Norimitsu Kurata

How useful is the ‘cocktail approach’ for evaluating human hepatic drug metabolizing capacity using cytochrome P450 phenotyping probes in vivo?

Relatively selective in vivo substrate probes have been developed for several major CYP isoforms involved in oxidative drug metabolism. There are basically two in vivo methods for identifying the phenotype. One method, the selective (CYP‐specific) phenotyping method, involves administering one single probe drug, whereas the other is a mixed phenotyping or ‘cocktail’ method involving the simultaneous administration of multiple probe drugs, specific for the individual P450. At present, caffeine and chlorzoxazone are used most often as probe drugs for CYP1A2 and CYP2E1, respectively, but these are not necessarily the best probe drugs. Of the potential probe drugs for CYP2C9, CYP2C19, CYP2D6 and CYP3A4, none is really useful. Despite current limitations, the cocktail method for obtaining information about multiple CYP activities in a single experimental session is likely to be more widely used as a screening or phenotyping method for humans in the future.

Effects of imidazole antimycotics on the liver microsomal cytochrome P450 isoforms in rats: comparison of in vitro and ex vivo studies.

SummaryWe have studied the effects of three imidazole derivatives, clotrimazole (CLO), ketoconazole (KET) and miconazole (MIC) on the liver microsomal diazepam (DZ) metabolism. In in vitro experiments using rats and human liver microsomes, significant inhibition of CYP3A in terms of DZ-3-hydroxylase activity was observed. The inhibition of DZ metabolism was seen 1 h after CLO dosing. On the other hand, the induction of certain cytochrome P450 (CYP) isozymes was observed in in vivo studies 24 h after dosing. That is, CYP1A, CYP2B and CYP3A2, but not CYP2E, were observed 24 h after CLO or KET or MIC treatment. Under these conditions, CLO was the most potent inducer of CYP3A and MIC was a more potent inducer of CYP1A and CYP2B. KET induced CYP1A and CYP2B whereas the inducibility of KET was less than those of CLO and MIC. All of the imidazole derivatives tested here showed significant inhibition of CYP isozymes which overcame the induction of the CYP isozymes by those drugs in the data of Western blot analysis.

Induction of cytochrome p-450 and related drug-oxidizing activities in muscone (3-methylcyclopentadecanone)-treated rats

In the present study, we investigated the effects of muscone on both in vitro and in vivo parameters of the hepatic microsomal drug-metabolizing enzyme system and other enzyme activities in rats. In the in vivo study, the serum dimethadione (DMO)/trimethadione (TMO) ratios at 2 hr after oral administration of TMO (100 mg/kg) were significantly increased in both male and female rats treated with 75 and 150 but not 40 mg muscone/kg. Antipyrine metabolite profile in 24 hr urine of rats pretreated with muscone (150 mg/kg) was examined. The results showed that the excretion of norantipyrine was significantly increased as compared to the control group. In the in vitro study, we found that the content of cytochrome P-450, and activities of aminopyrine, N-demethylase, aniline hydroxylase and delta-aminolevulinic acid (ALA) synthetase were significantly increased as compared to the controls in both male and female rats treated with muscone (75 and 150 mg/kg). This type of induction of the hepatic metabolizing enzymes was similar to that seen after treatment with a prototype drug, phenobarbital.

Involvement of cytochrome P450 2C9, 2E1 and 3A4 in trimethadione N-demethylation in human microsomes

Background and objectives:  Trimethadione (TMO), an antiepileptic drug, may be used as a candidate for estimating hepatic drug‐oxidizing activity. While TMO metabolism is mainly catalysed by CYP2C9, CYP2E1 and CYP3A4 the contribution of the different isoforms is unclear. In this study, we determined the percentage contribution of the three CYPs (CYP2C9, CYP2E1 and CYP3A4) to TMO N‐demethylation.

Inhibitory Effects of Gastrointestinal Drugs on CYP Activities in Human Liver Microsomes

OTC drugs have an important role in self-medication. However, the pharmacokinetic properties of some OTC drugs have not been fully investigated and reports concerning their drug interactions are insufficient. Several gastrointestinal drugs are available as OTC drugs. Because of their pharmacological properties, these drugs are often used concomitantly with other drugs. Therefore, it is important to predict the possible drug interactions among these drugs. In the current study, we investigated the inhibitory effects of five gastrointestinal drugs, namely loperamide, oxethazaine, papaverine, pirenzepine, and trimebutine, on CYP activities in human liver microsomes. Furthermore, we calculated the ratio of the intrinsic clearance of each CYP substrate in the presence or absence of the gastrointestinal drugs. The possibility of drug interactions in vivo was predicted by cut-off criteria. CYP3A4 activity was markedly inhibited by trimebutine, papaverine, and oxethazaine. Their inhibitory properties were competitive and the Ki values were 6.56, 12.8, and 3.08 µM, respectively. Alternative R values of CYP3A4 exceeded the cut-off level. These results suggested that drug interactions mediated by CYP3A4 may occur during treatment with these gastrointestinal drugs, necessitating the confirmation of the clinical significance of these drug interactions to prevent unexpected adverse effects.

Animal experiment ethics committee of Showa University

昭和大学における動物実験計画書の審査は，平成14年より実施している．審査は定期審査として年2回（9月，3月），その他緊急を要する計画に関しては，毎月臨時審査として実施している．審査を行う委員は医学部より4名（基礎，臨床各2名），歯学部，薬学部，保健医療学部より各2名，動物実験施設教員2名，動物実験施設長および附属病院教員1名の計14名より構成されている．過去5年間の年間審査件数は概ね370件であった．計画書の記載内容は，申請者の所属等に関する項目，共同実験者，動物種，系統，使用数，飼育期間，飼育条件などの飼育担当者に対する事務的内容の他に動物種，系統選択の理由，使用動物数決定の根拠，代替法の有無，実験動物に対する倫理的配慮，実験計画，安楽死法などの実験動物に加える負荷・苦痛の具体的内容およびその軽減，除去方法などの24項目から成り立っている．計画の申請は全てメール添付のWordファイルにて行い動物実験施設に提出する．申請された計画書は，受付時に記載の不備・不足等の確認を行い，加筆訂正が必要な場合には直接申請者と連絡を取り，審査会議までに必要記載事項を整える．内容確認が終了した計画書は，全て電子媒体に記録し14名の委員に審査の2週間前までに配布している．審査会議においては，動物に加えられる負荷苦痛の程度，その除去軽減に関する配慮等が主に論議される．これまでの審査によって，使用動物数の削減，処置後の動物の観察期間の短縮，薬物処置量や採血量の減量等につい申請者に計画変更を指示した例も多数出ている．これまでの所，審査については大きな問題は起きていないが，審査委員に動物の専門家，獣医等が加わっていない点は今後十分に配慮する必要があると考える．