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J Med Chem ; 60(14): 6166-6190, 2017 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-28635286


Agonism of the 5-HT2C receptor represents one of the most well-studied and clinically proven mechanisms for pharmacological weight reduction. Selectivity over the closely related 5-HT2A and 5-HT2B receptors is critical as their activation has been shown to lead to undesirable side effects and major safety concerns. In this communication, we report the development of a new screening paradigm that utilizes an active site mutant D134A (D3.32) 5-HT2C receptor to identify atypical agonist structures. We additionally report the discovery and optimization of a novel class of nonbasic heterocyclic amide agonists of 5-HT2C. SAR investigations around the screening hits provided a diverse set of potent agonists at 5-HT2C with high selectivity over the related 5-HT2A and 5-HT2B receptor subtypes. Further optimization through replacement of the amide with a variety of five- and six-membered heterocycles led to the identification of 6-(1-ethyl-3-(quinolin-8-yl)-1H-pyrazol-5-yl)pyridazin-3-amine (69). Oral administration of 69 to rats reduced food intake in an ad libitum feeding model, which could be completely reversed by a selective 5-HT2C antagonist.

Arginina/análogos & derivados , Flavonas/química , Receptor 5-HT2C de Serotonina/metabolismo , Agonistas do Receptor 5-HT2 de Serotonina/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Animais , Arginina/síntese química , Arginina/química , Arginina/farmacologia , Encéfalo/metabolismo , Células CACO-2 , Permeabilidade da Membrana Celular , Comportamento Alimentar/efeitos dos fármacos , Flavonas/síntese química , Flavonas/farmacologia , Células HEK293 , Humanos , Masculino , Membranas Artificiais , Camundongos Knockout , Microssomos Hepáticos/metabolismo , Mutação , Ratos Sprague-Dawley , Receptor 5-HT2A de Serotonina/metabolismo , Receptor 5-HT2B de Serotonina/metabolismo , Receptor 5-HT2C de Serotonina/genética , Agonistas do Receptor 5-HT2 de Serotonina/síntese química , Agonistas do Receptor 5-HT2 de Serotonina/farmacocinética , Agonistas do Receptor 5-HT2 de Serotonina/farmacologia , Relação Estrutura-Atividade
Drug Metab Dispos ; 41(8): 1566-74, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23723360


The purpose of this study was to determine the impact of CYP3A5 expression on inhibitory potency (Ki or IC50 values) of CYP3A inhibitors, using recombinant CYP3A4 and CYP3A5 (rCYP3A4 and rCYP3A5) and CYP3A5 genotyped human liver microsomes (HLMs). IC50 ratios between rCYP3A4 and rCYP3A5 (rCYP3A5/rCYP3A4) of ketoconazole (KTZ) and itraconazole (ITZ) were 8.5 and 8.8 for midazolam (MDZ), 4.7 and 9.1 for testosterone (TST), 1.3 and 2.8 for terfenadine, and 0.6 and 1.7 for vincristine, respectively, suggesting substrate- and inhibitor-dependent selectivity of the two azoles. Due to the difference in the IC50 values for CYP3A4 and CYP3A5, nonconcordant expression of CYP3A4 and CYP3A5 protein can significantly affect the observed magnitude of CYP3A-mediated drug-drug interactions in humans. Indeed, the IC50 values of KTZ and ITZ for CYP3A-catalyzed MDZ and TST metabolism were significantly higher in HLMs with CYP3A5*1/*1 and CYP3A5*1/*3 genotypes than in HLMs with the CYP3A5*3/*3 genotype, showing CYP3A5 expression-dependent IC50 values. Moreover, when IC50 values of KTZ and ITZ for different HLMs were kinetically simulated based on CYP3A5 expression level and enzyme-specific IC50 values, a good correlation between the simulated and the experimentally measured IC50 values was observed. Further simulation analysis revealed that both the Ki ratio (for inhibitors) and Vmax/Km ratio (for substrates) between CYP3A4 and CYP3A5 were major factors for CYP3A5 expression-dependent IC50 values. In conclusion, the present study demonstrates that CYP3A5 genotype and expression level have a significant impact on inhibitory potency for CYP3A-catalyzed drug metabolism, but that the magnitude of its effect is inhibitor-substrate pair specific.

Biocatálise , Inibidores do Citocromo P-450 CYP3A , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/fisiologia , Interações de Medicamentos , Feminino , Genótipo , Humanos , Itraconazol/farmacologia , Cetoconazol/farmacologia , Masculino , Microssomos Hepáticos/metabolismo , Midazolam/metabolismo , Terfenadina/metabolismo , Testosterona/metabolismo , Vincristina/metabolismo
Bioanalysis ; 4(14): 1747-61, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22877221


BACKGROUND: An integrated method that provides rates of both parent disappearance and metabolite formation was developed. RESULTS: Buspirone, mirtazapine and verapamil were used as model compounds in developing the method. Incubations were carried out on a robotic platform. Qualitative analysis of metabolites in 30 µM samples was conducted by data-dependent HPLC-MS/MS on a high-resolution instrument. Quantitative analysis of the parent compound and metabolites in 0.5 µM samples was conducted by full-scan MS(2) with product ion extraction using an ion trap mass spectrometer. Data generated for the compounds included half-life and intrinsic clearance of the parent molecule, characterization of metabolites and relative rates of metabolite formation. A correction factor was used to convert MS responses of metabolites in 0.5 µM samples to UV areas in order to compare relative metabolite concentrations. CONCLUSION: The approach allows for the investigation of a set of six compounds simultaneously, with a turnaround time of 1 week or less.

Técnicas de Química Analítica , Avaliação Pré-Clínica de Medicamentos/métodos , Farmacocinética , Animais , Automação , Biotransformação , Buspirona/análise , Buspirona/farmacocinética , Cromatografia Líquida de Alta Pressão/métodos , Cães , Meia-Vida , Humanos , Mianserina/análogos & derivados , Mianserina/análise , Mianserina/farmacocinética , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Mirtazapina , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas em Tandem/métodos , Verapamil/análise , Verapamil/farmacocinética
Rapid Commun Mass Spectrom ; 25(21): 3245-51, 2011 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-22006386


An early assessment of metabolite exposure in preclinical species can provide quantitative estimation on possible active or toxic metabolites. Frequently, synthetic metabolite standards are not available at the preclinical stage, precluding the quantitation of metabolites by means of calibration curves and quality control (QC) samples. We present here an approach to determine the extent of circulating metabolites using 'metabolite standards' generated by in vitro incubations in combination with the correction for mass spectrometry response based on UV response. The study was done by coupling ultra-high-performance liquid chromatography (UHPLC) to LTQ-Orbitrap high-resolution mass spectrometry, and the quantitation was based on full scan high-resolution accurate mass analysis in combination with retention time. First, we investigated the separation capacity of a 10.5 min UHPLC method and the quantitative capability of an LTQ-Orbitrap for full scan accurate mass quantitation by spiking chemical standards of buspirone and its six metabolites in blank plasma. Then we demonstrated the use of a UV correction approach to quantitatively estimate buspirone and its metabolites in plasma samples from a rat pharmacokinetics study. We compared the concentration versus time profiles of buspirone and its six metabolites in rat plasma samples obtained using three different approaches, including using UV correction, using individual standard curves for each metabolite prepared from the synthetic standard, and using a calibration curve of the parent compound buspirone. We demonstrated the estimated metabolite exposure of buspirone using this UV correction approach resulted in rank ordering of metabolite exposure within three-fold of the value obtained with metabolite standards, in contrast to eight-fold without UV correction. The approach presented in this paper provides a practical solution to an unmet bioanalytical need for quantitative information on metabolites without standards in preclinical in vivo studies.

Buspirona/química , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida de Alta Pressão/normas , Espectrometria de Massas/métodos , Espectrometria de Massas/normas , Animais , Biotransformação , Buspirona/metabolismo , Buspirona/farmacocinética , Calibragem , Masculino , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Espectrofotometria Ultravioleta
Toxicol Sci ; 84(2): 232-42, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15673846


4-(3-pentylamino)-2,7-dimethyl-8-(2-methyl-4-methoxyphenyl)-pyrazolo-[1,5-a]-pyrimidine (DMP 904) is a potent and selective antagonist of corticotropin releasing factor receptor-1 (CRF1 receptor) with an efficacious anxiolytic profile in preclinical animal models. In subchronic toxicity studies in Sprague-Dawley rats, DMP 904 produced thyroid follicular cell hypertrophy and hyperplasia, and a low incidence of follicular cell adenoma. The current investigations were designed to determine the mode of action by which DMP 904 disrupts thyroid homeostasis in male rats. Five-day treatment with DMP 904 (300 mg/kg/day) dramatically lowered serum thyroxine (T4) to levels below detectable limits (< 1 microg/dl) by 72 h, with concurrent decreases in triiodothyronine (T3, about a 70% decrease) and increases in thyroid stimulating hormone (TSH; about a three-fold increase). DMP 904 increased [125I]T4 total body clearance (Cl tb) (38.21 +/- 10.45 ml/h) compared to control (5.61 +/- 0.59 ml/h) and phenobarbital-treated rats (7.92 +/- 1.62 ml/h). This increase in Cl(tb) was associated with a significant increase in biliary clearance (Cl bile) of unconjugated [125I]T4 (nearly 80-times control rates) and increased liver:blood ratios of T4, suggestive of enhanced hepatic uptake of T4. A single dose of DMP 904 (200 mg/kg) increased mRNA levels of hepatic cytochrome P450s (CYP 3A1 and CYP 2B1) and UDP-glucuronosyltransferases (UGT 1A1 and UGT 1A2). DMP 904 also induced mRNAs of the canalicular transporter, multi-drug resistance protein-2 (Mrp2) and sinusoidal transporters, organic anion transporting proteins (Oatp1 and Oatp2) within 24 h. Western blot analysis confirmed DMP 904 related increases in Oatp2 protein expression. Collectively, these data suggest that DMP 904 is an agonist of the constitutive androstane receptor (CAR) and pregnane X receptor (PXR) and that the decreased serum levels of T4 and T3 resulted from increased hepatobiliary clearance. However, DMP 904 is distinguished from other compounds associated with similar effects on thyroid hormone homeostasis because its effects were primarily related to increased biliary excretion of unconjugated T4.

Hormônio Liberador da Corticotropina/antagonistas & inibidores , Homeostase/efeitos dos fármacos , Fígado/efeitos dos fármacos , Pirazóis/toxicidade , Pirimidinas/toxicidade , Glândula Tireoide/efeitos dos fármacos , Tiroxina/sangue , Tri-Iodotironina/metabolismo , Administração Oral , Animais , Autorradiografia , Bile/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Fígado/metabolismo , Masculino , Transportadores de Ânions Orgânicos , Proteínas de Transporte de Cátions Orgânicos/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Glândula Tireoide/metabolismo , Tireotropina/sangue