Farmakološka karakterizacija uticaja deuteracije izabranih pirazolohinolinona kao modulatora GABAA receptora

Abstract

Važno mesto u istraživanjima poslednjih godina zauzimaju GABAA receptori (GABAAR) sa α6podjedinicom u pentamernom receptorskom kompleksu. Ovi receptori imaju potencijalno važnuulogu u razvoju trigeminalnog orofacijalnog bola, migreni, neuropsihijatrijskim poremećajima sasenzorno-motornim deficitima kao što su tikovi, Tourette-ov sindrom, određeni simptomishizofrenije, opsesivno-kompulzivni poremećaj i poremećaji pažnje. Otkriveno je mesto vezivanjana α+β- međupovršini GABAAR označeno kao pirazolohinolinonsko (PQ) mesto, zajedno saprvim α6-selektivnim ligandom, PZ-II-029. Zatim je pronađeno još nekoliko PQ liganada kojideluju kao funkcionalno selektivni pozitivni modulatori na PQ mestu α6 GABAA receptora, dokneutralnu modulaciju ostvaruju posredstvom benzodiazepinskog mesta.Najpre smo sproveli eksperimente farmakokinetičke karakterizacije PQ jedinjenja iz tri strukturnegrupe (PZ-II-029 i srodni deuterisani i/ili nitrogenovani analozi DK-I-56-1, RV-I-029, DK-I-60-3i DK-I-86-1; LAU463 i srodni deuterisani i/ili nitrogenovani analozi DK-I-58-1 i DK-II-58-1; iDK-I-87-1) i još pet dodatnih liganada (jedan koji je meta izomer DK-I-87-1 (DK-I-59-1) i četirifluorirana 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-Dawleypacovi). Određena je apsolutna oralna biološka raspoloživost, kao i slobodne frakcije u plazmi imozgu dva liganda (PZ-II-029 i DK-I-56-1). Daljim istraživanjem procenjivali smo uticajnavedenih PQ liganada, samih ili u kombinaciji sa diazepamom, na ponašanje adultnih mužjakapacova Wistar i Sprague-Dawley soja, odnosno miševa C57BL soja. Supstanca DK-I-56-1, kojaje na osnovu prethodnih eksperimenata identifikovana kao vodeća molekula, ispitana je nakonhronične konstrikcione povrede infraorbitalnog živca kao modela trigeminalne neuropatije namužjacima pacova Wistar soja. Primena DK-I-56-1 u animalnom modelu trajala je 14 dana počevod prvog dana nakon operacije, a razvoj bolne preosetljivosti ispitivan je von Frey filamentima udefinisanim vremenskim periodima. Farmakokinetički profili i parametri nedvosmisleno ukazujuna optimizaciju i unapređenje farmakokinetike deuterisanih analoga u plazmi i mozgu, prirazličitim putevima primene, kao i pri različitim formulacijama ispitivanog liganda. Obe strategije,deuteracija i fluorovanje, povećale su metaboličku stabilnost, dok su razlike u farmakokineticiizmeđu deuterisanih i fluorovanih analoga bile izražene. Naime, fluorovani analozi pokazujuznačajno zadržavanje u organima za izlučivanje (jetra i bubreg), a uz podatke da pokazuju izvesnucitotoksičnost in vitro na ćelijskim linijama jetre i bubrega, kao i da su efikasnost i funkcionalnaselektivnost 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 sesuperiornom u odnosu na fluorovanje pirazolohinolinona u smislu održavanja α6-GABAAreceptorske selektivnosti uz povećanje metaboličke stabilnosti i biološke raspoloživosti...

GABAA receptors (GABAARs) containing the α6 subunit (α6-GABAARs) have recently obtainedan important place in research. They play a distinct role in trigeminal orofacial pain, migraine, andneuropsychiatric disorders with sensori-motor gating deficits (such as tic disorders, Tourette’ssyndrome, certain symptoms of schizophrenia, obsessive compulsive disorder and attention deficitdisorders). The binding site at the α+β- interface of GABAARs, designated as thepyrazoloquinolinone (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 positivemodulators at the PQ site of α6-containing GABAARs. PQs are also neutral modulators at thebenzodiazepine 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 andDK-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) andfour fluorinated analogues (DK-IV-19-1, DK-IV-20-1, DK-IV-22-1 and MM-I-10)), afterintraperitoneal, 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 asestimated free fractions in plasma and brain of two ligands (PZ-II-029 and DK-I-56-1), weredetermined. Through further research, we evaluated the influence of PQ ligands, alone or incombination with diazepam, on the behavior of adult male Wistar and Sprague-Dawley rats, orC57BL 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 oftrigeminal neuropathy in male Wistar rats. The application of DK-I-56-1 in the animal model lastedfor 14 days starting from the first day after surgery, and the development of pain hypersensitivitywas examined with von Frey filaments in defined time periods. Pharmacokinetic profiles andparameters clearly indicate optimization and improvement of pharmacokinetics of deuteratedanalogues in plasma and brain, with different routes of administration, as well as with differentformulations of the investigated ligand. While the strategy of deuteration and fluorinationincreased metabolic stability, the pharmacokinetic influence on plasma, brain, liver and kidneyexposure was dramatically different. Namely, the fluorinated analogues showed significantretention in the excretory organs (liver and kidney). Also, they exhibited a slightly increasedcytotoxic effect on liver and kidney cell lines in vitro. Efficacy and functional selectivity for theα6+β3- interface (PQ site) of α6-GABAARs was reduced for the fluorinated analogs in comparisonto the deuterated ligands. All these facts make them less suitable for further research. Thus, thedeuteration 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...