Takahiko Baba

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.

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.

Glucuronidation Converting Methyl 1-(3,4-Dimethoxyphenyl)-3-(3-ethylvaleryl)-4-hydroxy-6,7,8-trimethoxy-2-naphthoate (S-8921) to a Potent Apical Sodium-Dependent Bile Acid Transporter Inhibitor, Resulting in a Hypocholesterolemic Action

Methyl 1-(3,4-dimethoxyphenyl)-3-(3-ethylvaleryl)-4-hydroxy-6,7,8-trimethoxy-2-naphthoate (S-8921) is a novel inhibitor of the ileal apical sodium-dependent bile acid transporter (ASBT/SLC10A2) developed for the treatment of hypercholesterolemia. The present study investigated the hypocholesterolemic action of S-8921 glucuronide (S-8921G) in rats. The plasma concentration of S-8921G was higher than that of S-8921 after single oral administration of S-8921 in normal rats, and S-8921G was excreted into the bile (13% dose). Oral administration of either S-8921 or S-8921G reduced the serum total cholesterol, particularly nonhigh-density lipoprotein cholesterol, in hypercholesterolemic normal rats. In Gunn rats devoid of UDP glucuronosyltransferase-1A activity, S-8921G was undetectable both in the plasma and bile specimens, and only S-8921G administration significantly reduced the serum nonhigh-density lipoprotein cholesterol. An in vitro inhibition study showed that glucuronidation converts S-8921 to a 6000-fold more potent inhibitor of human ASBT (Ki = 18 nM versus 109 μM). S-8921G was detected both in the portal plasma and loop when S-8921 was administered into the loop of the rat jejunum, although the cumulative amount of S-8921G recovered in the bile was 5-fold greater than that in the loop. The uptake of S-8921G by freshly prepared rat hepatocytes was saturable, and sodium-dependent and -independent systems were involved. Organic anions, such as bromosulfophthalein, estrone 3-sulfate, and taurocholic acid, inhibited the uptake. These results suggest that UDP glucuronosyltransferase-1 isoforms play a critical role in the hypocholesterolemic action of S-8921 by converting S-8921 to a more potent ASBT inhibitor, and organic anion transporter(s) are also involved in its pharmacological action through the biliary excretion of S-8921G.

Identification of the Transporters Involved in the Hepatobiliary Transport and Intestinal Efflux of Methyl 1-(3,4-Dimethoxyphenyl)-3-(3-ethylvaleryl)-4-hydroxy-6,7,8-trimethoxy-2-naphthoate (S-8921) Glucuronide, a Pharmacologically Active Metabolite of S-8921

The glucuronide conjugate of methyl 1-(3,4-dimethoxyphenyl)-3-(3-ethylvaleryl)-4-hydroxy-6,7,8-trimethoxy-2-naphthoate (S-8921; S-8921G) is a 6000-fold more potent inhibitor of an ileal apical sodium-dependent bile acid transporter (SLC10A2) than S-8921 and is responsible for the hypocholesterolemic effect of S-8921 in rats. Because S-8921G is formed in the intestine and liver, the present study investigated the transporters involved in the secretion of S-8921G that govern its exposure to the target site and thereby play an important role in its pharmacological action. Organic anion transporting polypeptide (OATP) 1B1- and OATP1B3-expressing cells exhibited saturable accumulation of S-8921G with Km values (micromolar) of 1.9. The uptake of [14C]S-8921G by human cryopreserved hepatocytes was saturable and sodium-independent. Comparison of protein expression between the cDNA transfectants and hepatocytes suggests that the contribution of OATP1B1, OATP1B3, and Na+-taurocholate cotransporting polypeptide to the hepatic uptake of S-8921G is 63, 35, and 2.6%, respectively. The basal-to-apical transport of S-8921G was enhanced in Madin-Darby canine kidney cells expressing both OATP1B1 and multidrug resistance-associated protein (MRP) 2. In Mrp2-deficient mutant rats [Eisai hyperbilirubinemic rats (EHBR)], the biliary excretion clearance based on the plasma concentration was 20% of the normal value, whereas the pharmacokinetic parameters did not show any significant change in Bcrp-/- mice. Furthermore, the secretion clearance of S-8921G to the mucosal side was also significantly lower in everted jejunum sacs from EHBR (9.18 and 20.8 μl/min/g tissue). These results suggest that MRP2 is responsible for the secretion of S-8921G to the intestinal lumen and bile and that OATP1B1 and OATP1B3 account for the hepatic uptake. These transporters deliver S-8921G to the target site of its pharmacological action.

Sex differences in the metabolism of (+)-S-145, a novel thromboxane A2 receptor antagonist in rat

1. After the oral administration of 5 mg/kg S-1452 to rat, the plasma levels of (+)-S-145 were similar between the male and female, but there were sex differences in the profiles of its beta-oxidized and hydroxylated metabolites in plasma. 2. beta-Oxidation of (+)-S-145 determined in vitro was slightly higher in the female than in the male, and agreed with the plasma levels of the beta-oxidized metabolites. 3. 5-Hydroxylation activities of (+)-S-145 and beta-oxidized metabolites by rat liver microsomes were significantly higher in the male than in the female, but marked sex differences were not observed in 6-hydroxylation activities. These results revealed that differences in monooxygenase activities directly account for the sex differences in the plasma level of 5-hydroxylated metabolites, and that the peroxisomal beta-oxidation enzyme system also affected the plasma level of 6-hydroxylated metabolites. 4. Biliary excretion was higher in the male than in the female, and quantitative identification of metabolites in bile indicated that this was based on the prominent excretion of taurine conjugates in the male rat. This conclusion was supported by the fact that taurine conjugation activity was higher in male liver homogenates than in the female.

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

Synthesis and activities of oxidative metabolites of the anti-arthritic drug candidate S-2474

We have synthesized and characterized some oxidative metabolites of S-2474. In this study, we discovered a novel skeleton, the 2,3-dihydrobenzofuran derivative, which inhibited PGE(2) production at a very low concentration and was effective in the anti-carrageenin footpad edema assay.

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.