Genetic polymorphisms of drug metabolism

The molecular mechanisms of 3 genetic polymorphisms of drug metabolism have been studied at the level of enzyme activity, enzyme protein and RNA/DNA. As regards debrisoquine/sparteine polymorphism, cytochrome P-450IID6 was absent in livers of poor metabolizers; aberrant splicing of premRNA of P-450IID6 may be responsible for this. Moreover, 3 mutant alleles of the P-450IID6 locus on chromosome 22 associated with the poor metabolizer phenotype were identified by Southern analysis of leucocyte DNA. The presence of 2 identified mutant alleles allowed the prediction of the phenotype in approximately 25% of poor metabolizers. The additional gene-inactivating mutations which are operative in the remainder of poor metabolizers are now being studied. Regarding mephenytoin polymorphism, although the deficient reaction, S-mephenytoin 4'-hydroxylation, has been well defined in human liver microsomes, the mechanism of this polymorphism remains unclear. All antibodies prepared to date against cytochrome P-450 fractions with this activity recognize several structurally similar enzymes and several cDNAs related to these enzymes have been isolated and expressed in heterologous systems. However, which isozyme is affected by this polymorphism is not known. As regards N-acetylation polymorphism, N-acetyltransferases have been purified from human liver, specific antibodies prepared; it was observed that immunoreactive N-acetyltransferase is decreased or undetectable in liver of "slow acetylators". Two genes that encode functional N-acetyltransferase were characterized. The product of one of these genes has identical activity and characteristics as the polymorphic liver enzyme. Cloned DNA from rapid and slow acetylator individuals has been analyzed to identify the structural or regulatory defect that causes deficient N-acetyltransferase.     

Metabolism of digoxin, digoxigenin digitoxosides and digoxigenin in human hepatocytes and liver microsomes

In vitro metabolism of digoxin and its cleavage-related compounds was investigated using hepatocytes in primary culture and microsomal fractions both isolated from human livers. On these models, digoxin (DG3) and digoxigenin bisdigitoxoside (DG2) were not shown to be significantly metabolized in vitro in man. Therefore, it appeared that the stepwise cleavage of DG3 and DG2 sugars was not cytochrome P450 dependent. This enzymatic system probably plays a minor role in humans for this particular reaction. However, digoxigenin monodigitoxoside (DG1) and digoxigenin (DG0) which are known to be formed after intra-gastric hydrolysis of DG3, were extensively converted to polar compounds (mainly glucuronides). In addition, using human liver microsomes, a wide variability in UDP-glucuronyl transferase (UDPGT) activities responsible for DG1 glucuronidation was demonstrated. These results suggest that two main factors may contribute to the overall interindividual variability of digoxin biotransformation: 1), the individual intra-gastric pH which influences the sugar cleavage leading to DG1 and DG0; ii), a variability in the level of the hepatic UDPGT specific for digitalis compounds conjugation.     

Characterization of human cytochrome P450 isoenzymes involved in the metabolism of vinorelbine

Vinorelbine (VRL) (IV Navelbine) is a semi-synthetic vinca alkaloid, used in therapeutics for the treatment of non-small-cell lung cancer and advanced breast cancer. The aim of this study was to characterize the cytochrome P450 (CYP) isoenzymes involved in VRL metabolism. VRL was incubated at 1.28 x 10(-5) m for 90 min with human hepatic microsomes prepared from 14 donors (one woman and 13 men aged from 27 to 76 years old) and characterized for CYP1A2, CYP2D6, CYP2E1 and CYP3A4 activities. A four-combined-approach study was performed, including correlation between CYP activities and VRL metabolism among the 14 batches of microsomes, inhibition of VRL biotransformation by isoform-selective substrates and by specific inhibitory antibodies, and incubation with supersomes. Analysis of unchanged VRL and its metabolites was performed using an HPLC method coupled with both radioactive and UV detections. No correlation between CYP1A2 or CYP2E1 and VRL metabolism was observed in the 14 batches of microsomes used. A correlation was shown between VRL metabolism and CYP3A4 activity as determined with the dextromethorphan N-demethylase and nifedipine oxidase activities (r(2)=0.80 and 0.59, respectively). These results were strengthened by a correlation between the metabolism extent of VRL and CYP3A4 protein content determined by immunoblotting (r(2)=0.75). Furthermore, VRL biotransformation was inhibited by troleandomycine, the CYP3A4-specific inhibitor substrate (80% of inhibition) and by anti-CYP3A antibodies (36% of inhibition). On the contrary, a low correlation with CYP2D6 activity as determined by dextrometorphan O-demethylation (r(2)=0.31) was established. CYP2D6 supersomes did not metabolize the drug whereas 63.4% of VRL were metabolized by microsomes overexpressing CYP3A4 isoform. These data indicated that CYP3A4 is the main enzyme involved in the hepatic metabolism of VRL in human, whereas CYP2D6 is not involved.     

Impairment of drug metabolism in patients with liver cancer

Abstract. Drug metabolizing capacity was investigated in sixteen patients with cancer in the liver by comparing in vivo (antipyrine disappearance rate from plasma) and in vitro (cytochrome P-450, benzpyrene hydroxylase, and 7-ethoxycoumarin O-deethylase activities in liver biopsies) indices of drug metabolism with the condition of tumour-free liver parenchyma and with biochemical liver function tests. Drug metabolism was impaired as antipyrine hydroxylation was significantly lower than in matched controls. Impairment of antipyrine metabolism was related to the pathological changes in the liver; patients with metastatic liver cancer or with tumour and cirrhosis metabolized antipyrine at a reduced rate, whereas those with a hepatoma occupying from 20 to 80% of the liver volume had normal antipyrine metabolism. Liver enzyme activities were related to the site of the biopsy; values were low in and around the tumour tissue and high in normal parenchyma. The antipyrine half-life was related to serum albumin concentration but not to other liver function tests. The results demonstrate that in liver cancer patients the drug-hydroxylating capacity is dependent on the ability of the tumour-free parenchyma to metabolize drugs. Hence quantitative evaluation of liver changes together with tests measuring liver synthetic capacity might be of significance when predicting drug metabolism in patients with hepatic malignancy.     

Interspecies variability and drug interactions of clozapine metabolism by microsomes

Cytochrome P450 expression in liver is influenced by several factors, including species, sex and strain. We compared metabolism formation of clozapine in different species (rat, mouse, guinea-pig, dog, monkey and man) so as to choose between species to further validate interaction studies. Liver microsomes of male and female Sprague-Dawley rats, hairless rats, OF1 mice, Balb C mice and Dunkin-Hartley albino guinea-pigs, male beagle dogs, male cynomolgus monkeys and man were used to investigate in vitro metabolism of clozapine. This process was dependent on the presence of NADPH and on the presence of microsome protein. In addition, we observed the formation of desmethyl- and N-oxide metabolites, with the rate of formation of each of these compounds varying with species, sex and strain of microsomes incubated. The desmethyl- and N-oxide metabolites formed were statistically greater in male than in female rats, mice in the two strains studied, as well as for the guinea-pigs. Levels of desmethyl clozapine formed were high for the rats and no significant difference in clozapine biotransformation was observed between Sprague-Dawley and hairless rats. For man, the formation of metabolites of clozapine was comparable with guinea-pig, dog and monkey. In addition, we screened the effect of 52 molecules, representative of 11 different therapeutic classes, on the metabolism of clozapine by rat liver microsomes. We found that most of the calcium channel blockers (diltiazem, felodipine, isradipine, lacidipine, nicardipine and nitrendipine), antifungals (ketoconazole, miconazole) and two anticancer drugs (paclitaxel, teniposide) caused more than 50% inhibition of clozapine metabolism in vitro. The extent of inhibition was increased in a concentration-dependant manner. Complementary clinical and pharmacokinetic studies should be performed to confirm these results.     

A study of the in vitro interaction between lidocaine and premedications using human liver microsomes

OBJECTIVE: To investigate potential interactions between lidocaine (lignocaine) metabolism and premedication drugs, i.e. psychotropic and antianxiety agents (diazepam, midazolam), hypnotics (pentobarbital, thiamylal), depolarizing neuromuscular blocking agents (vecuronium, pancuronium and suxamethonium), an antihypertensive agent (clonidine) and an H2-receptor blocking agent (cimetidine) using human liver microsomes in vitro. METHODS: The interaction effects between lidocaine and premedication were examined using human liver microsomal preparations and monitored for enzyme activity. The lidocaine and its main metabolite (monoethylglycinexylide) were measured by HPLC/UV. RESULTS: Lidocaine metabolism was non-competitively inhibited by midazolam (Ki = 77.6 microM). Thiamylal was a competitive inhibitor of lidocaine metabolism (Ki = 885 microM). Cimethidine, pancuronium and vecuronium weakly inhibited lidocaine metabolism in a concentration-depend manner over the therapeutic range in human liver microsomes. On the contrary, suxamethonium, pentobarbital and clonidine did not inhibit lidocaine metabolism over the therapeutic range in human liver microsomes. CONCLUSION: These results show that the interactions between lidocaine and midazolam and thiamylal are of potential toxicological and clinical significance.     

Clinical consequences of polymorphic drug oxidation

Many characters are genetically regulated as polymorphisms. This means that discrete groups are seen within the distribution of a certain character. Drug metabolism is no exception and the polymorphism of acetylation is recognised since the 50's. Polymorphic drug oxidation was discovered in the 70's and has been extensively studied. There are two fully established polymorphisms in drug oxidation named as the debrisoquine/sparteine and the s-mephenytoin hydroxylation polymorphisms. The metabolism of a number of important drugs cosegregates with that of debrisoquine. Among these drugs are beta-blockers, antiarrhythmics, tricyclic antidepressants and neuroleptics. Apart from accumulation of parent drug and active metabolite, also reduced formation of active metabolite occur for some drugs in slow metabolisers. There are, however, few cases where the presence of polymorphic drug metabolism is of significant disadvantage. The polymorphisms will add to variability in drug clearance but the potential clinical importance should be evaluated for each drug. The cytochrome P-450 isozyme responsible for debrisoquine hydroxylation is of high affinity-low capacity character, which means that it can be saturated under certain circumstances. This will decrease the difference in drug metabolic rate between rapid and low metabolisers as will inhibitors of the debrisoquine isozyme like cimetidine, quinidine and propafenone. The debrisoquine isozyme is not readily inducible. In cases where a major metabolic route or the formation of an active metabolite are polymorphically controlled, knowledge about a patient's oxidator status might be of practical value for dose adjustments especially if there is a narrow therapeutic ratio or an established concentration-effect relationship. For some drugs it is difficult to differentiate between insufficient therapeutic effect and symptoms of overdosage. Tricyclic antidepressants and neuroleptics meet some of these criteria and patients who get recurrent treatment may benefit if the physician has knowledge about debrisoquine metabolic phenotype. Otherwise, the clinical consequences of polymorphisms in drug oxidation seem so far to be limited, considering that a number of disease conditions have not shown any clear association with oxidation status. The polymorphisms in drug metabolism should be considered as a part of natural variability which could in fact be larger with other drugs that do not show polymorphic elimination.     

Inhibition of drug metabolism by cimetidine in man: dependence on pretreatment microsomal liver function

The effect of cimetidine on hepatic microsomal drug metabolism was studied in six patients with normal liver and eleven patients with chronic liver disease using the aminopyrine breath test. Before administration of cimetidine, the elimination rate constant of 14CO2 from breath (Kb) was 28.3 +/- SD 1.3%/h in patients with normal liver and 13.5 +/- 7.7%/h in patients with liver disease (P less than 0.001). After 7 days of cimetidine therapy (1 g/day) Kb decreased to 23.3 +/- 5.2%/h (19.0 +/- 13.8% decrease; P less than 0.05) and 7.4 +/- 5.8%/h (50.5 +/- 14.4% decrease; P less than 0.001), respectively. Plasma levels of cimetidine were not significantly different (1.05 +/- 0.14% micrograms/ml v. 0.88 +/- 0.41; P greater than 0.05). The findings indicate that therapeutic doses of cimetidine lead to an inhibition of drug metabolism which is more pronounced in patients with impaired liver function than in liver normals. Therefore, patients with chronic liver disease may be at increased risk with respect to interactions between cimetidine and other drugs which are demethylated by the liver.     

In vitro inhibitory effects of Kampo medicines on metabolic reactions catalyzed by human liver microsomes

BACKGROUND: Although it is well known that drug-drug interactions may lead to toxicity and therapeutic failure, little is known about the incidence and consequences of herb-drug interactions in patients receiving Kampo medicines. METHODS: We evaluated the frequency of the combined use of Kampo medicines and Western drugs at Osaka University Hospital, and investigated the effects of these formulae on the metabolic activity of different cytochrome P450 (CYP) isoforms using pooled microsomes obtained from human liver. RESULTS: Twenty-two Kampo formulae were used together with 40 Western drugs catalyzed by the CYP isoforms CYP3A4, CYP2C9, CYP2D6 and CYP1A2. Among the Kampo medicines, HOCHUEKKI-TO, SHOSAIKO-TO, NINJINYOUEI-TO, SAIREI-TO and KAKKON-TO were most frequently used during the study period (1996-2000). These were co-administered with 11 categories of drugs, which are substrates for CYP3A4. HOCHUEKKI-TO and SAIREI-TO were competitive inhibitors of CYP3A4 with Ki values of 0.65 and 0.1 mg/mL, respectively. HOCHUEKKI-TO, SHOSAIKO-TO and SAIREI-TO inhibited the metabolic activities of CYP2C9, but had no effect on CYP2D6. HOCHUEKKI-TO and SAIREI-TO exhibited non-competitive inhibition of the metabolic activity of CYP2C9 with a similar Ki value (0.7-0.8 mg/mL). SAIRE-TO (0.25 mg/mL) was a potent inhibitor of CYP1A2 (inhibition > 68%). CONCLUSIONS: Frequently used Kampo medicines may interact with Western drugs, which are substrates for CYP3A4, CYP2C9 and CYP1A2. Their co-administration should be undertaken with care.     

Clearance of antipyrine-dependence of quantitative liver function

Abstract. The purpose of the present study was to assess the influence of liver disease on the hepatic drug-metabolizing enzyme systems. The clearance of antipyrine was found to be significantly decreased in 13 patients with liver disease compared with 9 control patients (18.5 and 58.6 ml/min. respectively). Among the patients with liver disease, those with a severely reduced capacity for work had significantly lower clearance of antipyrine than non incapacitated patients (12.5 and 25.4 ml/min. respectively). The clearance of antipyrine was significantly correlated with the galactose elimination capacity, the serum albumin and the prothrombin values. The results indicate that the drug-metabolizing capacity of the liver changes in parallel with the other metabolic functions, and the use of the clearance of antipyrine as a quantitative test of liver function is suggested.     

Ammonia and glutamine metabolism in human liver slices: new aspects on the pathogenesis of hyperammonaemia in chronic liver disease

Ammonia and glutamine metabolism was studied in slices from normal, fatty and cirrhotic human livers. The liver disease was evaluated by histological examination. With respect to ammonia removal, urea and glutamine synthesis in human liver represent low and high affinity systems with k0.5(NH4+) values of 3.6 and 0.11 mM, respectively. Compared with normal control livers, cirrhotic livers showed a decreased glutamine synthesis from NH4Cl by about 80%. The same was true for urea synthesis. Conversely, flux through hepatic glutaminase was increased in cirrhosis 4-6-fold. These changes in hepatic glutamine and ammonia metabolism were observed regardless of whether reference was made to liver wet weight, DNA or protein content. Acetazolamide inhibited urea synthesis in cirrhotic liver slices by about 50%, indicating that mitochondrial carbonic anhydrase is required for urea synthesis also in cirrhosis. There was a significant correlation between the in-vitro determined capacity for urea synthesis from NH4Cl and the in-vivo determined plasma bicarbonate concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

载脂蛋白A5基因-3A/G多态性对血脂代谢及其相关疾病的关系探讨

目的探讨载脂蛋白A5（apo A5）基因-3A/G单核苷酸多态性（SNP）与血脂代谢及其相关疾病的关系,为临床疾病诊断和早期干预提供实验数据。方法收集232名体检健康的汉族人群及209例冠心病（CHD）、132例糖尿病（DM）、107例脑梗塞（CI）和199例高血压（HTN）患者的血样标本,通过DNA提取、聚合酶链反应（PCR）扩增和DNA测序,检测apo A5基因-3A/G SNP。同时检测三酰甘油（TG）、总胆固醇（TC）、高密度脂蛋白胆固醇（HDL-C）和低密度脂蛋白胆固醇（LDL-C）等血脂水平,分析其相互之间的关系。结果 AA纯合子占52.5%,AG杂合子占39.7%,GG纯合子占7.8%。A等位基因分布频率为72.4%,G等位基因的分布频率为27.6%。GG基因型与AA型、AG型人群比较,TG和非高密度脂蛋白胆固醇（non-HDL-C）水平增高（P〈0.05）、HDL-C水平降低（P〈0.05）。对照组与疾病组比较,TG水平在AA和AG基因型中差异有统计学意义（P〈0.05）;GG基因型中对照组TG水平与疾病组比较差异无统计学意义（P〉0.05）。结论 apo A5基因-3A/GSNP对血脂代谢相关疾病的调控主要表现为GG基因型与高TG、低HDL-C和高non-HDL-C显著相关,是导致脂质代谢紊乱的重要候选基因之一。     

Ganglioside variations in human liver cirrhosis and hepatocellular carcinoma as shown by two-dimensional thin-layer chromatography

Gangliosides isolated from 5 cases of normal liver tissues, 11 cases of liver cirrhosis and 5 cases of hepatocellular carcinoma were compared in their concentrations and compositions. Quantitative analysis revealed no significant change of ganglioside levels between normal and cirrhotic liver tissues or hepatocellular carcinoma. There was also no significant difference (p greater than 0.05) between cirrhotic liver tissues and hepatocellular carcinoma. Two dimensional thin-layer chromatography of the total ganglioside preparations of liver tissues from both liver cirrhosis and hepatocellular carcinoma showed proliferation of GM2, GD3, GD1 and at least two unidentified components, named provisionally spots Nos. 1 and 2 in the present report, and loss of GM3. Sialidase treatment and thin-layer chromatography showed the components of these spots to be sialidase-labile monosialogangliosides and distinctly different from GD3 which was described elsewhere.     

急性百草枯中毒致药物性肝病影响因素的分析

目的：观察比较分析急性百草枯中毒致药物性肝病的影响因素。方法：选择2008-01-2013-12入住我院急诊科的急性百草枯中毒患者112例,根据急性百草枯中毒致肝损伤的的诊断标准判断患者是否发生药物性肝病,分为损伤组和对照组,对患者性别、年龄、口服剂量、是否接受超早期洗胃治疗、是否接受早期血液灌流治疗、是否接受CRRT治疗等因素对百草枯所致药物性肝病的影响进行Logistic回归分析。结果：2组患者性别、年龄差异无统计学意义,Logistic回归分析发现口服剂量、是否接受超早期洗胃治疗、是否接受早期血液灌流治疗、是否接受CRRT治疗与急性百草枯患者肝损伤的发生可能存在相关性（P〈0.05）。结论：百草枯的口服剂量对肝功能损伤的影响最大,及时给予超早期洗胃治疗、早期血液灌流治疗能减少患者肝功能损伤的发生。     

Rapid high performance liquid chromatography method for erlotinib quantification in vitro: Application to study the effect of resveratrol on metabolism and cellular uptake of erlotinib

Background: Erlotinib is a selective epidermal growth factor receptor inhibitor that is used for the treatment of non-small cell lung cancer and pancreatic cancer. Its metabolism is mainly mediated by cytochrome P450 3A (CYP 3A). Resveratrol, a natural compound found in many plants and supplements, is known to inhibit CYP3A enzyme, therefore, it may act as an inhibitor for the metabolism of erlotinib. Objective: Development of a rapid high performance liquid chromatography with photodiode array detection (HPLC-PDA) method for the quantification of erlotinib in liver microsomes and cancer cells and its application to study resveratrol effect on metabolism and cellular uptake of erlotinib. Methods: HPLC-PDA was used to develop an efficient bioanalytical method with a 2.5-min runtime preceded by a simple protein precipitation step. The method was validated according to the European Medicines Agency guidelines. Erlotinib metabolic stability and resveratrol effect on erlotinib metabolite formation were evaluated in rat liver microsomes. Furthermore, the method was used to measure the intracellular concentrations of erlotinib in cancer colorectal cells and investigating resveratrol effect on the cellular uptake of erlotinib. Results: A rapid HPLC-PDA method was developed and validated for the first time to address potential drug interaction of erlotinib with resveratrol. Resveratrol was a strong inhibitor of erlotinib metabolism in vitro with IC₅₀ = 4.03 μM. Resveratrol, however, had no effect on erlotinib cellular uptake after 1 h incubation in human colorectal cancer cells. Conclusion: The study suggests that resveratrol may produce a potential herb–drug interaction with erlotinib at the metabolism level and should be investigated in patients in the clinic.     

Modulation of cytochrome P450 isozymes in human liver, by ethanol and drug intake

Cytochromes P450 (P450) are a family of isozymes which play an important role in xenobiotic metabolism. The concentration of three P450 isozymes, namely P450-IIE1(A1c),-IIIA(NF) and -IIC8-10(MP) has been measured in human liver biopsies of patients with different alcohol and drug intake status. All these three P450s were expressed in all subjects. Ethanol intake increased P450-IIE1(A1c) content with no effect on the content of the two other P450s. Drug intake (barbiturates) increased both P450-IIIA(NF) and -IIC8-10(MP) content without any effect on P450-IIE1(A1c). This paper brought, at protein level, further evidence of the importance of environmental conditions on P450 isozyme pattern, and therefore, on drug metabolizing capacity of human liver.     

Role of gastrointestinal tract and liver in acetate metabolism in rat and man

Net acetate uptake/release by various tissues was studied in vivo in fed, starved and Paromomycin-treated rats and in patients with cirrhosis of the liver. In humans the portal vein, hepatic vein and hepatic arterial blood flow rates were determined simultaneously. In rats acetate is only intestinally produced and released into the portal vein. Intestinal production is decreased by 33% in starved and Paromomycin-treated rats compared to fed animals. Portal vein hepatic vein acetate differences are linearly related to the portal vein acetate concentration (r = 0.92). Acetate uptake from the portal vein by the liver was found when the portal venous concentration exceeded 180 mumol l-1. In humans the hepatic net acetate uptake from the portal vein/net acetate release into the hepatic vein, measured as mmol min-1, is linearly related to the portal vein acetate concentration (r = 0.96). The data indicate that the liver may homeostatically regulate the systemic acetate concentration in rat and man.     

老年代谢综合征与非酒精性脂肪肝患者载脂蛋白E基因多态性检测的意义

目的探讨老年代谢综合征(MS)与非酒精性脂肪肝(NAFL)的载脂蛋白E(apo E)基因多态性之间的关系及意义。方法本研究以60岁以上农村老年健康体检者筛检出的NAFL 126例、NAFL MS 105例、非脂肪肝125例及体检正常老年者95名为研究对象,其血液应用特异引物扩增人DNA apo E基因265 bp片段和特异探针杂交后,通过分析熔解曲线检测基因突变点及多态性。酶联免疫吸附试验(ELISA)检测apo E、apo E4基因浓度,并进行血脂、血糖、尿素、酶类、体质指数和血压分析。结果NAFL组和NAFL MS组以杂合子E2/3、E3/4基因型频率最高,分布频率分别为E2/3(36.5%、28.1%)、E3/4(27.2%、32.6%)、E3/3(32.3%、34.0%)、E2/4(2.4%、1.5%)、E4/4(0.8%、3.8%)、E2/2(0.8%、0.0%);NAFL MS组E4/4频率显著高于对照组,apoE基因总浓度明显升高(P<0.05),E2/3、E3/4 2组血脂等生化指标和体质指数及血压均有不同程度增高,差异有显著性(P<0.05)。结论apo E基因多态性与老年NAFL和MS存在相关性,携带E3/4、E2/3型或E4/4型的个体存在,提示在决定个体老年MS合并NAFL遗传易感性方面有重要作用,apo E基因112位和158位的氨基酸残基发生相应的变化可引起不同程度的脂质代谢障碍。     

Effects of oxazepam and acetaminophen on cicletanine metabolism in rat hepatocytes and liver microsomes

Cicletanine, a racemic furopyridine derivative synthesized as racemate, is used as an antihypertensive agent. Its two enantiomers are involved in the pharmacological effects of the drug. Cicletanine is metabolized by conjugation enzyme systems (phase II) into sulfoconjugated or glucuroconjugated enantiomers. As oxazepam and acetaminophen are widely prescribed, especially to elderly patients, these two drugs may be co-administered with cicletanine. The metabolic profile and the kinetics of biotransformation were studied by using rat hepatocytes and liver microsomes. Cicletanine was extensively metabolized by rat hepatocytes. More than 80% of the drug was biotransformed after a 3 h incubation. The formation of glucuroconjugated metabolites was characterized by the following kinetic parameters, i.e. Vmax = 2.05 +/- 0.21 nmol/min/mg protein and Km = 287 +/- 6.7 microM for (-)-cicletanine, and Vmax = 1.44 +/- 0.12 nmol/min/mg protein and K(m) = 171 +/- 4.1 microM for (+)-cicletanine. Oxazepam inhibited the glucuronidation of cicletanine in both rat hepatocytes and liver microsomes with a competitive-type inhibition, i.e. K(i) = 129 +/- 7.5 and 152 +/- 19.7 microM for (-)-cicletanine and (+)-cicletanine, respectively. The co-incubation of acetaminophen with cicletanine showed that only sulfoconjugation was inhibited in rat hepatocytes. Glucuronidation was not modified by acetaminophen. As natriuric activity is due to sulfoconjugated (+)-cicletanine, acetaminophen could potentially modulate in vivo the pharmacological effect of cicletanine. The data of the in vitro study reported here suggested an interaction between cicletanine and oxazepam or cicletanine and acetaminophen. However, the clinical impact of such a drug interaction needs further evaluation.