Dataset Information

Metabolic inhibition of meloxicam by specific CYP2C9 inhibitors in Cunninghamella blakesleeana NCIM 687: in silico and in vitro studies.

ABSTRACT: Specific inhibitors of Cytochrome P4502C9 enzyme (CYP2C9) viz. clopidogrel, fenofibrate fluvoxamine and sertraline at concentration of 50, 100, 150 and 200 µM were employed to investigate the nature of enzyme involved in bioconversion of meloxicam to its main metabolite 5-OH methyl meloxicam by Cunninghamella blakesleeana. Virtual screening for interaction of specific CYP2C9 inhibitors with human CYP2C9 enzyme was performed by molecular docking using Auto dock vina 4.2 version. The in silico studies were further substantiated by in vitro studies, which indicated fenofibrate to be a potent inhibitor of CYP2C9 enzyme followed by sertraline, clopidogrel and fluvoxamine, respectively. Two-stage fermentation protocol was followed to study metabolism of meloxicam and its inhibition by different CYP2C9 inhibitors. Meloxicam metabolites were identified using HPLC, LC-MS analysis and based on previous reports, as 5-OH methyl meloxicam (M1), 5-carboxy meloxicam (M2) and an unidentified metabolite (M3). All the inhibitors tested in the study showed a clear concentration dependent inhibition of meloxicam metabolism. The results suggest that the enzymes involved in metabolism of meloxicam in C. blakesleeana are akin to mammalian metabolism. Hence, C. blakesleeana can be used as a model organism in studying drug interactions and also in predicting mammalian drug metabolism.

SUBMITTER: Prasad GS

PROVIDER: S-EPMC4766174 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Metabolic inhibition of meloxicam by specific CYP2C9 inhibitors in Cunninghamella blakesleeana NCIM 687: in silico and in vitro studies.

Prasad G Shyam GS Srisailam K K Sashidhar R B RB

SpringerPlus 20160224

Specific inhibitors of Cytochrome P4502C9 enzyme (CYP2C9) viz. clopidogrel, fenofibrate fluvoxamine and sertraline at concentration of 50, 100, 150 and 200 µM were employed to investigate the nature of enzyme involved in bioconversion of meloxicam to its main metabolite 5-OH methyl meloxicam by Cunninghamella blakesleeana. Virtual screening for interaction of specific CYP2C9 inhibitors with human CYP2C9 enzyme was performed by molecular docking using Auto dock vina 4.2 version. The in silico stu ...[more]

PMID: 27026863

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Project description:Seven metabolites were obtained from the microbial transformation of anabolic-androgenic steroid mibolerone (1) with Cunninghamella blakesleeana, C. echinulata, and Macrophomina phaseolina. Their structures were determined as 10β,17β-dihydroxy-7α,17α-dimethylestr-4-en-3-one (2), 6β,17β-dihydroxy-7α,17α-dimethylestr-4-en-3-one (3), 6β,10β,17β-trihydroxy-7α,17α-dimethylestr-4-en-3-one (4), 11β,17β-dihydroxy-(20-hydroxymethyl)-7α,17α-dimethylestr-4-en-3-one (5), 1α,17β-dihydroxy-7α,17α-dimethylestr-4-en-3-one (6), 1α,11β,17β-trihydroxy-7α,17α-dimethylestr-4-en-3-one (7), and 11β,17β-dihydroxy-7α,17α-dimethylestr-4-en-3-one (8), on the basis of spectroscopic studies. All metabolites, except 8, were identified as new compounds. This study indicates that C. blakesleeana, and C. echinulata are able to catalyze hydroxylation at allylic positions, while M. phaseolina can catalyze hydroxylation of CH2 and CH3 groups of substrate 1. Mibolerone (1) was found to be a moderate inhibitor of β-glucuronidase enzyme (IC50 = 42.98 ± 1.24 μM) during random biological screening, while its metabolites 2-4, and 8 were found to be inactive. Mibolerone (1) was also found to be significantly active against Leishmania major promastigotes (IC50 = 29.64 ± 0.88 μM). Its transformed products 3 (IC50 = 79.09 ± 0.06 μM), and 8 (IC50 = 70.09 ± 0.05 μM) showed a weak leishmanicidal activity, while 2 and 4 were found to be inactive. In addition, substrate 1 (IC50 = 35.7 ± 4.46 μM), and its metabolite 8 (IC50 = 34.16 ± 5.3 μM) exhibited potent cytotoxicity against HeLa cancer cell line (human cervical carcinoma). Metabolite 2 (IC50 = 46.5 ± 5.4 μM) also showed a significant cytotoxicity, while 3 (IC50 = 107.8 ± 4.0 μM) and 4 (IC50 = 152.5 ± 2.15 μM) showed weak cytotoxicity against HeLa cancer cell line. Compound 1 (IC50 = 46.3 ± 11.7 μM), and its transformed products 2 (IC50 = 43.3 ± 7.7 μM), 3 (IC50 = 65.6 ± 2.5 μM), and 4 (IC50 = 89.4 ± 2.7 μM) were also found to be moderately toxic to 3T3 cell line (mouse fibroblast). Interestingly, metabolite 8 showed no cytotoxicity against 3T3 cell line. Compounds 1-4, and 8 were also evaluated for inhibition of tyrosinase, carbonic anhydrase, and α-glucosidase enzymes, and all were found to be inactive.

Project description:PURPOSE:The purpose of the study is to investigate the enzyme(s) responsible for siponimod metabolism and to predict the inhibitory effects of fluconazole as well as the impact of cytochrome P450 (CYP) 2C9 genetic polymorphism on siponimod pharmacokinetics (PK) and metabolism. METHODS:In vitro metabolism studies were conducted using human liver microsomes (HLM), and enzyme phenotyping was assessed using a correlation analysis method. SimCYP, a physiologically based PK model, was developed and used to predict the effects of fluconazole and CYP2C9 genetic polymorphism on siponimod metabolism. Primary PK parameters were generated using the SimCYP and WinNonlin software. RESULTS:Correlation analysis suggested that CYP2C9 is the main enzyme responsible for siponimod metabolism in humans. Compared with the CYP2C9*1/*1 genotype, HLM incubations from CYP2C9*3/*3 and CYP2C9*2/*2 donors showed ~ 10- and 3-fold decrease in siponimod metabolism, respectively. Simulations of enzyme contribution predicted that in the CYP2C9*1/*1 genotype, CYP2C9 is predominantly responsible for siponimod metabolism (~ 81%), whereas in the CYP2C9*3/*3 genotype, its contribution is reduced to 11%. The predicted exposure increase of siponimod with fluconazole 200 mg was 2.0-2.4-fold for CYP2C9*1/*1 genotype. In context of single dosing, the predicted mean area under the curve (AUC) is 2.7-, 3.0- and 4.5-fold higher in the CYP2C9*2/*2, CYP2C9*2/*3 and CYP2C9*3/*3 genotypes, respectively, compared with the CYP2C9*1/*1 genotype. CONCLUSION:.Enzyme phenotyping with correlation analysis confirmed the predominant role of CYP2C9 in the biotransformation of siponimod and demonstrated the functional consequence of CYP2C9 genetic polymorphism on siponimod metabolism. Simulation of fluconazole inhibition closely predicted a 2-fold AUC change (ratio within ~ 20% deviation) to the observed value. In silico simulation predicted a significant reduction in siponimod clearance in the CYP2C9*2/*2 and CYP2C9*3/*3 genotypes based on the in vitro metabolism data; the predicted exposure was close (within 30%) to the observed results for the CYP2C9*2/*3 and CYP2C9*3/*3 genotypes.

Dataset Information

Metabolic inhibition of meloxicam by specific CYP2C9 inhibitors in Cunninghamella blakesleeana NCIM 687: in silico and in vitro studies.

Publications

Metabolic inhibition of meloxicam by specific CYP2C9 inhibitors in Cunninghamella blakesleeana NCIM 687: in silico and in vitro studies.

Similar Datasets

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