Characterization of human liver cytochromes P-450 involved in theophylline metabolism.

Theophylline is metabolized in the liver by one or more cytochrome P-450 enzymes. To assess the amounts and types of these human cytochromes P-450, we incubated theophylline with microsomes prepared from 22 different human livers in the presence of NADPH, and measured simultaneous rates of 1- and 3-N-demethylations to 3-methylxanthine (3-MX) and 1-methylxanthine (1-MX), respectively; and 8-hydroxylation to 1,3-dimethyluric acid (1,3-DMU). Under optimal conditions, 3-MX, 1-MX, and 1,3-DMU formation proceeded with mean Km values of 2.05, 1.93, and 5.34 mM and Vmax values of 2.28, 2.48, and 23.4 pmol/mg/min, respectively. Formation of 3-MX and 1-MX correlated best with amounts of the immunoreactive protein HLd (P-450IA2) (p less than 0.05), whereas formation of 1,3-DMU correlated with the microsomal content of HLp (P-450IIIA3) and HLj (P-450IIE1). In immunoinhibition experiments, incubations conducted with a polyclonal anti-rat P-450c/d antibody, the formation of all the three theophylline metabolites (p less than 0.05) was significantly inhibited. However, addition of isoform-specific anti-rat-P-450d antibodies to the microsomal mixture significantly inhibited 1-N-demethylation, selectively, with little (if any) inhibition of 3-N-demethylation or 8-hydroxylation. Nonspecific cytochrome P-450 inhibition was ruled out by showing that erythromycin N-demethylation, an activity catalyzed by HLp, was unaffected by either anti-P-450c/d (P-450IA1/IA2) or anti-P-450d. Anti-rat-P-450p antibodies failed to block formation of theophylline metabolism, but did inhibit erythromycin N-demethylase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of decursinol angelate on the pharmacokinetics of theophylline and its metabolites in rats

Herb-drug interactions represent a serious problem as herbal medicine is used extensively in the modern world. This study investigated the effects of decursinol angelate on the pharmacokinetics of theophylline, a typical substrate of the cytochrome P450 1A2 enzyme, in rats. After 3 days of decursinol angelate pretreatment, on the fourth day, rats were administered decursinol angelate and theophylline concomitantly. Blood theophylline and its major metabolite [1-methylxanthine (1-MX), 3-methylxanthine (3-MX), 1-methyluric acid (1-MU), and 1,3-dimethyluric acid (1,3-DMU)] levels were monitored by liquid chromatography-tandem mass spectroscopy. The results indicated that theophylline clearance significantly decreased and the area under the concentration–time curve (AUC) increased in decursinol angelate (25 mg/kg)-pretreated rats administered theophylline (10 mg/kg). The elimination half-life (t1/2) of theophylline was increased by 20%. In the presence of decursinol angelate (25 mg/kg), the pharmacokinetic parameters of three metabolites (1-MX, 1,3-DMU, and 1-MU) were significantly altered (half-life for 1-MU, and AUC24 h for 1-MX, 1,3-DMU, and 1-MU). Our results suggest that patients receiving CYP1A2-metabolized drugs, such as caffeine and theophylline, should be advised of the potential herb-drug interaction to reduce the risk of therapeutic failure or increased toxicity of conventional drug therapy.     

Cytochrome P450 isoform selectivity in human hepatic theobromine metabolism

AIMS: The plasma clearance of theobromine (TB; 3,7-dimethylxanthine) is known to be induced in cigarette smokers. To determine whether TB may serve as a model substrate for cytochrome P450 (CYP) 1A2, or possibly other isoforms, studies were undertaken to identify the individual human liver microsomal CYP isoforms responsible for the conversion of TB to its primary metabolites. METHODS: The kinetics of formation of the primary TB metabolites 3-methylxanthine (3-MX), 7-methylxanthine (7-MX) and 3,7-dimethyluric acid (3,7-DMU) by human liver microsomes were characterized using a specific hplc procedure. Effects of CYP isoform-selective xenobiotic inhibitor/substrate probes on each pathway were determined and confirmatory studies with recombinant enzymes were performed to define the contribution of individual isoforms to 3-MX, 7-MX and 3,7-DMU formation. RESULTS: The CYP1A2 inhibitor furafylline variably inhibited (0-65%) 7-MX formation, but had no effect on other pathways. Diethyldithiocarbamate and 4-nitrophenol, probes for CYP2E1, inhibited the formation of 3-MX, 7-MX and 3,7-DMU by approximately 55-60%, 35-55% and 85%, respectively. Consistent with the microsomal studies, recombinant CYP1A2 and CYP2E1 exhibited similar apparent Km values for 7-MX formation and CYP2E1 was further shown to have the capacity to convert TB to both 3-MX and 3,7-DMU. CONCLUSIONS: Given the contribution of multiple isoforms to 3-MX and 7-MX formation and the negligible formation of 3,7-DMU in vivo, TB is of little value as a CYP isoform-selective substrate in humans.     

反相HPLC法同时测定人尿中茶碱及其两种代谢产物

目的:建立一种同时测定人尿中茶碱及其1,3-二甲基尿酸(1,3-DMU)和3-甲基噻嗪(3-MX)代谢产物的HPLC方法.方法:尿样用异丙醇/二氯甲烷(2/8)混合液提取,有机相在空气吹干,用流动相复溶后进行HPLC分析.色谱柱为Diamonsil ODS C_(18)5 μm,150 mm×4.6 mm I.D),流动相由0.1%甲酸液和乙腈(95:5)组成,流速1.0 mL/min,测定波长280 nm.测定12名受试者单剂量和多剂量口服茶碱后24 h内尿中茶碱及其代谢物累计排泄量.结果:尿中茶碱及其代谢物1,3 DMU和3-MX的线性范围分别为0.312～40.0、0.156～20.0、0.078～10.μg/mL,最低可定量浓度分别为0.312、0.156、0.078μg/mL.批间和批内的变异小于15%,回收率大于70%.结论:该方法的特异性、灵敏度能够满足临床上对人尿中茶碱及其代谢产物同时测定的要求.     

Renal clearance of theophylline and its major metabolites: Age and urine flow dependency in paediatric patients

Summary The renal clearance of theophylline (TH) and its metabolites 1,3-dimethyluric acid (1,3-DMU), 3-methylxanthine (3-MX), and 1-methyluric acid (1-MU) has been studied in 10 children aged 8 months to 14 years. Individual renal clearances were calculated from serum levels and amounts excreted in urine after i.v. administration of the parent drug. The clearance of 1,3-DMU was found to depend both upon urine flow rate and age, which are interrelated. An effect of urinary pH was expected, but was not studied. Consistent age-dependent changes in the relative quantities of metabolites excreted were not observed.     

Effect of fluconazole on theophylline disposition in humans

The effect of fluconazole, an antimycotic that inhibits cytochrome P-450-mediated drug metabolism, on theophylline kinetics and the production of its metabolites were compared with those of enoxacin in 5 healthy subjects. All subjects received a single oral dose of 240 mg theophylline (aminophylline, 300 mg) after they had been given oral fluconazole 100 mg every 12 h or enoxacin 200 mg every 8 h for three consecutive days.Pretreatment with enoxacin decreased the total clearance (CL_T) and elimination rate constant (K_el) of theophylline by 50% and 46%, respectively, without changing the volume of distribution (V_d), but there were no significant change in any pharmacokinetic parameter when fluconazole was administered. Enoxacin led to a 50% reduction in the metabolic clearance (CL_M) of theophylline and to decreases of 69%, 59% and 38% in the formation clearance of the three theophylline metabolites, 3-methylxanthine (3-MX), 1-methyluric acid (1-MU), and 1,3-dimethyluric acid (1,3-DMU), respectively, accompanied by significant changes in the urinary recovery of theophylline and its metabolites. In contrast, treatment with fluconazole led only to a slight decrease in the CL_M of theophylline (16%) and in the formation clearance of its metabolites (15%–18%), and there was no change in the renal clearance (CL_R) of theophylline.The results indicate that fluconazole is a minor inhibitor of theophylline disposition compared with enoxacin, and they suggest that the inhibitory action of fluconazole is selective for certain cytochrome P-450 isozymes, but not for the cytochrome P-4501A involved in theophylline metabolism.     

Caffeine and theophylline metabolism in newborn and adult human hepatocytes; comparison with adult rat hepatocytes

Cultured hepatocytes from newborn human (three samples), adult human (eight samples) and adult rat livers were used to study the metabolism of theophylline and caffeine, two drugs of which the metabolic pathways are known to be cytochrome P-450-dependent. Known metabolic pathways of caffeine in vivo were qualitatively maintained. However, only the primary metabolites were formed through oxidative N-demethylation giving theophylline, paraxanthine and theobromine and, through C-8 hydroxylation, giving 1,3,7-trimethyluric acid and a ring-opened compound the 6-amino-5[N-formylmethylamino]1,3-dimethyl uracil. The ratio of the three dimethylxanthine metabolites was dependent upon the species (human, rat), development stage (newborn, adult) and environmental factors. Similarly, theophylline was metabolized as in vivo by the demethylation pathway giving, preferentially, 3-methylxanthine and not 1-methylxanthine, and by a C-8 oxidation giving 1,3-dimethyluric acid. In newborn hepatocytes, all pathways were absent except the well-known methylation to caffeine. Moreover, such a methylation also occurred in adult human hepatocytes. This result was explained by the very low metabolic capacity of cultured cells, allowing the detection of only direct metabolites. Indeed, the overall biotransformation of both the methylxanthines by primary cultures of hepatocytes was remarkably weak, confirming previous studies with liver microsomal incubations. Thus the metabolism rate did not exceed about 30 nmoles/10(6) cells/24 hr in human adults, except for two subjects which were characterized by an extensive metabolism and a different metabolic profile. These two subjects were probably induced in vivo by environmental compounds. Both quantitative and qualitative data obtained from this study were roughly correlated with other in vivo and in vitro studies. Overall the experimental model of cultured human hepatocytes was shown to be capable of assessing the metabolic profile of two methylxanthines which is in agreement with the situation encountered in vivo. This example suggests that a breakthrough may be brought in new drugs development by the predictability from human hepatocyte culture model to the in vivo human situation.     

Effects of water deprivation on the pharmacokinetics of theophylline and one of its metabolites, 1,3-dimethyluric acid, after intravenous and oral administration of aminophylline to rats

It has been reported that the expressions of hepatic microsomal cytochrome P450 (CYP) 1A1/2, 2B1/2 and 3A1/2 were not changed in rats with water deprivation for 72 h (rat model of dehydration) compared with the controls. It has been also reported that 1,3-dimethyluric acid (1,3-DMU) was formed from theophylline via CYP1A1/2 in rats. Hence, it could be expected that the formation of 1,3-DMU could be comparable between the two groups of rats. As expected, after both intravenous and oral administration of theophylline at a dose of 5 mg/kg to the rat model of dehydration, the AUC of 1,3-DMU was comparable to the controls. After both intravenous and oral administration of theophylline to the rat model of dehydration, the Cl(r) of both theophylline and 1,3-DMU was significantly slower than the controls. This could be due to significantly smaller urinary excretions of both theophylline and 1,3-DMU since the AUC of both theophylline and 1,3-DMU were comparable between the two groups of rats. The smaller urinary excretion of both theophylline and 1,3-DMU could be due to urine flow rate-dependent timed-interval renal clearance of both theophylline and 1,3-DMU in rats.     

Effects of lansoprazole on pharmacokinetics and metabolism of theophylline

The effect of the new substituted benzimidazole proton pump inhibitor, lansoprazole, on pharmacokinetics and metabolism of theophylline has been studied in healthy adults given oral lansoprazole 30 mg once daily for 11 days. On Days 4 and 11 of 300 mg aminophylline was simultaneously administered orally and blood samples for theophylline analysis were taken over 24 h. Urine samples were collected for up to 24 h and were assayed for theophylline and its major metabolites 1,3-dimethyluric acid (1,3-DMU), 1-methyluric acid (1-MU) and 3-methylxanthine (3-MX). The pharmacokinetic parameters of theophylline were determined, and the urinary recovery of unchanged theophylline and its major metabolites were calculated.After administration of lansoprazole for 4 days, no significant alteration in the terminal elimination half-life (t _1/2) or the mean residence time (MRT) was detected. However, there was a significant decrease of about 13% in the area under the plasma concentration-time curve (AUC) and a significant increase of about 19% in the apparent clearance (CL_app). Lansoprazole treatment for 11 days caused a significant decrease of approximately 12% in t _1/2 and about 10% in the MRT of theophylline, although neither AUC nor CL_app showed a significant alteration. The excretion of 3-MX in the urine was significantly increased by about 20% after lansoprazole treatment for 4 and 11 days, although there was no significant alteration in the excretion of unchanged theophylline, 1,3-DMU or 1-MU.The results indicate that repeated administration of lansoprazole to humans induces the hepatic microsomal P-450-dependent drug oxidation system that mediates N-1-demethylation of theophylline, consequently increasing its metabolism.     

Effect of inducers of cytochrome P-450 on the metabolism of [3-14C]coumarin by rat hepatic microsomes

1. The metabolism of [3-14C]coumarin has been studied in rat hepatic microsomes and with two purified cytochrome P-450 isoenzymes. 2. [3-14C]Coumarin was converted by liver microsomes to several polar products including 3- and/or 5-hydroxycoumarin, omicron-hydroxyphenylacetic acid and a major unidentified novel coumarin metabolite. 3. [3-14C]Coumarin was also converted to reactive metabolite(s) as indicated by covalent binding to proteins, and by the depletion of reduced glutathione added to the microsomal incubations. 4. [3-14C]Coumarin metabolism to polar and covalently bound metabolites by rat liver microsomes was induced by pretreatment with phenobarbitone, 3-methylcholanthrene, beta-naphthoflavone, Aroclor 1254 and isosafrole; but not by dexamethasone or nafenopin. 5. The profile of [3-14C]coumarin metabolism to polar products was similar in control and pretreated liver microsomes and in incubations with purified cytochrome P450 IA1 and P450 IIB1 isoenzymes. 6. The results indicate that coumarin is a substrate for isoenzymes of the cytochrome P450 IA and P450 IIB subfamilies. The bioactivation of coumarin by rat hepatic microsomes is postulated to result in the formation of a coumarin 3,4-epoxide intermediate which may rearrange to 3-hydroxycoumarin, be further metabolized to a coumarin 3,4-dihydrodiol, or form a glutathione conjugate.     

Dose dependent pharmacokinetics of theophylline: Michaelis-Menten parameters for its major metabolic pathways.

Dose Dependency for pharmacokinetics of theophylline and the formation of its major metabolites, 3-methylxanthine (3-MX); 1-methyluric acid (1-MU); 1,3-dimethyluric acid (DMU), were examined by administering three single oral doses (250, 375, 500 mg) of theophylline to six healthy adult volunteers. The serum and urine concentrations of theophylline and the metabolites in serum and urine were determined by high-performance liquid chromatography. Total clearance of theophylline decreased and its half life increased over the range of doses administered (p<0.01). There was a significant dose related decrease in the fractional recovery of 3-MX and 1-MU (p<0.001) and a dose related increase in fractional excretion of DMU and unchanged theophylline (p<0.01 and p<0.001 respectively). No significant dose related changes were observed in the renal clearance of 3-MX, 1-MU and DMU, indicating linear urinary excretion kinetics of the metabolites. Theophylline metabolic clearance to 3-MX as well as to 1-MU decreased with increasing dose but clearance to DMU remained unnaffected by the size of dose. The individual Michaelis-Menten parameters Km and Vmax were estimated for six subjects receiving three different single doses. The Km values for theophylline metabolism to 3-MX, 1-MU and DMU were 2.4+/-0.6, 5.1+/-1.8+/- and 112.3+/-36.8 mg/L respectively and the Vmax values were 3.5+/-0.7, 7.5+/-2.6 and 112.3+/-36.8 mg/hr respectively. The Km values for the N-demethylation pathways (3MX and 1-MU) were lower corresponding to therapeutic serum concentrations of drug. These results suggest that the elimination kinetics of theophylline is nonlinear in the human in the therapeutic range of serum concenntrations and can be explained by saturable formation kinetics of 3-MX and 1-MU. In contrast to previous studies we didn't find obvious indication for nonlinear formation of DMU at therapeutic concentration range.     

8例中国病人茶碱代谢物的药物动力学

目的：研究病人常规剂量茶碱治疗后代谢物的动力学。方法：病人静滴茶碱（６．６μｍｏｌ·ｋｇ＾－１）。ＨＰＬＣ法测定给药前后２４ｈ茶碱及其代谢物：１，３－二甲基尿酸（ＤＭＵＡ），３－甲基黄嘌呤（３－ＭＸ），１－甲基尿酸（ＭＵＡ），中间代谢产物１－甲基黄嘌呤（１－ＭＸ）的浓度。结果：ＤＭＵＡ是代谢物中浓度最高的。３－ＭＸ的清除速率最低。１－ＭＸ很快转化成ＭＵＡ，体内浓度很低，但是，翌晨，１－ＭＸ又回升到     

Theophylline steady state pharmacokinetics is not altered by omeprazole

Summary The effect of omeprazole treatment on theophylline pharmacokinetics was studied in eight, non-smoking healthy male volunteers during repeated administration of a slow release formulation of theophylline. In a randomized double-blind cross-over study, the subjects received theophylline 5 mg·kg^–1 per day with omeprazole 20 mg per day or identical placebo during two periods, each of 7 days, separated by a washout period of 7 days.The oral clearance of theophylline remained unchanged whether it was administered alone or with omeprazole (54.2 ml·min^–1). The average urinary excretion of theophylline and its metabolites, 1,3 dimethyluric acid (1,3-DMU), 3-methylxanthine (3-MX), 1-methyluric acid (1-MU) amounted to 9%, 32%, 12% and 22% of the administered dose, respectively, and no significant change occured during concomitant treatment with omeprazole.Thus, the formation and clearance of the metabolites was not altered by omeprazole. Consequently, omeprazole in the recommended dose of 20 mg daily can safely be administered to patients on theophylline therapy.     

Characterization of triptolide hydroxylation by cytochrome P450 in human and rat liver microsomes

Triptolide, the primary active component of a traditional Chinese medicine Tripterygium wilfordii Hook F, has a wide range of pharmacological activities. In the present study, the metabolism of triptolide by cytochrome P450s was investigated in human and rat liver microsomes. Triptolide was converted to four metabolites (M-1, M-2, M-3, and M-4) in rat liver microsomes and three (M-2, M-3, and M-4) in human liver microsomes. All the products were identified as mono-hydroxylated triptolides by liquid chromatography-mass spectrometry (LC-MS). The studies with chemical selective inhibitors, complementary DNA-expressed human cytochrome P450s, correlation analysis, and enzyme kinetics were also conducted. The results demonstrate that CYP3A4 and CYP2C19 could be involved in the metabolism of triptolide in human liver, and that CYP3A4 was the primary isoform responsible for its hydroxylation.     

Characterization of triptolide hydroxylation by cytochrome P450 in human and rat liver microsomes

Triptolide, the primary active component of a traditional Chinese medicine Tripterygium wilfordii Hook F, has a wide range of pharmacological activities. In the present study, the metabolism of triptolide by cytochrome P450s was investigated in human and rat liver microsomes. Triptolide was converted to four metabolites (M-1, M-2, M-3, and M-4) in rat liver microsomes and three (M-2, M-3, and M-4) in human liver microsomes. All the products were identified as mono-hydroxylated triptolides by liquid chromatography-mass spectrometry (LC-MS). The studies with chemical selective inhibitors, complementary DNA-expressed human cytochrome P450s, correlation analysis, and enzyme kinetics were also conducted. The results demonstrate that CYP3A4 and CYP2C19 could be involved in the metabolism of triptolide in human liver, and that CYP3A4 was the primary isoform responsible for its hydroxylation.     

Selective serotonin reuptake inhibitors and theophylline metabolism in human liver microsomes: potent inhibition by fluvoxamine.

1. Fluvoxamine and seven other selective serotonin reuptake inhibitors (SRRI) were tested for their ability to inhibit a number of human cytochrome P450 isoforms (CYPs). 2. None of the drugs showed potent inhibition of CYP2A6 (coumarin 7-hydroxylase) or CYP2E1 (chlorzoxazone 6-hydroxylase), while norfluoxetine was the only potent inhibitor of CYP3A having IC50 values of 11 microM and 19 microM for testosterone 6 beta-hydroxylase and cortisol 6 beta-hydroxylase, respectively. 3. Norfluoxetine, sertraline and fluvoxamine inhibited CYP1A1 (7-ethoxyresorufin O-deethylase) in microsomes from human placenta (IC50 values 29 microM, 35 microM and 80 microM, respectively). Fluvoxamine was a potent inhibitor of CYP1A2-mediated 7-ethoxyresorufin O-deethylase activity (IC50 = 0.3 microM) in human liver. 4. In microsomes from three human livers fluvoxamine potently inhibited all pathways of theophylline biotransformation, the apparent inhibitor constant, Ki, was 0.07-0.13 microM, 0.05-0.10 microM and 0.16-0.29 microM for inhibition of 1-methylxanthine, 3-methylxanthine and 1,3-dimethyluric acid formation, respectively. Seven other SSRIs showed either weak or no inhibition of theophylline metabolism. 5. Ethanol inhibited the formation of 1,3-dimethyluric acid with K(i) value of 300 microM, a value which is consistent with inhibition of CYP2E1. Ethanol and fluvoxamine both inhibited 8-hydroxylation by about 45% and, in combination, the compounds decreased the formation of 1,3-dimethyluric acid by 90%, indicating that CYP1A2 and CYP2E1 are equally important isoforms for the 8-hydroxylation of theophylline. 6. It is concluded that pharmacokinetic interaction between fluvoxamine and theophylline is due to potent inhibition of CYP1A2.     

Contribution of CYP3A4, CYP2B6, and CYP2C9 isoforms to N-demethylation of ketamine in human liver microsomes.

Ketamine is a widely used drug for its anesthetic and analgesic properties; it is also considered as a drug of abuse, as many cases of ketamine illegal consumption were reported. Ketamine is N-demethylated by liver microsomal cytochrome P450 into norketamine. The identification of the enzymes responsible for ketamine metabolism is of great importance in clinical practice. In the present study, we investigated the metabolism of ketamine in human liver microsomes at clinically relevant concentrations. Liver to plasma concentration ratio of ketamine was taken into consideration. Pooled human liver microsomes and human lymphoblast-expressed P450 isoforms were used. N-demethylation of ketamine was correlated with nifedipine oxidase activity (CYP3A4-specific marker reaction), and it was also correlated with S-mephenytoin N-demethylase activity (CYP2B6-specific marker reaction). Orphenadrine, a specific inhibitor to CYP2B6, and ketoconazole, a specific inhibitor to CYP3A4, inhibited the N-demethylation of ketamine in human liver microsomes. In human lymphoblast-expressed P450, the activities of CYP2B6 were higher than those of CYP3A4 and CYP2C9 at three concentrations of ketamine, 0.005, 0.05, and 0.5 mM. When these results were extrapolated using the average relative content of these P450 isoforms in human liver, CYP3A4 was the major enzyme involved in ketamine N-demethylation. The present study demonstrates that CYP3A4 is the principal enzyme responsible for ketamine N-demethylation in human liver microsomes and that CYP2B6 and CYP2C9 have a minor contribution to ketamine N-demethylation at therapeutic concentrations of the drug.     

Characterization of the cytochrome P450 enzymes involved in the in vitro metabolism of rosiglitazone

AIMS: To identify the human cytochrome P450 enzyme(s) involved in the in vitro metabolism of rosiglitazone, a potential oral antidiabetic agent for the treatment of type 2 diabetes-mellitus. Method The specific P450 enzymes involved in the metabolism of rosiglitazone were determined by a combination of three approaches; multiple regression analysis of the rates of metabolism of rosiglitazone in human liver microsomes against selective P450 substrates, the effect of selective chemical inhibitors on rosiglitazone metabolism and the capability of expressed P450 enzymes to mediate the major metabolic routes of rosiglitazone metabolism. Result The major products of metabolism following incubation of rosiglitazone with human liver microsomes were para-hydroxy and N-desmethyl rosiglitazone. The rate of formation varied over 38-fold in the 47 human livers investigated and correlated with paclitaxel 6alpha-hydroxylation (P<0.001). Formation of these metabolites was inhibited significantly (>50%) by 13-cis retinoic acid, a CYP2C8 inhibitor, but not by furafylline, quinidine or ketoconazole. In addition, both metabolites were produced by microsomes derived from a cell line transfected with human CYP2C8 cDNA. There was some evidence for CYP2C9 playing a minor role in the metabolism of rosiglitazone. Sulphaphenazole caused limited inhibition (<30%) of both pathways in human liver microsomes and microsomes from cells transfected with CYP2C9 cDNA were able to mediate the metabolism of rosiglitazone, in particular the N-demethylation pathway, albeit at a much slower rate than CYP2C8. Rosiglitazone caused moderate inhibition of paclitaxel 6alpha-hydroxylase activity (CYP2C8; IC50=18 microm ), weak inhibition of tolbutamide hydroxylase activity (CYP2C9; IC50=50 microm ), but caused no marked inhibition of the other cytochrome P450 activities investigated (CYP1A2, 2A6, 2C9, 2C19, 2D6, 2E1, 3A and 4A). Conclusion CYP2C8 is primarily responsible for the hydroxylation and N-demethylation of rosiglitazone in human liver; with minor contributions from CYP2C9.     

Metabolism of coumarin by rat, gerbil and human liver microsomes.

1. o-Hydroxyphenylacetaldehyde was the major metabolite of coumarin (1 mM) in rat, gerbil and human liver microsomes. 2. Treatment of rats with phenobarbitone (PB) or beta-naphthoflavone increased the o-hydroxyphenylacetaldehyde formed. 3-Hydroxycoumarin was the other main metabolite produced by rat liver microsomes. 3. Liver microsomal metabolism of coumarin in gerbil was extensive with 3-, 5-, 6-, 7- and 8-hydroxycoumarins, and 3,7- and 6,7-dihydroxycoumarins produced, in addition to o-hydroxyphenylacetaldehyde. The profile of the hydroxy metabolites was altered by in vivo treatment of gerbils with cytochrome P-450 inducers, but there was no increase of coumarin metabolism. 4. Coumarin was metabolized by human liver microsomes to o-hydroxyphenylacetaldehyde, 7-hydroxycoumarin, 3-hydroxycoumarin, and trace amounts of 5-, 6- and 8-hydroxycoumarins. 5. At low substrate concentrations (0-10 microM) hepatic microsomal metabolism of coumarin in gerbil resembled that in man, with 7-hydroxycoumarin being a major metabolite. However, the production of o-hydroxyphenylacetaldehyde was greater in gerbil than human liver microsomes. 6. At higher substrate concentrations (1 mM) metabolism of coumarin by liver microsomes from PB-treated gerbils most closely resembled that by human liver microsomes. 7. The gerbil would appear to be a more appropriate animal model than rat for studies to assess the toxicological hazard of coumarin for man.     

Characterisation of theophylline metabolism in human liver microsomes.

1. A radiometric high performance liquid chromatographic method is described for the assay of theophylline metabolism in vitro by the microsomal fraction of human liver. 2. Formation of the three metabolites of theophylline (3-methylxanthine, 1-methylxanthine and 1,3-dimethyluric acid) were linear with protein concentrations to 4 mg ml-1 and with incubation times up to 180 min. 3. The coefficients of variation for the formation of 3-methylxanthine, 1-methylxanthine and 1,3-dimethyluric acid were 1.2%, 1% and 1.6%, respectively. 4. Theophylline is metabolised by microsomal enzymes with a requirement for NADPH. 5. The mean (n = 7) Km values for 1-demethylation, 3-demethylation and 8-hydroxylation were 545, 630 and 788 microM, respectively, and the mean Vmax values were 2.65, 2.84 and 11.23 pmol min-1 mg-1, respectively. 6. There was a high correlation between the Km and Vmax values for the two demethylation pathways suggesting that the demethylations are performed by the same enzyme. 7. Overall the in vitro studies are consistent with the in vivo results which suggest the involvement of two cytochrome P-450 isozymes in the metabolism of theophylline.