title: CYP3A Specifically Catalyzes 1β-Hydroxylation of Deoxycholic Acid: Characterization and Enzymatic Synthesis of a Potential Novel Urinary Biomarker for CYP3A Activity.
abstract: The endogenous bile acid metabolite 1β-hydroxy-deoxycholic acid (1β-OH-DCA) excreted in human urine may be used as a sensitive CYP3A biomarker in drug development reflecting in vivo CYP3A activity. An efficient and stereospecific enzymatic synthesis of 1β-OH-DCA was developed using a Bacillus megaterium (BM3) cytochrome P450 (P450) mutant, and its structure was confirmed by nuclear magnetic resonance (NMR) spectroscopy. A [(2)H4]-labeled analog of 1β-OH-DCA was also prepared. The major hydroxylated metabolite of deoxycholic acid (DCA) in human liver microsomal incubations was identified as 1β-OH-DCA by comparison with the synthesized reference analyzed by UPLC-HRMS. Its formation was strongly inhibited by CYP3A inhibitor ketoconazole. Screening of 21 recombinant human cytochrome P450 (P450) enzymes showed that, with the exception of extrahepatic CYP46A1, the most abundant liver P450 subfamily CYP3A, including CYP3A4, 3A5, and 3A7, specifically catalyzed 1β-OH-DCA formation. This indicated that 1β-hydroxylation of DCA may be a useful marker reaction for CYP3A activity in vitro. The metabolic pathways of DCA and 1β-OH-DCA in human hepatocytes were predominantly via glycine and, to a lesser extent, via taurine and sulfate conjugation. The potential utility of 1β-hydroxylation of DCA as a urinary CYP3A biomarker was illustrated by comparing the ratio of 1β-OH-DCA:DCA in a pooled spot urine sample from six healthy control subjects to a sample from one patient treated with carbamazepine, a potent CYP3A inducer; 1β-OH-DCA:DCA was considerably higher in the patient versus controls (ratio 2.8 vs. 0.4). Our results highlight the potential of 1β-OH-DCA as a urinary biomarker in clinical CYP3A DDI studies.
authors.affiliation: Cardiovascular and Metabolic Diseases, Drug Metabolism and Pharmacokinetics (M.A.H., X.-Q.L., T.B.A.) and Respiratory, Inflammation and Autoimmune Disease, Medicinal Chemistry (G.G.), Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Mölndal, Sweden; Division of Clinical Chemistry, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden (U.D.); Section of Pharmacogenetics, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (T.B.A.).
authors.forename: Martin A
authors.initials: MA
authors.lastname: Hayes
authors.affiliation: Cardiovascular and Metabolic Diseases, Drug Metabolism and Pharmacokinetics (M.A.H., X.-Q.L., T.B.A.) and Respiratory, Inflammation and Autoimmune Disease, Medicinal Chemistry (G.G.), Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Mölndal, Sweden; Division of Clinical Chemistry, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden (U.D.); Section of Pharmacogenetics, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (T.B.A.).
authors.forename: Xue-Qing
authors.initials: XQ
authors.lastname: Li
authors.affiliation: Cardiovascular and Metabolic Diseases, Drug Metabolism and Pharmacokinetics (M.A.H., X.-Q.L., T.B.A.) and Respiratory, Inflammation and Autoimmune Disease, Medicinal Chemistry (G.G.), Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Mölndal, Sweden; Division of Clinical Chemistry, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden (U.D.); Section of Pharmacogenetics, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (T.B.A.).
authors.forename: Gunnar
authors.initials: G
authors.lastname: Grönberg
authors.affiliation: Cardiovascular and Metabolic Diseases, Drug Metabolism and Pharmacokinetics (M.A.H., X.-Q.L., T.B.A.) and Respiratory, Inflammation and Autoimmune Disease, Medicinal Chemistry (G.G.), Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Mölndal, Sweden; Division of Clinical Chemistry, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden (U.D.); Section of Pharmacogenetics, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (T.B.A.).
authors.forename: Ulf
authors.initials: U
authors.lastname: Diczfalusy
authors.affiliation: Cardiovascular and Metabolic Diseases, Drug Metabolism and Pharmacokinetics (M.A.H., X.-Q.L., T.B.A.) and Respiratory, Inflammation and Autoimmune Disease, Medicinal Chemistry (G.G.), Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Mölndal, Sweden; Division of Clinical Chemistry, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden (U.D.); Section of Pharmacogenetics, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (T.B.A.) Tommy.B.Andersson@astrazeneca.com.
authors.forename: Tommy B
authors.initials: TB
authors.lastname: Andersson
mesh_terms: D055162:Biocatalysis; D015415:Biomarkers / Q000652:urine*; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism*; D003840:Deoxycholic Acid / Q000378:metabolism*; D006801:Humans; D006900:Hydroxylation
chemical_list: D015415:Biomarkers; D003840:Deoxycholic Acid; D051544:Cytochrome P-450 CYP3A