Effects of gut microbiota on in vivo metabolism and tissue accumulation of cytochrome P450 3A metabolized drug: Midazolam

Abstract

The link between drug‐metabolizing enzymes and gut microbiota is well established. In particular, hepatic cytochrome P450 (CYP) 3A activities are presumed to be affected by gut microbiota. However, there is no direct evidence that the gut microbiota affects CYP3A metabolism or the clearance of clinically relevant drugs in vivo. Our purpose was to evaluate the effects of gut microbiota on in vitro and in vivo drug metabolism and on the clearance of midazolam, which is a standard CYP3A metabolized drug. Hepatic Cyp3a activity and in vitro midazolam hydroxylase activity were compared using specific pathogen‐free (SPF) and germ‐free (GF) mice. In a pharmacokinetics (PK) study, SPF and GF mice were intraperitoneally injected with 60 mg/kg of midazolam, and plasma and tissue concentrations were measured. Hepatic Cyp3a activity and midazolam hydroxylase activity were significantly lower in GF mice than in SPF mice. Notably, in the PK study, the area under the plasma concentration–time curve from time zero to infinity and the elimination half‐life were approximately four‐fold higher in GF mice compared with SPF mice. Furthermore, the concentration of midazolam in the brain 180 min after administration was about 14‐fold higher in GF mice compared with SPF mice. Together, our results demonstrated that the gut microbiota altered the metabolic ability of Cyp3a and the tissue accumulation of midazolam.

Normal variation of the gut microbiota affects hepatic cytochrome P450 activity in mice

“…Hepatic microsomes were prepared using previously described methods. 10 The total protein concentration was measured using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Cyp3a, Cyp2b, and Cyp2c activities in hepatic microsomes were measured using the P450‐Glo™ Assay (Promega) according to the manufacturer's instructions and a previous report.…”

“…Cyp3a, Cyp2b, and Cyp2c activities in hepatic microsomes were measured using the P450‐Glo™ Assay (Promega) according to the manufacturer's instructions and a previous report. 10 In detail, a luminogenic substrate, i.e., luciferin IPA for Cyp3a, luciferin 2B6, for Cyp2b, and luciferin H for Cyp2c, and hepatic microsomes in 0.2 M potassium phosphate buffer (pH 7.4) were preincubated for 10 min at 37°C. After adding NADPH regeneration systems (Promega), the reaction mixtures were incubated for 10, 20, or 30 min to measure Cyp3a, Cyp2b, or Cyp2c activity, respectively, at 37°C.…”

“…Hepatic microsomes were prepared using previously described methods 10 . The total protein concentration was measured using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific).…”

“…Ample studies demonstrated the link between the gut microbiota and the hepatic activity of cytochrome P450 (Cyp), 6–8 a major drug‐metabolizing enzyme. In particular, previous papers comparing germ‐free (GF) and specific pathogen‐free (SPF) mice reported that in the absence of the gut microbiota, the expression and activity of hepatic Cyp3a were clearly depressed, 8,9 leading to decreased Cyp3a drug metabolism in vivo and increased drug concentrations in plasma and tissues 10 . CYP3A is the dominant isoform of CYP, and it plays a significant role in the metabolism of approximately 50% of marketed drugs, 11 therefore, it is possible that the gut microbiota affects the metabolism of many drugs.…”

Contribution of the Gut Microbiome to Drug Disposition, Pharmacokinetic and Pharmacodynamic Variability

“…In germ-free mice given LCA, CYP3A expression was significantly elevated suggesting that LCA activated farnesoid X receptor and PXR [13]. Hepatic CYP3A and the activity of the CYP3A substrate midazolam were significantly lower in germ-free mice compared with conventional mice, suggesting that gut microbes may alter the metabolic activity of CYP3A [37].…”