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.
Several studies revealed that substantial artificial changes in the gut microbiota resulted in modification of hepatic cytochrome P450 3a (Cyp3a) in mice. Consequently, we hypothesized that “normal” variation of the gut microbiota might also alter hepatic Cyp activity and lead to individual differences in drug metabolism. Therefore, this study investigated the effects of normal gut microbiota variation on hepatic Cyp activity under the same genetic and environmental conditions using ex‐germ‐free mice. Using the feces of three breeder BALB/c mice (Jcl, Slc, and Crj), germ‐free BALB/cYit mice were conventionalized (Yit‐Jcl, Yit‐Slc, and Yit‐Crj). The gut microbiota composition and hepatic Cyp activity of these donors and recipients were evaluated. 16S rRNA sequencing revealed clear differences of the gut microbiota among donors and among recipients. Cyp3a activity was significantly higher in Slc mice than in Jcl and Crj mice. Notably, among recipients, Cyp3a activity was significantly higher in Yit‐Slc and Yit‐Crj mice than in Yit‐Jcl mice. Cyp2b activity was significantly higher in Slc mice than in Jcl and Crj mice. Cyp2b activity was significantly higher in Yit‐Slc mice than in Yit‐Jcl mice. Additionally, in correlation analysis, some genera displayed significant positive or negative correlations with Cyp activity, particular the strong positive correlation between Clostridium sensu stricto 1 with Cyp3a activity. In conclusion, this study demonstrated that normal variation of the gut microbiota affected hepatic Cyp3a and Cyp2b activity, which might result in individual differences of drug metabolism.
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