Pharmacological characterization of the influence of deuteration of selected pyrazoloquinolinones as GABAA receptor modulatores

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..

Overcoming the low oral bioavailability of deuterated pyrazoloquinolinone ligand dk-i-60-3 by nanonization: A knowledge-based approach

Poor water solubility of new chemical entities is considered as one of the main obstacles in drug development, as it usually leads to low bioavailability after administration. To overcome these problems, the selection of the appropriate formulation technology needs to be based on the physicochemical properties of the drug and introduced in the early stages of drug research. One example of the new potential drug substance with poor solubility is DK-I-60-3, deuterated pyrazoloquinolinone, designed for the treatment of various neuropsychiatric disorders. In this research, based on preformulation studies, nanocrystal technology was chosen to improve the oral bioavailability of DK-I-60-3. Nanocrystal dispersions stabilized by sodium lauryl sulfate and polyvinylpyrrolidone were prepared by modified wet media milling technique, with the selection of appropriate process and formulation parameters. The nanoparticles characterization included particle size and zeta potential measurements, differential scanning calorimetry, X-ray powder diffraction, dissolution and solubility study, and in vivo pharmacokinetic experiments. Developed formulations had small uniform particle sizes and were stable for three months. Nanonization caused decreased crystallite size and induced crystal defects formation, as well as a DK-I-60-3 solubility increase. Furthermore, after oral administration of the developed formulations in rats, two to three-fold bioavailability enhancement was observed in plasma and investigated organs, including the brain

Pharmacological characterization of the influence of deuteration of selected pyrazoloquinolinones as GABAA receptor modulatores

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..

Behavioural interaction of pyrazoloquinolinone positive allosteric modulators at α6GABAA receptors and diazepam in rats: Anti-diazepam-induced ataxia action as a structure-dependent feature

Several pyrazoloquinolinone (PQ) ligands were recently discovered as func-tionally selective positive modulators at the PQ site ofα6-containing GABAAreceptors. PQs are also neutral modulators at the benzodiazepine site. Weassessed the influence of PQ compounds from three structural groups (PZ-II-029 and related deuterated analogues DK-I-56-1, RV-I-029, DK-I-60-3 and DK-I-86-1; LAU 463 and related analogues DK-I-58-1 and DK-II-58-1; and DK-I-87-1), alone and in combination with diazepam, on the behaviour of maleSprague–Dawley rats. An excellent behavioural safety profile of all tested PQswas demonstrated in the spontaneous locomotor activity, rotarod, loss of right-ing reflex and pentylenetetrazol tests. In interaction studies, only PZ-II-029and its analogues prevented the ataxic effects of the benzodiazepine, asassessed in the rotarod test and during monitoring of rat locomotor activityafter awakening from the loss of righting reflex. Published electrophysiologicalprofiles of PQ ligands imply that positive modulation elicited atα6-GABAAreceptors that contain theγ2 andδsubunit, rather than their neutral modula-tory action at the benzodiazepine site, may prevent the ataxic action of diaze-pam. Thus, PZ-II-029 and its deuterated analogues are not prone to untowardinteractions with benzodiazepines and may indeed completely abolish theirataxic action, seen at therapeutic, and especially toxic concentrations

Behavioural interaction of pyrazoloquinolinone positive allosteric modulators at α6GABAA receptors and diazepam in rats: Anti-diazepam-induced ataxia action as a structure-dependent feature

Several pyrazoloquinolinone (PQ) ligands were recently discovered as functionally selective positive modulators at the PQ site of α6-containing GABAA receptors. PQs are also neutral modulators at the benzodiazepine site. We assessed the influence of PQ compounds from three structural groups (PZ-II-029 and related deuterated analogues DK-I-56-1, RV-I-029, DK-I-60-3 and DK-I-86-1; LAU 463 and related analogues DK-I-58-1 and DK-II-58-1; and DK-I-87-1), alone and in combination with diazepam, on the behaviour of male Sprague–Dawley rats. An excellent behavioural safety profile of all tested PQs was demonstrated in the spontaneous locomotor activity, rotarod, loss of righting reflex and pentylenetetrazol tests. In interaction studies, only PZ-II-029 and its analogues prevented the ataxic effects of the benzodiazepine, as assessed in the rotarod test and during monitoring of rat locomotor activity after awakening from the loss of righting reflex. Published electrophysiological profiles of PQ ligands imply that positive modulation elicited at α6-GABAA receptors that contain the γ2 and δ subunit, rather than their neutral modulatory action at the benzodiazepine site, may prevent the ataxic action of diazepam. Thus, PZ-II-029 and its deuterated analogues are not prone to untoward interactions with benzodiazepines and may indeed completely abolish their ataxic action, seen at therapeutic, and especially toxic concentrations

High amount of lecithin facilitates oral delivery of a poorly soluble pyrazoloquinolinone ligand formulated in lipid nanoparticles: Physicochemical, structural and pharmacokinetic performances

Preclinical development of deuterated pyrazoloquinolinone ligands, promising drug candidates for various neuropsychiatric disorders, was hindered by unusually low solubility in water and oils. DK-I-60-3 (7-methoxy-d3- 2-(4-methoxy-d3-phenyl)-2,5-dihydro-3Hpyrazolo[4,3-c]quinolin-3-one) is one of such pyrazoloquinolinones, and we recently reported about increased oral bioavailability of its nanocrystal formulation (NC). Lipid nano- particles (LNP) with a high concentration of lecithin, which enhances loading capacity of the lipid matrix, may give rise to further improvement. After preformulation studies by differential scanning calorimetry and polarized light microscopy, LNP were prepared by the hot high pressure homogenization, and characterized in terms of particle size, morphology, and encapsulation efficacy. The layered structure visible on atomic force micrographs was confirmed by nuclear magnetic resonance. Obtained formulations were desirably stable, with small particle size (99 %). Lecithin was partially fluid and most probably located in the outer shell of the particle, together with DK-I-60-3. While the hydrophobic part of polysorbate 80 was completely immobilized, its hydrophilic part was free in the aqueous phase. In oral neuropharmacokinetic study in rats, an around 1.5-fold increase of area under the curve with LNP compared to NC was noticed both in brain and plasma. In bioavailability study, F value of LNP (34.7 ± 12.4 %) was 1.4-fold higher than of NC (24.5 ± 7.8 %); however, this difference did not reach statistical significance. Therefore, employment of LNP platform in preclinical formulation of DK-I-60-3 imparted an incremental improvement of its physicochemical as well as pharmacokinetic behavior

High amount of lecithin facilitates oral delivery of a poorly soluble pyrazoloquinolinone ligand formulated in lipid nanoparticles: Physicochemical, structural and pharmacokinetic performances

Preclinical development of deuterated pyrazoloquinolinone ligands, promising drug candidates for various neuropsychiatric disorders, was hindered by unusually low solubility in water and oils. DK-I-60-3 (7-methoxy-d3-2-(4-methoxy-d3-phenyl)-2,5-dihydro-3Hpyrazolo[4,3-c]quinolin-3-one) is one of such pyrazoloquinolinones, and we recently reported about increased oral bioavailability of its nanocrystal formulation (NC). Lipid nanoparticles (LNP) with a high concentration of lecithin, which enhances loading capacity of the lipid matrix, may give rise to further improvement. After preformulation studies by differential scanning calorimetry and polarized light microscopy, LNP were prepared by the hot high pressure homogenization, and characterized in terms of particle size, morphology, and encapsulation efficacy. The layered structure visible on atomic force micrographs was confirmed by nuclear magnetic resonance. Obtained formulations were desirably stable, with small particle size (99 %). Lecithin was partially fluid and most probably located in the outer shell of the particle, together with DK-I-60-3. While the hydrophobic part of polysorbate 80 was completely immobilized, its hydrophilic part was free in the aqueous phase. In oral neuropharmacokinetic study in rats, an around 1.5-fold increase of area under the curve with LNP compared to NC was noticed both in brain and plasma. In bioavailability study, F value of LNP (34.7 ± 12.4 %) was 1.4-fold higher than of NC (24.5 ± 7.8 %); however, this difference did not reach statistical significance. Therefore, employment of LNP platform in preclinical formulation of DK-I-60-3 imparted an incremental improvement of its physicochemical as well as pharmacokinetic behavior