 1 x cryobag A 3.3 x 106 cells/kg 6.6 x 107 cells 2 x cryobag B 1.65 x 108 cells 1 x cryobag A + 2 x cryobag B 3.3 x 108 cells 3 x cryobag A + 1 x cryobag B 1.0 x 107 cells/kg 2 x 108 cells 2 x cryobag A 5 x 108 cells 5 x cryobag A 1 x 109 cells 10 x cryobag A 1 Prilog 13: Primer standardne operativne procedure za izradu visoko manipulisanih !elija 2 Generic elements: PURPOSE AND SCOPE This method will describe the standard operating procedure for ! REFERENCES Add: standards, guidelines, publications and other external documents. DOCUMENTS Add: manufacturing and related standard operating procedures and other internal documents. DEFINITIONS Add: list of definitions. RESPONSIBILITIES Add: list of staff responsibilities. Product-specific elements: Example of standard operating procedure for the enrichment of antigen presenting dendritic cells from leukapheresis product. Procedure This protocol describes the enrichment of antigen presenting dendritic cells from leukapheresis product after depletion of xyz positive cells with the XYZ Instrument. Summary flow diagram of xyz isolation process Preparation of Leukapheresis Product (LP) 10-40 x 109 WBC ! Dilution of Leukapheresis Product 1:3 (e.g. 200mL LP+400mL Buffer) (200 x g, 15 min, RT, brake 0) ! Volume Adjustment to 90mL ! Labeling with Monoclonal Antibody (mAb) and Microbeads (5mL MicroBeads + 90mL LP) 30 min, RT, 25 rpm ! Removal of Excess Reagent: 1x wash (300 x g, 15 min, RT, brake 0) Preparation of the XYZ Instrument ! ! Volume Adjustment to 100mL Automated Selection of Target Cells XYZ LS Tubing Set DEPLETION 2.1 ! Selected Cells 2 ( depleted Cells) Removal of Excess buffer: 1x wash (300 x g, 15 min, RT, brake 0) ! Volume Adjustment to 95mL ! Labelling with XYZ Anti-Labelled MicroBeads (7,5mL Beads + 95mL LP) 30 min, RT, 25 rpm ! Removal of Excess Reagent: 2x wash ! 2 x (300 x g, 15 min, RT, brake 0) • Preparation of the XYZ Instrument ! ! Volume Adjustment to 100mL Automated Selection of Labelled Cells XYZ Tubing Set ENRICHMENT 3.1 ! Selected Cells ! Flow Cytometry-Analysis Notes Always carryout manipulations of the product in the biohazard safety cabinet, unless performing the XYZ run, sterile connects using the heal sealing procedures, incubations on the shaker or centrifugations. Place a sterile drape in the biohazard hood before starting the procedure. Change as appropriate. Always keep the same clamp and bag pair once recorded. Bag No. & Bag name 2 LEUKAPHERESIS PRODUCT (LP) 3 LP WASH SN 4 MICROBEAD WASH SN 5 CELL COLLECTION BAG (CCB) 8 POST ISOLATION WASH SN 9 1. POST ISOL. WASH 1 SN 10 2. POST ISOL. WASH 2 SN 11 LABELLED +VE CCB Preparation For xyz XYZ Isolation Preparation for production run-documentation/Records Complete, document, label and form issue list in the batch record (BR) Complete supplies checklist in the BR Complete equipment checklist in the BR 3 Labelling of bags and sample containers Label 8x 600ml transfer bags with pre-prepared labels (SOP# ) as below Label 14x 1.5ml eppendorf tubes with the pre-prepared labels (Labels in DC Production Process (2A, 2B, 3A, 4A, 5A, 5B, 6A, 7A, 8A, 9A, 10A, 11A, 12A, 13A) for cell counts/ flow cytometry Label 4x 15ml falcon tubes (Labels in DC Production Process SOP# ) (6A, 7A, 12A, 13A). Preparation of XYZ buffer Spray 3 bags of room temperature XYZ buffer with sterile 70% ethanol and place in biohazard hood. Spray Albumex-20 bottle with sterile 70% ethanol and place in biohazard hood Remove cap from one port each of the buffer bags Swap top of ports with disinfectant wipe Swap bung on Albumex-20 bottle with disinfectant wipe Using a 21g needle and 25 ml syringe remove 25ml of Albumex-20 and inject into one of the XYZ buffer bags using the swabbed port. Place needle and syringe in sharps bin Repeat steps 3.1.3.6 to 3.1.3.7 for the additional two buffer bags Label buffer bags with the pre-prepared labels (SOP#) buffer bag A, buffer bag B, buffer bag C Depletion of + cells: Antibody and Magnetic Labeling I Weigh Bag 2 with clamp. Record in BR Insert the spike on Bag 2 into Bag 1. Transfer the leukapheresis product into bag 2. Clamp off bag 2 and using the tube stripper remove, strip any remaining cellular product into bag 2 and remove the bag 1 using the heat sealer Reweigh bag 2 with clamp. Record in BR Determine the volume of leukapheresis product. Record in BR Insert a sample site coupler via port on bag 2. Remove a 500ul sample using a 21g needle plus 5ml syringe using the sample site coupler from Bag 2, place in Eppendorf 2A. Record in BR Send 2A sample to Pathology for automated cell count (SOP# ). Determine frequency of positive cells from the remaining sample (XYZ XYZ Staining SOP#). Swab the uncapped port of one XYZ buffer bag with a disinfectant wipe and insert a plasma transfer set with two spikes () Fuse XYZ buffer bag with bag 2 using Determine volume of buffer required to dilute the leukapheresis product (Bag 2) upto 400ml with XYZ Buffer using BR. Place bag 2 on balance with appropriate clamps and run XYZ buffer into the bag until the correct bag weight has been reached. Clamp off the tubing close to XYZ buffer bag, using the tube stripper remove, strip any remaining cellular product into bag 2 and clamp off close to bag 2. Record in BR Re-Weigh Bag 2 with a clamp. Record in BR Weight bag 3 with clamp. Record in BR Using the remove the XYZ buffer bag and attach bag 3 to bag 2. Place bag 2 with bag 3 attached in the centrifuge bucket taking care not to create folds in the bag as this will affect pelleting of the sample. Label of the bag must face to the outside of the centrifuge. Place bucket in centrifuge with appropriate balance and centrifuge sample (Bag 2) at 300g for 15 minutes, RT, Brake 1. Record in BR Remove supernatant using the plasma extractor into bag 3 taking care not to resuspend the pellet. Clamp off and disconnect bag 3 using heat sealer. Place to one side for further analysis. Using the tube stripper remove, strip any remaining cellular product into bag 2. Gently resuspend pellet by rubbing bag 2, using a low lint cloth, in a circular motion, whilst it is flat on the bench, Re-Weigh bag 2 with clamp. Record in BR Determine the volume of buffer required to adjust the volume to 90ml. Record in BR Connect XYZ buffer bag to bag 2 using the, place on balance and run XYZ buffer into bag 2 until the correct bag weight has been reached. Clamp off the tubing close to XYZ buffer bag, using the tube stripper remove, strip any remaining cellular product into bag 2 and clamp off close to bag 2. Record in BR Remove XYZ buffer bag using the heat sealer Wipe sampling site coupler on bag 2 with disinfectant wipe Gently mix XYZ Labelled +ve (Anti-xyz)-Labelled and XYZ MicroBead vials and remove lids using a clamp. Using a syringe and 21g needle take up the Microbeads, blow air into the vial first to help. Add the beads to bag 2 using the sampling site coupler. Record in BR Using a new syringe and needle repeat for the Labelled +ve labelled. Record in BR Mix bag 2 carefully. Incubate the cell preparation, bag 2, for 30 minutes at controlled room temperature (+19 to 25°C) on an orbital shaker at 25 rpm. Record in BR Re-weigh bag 3 Remove a 500ul sample using a 21g needle plus 5ml syringe from Bag 3, place in Eppendorf 3A. Record in BR Following incubation. Weigh bag 2. Record in BR Determine the amount of buffer required to make the volume in bag 2 up to 400mls using the BR. Record in BR Connect the XYZ buffer to bag 2 using the . Place on balance and run XYZ buffer into bag 2 until the correct bag weight has been reached. Clamp off the tubing close to XYZ buffer bag, using the tube stripper remove, strip any remaining cellular product into bag 2 and clamp off close to bag 2. Record in BR Weigh Bag 4 with and without a clamp. Record in BR Using the remove the XYZ buffer bag and attach bag 4 to bag 2. 4 Place bag 2 with bag 4 attached in the centrifuge bucket taking care not to create folds in the bag as this will affect pelleting of the sample. Label of the bag must face to the outside of the centrifuge. Place bucket in centrifuge with appropriate balance and centrifuge sample (Bag 2) at 300g for 15 minutes, RT, Brake 1. Record in BR Remove supernatant using plasma extractor into bag 4 taking care not to resuspend the pellet. Using the tube stripper remove, strip any remaining cellular product into bag 2 and clamp off close to bag 2 Using the , seal off and disconnect bag 4, connect a buffer bag. Gently resuspend pellet by rubbing bag 2, using a low lint cloth, in a circular motion, whilst it is flat on the bench. Re-weigh bag 2 and determine the volume of buffer required to adjust the volume to 95ml using the batch record. Record in BR Adjust bag 2 final volume to 95mls by placing bag 2 on balance with appropriate clamps and run the XYZ buffer into the bag until the correct bag weight has been reached. Using the tube stripper remove, strip any remaining cellular product into bag 2 and clamp off close to bag 2. Remove XYZ buffer bag using the heat sealer In the hood using a 21g needle and syringe take a 500ul sample from bag 2 and place in Eppendorf labelled 2B. (It is recommended to determine total cell number and viability). Re-weigh bag 4 Remove a 500ul sample using a 21g needle plus 5ml syringe from Bag 4, place in Eppendorf 4A. Record in BR Depletion Insert a luer/ spike interconnector in to one of the ports on a 600ml transfer bag Weigh this transfer bag with a clamp and label as cell collection bag (bag 5). Record in BR Connect Bag 5 to the cell collection port of the XYZ LS tubing set via the luer/spike Interconnector. Ensure the Spike Interconnector slide clamp is closed. Label & Weigh the Buffer Waste Bag (Bag 6, no clamp), the negative fraction bag (Bag 7, no clamp). Record in BR Clamp the tubing set just below each of the couplers (see diagram 1). Record in BR Wipe the surface of a port on the XYZ buffer bag with a disinfectant wipe and connect to the tubing set via the Buffer spike see diagram 1. Check that a clamp is firmly fitted so that the buffer cannot flow into the tubing set. Connect a pre-filter to the XYZ tubing set via Bubble trap spike see diagram 1. Ensure that the PALL logo will be facing you when the tubing is on the XYZ machine and is the correct way up. Ensure that the tubing is clamped to prevent the any cell suspension flowing into the tubing set and that the tubing on the cell bag (bag 2) is clamped then connect the cell bag (bag 2) to the pre-filter (see diagram 1). Switch on the XYZ Instrument and select the "DEPLETION 2.1” program. Confirm by pressing "ENT". Enter the Ref- number of the XYZ LS tubing set. Follow instructions given on the instrument screen for installation of the tubing set, keeping all clamps in place. Ensure that the buffer waste bag and the priming waste bag are higher than the cell preparation bag. Enter the WBC concentration determined from sample 2A. Record in BR Enter frequency of labelled (+) cells. Record in BR Enter Sample-loading volume (This should be 95ml). Record in BR Keep Bag 2 clamped, remove the clamp from below the buffer bag just before the priming cycle. Remove the clamp from below the cell bag (bag 2) and open the side clamp on the interconnect on the cell collection bag (Bag 5) respectively, when the program instructs the cell preparation bag to be connected and the line to the cell collection bag checked. Start the depletion process. Record in BR When the depletion process has finished (Record in BR), clamp the cell collection bag (bag 5) and heat seal off and remove the Buffer Waste Bag (Bag 6), the negative Fraction bag (Bag 7) and cell collection bag (bag 5). Also heat seal off and remove the XYZ buffer bag. Place bags 6 and 7 to one side for further analysis Follow the instructions on the XYZ machine and note the protocol process code. Record in BR Determine the weight of Cell Collection Bag (Bag 5), take a 500ul sample with a 5ml syringe via the leur/spike interconnector and place in the Eppendorf labelled 5A. Record in BR 5 !"##$%&'()& *%+,+-)&'()& !"''.$&/%(0&10+2$& !"''.$&3%(0& 4.(,0& 4.(,0& 4.(,0& *%$5 #+./$%& 6$..&1(,0.$& '()& !"#$%#&'()' 6 MAGNETIC LABELLING II (ENRICHMENT OF LABELLED +VE CELLS) Connect the XYZ buffer to bag 5 using the . Determine the amount of buffer required to make the volume in bag 5 up to 400mls using BR. Record in BR Run XYZ buffer into the bag 5 whilst it is on the balance until the correct bag weight has been reached. Clamp off the XYZ buffer bag. Using the tube stripper, strip any remaining cellular product into bag 5 and clamp off close to bag 5. Record in BR Weigh Bag 8 with a clamp Remove the XYZ buffer bag and attach bag 8 to bag 5 using the Place bag 5 with bag 8 attached in the centrifuge bucket taking care not to create folds in the bag as this will affect pelleting of the sample. Ensuring that the label of the sample bag faces to the outside of the centrifuge, place bucket in the centrifuge with appropriate balance and centrifuge the sample (Bag 5) at 300g for 15 minutes, RT, Brake 1, Acceleration 8. Record in BR Remove supernatant using plasma extractor into bag 8 taking care not to resuspend the pellet. Clamp off near bag 8 and using the tube stripper, strip any remaining cellular product into bag 5 and clamp off close to bag 5. Using the heat sealer, seal off and disconnect bag 8 place to one side for further analysis Resuspend pellet by gently massaging bag 5 in a circular motion, whilst bag is flat on the bench, using a low lint cloth Re-Weigh bag 5 Determine the volume of buffer required to adjust the volume to 95ml using batch record. • Connect XYZ buffer bag 5 using the • Run XYZ buffer into the bag 5 whilst it is on the balance until the correct bag weight has been reached. Clamp off the XYZ buffer bad and using the tube stripper, strip any remaining cellular product into bag 5 and clamp off close to bag 5. • Clamp off the tubing close to bag 5. Record in BR • Remove XYZ buffer bag using the heat sealer • Gently mix the XYZ Anti-Labelled MicroBeads vial. Using a 10ml syringe and 21g needle take up contents of vial. Add to bag 5 via the luer/ spike interconnector, which has been swabbed with a disinfectant wipe. Record in BR • Incubate bag 5 for 30 minutes at controlled room temperature (+19 to +25oC) on an orbital shaker at 25 rpm. Record in BR • Re-Weight of bag 6 and bag 7. Record in BR • Using a syringe take a 15ml sample from bag 6, put a 500ul sample into the Eppendorf labelled 6A and the place rest of sample in Falcon tube labelled 6A. • Repeat above step for bag 7, but use Eppendrof 7A and Falcon tube 7A • Re-weigh bag 8. Record in BR • Remove a 500ul sample using a 21g needle plus 5ml syringe from Bag 8, place in Eppendorf 8A. Record in BR • When incubation is complete re-weigh bag 5 • Connect the XYZ buffer to bag 5 with tubing set. • Determine the amount of buffer required to make the volume up to 400mls using batch record: • Place bag 5 on the balance and run in the determined weight of buffer. Clamp off near the XYZ bag and using the tube stripper remove, strip any remaining cellular product into bag 5 and clamp off close to bag 5. Record in BR • Weigh Bag 9 with a clamp. Record in BR • Using the remove the XYZ buffer bag and attach bag 9 to bag 5. • Place bag 5 with bag 9 attached in the centrifuge bucket taking care not to create folds in the bag as this will affect pelleting of the sample. Ensuring that the label of the sample bag faces to the outside of the centrifuge, place bucket in the centrifuge with appropriate balance and centrifuge the sample (Bag 5) at 300g for 15 minutes, RT, Brake 1, acceleration 8. Record in BR • Remove supernatant using plasma extractor into bag 9 taking care not to resuspend the pellet. Clamp off near bag 9 and using the tube stripper, strip any remaining cellular product into bag 5 and clamp off close to bag 5. • Using the , disconnect bag 9 and place to one side for further analysis and attach XYZ buffer bag. • Resuspend pellet by gently massaging bag 5 in a circular motion whilst bag is flat on the bench using a low lint cloth 7 • Re-weigh bag 5 with clamp. Record in BR • Determine the amount of buffer required to make the volume up to 400ml using BR • Place bag 5 on the balance and run in the determined weight of buffer. Clamp off near XYZ buffer bag and using the tube stripper, strip any remaining cellular product into bag 5 and clamp off close to bag 5. Record in BR • Weigh bag 10 with a clamp • Remove the XYZ buffer bag and attach bag 10. • Place bag 5 with bag 10 attached in the centrifuge bucket taking care not to create folds in the bag as this will affect pelleting of the sample. Ensuring that the label of the sample bag faces to the outside of the centrifuge, place bucket in the centrifuge with appropriate balance and centrifuge the sample (Bag 5) at 300g for 15 minutes, RT, Brake 1, acceleration 8. Record in BR • Re-weigh bag 9. Record in BR • Remove a 500ul sample using a 21g needle plus 5ml syringe from Bag 9, place in Eppendorf 9A. Record in BR • When centrifugation has finished, remove supernatant using plasma extractor into bag 10 taking care not to resuspend the pellet. Clamp off near bag 10 and using the tube stripper, strip any remaining cellular product into bag 5 and clamp off close to bag 5. • Using the Thermo remove bag 10, set to one side for further anaylsis and attach a XYZ buffer bag. • Resuspend pellet by gently massaging bag 5 in a circular motion whilst bag is flat on the bench using a low lint cloth • Using the batch record determine and add in XYZ buffer to make the volume up to 100mls. • Using a 21g needle and syringe take a 500ul sample from bag 5 to an Eppendorf labelled 5B. Record in BR • Re-weigh bag10. Record in BR • Remove a 500ul sample using a 21g needle plus 5ml syringe from Bag 10, place in Eppendorf 10A. Record in BR Labelled +ve ENRICHMENT • Insert a luer/spike interconnector in to one of the ports on a 600ml transfer bag • Weigh this 600ml transfer bag with clamp and label as Labelled +ve cell collection bag (bag 11). Record in BR • Connect Bag 11 via the Luer/Spike Interconnector to the cell collection port of a XYZ TS tubing set. Ensure that the Luer/Spike Interconnector slide clamp is closed. • Label & Weigh the Buffer Waste Bag (Bag 12, no clamp) and the negative Fraction bag (Bag 13, no clamp). Record in BR • Clamp the tubing set just below each of the couplers (see diagram 1). • Wipe the surface of a port on the XYZ buffer bag with a disinfectant wipe and connect to the tubing set via the Buffer spike (see diagram 1). Check that a clamp is firmly fitted so that the buffer cannot flow into the tubing set. • Connect a pre-filter to the XYZ tubing set via Bubble trap spike see diagram 1. Ensure that the PALL logo will be facing you when the tubing is on the XYZ machine and is the correct way up. • Ensure that the tubing is clamped to prevent the any cell suspension flowing into the tubing set and that the tubing on the cell bag (bag 5) is clamped then connect the cell bag (bag 5) to the pre-filter (see diagram 1). • Switch on XYZ Instrument and select the separation program called "ENRICHMENT 3.1". Confirm your choice by pressing "ENT" and enter the Ref-number of the XYZ tubing set. Follow instructions given on the instrument screen for installation of the tubing set and set up of the program, keeping all clamps in place. Ensure that the buffer waste bag and the priming waste bag are higher than the cell preparation bag. Keep Bag 5 clamped until after the system has been primed, remove the clamp from below the buffer bag just before the priming cycle. 8 • Remove the clamp from below the cell bag (bag 5) when the programme instructs the cell preparation bag to be connected. • Open the side clamp on the interconnect on the Labelled +ve cell collection bag (Bag 11) as instructed • Start the ENRICHMENT process. Record in BR • When the enrichment process has finished (Record in BR) clamp the cell collection bag (bag 11) and heat seal the tubing of the, Buffer Waste Bag (Bag 12), the negative Fraction bag (Bag 13) and cell collection bag (bag 11) remove and place in hood. Also heat seal off and remove the XYZ buffer bag. • Follow the instructions on the XYZ machine and note the protocol process code. Record in BR • Determine the weight of Cell Collection Bag (Bag 11), using a syringe take a 500ul sample via the leur/spike interconnector and put in the Eppendorf labelled 11A. Record in BR • Re-weigh bag 12 and bag 13. Record in BR • Using syringe take a 15ml sample from bag 12, put a 500ul sample into the Eppendorf labelled 12A and place the rest of the sample into the Faclon tube labelled 12A. • Repeat above step for bag 13, but use Eppendrof 13A and Falcon tube 13A. • Ensure that 500ul samples have been taken from samples 2A, 2B, 3A, 4A, 5A, 5B, 6A, 7A, 8A, 9A, 10A, 11A, 12A and 13A for pathology cell counting/flow cytometry. • Close Biohazard hood and run UV program • Take samples to Pathology and carry out differential cell counts according to SOP#. Record in BR • Take samples for Flow cytometry analysis according to SOP#. Record relevant results in BR Antigen loading of Labelled +ve+ enriched cell population. • For rest of process use sterile gloves, changing on every entry into the biohazard hood. • Clean biohazard hood with 70% alcohol, put down sterile drapes and put out settle and contact plates for in-process monitoring as SOP # • Label a 50ml Falcon tube AL1 using pre-prepared label SOP# for the sample antigen loading • Label two eppendorf tubes with AL2 and AL3 for cell counting samples • Remove peptides and KLH from +4°C fridge, swab with 70% alcohol and place in biohazard hood • Using a 50ml syringe remove cells from bag 11 via leur/spike interconnector. Record in BR • Place in 50ml Falcon tube 1A • Using a syringe, add 10ml Normal saline to bag 11 to wash out bag. Use a cannula to load the syringe. Record in BR • Remove saline from bag 11, via leur/spike interconnector using a syringe and add to 50 ml Falcon tube 1A. Record in BR • Make volume up to 50 mls with X-VIVO 15 media. Determine required volume using the batch record. Record in BR • Take a 100ul sample from Falcon tube 1A using a autoclaved pipette and sterile filter tips • Carry out a viable cell count using (Cell Counting SOP #) Record in BR • Centrifuge 50ml Falcon tube AL1 at 400g for 5 min, Brake 9, Acceleration 8. RT • Without disturbing the cell pellet, remove supernatant using a 60ml syringe and cannula. • Re-cap tube and flick pellet to resuspend add required volume of X-vivo 15 medium to give 1x106 cells/ml using a sterile syringe. Determine volume using batch record. Record in BR 9 • Using an autoclaved P20 or P200 pipette add P1, P2, P3 and P4 peptides to a final concentration of 1ug/ml and KLH to give final required concentration of 25ug/ml. • Incubate cell preparation at RT for 1 hr, in the biohazard cabinet. Record in BR • Make volume up to 50ml in sterile normal saline. Use batch record to determine volume. Record in BR • Centrifuge sample at 400g for 5 min, Brake 9, RT. Record in BR • Without disturbing the cell pellet, remove supernatant using a 60ml syringe and cannula. Inoculate a Thioglycollate broth bottle obtained from MAH microbiology department with 5mls of the supernatant- label with prepared label- AL WASH 1 • Make volume up to 50ml in sterile normal saline. Use batch record to determine volume. Record in BR • Centrifuge sample at 400g for 5 min, Brake 9, RT. Record in BR • Without disturbing the cell pellet, remove supernatant using a 60ml syringe and cannula. Inoculate a Thioglycollate broth bottle obtained from MAH microbiology department with 5mls of the supernatant- label with prepared label- AL WASH 2 • Resuspend cell pellet in 1ml normal saline and carry out a viable cell count (Cell Counting SOP #) Record in BR • Use one of the following to load the administration syringe depending on the product schedule of the patient the product is being manufactured for. • 1x106 xyz ID- o Remove required volume of LABELLED +VE+)/-- work out the volume required using the FACS purity data and calculation in BR using a autoclaved pipette and sterile filter tips o Place in a sterile 5ml tube; add sterile saline to give a 4ml volume and load 1ml into each of 4x 1ml syringes. Record in BR • 1x106 xyz IV- o Remove required volume of LABELLED +VE+)/-- work out the volume required using the FACS purity data and calculation in BR using a autoclaved pipette and sterile filter tips o Place in a sterile 5ml tube; add sterile saline to give a 1ml volume and load into a 1ml syringe. Record in BR • 1x106- 1x107 xyz ID- o If participant is on the 1x107 LABELLED +VE+)/-dose schedule then this number of cells should be used if possible. If this cannot be achieved then the patients will receive a dose, which is as close as possible to the target dose. o Remove required volume of LABELLED +VE+)/- work out the volume required using the FACS purity data and calculation in BR using a autoclaved pipette and sterile filter tips o Place in a sterile 5ml tube; add sterile saline to give a 4ml volume and load 1ml into each of 4x 1ml syringes. Record in BR • 1x106- 1x107 xzy IV- o If participant is on the 1x107 LABELLED +VE+)/-dose schedule then this number of cells should be used if possible. If this cannot be achieved then the patients will receive a dose, which is as close as possible to the target dose. o Remove required volume of LABELLED +VE+)/-- work out the volume required using the FACS purity data and calculation in BR using a autoclaved pipette and sterile filter tips o Place in a sterile 5ml tube; add sterile saline to give a 1ml volume and load into a 1ml syringe. Record in BR • Cryopreserve remaining cells according to Cryopreservation of DC Product SOP. END OF DOCUMENT Document Review Record Date Reviewed Reviewed By Reason for Review 1 Prilog 14: Pregled najva!nijih GMP elemenata u izradi visoko manipulisanih "elija za primenu u klini#ke svrhe Tabela P14.1: Pregled najva!nijih elemenata u izradi visoko manipulisanih "elija za primenu u klini#ke svrhe (primer mezenhimskih "elija izolovanih iz placente). Good Manufacturing Practice Code Requirements Quality Management System (QMS) Requirements Assigned Priority Time Allocation Increase of Resources Required Requirements level Policies Procedures Records High, Medium or Low A, B, C, D ** Yes/No QUALITY MANAGEMENT SYSTEM Quality Objectives, Quality System Requirements High A No Organisational Structure: Director, Quality Nominee and Production Nominee(s) High A No Resources and Personnel High D Yes Monitoring Systems, Internal Audits, Corrective Actions and Management Review Medium D No Change Control Low D No PERSONNEL AND TRAINING General Personnel Objectives Medium A No Initial Training and Induction High A No Laboratory Training High C No QA and QMS Training Medium C No Annual Training, Task-based Training Medium B No BUILDINGS AND FACILITIES General Building and Facilities Objectives, Floors, Walls and Fittings High C Yes Environmental Control High D Yes Cleaning and Pest Control High D Yes Goods receipt and Storage Areas Medium C No Manufacturing Areas including General, Donor areas, Despatch Medium C No EQUIPMENT General Equipment Objectives, Preventative Maintenance High D Yes Qualification and Validation including IQ, OQ, PQ# (see definitions below) Medium A Yes Calibration High B Yes Specific Equipment including Refrigeration equipment, Irradiation equipment, Barcode equipment High A Yes # IQ, Installation Qualification; OQ, Operational Qualification; PQ, Process Qualification. DOCUMENTATION General Document Control Objectives High D No Control of Documentation High D No Storage and Retention Medium B No ** A (first 1-3 months only); B (once or less than 3 months at any stage); C (6-12 months); D (over 12 months) 2 cont’d. Good Manufacturing Practice Code Requirements Quality Management System Requirements Assigned Priority Time Allocation Increase of Resources Required Requirements level Policies Procedures Records High, Medium or Low A, B, C, D ** Yes/ No RECORDS General Record Keeping Objectives, Donor Sessions, Donor Deferral High D No Collection, Retrieval and Processing Records High D No Final Product and Batch Record High D No Storage, retention and Archiving High B Yes Product Complaints and Recall, Corrective Action High D No Computer Records including General Computer Records Requirements, Documentation, Validation Data, Data Control Medium C Yes CONTROL OF MATERIEL General Control of Materiel Objectives High D No Starting Materiel including General, Reagents, Collection Packs, Labels/ High C Yes In-Process Materiel High C Yes Suppliers and Sub-Contractors Review Medium B No Non-Conforming Materiel, Recall of Products High C No DONOR SELECTION, DONATION AND TESTING^ General Donor Selection Requirements including Pre-Donation, Donation, Post Donation High C No Tissue Donors including Pre-Donation, Donation, Post Donation High C No Testing of Donor Samples including General, Contracted Laboratories High C Yes ^Includes Additional Requirements of both Australian Red Cross and Auscord (The Australian Cord Blood Registry) Guidelines for Donor Selection and Testing PROCESS CONTROL General Process Control Requirements High D No Validation Medium A Yes Quality Control, Monitoring Medium D No Product release High C No STORAGE, PACKAGING AND TRANSPORT Storage includingGeneral, Labeling, Quarantine, Discard High C No Packaging including General, Labelling, Records High B Yes Transport Medium B Yes ** A (first 1-3 months only); B (once or less than 3 months at any stage); C (6-12 months); D (over 12 months) 3 Tabela P14.2: Pregled najva!nijih elemenata u izradi visoko manipulisanih "elija za primenu u klini#ke svrhe (primer mezenhimskih "elija izolovanih iz placente): posebna razmatranja. Stage of manufacturing and/or cGMP area: Special considerations in manufacturing process Obtaining and control of clinical-grade or GMP grade critical materiel: - A major obstacle has been a lack of clinical-grade cGMP reagents in the cell isolation steps. If clinical grade consumables are available, these should given priority (e.g. Ficoll-Paque is produced as a cGMP reagent whereas Percoll is not). Any change in reagent should be validated, e.g. we have found that Ficoll-Paque produces equivalent results for isolation of placental cells in our pre-clinical data. - Consumables with a sterility testing certificate and/or a Certificate of Analysis (COA) stating the minimal level of “contaminants” should also be considered (e.g. collagenase I remains a relatively crude isolation fraction containing a range of proteins and enzymes such as collagenase, trypsin and dispase). - Control of critical material (that directly or indirectly may influence the quality of the cell product) is obtained according to specific requirements outlined in procedural documents. This includes all items used in the production process (such as media, enzymes, flasks and tubes), but also items such as 70% ethanol, sterile drapes, sterile gloves and disposable laboratory gowns. Transport and handling of placenta: - We currently use elective caesarean sections as our placenta source as, although the operating procedures are open, they are performed in relatively sterile environment. The placenta is subsequently double bagged in plastic draw string bags. After weighing, the placenta is placed in a cool box and transferred to our manufacturing facilities on the same campus for processing. Appropriate documentation is signed (donor details and ID check, date, time, witnessing process). The placenta must never be left unattended; it must be given directly to the manufacturing staff to be placed into the laboratory and attended immediately by the manufacturing team. Although class II safety cabinets are used in our production of MSC, this still represents an “open” system of manufacture. Even with a quality system in place and numerous safety precautions, there remains a greater chance of microbial contamination in an open system versus a closed system. Our hospital manufacturing laboratory is not classified as a clean room and is therefore not required to meet defined levels for air particles or viable organisms as set out by appropriate regulatory authorities for clean room standards. However, in order to provide a highly controlled environment and to ensure processing is carried out in an environment where microbial contamination levels are known, environmental monitoring is conducted on a regular basis. - If cord blood has not been drained prior to receiving placenta, blood in the placenta will still be at foetal arterial pressure. Care should be taken when taking the cord blood and placental anatomy should be known in order to find the cord vein. Relevant safety equipment such as eye protection, mask and gloves should always be used. Documentation: - The Batch Record Form and procedural documents (e.g. Standard Operating Procedures, SOP) are subject to strict document control; each version is reviewed, approved and released. Archived versions are stored and made available, if required. - It is useful to have a brief check-list of tasks to be completed prior to and on the day of production. This list enables staff to focus and perform their tasks according to procedural requirements and record any non-conformances occurring in the manufacturing process. 4 Stage of manufacturing and/or cGMP area: Special considerations in manufacturing process Equipment and facilities: - Equipment servicing and calibration is obtained on a regular basis and along with the cleaning records kept on the premises; it is required that all equipment used in the production run is up-to-date with regards to maintenance and calibration (including items such as thermometers, timers, automatic pipettes, data-loggers monitoring temperature and humidity). - Equipment status can be recorded on separate documents linked to the Batch Record. - Production facility storage cupboards are temperature mapped, temperature and humidity monitored and access-controlled. - Requirements for each equipment item maintenance, cleaning and sterilisation are outlined in equipment- specific procedures (SOP). - Production facility maintenance requirements are outlined and recorded in separate documents that may be directly linked to the Batch Record Form or cross-referenced (by using date, production ID, unique product identifier or similar). Environmental monitoring: - Environmental monitoring shall at a minimum include temperature, humidity and microbial contamination. Acceptable limits (alert vs. action limits) are set according to historical data and/or literature data. - Environmental monitoring data outside the acceptable limits are considered to be a non-conformance with the QMS requirements and addressed as such (including root cause analysis, corrective and preventative action implementation). Labelling: - Labels and related procedural documents are document controlled. A label reconciliation process is implemented for each production run. Unique identifiers are devised and no numbers can be repeated in any other production process. Patient confidentiality is maintained throughout the process. - Accurate labelling of products is an important component of Good Clinical Practice (GCP) and is required to ensure compliance with the local Quality Management System Process. Labels must be compatible with the use/handling and storage conditions of the labelled sample and must not detach when stored in incubators or in the vapour phase of liquid nitrogen, or if immersed in water (e.g. use cryovials labels that can be stored at -180°C and remain attached to the cryovial). - Labels should be affixed in such a way as to allow all information to be read clearly. Labels must detail that it is label X of Y e.g. 4 of 12, to allow traceability; labels must be used in sequential number order. - Extra copies of labels must be destroyed or defaced. Copies of labels should be attached to the label reconciliation form and where possible to batch records, storage logs etc as a confirmation of the identity of the product that was being handled. Production approval and decision making: - Requirements for production approval are outlined in a separate procedure; cGMP requirements are followed in terms of responsibilities and Quality Assurance staff and production staff act independently (“arm’s length” - production nominee vs. quality nominee). The ultimate decision-maker is the Director (both production and quality staff independently report to the Director). This also provides a relevant Risk Management framework and decision making structure. - Conditions of the cryostorage should be clearly defined and action limits set. Cryostorage conditions should be continuously monitored and alarmed. Cellular product specifications have been devised in order to provide a defined set of parameters for their storage, transport and testing prior to use. Maintenance of the vapour phase liquid N2 tank is required, including regular refill of the liquid N2 by an 5 Cryostorage, cell release and dispatch: approved supplier. - The cell release process consists of two phases. The first step is production release according to production release criteria (Table 2). These tests are obtained in an accredited Pathology laboratory and it is recommended to outsource them rather than doing in-house testing for release criteria purposes. The second step is release prior to infusion obtained at the clinical site, externally or internally, with the testing of sterility by 14-day microbiology culture and viability >70% by Trypan Blue exclusion. Cells have to pass both of these tests in order to be administered to a patient. - Dispatch of cells is by an approved courier only, in a validated and approved dry shipper (that maintains required temperature during the entire transport process). Accompanying documentation (instruction for handling, storage and testing of the cells) is required and regular liaison with clinical site staff, externally or internally, occurs. !! 1 Prilog 15: Primer liste za GMP inspekciju ! ! 1. QUALITY SYSTEM Rationale and General Completed Review Required Partially Completed Not Completed Quality Objectives Completed Review Required Partially Completed Not Completed Organisational Structure Completed Review Required Partially Completed Not Completed General • Director • QA and Production Nominees • Resources and Personnel Quality System requirements Completed Review Required Partially Completed Not Completed • Documentation is mostly completed or under review • Change Control is not implemented Monitoring Systems Completed Review Required Partially Completed Not Completed • General • Internal Audit • Corrective Action Management Review Completed Review Required Partially Completed Not Completed 2. PERSONNEL AND TRAINING Rationale Completed Review Required Partially Completed Not Completed General Completed Review Required Partially Completed Not Completed Training Completed Review Required Partially Completed Not Completed Records Completed Review Required Partially Completed Not Completed 3. BUILDINGS AND FACILITIES Rationale and General Completed Review Required Partially Completed Not Completed Environmental Control Completed Review Required Partially Completed Not Completed Floors, Walls and Fittings Completed Review Required Partially Completed Not Completed Cleaning Completed Review Required Partially Completed Not Completed Goods receipt and Storage Areas Completed Review Required Partially Completed Not Completed Manufacturing Areas Completed Review Required Partially Completed Not Completed • General • Donor areas • Despatch • Mobile blood collection sites 4. EQUIPMENT Rationale and General Completed Review Required Partially Completed Not Completed Qualification and Validation Completed Review Required Partially Completed Not Completed Preventative Maintenance Completed Review Required Partially Completed Not Completed Calibration and Validation Completed Review Required Partially Completed Not Completed !! 2 Specific Equipment Completed Review Required Partially Completed Not Completed • Refrigeration equipment • Irradiation equipment • Barcode equipment 5. DOCUMENTATION Rationale and General Completed Review Required Partially Completed Not Completed Control of Documentation Completed Review Required Partially Completed Not Completed Storage and Retention Completed Review Required Partially Completed Not Completed 6. RECORDS Rationale and General Completed Review Required Partially Completed Not Completed Donor sessions Completed Review Required Partially Completed Not Completed Collection/Retrieval and Processing Records Completed Review Required Partially Completed Not Completed Final Product Completed Review Required Partially Completed Not Completed Storage, retention and Archiving Completed Review Required Partially Completed Not Completed Product Complaints and Recall Completed Review Required Partially Completed Not Completed Donor Deferral Completed Review Required Partially Completed Not Completed Corrective Action Completed Review Required Partially Completed Not Completed Security Completed Review Required Partially Completed Not Completed Computer Records Completed Review Required Partially Completed Not Completed • General • Documentation • Validation Data • Data Control 7. CONTROL OF MATERIEL Rationale and General Completed Review Required Partially Completed Not Completed Suppliers and Sub-Contractors Completed Review Required Partially Completed Not Completed Starting Materiel Completed Review Required Partially Completed Not Completed • General • Reagents • Collection Packs • Labels In-Process Materiel Completed Review Required Partially Completed Not Completed Non-Conforming Materiel Completed Review Required Partially Completed Not Completed Recall of Products Completed Review Required Partially Completed Not Completed 8. DONOR SELECTION, DONATION AND TESTING Rationale and General Completed Review Required Partially Completed Not Completed • Pre-Donation • Donation • Post Donation Whole Blood Donors – N/A Apheresis Donors Completed Review Required Partially Completed Not Completed !! 3 • Donation Tissue Donors Completed Review Required Partially Completed Not Completed • Pre-Donation • Donation • Post Donation Testing of Donor Samples Completed Review Required Partially Completed Not Completed • General • Contracted Laboratories 9. PROCESS CONTROL Rationale Completed Review Required Partially Completed Not Completed General Completed Review Required Partially Completed Not Completed Validation Completed Review Required Partially Completed Not Completed • General • Quality Control • Monitoring Product release Completed Review Required Partially Completed Not Completed 10. STORAGE, PACKAGING AND TRANSPORT Rationale Completed Review Required Partially Completed Not Completed Storage Completed Review Required Partially Completed Not Completed • General • Labelling • Quarantine • Discard Packaging Completed Review Required Partially Completed Not Completed • General • Labelling • Records Transport Completed Review Required Partially Completed Not Completed !!! ! 1 Prilog 16: Rezultati korelacione analize dokumenata. Slika 4.5: Korelaciona analiza (3D grafi!ki prikaz) odnosa izmedju koncepata. Lista svih dokumenata je prikazana u Tabeli 3.2 u poglavlju: Materijali. A Konceptualni parovi i u!estalost njihovog zajedni!kog pojavljivanja u tekstu dokumenata su prikazani za dokumenta agencije FDA (A). Razli!ite boje na grafi!kom prikazu reprezentuju relativne frekvencije koncepata koji se zajedno pojavljuju kroz tekst. 2 B Konceptualni parovi i u!estalost njihovog zajedni!kog pojavljivanja u tekstu dokumenata su prikazani za dokumenta agencije TGA (B). Razli!ite boje na grafi!kom prikazu reprezentuju relativne frekvencije koncepata koji se zajedno pojavljuju kroz tekst. 3 C Konceptualni parovi i u!estalost njihovog zajedni!kog pojavljivanja u tekstu dokumenata su prikazani za dokumenta agencije EMA (C). Razli!ite boje na grafi!kom prikazu reprezentuju relativne frekvencije koncepata koji se zajedno pojavljuju kroz tekst. 4 D Konceptualni parovi i u!estalost njihovog zajedni!kog pojavljivanja u tekstu dokumenata su prikazani za regulatorne okvire GMP(1) i GMP(2) (D). Razli!ite boje na grafi!kom prikazu reprezentuju relativne frekvencije koncepata koji se zajedno pojavljuju kroz tekst. 5 E Konceptualni parovi i u!estalost njihovog zajedni!kog pojavljivanja u tekstu dokumenata su prikazani za GCP dokumenta (E). Razli!ite boje na grafi!kom prikazu reprezentuju relativne frekvencije koncepata koji se zajedno pojavljuju kroz tekst. Prilog 17: Dodatni rezultati iz analize intervjua. Tabela P17.1: Rezultati vezani za razvojne pristupe kao !to su obuka osoblja, usvajanje novih tehnologija ili novih ideja kao i za zna"ajne pouke nastale u procesu razvoja i saradnju sa partnerima (engl. Staff Training & Access to New People, New Technology, New Ideas; Important Lessons Learned; On Partnerships). ! KNOWLEDGE, PRACTICE & EXPERIENCE VIEW, PERCEPTION & OPINION Excerpts from the interview transcripts related to the topic S ta ff Tr ai ni ng & A cc es s to N ew P eo pl e, N ew T ec hn ol og y, N ew Id ea s - “conferences, publishing; a formal training and assessing program which is actually sub-contracted[CTE01]. -follow medical literature; stem cell meetings and conferences; team meetings, seminars, publishing” [CTE02]. - there's a very conscious effort to stay in touch; it's staying in touch, continuous dialogue ! it's a two-way street; regular meeting at conferences, the participation on committees, in societies ! give you that opportunity to swap stories, learn, play catch up and, on the academic sense, quite often it'll lead to joint papers being written” [CTE03]. - “one external education opportunity per year ! also an internal staff training programme (ongoing) [and] an intra-company education programme, divisions and individuals present on what they're doing, what their challenges are and what they face on a daily basis ! so that everyone is continuing to be educated at a professional level but also at a company level [formal and informal]” [CTE05]. - “predominantly we try and hire staff who have a basic skill set in this industry or certainly have a skill set working within the GMP environment. From there we take people and mould them into what we do within this organisation ! we have a particular culture that requires that we gel together pretty well. Our training and development tends to be group based where we, team building in essence, in order to keep the group together. In doing so keep them multi-skilled and keep them up to date with GMP so that we have as much flexibility as we possibly can have within a facility of this size” [CTE22]. Im po rta nt L es so ns L ea rn ed - “keeping consistency, aiming above the regulatory minimum” [CTE01]. - “team work is essential” [CTE02]. - “independence, speed of response and key personnel are success factors; not to take the exemptions [since many] have attempted to take every exemption they could in order not to have to be compliant; in doing so, they ended up with a research capability that had an inability to move forward” [CTE03]. - “ability to interact [with] different sector, from businessmen to top scientist, from regulatory body to patients” [CTE04]. - “cost economies [understanding and application]” [CTE05]. - “business models are not yet clearly defined, there are not really so many big successes, but we have to be realistic, nothing goes so fast if you're dealing with drugs... so this industry is now, I think, in the beginning of the higher growth and in [the next]15 years, we can see some major disappointments and also some major successes, we have to adapt our curve all the time” [CTE11]. - “have contingency planning, for example, double the time and multiply the cost by four or the other way around – for a successful completion of a project” [CTE13]. - “the main thing that's really critical to taking scientific advancement forward is the relationship between the scientists and the clinicians! no scientific development will really make it through without clinical champions (ones who know the patient and understand the benefit to their patient, but also can understand the science, not just accept the word) ! without that strong relationship, the regulatory environment is really just - it doesn't even come into the equation” [CTE19]. - “scientists are figuring out, okay well this cell if we add this chemical to it in vivo we can make this cell do something else ! it is a massively innovative space ! people from all different disciplines are attracted to the field and are coming into it bringing different points of view and their own expertise which is really driving things forward” [CTE23]. On partnerships: ! “The word serendipity comes to mind. I'm not sure that it's appropriate but I'm of the ilk that if you are in and around the marketplace on a regular basis and, if you're relatively open, those opportunities come. You have to recognise them and grab them with both hands when they're there. So you need to put yourself in the right place, and that's the planned bit. The unplanned bit is what happens. So we make sure that we are in and around and regularly exposed, and rely on our purely opportunistic capabilities and the fact that it's a dynamic field. It's not a stagnant field.” [CTE03] ! “Our partnerships have matured as our company matured. So from scientific collaborations and partnerships to clinical [research] collaborations and partnerships and now we're moving more towards corporate collaborations and relationships.” [CTE05] ! “There were [always] partnerships and alliances, they were on a smaller scale. Now we need to scale them up because there are areas of great expertise, which could theoretically help and influence like the nanotechnology groups may be able to help. The materials groups, the smart surface groups and so on. So you can sort of start to conceive of groups with different disciplines, backgrounds and expertise coming together to create new institutions that have feet in the more traditional universities and research institutes. [It all appears to be] very positive and people are much more accepting today of these cross-institutional arrangements.” [CTE14] ! “There's a lot of unknowns, so that whole scenario, around developing a cell therapy pipeline platform(s) – we're making that up as we go along because nobody really knows what is needed. The regulatory bodies are looking at this, not quite sure what a cell production facility would look like and what you would have to have in place in terms of safety and composition; a lot of it is a work in progress and the other thing that makes it really interesting is people are approaching it from a whole variety of different perspectives.” [CTE21] "#$%&'(#%()*'+$,&'()(,+-.%/,0%$1)2(34567(!(34589:((( ( Prilog 18: Dodatne mape iz pretrage podataka. Slika P18.1: Mapa klini!kih istra"ivanja uz upotrebu #elijskih terapija u Australiji (pretraga uz klju!ne re!i “cell therapies”) u januaru 2011. godine. Slika P18.2: Mapa klini!kih istra"ivanja uz upotrebu #elijskih terapija u SAD-u (pretraga uz klju!ne re!i “cell therapies”) u januaru 2011. godine. Slika P18.3: Mapa klini!kih istra"ivanja uz upotrebu dendriti!nih #elija na svetskom nivou (pretraga uz klju!ne re!i “cell therapies AND dendritic cells”) u januaru 2011. godine. Ostali prilozi tezi: Prilog 19: Biografija autora Prilog 20: Izjava o autorstvu Prilog 21: Izjava o istovetnosti !tampane i elektronske verzije doktorske disertacije Prilog 22: Izjava o kori!"enju Biografija autora Dragica KNE!EVI" ILI" ro#ena je 03.12.1968. godine u Rumi. Osnovnu $kolu je pohadjala u Rumi i Beogradu, a srednju medicinsku $kolu, farmaceutski smer, zavr$ila je 1987. godine u Beogradu. Diplomirala je 1995. godine na Biolo$kom fakultetu, studijska grupa molekularna biologija i fiziologija. Ovim je stekla stru%ni naziv diplomiranog molekularnog biologa i fiziologa. Poslediplomske studije na Biolo$kom fakultetu upisala je $kolske 1995/96. godine. Zvanje magistra biolo$kih nauka stekla je 1998. godine, odbranom teme „Identifikacija genoma hepatitis C virusa u humanim tkivima pomo&u RT PCR metode“. Zvanje Medical Scientist dodeljeno joj je od strane Australian Institute of Medical Scientists (AIMS) 2003. godine. Poslediplomske studije u oblasti menad'menta na University of Leicester (UK) upisala je 2004/05. godine. Zvanje Master of Business Administration, MBA stekla je na istom univerzitetu 2008. godine. Poslediplomski kurs iz oblasti zakonskih regulativa u klini%kim istra'ivanjima (engl. Regulatory Affairs) zavr$ila je sa zapa'enim rezultatima 2006. godine na University of Queensland, School of Pharmacy, Brisbane, Australia. Radila je na Biolo$kom fakultetu Univerziteta u Beogradu kao stru%ni saradnik i asistent u nastavi. Bila je zaposlena na Vojnomedicinskoj akademiji u Beogradu kao molekularni biolog u Centralnoj laboratoriji na odeljenju za ispitivanja u oblasti virusologije, da bi u junu 2000. godine bila pozvana od strane University of Malta da u%estvuje u radu njihovog tima kao saradnik (engl. Research Fellow) u istra'iva%koj i dijagnosti%koj laboratoriji za molekularnu genetiku (engl. Molecular Genetics Laboratory). Nakon toga bila je zaposlena kao Research Scientist u tada novoosnovanoj kompaniji Atheneum Biotechnology, Malta, kasnije Synergene Ltd, Malta. Poslove tehni%kog $efa laboratorije (engl. Laboratory and Technical Manager) obavljala je u istoj kompaniji za potrebe molekularne dijagnostike, genotipizacije i primene molekularne dijagnostike u sudskoj medicini. Od juna 2005. godine zaposlena je u univerzitetskoj bolnici Mater Health Services (MHS), Brisbane, Australia, kao vi$i menad'er za kvalitet i primenu zakonskih regulativa (engl. Senior Quality and Regulatory Affairs Manager) u unapredjenju konvencionalnih i razvoju novih &elijskih terapija za klini%ke potrebe i klini%ka istra'ivanja. U medjuvremenu je, godinu dana radila u timu za akreditaciju kao stru%ni savetnik jednog od izvr$nih direktora u istoj univerzitetskoj bolnici. Osim engleskog jezika koji fluentno pi$e, %ita i govori, slu'i se italijanskim jezikom i poznaje i uspe$no koristi niz programskih paketa (npr. Microsoft, Macintosh, Leximancer, Nvivo, QPulse). U svom radu stekla je zna%ajno iskustvo, pre svega, u primeni novih nau%nih dostignu&a u klini%koj praksi (za potrebe dijagnostike i le%enja) kao i u oblasti zakonsko regulatornih principa i akreditacijskih sistema koji se primenjuju u proizvodnji bioterapeutskih sredstava u Evropi, Australiji i Americi. (lan je Nacionalne asocijacije za unapredjenje i razvoj regenerativne medicine u Srbiji (NAURRM), Evropskog dru$tva za genske i &elijske terapije (engl. European Society of Gene and Cell Therapy, ESGCT), Internacionalnog udru'enja za &elijske terapije (engl. International Society for Cellular Therapy, ISCT) i Asocijacije za klini%ke i regulatorne nauke u Australiji (engl. Association of Regulatory and Clinical Scientists Australia, ARCS). Od 2005. godine sve radove i publikacije objavljuje pod svojim skra&enim imenom Nina ILIC. Изјава о ауторству Потписани-a mr Dragica Knežević Ilić број индекса - Изјављујем да је докторска дисертација под насловом KOMPARATIVNA ANALIZA PROCESA, SISTEMA I REGULATIVA U IZRADI I PRIMENI MATI!NIH "ELIJA I DRUGIH "ELIJSKIH TERAPIJA • резултат сопственог истраживачког рада, • да предложена дисертација у целини ни у деловима није била предложена за добијање било које дипломе према студијским програмима других високошколских установа, • да су резултати коректно наведени и • да нисам кршио/ла ауторска права и користио интелектуалну својину других лица. Потпис докторанда У Београду, 15.10.2013. godine _________________________ Изјава o истоветности штампане и електронске верзије докторског рада Име и презиме аутора _________ mr Dragica Knežević Ilić _________________ Број индекса _______________________-________________________________ Студијски програм ________________ Farmacija _________________________ Наслов рада KOMPARATIVNA ANALIZA PROCESA, SISTEMA I REGULATIVA U IZRADI I PRIMENI MATI!NIH "ELIJA I DRUGIH "ELIJSKIH TERAPIJA Ментор __ Prof. dr Snežana Savić i Prof. dr Ljiljana Tasić, Farmaceutski fakultet __ Потписани/а ______ mr Dragica Knežević Ilić _______ Изјављујем да је штампана верзија мог докторског рада истоветна електронској верзији коју сам предао/ла за објављивање на порталу Дигиталног репозиторијума Универзитета у Београду. Дозвољавам да се објаве моји лични подаци везани за добијање академског звања доктора наука, као што су име и презиме, година и место рођења и датум одбране рада. Ови лични подаци могу се објавити на мрежним страницама дигиталне библиотеке, у електронском каталогу и у публикацијама Универзитета у Београду. Потпис докторанда У Београду, 15.10.2013. godine _________________________ 3. Ауторство – некомерцијално – без прераде ! Изјава о коришћењу Овлашћујем Универзитетску библиотеку „Светозар Марковић“ да у Дигитални репозиторијум Универзитета у Београду унесе моју докторску дисертацију под насловом: KOMPARATIVNA ANALIZA PROCESA, SISTEMA I REGULATIVA U IZRADI I PRIMENI MATI!NIH "ELIJA I DRUGIH "ELIJSKIH TERAPIJA која је моје ауторско дело. Дисертацију са свим прилозима предао/ла сам у електронском формату погодном за трајно архивирање. Моју докторску дисертацију похрањену у Дигитални репозиторијум Универзитета у Београду могу да користе сви који поштују одредбе садржане у одабраном типу лиценце Креативне заједнице (Creative Commons) за коју сам се одлучио/ла. 1. Ауторство 2. Ауторство - некомерцијално 4. Ауторство – некомерцијално – делити под истим условима 5. Ауторство – без прераде 6. Ауторство – делити под истим условима (Молимо да заокружите само једну од шест понуђених лиценци, кратак опис лиценци дат је на полеђини листа). Потпис докторанда У Београду, 15.10.2013. godine ____________________ 1. Ауторство - Дозвољавате умножавање, дистрибуцију и јавно саопштавање дела, и прераде, ако се наведе име аутора на начин одређен од стране аутора или даваоца лиценце, чак и у комерцијалне сврхе. Ово је најслободнија од свих лиценци. 2. Ауторство – некомерцијално. Дозвољавате умножавање, дистрибуцију и јавно саопштавање дела, и прераде, ако се наведе име аутора на начин одређен од стране аутора или даваоца лиценце. Ова лиценца не дозвољава комерцијалну употребу дела. 3. Ауторство - некомерцијално – без прераде. Дозвољавате умножавање, дистрибуцију и јавно саопштавање дела, без промена, преобликовања или употребе дела у свом делу, ако се наведе име аутора на начин одређен од стране аутора или даваоца лиценце. Ова лиценца не дозвољава комерцијалну употребу дела. У односу на све остале лиценце, овом лиценцом се ограничава највећи обим права коришћења дела. 4. Ауторство - некомерцијално – делити под истим условима. Дозвољавате умножавање, дистрибуцију и јавно саопштавање дела, и прераде, ако се наведе име аутора на начин одређен од стране аутора или даваоца лиценце и ако се прерада дистрибуира под истом или сличном лиценцом. Ова лиценца не дозвољава комерцијалну употребу дела и прерада. 5. Ауторство – без прераде. Дозвољавате умножавање, дистрибуцију и јавно саопштавање дела, без промена, преобликовања или употребе дела у свом делу, ако се наведе име аутора на начин одређен од стране аутора или даваоца лиценце. Ова лиценца дозвољава комерцијалну употребу дела. 6. Ауторство - делити под истим условима. Дозвољавате умножавање, дистрибуцију и јавно саопштавање дела, и прераде, ако се наведе име аутора на начин одређен од стране аутора или даваоца лиценце и ако се прерада дистрибуира под истом или сличном лиценцом. Ова лиценца дозвољава комерцијалну употребу дела и прерада. Слична је софтверским лиценцама, односно лиценцама отвореног кода. !