Farmakološka karakterizacija uticaja deuteracije izabranih pirazolohinolinona kao modulatora GABAA receptora

Abstract

Važno mesto u istraživanjima poslednjih godina zauzimaju GABAA receptori (GABAAR) sa α6 podjedinicom u pentamernom receptorskom kompleksu. Ovi receptori imaju potencijalno važnu ulogu u razvoju trigeminalnog orofacijalnog bola, migreni, neuropsihijatrijskim poremećajima sa senzorno-motornim deficitima kao što su tikovi, Tourette-ov sindrom, određeni simptomi shizofrenije, opsesivno-kompulzivni poremećaj i poremećaji pažnje. Otkriveno je mesto vezivanja na α+β- međupovršini GABAAR označeno kao pirazolohinolinonsko (PQ) mesto, zajedno sa prvim α6-selektivnim ligandom, PZ-II-029. Zatim je pronađeno još nekoliko PQ liganada koji deluju kao funkcionalno selektivni pozitivni modulatori na PQ mestu α6 GABAA receptora, dok neutralnu modulaciju ostvaruju posredstvom benzodiazepinskog mesta. Najpre smo sproveli eksperimente farmakokinetičke karakterizacije PQ jedinjenja iz tri strukturne grupe (PZ-II-029 i srodni deuterisani i/ili nitrogenovani analozi DK-I-56-1, RV-I-029, DK-I-60-3 i DK-I-86-1; LAU463 i srodni deuterisani i/ili nitrogenovani analozi DK-I-58-1 i DK-II-58-1; i DK-I-87-1) i još pet dodatnih liganada (jedan koji je meta izomer DK-I-87-1 (DK-I-59-1) i četiri fluorirana analoga (DK-IV-19-1, DK-IV-20-1, DK-IV-22-1 i MM-I-10)) nakon intraperitonealne, oralne ili intravenske primene. Osim puta primene, faktor varijacije predstavljala je i formulacija (suspenzija, rastvor ili nanoemulzija) kao i soj životinja (C57BL miševi, Wistar i Sprague-Dawley pacovi). Određena je apsolutna oralna biološka raspoloživost, kao i slobodne frakcije u plazmi i mozgu dva liganda (PZ-II-029 i DK-I-56-1). Daljim istraživanjem procenjivali smo uticaj navedenih PQ liganada, samih ili u kombinaciji sa diazepamom, na ponašanje adultnih mužjaka pacova Wistar i Sprague-Dawley soja, odnosno miševa C57BL soja. Supstanca DK-I-56-1, koja je na osnovu prethodnih eksperimenata identifikovana kao vodeća molekula, ispitana je nakon hronične konstrikcione povrede infraorbitalnog živca kao modela trigeminalne neuropatije na mužjacima pacova Wistar soja. Primena DK-I-56-1 u animalnom modelu trajala je 14 dana počev od prvog dana nakon operacije, a razvoj bolne preosetljivosti ispitivan je von Frey filamentima u definisanim vremenskim periodima. Farmakokinetički profili i parametri nedvosmisleno ukazuju na optimizaciju i unapređenje farmakokinetike deuterisanih analoga u plazmi i mozgu, pri različitim putevima primene, kao i pri različitim formulacijama ispitivanog liganda. Obe strategije, deuteracija i fluorovanje, povećale su metaboličku stabilnost, dok su razlike u farmakokinetici između deuterisanih i fluorovanih analoga bile izražene. Naime, fluorovani analozi pokazuju značajno zadržavanje u organima za izlučivanje (jetra i bubreg), a uz podatke da pokazuju izvesnu citotoksičnost in vitro na ćelijskim linijama jetre i bubrega, kao i da su efikasnost i funkcionalna selektivnost za α6+β3- PQ mesto α6-GABAAR smanjeni u poređenju sa deuterisanim ligandima, čini ih manje pogodnim za dalje faze istraživanja. Dakle, strategija deuteracije pokazala se superiornom u odnosu na fluorovanje pirazolohinolinona u smislu održavanja α6-GABAA receptorske selektivnosti uz povećanje metaboličke stabilnosti i biološke raspoloživosti...

GABAA receptors (GABAARs) containing the α6 subunit (α6-GABAARs) have recently obtained an important place in research. They play a distinct role in trigeminal orofacial pain, migraine, and neuropsychiatric disorders with sensori-motor gating deficits (such as tic disorders, Tourette’s syndrome, certain symptoms of schizophrenia, obsessive compulsive disorder and attention deficit disorders). The binding site at the α+β- interface of GABAARs, designated as the pyrazoloquinolinone (PQ) site, was discovered, along with the first α6-selective ligand, PZ-II-029. Subsequent research led to the development of a series of PQs, as functionally selective positive modulators at the PQ site of α6-containing GABAARs. PQs are also neutral modulators at the benzodiazepine site. Firstly, we conducted pharmacokinetic characterization of PQs from three structural groups (PZ- II-029 and related deuterated and/or nitrogenated analogs DK-I-56-1, RV-I-029, DK-I-60-3 and DK-I-86-1; LAU 463 and related deuterated and/or nitrogenated analogs DK-I-58-1 and DK-II- 58-1; and DK-I-87-1) and five additional ligands (one meta isomer of DK-I-87-1 (DK-I-59-1) and four fluorinated analogues (DK-IV-19-1, DK-IV-20-1, DK-IV-22-1 and MM-I-10)), after intraperitoneal, oral or intravenous administration. Beside the route of administration, formulation (suspension, solution or nanoemulsion) and the animal strain (C57BL mice, Wistar and Sprague- Dawley rats) were additional factors of variation. Absolute oral bioavailability, as well as estimated free fractions in plasma and brain of two ligands (PZ-II-029 and DK-I-56-1), were determined. Through further research, we evaluated the influence of PQ ligands, alone or in combination with diazepam, on the behavior of adult male Wistar and Sprague-Dawley rats, or C57BL mice. On the basis of previous experiments, DK-I-56-1 was identified as a "lead" compound and tested after chronic constriction injury of the infraorbital nerve as a model of trigeminal neuropathy in male Wistar rats. The application of DK-I-56-1 in the animal model lasted for 14 days starting from the first day after surgery, and the development of pain hypersensitivity was examined with von Frey filaments in defined time periods. Pharmacokinetic profiles and parameters clearly indicate optimization and improvement of pharmacokinetics of deuterated analogues in plasma and brain, with different routes of administration, as well as with different formulations of the investigated ligand. While the strategy of deuteration and fluorination increased metabolic stability, the pharmacokinetic influence on plasma, brain, liver and kidney exposure was dramatically different. Namely, the fluorinated analogues showed significant retention in the excretory organs (liver and kidney). Also, they exhibited a slightly increased cytotoxic effect on liver and kidney cell lines in vitro. Efficacy and functional selectivity for the α6+β3- interface (PQ site) of α6-GABAARs was reduced for the fluorinated analogs in comparison to the deuterated ligands. All these facts make them less suitable for further research. Thus, the deuteration strategy proved superior to fluorination of these PQs in terms of maintaining α6- GABAAR selectivity while enhancing the metabolic stability and bioavailability. These α6- GABAAR ligands were devoid of observable detrimental effects...