Molekulske osnove delovanja atipičnih antipsihotika klozapina, sertindola i ziprazidona na jetru pacova i eritrocite čoveka

Abstract

Plejotropni efekti lekova obuhvataju delovanja na mesta i strukture u telu za koje oni nisu posebno razvijeni i obično su neočekivani. Atipični antipsihotici podležu intenzivnom metabolizmu u jetri, uz stvaranje toksičnih slobodno-radikalskih intermedijera kao jedan od predloženih mehanizama oštećenja jetre kod pacijenata hronično tretiranim ovim psihofarmacima. Cilj prvog dela disertacije bio je da istraži da li atipični antipsihotici klozapin, sertindol ili ziprazidon utiču na enzimski sistem antioksidativne zaštite u jetri. Ekspresija i aktivnost superoksid-dismutaze (SOD), katalaze (CAT), glutation-peroksidaze (GPx), glutation-reduktaze (GR) i glutation-S-transferaze (GST) su izmereni u jetri pacova pri dozama koje odgovaraju ljudskoj terapiji antipsihoticima. Klozapin je povećao aktivnost SOD tipova 1 i 2, GR i GST, uz smanjenje CAT aktivnosti, dok je tretman sertindolom povećao aktivnost obe SOD. U ziprazidonom-tretiranoj grupi pacova jedino je nađeno smanjenje CAT aktivnosti. Sva tri antipsihotika su proizvela blage do umerene histopatološke promene, okarakterisane kao regenerativne promene jetre. Nisu primećeni očigledni znaci infiltracije imunskih ćelija, mikrovezikularnih ili makrovezikularnih masnih promena, ili hepatociti u mitozi. U zaključku, tretman atipičnim antipsihoticima u trajanju od 28 dana promenio je aktivnost zaštitnih antioksidativnih enzima i izazvao histopatološke promene u jetri, pre svega kod pacova tretiranih klozapinom. Rezultati povrđuju da poremećaji u redoks-homeostazi mogu da doprinose disfunkciji jetre nakon dugotrajnog lečenja atipičnim antipsihoticima. Prepuni hemoglobinom, esencijalnim proteinom za transport kiseonika kroz telo, eritrociti čoveka su takođe pogodan model sistem za testiranje plejotropnih efekata lipofilnih lekova. U drugom delu disertacije najpre je ispitano vezivanje klozapina, ziprazidona i sertindola za humani hemoglobin pod simuliranim fiziološkim uslovima. Analiza gašenja (kvenčinga) fluorescencije proteina na različitim temperaturama i podaci iz van't Hoff-ovog dijagrama i molekulskog dokinga pokazali su statični tip kvenčinga, da tetramerni hemoglobin ima jedno vezivno mesto za sve lekove u centralnoj šupljini u blizini αβ interfejsa, te da hidrofobne sile pre svega učestvuju u stabilizaciji nagrađenih protein–ligand kompleksa. Konstante asocijacije bile su umerene jačine (~104 M–1), najveće za klozapin (2,2 x 104 M–1 na 25°C). Vezivanje klozapina pokazalo je "prijateljske" učinke: povećan sadržaj α-heliksa, viša tačka topljenja i zaštita proteina od oksidacije posredovane slobodnim radikalima. S druge strane, vezani ziprazidon i sertindol imali su blago pro-oksidativni učinak, povećavajući sadržaj ferihemoglobina, oblika koji ne vezuje kiseonik: mogući "neprijatelji". Niti jedan antipsihotik nije pokazao značajniji efekat na fluidnost plazmine membrane eritrocita, u EPR studiji sa spinskim probama. Sva tri leka su efikasno štitila integritet eritrocita u oksidativnom okruženju koje favorizuje njihovu hemolizu, a pre svega klozapin. Budući da interakcije proteina sa lekovima utiču na njihova farmakokinetička i farmakodinamička svojstva, dobijeni rezultati ukazuju na mogući značaj hemoglobina u ovim procesima in vivo.

Pleiotropic effects of drugs include actions on places and structures in the body for which they have not been specifically developed and are usually unexpected. Atypical antipsychotics are subject to intensive metabolism in the liver, with the formation of toxic free-radical intermediates as one of the proposed mechanisms of liver damage in patients chronically treated with these psychopharmaceuticals. The aim of the first part of the dissertation was to investigate whether the atypical antipsychotics clozapine, sertindole, or ziprasidone affect the enzyme system of antioxidant protection in the liver. Expression and activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione-S-transferase (GST) were measured in rat liver at doses corresponding to human antipsychotic therapy. Clozapine increased the activity of SOD types 1 and 2, GR and GST while decreasing CAT activity, while sertindole treatment increased the activity of both SODs. Only a decrease in CAT activity was found in the ziprasidone-treated group of rats. All three antipsychotics produced mild to moderate histopathological changes characterized as regenerative liver alterations. No obvious signs of immune cell infiltration, micro-vesicular or macrovesicular fatty changes, or hepatocytes in mitosis were observed. In conclusion, treatment with atypical antipsychotics for 28 days changed the activity of the protective antioxidant enzyme and caused histopathological changes in the liver, primarily in rats treated with clozapine. The results suggest that disturbances in redox homeostasis may contribute to liver dysfunction after long-term treatment with atypical antipsychotics. Loaded with hemoglobin, an essential protein for transporting oxygen throughout the body, human erythrocytes are also a convenient model system for testing the pleiotropic effects of lipophilic drugs. In the second part of the dissertation, the binding of clozapine, ziprasidone, and sertindole to human hemoglobin was first examined under simulated physiological conditions. Analysis of protein fluorescence quenching at different temperatures and data obtained from the van't Hoff diagram and molecular docking showed the static type of quenching that tetrameric hemoglobin has one binding site for all drugs in the central cavity near the αβ interface, and that hydrophobic forces dominantly mediate the protein-ligand complex stabilization. Association constants were of lower-moderate strength (~104 M–1), the highest for clozapine (2.2 x 104 M–1 at 25°C). Clozapine binding showed "friendly" effects: increased α-helix content, higher melting point, and protein protection from free radical-mediated oxidation. On the other hand, bound ziprasidone and sertindole had a slightly pro-oxidative effect, increasing the content of ferrihemoglobin: possible "enemies". None of the antipsychotics showed a significant effect on the fluidity of the erythrocyte plasma membrane in an EPR study with spin probes. All three drugs effectively protected the integrity of erythrocytes in an oxidative environment that favors their hemolysis, especially clozapine. Since protein interactions with drugs affect their pharmacokinetic and pharmacodynamic properties, the obtained results indicate the importance of hemoglobin in these processes in vivo.