Oxidative metabolism of zolpidem by human liver cytochrome P450S

The aim of this study was to identify the form(s) of cytochrome P450 (CYP) responsible for the biotransformation of zolpidem to its alcohol derivatives which, after rapid conversion to carboxylic acids, represents the main way of metabolism in humans. In human liver microsomes, zolpidem was converted to alcohol derivatives. Production of these correlated with the level of CYP3A4 and with cyclosporin oxidation and erythromycin N-demethylation activities, but not with the level of CYP1A2 nor with ethoxyresorufin O-deethylation or S-mephenytoin 4'-hydroxylation activities. Liver microsomes from CYP2D6-deficient patients exhibited normal activity. Production of alcohol derivatives was significantly inhibited by anti-CYP3A antibodies and by ketoconazole. Antibodies directed against other CYP forms (including CYP1A1, CYP1A2, CYP2A6, CYP2B4, and CYP2C8), and CYP-specific substrates or inhibitors (including propranolol, coumarin, mephenytoin, sulfaphenazole, quinidine, aniline, and lauric acid) produced a moderate or no inhibitory effect. cDNA-expressed CYP3A4 and CYP1A2 generated significant amounts of one of the alcohol derivatives, whereas CYP2D6 generated both of them in similar amounts. In human hepatocytes in primary culture, zolpidem was extensively and almost exclusively converted to one of the carboxylic acid derivatives, the main species identified in vivo. Treatment of cells with inducers of CYP1A (beta-naphthoflavone) and CYP3A (rifampicin and phenobarbital) greatly increased the rate of production of this metabolite. We conclude that the formation of alcohol derivatives of zolpidem is rate-limiting and principally mediated by CYP3A4. Both CYP1A2 and CYP2D6 participate in alcohol formation; but, because of their low relative level of expression in the human liver, their contribution is minor.     

Presence and activity of cytochrome P450 isoforms in minipig liver microsomes. Comparison with human liver samples

The average tritiated water concentration in the indoor air of the occupationally exposed worker's residence (55 Bq m(-3), range 53-59 Bq m(-3)) was higher than the indoor air of control residences (0.7 Bq m(-3), range 0.4-0.8 Bq m(-3)). The worker had an average concentration of tritium-in-urine of 30 kBq L(-1) from chronic intakes of occupational levels of tritiated water. Higher residential concentrations of tritiated water vapor were due to tritium transferred by the worker. Urine samples from an adult co-occupant were collected and had tritiated water concentrations between 89 and 345 Bq L(-1). These concentrations were higher than for individuals (range, 6-32 Bq L(-1)) living in other residences having similar outdoor and indoor concentrations of tritiated water in air. The range of measured tritiated water in urine was in agreement with the prediction of biokinetic models for tritium intakes as recommended by the International Commission on Radiological Protection Publication 56. The tritiated water vapor in the indoor air of the exposed worker's residence contributed about 96% of the daily tritium intakes. The annual average tritium dose to the family member (7 microSv) was well below the International Commission on Radiological Protection Publication 60 recommended annual dose limit (1 mSv) for members of the public. We conclude that, for a few members of the public living near a heavy-water research reactor facility, daily intakes of tritium will relate to tritiated water dispersed by the exposed worker, as well as to tritium transported by the atmosphere from the reactor site.     

A convenient method to discriminate between cytochrome P450 enzymes and flavin-containing monooxygenases in human liver microsomes

Liver microsomes are a frequently used probe to investigate the phase I metabolism of xenobiotics in vitro. Structures containing nucleophilic hetero-atoms are possible substrates for cytochrome P450 enzymes (P450) and flavin-containing monooxygenases (FMO). Both enzymes are located in the endoplasmatic reticulum of hepatocytes and both need oxygen and NADPH as cofactors. The common method to distinguish between the two enzyme systems is to use the thermal inactivation of FMO and to inhibit P450 completely with carbon monoxide, N-octylamine or N-benzylimidazole. In the literature no indication could be found that the heat inactivation of FMO does not affect any of the human P450 enzymes or that the overall P450 inhibitors inhibit the different human P450 enzymes sufficiently and do not affect the FMO. The effect of N-benzylimidazole and heat inactivation was tested on specific activities of seven P450 enzymes in human liver microsomes, 1A2, 2A6, 2C9, 2C19, 2D6, 3A4/5, and 2E1, using methoxyresorufin O-demethylation, coumarin 7-hydroxylation, (S)-warfarin 4-hydroxylation, (S)-(+)-mephenytoin 4-hydroxylation, dextrometorphan O-demethylation, oxidation of denitronifedipine, and chlorzoxazone 6-hydroxylation respectively. The sulfoxidation of methimazole (MMI) was used as a specific probe for the determination of FMO activity. Methimazole sulfoxidation was compared with the well known assay for FMO metabolism, the formation of N,N-dimethylaniline (DMA) N-oxide, to be confirmed as an exclusively FMO mediated reaction. The participation of P450 and FMO in the sulfoxidation of four sulfur containing peptides, ametryne; terbutryne, prometryne and methiocarb was investigated using human liver microsomes. All four reactions were demonstrated to be catalysed predominantly by cytochrome P450.     

Integrated cytochrome P450 reaction phenotyping: attempting to bridge the gap between cDNA-expressed cytochromes P450 and native human liver microsomes

With the increased availability of human liver tissue, recombinant (cDNA-expressed) cytochrome P450 proteins (rCYPs), and knowledge of the human CYP pool (e.g. immunoquantitated levels of each CYP form in native liver microsomes), it is now possible to carry out in vitro "CYP reaction phenotyping" in an integrated manner. Reaction phenotyping allows one to identify which CYP form(s) is (are) involved in the metabolism of a given drug, using a combination of data obtained with native human liver microsomes and rCYP proteins. The following describes how one can attempt to integrate such data. A total of ten drugs are included in the analysis, represented by twelve reactions (six hydroxylations, two O-demethylations, one N-demethylation, one O-deethylation, and two sulfoxidations) that are largely catalyzed (> or =20%) by various combinations of CYPs (CYP3A4, CYP2C9, CYP1A2, and CYP2D6), and characterized by a wide range of apparent Km values (12-820 microM). Briefly, reaction rates measured with individual rCYPs are normalized with respect to the nominal specific content of the corresponding CYP in native human liver microsomes. In turn, the normalized rates for each rCYP are summed, yielding a "total normalized rate" (TNR), and the normalized rate for each rCYP is expressed as a percent of the TNR (% TNR). Finally, % TNR is related to inhibition (percent inhibition in the presence of CYP form selective chemical inhibitors; % I) and univariate regression analysis (r > or = 0.63; P < or = 0.05; N > or = 10 different livers) data obtained with native human liver microsomes. Therefore, the reaction phenotype of a drug is assigned by integrating all three data sets (r, % TNR, and % I).     

Examination of metabolic pathways and identification of human liver cytochrome P450 isozymes responsible for the metabolism of barnidipine, a calcium channel blocker

1. In a human liver microsomal system, barnidipine was converted into three primary metabolites, an N-debenzylated product (M-1), a hydrolyzed product of the benzyl-pyrrolidine ester (M-3) and an oxidized product of the dihydropyridine ring (M-8). 2. Involvement of CYP3A in the three primary metabolic pathways was revealed by the following studies: (a) inhibition of CYP3A, (b) a correlation study using 10 individual human liver microsomes and (c) cDNA-expression studies. The secondary metabolites, M-2 and M-4 (pyridine forms of M-1 and M-3), were most likely generated from M-8 but were unlikely from M-1 or M-3. Involvement of CYP3A in the secondary pathways of metabolism is also suggested. 3. The possibility of interactions between barnidipine and coadministered drugs was examined in vitro. The formation rate of the primary metabolites was little affected by warfarin, theophylline, phenytoin, diclofenac and amitriptyline at concentrations of 200 microM, but was inhibited by glibenclamide, simvastatin and cyclosporin A. IC50 for the latter drugs was estimated to be > 200, 200 and 20 microM respectively, which was roughly > 200, 6000 and 50 times higher than their respective therapeutic plasma levels, suggesting that interactions with cyclosporin A, a CYP3A inhibitor, are of possible clinical relevance.     

Identification of human liver cytochrome P450 isoforms involved in the in vitro metabolism of cyclobenzaprine

Cyclobenzaprine (Flexeril) is a muscle relaxant, possessing a tricyclic structure. Numerous therapeutic agents containing this structure are known to be metabolized by polymorphic cytochrome P4502D6. The aim of this study was to determine if cytochrome P4502D6 and other isoforms are involved in the metabolism of cyclobenzaprine in human liver microsomes. Selective cytochrome P450 inhibitors for CYP1A1/2 (furafylline and 7,8-benzoflavone) and CYP3A4 (troleandomycin, gestodene, and ketoconazole) inhibited the formation of desmethylcyclobenzaprine, a major metabolite of cyclobenzaprine, in human liver microsomes. Antibodies directed against CYP1A1/2 and CYP3A4 inhibited the demethylation reaction whereas anti-human CYP2C9/10, CYP2C19, and CYP2E1 antibodies did not show any inhibitory effects. When a panel of microsomes prepared from human B-lymphoblastoid cells that expressed specific human cytochrome P450 isoforms were used, only microsomes containing cytochromes P4501A2, 2D6, and 3A4 catalyzed N-demethylation. In addition, demethylation catalyzed by these recombinant cytochromes P450 can be completely inhibited with selective inhibitors at concentrations as low as 1 to 20 microM. Interestingly, cyclobenzaprine N-demethylation was significantly correlated with caffeine 3-demethylation (1A2) and testosterone 6 beta-hydroxylation (3A4) but not with dextromethorphan O-demethylation (2D6) in human liver microsomes. To further determine the involvement of cytochrome P4502D6 in cyclobenzaprine metabolism, liver microsomes from a human that lacked CYP2D6 enzyme activities was included in this study. The data showed that cyclobenzaprine N-demethylation still occurred in the incubation with this microsome. These results suggested that cytochrome P4502D6 plays only a minor role in cyclobenzaprine N-demethylation whereas 3A4 and 1A2 are primarily responsible for cyclobenzaprine metabolism in human liver microsomes. Due to the minimum involvement of CYP2D6 in the vitro metabolism of cyclobenzaprine, the polymorphism of cytochrome P4502D6 in man should not be of muci concern in the clinical use of cyclobenzaprine.     

Nutritional status modulates rat liver cytochrome P450 arachidonic acid metabolism

Alterations in nutritional status affect hepatic cytochrome P450 levels. Since cytochromes P450 participate in the metabolism of arachidonic acid, we hypothesized that changes in liver P450 arachidonic acid metabolism occur during fasting and refeeding. Male Fisher 344 rats were either fed, fasted 48 hr (F48), fasted 48 hr and then refed 6 hr (F48/R6), or fasted 48 hr and then refed 24 hr (F48/R24). F48 rats had reduced body weight, increased plasma beta-hydroxybutyrate, and reduced plasma insulin compared with the other groups. Although there was no significant change in total liver P450 content, there was a significant 20%, 48%, and 24% reduction in total hepatic microsomal arachidonic acid metabolism in F48, F48/R6, and F48/R24 rats, respectively, compared with fed rats. Epoxygenase activity decreased by 28%, 51%, and 26% in F48, F48/R6, and F48/R24 rats, respectively. In contrast, omega-1 hydroxylase activity increased by 126% in F48 rats compared with fed rats. Immunoblotting revealed that levels of CYP2C11 protein were markedly reduced, whereas levels of CYP2E1 protein were markedly increased in the F48 and F48/R6 groups. In contrast, levels of CYP1A1, CYP1A2, CYP2B1, CYP2J3, CYP4A1, and CYP4A3 were unchanged with fasting/refeeding. Northern blots revealed that levels of CYP2C11 mRNAs were decreased, whereas CYP2E1 mRNAs were increased in F48 and F48/R6 rats. Recombinant CYP2C11 metabolized arachidonic acid primarily to epoxides with preference for the 14(S),15(R)-, 11(R), 12(S)-, and 8(S),9(R)- epoxyeicosatrienoic acid enantiomers. We conclude that (1) nutritional status affects hepatic microsomal arachidonic acid metabolism, (2) reduced epoxygenase activity in F48 and F48/R6 rats is accompanied by decreased levels of CYP2C11, (3) increased omega-1 hydroxylase activity is accompanied by augmented levels of CYP2E1, and (4) the effects of fasting on CYP2C11 and CYP2E1 expression occur at the pretranslational level.     

Amiodarone N-deethylation in human liver microsomes: involvement of cytochrome P450 3A enzymes (first report)

Experiments were conducted on three different human liver samples to identify the cytochrome P450 isozyme which is involved in the biotransformation of the class III antiarrhythmic agent, amiodarone, into its major metabolite, desethylamiodarone (DEA). The classic P450 inhibitors, SKF 525A, metyrapone, and carbon monoxide provided a significant reduction in the in vitro formation of DEA by human hepatic microsomes. Amiodarone N-deethylase activities expressed by intrinsic clearance values were similar in all the livers used, although two livers were genotyped as extensive and one as a poor metabolizer for the cytochrome P450 CYP2D6 gene. DEA production was strongly inhibited (more than 80%) by the anti-P450 3A4 antibody, but not by anti-LKM1-positive serum. It seems therefore that the P450 3A subfamily is certainly implicated in human hepatic amiodarone N-deethylation.     

Novobiocin inhibits both UDP-glucuronosyltransferase and cytochrome P450-mediated enzyme activities in pig liver microsomes

The effects of novobiocin (range 0.0125-2 mmol/L) on the hydroxylation of testosterone, the N-demethylation of erythromycin, and the glucuronidation of alpha-naphthol and paracetamol were studied using pig hepatic microsomes, pooled from five animals. The final concentrations of these substrates in the incubation mixtures were selected to meet Vmax conditions. Novobiocin caused a concentration-dependent inhibition of the glucuronidation of paracetamol; the formation of alpha-naphthol-glucuronide was reduced to a lesser degree. These results confirm and extend earlier findings in laboratory animal species that novobiocin inhibits UDP-glucuronosyltransferases (UDPGTs). Moreover, novobiocin strongly inhibited 6 beta-hydroxylation of testosterone. The microsomal N-demethylation of erythromycin and hydroxylation of testosterone at the 15 alpha position were less affected by novobiocin. These results suggest that novobiocin inhibits not only UDPGTs, but also cytochrome P450 (CYP) enzyme activities, probably those belonging to the CYP3A subfamily. More research is needed to reveal which CYPs and UDPGTs are affected by novobiocin in vivo, in order to improve the understanding the probably the predictability of potential drug interactions with this antibiotic.     

Trimethadione metabolism by human liver cytochrome P450: evidence for the involvement of CYP2E1

1. Caucasian liver samples were used in this study. N-demethylation of trimethadione (TMO) to dimethadione (DMO) was monitored in the presence of chemical inhibitors of CYPs, such as fluconazole, quinidine, dimethyl-nitrosamine, acetaminophen, phenacetin, chlorzoxazone and mephenytoin. Trimethadione N-demethylation was selectively inhibited by dimethylnitrosamine and chlorzoxazone (> 50%) and weakly inhibited by tolbutamide (12%) and fluconazole (22%), whereas other inhibitors showed no effect. This result suggested that TMO metabolism to DMO is mainly mediated by CYP2E1 and marginally by CYP2C and CYP3A4. 2. Fifteen human livers were screened and interindividual variability of TMO N-demethylation activity was 3-fold. Chlorzoxazone 6-hydroxylation activity was also measured and both activities were significantly correlated (r=0.735, p < 0.01). 3. DMO production by human cDNA expressed CYP enzymes was observed mainly for CYP2E1 (10.8 nmol/tube), marginally for CYP2C8 (0.22 nmol/tube) and not detectable for other CYP enzymes. 4. These results indicate that TMO metabolism is primarily catalysed by CYP2E1 and that trimethadione would be a suitable selective probe drug for the estimation of human CYP2E1 activity in vivo.     

Identification of human cytochrome P450 isoforms involved in the 3-hydroxylation of quinine by human live microsomes and nine recombinant human cytochromes P450

Studies using human liver microsomes and nine recombinant human cytochrome P450 (CYP) isoforms (CYP1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1 and 3A4) were performed to identify the CYP isoform(s) involved in the major metabolic pathway (3-hydroxylation) of quinine in humans. Eadie-Hofstee plots for the formation of 3-hydroxyquinine exhibited apparently monophasic behavior for all of the 10 different microsomal samples studies. There was interindividual variability in the kinetic parameters, as follows: 1.8-, 3.2- and 3.5-fold for K(m) Vmax and Vmax/K(m), respectively. The mean +/- S.D. values for K(m), Vmax and Vmax/K(m) were 106.1 +/- 19.3 microM, 1.33 +/- 0.48 nmol/mg protein/min and 12.8 +/- 5.1 microliters/mg protein/min, respectively. With 10 different human liver microsomes, the relationships between the 3-hydroxylation of quinine and the metabolic activities for substrates of the respective CYP isoforms were evaluated. The 3-hydroxylation of quinine showed an excellent correlation (r = 0.986, P < .001) with 6 beta-hydroxylation of testosterone, a marker substrate for CYP3A4. A significant correlation (r = 0.768, P < .01) between the quinine 3-hydroxylase and S-mephenytoin 4'-hydroxylase activities was also observed. However, no significant correlation existed between the 3-hydroxylation of quinine and the oxidative activities for substrates for CYP1A2 (phenacetin), 2C9 (diclofenac), 2D6 (desipramine) and 2E1 (chlorzoxazone). Ketoconazole and troleandomycin (inhibitors of CYP3A4) inhibited the 3-hydroxylation of quinine by human liver microsomes with respective mean IC50 values of 0.026 microM and 28.9 microM. Anti-CYP3A antibodies strongly inhibited quinine 3-hydroxylation, whereas weak inhibition was observed in the presence of S-mephenytoin or anti-CYP2C antibodies. Among the nine recombinant human CYP isoforms, CYP3A4 exhibited the highest catalytic activity with respect to the 3-hydroxylation of quinine, compared with the minor activity of CYP2C19 and little discernible or no effect of other CYP isoforms. Collectively, these data suggest that the 3-hydroxylation of quinine is mediated mainly by CYP3A4 and to a minor extent by CYP2C19. Other CYP isoforms used herein appear to be of negligible importance in this major pathway of quinine in humans.     

Metabolic interactions of putative cytochrome P4503A substrates with alternative pathways of dapsone metabolism in human liver microsomes

The cytochrome P4503A (CYP3A) subfamily of enzymes are responsible for the metabolism of a large number of endogenous and exogenous compounds, and activation of some procarcinogens; but the activity is not well understood. N-Hydroxylation of dapsone in human liver microsomes has been shown to be mediated largely by CYP3A4. We have also observed the formation of an as yet unidentified metabolite of dapsone, whose formation is inhibited by antibody to CYP3A4, by these microsomes. This study investigated the influence of various (22) CYP3A putative substrates on the formation of both metabolites of dapsone in human liver microsomes. The compounds fall into four different categories on the basis of the pattern of their inhibitory interaction with the formation of both metabolites: those that inhibited both metabolites; those that inhibited N-hydroxylamine alone; those that inhibited the unidentified metabolite alone; and those with no significant effect on either metabolite. Some others were stimulatory. These results are consistent with two alternative but not mutually exclusive hypotheses: 1) different isoforms of CYP3A are involved in the formation of the alternative metabolites and the pattern of interaction observed was caused by the particular isoform(s) that each compound interacted with; or 2) formation of the alternative metabolites is a result of dapsone's interaction with and orientation at the enzyme's active site and the pattern of interaction observed is a consequence of changes in orientation caused by these compounds. This study provides relevant observations that must be considered in understanding mechanisms of CYP3A-mediated metabolism.     

Cytochrome P450-mediated metabolism of the HIV-1 protease inhibitor ritonavir (ABT-538) in human liver microsomes

The HIV-1 protease inhibitor ritonavir (ABT-538) undergoes cytochrome P450-mediated biotransformation in human liver microsomes to three major metabolites, Ml, M2 and M11, with wide interindividual variation in the rates of metabolite formation. The structures of these metabolites were determined with the use of electrospray ionization mass spectrometry. Chemical inhibition, metabolic correlation, immunoinhibition and metabolism by microsomes derived from specific CYP cDNA-transfected B-lymphoblastoid cell lines indicated that the CYP3A subfamily of enzymes was the major contributor to the formation of M1 and M11, whereas both CYP3A and CYP2D6 contributed to the formation of M2. None of the typical CYP3A substrates/inhibitors (e.g., ketoconazole, troleandomycin) were able to completely inhibit ritonavir metabolism, even at high concentrations. Ritonavir was found to be a potent inhibitor of CYP3A-mediated biotransformations (nifedipine oxidation, IC50) = 0.07 microM; 17alpha-ethynylestradiol 2-hydroxylation, IC50 = 2 microM; terfenadine hydroxylation, IC50 = 0.14 microM). Ritonavir was also found to be an inhibitor of the reactions mediated by CYP2D6 (IC50 = 2.5 microM) and CYP2C9/10 (IC50 = 8.0 microM). The results of this study indicate the potential for in vivo inhibition of the metabolism by ritonavir of drugs that are CYP3A, CYP2D6 and, to a lesser extent, CYP2C9/10 substrates.     

Identification of the human liver cytochrome P450 enzymes involved in the in vitro metabolism of a novel 5-lipoxygenase inhibitor

In vitro studies were conducted to identify the hepatic cytochrome P450 (CYP) forms involved in the oxidative metabolism of [14C]ABT-761 and its N-dehydroxylated metabolite, [14C]ABT-438, by human liver microsomes. The two compounds were metabolized by parallel pathways, to form the corresponding methylene bridge hydroxy metabolites. There was no evidence of sulfoxidation and/or ring hydroxylation. Over the ABT-761 and ABT-438 concentration ranges studied (1-300 microM), the rate of NADPH-dependent hydroxylation was linear with respect to substrate concentration ([S]) and did not conform to saturable Michaelis-Menten kinetics. Under these conditions ([S] < KM), the intrinsic clearance (Vmax/KM) of ABT-438 was 10-fold higher than that of ABT-761 (1.7 +/- 0.8 vs. 0.17 +/- 0.06 microl/min/mg, mean +/- SD, N = 3 livers). The hydroxylation of both compounds was shown to be highly correlated (r = 0.83, p < 0.01, N = 11 different human livers) with CYP3A-selective erythromycin N-demethylase activity, and the correlation between ABT-761 hydroxylation and tolbutamide hydroxylase (CYP2C9-selective) activity (r = 0.63, p < 0.05, N = 10) was also statistically significant. Ketoconazole (2.0 microM), a CYP3A-selective inhibitor, inhibited the hydroxylation of both compounds by 53-67%, and sulfaphenazole (CYP2C9-selective) decreased activity by 10-20%. By comparison, alpha-naphthoflavone, a known activator of CYP3A, stimulated the hydroxylation of ABT-761 (8-fold) and ABT-438 (4-fold). In addition, the abundance-normalized rates of cDNA-expressed CYP-dependent metabolism indicated that hydroxylation was largely mediated (66-86%) by CYP3A(4). Therefore, it is concluded that the hydroxylation of ABT-761 and ABT-438 (相似文献   

Mechanism-based inactivation of human liver cytochrome P450 2A6 by 8-methoxypsoralen

The P450 2A6 catalyzed 7-hydroxylation of coumarin proceeded with a mean Km of 0.40 (+/-0.13) microM and Vmax of 6.34 nmol/nmol P450/min (36-fold variation) in microsomal preparations from a panel of 12 human livers. Substrate depletion was avoided during the kinetic determinations. 8-Methoxypsoralen (8-MOP) is a potent mechanism-based inactivator of human liver P450 2A6 and reconstituted purified recombinant P450 2A6 based on the following evidence: 1) 8-MOP causes time, concentration, and NADPH-dependent loss of P450 2A6 activity that is not reversed by potassium ferricyanide or extensive dialysis, 2) loss of P450 2A6 activity is associated with a loss of spectrally observable P450, 3) addition of nucleophiles or reactive oxygen scavengers to the incubations does not prevent inactivation of P450 2A6, and 4) 8-MOP-dependent P450 2A6 inactivation is inhibited (concentration dependent) by the addition of a competitive inhibitor (pilocarpine). Inactivation is selective for P450 2A6 at low concentrations of 8-MOP (2.5 microM) after short incubation time periods (3 min) and was characterized by a KI of 0.8 and 1.9 microM in a reconstituted and microsomal system, respectively, and a kinact of 1 min-1 and 2 min-1 in a reconstituted and microsomal system, respectively. A substrate depletion partition ratio of 21 was calculated for the inactivation of recombinant P450 2A6. Potency and selectivity suggest that 8-MOP could be a useful tool in vitro for evaluating P450 2A6 activity in various enzyme preparations.     

High catalytic activity of human cytochrome P450 co-expressed with human NADPH-cytochrome P450 reductase in Escherichia coli

Forms of human cytochrome P450 (P450 or CYP), such as CYP1A1, CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4, were expressed or co-expressed together with human NADPH-P450 reductase in Escherichia coli. When P450 was expressed alone in E. coli, the expression level of holo-P450 ranged from 310 to 1620 nmol/L of culture. The expression level of holo-P450 decreased by co-expression with the reductase, and the level ranged from 66 to 381 nmol/L of culture. The expression level of the reductase varied depending on the forms of P450 co-expressed, and ranged from 204 to 937 U/L of culture. We assayed the catalytic activity of P450 using E. coli cells disrupted by freeze-thaw. When co-expressed with the reductase, human P450 catalyzed the oxidation of representative substrates at efficient rates. The rates appeared comparable to the reported activities of P450 in a reconstituted system containing purified preparations of P450 and the reductase.     

In vitro identification of the P450 enzymes responsible for the metabolism of ropinirole

The in vitro metabolism of ropinirole was investigated with the aim of identifying the cytochrome P450 enzymes responsible for its biotransformation. The pathways of metabolism after incubation of ropinirole with human liver microsomes were N-despropylation and hydroxylation. Enzyme kinetics demonstrated the involvement of at least two enzymes contributing to each pathway. A high affinity component with a K(M) of 5-87 microM and a low affinity component with a K(M) of approximately two orders of magnitude greater were evident. The high affinity component could be abolished by pre-incubation of the microsomes with furafylline. Additionally, incubation of ropinirole with microsomes derived from CYP1A2 transfected cells readily produced the N-despropyl and hydroxy metabolites. Some inhibition of ropinirole metabolism was also observed with ketoconazole, indicating a minor contribution by CYP3A. Multivariate correlation data were consistent with the involvement of the cytochrome P450 enzymes 1A2 and 3A in the metabolism of ropinirole. Thus, it could be concluded that the major P450 enzyme responsible for ropinirole metabolism at lower (clinically relevant) concentrations is CYP1A2 with a contribution from CYP3A, particularly at higher concentrations.