No 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.

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.

Several studies revealed that gut microbiota affects the hepatic drug-metabolizing enzyme cytochrome P450 (Cyp). We hypothesized that individual gut microbiota variations could contribute to CYP activity. Human flora-associated (HFA) mice are established from germ-free mice using human feces and are often used to determine the effect of the human gut microbiota on the host. This study generated two groups of HFA mice using feces from two healthy individuals. Then, the composition of gut microbiota and hepatic Cyp activity was compared to analyze the effects of gut microbiota in healthy individuals on hepatic Cyp activity. A principal coordinate analysis based on the UniFrac distance for the composition of the cecal and fecal microbiota revealed apparent differences between the recipient groups. Hepatic Cyp, which is a marked difference in Cyp3a activity and Cyp3a11 gene expression, was observed between the recipient groups. Cyp2c and Cyp1a activities did not differ between recipient groups, with significantly lower enzymatic activities in recipients than in germ-free mice. These results indicate that the human gut microbiota affects hepatic Cyp activity. Especially, human gut microbiota composition differences have a pronounced effect on Cyp3a activity via Cyp3a11 gene expression regulation. Therefore, human gut microbiota variations among individuals may affect numerous drug metabolism, leading to drug efficacy and toxicity.

An incident reporting system (IRS) prevents possible adverse events by collecting and analyzing incidents that occur. However, few studies are available regarding IRSs in the laboratory animal field. This study aimed to develop an incident severity classification for laboratory animals (ISCLA) to evaluate the usefulness of the IRS in laboratory animal facilities. Twenty-three incidents reported from March 2019 to February 2020 on our IRS were retrospectively reviewed. Three of the 23 incidents failed to obtain some experimental data. Two of these incidents were harmless to animals, but the other caused the animals moderate distress. In addition, two of the three incidents made animals unsuitable for experiments. Since the inconsistent impact of incidents on animals and experiments prevented the comparison of the severity of individual incidents, we developed the ISCLA. According to the ISCLA, the above three incidents were classified into Category 3b and 4a. The others were classified into Category 0 (n = 5), 1 (n = 6), 2 (n = 3), and 3a (n = 6) in ascending order of severity. No incident was classified into Category 4b and 5. Furthermore, incidents occurring in the animal housing area were more severe than those occurring in the supporting area (P = 0.002).This study showed that incident occurrences had characteristics that were not visible from individual incidents alone. Moreover, the ISCLA was considered useful when conducting the IRS and taking improvement measures in laboratory animal facilities.