Recombinant CYP2C9 Research Articles

EVALUATION OF TIME-DEPENDENT CYTOCHROME P450 INHIBITION USING CULTURED HUMAN HEPATOCYTES

Primary human hepatocytes in culture are commonly used to evaluate cytochrome P450 (P450) induction via an enzyme activity endpoint. However, other processes can confound data interpretation. To this end, the impact of time-dependent P450 inhibition in this system was evaluated. Using a substrate-cassette approach, P450 activities were determined after incubation with the prototypic inhibitors tienilic acid (CYP2C9), erythromycin, troleandomycin, and fluoxetine (CYP3A4). Kinetic analysis of enzyme inactivation in hepatocytes was used to describe the effect of these time-dependent inhibitors and derive the inhibition parameters kinact and KI) which generally were in good agreement with the values derived using recombinant P450s and human liver microsomes (HLMs). Tienilic acid selectively inhibited CYP2C9-dependent diclofenac 4'-hydroxylation activity, and erythromycin, troleandomycin, and fluoxetine inhibited CYP3A4-dependent midazolam 1'-hydroxylation in a time- and concentration-dependent manner. Fluoxetine also inhibited CYP2C19-dependent S-mephenytoin 4'-hydroxylation in a time- and concentration-dependent manner in hepatocytes, HLMs, and recombinant CYP2C19 (KI 0.4 microM and kinact 0.5 min(-1)). As expected, the effect of fluoxetine on CYP2D6 in hepatocytes was consistent with potent yet reversible inhibition. A very weak time-dependent CYP2C9 inhibitor (AZ1, a proprietary AstraZeneca compound; KI 30 microM and kinact 0.02 min(-1)) effectively abolished CYP2C9 activity over 24 h at low (micromolar) concentrations in primary cultured human hepatocytes. This work demonstrates that caution is warranted in the interpretation of enzyme induction studies with metabolically stable, weak time-dependent inhibitors, which may have dramatic inhibitory effects on P450 activity in this system. Therefore, in addition to enzyme activity, mRNA and/or protein levels should be measured to fully evaluate the P450 induction potential of a drug candidate.

Optimization of Recombinant Cytochrome P450 2C9 Protein Production in Escherichia coli DH5α by Statistically-Based Experimental Design

Cytochrome P450 (CYP) 2C9 is of major importance in drug metabolism. However, the low yield of recombinant CYP2C9 protein in E. coli strains prevents its extensive use in the study of in vitro drug metabolism. In the present study, Taguchi design and desirability function were first used to investigate the effect of medium components (glycerol, δ-ALA, IPTG, ampicillin, chloramphenicol, inoculum density, peptone, thiamine, trace elements, NH4Cl, and MgSO4) on recombinant human CYP2C9 production by E. coli DH5α. An L12 (211) orthogonal array was used to design the experiments to screen out the most influential factors. The CYP concentration and the specific content of CYP were considered as two product quality variables. A desirability function was applied to combine these two qualities as a single objective function. Optimization via central composite design (CCD) was then undertaken to yield the best performance. The confirmation experiments indicated that the expression performance under the optimized conditions was better than those obtained under other conditions. A compromise between conflicting goals, such as achievement of good yield of recombinant CYP2C9 and facility of the following purification, was found by means of the desirability function D. This is the first report that combined Taguchi design and CCD, and performed experiments in a multiresponse framework to optimize the production of human CYP in a recombinant E. coli strain.

Oxidative metabolism of 5-methoxy- N, N-diisopropyltryptamine (Foxy) by human liver microsomes and recombinant cytochrome P450 enzymes

In vitro quantitative studies of the oxidative metabolism of (5-methoxy- N, N-diisopropyltryptamine, 5-MeO-DIPT, Foxy) were performed using human liver microsomal fractions and recombinant CYP enzymes and synthetic 5-MeO-DIPT metabolites. 5-MeO-DIPT was mainly oxidized to O-demethylated (5-OH-DIPT) and N-deisopropylated (5-MeO-IPT) metabolites in pooled human liver microsomes. In kinetic studies, 5-MeO-DIPT O-demethylation showed monophasic kinetics, whereas its N-deisopropylation showed triphasic kinetics. Among six recombinant CYP enzymes (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) expressed in yeast or insect cells, only CYP2D6 exhibited 5-MeO-DIPT O-demethylase activity, while CYP1A2, CYP2C8, CYP2C9, CYP2C19 and CYP3A4 showed 5-MeO-DIPT N-deisopropylase activities. The apparent K m value of CYP2D6 was close to that for 5-MeO-DIPT O-demethylation, and the K m values of other CYP enzymes were similar to those of the low- K m (CYP2C19), intermediate- K m (CYP1A2, CYP2C8 and CYP3A4) and high- K m phases (CYP2C9), respectively, for N-deisopropylation in human liver microsomes. In inhibition studies, quinidine (1 μM), an inhibitor of CYP2D6, almost completely inhibited human liver microsomal 5-MeO-DIPT O-demethylation at a substrate concentration of 10 μM. Furafylline, a CYP1A2 inhibitor, quercetin, a CYP2C8 inhibitor, sulfaphenazole, a CYP2C9 inhibitor and ketoconazole, a CYP3A4 inihibitor (5 μM each) suppressed about 60%, 45%, 15% and 40%, respectively, of 5-MeO-DIPT N-deisopropylation at 50 μM substrate. In contrast, omeprazole (10 μM), a CYP2C19 inhibitor, suppressed only 10% of N-deisopropylation by human liver microsomes, whereas at the same concentration the inhibitor suppressed the reaction by recombinant CYP2C19 almost completely. These results indicate that CYP2D6 is the major 5-MeO-DIPT O-demethylase, and CYP1A2, CYP2C8 and CYP3A4 are the major 5-MeO-DIPT N-deisopropylase enzymes in the human liver.

Effect of Glimepiride and Glibenclamide on S-Warfarin 7-Hydroxylation by Human Liver Microsomes, Recombinant Human CYP2C9.1 and CYP2C9.3

The effect of glimepiride on metabolism of S-warfarin to 7-hydroxywarfarin was studied using human liver microsomes and recombinant cytochrome P450 2C9 microsomes (CYP2C9.1 and CYP2C9.3), and was compared with the results from the experiments using glibenclamide as an inhibitor. S-Warfarin 7-hydroxylation by recombinant CYP2C9.1 and CYP2C9.3 was inhibited by glimepiride competitively. The apparent K(i) value of glimepiride was lower at CYP2C9.3 than at CYP2C9.1. Glimepiride also inhibited 7-hydroxylation of S-warfarin in a competitive manner by microsomes from human liver which showed the genotypes of CYP2C9, as CYP2C9*1/*1 or CYP2C9*1/*3. The apparent K(i) value of glimepiride was lower than that of glibenclamide. These results may provide valuable information for optimizing the anticoagulant activity of warfarin when glimepiride is co-administered to patients.

Metabolism of Δ³-Carene by Human Cytochrome P450 Enzymes: Identification and Characterization of Two New Metabolites

The metabolism of the bicyclic monoterpene Delta(3)-carene was investigated in vitro using human liver microsomes as well as human smoker/non-smoker lung microsomes and 12 different recombinant cytochrome P450 enzymes coexpressed with human CYP-reductase in Escherichia coli cells. We detected two metabolites using GC-MS analysis. The mass fragmentation indicated for one metabolite hydroxylation in the allyl position and for the other metabolite epoxidation at the double bond. For clear identification the suggested metabolites were synthesized in a four-step reaction. Comparison of GC retention times and mass spectra lead to the identification of the metabolites as Delta(3)-carene-10-ol ((1S, 6R)-7,7-Dimethylbicyclo[4.1.0]hept-3-en-3-yl-methanol) and Delta(3)-carene-epoxide ((1S, 3S, 5R, 7R)-3,8,8-Trimethyl-4-oxa-tricyclo[5.1.0.0(3,5)]octane). Delta(3)-carene-10-ol was formed by human liver microsomes and recombinant human CYP2B6, CYP2C19 and CYP2D6. Delta(3)-Carene-epoxide was obviously catalyzed only by CYP1A2. In both cases there was a clear correlation between the metabolite formation, incubation time and enzyme concentration, respectively. Further kinetic analysis revealed that CYP2B6 exhibited the highest activity for Delta(3)-carene 10-hydroxylation. Michaelis-Menten K(m) and V(max) for oxidation of Delta(3)-carene were 0.6 mM and 28.4 nmol/min/nmol P450 using human CYP2B6. For the formation of Delta(3)-carene-epoxide 98.2 mM and 3.9 nmol/min/nmol P450 were determined as K(m) and V(max) by using human CYP1A2. To our knowledge, this is the first time that Delta(3)-carene-10-ol and Delta(3)-carene-epoxide are described as human metabolites of Delta(3)-carene.

GLUCURONIDATION CONVERTS GEMFIBROZIL TO A POTENT, METABOLISM-DEPENDENT INHIBITOR OF CYP2C8: IMPLICATIONS FOR DRUG-DRUG INTERACTIONS

Gemfibrozil more potently inhibits CYP2C9 than CYP2C8 in vitro, and yet the opposite inhibitory potency is observed in the clinic. To investigate this apparent paradox, we evaluated both gemfibrozil and its major metabolite, an acyl-glucuronide (gemfibrozil 1-O-beta-glucuronide) as direct-acting and metabolism-dependent inhibitors of the major drug-metabolizing cytochrome P450 enzymes (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) in human liver microsomes. Gemfibrozil most potently inhibited CYP2C9 (IC50 of 30 microM), whereas gemfibrozil glucuronide most potently inhibited CYP2C8 (IC50 of 24 microM). Unexpectedly, gemfibrozil glucuronide, but not gemfibrozil, was found to be a metabolism-dependent inhibitor of CYP2C8 only. The IC50 for inhibition of CYP2C8 by gemfibrozil glucuronide decreased from 24 microM to 1.8 microM after a 30-min incubation with human liver microsomes and NADPH. Inactivation of CYP2C8 by gemfibrozil glucuronide required NADPH, and proceeded with a K(I) (inhibitor concentration that supports half the maximal rate of enzyme inactivation) of 20 to 52 microM and a k(inact) (maximal rate of inactivation) of 0.21 min(-1). Potent inhibition of CYP2C8 was also achieved by first incubating gemfibrozil with alamethicin-activated human liver microsomes and UDP-glucuronic acid (to form gemfibrozil glucuronide), followed by a second incubation with NADPH. Liquid chromatography-tandem mass spectrometry analysis established that human liver microsomes and recombinant CYP2C8 both convert gemfibrozil glucuronide to a hydroxylated metabolite, with oxidative metabolism occurring on the dimethylphenoxy moiety (the group furthest from the glucuronide moiety). The results described have important implications for the mechanism of the clinical interaction reported between gemfibrozil and CYP2C8 substrates such as cerivastatin, repaglinide, rosiglitazone, and pioglitazone.

Metabolism of Repaglinide by CYP2C8 and CYP3A4 in vitro: Effect of Fibrates and Rifampicin

Repaglinide is an antidiabetic drug metabolised by cytochrome P450 (CYP) 2C8 and CYP3A4 enzymes. To clarify the mechanisms of observed repaglinide drug interactions, we determined the contribution of the two enzymes to repaglinide metabolism at different substrate concentrations, and examined the effect of fibrates and rifampicin on CYP2C8, CYP3A4 and repaglinide metabolism in vitro. We studied repaglinide metabolism using pooled human liver microsomes, recombinant CYP2C8 and recombinant CYP3A4 enzymes. The effect of quercetin and itraconazole on repaglinide metabolism, and of gemfibrozil, bezafibrate, fenofibrate and rifampicin on CYP2C8 (paclitaxel 6alpha-hydroxylation) and CYP3A4 (midazolam 1-hydroxylation) activities and repaglinide metabolism were studied using human liver microsomes. At therapeutic repaglinide concentrations (<0.4 microM), CYP2C8 and CYP3A4 metabolised repaglinide at similar rates. Quercetin (25 microM) and itraconazole (3 microM) inhibited the metabolism of 0.2 microM repaglinide by 58% and 71%, and that of 2 microM repaglinide by 56% and 59%, respectively. The three fibrates inhibited CYP2C8 (Ki: bezafibrate 9.7 microM, gemfibrozil 30.4 microM and fenofibrate 92.6 microM) and repaglinide metabolism (IC50: bezafibrate 37.7 microM, gemfibrozil 111 microM and fenofibrate 164 microM), but had no effect on CYP3A4. Rifampicin inhibited CYP2C8 (Ki 30.2 microM), CYP3A4 (Ki 18.5 microM) and repaglinide metabolism (IC50 13.7 microM). In conclusion, both CYP2C8 and CYP3A4 are important in the metabolism of therapeutic concentrations of repaglinide in vitro, but their predicted contributions in vivo are highly dependent on the scaling factor used. Gemfibrozil is only a moderate inhibitor of CYP2C8 and does not inhibit CYP3A4; inhibition of CYP-enzymes by parent gemfibrozil alone does not explain its interaction with repaglinide in vivo. Rifampicin competitively inhibits both CYP2C8 and CYP3A4, which can counteract its inducing effect in humans.

Enantiomer/Enantiomer Interactions between theS- andR- Isomers of Omeprazole in Human Cytochrome P450 Enzymes: Major Role of CYP2C19 and CYP3A4

We investigated the enzyme kinetic basis for the stereoselective disposition of R- and S-omeprazole (OME) and racemic OME in human liver microsomes. OME is primarily metabolized by the hepatic cytochrome P450 enzyme system (CYP2C19 and 3A4). The metabolism of each enantiomer and pseudoracemic OME was studied using unlabeled and (13)C(7)-labeled enantiomers. The enantiomers inhibited each other's metabolism competitively in human liver microsomes and in recombinant CYP2C19 and 3A4. The results obtained with the individual enantiomers allowed successful prediction of the enzyme kinetics for the pseudoracemate. The intrinsic clearance of each enantiomer in a pseudoracemic mixture remained the same as those of the individually incubated enantiomers, although K(m) and V(max) decreased. In the pseudoracemate, the relative contribution of CYP2C19 and 3A4 to 5-hydroxylation and 5'-O-demethylation of R-OME was comparable to that obtained for the incubation of R-OME alone. For S-OME, however, the presence of its antipode greatly increased the contribution of CYP3A4, with increasing concentrations, compared with that obtained when incubating S-OME alone. The results of our in vitro study clearly show metabolic interactions between the OME enantiomers, which may also occur in vivo. Because the enantiomers of OME produce similar pharmacological effects, the enantiomer interactions should not significantly affect the pharmacodynamics. On the other hand, the use of the S-enantiomer results in less complex enzyme kinetics than those of the racemate; thus, the outcome of its clinical use is more predictable.

Identification of human liver cytochrome P450 enzymes involved in the metabolism of SCH 351125, a CCR5 antagonist

The identification and relative contribution of human cytochrome P450 enzyme(s) involved in the metabolism of SCH 351125 were investigated. In human liver microsomes, O-deethylation was the major metabolic pathway, whereas aromatization of a piperidine ring to pyridine and the reduction of the N-oxide moiety were minor routes. Recombinant human CYP3A4 and CYP2C9 both exhibited catalytic activity with respect to the formation of rotameric O-deethylated metabolites (M12, M13), the metabolites resulting from aromatization (M22/M24) and N-oxide reduction (M31). Using the relative activity factor (RAF) approach, the relative contributions of CYP3A4 and CYP2C9 to M13 formation were estimated to be 76 and 24%, respectively. There was a high correlation (r > 0.96) between the rate of formation of M12 and M13 and 6β-hydroxylation of testosterone catalysed by CYP3A4/5. Ketoconazole (2 µM) and CYP3A4/5-specific inhibitory monoclonal antibody inhibited the formation of M12 and M13 from human liver microsomes by approximately 60 and 71%, respectively. The results demonstrate that the in vitro metabolism of SCH 351125 is mediated primarily via CYP3A4 and that CYP2C9 plays a minor role. Clinical study designs should encompass these enzymology data to address any potential drug interactions.

Role of Human Liver Cytochrome P450 2C9 in the Metabolism of a Novel α4β1/α4β7 Dual Antagonist, TR-14035

The metabolism of a novel dual antagonist for alpha4beta1/alpha4beta7 integrin, TR-14035, and the role of polymorphic enzyme responsible for this metabolism were investigated. Human liver microsomes catalyzed the NADPH-dependent metabolism of TR-14035 to a primary metabolite, O-desmethyl TR-14035. This formation was completely blocked by both sulfaphenazole, a selective CYP2C9 inhibitor, and CYP2C9 antibody, whereas potent inhibitors selective for other CYPs exhibited little effects. Of 12 recombinant CYPs examined, O-desmethyl metabolite was principally formed by CYP2C9. CYP1A1, an extrahepatic enzyme, also had this activity (about one-fourth of CYP2C9). Utilizing recombinant CYP2C9*1, K(m) and V(max)/K(m) values of 23.3 microM and 0.284 microL/min/pmol CYP2C9, respectively, were obtained for the O-desmethyl formation, which were quite similar to those in CYP2C9*2 enzyme. In contrast, V(max)/K(m) value in recombinant CYP2C9*3 was approximately one-sixth of CYP2C9*1 and *2. In agreement, kinetics studies using human liver microsomes with CYP2C9*1/*1, *2/*2 and *3/*3 genotypes revealed that the V(max)/K(m) value in *2/*2 microsomes was comparable to that in wild type microsomes, in contrast, that in *3/*3 microsomes was reduced. These results demonstrate CYP2C9 is a primary enzyme mediating the O-desmethylation of TR-14035 in human liver. In homozygotes of CYP2C9*3, the metabolic clearance of TR-14035 should be decreased compared with homozygotes of CYP2C9*1 or 2.

Examination of 209 Drugs for Inhibition of Cytochrome P450 2C8

Cytochrome P450 2C8 is involved in the metabolism of drugs such as paclitaxel, repaglinide, rosiglitazone, and cerivastatin, among others. An in vitro assessment of 209 frequently prescribed drugs and related xenobiotics was carried out to examine their potential to inhibit CYP2C8. A validated sensitive, moderate-throughput high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) assay was used to detect N-desethylamodiaquine, the CYP2C8-derived major metabolite of amodiaquine metabolism, using heterologously expressed recombinant CYP2C8 (rhCYP2C8) and pooled human liver microsomes. The 209 drugs were first tested at 30 muM for their ability to inhibit rhCYP2C8. Forty-eight compounds exhibited greater than 50% inhibition and were further evaluated for measurement of IC50. The six most potent inhibitors (IC50 <1 microM) from this set were measured for IC50 in pooled human liver microsomes, and the most potent inhibitor identified was the leukotriene receptor antagonist, montelukast (IC50 = 19.6 nM). Inhibitors of CYP2C8 were identified from a wide variety of therapeutic classes, with no single class predominating. Other potent inhibitors included candesartan cilexetil (cyclohexylcarbonate ester prodrug of candesartan), zafirlukast, clotrimazole, felodipine, and mometasone furoate. Seventeen moderate inhibitors of rhCYP2C8 (1 < IC50 < 10 microM) included salmeterol, raloxifene, fenofibrate, ritonavir, levothyroxine, tamoxifen, loratadine, quercetin, oxybutynin, medroxyprogesterone, simvastatin, ketoconazole, ethinyl estradiol, spironolactone, lovastatin, nifedipine, and irbesartan. These in vitro data were used along with clinical pharmacokinetic information in predicting potential drug-drug interactions that could occur by inhibition of CYP2C8. Although almost all drugs tested are not expected to cause drug interactions via inhibition of CYP2C8, montelukast was identified as being of concern as a potential inhibitor of clinical relevance. These findings are discussed in context to potential drug interactions that could be observed between these agents and drugs for which CYP2C8 is involved in metabolism and warrant investigation of the possibility of clinical drug interactions mediated by inhibition of this enzyme.

LANSOPRAZOLE ENANTIOMER ACTIVATES HUMAN LIVER MICROSOMAL CYP2C9 CATALYTIC ACTIVITY IN A STEREOSPECIFIC AND SUBSTRATE-SPECIFIC MANNER

We recently proposed a possible stereoselective activation by lansoprazole of CYP2C9-catalyzed tolbutamide hydroxylation, as well as stereoselective inhibition of several cytochrome P450 (P450) isoforms. This study evaluated the effects of lansoprazole enantiomers on CYP2C9 activity in vitro, using several probe substrates. For tolbutamide 4-methylhydroxylation and phenytoin 4-hydroxylation, R-lansoprazole was an activator (140 and 550% of control at 100 microM R-lansoprazole, EC50 values of 19.9 and 30.2 microM, respectively). R-Lansoprazole-mediated activation of the formation of 4-hydroxyphenytoin was also seen with recombinant human CYP2C9. R-Lansoprazole increased the Michaelis-Menten-derived V(max) of phenytoin 4-hydroxylation from 0.024 to 0.121 pmol/min/pmol P450, and lowered its K(m) from 20.5 to 15.0 microM, suggesting that R-lansoprazole activates CYP2C9-mediated phenytoin metabolism without displacing phenytoin from the active site. Kinetic parameters were also estimated using the two-site binding equation, with alpha values <1 and beta values >1, indicative of activation. Additionally, phenytoin at 10 to 200 microM had no reciprocal effect on the hydroxylation of R-lansoprazole. Meanwhile, R-lansoprazole had no activation effect on diclofenac and S-warfarin metabolism in the incubation study using both recombinant CYP2C9 and human liver microsomes. These substrate-dependent activation effects suggest that phenytoin has a different binding orientation compared with diclofenac and S-warfarin. Overall, these results suggest that R-lansoprazole activates CYP2C9 in a stereospecific and substrate-specific manner, possibly by binding within the active site and inducing positive cooperativity. This is the first report to describe stereoselective activation of this cytochrome P450 isoform.

Genetic polymorphisms of cytochrome P450 2C9 causing reduced phenprocoumon (S)-7-hydroxylation in vitro and in vivo

The effect of cytochrome P450 (CYP) 2C9 polymorphisms on the stereoselective biotransformation of the oral anticoagulant phenprocoumon (PPC) to inactive, monohydroxylated metabolites was studied in vitro and in vivo.In human liver microsomes, the (S)-7-hydroxylation — being the major metabolic pathway — was significantly compromised in a gene–dose-dependent manner in samples expressing the CYP2C9*2 or CYP2C9*3 allele. The CYP2C9*3/*3 genotype corresponded to an almost fourfold lower (S)-7-hydroxylation rate than CYP2C9*1/*1 (wild-type).The intrinsic clearance of human recombinant CYP2C9*2 and CYP2C9*3 for the (S)-7-hydroxylation was 28.9 and 50.9% lower than of CYP2C9*1, respectively.The area under the plasma concentration–time curve (AUC) of PPC metabolites after oral intake of 12 mg racemic PPC was significantly lower in volunteers expressing the CYP2C9*2 or CYP2C9*3 allele. Increasing plasma AUC metabolic ratios (parent compound/metabolite) in CYP2C9*2 and CYP2C9*3 variant allele carriers were found for each hydroxylation reaction and the CYP2C9*3/*3 genotype corresponded to an about 10-fold higher metabolic ratio of PPC (S)-7-hydroxylation relative to CYP2C9*1/*1.CYP2C9 polymorphisms cause a markedly compromised PPC (S)-7-hydroxylation. However, PPC metabolism appears overall less influenced by CYP2C9 genotype compared with other oral anticoagulants and it may thus be a valuable alternative for therapeutic anticoagulation of patients expressing CYP2C9 variant alleles.

Mechanism-based inactivation of human cytochrome P4502C8 by drugs in vitro.

Studies were conducted to evaluate the potential mechanism-based inactivation of recombinant and human liver microsomal CYP2C8 by clinically used drugs. Several tricyclic antidepressants, calcium channel blockers, monoamine oxidase inhibitors, and various other known CYP3A4 inhibitors exhibited greater inhibition of CYP2C8 (paclitaxel 6alpha-hydroxylation) following preincubation, consistent with mechanism-based inactivation. Inactivation of recombinant CYP2C8 by phenelzine, amiodarone, verapamil, nortriptyline, fluoxetine, and isoniazid was of the pseudo-first order type and was characterized by respective inactivation kinetic constants (KI and kinact) of 1.2 microM and 0.243 min(-1), 1.5 microM and 0.079 min(-1), 17.5 microM and 0.065 min(-1), 49.9 microM and 0.036 min(-1), 294 microM and 0.083 min(-1), and 374 microM and 0.042 min(-1). Spectral scanning of recombinant CYP2C8 demonstrated the formation of metabolite-intermediate complexes with verapamil, nortriptyline, fluoxetine, and isoniazid, but not amiodarone. In contrast, inactivation by phenelzine resulted from heme destruction by free radicals. Studies with human liver microsomes (HLMs) revealed that nortriptyline, verapamil, and fluoxetine were not mechanism-based inactivators (MBIs) of CYP2C8. Simultaneous inactivation of CYP2C8 and CYP3A4 (paclitaxel 3'-phenyl-hydroxylation) was observed using amiodarone, isoniazid, and phenelzine with the efficiency of inactivation greater for the CYP3A4 pathway. With the exception of phenelzine, glutathione and superoxide dismutase failed to protect CYP2C8 (recombinant and HLMs) or CYP3A4 from inactivation by MBIs. However, the alternate CYP2C8 substrate, torsemide, prevented CYP2C8 inactivation in all cases. These data are consistent with mechanism-based inactivation of CYP2C8 by a range of commonly prescribed drugs, several of which have been implicated in clinically important drug-drug interactions.

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.

High-Throughput Screening Assays for the Assessment of CYP2C9*1, CYP2C9*2, and CYP2C9*3 Metabolism Using Fluorogenic Vivid® Substrates

CYP2C9 is a genetically polymorphic human cytochrome P450 isozyme involved in the oxidative metabolism of many drugs, including nonsteroidal anti-inflammatory compounds. Individuals genotyped heterozygous or homozygous for CYP2C9 allelic variants have demonstrated altered metabolism of some drugs primarily metabolized by CYP2C9. The ability to expand screening of CYP2C9 allelic variants to a larger set of drugs and pharmaceutical agents would contribute to a better understanding of the significance of CYP2C9 polymorphisms in the population and to predictions of possible outcomes. The authors report the development of an in vitro fluorescence-based assay employing recombinant CYP2C9 variants (CYP2C9*1, CYP2C9*2, and CYP2C9*3) and fluorogenic Vivid(R) CYP2C9 substrates to explore the effects of CYP2C9 polymorphisms on drug metabolism, using drugs primarily metabolized by CYP2C9. Several chemically diverse fluorogenic substrates (Vivid(R) CYP2C9 blue, green, and red substrates) were used as prototypic probes to obtain in vitro CYP2C9 metabolic rates and kinetic parameters, such as apparent K(m), V(max), and V(max)/K(m) ratios for each allelic variant. In addition, a diverse panel of drugs was screened as assay modifiers with CYP2C9*1, CYP2C9*2, CYP2C9*3, and the fluorogenic Vivid(R) CYP2C9 substrates. The inhibitory potential of this large group of chemically diverse drugs and compounds has been assessed on the basis of their ability to compete with Vivid(R) CYP2C9 substrates in fluorescent reporter assays, thus providing a sensitive and quick assessment of polymorphism-dependent changes in CYP2C9 metabolism.

Active-site characteristics of CYP2C19 and CYP2C9 probed with hydantoin and barbiturate inhibitors

Three series of N-3 alkyl substituted phenytoin, nirvanol, and barbiturate derivatives were synthesized and their inhibitor potencies were tested against recombinant CYP2C19 and CYP2C9 to probe the interaction of these ligands with the active sites of these enzymes. All compounds were found to be competitive inhibitors of both enzymes, although the degree of inhibitory potency was generally much greater towards CYP2C19. Inhibitor stereochemistry did not markedly influence K i towards CYP2C9, and log P adequately predicted inhibitor potency for this enzyme. In contrast, stereochemistry was an important factor in determining inhibitor potency towards CYP2C19. ( S)-(+)- N-3-Benzylnirvanol and ( R)-(−)- N-3-benzylphenobarbital emerged as the most potent and selective CYP2C19 inhibitors, with K i values of <250 nM—at least two orders of magnitude greater inhibitor potency than towards CYP2C9. Both inhibitors were metabolized preferentially at their C-5 phenyl substituents, indicating that CYP2C19 prefers to orient the N-3 substituents away from the active oxygen species. These features were incorporated into expanded CoMFA models for CYP2C9, and a new, validated CoMFA model for CYP2C19.

Identification and relative contributions of human cytochrome P450 isoforms involved in the metabolism of glibenclamide and lansoprazole: evaluation of an approach based on the in vitro substrate disappearance rate

1. The identification and relative contributions of human cytochrome P450 (CYP) enzymes involved in the metabolism of glibenclamide and lansoprazole in human liver microsomes were investigated using an approach based on the in vitro disappearance rate of unchanged drug.2. Recombinant CYP2C19 and CYP3A4 catalysed a significant disappearance of both drugs. When the contribution of CYPs to the intrinsic clearance (CLint) of drugs in pooled human microsomes was estimated by relative activity factors, contributions of CYP2C19 and CYP3A4 were determined to be 4.6 and 96.4% for glibenclamide, and 75.1 and 35.6% for lansoprazole, respectively.3. CLint of glibenclamide correlated very well with CYP3A4 marker activity, whereas the CLint of lansoprazole significantly correlated with CYP2C19 and CYP3A4 marker activities in human liver microsomes from 12 separate individuals. Effects of CYP-specific inhibitors and anti-CYP3A serum on the CLint of drugs in pooled human liver microsomes reflected the relative contributions of CYP2C19 and CYP3A4.4. The results suggest that glibenclamide is mainly metabolized by CYP3A4, whereas lansoprazole is metabolized by both CYP2C19 and CYP3A4 in human liver microsomes. This approach, based on the in vitro drug disappearance rate, is useful for estimating CYP identification and their contribution to drug discovery.

Identification of cytochromes P450 2C9 and 3A4 as the major catalysts of phenprocoumon hydroxylation in vitro.

This in-vitro study aimed at an identification of cytochrome P(450) (CYP) enzymes catalysing the (S)- and (R)-hydroxylation of the widely used anticoagulant phenprocoumon (PPC) to its major, inactive metabolites. Relevant catalysts were identified by kinetic, correlation and inhibition experiments using human liver microsomes and recombinant enzymes. Kinetics revealed (S)-7-hydroxylation as quantitatively most important. Biphasic Eadie-Hofstee plots indicated more than one catalyst for the 4'-, 6- and 7-hydroxylation of both enantiomers with mean K(m1) and K(m2) of 144.5+/-34.9 and 10.0+/-6.49 microM, respectively. PPC hydroxylation rates were significantly correlated with CYP2C9 and CYP3A4 activity and expression analysing 11 different CYP-specific probes. Complete inhibition of PPC hydroxylation was achieved by combined addition of the CYP3A4-specific inhibitor triacetyloleandomycin (TAO) and a monoclonal, inhibitory antibody (mAb) directed against CYP2C8, 9, 18 and 19, except for the (R)-4'-hydroxylation that was, however, inhibited by ~80% using TAO alone. (S)-PPC hydroxylation was reduced by approximately 2/3 and approximately 1/3 using mAb2C8-9-18-19 and TAO, respectively, but (R)-6- and 7-hydroxylation by approximately 50% each. Experiments with mAbs directed against single CYP2C enzymes clearly indicated CYP2C9 as a major catalyst of the 6- and 7-hydroxylation for both enantiomers. However, CYP2C8 was equally important regarding the (S)-4'-hydroxylation. Recombinant CYP2C8 and CYP2C9 were high-affinity catalysts (K(m) <5 microM), whereas CYP3A4 operated with low affinity (K(m) >100 microM). CYP2C9 and CYP3A4 are major catalysts of (S)- and (R)-PPC hydroxylation, while CYP2C8 partly catalysed the (S)-4'-hydroxylation. Increased vigilance is warranted when PPC treatment is combined with substrates, inhibitors, or inducers of these enzymes.

The metabolism of diclofenac--enzymology and toxicology perspectives.

Diclofenac is a nonsteroidal anti-inflammatory drug bearing a carboxylic acid functional group. As a result, the metabolism of diclofenac in humans partitions between acyl glucuronidation and phenyl hydroxylation, with the former reaction catalyzed primarily by uridine 5'-diphosphoglucuronosyl transferase 2B7 while the latter is catalyzed by cytochrome P450 (CYP)2C9 and 3A4. Further hydroxylation of diclofenac glucuronide was shown to occur in vitro with recombinant CYP2C8, which may be of clinical significance in terms of defining major metabolic routes involved in the elimination of diclofenac in humans. The 4'-hydroxylation of the drug appears to represent a feature reaction for CYP2C9 catalysis, and this regioselective oxidation is presumably dictated by interactions of the carboxylate moiety of the substrate with a putative cationic residue of the enzyme. Several other residues of CYP2C9 were identified in studies with site-directed mutants that influence substrate binding affinity and specificity, including Arg97, Phe114, Asn289 and Ser286. The 5-hydroxylation of diclofenac is subject to CYP3A4 cooperativity elicited by quinidine. In this case, enhancement by quinidine of diclofenac metabolism in vitro was attributed to increases in the V(max) with little contribution from changes in the K(m) value. These cooperative interactions in recombinant systems, however, appeared to be influenced by enzyme host membranes of various cDNA-directed expressing CYP3A4. Nevertheless, the in vivo significance of CYP3A cooperativity was demonstrated in a pharmacokinetic study in monkeys, wherein the hepatic clearance of diclofenac increased 2-fold when quinidine was co-administered. Therapeutic use of diclofenac is associated with rare but sometimes fatal hepatotoxicity characterized by delayed onset of symptoms and lack of a clear dose-response relationship. The toxicity has consequently been categorized as metabolic idiosyncrasy. In this regard, the acyl glucuronide of the drug was demonstrated to be reactive and capable of covalent modification of cellular proteins, with covalent binding to liver proteins in rats depending on the activity of multidrug resistance protein 2, a hepatic canalicular transporter. One of the modified proteins was identified as dipeptidyl peptidase IV. Formation of protein adducts also was evident following the oxidative metabolism of diclofenac catalyzed by CYP enzymes. The reactive intermediates in this case were presumably diclofenac 1',4'- and 2,5-quinone imines, both of which were trapped by conjugation with glutathione and identified as glutathione adducts. These same glutathione adducts were detected in rats as well as in human hepatocytes treated with diclofenac, and a corresponding mercapturic acid derivative was identified in urine from patients administered the drug. It is conceivable that the acyl glucuronide and benzoquinone imines derived from diclofenac modify proteins covalently and thereby produce toxicity in susceptible patients via either direct disruption of critical cellular functions or elicitation of immunological responses.