Human Cytochrome P450 2C9 Research Articles

Sequence diversity and functional characterization of the 5'-regulatory region of human CYP2C19.

Cytochrome p4502C19 (CYP2C19) plays an important role in drug biotransformation and has been shown to be genetically polymorphic. While polymorphisms in the coding region that have large effects on activity are well described, until recently, a lack of knowledge of the promoter sequence has hindered efforts to study it. Genetic variants in the promoter region have not been described and factors that influence its gene expression via promotor regulation remain largely undefined. We have cloned and sequenced 1.8 kb of the human CYP2C19 promoter. This promoter contains a number of putative transcription factor sites, including HepG2-specific factor 1, glucocorticoid response element, estrogen receptor element, constitutive androstane receptor and peroxisome proliferator-activated receptor. Sequencing of DNA obtained from 67 individuals identified eight single nucleotide polymorphisms within this region. No sequence of a known human pregnane X receptor response element was found in this section of the CYP2C19 promoter, despite the known effect of rifampin on the expression of this gene. A plasmid containing the 1.8-kb CYP2C19 promoter coupled to a luciferase reporter gene has been constructed and demonstrated to be functional and sensitive to induction by omeprazole in HuH7 cells. Nested deletions of CYP2C19 promoter were generated and the ability of serial promoter deletion constructs to activate luciferase expression in the HepG2 cell line was analysed. These data make possible future studies to elucidate the molecular mechanisms by which CYP2C19 can be induced in clinical settings and the consequences of genetic variability in its promoter.

Role of conserved Asp293 of cytochrome P450 2C9 in substrate recognition and catalytic activity.

Human cytochrome P450 2C9 (CYP2C9) is important in the metabolism of non-steroidal anti-inflammatory compounds such as diclofenac, the antidiabetic agent tolbutamide and other clinically important drugs, many of which are weakly acidic. Multiple sequence alignment of CYPs identified CYP2C9 Asp(293) as corresponding to Asp(301) of CYP2D6, which has been suggested to play a role in the binding of basic substrates to the latter enzyme. Replacement of Asp(293) with Ala (D293A) decreased activity by more than 90%, and led to an approx. 3- to 10-fold increase in K (m) values for the three test substrates tolbutamide, dextromethorphan and diclofenac. Conservative replacement of the carboxyl side chain in a Glu (D293E) mutant produced no significant changes in K (m) values and slight increases in k (cat) values. Changes in regiospecificity were observed for both the Ala and Glu substitutions; low levels of both dextromethorphan O- and N-demethylation were observed in the D293A mutant, whereas increased preference for O-demethylation was observed for the D293E mutant. Expression of constructs coding for Asn (D293N) and Gln (D293Q) substitutions failed to form a P450 correctly. Our analysis suggests a structural role for the carboxyl side chain of Asp(293) in CYP2C9 substrate binding and catalysis. The conservation of an Asp residue in other CYP families in a position equivalent to Asp(293) indicates a common mechanism for maintaining the active-site architecture.

In vivo and in vitro disposition profile of tezosentan, an intravenous dual endothelin receptor antagonist, in humans

1. The plasma concentration-time profile of the 14 C-labelled endothelin receptor antagonist tezosentan in healthy male volunteers after a 1-h infusion at 100 mg h − 1 followed a biphasic decline with half-lives of 3-5 min for the initial disposition phase and approximately 4 h for the terminal phase. 2. Tezosentan was predominantly excreted unchanged into faeces, whereas less than 5% of the dose was excreted as unchanged drug in urine. Two isomeric, hydroxylated metabolites (M1, M2) were detected in faeces representing 2-5% of the total radioactivity. 3. In vitro, with human liver microsomes and primary hepatocytes, tezosentan was metabolized at very low rates. Upon prolonged incubation with human hepatocytes for 24 h, formation of the hydroxylated metabolite M1 and a glucuronic acid conjugate, M3, was observed. 4. No relevant inhibition of the human cytochrome P450 (CYP) forms, CYP1A2, 2C9, 2C19, 2D6 and 3A4, was observed in vitro at tezosentan concentrations largely exceeding those observed in clinical trials. 5. In human blood, tezosentan was highly bound to plasma proteins, mainly albumin, and hardly penetrated into red blood cells.

Substrate selectivity of human cytochrome P450 2C9: importance of residues 476, 365, and 114 in recognition of diclofenac and sulfaphenazole and in mechanism-based inactivation by tienilic acid.

A series of six site-directed mutants of CYP 2C9 were constructed with the aim to better define the amino acid residues that play a critical role in substrate selectivity of CYP 2C9, particularly in three distinctive properties of this enzyme: (i) its selective mechanism-based inactivation by tienilic acid (TA), (ii) its high affinity and hydroxylation regioselectivity toward diclofenac, and (iii) its high affinity for the competitive inhibitor sulfaphenazole (SPA). The S365A mutant exhibited kinetic characteristics for the 5-hydroxylation of TA very similar to those of CYP 2C9; however, this mutant did not undergo any detectable mechanism-based inactivation by TA, which indicates that the OH group of Ser 365 could be the nucleophile forming a covalent bond with an electrophilic metabolite of TA in TA-dependent inactivation of CYP 2C9. The F114I mutant was inactive toward the hydroxylation of diclofenac; moreover, detailed analyses of its interaction with a series of SPA derivatives by difference visible spectroscopy showed that the high affinity of SPA to CYP 2C9 (K(s)=0.4 microM) was completely lost when the phenyl substituent of Phe 114 was replaced with the alkyl group of Ile (K(s)=190+/-20 microM), or when the phenyl substituent of SPA was replaced with a cyclohexyl group (K(s)=120+/-30 microM). However, this cyclohexyl derivative of SPA interacted well with the F114I mutant (K(s)=1.6+/-0.5 microM). At the opposite end, the F94L and F110I mutants showed properties very similar to those of CYP 2C9 toward TA and diclofenac. Finally, the F476I mutant exhibited at least three main differences compared to CYP 2C9: (i) big changes in the k(cat) and K(m) values for TA and diclofenac hydroxylation, (ii) a 37-fold increase of the K(i) value found for the inhibition of CYP 2C9 by SPA, and (iii) a great change in the regioselectivity of diclofenac hydroxylation, the 5-hydroxylation of this substrate by CYP 2C9 F476I exhibiting a k(cat) of 28min(-1). These data indicate that Phe 114 plays an important role in recognition of aromatic substrates of CYP 2C9, presumably via Pi-stacking interactions. They also provide the first experimental evidence showing that Phe 476 plays a crucial role in substrate recognition and hydroxylation by CYP 2C9.

Amino acid residues affecting the activities of human cytochrome P450 2C9 and 2C19.

The amino acid residues affecting the substrate specificity of human cytochrome P450 CYP2C9 and CYP2C19 for their metabolic activities were investigated using chimeras and mutant enzymes, which were constructed by replacing the corresponding residues. Although CYP2C19 showed nearly the same tolbutamide hydroxylase activity as CYP2C9, the activities for the CYP2C19(H99I) mutant and the chimeras that replaced residues 1-212 were much lower than those for CYP2C19. The activities of the CYP2C19(H99I) mutant and the chimeras that replaced residues 228-340 were lower than those for CYP2C19 toward S-mephenytoin, aminopyrine, and testosterone. These results suggest that residues in substrate recognition site (SRS) 3 and 4 are important for the substrate specificity, whereas His99 is important in the substrate binding of CYP2C19. For the 4'-hydroxylation of diclofenac, CYP2C9 had a lower K(m) and a higher V(max) than CYP2C19. Although the V(max) values for the CYP2C9(1-288)/CYP2C19(289-490) chimera and the CYP2C9(I99H, V292A, F295L, I331V) mutant were comparable to those for CYP2C9, its K(m) value was comparable to that for CYP2C19. The V(max) and K(m) values for the CYP2C19(1-288)/CYP2C9(289-490) chimera were comparable to those for CYP2C19, and the activity by CYP2C9(1-230)/CYP2C19(231-490) chimera was negligible. These results suggest that the residues 292, 295, and/or 331 of CYP2C9 are essential for the recognition of substrate in CYP2C9 and that the residues of 231-288 including SRS 3 are important for the metabolizing capacity of CYP2C9.

Cloning of cytochrome P-450 2C9 cDNA from human liver and its expression in CHL cells.

Using bacterial, yeast, or mammalian cell expressing a human drug metabolism enzyme would seem good way to study drug metabolism-related problems. Human cytochrome P-450 2C9(CYP2C9) is a polymorphic enzyme responsible for the metabolism of a large number of clinically important drugs. It ranks among the most important drug metabolizing enzymes in humans. In order to provide a sufficient amount of the enzyme for drug metabolic research, the CYP2C9 cDNA was cloned and expressed stably in CHL cells. After extraction of total RNA from human liver tissue, the human CYP2C9 cDNA was amplified with reverse transcription-polymerase chain reaction (RT-PCR), and cloned into cloning vector pGEM-T. The cDNA fragment was identified by DNA sequencing and subcloned into a mammalian expression vector pREP9. A transgenic cell line was established by transfecting the recombinant vector of pREP9-CYP2C9 into CHL cells. The enzyme activity of CYP2C9 catalyzing oxidation of tolbutamide to hydroxy tolbutamide in S9 fraction of the cell was determined by high performance liquid chromatography(HPLC). The amino acid sequence predicted from the cDNA segment was identical to that of CYP2C9*1, the wild type CYP2C9. However, there were two base differences, i.e. 21T>C, 1146C>T, but the encoding amino acid sequence was the same, L7, P382. The S9 fraction of the established cell line metabolizes tolbutamide to hydroxy tolbutamide; tolbutamide hydroxylase activity was found to be 0.465 +/- 0.109 micromol.min(-1).g(-1) S9 protein or 8.62 +/- 2.02mol.min(-1).mol(-1) CYP, but was undetectable in parental CHL cell. The cDNA of human CYP2C9 was successfully cloned and a cell line of CHL- CYP2C9, efficiently expressing the protein of CYP2C9, was established.

Comparison of rat and human cytochrome P450 (CYP) sources of N-alkylprotoporphyrin IX. Formation after interaction with porphyrinogenic xenobiotics: studies with cDNA-expressed single CYP enzymes

1. The porphyrinogenicity of certain xenobiotics is due to mechanism-based inactivation of selected cytochrome P450 (CYP) enzymes, with concurrent formation of N -alkylprotoporphyrins (N-alkylPPs), which disrupt control of haem biosynthesis. An ambiguity arises when extrapolating results obtained with such porphyrinogenic xenobiotics in animals to humans owing to species' differences in CYP enzymes. The objective was to use cDNA-expressed individual rat CYP enzyme preparations in microsomes prepared from baculovirus-infected insect cells to determine which rat CYP enzymes were the source of N-alkylPPs after interaction with three porphyrinogenic xenobiotics and to compare the results with formation of N-alkylPPs in individual human CYP enzyme orthologues. 2. A sensitive fluorometric technique was employed to quantitate N-alkylPP formation after interaction of individual CYP enzymes with a porphyrinogenic xenobiotic. 3. N-alkylPP formation was found following the interaction of three porphyrinogenic xenobiotics with CYP1A2, 2B1, 2C6, 2C11 and 3A2, in amounts ranging from 0.45 to 0.07 nmol N-alkylPP nmol−1 CYP. The results obtained with rat CYP1A2, 2C6, 2C11 and 3A2 were compared with those previously obtained with the human CYP orthologues 1A2, 2C9 and 3A4. 4. Some results corresponded while others did not, reinforcing a previous recommendation that when dealing with xenobiotics whose porphyrinogenicity depends upon interaction with CYP enzymes resulting in N-alkylPP formation, animal experiments should be augmented with studies using human CYP preparations.

Ticlopidine as a selective mechanism-based inhibitor of human cytochrome P450 2C19.

Experiments using recombinant yeast-expressed human liver cytochromes P450 confirmed previous literature data indicating that ticlopidine is an inhibitor of CYP 2C19. The present studies demonstrated that ticlopidine is selective for CYP 2C19 within the CYP 2C subfamily. UV-visible studies on the interaction of a series of ticlopidine derivatives with CYP 2C19 showed that ticlopidine binds to the CYP 2C19 active site with a K(s) value of 2.8 +/- 1 microM. Derivatives that do not involve either the o-chlorophenyl substituent, the free tertiary amine function, or the thiophene ring of ticlopidine did not lead to such spectral interactions and failed to inhibit CYP 2C19. Ticlopidine is oxidized by CYP 2C19 with formation of two major metabolites, the keto tautomer of 2-hydroxyticlopidine (1) and the dimers of ticlopidine S-oxide (TSOD) (V(max) = 13 +/- 2 and 0.4 +/- 0.1 min(-1)). During this oxidation, CYP 2C19 was inactivated; the rate of its inactivation was time and ticlopidine concentration dependent. This process meets the chemical and kinetic criteria generally accepted for mechanism-based enzyme inactivation. It occurs in parralel with CYP 2C19-catalyzed oxidation of ticlopidine, is inhibited by an alternative well-known substrate of CYP 2C19, omeprazole, and correlates with the covalent binding of ticlopidine metabolite(s) to proteins. Moreover, CYP 2C19 inactivation is not inhibited by the presence of 5 mM glutathione, suggesting that it is due to an alkylation occurring inside the CYP 2C19 active site. The effects of ticlopidine on CYP 2C19 are very analogous with those previously described for the inactivation of CYP 2C9 by tienilic acid. This suggests that a similar electrophilic intermediate, possibly a thiophene S-oxide, is involved in the inactivation of CYP 2C19 and CYP 2C9 by ticlopidine and tienilic acid, respectively. The kinetic parameters calculated for ticlopidine-dependent inactivation of CYP 2C19, i.e., t(1/2max) = 3.4 min, k(inact) = 3.2 10(-3) s(-1), K(I) = 87 microM, k(inact)/K(I) = 37 L.mol(-1).s(-1), and r (partition ratio) = 26 (in relation with formation of 1 + TSOD), classify ticlopidine as an efficient mechanism-based inhibitor although somewhat less efficient than tienilic acid for CYP 2C9. Importantly, ticlopidine is the first selective mechanism-based inhibitor of human liver CYP 2C19 and should be a new interesting tool for studying the topology of the active site of CYP 2C19.

In-vitro metabolism of celecoxib, a cyclooxygenase-2 inhibitor, by allelic variant forms of human liver microsomal cytochrome P450 2C9: correlation with CYP2C9 genotype and in-vivo pharmacokinetics.

In-vitro studies were conducted to assess the impact of CYP2C9 genotype on the metabolism (methyl hydroxylation) and pharmacokinetics of celecoxib, a novel cyclooxygenase-2 inhibitor and CYP2C9 substrate. When compared to cDNA-expressed wild-type CYP2C9 (CYP2C9*1), the Vmax/Km ratio for celecoxib methyl hydroxylation was reduced by 34% and 90% in the presence of recombinant CYP2C9*2 and CYP2C9*3, respectively. These data indicated that the amino acid substitution at position 359 (Ile to Leu) elicited a more pronounced effect on the metabolism of celecoxib than did a substitution at position 144 (Arg to Cys). The Vmax/Km ratio was also decreased in microsomes of livers genotyped CYP2C9*1/*2 (47% decrease, mean of two livers), or CYP2C9*1/*3 (59% decrease, one liver). In all cases, these changes were largely reflective of a decrease in Vmax, with a minimal change in Km. Based on simulations of the in-vitro data obtained with the recombinant CYP2C9 proteins, it was anticipated that the pharmacokinetics of celecoxib (as a much as a five-fold increase in plasma AUC) would be altered (versus CYP2C9*1/*1 subjects) in subjects genotyped heterozygous or homozygous for the CYP2C9*2 (Cys144) or CYP2C9*3 (Leu359) allele. In a subsequent clinical study, the AUC of celecoxib was increased (versus CYP2C9*1/*1 subjects) approximately 2.2-fold (range, 1.6-3-fold) in two CYP2C9*1/*3 subjects and one CYP2C9*3/*3 subject receiving a single oral dose (200 mg) of the drug. In contrast, there was no significant change in celecoxib AUC in two subjects genotyped CYP2C9*1/*2.

Cytochrome P4502B6 and 2C9 do not metabolize midazolam: kinetic analysis and inhibition study with monoclonal antibodies

We determined the contribution of cytochrome P450 (CYP) isoforms to the metabolism of midazolam by kinetic analysis of human liver microsomes and CYP isoforms and by examining the effect of chemical inhibitors and monoclonal antibodies against CYP isoforms in vitro. Midazolam was metabolized to 1'-hydroxymidazolam (1'-OH MDZ) by human liver microsomes with a Michaelis-Menten constant (Km) of 4.1 (1.0) (mean (SD)) micromol litre(-1) and a maximum rate of metabolism (Vmax) of 5.5 (1.1) nmol min(-1) mg protein(-1) (n = 6). Of the nine representative human liver CYP isoforms, CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4 and 3A5, three (CYP2B6, 3A4 and 3A5) showed midazolam 1'-hydroxylation activity, with Kms of 40.7, 1.7 and 3.0 micromol litre(-1), respectively, and Vmax values of 12.0, 3.3 and 13.2 nmol min(-1) nmol P450(-1), respectively (n = 4). Midazolam 1'-hydroxylation activity of human liver microsomes correlated significantly with testosterone 6beta-hydroxylation activity, a marker of CYP3A activity (r2 = 0.77, P = 0.0001), but not with S-mephenytoin N-demethylation activity, a marker of CYP2B6 activity (r2 < 0.01, P = 0.84) (n = 11). Troleandomycin and orphenadrine, chemical inhibitors of CYP isoforms, inhibited the formation of 1'-OH MDZ by human liver microsomes. Monoclonal antibody against CYP3A4 inhibited the formation of 1'-OH MDZ by 79%, whereas monoclonal antibody against CYP2B6 had no effect on midazolam 1'-hydroxylation by human liver microsomes (n = 5). These results indicate that only CYP3A4, but not CYP2B6 or CYP2C, is involved in the metabolism of midazolam in vitro.

Use of isotopes and LC-MS-ESI-TOF for mechanistic studies of tienilic acid metabolic activation.

Tienilic Acid (TA) is a uricosuric drug marketed in 1978 and which caused a number of rare immunoallergic hepatitis. It was withdrawn in US in 1980, in France in 1992. Early batches of tienilic acid also contained 0.1–0.5% tienilic acid isomer. Tienilic acid isomer (TAI), has been shown to be metabolised by Cytochrome P450 into a reactive thiophene 1-oxide which either binds to proteins, or can be trapped by sulfur nucleophiles (Valadon et al. 1996). Tienilic acid is metabolized by human cytochrome P450 2C9 into 5-hydroxytienilic acid (a major metabolite representing 70% of the dose excreted in human urine) but it also forms (a) reactive metabolite(s) which binds covalently to CYP 2C9 and it is a mechanism based inhibitor of CYP 2C9. Adding glutathione to incubations decreases the covalent binding, but only to 1 mol/mol P450. However the reactive metabolite of tienilic acid is still unknown. Recently Koenigs et al. have shown using ESI-LC-MS that CYP 2C9 binds ≈ 2 mol of TA in absence of GSH and only one in presence of 3 mM GSH (Koenigs et al. 1999).

Hydroxylation of diclofenac: an illustration of the complementary roles of biomimetic metalloporphyrin catalysts and yeasts expressing human cytochromes P450 in drug metabolism studies

Abstract Preparative hydroxylation of aromatic compounds such as drugs remain a difficult challenge. Here we show that the two main human metabolites of the anti-inflammatory drug diclofenac, 5-hydroxy- and 4′-hydroxy-diclofenac, can be prepared from diclofenac in high yields by using two complementary model systems for human cytochromes P450. The former, which results from hydroxylation of the most reactive aromatic position of diclofenac, was obtained by using H2O2 or t-BuOOH in the presence of an iron porphyrin. The latter could only be prepared from bioconversion of diclofenac with a recombinant yeast expressing human cytochrome P450 2C9.

Arginines 97 and 108 in CYP2C9 Are Important Determinants of the Catalytic Function

Human cytochrome P450 2C9 (CYP2C9) is one of the major drug metabolising enzymes which exhibits a broad substrate specificity. The B-C loop is located in the active-site but has been difficult to model, owing to its diverse and flexible structure. To elucidate the function of the B-C loop we used homology modelling based on the Cyp102 structure in combination with functional studies of mutants using diclofenac as a model substrate for CYP2C9. The study shows the importance of the conserved arginine in position 97 and the arginine in position 108 for the catalytic function. The R97A mutant had a 13-fold higher Km value while the Vmax was in the same order as the wild type. The R108 mutant had a 100-fold lower activity with diclofenac compared to the wild-type enzyme. The other six mutants (S95A, F100A, L102A, E104A, R105A, and N107A) had kinetic parameters similar to the CYP2C9 wild-type. Our homology model based on the CYP102 structure as template indicates that R97, L102, and R105 are directed into the active site, whereas R108 is not. The change in catalytic function when arginine 97 was replaced with alanine and the orientation of this amino acid in our homology model indicates its importance for substrate interaction.

Phenobarbital increases DNA adduct and metabolites formed by ochratoxin A: role of CYP 2C9 and microsomal glutathione-S-transferase.

Ochratoxin A (OTA), a mycotoxin that induces nephrotoxicity and urinary tract tumors, is genotoxic and can be metabolized not only by different cytochromes P450 (CYP) but also by peroxidases involved in the arachidonic cascade, although the exact nature of the metabolites involved in the genotoxic process is still unknown. In order to establish the relation between OTA genotoxicity and the formation of metabolites, we chose three experimental models: kidney microsomes from rabbit, human bronchial epithelial cells, and microsomes from yeast that specifically express the human cytochrome P450 2C9 or 2B6 genes. OTA-DNA adducts were analyzed by (32)P postlabeling and the OTA derivatives formed were isolated by HPLC after incubation of OTA in the presence of: (1) kidney microsomes from rabbit pretreated or not with phenobarbital (PB); (2) human pulmonary epithelial cells simultaneously pretreated (or not) with PB alone or in the presence of ethacrynic acid (EA); (3) microsomes expressing CYP 2B6 and 2C9. PB pretreatment significantly increased DNA adducts formed after OTA treatment, both in the presence of kidney microsomes and bronchial epithelial cells, and induced the formation of new adducts. Ethacrynic acid, which inhibits microsomal glutathione-S-transferase, reduced DNA adduct level. DNA adducts were detected when OTA were incubated with microsomes expressing human CYP 2C9 but not with those expressing CYP 2B6. Several metabolites detected by HPLC were increased after PB treatment. Some of them could be related to DNA-adduct formation. In conclusion, OTA biotransformation, enhanced by PB pretreatment, increased DNA-adduct formation through pathways involving microsomal glutathion-S-transferase and CYP 2C9.

Phenobarbital increases DNA adduct and metabolites formed by ochratoxin A: Role of CYP 2C9 and microsomal glutathione-S-transferase

Ochratoxin A (OTA), a mycotoxin that induces nephrotoxicity and urinary tract tumors, is genotoxic and can be metabolized not only by different cytochromes P450 (CYP) but also by peroxidases involved in the arachidonic cascade, although the exact nature of the metabolites involved in the genotoxic process is still unknown. In order to establish the relation between OTA genotoxicity and the formation of metabolites, we chose three experimental models: kidney microsomes from rabbit, human bronchial epithelial cells, and microsomes from yeast that specifically express the human cytochrome P450 2C9 or 2B6 genes. OTA-DNA adducts were analyzed by 32P postlabeling and the OTA derivatives formed were isolated by HPLC after incubation of OTA in the presence of: (1) kidney microsomes from rabbit pretreated or not with phenobarbital (PB); (2) human pulmonary epithelial cells simultaneously pretreated (or not) with PB alone or in the presence of ethacrynic acid (EA); (3) microsomes expressing CYP 2B6 and 2C9. PB pretreatment significantly increased DNA adducts formed after OTA treatment, both in the presence of kidney microsomes and bronchial epithelial cells, and induced the formation of new adducts. Ethacrynic acid, which inhibits microsomal glutathione-S-transferase, reduced DNA adduct level. DNA adducts were detected when OTA were incubated with microsomes expressing human CYP 2C9 but not with those expressing CYP 2B6. Several metabolites detected by HPLC were increased after PB treatment. Some of them could be related to DNA-adduct formation. In conclusion, OTA biotransformation, enhanced by PB pretreatment, increased DNA-adduct formation through pathways involving microsomal glutathion-S-transferase and CYP 2C9. Environ. Mol. Mutagen. 35:123–131, 2000 © 2000 Wiley-Liss, Inc.

An inhibitory monoclonal antibody to human cytochrome P450 that specifically binds and inhibits P4502C9II, an allelic variant of P4502C9 having a single amino acid change Arg144 Cys

A monoclonal antibody (MAb 292-2-3) has been isolated that binds specifically to a single allele of three expressed human cytochrome P4502C9 alleles. The MAb binds to 2C9

Opposite Behaviors of Reactive Metabolites of Tienilic Acid and Its Isomer toward Liver Proteins: Use of Specific Anti-Tienilic Acid−Protein Adduct Antibodies and the Possible Relationship with Different Hepatotoxic Effects of the Two Compounds

Tienilic acid (TA) is responsible for an immune-mediated drug-induced hepatitis in humans, while its isomer (TAI) triggers a direct hepatitis in rats. In this study, we describe an immunological approach developed for studying the specificity of the covalent binding of these two compounds. For this purpose, two different coupling strategies were used to obtain TA-carrier protein conjugates. In the first strategy, the drug was linked through its carboxylic acid function to amine residues of carrier proteins (BSA-N-TA and casein-N-TA), while in the second strategy, the thiophene ring of TA was attached to proteins through a short 3-thiopropanoyl linker, the corresponding conjugates (BSA-S-5-TA and betaLG-S-5-TA) thus preferentially presenting the 2, 3-dichlorophenoxyacetic moiety of the drug for antibody recognition. The BSA-S-5-TA conjugate proved to be 30 times more immunogenic than BSA-N-TA. Anti-TA-protein adduct antibodies were obtained after immunization of rabbits with BSA-S-5-TA (1/35000 titer against betaLG-S-5-TA in ELISA). These antibodies strongly recognized the 2, 3-dichlorophenoxyacetic moiety of TA but poorly the part of the drug engaged in the covalent binding with the proteins. This powerful tool was used in immunoblots to compare TA or TAI adduct formation in human liver microsomes as well as on microsomes from yeast expressing human liver cytochrome P450 2C9. TA displayed a highly specific covalent binding focused on P450 2C9 which is the main cytochrome P450 responsible for its hepatic activation in humans. On the contrary, TAI showed a nonspecific alkylation pattern, targeting many proteins upon metabolic activation. Nevertheless, this nonspecific covalent binding could be completely shifted to a thiol trapping agent like GSH. The difference in alkylation patterns for these two compounds is discussed with regard to their distinct toxicities. A relationship between the specific covalent binding of P450 2C9 by TA and the appearance of the highly specific anti-LKM2 autoantibodies (known to specifically recognize P450 2C9) in patients affected with TA-induced hepatitis is strongly suggested.

Role of cDNA-Expressed Human Cytochromes P450 in the Metabolism of Diazepam

The metabolic conversion of diazepam (DZ) to temazepam (TMZ, a C3-hydroxylation product of DZ) and N-desmethyldiazepam (NDZ, an N1-demethylation product of DZ) was studied using cDNA-expressed human cytochrome P450 (CYP) isozymes 1A2, 2B6, 2C8, 2C9, 2C9 R144C, 2E1, 3A4, and 3A5 and human liver microsomes from five organ donors. Of the CYPs examined, 3A5, 3A4, and 2B6 exhibited the highest enzymatic activities with turnovers ranging from 7.5 to 12.5 nmol of product formed/min/nmol for the total metabolism of DZ, while 2C8, 2C9, and 2C9 R144C showed lesser and moderate activities. 1A2 and 2E1 produced insignificant amounts of metabolites of DZ. The regioselectivity of CYPs was determined, and 2B6 was found to catalyze exclusively and 2C8, 2C9, and 2C9 R144C preferentially the N1-demethylation of DZ to form NDZ. 3A4 and 3A5 catalyzed primarily the C3-hydroxylation of DZ, which was more extensive than the N1-demethylation. The ratios of TMZ to NDZ formed in the metabolism of DZ by 3A4 and 3A5 were approximately 4:1. Enzyme kinetic studies indicated that 2B6- and 2C9-catalyzed DZ metabolism followed Michaelis–Menten kinetics, whereas 3A4 and 3A5 displayed atypical and non-linear curves in Lineweaver–Burk plots. Human liver microsomes converted DZ to both TMZ and NDZ at a ratio of 2:1. Our results suggest that hepatic CYP3A, 2C, and 2B6 enzymes have an important role in the metabolism of DZ by human liver.

Identification of a novel transcriptional silencer in the protein-coding region of the human CYP2C9 gene

A novel regulatory element (27 bp) which confers transcriptional repression was identified within the protein-coding region immediately after the translation start codon in the human cytochrome P450 (CYP) 2C9 gene. Deletion of this element increased transcriptional activity in HepG2 cells by transient transfection assay. Nuclear protein extracts from HepG2 cells and human liver were found in electrophoretic mobility shift assays to bind specifically to the 27 bp element. A putative binding protein was partially purified by DNA-affinity chromatography and was determined by Southwestern blotting to have a molecular weight of approx. 100 kDa. Studies with mutated competitor oligonucleotides established that binding of the nuclear protein to the 27 bp cis-element was dependent upon two 6 bp direct repeats (5′-CTTGTG-3′) that were separated by three bases. It is possible that this novel cis-acting element may be involved in the negative regulation of CYP2C9.

Five distinct human cytochromes mediate amitriptyline N-demethylation in vitro: dominance of CYP 2C19 and 3A4.

The human cytochromes P450 (CYPs) mediating amitriptyline N-demethylation have been identified using a combination of enzyme kinetic and chemical inhibition studies. Amitriptyline was N-demethylated to nortriptyline by microsomes from cDNA transfected human lymphoblastoid cells expressing human CYPs 1A2, 2C9, 2C19, 2D6, and 3A4. CYP 2E1 showed no detectable activity. While CYP 2C19 and CYP 2D6 showed high affinity, CYP 3A4 showed low affinity; CYP 2C9 and 1A2 showed intermediate affinities. Based on these kinetic parameters and estimated relative abundance of the different CYPs in human liver, CYP 2C19 was identified as the major amitriptyline N-demethylase at low (therapeutically relevant) amitriptyline concentrations, whereas CYP 3A4 may be more important at higher amitriptyline concentrations. Chemical inhibition studies with ketoconazole and omeprazole indicate that CYP 3A4 is the major amitriptyline N-demethylase at 100 mumol/L amitriptyline, while CYP 2C19 is equally important at a substrate concentration of 5 mumol/L. The CYP 1A2 inhibitor alpha-naphthoflavone and the CYP 2C9 inhibitor sulfaphenazole produced much less inhibition of amitriptyline N-demethylation at both substrate concentrations. Quinidine produced no detectable inhibition. The kinetics of amitriptyline N-demethylation by human liver microsomes were consistent with a two enzyme model, with the high affinity component exhibiting Michaelis Menten kinetics and the low affinity component exhibiting Hill enzyme kinetics. No difference was apparent in the kinetics of amitriptyline N-demethylation in two liver samples with low levels of CYP 2C19 activity compared with two other samples with relatively normal 2C19 activity. This may reflect the importance of higher substrate concentration values in estimation of kinetic parameters in vitro.