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1.
Xenobiotica ; 49(8): 877-886, 2019 Aug.
Article in English | MEDLINE | ID: mdl-30118378

ABSTRACT

Metabolite identification is an integral part of both preclinical and clinical drug discovery and development. Synthesis of drug metabolites is often required to support definitive identification, preclinical safety studies and clinical trials. Here we describe the use of microbial biotransformation as a tool to produce drug metabolites, complementing traditional chemical synthesis and other biosynthetic methods such as hepatocytes, liver microsomes and recombinant human drug metabolizing enzymes. A workflow is discussed whereby microbial strains are initially screened for their ability to form the putative metabolites of interest, followed by a scale-up to afford quantities sufficient to perform definitive identification and further studies. Examples of the microbial synthesis of several difficult-to-synthesize hydroxylated metabolites and three difficult-to-synthesize glucuronidated metabolites are described, and the use of microbial biotransformation in drug discovery and development is discussed.


Subject(s)
Bacteria/metabolism , Pharmaceutical Preparations/metabolism , Biotransformation , Humans , Metabolome , Oxidation-Reduction , Pharmaceutical Preparations/chemistry
2.
Drug Metab Dispos ; 41(3): 668-81, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23293300

ABSTRACT

The inhibitory effect of boceprevir (BOC), an inhibitor of hepatitis C virus nonstructural protein 3 protease was evaluated in vitro against a panel of drug-metabolizing enzymes and transporters. BOC, a known substrate for cytochrome P450 (P450) CYP3A and aldo-ketoreductases, was a reversible time-dependent inhibitor (k(inact) = 0.12 minute(-1), K(I) = 6.1 µM) of CYP3A4/5 but not an inhibitor of other major P450s, nor of UDP-glucuronosyltransferases 1A1 and 2B7. BOC showed weak to no inhibition of breast cancer resistance protein (BCRP), P-glycoprotein (Pgp), or multidrug resistance protein 2. It was a moderate inhibitor of organic anion transporting polypeptide (OATP) 1B1 and 1B3, with an IC(50) of 18 and 4.9 µM, respectively. In human hepatocytes, BOC inhibited CYP3A-mediated metabolism of midazolam, OATP1B-mediated hepatic uptake of pitavastatin, and both the uptake and metabolism of atorvastatin. The inhibitory potency of BOC was lower than known inhibitors of CYP3A (ketoconazole), OATP1B (rifampin), or both (telaprevir). BOC was a substrate for Pgp and BCRP but not for OATP1B1, OATP1B3, OATP2B1, organic cation transporter, or sodium/taurocholate cotransporting peptide. Overall, our data suggest that BOC has the potential to cause pharmacokinetic interactions via inhibition of CYP3A and CYP3A/OATP1B interplay, with the interaction magnitude lower than those observed with known potent inhibitors. Conversely, pharmacokinetic interactions of BOC, either as a perpetrator or victim, via other major P450s and transporters tested are less likely to be of clinical significance. The results from clinical drug-drug interaction studies conducted thus far are generally supportive of these conclusions.


Subject(s)
Antiviral Agents/metabolism , Enzyme Inhibitors/metabolism , Enzymes/metabolism , Liver/enzymology , Membrane Transport Modulators/metabolism , Membrane Transport Proteins/metabolism , Proline/analogs & derivatives , Animals , Antiviral Agents/toxicity , Biotransformation , CHO Cells , Cricetinae , Cricetulus , Cytochrome P-450 CYP3A/metabolism , Cytochrome P-450 CYP3A Inhibitors , Dogs , Dose-Response Relationship, Drug , Drug Interactions , Enzyme Inhibitors/toxicity , Enzymes/genetics , Female , Glucuronosyltransferase/metabolism , Humans , Kinetics , LLC-PK1 Cells , Liver/drug effects , Liver-Specific Organic Anion Transporter 1 , Madin Darby Canine Kidney Cells , Male , Membrane Transport Modulators/toxicity , Membrane Transport Proteins/drug effects , Membrane Transport Proteins/genetics , Microsomes, Liver/enzymology , Organic Anion Transporters/antagonists & inhibitors , Organic Anion Transporters/metabolism , Oxidoreductases/metabolism , Proline/metabolism , Proline/toxicity , Recombinant Proteins/metabolism , Swine , Transfection
3.
Drug Metab Dispos ; 39(5): 763-70, 2011 May.
Article in English | MEDLINE | ID: mdl-21325431

ABSTRACT

Setileuton [4-(4-fluorophenyl)-7-[({5-[(1S)-1-hydroxy-1-(trifluoromethyl)propyl]-1,3,4-oxadiazol-2-yl}amino)methyl]-2H-1-benzopyran-2-one] is a selective inhibitor of the 5-lipoxygenase enzyme, which is under investigation for the treatment of asthma and atherosclerosis. During the development of setileuton, a metabolite (M5) was identified in incubations with rat, dog, and human liver microsomes that represented the addition of 18 Da to the 1,3,4-oxadiazole portion of the molecule. Based on mass spectral data, a ring opened structure was proposed and confirmed through comparison with a synthetic standard. The metabolic ring opening was examined in vitro in rat liver microsomes and was determined to be mediated by cytochrome P450s (P450s). Upon examination of the specific P450s involved using cDNA-expressed rat P450s, it was shown that CYP1A2 likely was the major isoform contributing to the formation of M5. Studies using stable labeled molecular oxygen and water demonstrated that the oxygen was incorporated from molecular oxygen, rather than water, and confirmed that the metabolic formation was oxidative. An alternative, comparatively slow pathway of chemical hydrolysis also was identified and described. Three potential mechanisms for the two-step metabolic ring opening of the 1,3,4-oxadizole are proposed.


Subject(s)
Coumarins/chemistry , Coumarins/metabolism , Cytochrome P-450 Enzyme System/metabolism , Lipoxygenase Inhibitors/chemistry , Lipoxygenase Inhibitors/metabolism , Microsomes, Liver/enzymology , Oxadiazoles/chemistry , Animals , Arachidonate 5-Lipoxygenase/metabolism , Asthma/drug therapy , Atherosclerosis/drug therapy , Coumarins/analysis , Coumarins/pharmacology , Cytochrome P-450 Enzyme System/chemistry , Lipoxygenase Inhibitors/analysis , Lipoxygenase Inhibitors/pharmacology , Male , Microsomes, Liver/metabolism , Oxadiazoles/analysis , Oxadiazoles/metabolism , Oxadiazoles/pharmacology , Rats
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