Microbial Metabolism and Inhibition Studies of Phenobarbital

Fungi is a well-known model used to study drug metabolism and its production in in vitro condition. We aim to screen the most efficient strain of Cunninghamella sp. among C. elegans, C. echinulata and C. blakesleeana for bromhexine metabolites production. We characterized the metabolites produced using various analytical tools and compared them with mammalian metabolites in Rat liver microsomes (RLM). The metabolites were collected by two-stage fermentation of bromhexine with different strains of Cunninghamella sp. followed by extraction. Analysis was done by thin layer chromatography, high performance thin layer chromatography, Fourier transform infrared spectroscopy, high performance liquid chromatography and Liquid chromatography–mass spectrometry. The role of Cytochrome P3A4 (CYP3A4) enzymes in bromhexine metabolism was studied. Fungal incubates were spiked with reference standard – clarithromycin to confirm the role of CYP3A4 enzyme in bromhexine metabolism. Three metabolites appeared at 4.7, 5.5 and 6.4 min retention time in HPLC. Metabolites produced by C. elegans and RLM were concluded to be similar based on their retention time, peak area and peak response of 30.05%, 21.06%, 1.34%, and 47.66% of three metabolites and bromhexine in HPLC. The role of CYP3A4 enzyme in metabolism of bromhexine and the presence of these enzymes in Cunninghamella species was confirmed due to absence of peaks at 4.7, 5.4 and 6.7 min when RLM were incubated with a CYP3A4 enzyme inhibitor – clarithromycin.

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“…Aliquots (25 μL each) were injected into the HPLC system for analysis. Controls were also prepared to provide suitable blanks 8, 18…”

Section: Methodsmentioning

confidence: 99%

Biotransformation of bromhexine by Cunninghamella elegans , C. echinulata and C. blakesleeana

Dube

Kumar

2017

Brazilian Journal of Microbiology

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Fungal Biotransformation of Chlordiazepoxide and Evaluation of Type and Kinetics of Fungal Enzyme

Keerthana¹,

Vidyavathi²

2022

J. Pure Appl. Microbiol.

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To produce an active metabolite of Chlordiazepoxide by fungal biotransformation in an easy and economic way and also to develop microbial models for drug metabolism studies. Chlordiazepoxide is metabolized in the liver by CYP3A4 and forms major active metabolite N-desmethyl chlordiazepoxide. The focus of the study was to explore the ability of six distinct fungi to biotransform the drug Chlordiazepoxide to its metabolites. Induction, Inhibition and kinetic studies were also conducted to find out the type and capability of enzyme involved in fungal biotransformation. The screening studies were performed and fermentation protocol was designed with two controls (culture control and drug control) and one sample. Extract metabolite samples were reconstituted and analysed using HPLC. Induction, Inhibition studies were conducted similarly by maintaining its respective controls using CYP3A4 inducer (Carbamazepine) and inhibitor (Fluoxetine), further kinetic studies were performed to find Km and Vmax of fungal biotransformation of Chlordiazepoxide. Among six organisms Aspergillus ochreus has shown an extra peak at 6.9 min. in HPLC when compared with its controls indicated the formation of metabolite. The metabolite thus formed was identified, isolated and structure was confirmed by mass spectrometry and NMR spectroscopy as Nor-chlordiazepoxide. During inhibition and induction studies, it was found that quantity of the metabolite was increased with inducer and decreased with inhibitor. The Km and Vmax of fungal metabolism of Chlordiazepoxide was 1.928 µg/ml and 0.1802 µg/ml/hr respectively. Aspergillus ochreus has the ability to biotransform the Chlordiazepoxide to its active metabolite by CYP3A4 like enzyme and it followed Michaelis-Menten kinetics.

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Microbial Metabolism and Inhibition Studies of Phenobarbital

Cited by 2 publications

References 14 publications

Biotransformation of bromhexine by Cunninghamella elegans , C. echinulata and C. blakesleeana

Biotransformation of bromhexine by Cunninghamella elegans , C. echinulata and C. blakesleeana

Fungal Biotransformation of Chlordiazepoxide and Evaluation of Type and Kinetics of Fungal Enzyme

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