Yoshitaka Yamaguchi

Assessment of the hepatic and intestinal first-pass metabolism of midazolam in a CYP3A drug-drug interaction model rats

In the current study, to understand the characteristics of dexamethasone (DEX)-treated female rats as an animal model for drug–drug interactions, a double-cannulation method was applied and separately assessed for the intestinal and hepatic first-pass metabolism of midazolam. Midazolam was administered intravenously or orally to the animals, and midazolam concentrations in the portal and systemic plasma were simultaneously determined. Next, the rates of elimination from the intestine and liver were estimated using the AUC values. After oral administration of midazolam, the entire drug was absorbed without intestinal first-pass metabolism, and 93% of the administered midazolam was extracted in the liver of the DEX-treated female rats. Seven per cent of the midazolam administered reached the systemic circulation. When ketoconazole was given orally to the animals, in conjunction with midazolam, the extraction ratio in the liver decreased from 93% to 77% in the control rats, and the bioavailability of midazolam increased to 23%. On the other hand, after intravenous administration, the elimination half-life of midazolam was not changed by ketoconazole pretreatment. These results indicated that midazolam is only extracted in the liver of DEX-treated female rats and that ketoconazole inhibits the hepatic first-pass metabolism, but not the systemic metabolism. In conclusion, DEX-treated female rats can be used as a drug–drug interaction model via CYP3A4 enzyme inhibition, especially for the hepatic first-pass metabolism of orally administered drugs.

Model for the drug–drug interaction responsible for CYP3A enzyme inhibition. I: evaluation of cynomolgus monkeys as surrogates for humans

1. Anti-human cytochrome P450 (CYP) 3A4 antiserum completely inhibited midazolam metabolism in monkey liver microsomes, suggesting that midazolam was mainly metabolized by CYP3A enzyme(s) in monkey liver microsomes. 2. Midazolam metabolism was also inhibited in vitro by typical chemical inhibitors of CYP3A, such as ketoconazole, erythromycin and diltiazem, and the apparent Ki values for ketoconazole, erythromycin and diltiazem were 0.127, 94.2 and 29.6 μM, respectively. 3. CYP3A inhibitors increased plasma midazolam concentrations when midazolam and CYP3A inhibitors were co-administered orally. However, the pharmacokinetic parameters of midazolam were not changed by treatment with CYP3A inhibitors when midazolam was given intravenously. This suggests that CYP3A inhibitors modified the first-pass metabolism in the liver and/or intestine, but not systemic metabolism. 4. The drug–drug interaction responsible for CYP3A enzyme(s) inhibition was observed when midazolam and inhibitors were co-administrated orally. Therefore, it was concluded that monkeys given midazolam orally could be useful models for predicting drug–drug interactions in man based on CYP3A enzyme inhibition.

Induction and regulation of cytochrome P450 K-5 (lauric acid hydroxylase) in rat renal microsomes by starvation

The effects of starvation on rat renal cytochrome P-450s were studied. The content of spectrally measured cytochrome P-450 in the renal microsomes of male rats increased 2-fold with 72 h starvation, but cytochrome b5 and NADPH-cytochrome P-450 reductase were not induced. 7-Ethoxycoumarin O-dealkylation and aniline hydroxylation activities of the renal microsomes of control male rats were very low but were induced 2.5-3-fold by 72 h starvation. Aminopyrine N-demethylation and lauric acid hydroxylation activities were induced 1.5-2-fold by 72 h starvation. The changes in catalytic activities suggested that the contents of individual cytochrome P-450s in the renal microsomes were altered by starvation. The contents of some cytochrome P-450s were measured by Western blotting. P450 DM (P450IIE1), a typical form of cytochrome P-450 induced by starvation in rat liver, was barely detected in rat kidney and was induced 2-fold by 72 h starvation. P450 K-5, a typical renal cytochrome P-450 and lauric acid hydroxylase, accounted for 81% of the spectrally measured cytochrome P-450 in the renal microsomes of control male rats and was induced 2-fold by 72 h starvation. P450 K-5 was not induced in rat kidney by treatment with chemicals such as acetone or clofibrate. The renal microsomes of male rats contained 6-times as much P450 K-5 as those of female rats. These results suggest that P450 K-5 is regulated by an endocrine factor.

Model for the drug–drug interaction responsible for CYP3A enzyme inhibition. II: establishment and evaluation of dexamethasone-pretreated female rats

1. Cytochrome P450 (CYP) 3A catalysis of testosterone 6β-hydroxylation in female rat liver microsomes was significantly induced, then reached a plateau level after pretreatment with 80 mg kg−1 day−1 dexamethasone (DEX) for 3 days. 2. Midazolam was mainly metabolized by CYP3A in DEX-treated female rat liver microsomes from an immuno-inhibition study, and the apparent Km was 1.8 μM, similar to that in human microsomes. 3. Ketoconazole and erythromycin, typical CYP3A inhibitors, demonstrated extensive inhibition of midazolam metabolism in DEX-treated female rat liver microsomes, and the apparent Ki values were 0.088 and 91.2 μM, respectively. The values were similar to those in humans, suggesting that DEX-treated female rat liver microsomes have properties similar to those of humans. 4. After oral administration of midazolam, the plasma midazolam concentration in DEX-treated female rats significantly decreased compared with control female rats. The area under the plasma concentration curve (AUC) and elimination half-life were one-11th and one-20th of those of control female rats, respectively. 5. Using DEX-treated female rats, the effect of CYP3A inhibitors on midazolam pharmacokinetics was evaluated. The AUC and maximum concentration in plasma (Cmax) increased when ketoconazole was co-administered with midazolam. 6. It was shown that the drug–drug interaction that occurs in vitro is also observed in vivo after oral administration of midazolam. In conclusion, the DEX-treated female rat could be a useful model for evaluating drug–drug interactions based on CYP3A enzyme inhibition.

Structure-activity relationship studies and discovery of a potent transient receptor potential vanilloid (TRPV1) antagonist 4-[3-chloro-5-[(1S)-1,2-dihydroxyethyl]-2-pyridyl]-N-[5-(trifluoromethyl)-2-pyridyl]-3,6-dihydro-2H-pyridine-1-carboxamide (V116517) as a clinical candidate for pain management.

A series of novel tetrahydropyridinecarboxamide TRPV1 antagonists were prepared and evaluated in an effort to optimize properties of previously described lead compounds from piperazinecarboxamide series. The compounds were evaluated for their ability to block capsaicin and acid-induced calcium influx in CHO cells expressing human TRPV1. The most potent of these TRPV1 antagonists were further characterized in pharmacokinetic, efficacy, and body temperature studies. On the basis of its pharmacokinetic, in vivo efficacy, safety, and toxicological properties, compound 37 was selected for further evaluation in human clinical trials.

Non-clinical evaluation of the metabolism, pharmacokinetics and excretion of S-777469, a new cannabinoid receptor 2 selective agonist.

Abstract 1. The drug metabolism and pharmacokinetics of S-777469 were investigated in in vitro (rat, dog and human) and in in vivo (rats and dogs). 2. S-777469 was rapidly and well absorbed, with bioavailability values ranging from 50 to 70% in rats and dogs, almost all drug radioactivity was excreted into the feces via bile within 48 h. Thus, good pharmacokinetics of S-777469 (e.g. systemic exposure and excretion rate) would be anticipated in humans. 3. In vitro metabolism of S-777469 was qualitatively similar in rat, dog and human hepatocytes. S-777469 acyl glucuronide, S-777469 5-hydroxymethyl and S-777469 4-hydroxycyclohexane were the main metabolites in rats, dogs and humans. In vivo metabolism in rats and dogs showed good qualitative agreement with in vitro metabolism, and no metabolites exceeded 10% of total radioactivity in rat and dog plasma. 4. No unique metabolites were observed in human hepatocytes. Therefore, rats and dogs were thought to be appropriate species for non-clinical toxicity studies. 5. In conclusion, these data should be useful for the characterization of the pharmacokinetic properties of S-777469 and the estimation of its pharmacokinetic fate in humans.

Investigation of drug–drug interaction via mechanism‐based inhibition of cytochrome P450 3A by macrolides in dexamethasone‐treated female rats

The in vitro and in vivo inhibition of cytochrome P450 (CYP) 3A with mechanism‐based inhibition (MBI) by macrolides was investigated using dexamethasone‐treated female rats (DEX‐female rats). In the in vitro CYP inhibition studies using erythromycin (ERM) and clarithromycin (CAM), similar inhibition responses were observed between human and DEX‐female rat liver microsomes, however, there were fewer effects in intact male rats. The ex vivo study showed that midazolam (MDZ) metabolism in liver microsomes of DEX‐female rats was reduced by ERM administration and the inhibitory effect was increased with increasing ERM doses, indicating that metabolite intermediate complex formation caused irreversible inhibition of CYP3A activity in DEX‐female rats as well as in humans. In the in vivo studies, ERM and CAM significantly increased the area under the plasma concentration–time curve of MDZ and decreased the total clearance in DEX‐female rats. It was concluded that the DDIs via MBI of CYP3A following macrolide administration in humans could be reproduced in female rats, suggesting that DEX‐female rats can serve as an in vivo model for assessing this DDI in humans. Copyright

Species differences in β-oxidative metabolism of a thromboxane A2-receptor antagonist [(+)-S-145] in rat, dog and monkey

1. The formation of β-oxidized metabolites from (+ )-S-145 [(+ )-(Z)-7-[(lR, IS, 3S, 4S)-3-(benzenesulphonamide)bicyclo-[2.2.l]-hept-2-yl]-5-heptenob acid] by liver homo- genates were compared between rat, dog and monkey. Species differences were found in hepatic β-oxidation capacities. The results agree with the qualitative and quantitative differences in β-oxidized metabolite proportions among these species observed in vivo. 2. The activities of microsomal (+ )-S-145-CoA synthesis, the initial step of the β- oxidation, were determined. Species differences in their intrinsic clearances primarily agreed with those of the β-oxidized metabolite formation. 3. (+ )-S-145-CoA oxidation activities towards (+ )-S-145-CoA by liver homogenates were much higher than the β-oxidized metabolite formation in all species, indicating that formed (+ )-S-145-CoA was immediately β-oxidized in peroxisomes. The species differences were inconsistent with those of β-oxidized metabolite formation in vitro. 4. Therefore, quantitative differences of hepatic (+ )-S-145 β-oxidation capacity in rat, dog and monkey were considered to be mainly due to the species difference in (+ )-S- 145-CoA formation.

ELUCIDATION OF METABOLIC PATHWAY OF THROMBOXANE A2 RECEPTORANT ANTAGONIST, (+)-S-145, IN RAT

Metabolism of (+)-S-145 was investigated within vitro studies to elucidate the metabolic pathway and responsible enzymes therein. Co-factor requirements and subcellular distribution indicated that a-side chain of (+)-S-145 was β-oxidized by peroxisomal enzymes, and that hydroxylation at C-5 or C-6 position of bicyclo-ring was catalyzed by cytochrome P-450s in microsomes. Results of these studies revealed that the most of (+)-S 145 incorporated into liver was activated to its acyl-CoA ester, and that β-oxidation was major pathway in metabolism of (+)-S-145. In peroxisome, there were two independent pathways in β-oxidation, thus (+)-S 145-CoA was generally β-oxidized to Bisnor-(+)S-145 and Tetranor-(+)-S 145, meanwhile its a-side chain was saturated by Δ5-reduction to form DH(+)-S-145 by NADPH dependent manner, then it was β-oxidized to DH-bisnor-(+)-S-145. As OH-(+)S-145 could never be n-oxidized, it was concluded that OH-Tetranor-(+)-S-145, one of major metabolites in vivo, was produced in the hydroxylation of Tetranor-(+)-S-145 catalyzed mainly by P-450 3A1/2.