RESUMO
XBD173 and etifoxine are translocator protein (TSPO) ligands that modulate inflammatory responses in preclinical models. Limited human pharmacokinetic data is available for either molecule, and the binding affinity of etifoxine for human TSPO is unknown. To allow for design of human challenge experiments, we derived pharmacokinetic data for orally administered etifoxine (50 mg 3 times daily) and XBD173 (90 mg once daily) and determined the binding affinity of etifoxine for TSPO. For XBD173, maximum plasma concentration and free fraction measurements predicted a maximal free concentration of 1.0 nM, which is similar to XBD173 binding affinity. For etifoxine, maximum plasma concentration and free fraction measurements predicted a maximal free concentration of 0.31 nM, substantially lower than the Ki for etifoxine in human brain derived here (7.8 µM, 95% CI 4.5-14.6 µM). We conclude that oral XBD173 dosing at 90 mg once daily will achieve pharmacologically relevant TSPO occupancy. However, the occupancy is too low for TSPO mediated effects after oral dosing of etifoxine at 50 mg 3 times daily.
Assuntos
Purinas , Receptores de GABA , Proteínas de Transporte/metabolismo , Humanos , Oxazinas/farmacocinética , Purinas/farmacologia , Receptores de GABA/metabolismoRESUMO
Herein we describe a series of tetrahydrobenzotriazoles as novel, potent metabotropic glutamate receptor subtype 5 (mGlu5) positive allosteric modulators (PAMs). Exploration of the SAR surrounding the tetrahydrobenzotriazole core ultimately led to the identification of 29 as a potent mGlu5 PAM with a low maximal glutamate potency fold shift, acceptable in vitro DMPK parameters and in vivo PK profile and efficacy in the rat novel object recognition (NOR) assay. As a result 29 was identified as a suitable compound for progression to in vivo safety evaluation.
Assuntos
Antipsicóticos/química , Receptor de Glutamato Metabotrópico 5/antagonistas & inibidores , Triazóis/química , Regulação Alostérica/efeitos dos fármacos , Animais , Antipsicóticos/metabolismo , Antipsicóticos/farmacologia , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Cognição/efeitos dos fármacos , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Meia-Vida , Humanos , Microssomos/metabolismo , Ratos , Receptor de Glutamato Metabotrópico 5/metabolismo , Relação Estrutura-Atividade , Triazóis/metabolismo , Triazóis/farmacologiaRESUMO
Using a parallel synthesis approach to target a non-conserved region of the PI3K catalytic domain a pan-PI3K inhibitor 1 was elaborated to provide alpha, delta and gamma isoform selective Class I PI3K inhibitors 21, 24, 26 and 27. The compounds had good cellular activity and were selective against protein kinases and other members of the PI3K superfamily including mTOR and DNA-PK.
Assuntos
Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Tiazóis/química , Tiazóis/farmacologia , Animais , Domínio Catalítico , Feminino , Humanos , Camundongos , Modelos Moleculares , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/metabolismo , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Inibidores de Proteínas Quinases/farmacocinética , Ratos , Transdução de Sinais/efeitos dos fármacos , Tiazóis/farmacocinéticaRESUMO
Scaffold hopping from a non-basic series of 5-HT(2A) receptor antagonists developed in-house that possessed reduced activity in vivo enabled the discovery of a novel series of diaryl sulfones that gave excellent occupancy on oral dosing. Not only does this work further demonstrate that oral bioavailability of a given series can be enhanced by improving physicochemical parameters such as log P, but it corroborates the growing evidence that a protonated amine is not essential for affinity at aminergic GPCRs.
Assuntos
Antagonistas do Receptor 5-HT2 de Serotonina , Sulfonas/síntese química , Administração Oral , Aminas , Animais , Disponibilidade Biológica , Descoberta de Drogas , Humanos , Ligantes , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Agonistas do Receptor de Serotonina , Transtornos do Sono-Vigília/tratamento farmacológico , Sulfonas/farmacologia , Sulfonas/uso terapêuticoRESUMO
3-tert-Butyl-7-(5-methylisoxazol-3-yl)-2-(1-methyl-1H-1,2,4-triazol-5-ylmethoxy)-pyrazolo[1,5-d][1,2,4]triazine (MRK-016) is a pyrazolotriazine with an affinity of between 0.8 and 1.5 nM for the benzodiazepine binding site of native rat brain and recombinant human alpha1-, alpha2-, alpha3-, and alpha5-containing GABA(A) receptors. It has inverse agonist efficacy selective for the alpha5 subtype, and this alpha5 inverse agonism is greater than that of the prototypic alpha5-selective compound 3-(5-methylisoxazol-3-yl)-6-[(1-methyl-1,2,3-triazol-4-hdyl)methyloxy]-1,2,4-triazolo[3,4-a]phthalazine (alpha5IA). Consistent with its greater alpha5 inverse agonism, MRK-016 increased long-term potentiation in mouse hippocampal slices to a greater extent than alpha5IA. MRK-016 gave good receptor occupancy after oral dosing in rats, with the dose required to produce 50% occupancy being 0.39 mg/kg and a corresponding rat plasma EC(50) value of 15 ng/ml that was similar to the rhesus monkey plasma EC(50) value of 21 ng/ml obtained using [(11)C]flumazenil positron emission tomography. In normal rats, MRK-016 enhanced cognitive performance in the delayed matching-to-position version of the Morris water maze but was not anxiogenic, and in mice it was not proconvulsant and did not produce kindling. MRK-016 had a short half-life in rat, dog, and rhesus monkey (0.3-0.5 h) but had a much lower rate of turnover in human compared with rat, dog, or rhesus monkey hepatocytes. Accordingly, in human, MRK-016 had a longer half-life than in preclinical species ( approximately 3.5 h). Although it was well tolerated in young males, with a maximal tolerated single dose of 5 mg corresponding to an estimated occupancy in the region of 75%, MRK-016 was poorly tolerated in elderly subjects, even at a dose of 0.5 mg, which, along with its variable human pharmacokinetics, precluded its further development.
Assuntos
Agonistas GABAérgicos/farmacologia , Agonistas de Receptores de GABA-A , Isoxazóis/farmacologia , Triazinas/farmacologia , Animais , Ansiedade/psicologia , Comportamento Animal/efeitos dos fármacos , Convulsivantes/farmacologia , Cães , Relação Dose-Resposta a Droga , Estimulação Elétrica , Eletrofisiologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Fibroblastos , Flumazenil/metabolismo , Agonistas GABAérgicos/metabolismo , Agonistas GABAérgicos/farmacocinética , Moduladores GABAérgicos/metabolismo , Hepatócitos/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Humanos , Macaca mulatta , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Técnicas de Patch-Clamp , Equilíbrio Postural/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores de GABA-A/metabolismo , Adulto JovemRESUMO
This report describes the effect of replacing the central basic amine present in many known 5-HT(2A) ligands with an aromatic residue. We targeted the isomeric phenethylpyridines 2 and 3 and these compounds proved to be excellent leads, possessing good 5-HT(2A) receptor binding affinity and selectivity over the 5-HT(2C) subtype. Optimization of one isomer led to the identification of 25, a compound with sub-nanomolar 5-HT(2A) affinity and selectivity over 5-HT(2C) of greater than 4600-fold.
Assuntos
Química Farmacêutica/métodos , Piridinas/química , Piridinas/síntese química , Receptor 5-HT2A de Serotonina/química , Antagonistas da Serotonina/química , Antagonistas da Serotonina/síntese química , Animais , Desenho de Fármacos , Humanos , Cinética , Ligantes , Modelos Químicos , Conformação Molecular , Piridinas/farmacologia , Ratos , Antagonistas da Serotonina/farmacologia , Sulfonas/químicaRESUMO
The capability of ultra performance liquid chromatography coupled with time-of-flight mass spectrometry (UPLC/TOFMS) in the high-throughput quantitative analysis of a drug candidate in plasma has been investigated. Data obtained were compared with results from conventional analysis by high-performance liquid chromatography with tandem mass spectrometric detection on a triple quadrupole instrument (HPLC/MS/MS). The accuracies and precisions of the two approaches were comparable. The UPLC/TOFMS system displayed excellent robustness over the course of 276 injections of protein-precipitated plasma samples. With the instrumentation used, the limits of detection and quantification were approximately five-fold higher with UPLC/TOFMS than for HPLC/MS/MS. Nevertheless, the UPLC/TOFMS system proved adequate to quantify plasma concentrations of a drug molecule administered orally to rats at a pharmacologically relevant dose of 4 mg/kg. As well as providing quantitative data on the test compound, it was also possible to extract data for eight different metabolites, including several isomeric species (three +O and three +2O) from the UPLC/TOFMS data sets, using an analytical method with a 2.5-minute run time. Selectivity for the test compound and its metabolites was derived from the accurate mass capabilities of the TOF instrument, and no MS method development was required.
Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Nootrópicos/sangue , Nootrópicos/metabolismo , Animais , Masculino , Estrutura Molecular , Nootrópicos/administração & dosagem , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
The application of ultra-performance liquid chromatography coupled to time-of-flight mass spectrometry (UPLC/TOFMS) for high-throughput analysis of a 96-well plate based metabolic stability assay has been investigated. Full-scan data were acquired, with run times of 2.5-3.5 min, from which narrow window extracted ion chromatograms were generated, producing quantitative data for the test compound equivalent to that obtained by high-performance liquid chromatography with tandem mass spectrometric detection on a triple quadrupole instrument (HPLC/MS/MS). Sensitivity and mass accuracy were maintained over the analysis of >300 samples. Additionally, the UPLC/TOFMS datasets obtained gave access to metabolic route information, at no cost in terms of sensitivity for the test compound.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Diazepam/metabolismo , Encefalina Leucina/metabolismo , Imipramina/metabolismo , Neurotransmissores/metabolismo , Psicotrópicos/metabolismo , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Avaliação Pré-Clínica de Medicamentos/métodos , Estabilidade de Medicamentos , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
(3-tert-Butyl-7-(5-methylisoxazol-3-yl)-2-(1-methyl-1H-1,2,4-triazol-5-ylmethoxy)pyrazolo[1,5-d][1,2,4]triazine (1) was recently identified as a functionally selective, inverse agonist at the benzodiazepine site of GABA(A) alpha5 receptors and enhances performance in animal models of cognition. The routes of metabolism of this compound in vivo in rat have been well characterised, the identities of the major metabolites are confirmed by synthesis and their biological profiles were evaluated. An unusual oxidation of the pyrazolo[1,5-d][1,2,4]triazine core to the corresponding pyrazolo[1,5-d][1,2,4]triazin-4(5H)-one scaffold by aldehyde oxidase has been observed.
Assuntos
Agonistas GABAérgicos/metabolismo , Agonistas GABAérgicos/farmacocinética , Agonistas de Receptores de GABA-A , Isoxazóis/metabolismo , Isoxazóis/farmacocinética , Subunidades Proteicas/agonistas , Triazinas/metabolismo , Triazinas/farmacocinética , Animais , Cães , Relação Dose-Resposta a Droga , Agonistas GABAérgicos/síntese química , Hidrólise , Isoxazóis/síntese química , Macaca mulatta , Camundongos , Conformação Molecular , Ratos , Estereoisomerismo , Relação Estrutura-Atividade , Distribuição Tecidual , Triazinas/síntese químicaRESUMO
Screening of the Merck compound collection identified 6 as an unusually simple, low molecular weight hit with moderate affinity for GABAA receptors. The structural novelty of 6, compared to our advanced series of GABAA alpha5 inverse agonists, made it an attractive molecule for further exploration. This paper will describe the evolution of 6 into a new series of ligands with nanomolar affinity and functional selectivity for GABAA alpha5 receptor subtypes.
Assuntos
Piridazinas/síntese química , Receptores de GABA-A/efeitos dos fármacos , Animais , Linhagem Celular , Humanos , Técnicas In Vitro , Ligantes , Masculino , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Técnicas de Patch-Clamp , Subunidades Proteicas/fisiologia , Piridazinas/farmacocinética , Piridazinas/farmacologia , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Receptores de GABA-A/fisiologia , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
The resource investment required to characterise the metabolic fate of a compound is relatively large, meaning that within a drug discovery environment relatively few compounds are characterised in depth. Rate-limiting steps include the setting up of a complex array of mass spectrometry experiments and the subsequent analysis of the large data sets produced. We describe here a strategy for the evaluation of metabolic routes using full-scan high-resolution liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/QToFMS) with automated data analysis using Metabolynx, a commercially available software package. Data from several structurally diverse compounds taken from the literature illustrate that, with careful setting of key parameters, this approach is able to indicate the presence of a wide range of metabolites with only a limited requirement for manual intervention.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Preparações Farmacêuticas/química , Preparações Farmacêuticas/metabolismo , Animais , Automação , Cromatografia Líquida , Diazepam/química , Diazepam/metabolismo , Indinavir/química , Indinavir/metabolismo , Espectrometria de Massas , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Ratos , Padrões de Referência , Sensibilidade e Especificidade , Fatores de TempoRESUMO
The removal of bottlenecks in discovery stage metabolite identification studies is an ongoing challenge for the pharmaceutical industry. We describe the use of an 'All-in-One' approach to metabolite characterization that leverages the fast scanning and high mass accuracy of hybrid quadrupole time-of-flight mass spectrometry (QqToFMS) instruments. Full-scan MS and MS/MS data is acquired using collision energy switching without the preselection, either manually or in a data-dependent manner, of precursor ions. The acquisition of 'clean' MS/MS data is assisted by the use of ultrahigh-performance chromatography. Data acquired using this method can then be mined post-acquisition in a number of ways. These include using narrow window extracted ion chromatograms (nwXICs) for expected biotransformations, XICs for the product ions of the parent compound and/or expected modification of these product ions, and neutral loss chromatograms. This approach has the potential to be truly comprehensive for the determination of in vitro biotransformations in a drug discovery environment.
Assuntos
Espectrometria de Massas/métodos , Verapamil/análise , Verapamil/metabolismo , Animais , Cromatografia Líquida , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Ratos , Verapamil/químicaRESUMO
Incorporation of fluorine at the 4-position of an existing series of sulfonyl piperidine 5-HT2A antagonists gave compounds with increased selectivity over the IKr potassium channel. This work led to the identification of 3b, a compound that gave no increase in QTc in the anesthetized dog up to plasma levels as high as 148 microM. Furthermore, 3b has been shown to increase slow-wave sleep bout duration and to decrease the number of awakenings in rats, indicating the potential utility of 5-HT2A antagonists in the treatment of insomnia.
Assuntos
Piperidinas/farmacologia , Piperidinas/uso terapêutico , Antagonistas do Receptor 5-HT2 de Serotonina , Distúrbios do Início e da Manutenção do Sono/tratamento farmacológico , Animais , Cães , Avaliação Pré-Clínica de Medicamentos , Ligantes , Estrutura Molecular , Piperidinas/síntese química , Ratos , Relação Estrutura-Atividade , Fatores de TempoRESUMO
It is well known that modafinil is an effective wake-promoting agent, but there is growing evidence to suggest that modafinil may also enhance some aspects of cognition. In man, modafinil has been shown to enhance vigilance in sleep-deprived and/or narcoleptic subjects and also to improve executive-type functioning (predominantly inhibitory response control processes) across a variety of human patient population groups. Preclinically, a delay-dependent improvement has been reported with modafinil in a mouse T-maze test of working memory. To investigate further the role of modafinil as a potential cognition enhancer, the effects of modafinil on attentional processes were assessed in the rat. The aim of the present study was to evaluate the potential of modafinil to enhance five-choice serial reaction time test (5-CSRT) performance. Lister Hooded rats received 32-128 mg/kg modafinil and 5-CSRT performance was assessed under standard and test parametric conditions in which the attentional load was increased, and also under conditions of scopolamine pre-treatment. Modafinil failed to significantly enhance 5-CSRT performance under standard conditions. Similarly, modafinil was unable to reverse the deficits in accuracy and/or increased omission errors induced by either parametric or pharmacological manipulations. Indeed, at higher doses, modafinil caused an increase in premature responding under certain test conditions, suggestive of increased impulsivity. The present findings suggest that, although modafinil may enhance vigilance in sleep-deprived human subjects, attentional processes in normal awake rats remain unaffected. No evidence was found to support a modafinil-induced improvement in response control; rather, under conditions of increased attentional load, modafinil appeared to facilitate impulsive responding. Finally, the failure of modafinil to improve a scopolamine-induced performance deficit suggests that modafinil does not act on the cholinergic system directly.
Assuntos
Atenção/efeitos dos fármacos , Compostos Benzidrílicos/farmacologia , Estimulantes do Sistema Nervoso Central/farmacologia , Tempo de Reação/efeitos dos fármacos , Animais , Condicionamento Operante/efeitos dos fármacos , Relação Dose-Resposta a Droga , Masculino , Modafinila , Parassimpatolíticos/farmacologia , Parassimpatomiméticos/farmacologia , Estimulação Luminosa , Fisostigmina/farmacologia , Desempenho Psicomotor/efeitos dos fármacos , Ratos , Escopolamina/farmacologia , Aprendizagem Seriada/efeitos dos fármacosRESUMO
(3-tert-Butyl-7-(5-methylisoxazol-3-yl)-2-(1-methyl-1H-1,2,4-triazol-5-ylmethoxy)pyrazolo[1,5-d][1,2,4]triazine (13) has been identified as a functionally selective, inverse agonist at the benzodiazepine site of GABA(A) alpha5 receptors. 13 is orally bioavailable, readily penetrates the CNS, and enhances performance in animal models of cognition. It does not exhibit the convulsant, proconvulsant, or anxiogenic activity associated with nonselective GABA(A) inverse agonists.
Assuntos
Cognição/efeitos dos fármacos , Agonistas de Receptores de GABA-A , Isoxazóis/síntese química , Nootrópicos/síntese química , Triazinas/síntese química , Administração Oral , Animais , Sítios de Ligação , Disponibilidade Biológica , Cães , Isoxazóis/efeitos adversos , Isoxazóis/farmacologia , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Nootrópicos/efeitos adversos , Nootrópicos/farmacologia , Técnicas de Patch-Clamp , Subunidades Proteicas , Ensaio Radioligante , Ratos , Receptores de GABA-A/metabolismo , Triazinas/efeitos adversos , Triazinas/farmacologiaRESUMO
Nonselective inverse agonists at the gamma-aminobutyric acid(A) (GABA-A) benzodiazepine binding site have cognition-enhancing effects in animals but are anxiogenic and can precipitate convulsions. Herein, we describe novel GABA-A alpha5 subtype inverse agonists leading to the identification of 16 as an orally active, functionally selective compound that enhances cognition in animals without anxiogenic or convulsant effects. Compounds of this type may be useful in the symptomatic treatment of memory impairment associated with Alzheimer's disease and related dementias.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/síntese química , Agonistas de Receptores de GABA-A , Nootrópicos/síntese química , Ftalazinas/síntese química , Triazóis/síntese química , Animais , Ligação Competitiva , Disponibilidade Biológica , Compostos Bicíclicos Heterocíclicos com Pontes/química , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Convulsivantes/síntese química , Convulsivantes/química , Convulsivantes/farmacologia , Cães , Humanos , Macaca mulatta , Camundongos , Nootrópicos/química , Nootrópicos/farmacologia , Oócitos/metabolismo , Técnicas de Patch-Clamp , Ftalazinas/química , Ftalazinas/farmacologia , Ensaio Radioligante , Ratos , Receptores de GABA-A/fisiologia , Relação Estrutura-Atividade , Triazóis/química , Triazóis/farmacologia , Xenopus laevisRESUMO
Studies with our screening lead 5 and the literature compound 6 led to the identification of 6-benzyloxy-3-(4-methoxy)phenyl-1,2,4-triazolo[3,4-a]phthalazine 8 as a ligand with binding selectivity for the gamma-aminobutyric acid-A (GABA-A) alpha 3- and alpha 5-containing receptor subtypes over the GABA-A alpha 1 subtype (K(i): alpha 2 = 850 nM, alpha 3 = 170 nM, alpha 5 = 72 nM, alpha 1 = 1400 nM). Early optimization studies identified the close analogue 10 (K(i): alpha 2 = 16 nM, alpha 3 = 41 nM, alpha 5 = 38 nM, alpha 1 = 280 nM) as a suitable lead for further study. High-affinity ligands were identified by replacing the 6-benzyloxy group of compound 10 with 2-pyridylmethoxy (compound 29), but binding selectivity was not enhanced (K(i): alpha 2 = 1.7 nM, alpha 3 = 0.71 nM, alpha 5 = 0.33 nM, alpha 1 = 2.7 nM). Furthermore, on evaluation in xenopus oocytes,(22) 29 was discovered to be a weak to moderate inverse agonist at all four receptor subtypes (alpha 1, -7%; alpha 2, -5%; alpha 3, -16%; alpha 5, -5%). Replacement of the 3-phenyl group of 29 with alternatives led to reduced affinity, and smaller 3-substituents led to reduced efficacy. Methyl substitution of the benzo-fused ring of 29 at the 7-, 8-, and 10-positions resulted in increased efficacy although selectivity was abolished. Increased efficacy and retention of selectivity for alpha 3 over alpha 1 was achieved with the 7,8,9,10-tetrahydro-(7,10-ethano)-phthalazine 62. Compound 62 is currently one of the most binding selective GABA-A alpha 3-benzodiazepine-site partial agonists known, and although its selectivity is limited, its good pharmacokinetic profile in the rat (33% oral bioavailability after a 3 mg/kg dose, reaching a peak plasma concentration of 179 ng/mL; half-life of 1 h) made it a useful pharmacological tool to explore the effect of a GABA-A alpha 2/alpha 3 agonist in vivo.
Assuntos
Agonistas de Receptores de GABA-A , Ftalazinas/síntese química , Triazóis/síntese química , Animais , Ansiolíticos/síntese química , Ansiolíticos/química , Ansiolíticos/farmacologia , Ligação Competitiva , Disponibilidade Biológica , Linhagem Celular , Humanos , Espectroscopia de Ressonância Magnética , Aprendizagem em Labirinto/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Ftalazinas/química , Ftalazinas/farmacologia , Ensaio Radioligante , Ratos , Receptores de GABA-A/metabolismo , Receptores de GABA-A/fisiologia , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Triazóis/química , Triazóis/farmacologia , XenopusRESUMO
"Reaction phenotyping" studies were performed with eletriptan (ETT) to determine its propensity to interact with coadministered medications. Its ability to serve as a substrate for human P-glycoprotein (P-gp) was also investigated since a central mechanism of action has been proposed for this "triptan" class of drug. In studies with a characterized bank of human liver microsome preparations, a good correlation (r2 = 0.932) was obtained between formation of N-desmethyl eletriptan (DETT) and CYP3A4-catalyzed testosterone 6 beta-hydroxylation. DETT was selected to be monitored in our studies since it represents a significant ETT metabolite in humans, circulating at concentrations 10 to 20% of those observed for parent drug. ETT was metabolized to DETT by recombinant CYP2D6 (rCYP2D6) and rCYP3A4, and to a lesser extent by rCYP2C9 and rCYP2C19. The metabolism of ETT to DETT in human liver microsomes was markedly inhibited by troleandomycin, erythromycin, miconazole, and an inhibitory antibody to CYP3A4, but not by inhibitors of other major P450 enzymes. ETT had little inhibitory effect on any of the P450 enzymes investigated. ETT was determined to be a good substrate for human P-gp in vitro. In bidirectional transport studies across LLC-MDR1 and LLC-Mdr1a cell monolayers, ETT had a BA/AB transport ratio in the range 9 to 11. This finding had significance in vivo since brain exposure to ETT was reduced 40-fold in Mdr1a+/+ relative to Mdr1a-/- mice. ETT metabolism to DETT is therefore catalyzed primarily by CYP3A4, and plasma concentrations are expected to be increased when coadministered with inhibitors of CYP3A4 and P-gp activity.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Indóis/metabolismo , Pirrolidinas/metabolismo , Animais , Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Citocromo P-450 CYP2D6/genética , Citocromo P-450 CYP2D6/metabolismo , Citocromo P-450 CYP3A , Inibidores das Enzimas do Citocromo P-450 , Eritromicina , Genes MDR/efeitos dos fármacos , Genes MDR/fisiologia , Humanos , Indóis/administração & dosagem , Indóis/análise , Indóis/antagonistas & inibidores , Indóis/farmacocinética , Indóis/farmacologia , Injeções Intravenosas , Masculino , Camundongos , Camundongos Mutantes , Miconazol , Microssomos Hepáticos/enzimologia , Oxazolidinonas/administração & dosagem , Oxazolidinonas/farmacocinética , Oxirredutases N-Desmetilantes/antagonistas & inibidores , Oxirredutases N-Desmetilantes/genética , Oxirredutases N-Desmetilantes/metabolismo , Fenótipo , Piperidinas/administração & dosagem , Piperidinas/farmacocinética , Pirrolidinas/análise , Pirrolidinas/antagonistas & inibidores , Pirrolidinas/farmacologia , Proteínas Recombinantes/metabolismo , Estatística como Assunto , Sumatriptana/administração & dosagem , Sumatriptana/farmacocinética , Triazóis/administração & dosagem , Triazóis/farmacocinética , Troleandomicina , TriptaminasRESUMO
This report presents a highly automated procedure for the determination of drug concentrations in plasma samples. The method is generic, in that it has been applied without adaptation to many different drug candidate molecules, but is also flexible, in that variations in the nature and number of samples to be analyzed can be readily accommodated. The method includes preparation of dilutions of analyte stock solutions, spiking these into control plasma to generate analytical standards, and preparation of samples suitable for analysis by high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) by precipitation of plasma proteins with acetonitrile, centrifugation, and dilution of the supernatants with HPLC buffer. All of these steps, apart from centrifugation, are performed without manual intervention on an automated liquid-handling workstation using 96-well plates. Analysis is by HPLC/MS/MS, using a generic HPLC gradient. Commercially available software was used for optimization of parameters for analysis by HPLC/MS/MS, integration of chromatographic peaks, and quantification of drug concentrations. The use of this methodology in our laboratory has greatly facilitated the analysis of small sample sets for a large number of analytes, a situation regularly encountered in an early drug discovery environment.
Assuntos
Proteínas Sanguíneas/química , Cromatografia Líquida de Alta Pressão/métodos , Avaliação de Medicamentos/métodos , Espectrometria de Massas/métodos , Preparações Farmacêuticas/sangue , Humanos , SoftwareRESUMO
Bioanalytical groups in the pharmaceutical industry provide quantitative data to support all stages of drug discovery. The increased use of 96-well plates and robotic liquid handling systems, the availability of robust triple quadruple mass spectrometers, and developments in chromatographic and samples preparation techniques, have all increased the rate at which this data can be generated. This review describes currently used methods and emerging technologies for automation of high-throughput quantitative bioanalysis. The focus is on recent applications of sample preparation and chromatography techniques compatible with detection by triple quadruple mass spectrometers.