Design and optimization of quinazoline derivatives as melanin concentrating hormone receptor 1 (MCHR1) antagonists.

Melanin concentrating hormone (MCH) is an important mediator of energy homeostasis and plays a role in metabolic and CNS disorders. The modeling-supported design, synthesis and multi-parameter optimization (biological activity, solubility, metabolic stability, hERG) of novel quinazoline derivatives as MCHR1 antagonists are described. The in vivo proof of principle for weight loss with a lead compound from this series is exemplified. Clusters of refined hMCHR1 homology models derived from the X-ray structure of the ß2-adrenergic receptor, including extracellular loops, were developed and used to guide the design.

Discovery of novel, orally available benzimidazoles as melanin concentrating hormone receptor 1 (MCHR1) antagonists.

Melanin concentrating hormone (MCH) is an important mediator of energy homeostasis and plays role in several disorders such as obesity, stress, depression and anxiety. The synthesis and biological evaluation of novel benzimidazole derivatives as MCHR1 antagonists are described. The in vivo proof of principle for weight loss with a lead compound from this series is exemplified.

Concurrent determination of ezetimibe and its phase-I and II metabolites by HPLC with UV detection: quantitative application to various in vitro metabolic stability studies and for qualitative estimation in bile.

Simultaneous separation and quantification of ezetimibe (EZM) and its phase-I metabolite i.e., ezetimibe ketone (EZM-K) and phase-II metabolite i.e., ezetimibe glucuronide (EZM-G) in various matrices was accomplished by gradient HPLC with UV detection. The assay procedure involved deproteinization of 500 microL of either incubation or bile sample containing analytes and internal standard (IS, theophylline) with 75 microL acetonitrile containing 25% perchloric acid. An aliquot of 100 microL supernatant was injected onto a C18 column. The chromatographic separation was achieved by gradient elution consisting of 0.05 M formic acid:acetonitrile:methanol:water at a flow rate of 1.0 mL/min. The detection of analyte peaks were achieved by monitoring the eluate using an UV detector set at 250 nm. Nominal retention times of IS, EZM-G, ezetimibe ketone glucuronide (EZM-KG), EZM and EZM-K were 9.39, 24.23, 27.82, 29.04 and 30.56 min, respectively. Average extraction efficiencies of EZM, EZM-G and IS was >75-80% and for EZM-K was >50% from all the matrices tested. Limit of quantitation (LOQ) for EZM, EZM-K and EZM-G was 0.02 microg/mL. Due to the lack of availability of reference standard of EZM-KG, the recovery and LOQ aspects for this metabolite were not assessed. Overall, the method is suitable for simultaneous measurement of EZM, and its phase-I and phase-II metabolite (EZM-G) in in vitro and in vivo studies.

Glucuronidation of DRF-6574, hydroxy metabolite of DRF-4367 (a novel COX-2 inhibitor) by pooled human liver, intestinal microsomes and recombinant human UDP-glucuronosyltransferases (UGT): role of UGT1A1, 1A3 and 1A8.

DRF-4367 is a novel COX-2 inhibitor, which showed good efficacy in several animal models of inflammation. In a comparative in vitro metabolism in various liver microsomes, DRF-4367 forms a hydroxy metabolite (DRF-6574) mediated by CYP2D6 and 2C19 isoenzymes. DRF-6574 readily undergoes Phase-II metabolism and forms glucuronide and sulfate conjugates both in vitro and in vivo. The objective of the present study was two folds: to study the glucuronidation of DRF-6574 in human liver and intestinal microsomes and to identify the recombinant human liver and intestinal UDP-glucuronosyltransferase (UGT) enzymes responsible for glucuronidation of DRF-6574. Of twelve recombinant UGTs tested, two hepatic UGTs viz., UGT1A1 and 1A3 and an extra hepatic UGT i.e., UGT1A8 showed the catalytic activity. The enzyme kinetics in pooled human liver, intestinal and recombinant UGT microsomes showed a typical Michaelis-Menten plot. The apparent Km and Vmax value for DRF-6574 was found to be 116 +/- 24 microM and 2.07 +/- 0.12 microg/min/mg protein and 142 +/- 17 microM and 3.83 +/- 0.15 microg/min/mg protein in pooled human liver and intestinal microsomes, respectively. The intrinsic clearance (Vmax/Km) value for DRF-6574 was estimated to be 0.043 and 0.065 ml/min/mg protein, respectively in pooled human liver and intestinal microsomes. Moreover we have determined the Km and Vmax and intrinsic clearance values for specific UGTs viz., UGT 1A1, 1A3 and 1A8. The apparent Km and Vmax values are 23 +/- 7.2 microM, 3.44 +/- 0.17 microg/min/mg protein for UGT1A1, 60 +/- 7.9 microM, 3.67 +/- 0.11 microg/min/mg protein for UGT1A3, 96 +/- 8.0 microM, 2.95 +/- 0.06 microg/min/mg protein for UGT1A8. The intrinsic clearance values (Vmax/Km) estimated were 0.367, 0.148, 0.074 ml/min/mg protein for UGT1A1, 1A3 and 1A8, respectively. The intrinsic clearance value in UGT1A8 was very close to that in human intestinal and liver microsomes. The formation of DRF-6574 glucuronide by human liver, intestinal and UGT1A1, 1A3 and 1A8 microsomes was effectively inhibited by phenylbutazone.

Analysis of five HMG-CoA reductase inhibitors-- atorvastatin, lovastatin, pravastatin, rosuvastatin and simvastatin: pharmacological, pharmacokinetic and analytical overview and development of a new method for use in pharmaceutical formulations analysis and in vitro metabolism studies.

A specific, accurate, precise and reproducible high-performance liquid chromatographic (HPLC) method was developed and validated for the simultaneous quantitation of five 3-hydroxy-3-methyglutaryl coenzyme A (HMG-CoA) reductase inhibitors, viz. atorvastatin, lovastatin, pravastatin, rosuvastatin and simvastatin, in pharmaceutical formulations and extended the application to in vitro metabolism studies of these statins. Ternary gradient elution at a flow rate of 1 mL/min was employed on an Intertisl ODS 3V column (4.6 x 250 mm, 5 microm) at ambient temperature. The mobile phase consisted of 0.01 m ammonium acetate (pH 5.0), acetonitrile and methanol. Theophylline was used as an internal standard (IS). The HMG-CoA reductase inhibitors and their metabolites were monitored at a wavelength of 237 nm. Drugs were found to be 89.6-105.6% of their label's claim in the pharmaceutical formulations. For in vitro metabolism studies the reaction mixtures were extracted with simple liquid-liquid extraction using ethyl acetate. Baseline separation of statins and their metabolites along with IS free from endogenous interferences was achieved. Nominal retention times of IS, atorvastatin, lovastatin, pravastatin, rosuvastatin and simvastatin were 7.5, 17.2, 21.6, 28.5, 33.5 and 35.5 min, respectively. The proposed method is simple, selective and could be applicable for routine analysis of HMG-CoA reductase inhibitors in pharmaceutical preparations as well as in vitro metabolism studies.

Effect of ultraviolet B (302 nm) irradiation on viability, metabolic and detoxification functions of goat hepatocytes--in vitro study.

The object of the present study was to investigate the effect(s) of UV-B irradiation on the functional integrity, metabolic and detoxifying capacity of the isolated goat hepatocytes. Isolated goat hepatocytes were subjected to UV-B irradiation invitro for 0, 250, 500, 1250, 2500 and 7500 Joules/m2 which correspond to the irradiation time of 0, 1, 2, 5, 10 and 30 min. Cells were then analysed for Viability (Trypan blue exclusion test [TBE], 3-[4,5-dimethylthiozol-2yl]-2,5-diphenyltetrazolium bromide [MTT] assay, Membrane integrity (Lactate dehydrogenase [LDH] leakage, Lipid peroxidation) Detoxification (Ureagenesis, Cytochrome P450 activity [CYP450, Diazepam metabolism] and Glutathione-S-Transferase [GST] activity. The results show that there was no difference in functional, metabolic as well as detoxifying parameters of the hepatocytes when irradiated from 0-1250 Joules/m2, whereas a significant alteration was appreciable in the parameters such as LDH leakage, lipid peroxidation, and CYP450 activity when irradiated beyond 1250 Joules/m2. Our present findings suggest that the biologically compatible and feasible dose of UV-B irradiation for xenotransplantation appears to be 1250 Joules/m2.