Approach to the prediction of the contribution of major cytochrome P450 enzymes to drug metabolism in the early drug-discovery stage

It is important to determine the cytochrome P450 (CYP) contribution of certain drugs by taking into consideration the attrition due to issues such as genetic polymorphism and inter-individual variation. In many cases in the early discovery stage, the metabolites of a new chemical have not been identified. Therefore, the present paper devised an approach in which the in vitro intrinsic clearance (CL_int) value for new chemicals was determined by measuring substrate depletion. The following prediction methods were compared to calculate CL_int using data from recombinant CYP enzymes: (1) the relative CYP content in human liver microsomes; (2) the relative activity factor (RAF) based on the V_max value; and (3) the RAF value based on the CL_int value. The most accurate prediction method was RAF based on CL_int. This method would be useful in the early drug-discovery process in cases in which the main metabolite is not identified.     

Approach to predict the contribution of cytochrome P450 enzymes to drug metabolism in the early drug-discovery stage: the effect of the expression of cytochrome b(5) with recombinant P450 enzymes

In order to evaluate the potential adverse effects due to genetic polymorphism and/or inter-individual variation, it is necessary to calculate the cytochrome P450 (CYP) contribution to the metabolism of new drugs. In the current study, the in vitro intrinsic clearance (CL(int)) values of marker substrates and drugs were determined by measuring metabolite formation and substrate depletion, respectively. Recombinant CYP microsomes expressing CYP2C9, CYP2C19 and CYP3A4 with co-expressed cytochrome b(5) were used, but those expressing CYP1A2 and CYP2D6 did not have co-expressed cytochrome b(5). The following prediction methods were compared to determine the CL(int) value using data from recombinant CYP enzymes: (1) relative CYP enzyme content in human liver microsomes; (2) relative activity factor (RAF) estimated from the V(max) value; and (3) RAF estimated from the CL(int) value. Estimating RAF from CL(int) proved the most accurate prediction method among the three tested, and differences in the CYP3A4 marker reactions did not affect its accuracy. The substrate depletion method will be useful in the early drug-discovery stage when the main metabolite and/or metabolic pathway has not been identified. In addition, recombinant CYP microsomes co-expressed with cytochrome b(5) might be suitable for the prediction of the CL(int) value.     

Approach to the prediction of the contribution of major cytochrome P450 enzymes to drug metabolism in the early drug-discovery stage

It is important to determine the cytochrome P450 (CYP) contribution of certain drugs by taking into consideration the attrition due to issues such as genetic polymorphism and inter-individual variation. In many cases in the early discovery stage, the metabolites of a new chemical have not been identified. Therefore, the present paper devised an approach in which the in vitro intrinsic clearance (CLint) value for new chemicals was determined by measuring substrate depletion. The following prediction methods were compared to calculate CLint using data from recombinant CYP enzymes: (1) the relative CYP content in human liver microsomes; (2) the relative activity factor (RAF) based on the Vmax value; and (3) the RAF value based on the CLint value. The most accurate prediction method was RAF based on CLint. This method would be useful in the early drug-discovery process in cases in which the main metabolite is not identified.     

Contribution of cytochrome P450 3A4 and 3A5 to the metabolism of atorvastatin

Atorvastatin is a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor that is mainly metabolized by cytochrome P450 (CYP) 3A4. A recent study showed that the lipid-lowering effect of statins is affected by the CYP3A5 polymorphism. Therefore, it was investigated whether CYP3A5 contributes to the metabolism of atorvastatin. Two metabolites of atorvastatin, para- and ortho-hydroxyatorvastatin, were produced by human liver microsomes and human recombinant CYP3A enzymes, and the enzyme kinetic pattern exhibited substrate inhibition. The intrinsic clearance (CL_int) rates of para- and ortho-hydroxyatorvastatin by CYP3A4 were 2.4- and 5.0-fold of the respective CL_int rates of CYP3A5, indicating that CYP3A4 is the major P450 isoform responsible for atorvastatin metabolism. These results suggest that atorvastatin is preferentially metabolized by CYP3A4 rather than by CYP3A5, and thus the genetic CYP3A5 polymorphism might not be an important factor in the inter-individual variation of atorvastatin disposition and pharmacodynamics in human.     

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 (CL_int) 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.?CL_int of glibenclamide correlated very well with CYP3A4 marker activity, whereas the CL_int 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 CL_int 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.     

Contributions of human cytochrome P450 enzymes to glyburide metabolism

Glyburide (GLB) is a widely used oral sulfonylurea for the treatment of gestational diabetes. The therapeutic use of GLB is often complicated by a substantial inter‐individual variability in the pharmacokinetics and pharmacodynamics of the drug in human populations, which might be caused by inter‐individual variations in factors such as GLB metabolism. Therefore, there has been a continued interest in identifying human cytochrome P450 (CYP) isoforms that play a major role in the metabolism of GLB. However, contrasting data are available in the present literature in this regard. The present study systematically investigated the contributions of various human CYP isoforms (CYP3A4, CYP3A5, CYP2C8, CYP2C9 and CYP2C19) to in vitro metabolism of GLB. GLB depletion and metabolite formation in human liver microsomes were most significantly inhibited by the CYP3A inhibitor ketoconazole compared with the inhibitors of other CYP isoforms. Furthermore, multiple correlation analysis between GLB depletion and individual CYP activities was performed, demonstrating a significant correlation between GLB depletion and the CYP3A probe activity in 16 individual human liver microsomal preparations, but not between GLB depletion and the CYP2C19, CYP2C8 or CYP2C9 probe activity. By using recombinant supersomes overexpressing individual human CYP isoforms, it was found that GLB could be depleted by all the enzymes tested; however, the intrinsic clearance (V_max/K_m) of CYP3A4 for GLB depletion was 4–17 times greater than that of other CYP isoforms. These results confirm that human CYP3A4 is the major enzyme involved in the in vitro metabolism of GLB. Copyright © 2010 John Wiley & Sons, Ltd.     

Metabolism of deltamethrin and cis- and trans-permethrin by human expressed cytochrome P450 and carboxylesterase enzymes

1. The metabolism of the pyrethroids deltamethrin (DLM), cis-permethrin (CPM) and trans-permethrin (TPM) was studied in human expressed cytochrome P450 (CYP) and carboxylesterase (CES) enzymes.

2. DLM, CPM and TPM were metabolised by human CYP2B6 and CYP2C19, with the highest apparent intrinsic clearance (CL_int) values for pyrethroid metabolism being observed with CYP2C19. Other CYP enzymes contributing to the metabolism of one or more of the three pyrethroids were CYP1A2, CYP2C8, CYP2C9*1, CYP2D6*1, CYP3A4 and CYP3A5. None of the pyrethroids were metabolised by CYP2A6, CYP2E1, CYP3A7 or CYP4A11.

3. DLM, CPM and TPM were metabolised by both human CES1 and CES2 enzymes.

4. Apparent CL_int values for pyrethroid metabolism by CYP and CES enzymes were scaled to per gram of adult human liver using abundance values for microsomal CYP enzymes and for CES enzymes in liver microsomes and cytosol. TPM had the highest and CPM the lowest apparent CL_int values for total metabolism (CYP and CES enzymes) per gram of adult human liver.

5. Due to their higher abundance, all three pyrethroids were extensively metabolised by CES enzymes in adult human liver, with CYP enzymes only accounting for 2%, 10% and 1% of total metabolism for DLM, CPM and TPM, respectively.

     

Assessment of multiple cytochrome P450 activities in metabolically inactivated human liver microsomes and roles of P450 2C isoforms in reaction phenotyping studies

The fraction of substrate metabolized (f_m) can be used to estimate drug interactions and can be determined by comparison of the intrinsic clearances (CL_int) of victim drugs obtained from inhibited and uninhibited hepatic enzymes_. Commercially available human liver microsomes were recently developed in which one cytochrome P450 (P450) isoform is selectively inactivated. These inactivated liver microsomes were used to evaluate the roles of P450 2C isoforms in the depletion and oxidation of probe substrates. Determination of CL_int with sets of control and P450 2C9‐inactivated liver microsomes yielded f_{m,P450 2C9} values of 0.69–1.0 for celecoxib, diclofenac and warfarin. Apparent minor contributions of P450 1A2/2C8/3A4 were seen in depletion assays, yielding ~1 for the sum of the f_m values. Selectively inactivated liver microsomes were thereby shown to be potentially useful for determining the in vitro f_m values for major P450 2C9 contributions to substrate oxidations. Metabolite formations from diclofenac and warfarin were suppressed by 62–84% by the replacement of control liver microsomes with P450 2C9‐inactivated liver microsomes. R‐, S‐ and racemic omeprazole and troglitazone oxidation activities by liver microsomes at multiple substrate concentrations were suppressed by 26–36% and 22–50%, respectively, when P450 2C19‐ and 2C8‐inactivated liver microsomes were used in place of control liver microsomes. This study provides important information to help elucidate the different roles of P450 isoforms in metabolite formation at different substrate concentrations. The data obtained allow the fractions metabolized to be calculated for victim drugs.     

Species differences in hepatic and intestinal metabolic activities for 43 human cytochrome P450 substrates between humans and rats or dogs

Abstract

1. Prediction of human pharmacokinetics might be made more precise by using species with similar metabolic activities to humans. We had previously reported the species differences in intestinal and hepatic metabolic activities of 43 cytochrome P450 (CYP) substrates between cynomolgus monkeys and humans. However, the species differences between humans and rats or dogs had not yet been determined using comparable data sets with sufficient number of compounds.

2. Here, we investigated metabolic stabilities in intestinal and liver microsomes obtained from rats, dogs and humans using 43 substrates of human CYP1A2, CYP2J2, CYP2C, CYP2D6 and CYP3A.

3. Hepatic intrinsic clearance (CL_int) values for most compounds in dogs were comparable to those in humans (within 10-fold), whereas in rats, those for the human CYP2D6 substrates were much higher and showed low correlation with humans. In dog intestine, as with human intestine, CL_int values for almost all human CYP1A2, CYP2C, CYP2D6 substrates were not determined because they were very low. Intestinal CL_int values for human CYP3A substrates in rats and dogs appeared to be lower for most of the compounds and showed moderate correlation with those in humans.

4. In conclusion, dogs showed the most similar metabolic activity to humans.     

Comparative study of the metabolism of drug substrates by human cytochrome P450 3A4 expressed in bacterial,yeast and human lymphoblastoid cells

1. The aim was to compare the metabolic activity of human CYP3A4 expressed in bacteria (E. coli), yeast (S. cerevisiae) and human lymphoblastoid cells (hBl), with the native CYP3A4 activity observed in a panel of human livers. 2. Three CYP3A4 substrates were selected for study: dextromethorphan (DEM), midazolam (MDZ) and diazepam (DZ). The substrate metabolism in each of the four systems was characterized by deriving the kinetic parameters K_m or S₅₀, V_max and intrinsic clearance (CL_int) or maximum clearance (CL_max) from the kinetic profiles; the latter differing by 100-fold across the three substrates. 3. The K_m or S₅₀ for the formation of metabolites 3-methoxymorphinan (MEM), 1'-hydroxymidazolam (1'-OH MDZ) and 3-hydroxydiazepam (3HDZ) compared well in all systems. For CYP3A4-mediated metabolism of DEM, MDZ and DZ, the V_max for hBl microsomes were generally 2-9-fold higher than the respective yeast and human liver microsomes and E. coli membrane preparations, resulting in greater CL_int or CL_max. In the case of 3HDZ formation, non-linear kinetics were observed for E. coli, hBl microsomes and human liver microsomes, whereas the kinetics observed for S. cerevisiae were linear. 4. The use of native human liver microsomes for drug metabolic studies will always be preferable. However, owing to the limited availability of human tissues, we find it is reasonable to use any of the recombinant systems described herein, since all three recombinant systems gave good predictions of the native human liver enzyme activities.     

Identification of human cytochrome P450 enzymes involved in the formation of 4-hydroxyestazolam from estazolam

To predict drug interactions with estazolam, the biotransformation of estazolam to its major hydoxylated metabolite, 4-hydroxyestazolam was studied in vitro using pooled human liver microsomes and individual expressed human cytochrome P450 (CYP) enzymes. Estazolam was metabolized to 4-hydroxyestazolam according to the Hill kinetic model in pooled human liver microsomes. The K_m value for the 4-hydroxylation of estazolam was 24.1?µM, and the V_max value was 52.6?pmol?min^?1?mg^?1 protein. The formation of 4-hydroxyestazolam from estazolam in pooled human liver microsomes was significantly inhibited by itraconazole and erythromycin, specific CYP3A4 inhibitors, in a dose-dependent manner, with IC₅₀ values of 1.1 and 12.8?µM, respectively. When estazolam was incubated with expressed human CYP enzymes (CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4), it was metabolized only by CYP3A4. In conclusion, the biotransformation of estazolam to 4-hydroxyestazolam was catalyzed by CYP3A4.     

Identification of the cytochrome P450 enzymes involved in the metabolism of domperidone

1.?The objective was to identify the major cytochrome P450 enzyme(s) involved in the metabolism of domperidone.

2.?Experiments were performed using human liver microsomes (HLMs), recombinant human cytochrome P450 enzymes, cytochrome P450 chemical inhibitors and monoclonal antibodies directed against cytochrome P450 enzymes.

3.?Four metabolites were identified from incubations performed with HLMs and excellent correlations were observed between the formation of domperidone hydroxylated metabolites (M1, M3 and M4), N-desalkylated domperidone metabolite (M2) and enzymatic markers of CYP3A4/5 (r²?=?0.9427, 0.951, 0.9497 and 0.8304, respectively).

4.?Ketoconazole (1?μM) decreased the formation rate of M1, M2, M3 and M4 by 83, 78, 75 and 88%, respectively, whereas the effect of other inhibitors (quinidine, furafylline and sulfaphenazole) was minimal. Important decreases in the formation rate of M1 (68%), M2 (64%) and M3 (54%) were observed with anti-CYP3A4 antibodies.

5.?Formation of M1, M2 and M3 in HLMs exhibited Michaelis–Menten kinetics (K_m: 166, 248 and 36?μM, respectively). Similar K_m values were observed for M1, M2 and M3 when incubations were performed with recombinant human CYP3A4 (K_m: 107, 273 and 34?μM, respectively).

6.?The data suggest that CYP3As are the major enzymes involved in the metabolism of domperidone.     

Effect of 4-tert-octylphenol on cytochrome P450 enzymes in rat liver

The effects were studied of 4-tert-octylphenol (OP) on hepatic cytochrome P450 enzymes in rats. Rats were treated intraperitoneally with OP twice, at doses of 5, 10, and 20 mg/kg. Among the cytochrome P450-dependent monooxygenase activities, testosterone 2α-hydroxylase activity, which is associated with CYP2C11, was significantly decreased by OP at all doses. The level relative to control activity was 67–22%. CYP3A2-dependent monooxygenase, testosterone 6β-hydroxylase activity was also decreased by 51% by OP at 20 mg/kg. Furthermore, immunoblotting showed that OP (10 or 20 mg/kg) significantly decreased CYP2C11/6 and CYP3A2/1 protein levels. However, the reduction ratio of CYP2C11/6 and CYP3A2/1 protein levels by OP treatment was lower than that of testosterone 2α-hydroxylase and testosterone 6β-hydroxylase activities. The Cl _int (V _max/K _m) value for testosterone 2α-hydroxylase was significantly decreased by OP at all doses, whereas the Cl _int value for testosterone 6β-hydroxylase was only decreased by OP at 20 mg/kg. In addition, 7-ethoxycoumarin O-deethylase activity was significantly decreased by 32% by the highest dose of OP. By contrast, CYP1A1-, CYP1A2-, CYP2A1-, CYP2B1/2-, CYP2D1-, CYP2E1- and CYP4A1/2/3- dependent monooxygenase activities were not affected by OP at any dose. These results suggest that OP changes the male-specific cytochrome P450 isoforms in rat liver, and that these changes closely relate to the toxicity of OP. Received: 29 September 1999 / Accepted: 18 October 1999     

Predicted contributions of cytochrome P450s to drug metabolism in human liver microsomes using relative activity factor were dependent on probes

1. Contributions of cytochrome P450 (CYP450) isoforms to drug metabolism are often predicted using relative activity factor (RAF) method, assuming RAF values were independent of probe. We aimed to report probe-dependent characteristic of RAF values using CYP3A4 or CYP2C9 probes.

2. Metabolism of four CYP3A4 probes (testosterone, midazolam, verapamil and atorvastatin) and three CYP2C9 probes (tolbutamide, diclofenac and S-warfarin) in human liver microsomes (HLM) and cDNA-expressed recombinant CYP450 (Rec-CYP450) systems were characterized and RAF_CL value was estimated as ratio of probe intrinsic clearance in HLM to that in Rec-CYP450. CYP450i contributions to metabolic reaction of a probe were predicted using other probes and compared with data from specific inhibitions. Contributions of CYP3A4 and CYP2C9 to metabolism of deoxypodophyllotoxin and nateglinide were also predicted.

3. RAF values were dependent on probes, leading to probe-dependently predicted contributions. Predicted contributions of CYP3A4 to formations of 6β-hydroxytestosterone, 1′-hydroxymidazolam, norverapamil, ortho-hydroxyatorvastatin and para-hydroxyatorvastatin using other probes were 47.46–219.46%, 21.62–98.87%, 186.49–462.44%, 21.87–101.15% and 53.62–247.97%, respectively. Predicted contributions of CYP3A4 and CYP2C9 to nateglinide metabolism were 8.18–37.84% and 36.08–94.04%, separately.

4. In conclusion, CYP450i contribution to drug metabolism in HLM estimated using RAF approach were probe-dependent. Therefore, contribution of each isoform must be confirmed by multiple probes.

     

Functional characterization of human cytochrome P450 2E1 allelic variants: in vitro metabolism of benzene and toluene by recombinant enzymes expressed in yeast cells

Benzene and toluene are common organic solvents currently in worldwide industrial usage, which are metabolized mainly by hepatic cytochrome P450 2E1 (CYP2E1) in humans. Genetic polymorphism of CYP2E1 in 5′-flanking and coding regions has been found previously in Caucasian and Chinese populations. In this study, the effects of CYP2E1 alleles causing amino acid substitutions (CYP2E1*2, CYP2E1*3 and CYP2E1*4; wild-type, CYP2E1.1A) on benzene hydroxylation and toluene methylhydroxylation were studied using recombinant CYP2E1 enzymes of wild-type (CYP2E1.1) and variants (CYP2E1.2 having Arg76His, CYP2E1.3 having Val389Ile and CYP2E1.4 having Val179Ile) expressed in yeast cells. The K _m, V _max and CL _int values of CYP2E1.1 were 10.1 mM, 9.38 pmol/min/pmol CYP and 0.99 nL/min/pmol CYP for benzene hydroxylation, and 3.97 mM, 19.9 pmol/min/pmol CYP and 5.26 nL/min/pmol CYP for toluene methylhydroxylation, respectively. The K _m, V _max and CL _int values for benzene and toluene metabolism of CYP2E1.2, CYP2E1.3 and CYP2E1.4 were comparable to those of wild-type CYP2E1. These findings may mean that the polymorphic alleles of CYP2E1 causing amino acid substitutions are not directly associated with the metabolic activation of benzene and toluene. The information gained in this study should help to identify the variations in the toxicity of environmental pollutants.     

甘草次酸在人细胞色素CYP450中体外代谢研究(英)

甘草根是中医临床常用解毒草药, 其活性成分甘草次酸主要是通过肝脏代谢。本文研究了人肝微粒体以及人源性CYP450s对甘草次酸的体外代谢影响, 以及甘草次酸对几种CYP450酶活性的影响。实验结果表明, 甘草次酸体外主要代谢酶为CYP3A4。体外药代动力学参数K_m, V_max和CL_int分别为18.6 μmol·L⁻¹, 4.4 nmol·mg⁻¹(protein)·min⁻¹和0.237 mL·mg⁻¹(protein)·min⁻¹。体外抑制试验显示, 50 μmol·L⁻¹甘草次酸可以抑制CYP2C19、CYP2C9、CYP3A4酶的活性, 其抑制率可高达50%以上。     

Identification of cytochrome P450 isoforms involved in the metabolism of Syl930, a selective S1PR1 agonist acting as a potential therapeutic agent for autoimmune encephalitis

Syl930 is a novel sphingosine-1-phosphate receptor subtype 1 (S1PR₁) agonist for the treatment of autoimmune encephalitis with promising receptor selectivity and little risk of bradycardia. Syl930 could be reversibly converted to its phosphorylated metabolite, acting as the active form to provide therapeutic effects, but eliminated principally in the form of oxidative metabolites. The aim of the present study was to identify the cytochrome P450 isoforms (CYPs) responsible for the oxidative metabolism of Syl930. Considerable production of hydroxylated metabolite (Syl930-M1) was found in both rat blood and tissue homogenates in vivo and in vitro. Moreover, another hydroxylated metabolite, Syl930-M2, was detected in human, beagle dog and cynomolgus monkey liver microsomes with significant differences in the K_m, V_max and CL_int of the metabolites among species. CYP1A1, CYP2J2, CYP4F2 and CYP3A4 were identified to be the major CYPs mediated in the hydroxylation of Syl930 by using 14 recombinant human CYPs, selective chemical inhibitors and monoclonal antibodies against CYPs. The multiple CYPs mediated oxidation was believed to be one of the reasons for the relatively short elimination half-life of Syl930.     

In vitro metabolism of the anti-inflammatory clerodane diterpenoid polyandric acid A and its hydrolysis product by human liver microsomes and recombinant cytochrome P450 and UDP-glucuronosyltransferase enzymes

1.?The metabolism of the anti-inflammatory diterpenoid polyandric acid A (PAA), a constituent of the Australian Aboriginal medicinal plant Dodonaea polyandra, and its de-esterified alcohol metabolite, hydrolysed polyandric acid A (PAAH) was studied in vitro using human liver microsomes (HLM) and recombinant UDP-glucuronosyltransferase (UGT) and cytochrome P450 (CYP) enzymes.

2.?Hydrolysis of PAA to yield PAAH occurred upon incubation with HLM. Further incubations of PAAH with HLM in the presence of UGT and CYP cofactors resulted in significant depletion, with UGT-mediated depletion as the major pathway.

3.?Reaction phenotyping utilising selective enzyme inhibitors and recombinant human UGT and CYP enzymes revealed UGT2B7 and UGT1A1, and CYP2C9 and CYP3A4 as the major enzymes involved in the metabolism of PAAH.

4.?Analysis of incubations of PAAH with UDP-glucuronic acid-supplemented HLM and recombinant enzymes by UPLC/MS/MS identified three glucuronide metabolites. The metabolites were further characterised by β-glucuronidase and mild alkaline hydrolysis. The acyl glucuronide of PAAH was shown to be the major metabolite.

5.?This study demonstrates the in vitro metabolism of PAA and PAAH and represents the first systematic study of the metabolism of an active constituent of an Australian Aboriginal medicinal plant.     

Cytochromes of the P450 2C subfamily are the major enzymes involved in the O-demethylation of verapamil in humans

The calcium channel blocker verapamil [2,8-bis-(3,4-dimethoxyphenyl)-6-methyl-2-isopropyl-6-azaoctanitrile] undergoes extensive biotransformation in man. We have previously demonstrated cytochrome P450 (CYP) 3A4 and 1A2 to be the enzymes responsible for verapamil N-dealkylation (formation of D-617 [2-(3,4-dimethoxyphenyl)-5-methylamino-2-isopropylvaleronitrile]), and verapamil N-demethylation (formation of norverapamil [2,8-bis(3,4-dimethoxyphenyl)-2-isopropyl-6-azaoctanitrile]), while there was no involvement of CYP3A4 and CYP1A2 in the third initial metabolic step of verapamil, which is verapamil O-demethylation. This pathway yields formation of D-703 [2-(4-hydroxy-3-methoxyphenyl)-8-(3,4-dimethoxyphenyl)-6-methyl-2-isopropyl-6-azaoctanitrile] and D-702 [2-(3,4-dimethoxyphenyl)-8-(4-hydroxy-3-methoxyphenyl)6-methyl-2-isopropyl-6-azaoctanitrile]. The enzymes catalyzing verapamil O-demethylation have not been characterized so far. We have therefore identified and characterized the enzymes involved in verapamil O-demethylation in humans by using the following in vitro approaches: (I) characterization of O-demethylation kinetics in the presence of the microsomal fraction of human liver, (II) inhibition of verapamil O-demethylation by specific antibodies and selective inhibitors and (111) investigation of metabolite formation in microsomes obtained from yeast strain Saccharomyces cerevisiae W(R), that was genetically engineered for stable expression of human CYP2C8, 2C9 and 2C18.In human liver microsomes (n=4), the intrinsic clearance (CL_int), as derived from the ratio of V _max/K_m, was significantly higher for O-demethylation to D-703 compared to formation of D-702 following incubation with racemic verapamil (13.9±1.0 vs 2.4±0.6 ml^*min^{-1 *}g^-1 mean±SD; p<0.05), S-Verapamil (16.8±3.3 vs 2.2±1.2 ml^* mini^*g^-1, p<0.05) and R-verapamil (12.1±2.9 vs 3.6 ±1.3 ml^*min^{-1 *} g^-1; p<0.05), thus indicating regioselectivity of verapamil O-demethylation process. The CL_int of D-703 formation in human liver microsomes showed a modest but significant degree of stereo selectivity (p<0.05) with a S/R-ratio of 1.41±0.17. Anti-LKM2 (anti-liver/kidney microsome) autoantibodies (which inhibit CYP2C9 and 2C19) and sulfaphenazole (a specific CYP2C9 inhibitor) reduced the maximum rate of formation of D-703 by 81.5±4.5% and 45%, that of D-702 by 52.7±7.5% and 72.5%, respectively. Both D-703 and D-702 were formed by stably expressed CYP2C9 and CYP2C18, whereas incubation with CYP2C8 selectively yielded D-703.In conclusion, our results show that enzymes of the CYP2C subfamily are mainly involved in verapamil O-demethylation. Verapamil therefore has the potential to interact with other drugs which inhibit or induce these enzymes.     

Interindividual variability in catalytic activity and immunoreactivity of three major human liver cytochrome P450 isozymes

Objective: Interindividual variations in immunoreactivity and function of three major human drug metabolising P450 monooxygenases has been investigated in liver microsomes from 42 Caucasians (kidney donors or liver biopsies). Methods: Diclofenac 4′-hydroxylation, dextromethorphan O-demethylation and midazolam 1′-hydroxylation, measured by HPLC in incubates, were used as probes to determine CYP2C9, CYP2D6 and CYP3A4 function kinetics, respectively. Immunoquantification of the three isoforms was achieved by Western blotting, using rabbit polyclonal antibodies raised against human CYP2C9 and human CYP3A4, and mouse monoclonal antibody raised against human CYP2D6. Results: Diclofenac 4′-hydroxylation exhibited Michaelis-Menten kinetics with k_M= 3.4 μmol ⋅l⁻¹ and V_max = 45 nmole ⋅mg⁻¹P ⋅h⁻¹. Relative immunoreactivity of CYP2C9 was correlated with V_max and CL_int. Dextromethorphan O-demethylation in EM (extensive metabolisers) liver microsomes also showed Michaelis-Menten kinetics, with k_M = 4.4 μmol ⋅l⁻¹ and V_max = 5.0 nmol ⋅mg⁻¹P ⋅h⁻¹. Relative immunoreactivity of CYP2D6 was correlated with V_max and CL_int. Midazolam 1′-hydroxylation also exhibited Michaelis-Menten kinetics with k_M = 3.3 μmol ⋅l⁻¹ and V_max = 35 nmol ⋅mg⁻¹P ⋅h⁻¹. Relative immunoreactivity of CYP3A4 was correlated with V_max and CL_int. Immunoreactivity and function were correlated for each isozyme, but there was no cross correlation between isozymes. Conclusion: The velocity of metabolite formation (V_max) by the three major human drug metabolising P450 monooxygenases is correlated with their immunoreactivity in liver microsomes. Interindividual variation was much larger for V_max than k_M. Interindividual variability was more pronounced for CYP2D6, probably due to the presence of several different functional alleles in the population of extensive metabolisers. Received: 27 December 1995/Accepted in revised form: 29 March 1996