In vitro metabolism of chloroquine: identification of CYP2C8, CYP3A4, and CYP2D6 as the main isoforms catalyzing N-desethylchloroquine formation.

In humans, the antimalarial drug chloroquine (CQ) is metabolized into one major metabolite, N-desethylchloroquine (DCQ). Using human liver microsomes (HLM) and recombinant human cytochrome P450 (P450), we performed studies to identify the P450 isoform(s) involved in the N-desethylation of CQ. In HLM incubated with CQ, only DCQ could be detected. Apparent Km and Vmax values (mean +/- S.D.) for metabolite formation were 444 +/- 121 microM and 617 +/- 128 pmol/min/mg protein, respectively. In microsomes from a panel of 16 human livers phenotyped for 10 different P450 isoforms, DCQ formation was highly correlated with testosterone 6beta-hydroxylation (r = 0.80; p < 0.001), a CYP3A-mediated reaction, and CYP2C8-mediated paclitaxel alpha-hydroxylation (r = 0.82; p < 0.001). CQ N-desethylation was diminished when coincubated with quercetin (20-40% inhibition), ketoconazole, or troleandomycin (20-30% inhibition) and was strongly inhibited (80% inhibition) by a combination of ketoconazole and quercetin, which further corroborates the contribution of CYP2C8 and CYP3As. Of 10 cDNA-expressed human P450s examined, only CYP1A1, CYP2D6, CYP3A4, and CYP2C8 produced DCQ. CYP2C8 and CYP3A4 constituted low-affinity/high-capacity systems, whereas CYP2D6 was associated with higher affinity but a significantly lower capacity. This property may explain the ability of CQ to inhibit CYP2D6-mediated metabolism in vitro and in vivo. At therapeutically relevant concentrations ( approximately 100 microM CQ in the liver), CYP2C8, CYP3A4, and, to a much lesser extent, CYP2D6 are expected to account for most of the CQ N-desethylation.     

Characterisation of the cytochrome P450 enzymes involved in the in vitro metabolism of granisetron.

1. The metabolism of granisetron was investigated in human liver microsomes to identify the specific forms of cytochrome P450 responsible. 2. 7-hydroxy and 9'-desmethyl granisetron were identified as the major products of metabolism following incubation of granisetron with human liver microsomes. At low, clinically relevant, concentrations of granisetron the 7-hydroxy metabolite predominated. Rates of granisetron 7-hydroxylation varied over 100-fold in the human livers investigated. 3. Enzyme kinetics demonstrated the involvement of at least two enzymes contributing to the 7-hydroxylation of granisetron, one of which was a high affinity component with a Km of 4 microM. A single, low affinity, enzyme was responsible for the 9'-desmethylation of granisetron. 4. Granisetron caused no inhibition of any of the cytochrome P450 activities investigated (CYP1A2, CYP2A6, CYP2B6, CYP2C9/8, CYP2C19, CYP2D6, CYP2E1 and CYP3A), at concentrations up to 250 microM. 5. Studies using chemical inhibitors selective for individual P450 enzymes indicated the involvement of cytochrome P450 3A (CYP3A), both pathways of granisetron metabolism being very sensitive to ketoconazole inhibition. Correlation data were consistent with the role of CYP3A3/4 in granisetron 9'-desmethylation but indicated that a different enzyme was involved in the 7-hydroxylation.     

In vitro biotransformation of sildenafil (Viagra): identification of human cytochromes and potential drug interactions.

The in vitro biotransformation of sildenafil to its major circulating metabolite, UK-103,320, was studied in human liver microsomes and in microsomes containing heterologously expressed human cytochromes. In human liver microsomes, the mean K(m) (+/-S.E. ) was 14.4 +/- 2.0 microM. A screen of the chemical inhibitors omeprazole (10 microM), quinidine (10 microM), sulfaphenazole (10 microM), and ketoconazole (2.5 microM) only revealed detectable inhibition with ketoconazole. Sildenafil biotransformation (36 microM) was inhibited by increasing concentrations of ketoconazole and ritonavir (IC(50) values less than 0.02 microM), which are established cytochrome P450 (CYP) 3A4 inhibitors. Using microsomes containing cDNA-expressed cytochromes, UK-103,320 formation was found to be mediated by four cytochromes: CYP3A4, -2C9, -2C19, and -2D6. Estimated relative contributions to net intrinsic clearance were 79% for CYP3A4 and 20% for CYP2C9; for CYP2C19 and -2D6, estimated contributions were less than 2%. These results demonstrate that CYP3A4 is the primary cytochrome mediating UK-103,320 formation and that drugs that inhibit CYP3A4 are likely to impair sildenafil biotransformation.     

Metabolism of the active metabolite of quetiapine, N-desalkylquetiapine in vitro

The antipsychotic drug quetiapine has been approved for the treatment of unipolar and bipolar depression. The antidepressant activity is considered to be mediated by the active metabolite N-desalkylquetiapine, which is mainly formed by CYP3A4. Little is known about the subsequent elimination of this metabolite. Therefore, this study investigated the possible involvement of cytochrome P450 (P450) enzymes in the metabolism of N-desalkylquetiapine. Screening for and interpretation of metabolites were performed by incubating N-desalkylquetiapine in human liver microsomes (HLM) followed by liquid chromatography-tandem mass spectrometry. The possible involvement of P450 enzymes in N-desalkylquetiapine metabolism was evaluated by coincubation of selective P450 inhibitors in HLM and subsequent experiments with recombinant human P450 enzymes. In HLM experiments, three chromatographic peaks were interpreted as possible metabolites of N-desalkylquetiapine, namely, N-desalkylquetiapine sulfoxide, 7-hydroxy-N-desalkylquetiapine, and an unrecognized metabolite (denoted M3). Inhibition of CYP2D6 (by quinidine) reduced formation of 7-hydroxy-N-desalkylquetiapine by 81%, whereas the CYP3A4 inhibitor ketoconazole inhibited formation of N-desalkylquetiapine sulfoxide and M3 by 65 and 34%, respectively. Inhibitors of CYP1A2, CYP2C9, and CYP2C19 showed only limited changes in metabolite formation. In recombinant systems, 7-hydroxy-N-desalkylquetiapine was exclusively formed by CYP2D6, whereas N-desalkylquetiapine sulfoxide and M3 were formed by both CYP3A4 and CYP2D6. Overall, intrinsic clearance of N-desalkylquetiapine was 12-fold higher by recombinant CYP2D6 relative to CYP3A4. In conclusion, N-desalkylquetiapine is metabolized by both CYP2D6 and CYP3A4 in vitro with preference for the former enzyme. The pharmacologically active metabolite, 7-hydroxy-N-desalkylquetiapine, was exclusively formed by CYP2D6, whereas the two other metabolites were mainly formed by CYP3A4.     

Application of semi-automated metabolite identification software in the drug discovery process for rapid identification of metabolites and the cytochrome P450 enzymes responsible for their formation

Rapid identification of metabolites of compound X using data dependent scan function of a quadrupole ion trap mass spectrometer and semi-automated metabolite identification software is described. Compound X is metabolized via monooxygenation and desmethylation. LC-ESI-MS spectra obtained, following incubations of Compound X with microsomes in the presence and absence of chemical inhibitors specific for CYP1A2, CYP3A4, CYP2D6, CYP2C9 and CYP2E1, were processed using semi-automated metabolite identification software to extract information and to identify the cytochrome P450 enzymes responsible for metabolite formation. Chemical inhibition data suggests that the primary cytochrome P450 (CYP450) isozyme responsible for the metabolism of compound X is CYP3A4 with a minor contribution from both CYP2D6 and CYP2E1. Additionally, neither CYP2C9 nor CYP1A2 appears to contribute to the metabolism of compound X.     

Interactions of two major metabolites of prasugrel, a thienopyridine antiplatelet agent, with the cytochromes P450.

The biotransformation of prasugrel to R-138727 (2-[1-2-cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl]-4-mercapto-3-piperidinylidene]acetic acid) involves rapid deesterification to R-95913 (2-[2-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl]-1-cyclopropyl-2-(2-fluorophenyl)ethanone) followed by cytochrome P450 (P450)-mediated formation of R-138727, the metabolite responsible for platelet aggregation. For identification of the P450s responsible for the formation of the active metabolite, the current studies were conducted with R-95913 as the substrate. Incubations required supplementation with reduced glutathione. Hyperbolic kinetics (K(m) 21-30 microM), consistent with a single enzyme predominating, were observed after incubations with human liver microsomes. Correlation analyses revealed a strong relationship between R-138727 formation and CYP3A-mediated midazolam 1'-hydroxylation (r(2) = 0.98; p < 0.001) in a bank of characterized human liver microsomal samples. The human lymphoblast-expressed enzymes capable of forming R-138727, in rank order of rates, were CYP3A4>CYP2B6>CYP2C19 approximately CYP2C9>CYP2D6. A monoclonal antibody to CYP2B6 and the CYP3A inhibitor ketoconazole substantially inhibited R-138727 formation, whereas inhibitors of CYP2C9 (sulfaphenazole) and CYP2C19 (omeprazole) did not. Scaling of in vitro intrinsic clearance values from expressed enzymes to the whole liver using a relative abundance approach indicated that either CYP3A4 alone or CYP3A4 and CYP2B6 are the major contributors to R-138727 formation. R-95913 and R-138727 were also examined for their ability to inhibit metabolism mediated by five P450s. R-138727 did not inhibit the P450s tested. In vitro, R-95913 inhibited CYP2C9, CYP2C19, CYP2D6, and CYP3A, with K(i) values ranging from 7.2 microM to 82 microM, but did not inhibit CYP1A2. These K(i) values exceed circulating concentrations in humans by 3.8- to 43-fold. Therefore, neither R-95913 nor R-138727 is expected to substantially inhibit the P450-mediated metabolism of coadministered drugs.     

CYP2B6, CYP3A4, and CYP2C19 are responsible for the in vitro N-demethylation of meperidine in human liver microsomes.

Meperidine is an opioid analgesic metabolized in the liver by N-demethylation to normeperidine, a potent stimulant of the central nervous system. The purpose of this study was to identify the human cytochrome P450 (P450) enzymes involved in normeperidine formation. Our in vitro studies included 1) screening 16 expressed P450s for normeperidine formation, 2) kinetic experiments on human liver microsomes and candidate P450s, and 3) correlation and inhibition experiments using human hepatic microsomes. After normalization by its relative abundance in human liver microsomes, CYP2B6, CYP3A4, and CYP2C19 accounted for 57, 28, and 15% of the total intrinsic clearance of meperidine. CYP3A5 and CYP2D6 contributed to < 1%. Formation of normeperidine significantly correlated with CYP2B6-selective S-mephenytoin N-demethylation (r = 0.88, p < 0.0001 at 75 > microM meperidine, and r = 0.89, p < 0.0001 at 350 microM meperidine, n = 21) and CYP3A4-selective midazolam 1'-hydroxylation (r = 0.59, p < 0.01 at 75 microM meperidine, and r = 0.55, p < 0.01 at 350 microM meperidine, n = 23). No significant correlation was observed with CYP2C19-selective S-mephenytoin 4'-hydroxylation (r = 0.36, p = 0.2 at 75 microM meperidine, and r = 0.02, p = 0.9 at 350 microM meperidine, n = 13). An anti-CYP2B6 antibody inhibited normeperidine formation by 46%. In contrast, antibodies inhibitory to CYP3A4 and CYP2C8/9/18/19 had little effect (<14% inhibition). Experiments with thiotepa and ketoconazole suggested inhibition of microsomal CYP2B6 and CYP3A4 activity, whereas studies with fluvoxamine (a substrate of CYP2C19) were inconclusive due to lack of specificity. We conclude that normeperidine formation in human liver microsomes is mainly catalyzed by CYP2B6 and CYP3A4, with a minor contribution from CYP2C19.     

Effects of propofol on human hepatic microsomal cytochrome P450 activities

1. The potential of propofol to inhibit the activity of major human cytochrome P450 enzymes has been examined in vitro using human liver microsomes. Propofol produced inhibition of CYP1A2 (phenacetin O -deethylation), CYP2C9 (tolbutamide 4-hydroxylation), CYP2D6 (dextromethorphan O -demethylation) and CYP3A4 (testosterone 6 beta hydroxylation) activities with IC = 40, 49, 213 and 32 mu M respectively. K for propofol against all of these enzymes with the exception of CYP2D6, where propofolishowed little inhibitory activity, was 30, 30 and 19 mu M respectively for CYPs 1A2, 2C9 and 3A4. 2. Furafylline, sulphaphenazole, quinidine and ketoconazole, known selective inhibitors of CYPs 1A2, 2C9, 2D6 and 3A4 respectively, were much more potent than propofol having IC = 0.8, 0.5, 0.2 and 0.1 mu M; furafylline and sulphaphenazole yielded K = 0.6 and 0.7 mu M respectively. i 3. The therapeutic blood concentration of propofol (20 mu M; 3-4 mu?g ml) together with the in vitro K estimates for each of the major human P450 enzymes have been used to i estimate the extent of cytochrome P450 inhibition, which may be produced in vivo by propofol. This in vitro - in vivo extrapolation indicates that the degree of inhibition of CYP1A2, 2C9 and 3A4 activity which could theoretically be produced in vivo by propofol is relatively low (40-51%); this is considered unlikely to have any pronounced clinical significance. 4. Although propofol has now been used in 190 million people since its launch in 1986,thereare onlysinglereportsofpossible druginteractions between propofoland either alfentanil or warfarin. Consequently, it is difficult to conclude from either the published literature or the ZENECA safety database whether there is any evidence to indicate that propofol produces clinically significant drug interactions through inhibition of cytochrome P450-related drug metabolism.     

Halofantrine metabolism in microsomes in man: major role of CYP 3A4 and CYP 3A5.

We have clarified the contribution of the different enzymes involved in the N-debutylation of halofantrine in liver microsomes in man. The effect of ketoconazole and cytochrome P450 (CYP) 3A substrates on halofantrine metabolism has also been studied. The antimalarial drug halofantrine is metabolized into one major metabolite, N-debutylhalofantrine. In microsomes from nine livers from man, N-debutylation of halofantrine was highly variable with apparent Michaelis-Menten constant V(max) and K(m) values of 215+/-172 pmol min(-1) mg(-1) and 48+/-26 micromol L(-1), respectively, (mean+/-standard deviation). Formation of N-debutylhalofantrine was cytochrome P450 (CYP)-mediated. Studies using selective inhibitors of individual CYPs revealed the role of CYP 3As in the formation of N-debutylhalofantrine. alpha-Naphthoflavone, a CYP 3A activator, increased metabolite formation. In microsomes from 12 livers from man the rate of N-debutylation of halofantrine correlated strongly with CYP 3A4 relative levels (P = 0.002) and less strongly, but significantly, with CYP 2C8 levels (P = 0.025). To characterize CYP-mediated metabolism of halofantrine further, incubations were performed with yeast microsomes expressing specific CYP 3A4, CYP 3A5, CYP 2D6, CYP 2C8 and CYP 2C19 from man. The rate of formation of N-debutylhalofantrine was six- and twelvefold with CYP 3A4 than with CYP 3A5 and CYP 2C8, respectively. CYP 2D6 and CYP 2C19 did not mediate the N-debutylation of halofantrine, but, because in-vivo CYP 2C8 is present at lower concentrations than CYP 3A in the liver in man, the involvement of CYP 3As would be predominant. Diltiazem, erythromycin, nifedipine and cyclosporin (CYP 3A substrates) inhibited halofantrine metabolism. Similarly, ketoconazole inhibited, non-competitively, formation of N-debutylhalofantrine with an inhibition constant, K(i), of 0.05 microM. The theoretical percentage inhibition of halofantrine metabolism in-vivo by ketoconazole was estimated to be 99%. These results indicate that both CYP 3A4 and CYP 3A5 metabolize halofantrine, with major involvement of CYP 3A4. In-vivo, the other CYPs have a minor role only. Moreover, strong inhibition, and consequently increased halofantrine cardiotoxicity, might occur with the association of ketoconazole or other CYP 3A4 substrates.     

Cytochrome P450 3A4 is the major enzyme involved in the metabolism of the substance P receptor antagonist aprepitant.

The contribution of human cytochrome P450 (P450) isoforms to the metabolism of aprepitant in humans was investigated using recombinant P450s and inhibition studies. In addition, aprepitant was evaluated as an inhibitor of human P450s. Metabolism of aprepitant by microsomes prepared from baculovirus-expressed human P450s was observed only when CYP1A2, CYP2C19, or CYP3A4 was present in the expression system. Incubation with CYP1A2 and CYP2C19 yielded only products of O-dealkylation, whereas CYP3A4 catalyzed both N- and O-dealkylation reactions. The metabolism of aprepitant by human liver microsomes was inhibited completely by ketoconazole or troleandomycin. No inhibition was observed with other P450 isoform-selective inhibitors. Aprepitant was evaluated also as a P450 inhibitor in human liver microsomes. No significant inhibition of CYP1A2, CYP2B6, CYP2C8, CYP2D6, and CYP2E1 was observed in experiments with isoform-specific substrates (IC50 > 70 microM). Aprepitant was a moderate inhibitor of CYP3A4, with Ki values of approximately 10 microM for the 1'- and 4-hydroxylation of midazolam, and the N-demethylation of diltiazem, respectively. Aprepitant was a very weak inhibitor of CYP2C9 and CYP2C19, with Ki values of 108 and 66 microM for the 7-hydroxylation of warfarin and the 4'-hydroxylation of S-mephenytoin, respectively. Collectively, these results indicated that aprepitant is both a substrate and a moderate inhibitor of CYP3A4.     

Ilaprazole, a new proton pump inhibitor, is primarily metabolized to ilaprazole sulfone by CYP3A4 and 3A5

Ilaprazole is a new proton pump inhibitor, designed for treatment of gastric ulcers, and developed by Il-Yang Pharmaceutical Co (Seoul, Korea). It is extensively metabolised to the major metabolite ilaprazole sulfone. In the present study, several in vitro approaches were used to identify the cytochrome P450 (CYP) enzymes responsible for ilaprazole sulfone formation. Concentrations of ilaprazole sulfone were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Incubation of ilaprazole with cDNA-expressed recombinant CYPs indicated that CYP3A was the major enzyme that catalyses ilaprozole to ilaprazole sulfone. This reaction was inhibited significantly by ketoconazole, a CYP3A inhibitor, and azamulin, a mechanism-based inhibitor of CYP3A, while no substantial effect was observed using selective inhibitors for eight other P450s (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP2E1). In addition, the formation of ilaprazole sulfone correlated well with CYP3A-catalysed testosterone 6β-hydroxylation and midazolam 1'-hydroxylation in 20 different human liver microsome panels. The intrinsic clearance of the formation of ilaprazole sulfone by CYP3A4 was 16-fold higher than that by CYP3A5. Collectively, these results indicate that the formation of the major metabolite of ilaprazole, ilaprazole sulfone, is predominantly catalysed by CYP3A4/5.     

Oxidative metabolism of the alkaloid rutaecarpine by human cytochrome P450.

Rutaecarpine is the main active alkaloid of the herbal medicine, Evodia rutaecarpa. To identify the major human cytochrome P450 (P450) participating in rutaecarpine oxidative metabolism, human liver microsomes and bacteria-expressed recombinant human P450 were studied. In liver microsomes, rutaecarpine was oxidized to 10-, 11-, 12-, and 3-hydroxyrutaecarpine. Microsomal 10- and 3-hydroxylation activities were strongly inhibited by ketoconazole. The 11- and 12-hydroxylation activities were inhibited by alpha-naphthoflavone, quinidine, and ketoconazole. These results indicated that multiple hepatic P450s including CYP1A2, CYP2D6, and CYP3A4 participate in rutaecarpine hydroxylations. Among recombinant P450s, CYP1A1 had the highest rutaecarpine hydroxylation activity. Decreased metabolite formation at high substrate concentration indicated that there was substrate inhibition of CYP1A1- and CYP1A2-catalyzed hydroxylations. CYP1A1-catalyzed rutaecarpine hydroxylations had V(max) values of 1,388 to approximately 1,893 pmol/min/nmol P450, K(m) values of 4.1 to approximately 9.5 microM, and K(i) values of 45 to approximately 103 microM. These results indicated that more than one molecule of rutaecarpine is accessible to the CYP1A active site. The major metabolite 10-hydroxyrutaecarpine decreased CYP1A1, CYP1A2, and CYP1B1 activities with respective IC(50) values of 2.56 +/- 0.04, 2.57 +/- 0.11, and 0.09 +/- 0.01 microM, suggesting that product inhibition might occur during rutaecarpine hydroxylation. The metabolite profile and kinetic properties of rutaecarpine hydroxylation by human P450s provide important information relevant to the clinical application of rutaecarpine and E. rutaecarpa.     

The involvement of CYP3A4 and CYP2C9 in the metabolism of 17 alpha-ethinylestradiol.

The role of specific cytochrome P450 (P450) isoforms in the metabolism of ethinylestradiol (EE) was evaluated. The recombinant human P450 isozymes CYP1A1, CYP1A2, CYP2C9, CYP2C19, and CYP3A4 were found to be capable of catalyzing the metabolism of EE (1 microM). Without exception, the major metabolite was 2-hydroxy-EE. The highest catalytic efficiency (Vmax/Km) was observed with rCYP1A1, followed by rCYP3A4, rCYP2C9, and rCYP1A2. The P450 isoforms 3A4 and 2C9 were shown to play a significant role in the formation of 2-hydroxy-EE in a pool of human liver microsomes by using isoform-specific monoclonal antibodies, in which the inhibition of formation was approximately 54 and 24%, respectively. The involvement of CYP3A4 and CYP2C9 was further confirmed by using selective chemical inhibitors (i.e., ketoconazole and sulfaphenazole). The relative contribution of each P450 isoform to the 2-hydroxylation pathway was obtained from the catalytic efficiency of each isoform normalized by its relative abundance in the same pool of human liver microsomes, as determined by quantitative Western blot analysis. Collectively, these results suggested that multiple P450 isoforms were involved in the oxidative metabolism of EE in human liver microsomes, with CYP3A4 and CYP2C9 as the major contributing enzymes.     

Identification of human cytochrome P450 isoforms that contribute to all-trans-retinoic acid 4-hydroxylation

The role of specific human cytochrome P450 (CYP) isoforms in the oxidative metabolism of all-trans-retinoic acid was investigated by studies in human liver microsomes using isoform-specific chemical inhibitors and inhibitory antibodies. Studies using individual isoforms expressed in lymphoblastoid cells and correlation analysis using different microsome preparations were also performed. With expressed isoforms, evidence for a role for CYP2C8, CYP3A4, CYP2C9, and CYP1A1 in 4-hydroxylation was obtained, with the highest catalytic efficiency being observed for CYP2C8. Using inhibition studies and correlation analysis, we also concluded that CYP2C8 was the major all-trans-retinoic acid 4-hydroxylating cytochrome P450 in human liver microsomes, though CYP3A4 and, to a lesser extent CYP2C9, also made a contribution. In addition, we compared the rate of retinoic acid degredation in HepG2 cells when cultured in the absence and presence of 3-methylcholanthrene or all-trans-retinoic acid. Culture in the presence of all-trans-retinoic acid decreased the half-life twofold and resulted in an increased sensitivity of retinoic acid degredation to ketoconazole. Since no induction of either CYP1A1, CYP2C8, CYP2C9, or CYP3A4 was detected using immunoblotting and as mRNA encoding another cytochrome P450 enzyme, CYP26, has been previously demonstrated to be induced by retinoic acid treatment of HepG2 cells and to be highly sensitive to ketoconazole, this enzyme in addition to CYP2C8, CYP2C9 and CYP3A4 likely plays a role in all-trans-retinoic acid oxidation in the liver at high retinoic acid levels.     

Cytochrome P450-dependent metabolism of gefitinib

The in vitro metabolism of [(14)C]-gefitinib (1-3 microM) was investigated using human liver microsomes and a range of expressed human cytochrome P450 enzymes, with particular focus on the formation of O-desmethyl-gefitinib (M523595), the major metabolite observed in human plasma. High-performance liquid chromatography with ultraviolet light, radiochemical and mass spectral analysis, together with the availability of authentic standards, enabled quantification and structural identification of metabolites. On incubation with pooled human liver microsomes, [(14)C]-gefitinib underwent rapid and extensive metabolism to a number of metabolites, although M523595 was only a minor microsomal product. Formation of most metabolites was markedly decreased by ketoconazole, but M523595 production was inhibited only by quinidine. Gefitinib was metabolized extensively by expressed CYP3A4, producing a similar range of metabolites to liver microsomes, but M523595 was not formed. CYP1A2, 2C9 and 2C19 produced no measurable metabolism of gefitinib, while CYP3A5 produced a range of metabolites similar to CYP3A4, but to a much lower degree. In contrast, CYP2D6 catalysed rapid and extensive metabolism of gefitinib to M523595. While formation of M523595 was CYP2D6 mediated, the overall metabolism of gefitinib was dependent primarily on CYP3A4, and this was not obviously diminished in liver microsomes from CYP2D6 poor metabolizers.     

In vitro human metabolism and interactions of repellent N,N-diethyl-m-toluamide.

Oxidative metabolism of the insect repellent N,N-diethyl-m-toluamide (DEET) by pooled human liver microsomes (HLM), rat liver microsomes (RLM), and mouse liver microsomes (MLM) was investigated. DEET is metabolized by cytochromes P450 (P450s) leading to the production of a ring methyl oxidation product, N,N-diethyl-m-hydroxymethylbenzamide (BALC), and an N-deethylated product, N-ethyl-m-toluamide (ET). Both the affinities and intrinsic clearance of HLM for ring hydroxylation are greater than those for N-deethylation. Pooled HLM show significantly lower affinities (K(m)) than RLM for metabolism of DEET to either of the primary metabolites (BALC and ET). Among 15 cDNA-expressed P450 enzymes examined, CYP1A2, 2B6, 2D6*1 (Val(374)), and 2E1 metabolized DEET to the BALC metabolite, whereas CYP3A4, 3A5, 2A6, and 2C19 produced the ET metabolite. CYP2B6 is the principal cytochrome P450 involved in the metabolism of DEET to its major BALC metabolite, whereas CYP2C19 had the greatest activity for the formation of the ET metabolite. Use of phenotyped HLMs demonstrated that individuals with high levels of CYP2B6, 3A4, 2C19, and 2A6 have the greatest potential to metabolize DEET. Mice treated with DEET demonstrated induced levels of the CYP2B family, increased hydroxylation, and a 2.4-fold increase in the metabolism of chlorpyrifos to chlorpyrifos-oxon, a potent anticholinesterase. Preincubation of human CYP2B6 with chlorpyrifos completely inhibited the metabolism of DEET. Preincubation of human or rodent microsomes with chlorpyrifos, permethrin, and pyridostigmine bromide alone or in combination can lead to either stimulation or inhibition of DEET metabolism.     

Fully automated analysis of activities catalysed by the major human liver cytochrome P450 (CYP) enzymes: assessment of human CYP inhibition potential

1. Fully automated inhibition screens for the major human hepatic cytochrome P450s have been developed and validated. Probe assays were the fluorometric-based ethoxyresorufin O-deethylation for CYP1A2 and radiometric analysis of erythromycin N-demethylation for CYP3A4, dextromethorphan O-demethylation for CYP2D6, naproxen O-demethylation for CYP2C9 and diazepam N-demethylation for CYP2C19. For the radiometric assays > 99.7% of 14C-labelled substrate was routinely extracted from incubations by solid-phase extraction. 2. Furafylline, sulphaphenazole, omeprazole, quinidine and ketoconazole were identified as specific markers for the respective CYP1A2 (IC50 = 6 microM), CYP2C9 (0.7 microM), CYP2C19 (6 microM), CYP2D6 (0.02 microM) and CYP3A4 (0.2 microM) inhibition screens. 3. For the radiometric methods, a two-point IC50 estimate was validated by correlating the IC50 obtained with a full (seven-point) assay (r2 = 0.98, p < 0.001). The two-point IC50 estimate is useful for initial screening, while the full IC50 method provides more definitive quantitation, where required. 4. IC50 determined for a series of test compounds in human liver microsomes and cytochrome P450 cDNA-expressed enzymes were similar (r2 = 0.89, p < 0.001). In particular, the CYP1A2, CYP2D6 and CYP3A4 screens demonstrated the flexibility to accept either enzyme source. As a result of incomplete substrate selectivity, expressed enzymes were utilized for analysis of CYP2C9 and CYP2C19 inhibition. Good agreement was demonstrated between IC50 determined in these assays to IC50 published by other laboratories using a wide range of analytical techniques, which provided confidence in the universality of these inhibition screens. 5. These automated screens for initial assessment of P450 inhibition potential allow rapid determination of IC50. The radiometric assays are flexible, sensitive, robust and free from analytical interference, and they should permit the identification and eradication of inhibitory structural motifs within a series of potential drug candidates.     

Metabolism of tamoxifen by recombinant human cytochrome P450 enzymes: formation of the 4-hydroxy, 4'-hydroxy and N-desmethyl metabolites and isomerization of trans-4-hydroxytamoxifen.

The cytochrome P450 (P450)-mediated biotransformation of tamoxifen is important in determining both the clearance of the drug and its conversion to the active metabolite, trans-4-hydroxytamoxifen. Biotransformation by P450 forms expressed extrahepatically, such as in the breast and endometrium, may be particularly important in determining tissue-specific effects of tamoxifen. Moreover, tamoxifen may serve as a useful probe drug to examine the regioselectivity of different forms. Tamoxifen metabolism was investigated in vitro using recombinant human P450s. Forms CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7 were coexpressed in Escherichia coli with recombinant human NADPH-cytochrome P450 reductase. Bacterial membranes were harvested and incubated with tamoxifen or trans-4-hydroxytamoxifen under conditions supporting P450-mediated catalysis. CYP2D6 was the major catalyst of 4-hydroxylation at low tamoxifen concentrations (170 +/- 20 pmol/40 min/0.2 nmol P450 using 18 microM tamoxifen), but CYP2B6 showed significant activity at high substrate concentrations (28.1 +/- 0.8 and 3.1 +/- 0.5 nmol/120 min/0.2 nmol P450 for CYP2D6 and CYP2B6, respectively, using 250 microM tamoxifen). These two forms also catalyzed 4'-hydroxylation (13.0 +/- 1.9 and 1.4 +/- 0.1 nmol/120 min/0.2 nmol P450, respectively, for CYP2B6 and CYP2D6 at 250 microM tamoxifen; 0.51 +/- 0.08 pmol/40 min/0.2 nmol P450 for CYP2B6 at 18 microM tamoxifen). Tamoxifen N-demethylation was mediated by CYP2D6, 1A1, 1A2, and 3A4, at low substrate concentrations, with contributions by CYP1B1, 2C9, 2C19 and 3A5 at high concentrations. CYP1B1 was the principal catalyst of 4-hydroxytamoxifen trans-cis isomerization but CYP2B6 and CYP2C19 also contributed.     

Studies on the inhibition of human cytochromes P450 by selenocysteine Se-conjugates

1. To investigate whether cytochrome P450 (P450) inhibition can contribute to the chemopreventive activity of selenocysteine Se-conjugates (SeCys conjugates), 21 SeCys conjugates were screened for their inhibitory potency towards seven of the most important human P450s. 2. The majority of the SeCys conjugates produced near complete inhibition of CYP1A1 at a concentration of 250 microm. The most potent inhibitor, Se-benzyl-L-selenocysteine, displayed an IC50 of 12.8 +/- 1.2 microm. CYP2C9, -2C19 and -2D6 were moderately (50-60%) inhibited by the SeCys conjugates. CYP1A2, -2E1 and -3A4 were least inhibited. 3. Studies on the susceptibility of CYP1A1 to SeCys conjugates implicated a thiol-reactive intermediate, as evidenced by reduced inhibition levels in the presence of glutathione and N-acetyl cysteine. Uncoupling of the P450-catalytic cycle was of no importance as ROS scavengers did not influence inhibition levels. 4. P450 inhibition by two physiologically relevant metabolite classes of SeCys conjugates was also studied. N-acetylation of SeCys conjugates consistently increased the inhibitory potency towards CYP1A2, -2C19, -2E1 and -3A4. Beta-lyase catalysed bioactivation of alkyl-substituted SeCys conjugates or Se-benzyl-L-selenocysteine produced little or no additional inhibition of P450 activity. For Se-phenyl-L-selenocysteine, however, significant increases in P450 inhibition were obtained by beta-lyase pre-incubation. 5. It is concluded that the potent and relatively selective CYP1A1 inhibition exerted by SeCys conjugates may contribute to their chemopreventive activity.     

The oxidative metabolism of dimemorfan by human cytochrome P450 enzymes

To characterize the human cytochrome P450 (P450) forms involved in dimemorfan oxidation (DFO), human liver microsomes, and recombinant P450s were investigated. Liquid chromatography-mass spectral analysis suggested that metabolite (M)1 ([M + H]⁺ m/z at 272.200) and M2 ([M + H]⁺ m/z at 242.190) were d-3-hydroxymethyl-N-methylmorphinan and d-3-methylmorphinan, respectively. Kinetic analyses of microsomal DFO showed that the substrate concentration showing a half-maximal velocity (S₅₀) of M1 formation was less than that of M2. Microsomal M1 and M2 formation activities correlated significantly with the CYP2D6 marker, dextromethorphan O-demethylation activity. The M2 formation activity was also correlated with the CYP3A4 marker, nifedipine oxidation activity. Microsomal M1 and M2 formation was most sensitive to the inhibition by a CYP2D6 inhibitor, paroxetine and a CYP3A4 inhibitor, ketoconazole, respectively. The immunoinhibition-defined P450 contributions indicated the participation of CYP2C9, CYP2C19, and CYP2D6 in the M1 formation and CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 in the M2 formation. Among recombinant P450s, CYP2D6 had the highest intrinsic clearance with a K_m value of 0.02 mM in forming M1. CYP2B6, CYP2C9, and CYP2C19 had the K_m or S₅₀ values smaller than those (1 mM) of CYP2D6 and CYP3A4 in forming M2. These results indicated the participation of multiple P450 forms in DFO. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:1063–1077, 2010