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.     

In vitro effect of fluoroquinolones on theophylline metabolism in human liver microsomes.

Some quinolone antibiotics cause increases in levels of theophylline in plasma that lead to serious adverse effects. We investigated the mechanism of this interaction by developing an in vitro system of human liver microsomes. Theophylline (1,3-dimethylxanthine) was incubated with human liver microsomes in the presence of enoxacin, ciprofloxacin, norfloxacin, or ofloxacin. Theophylline, its demethylated metabolites (3-methylxanthine and 1-methylxanthine), and its hydroxylated metabolite (1,3-dimethyluric acid) were measured by high-pressure liquid chromatography, and Km and Vmax values were estimated. Enoxacin and ciprofloxacin selectively blocked the two N demethylations; they significantly inhibited the hydroxylation only at high concentrations. Norfloxacin and ofloxacin caused little or no inhibition of the three metabolites at comparable concentrations. The extent of inhibition was reproducible in five different human livers. Inhibition enzyme kinetics revealed that enoxacin caused competitive and mixed competitive types of inhibition. The oxo metabolite of enoxacin caused little inhibition of theophylline metabolism and was much less potent than the parent compound. Nonspecific inhibition of cytochrome P-450 was ruled out since erythromycin N demethylation (cytochrome P-450 mediated) was unaffected in the presence of enoxacin. These in vitro data correlate with the clinical interaction described for these quinolones and theophylline. We conclude that some quinolones are potent and selective inhibitors of specific isozymes of human cytochrome P-450 that are responsible for theophylline metabolism. This in vitro system may be useful as a model to screen similar compounds for early identification of potential drug interactions.     

Induction and inhibition of cicletanine metabolism in cultured hepatocytes and liver microsomes from rats

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. This study reports on the use of both the induction with 3-methylcholanthrene (3-MC) or phenobarbital (PB) and inhibition with selective compounds to determine and identify UGT isoenzymes involved in the metabolism of cicletanine enantiomers. PB and 3-MC both enhanced the cicletanine enantiomer glucuronidation. These two compounds being known as inducing agents of UGT2B1 and UGTIA6 isoforms, respectively, this suggests an implication of UGT2B1 and UGT1A6 isoforms in the metabolism of the two cicletanine enantiomers: ( + )-cicletanine and ( - )-cicletanine. The use of selective compounds for inhibition study evidenced, in addition to UGT2B1 and UGT1A6 isoforms, the involvement of other UGT isoforms such as UGT1A1, UGT2B7 and UGT2B15 in cicletanine metabolism.     

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.     

NADPH-dependent metabolism of estrone by human liver microsomes

We characterized the NADPH-dependent metabolism of estrone (E1) by liver microsomes of 21 male and 12 female human subjects. The structures of 11 hydroxylated or keto metabolites of E1 formed by human liver microsomes were identified by chromatographic and mass spectrometric analyses. 2-Hydroxylation of E1 was the dominant metabolic pathway with all human liver microsomes tested. E1 is more prone to form catechol estrogens (particularly 4-OH-E1) than 17beta-estradiol (E2) and the average ratio of E1 4-hydroxylation to 2-hydroxylation (0.24) was slightly higher than the ratio of E2 4- to 2-hydroxylation (0.20, P < 0.001). An unidentified monohydroxylated E1 metabolite (y-OH-E1) was found to be one of the major metabolites formed by human liver microsomes of both genders. 6beta-OH-E1, 16alpha-OH-E1, and 16beta-OH-E1 were also formed in significant quantities. 16alpha-hydroxylation was not a major pathway for E1 metabolism. The overall profiles for the E1 metabolites formed by male and female human liver microsomes were similar, and their average rates were not significantly different. Hepatic CYP3A4/5 activity in both male and female liver microsomes correlated strongly with the rates of formation of several hydroxyestrogen metabolites. The dominant role of hepatic CYP3A4 and CYP3A5 in the formation of these hydroxyestrogen metabolites was further confirmed by incubations of human CYP3A4 or CYP3A5 with [3H]E1 and NADPH. Notably, human CYP3A5 has very high relative activity for E1 4-hydroxylation, exceeding its activity for E1 2-hydroxylation by approximately 100%. It will be of interest to determine the potential biological functions associated with any of the E1 metabolites identified in our present study.     

Stereoselective metabolism of bufuralol racemate and enantiomers in human liver microsomes

A new HPLC method was developed using a chiral column to efficiently separate four 1"-hydroxybufuralol (1"-OH-BF) diastereomers that are major metabolites of bufuralol (BF). Employing this method, we examined diastereomer selectivity in the formation of 1"-OH-BF from BF racemate or enantiomers in four individual samples of human liver microsomes. Three different human liver microsomes showed a selectivity of 1"R-OH < 1"S-OH for BF enantiomers, which was similar to that of recombinant CYP2D6 expressed in insect cell microsomes, whereas one human liver microsomal fraction yielded a selectivity of 1"R-OH > 1"S-OH for BF enantiomers, which was similar to those of recombinant CYP2C19 expressed in insect cell microsomes. Recombinant CYP1A2 and CYP3A4 showed a selectivity similar to that of CYP2D6, but their BF 1"-hydroxylase activities were much lower than those of CYP2D6. In inhibition studies, quinidine, a known CYP2D6 inhibitor, markedly inhibited BF 1"-hydroxylation in the fractions of human liver microsomes that showed the CYP2D6-type selectivity. Furthermore, omeprazole, a known CYP2C19 inhibitor, efficiently suppressed the formation of 1"-OH-BF diastereomers from BF in the microsomal fraction that showed the CYP2C19-type selectivity. From these results, we concluded that the diastereomer selectivity in the formation of 1"-OH-BF from BF differs between CYP2D6 and CYP2C19, both of which can be determinant enzymes in the diastereoselective 1"-hydroxylation of BF in human liver microsomes.     

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.     

Voriconazole inhibition of the metabolism of tacrolimus in a liver transplant recipient and in human liver microsomes

The purpose of this study was to assess the effect of voriconazole on the blood tacrolimus concentration in a liver transplant recipient and to examine the interaction between voriconazole and tacrolimus by using human liver microsomes. Two subjects were enrolled in the clinical study: one received voriconazole, and the other received a placebo. Tacrolimus metabolism was evaluated in human liver microsomes at various concentrations in the absence and presence of various concentrations of voriconazole. Coadministration of voriconazole and tacrolimus resulted in elevated (nearly 10-fold-higher) trough tacrolimus blood concentrations in the liver transplant patient. In the in vitro study, voriconazole at a concentration of 10.4 +/- 4.3 micro g/ml inhibited the metabolism of tacrolimus by 50%. Clinically relevant concentrations of voriconazole inhibited the metabolism of tacrolimus in human liver microsomes. Close monitoring of the blood concentration and adjustment in the dose of tacrolimus are warranted in transplant recipients treated with voriconazole.     

Effect of age on in vitro triazolam biotransformation in male human liver microsomes

We studied age-related changes in enzyme kinetic parameters in human liver microsomes (HLMs) in vitro, using triazolam (TRZ), an index of CYP3A activity. HLMs were prepared from male livers from four age groups, n = 5 per group: A (14-20 years), B (21-40 years), C (41-60 years), and D (61-72 years). Mean V(max) values in groups B and C for both 1-hydroxytriazolam (1-OH-TRZ) and 4-hydroxy-triazolam (4-OH-TRZ) formation were significantly greater as compared with groups A and D individually, as well as the net intrinsic clearance (sum of the two pathways). The mean net intrinsic clearance (Cl(int)) values were 25.2, 89.8, 78, and 20.6 nl/min/mg protein in A, B, C, and D, respectively. TRZ Cl(int) correlated well with total CYP3A content (r(s) = 0.84; P < 0.0001). Testosterone (TST) inhibited 1-OH-TRZ formation and activated 4-OH-TRZ formation in all age groups, with no significant differences among the groups; this suggests that the drug-drug interaction potential using TRZ and TST as index CYP3A substrates may not change with age. Reduced V(max) and Cl(int) for TRZ hydroxylation and CYP3A protein in livers from elderly men suggest reduced CYP3A gene expression in this group.     

用新生大鼠肝细胞体外构建工程化肝组织

目的:在供体肝短缺的情况下,构建可植入的工程化肝组织具有重要的临床意义.实验尝试以新生大鼠肝细胞为种子细胞体外构建工程化肝组织,以期进一步的体内植入.方法:实验于2007-04/08在解放军总医院普通外科研究所完成.①实验材料:SPF级、出生24 h以内的雄性SD新生大鼠.②实验过程:采用胰酶消化法获取新生大鼠肝细胞;以2×L-DMEM液(添加地塞米松10 μg/L、表皮生长因子20 μg/L、肝细胞生长因子40 μg/L及胰岛素0.04 U/mL)与液态鼠尾胶原等比例混合;再将肝细胞与胶原凝胶复合构建细胞/凝胶复合物,接种于培养板进行培养.③实验评估:培养后第1,3,5,7,9天,采用相差显微镜观察、四甲基偶氮唑盐比色法、苏木精-伊红染色和免疫组织化学染色分别对工程化组织的生长情况及组织形态特征进行观察,并对工程化组织的白蛋白合成功能及尿素的代谢水平进行评价.结果:①肝细胞与胶原凝胶复合后,细胞均匀的分布在复合物中.在整个培养过程中,肝细胞保持着稳定的细胞形态.②四甲基偶氮唑盐检测显示肝细胞活性在生长初期平缓下降,直至第5天时仍然保持75%的细胞活性,之后快速下降.③体外培养5 d后,相差显微镜下观察显示肝细胞在胶原凝胶中呈三维立体生长,并保持肝细胞胞体圆形,核大而圆的特异形态;免疫组织化学证实这些肝细胞抗白蛋白抗体染色呈强阳性.④对培养上清中白蛋白和尿素的含量测定表明胶原凝胶中的肝细胞在培养初期保持着稳定的代谢合成功能.结论:用新生大鼠肝细胞及胶原构建出一种有功能的工程化肝组织模型,这种模型可以应用于今后的工程化肝组织研究.     

Lidocaine metabolism in human liver microsomes by cytochrome P450IIIA4

The metabolism of lidocaine to its major metabolite monoethylglycinexylidide (MEGX) was studied in human liver microsomes of 13 kidney transplant donors and of one patient with liver cirrhosis. Interindividual variation in metabolite formation was considerable. Biphasic kinetics indicated the involvement of at least two distinct enzymatic activities. With use of a series of antisera that recognize different human cytochrome P450 isozymes, we were able to identify an enzyme of the P450III gene family as one of two enzymes. By expressing human P450IIIA4 complementary deoxyribonucleic acid (cDNA) in HepG2 cells, we directly demonstrated lidocaine-deethylase activity for this P450 isozyme. These data suggest that P450IIIA4 is at least in part responsible for microsomal MEGX formation.     

Effects of MCPA and other phenoxyacid compounds on hepatic xenobiotic metabolism in rats.

The effects of ethyl 4-chloro-2-methylphenoxyacetate (MCPA) and other phenoxyacid compounds on hepatic xenobiotic metabolizing enzymes were studied in male rats. These compounds were administered orally 200 mg/kg/day to the rats for 2 weeks. Both MCPA and clofibrate increased the hepatic level of cytochrome P-450. In the MCPA-treated group, the activities of aniline hydroxylase and 7-ethoxycoumarin O-deethylase increased by 15% and 1.5-fold, respectively. The free acid form of MCPA increased these activities more potently than MCPA. Both MCPA and its free acid did not change the activity of aminopyrine N-demethylase. A marked increase in the activity of aniline hydroxylase was noted in the 2,4-dichlorophenoxyacetic acid-treated group, whereas the aminopyrine N-demethylase activity significantly decreased in the same group. Clofibrate also increased the activities of hepatic microsomal cytochrome P-450-mediated oxidation tested, but to a lesser extent when compared with the effects of MCPA. These results indicate that MCPA may have a potent effect on the hepatic metabolizing enzymes in rats, and also that the induction of xenobiotic metabolizing enzymes may change when the chemical moiety of phenoxyacid compounds is modified.     

Effect of CYP3A5 expression on vincristine metabolism with human liver microsomes

Vincristine is preferentially metabolized to a secondary amine, M1, by CYP3A5 with a 9- to 14-fold higher intrinsic clearance than CYP3A4 using cDNA-expressed enzymes. The genetically polymorphic expression of CYP3A5 may contribute to interindividual variability in vincristine efficacy and toxicity. The current study quantifies the contribution of cytochromes P450 (P450s), including CYP3A4 and CYP3A5, to vincristine metabolism with a bank of human liver microsomes (HLMs). M1 was the major metabolite formed with HLMs, and selective chemical inhibition of P450s confirmed that CYP3A was the major metabolizing subfamily. The liver tissues were genotyped for low expression alleles, CYP3A5*3,*6, and *7, and the HLMs were phenotyped for CYP3A4 and CYP3A5 expression by Western blot. Testosterone 6beta-hydroxylation and itraconazole hydroxylation were used to quantify CYP3A4 activity in the HLMs. For each CYP3A5 high expresser (n=10), the rate of M1 formation from vincristine due to CYP3A5 was quantified by subtracting the CYP3A4 contribution as determined by linear regression with CYP3A5*3/*3 samples. For CYP3A5 high expressers, the contribution of CYP3A5 to the metabolism of vincristine was 54 to 95% of the total activity, and the rate of M1 formation mediated by CYP3A5 correlated with CYP3A5 protein content (r2=0.95). Selective inhibition of CYP3A4 demonstrated that the M1 formation rate with CYP3A5 high expressers was differentially inhibited based on CYP3A4 activity. Using median values, the estimated hepatic clearances were 5-fold higher for CYP3A5 high expressers than low expressers. We conclude that polymorphic expression of CYP3A5 may be a major determinant in the P450-mediated clearance of vincristine.     

Effects of age on in vitro midazolam biotransformation in male CD-1 mouse liver microsomes

To study age-related changes in drug metabolism, we examined the in vitro biotransformation of midazolam (MDZ), a human cytochrome P-450 (CYP) 3A substrate, using liver microsomes from three age groups of male CD-1 mice ranging from 6 weeks to 2 years old. MDZ was metabolized to two major products, alpha-OH- and 4-OH-MDZ, which were quantified by HPLC. For both metabolites, V(max) values were reduced in old livers (P <.05), while K(m) values did not change with age. The net intrinsic clearance (the sum of V(max)/K(m) for both pathways) also was reduced in the old animals (P <.05). The capacity of ketoconazole, a CYP3A inhibitor in humans, to inhibit the biotransformation of MDZ and of alprazolam, another human CYP3A substrate, did not differ significantly with age. At 100 microM alprazolam, 0.5 microM ketoconazole inhibited metabolite formation by >80%. At 30 microM MDZ, 2.5 microM ketoconazole impaired 4-OH-MDZ formation by 88%, whereas it reduced alpha-OH-MDZ formation by only 46%. Immunoinhibition studies with polyclonal anti-rat CYP3A1/2 and CYP2C11 antibodies confirmed that 4-OH-MDZ formation was largely CYP3A-dependent, while alpha-OH-MDZ formation was mediated by CYP3A and -2C isoforms. Western blot analysis revealed decreased microsomal content of CYP3A in old livers. Net intrinsic clearance of MDZ was correlated with total CYP3A content (P <.001). These results demonstrate a reduction in MDZ biotransformation in old male mice, which may be attributable, in part, to decreased CYP3A content in old livers. Changes in expression and activity of CYP2C isoforms also may contribute to age-related changes in MDZ biotransformation, but this requires more investigation.     

模拟构建人肝衰竭血清对体外培养肝细胞功能的影响

背景:生物人工肝核心材料为肝细胞,其功能好坏直接影响临床效果.目的:模拟构建一种能够和肝衰竭血清对体外培养肝细胞具有相同生物学效应的组合物.方法:观察和对比模拟肝衰竭血清、人肝衰竭血清和体积分数10%胎牛血清对培养的 CL-1 细胞形态学和细胞活力的变化,检测 CL-1 细胞酶包括乳酸脱氢酶、谷丙转氨酶、谷草转氨酶及谷胱甘肽的分泌量.结果与结论:模拟肝衰竭血清和人肝衰竭血清对培养的 CL-1 细胞活力,乳酸脱氢酶、谷丙转氨酶、谷草转氨酶和谷胱甘肽的分泌量的影响差异无显著性意义(P>0.05),CL-1细胞的生长均受到抑制,体积分数10%胎牛血清细胞功能良好.结果提示,模拟肝衰竭血清组合物与人肝衰竭血清对CL-1细胞的生物学效应基本相同.