Masahiro Koike

Quantitative determination of cytochrome P-450 in rat liver homogenate

Abstract Cytochrome P -450 in whole liver homogenates, which contain an appreciable amount of hemoglobin, is detected by dithionite-difference spectroscopy of CO-bubbled homogenates. The molar extinction difference of cytocrhome P -450 by this method was determined to be 104 m m −1 cm −1 by comparative observations of the absorbance change in the dithionite- and CO-difference spectra of the membrane-bound hemoprotein. The content of cytochrome P -450 in normal rat liver was estimated to be 50 nmol/g wet weight of liver, and increased significantly after pretreatment of the animals with either phenobarbital or 3-methylcholanthrene.

Mechanism of the drug interaction between valproic acid and carbapenem antibiotics in monkeys and rats.

The Ministry of Health and Welfare, Japan banned coadministration of carbapenems, such as panipenem/betamipron (PAPM), meropenem (MEPM), and valproic acid (VPA) because clinical reports have indicated that the coadministration caused seizures in epileptic patients due to lowered plasma levels of VPA. In this study, we have clarified the mechanism of the drug-drug interaction using PAPM, MEPM, and doripenem [S-4661; (+)-(4R,5S,6S)-6-[(1R)-1-hydroxyethyl]-4-methyl-7-oxo-3-[[(3S,5S)-5-[(sulfamoylamino)methyl]-3-pyrrolidinyl]thio]-1-azabicyclo[3.2.0]hept-2-ene-2-caboxylic acid monohydrate], a newly synthesized carbapenem. In vitro experiments using monkey liver slices suggested that the apparent synthetic rate of VPA glucuronide (VPA-G) increased in the presence of carbapenems. However, no such increase was observed in the experiment using monkey liver microsomes. Although no increase of uridine 5′-diphosphate d-glucuronic acid was found in monkey liver slices in the presence of carbapenems, potent inhibitory activity of carbapenems for the hydrolysis of VPA-G was found in monkey and rat liver homogenate. In vivo hydrolysis of VPA-G was clearly shown by the existence of VPA in plasma after dosing of VPA-G to rats, and its inhibition by carbapenems was also clearly shown by the negligible levels of VPA in rat plasma after coadministration of carbapenems and VPA-G. These results clearly indicate one of the important causes of drug interaction as follows: carbapenems would inhibit the hydrolytic enzyme, which is involved in the hydrolysis of VPA-G to VPA, resulting in a decrease of plasma concentration of VPA.

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.u2002Anti-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.u2002Midazolam 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 29.6u2009μM, respectively. 3.u2002CYP3A 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.u2002The 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.u2002Cytochrome P450 (CYP) 3A catalysis of testosterone 6β-hydroxylation in female rat liver microsomes was significantly induced, then reached a plateau level after pretreatment with 80u2009mgu2009kg−1u2009day−1 dexamethasone (DEX) for 3 days. 2.u2002Midazolam was mainly metabolized by CYP3A in DEX-treated female rat liver microsomes from an immuno-inhibition study, and the apparent Km was 1.8u2009μM, similar to that in human microsomes. 3.u2002Ketoconazole 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 91.2u2009μM, 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.u2002After 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.u2002Using 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.u2002It 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 determination of metabolites of rilmazafone, a 1H-1,2,4-triazolyl benzophenone derivative in monkey urine

1. The metabolism of a new hypnotic 5-[(2-aminoacetamido)methyl]- 1-[4-chloro-2-(o-chlorobenzoyl)phenyl]-N,N-dimethyl-1H-1,2,4-tr iaz ole-3-carboxamide hydrochloride dihydrate (rilmazafone hydrochloride) was studies in female cynomolgus monkeys. 2. The structures of ten urinary metabolites were determined by mass spectrometry, and confirmed by comparison with synthetic authentic compounds. 3. Pathways of metabolism are postulated indicating that rilmazafone is desglycylated and cyclized to M-1, demethylated successively to M-2 and M-3, then hydrolysed to M-4, or hydroxylated at the 4-position of benzodiazepine ring or the p-position of the o-chlorophenyl group.