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CYP3A4-Mediated carbamazepine (CBZ) metabolism: formation of a covalent CBZ-CYP3A4 adduct and alteration of the enzyme kinetic profile.

ABSTRACT: Carbamazepine (CBZ) is a widely prescribed anticonvulsant whose use is often associated with idiosyncratic hypersensitivity. Sera of CBZ-hypersensitive patients often contain anti-CYP3A antibodies, including those to a CYP3A23 K-helix peptide that is also modified during peroxidative CYP3A4 heme-fragmentation. We explored the possibility that cytochromes P450 (P450s) such as CYP3A4 bioactivate CBZ to reactive metabolite(s) that irreversibly modify the P450 protein. Such CBZ-P450 adducts, if stable in vivo, could engender corresponding serum P450 autoantibodies. Incubation with CBZ not only failed to inactivate functionally reconstituted, purified recombinant CYP3A4 or CYP3A4 Supersomes in a time-dependent manner, but the inclusion of CBZ (0-1 mM) also afforded a concentration-dependent protection to CYP3A4 from inactivation by NADPH-induced oxidative uncoupling. Incubation of CYP3A4 Supersomes with (3)H-CBZ resulted in its irreversible binding to CYP3A4 protein with a stoichiometry of 1.58 +/- 0.15 pmol (3)H-CBZ bound/pmol CYP3A4. Inclusion of glutathione (1.5 mM) in the incubation reduced this level to 1.09. Similar binding (1.0 +/- 0.4 pmol (3)H-CBZ bound/pmol CYP3A4) was observed after (3)H-CBZ incubation with functionally reconstituted, purified recombinant CYP3A4(His)(6). The CBZ-modified CYP3A4 retained its functional activity albeit at a reduced level, but its testosterone 6beta-hydroxylase kinetics were altered from sigmoidal (a characteristic profile of substrate cooperativity) to near-hyperbolic (Michaelis-Menten) type, suggesting that CBZ may have modified CYP3A4 within its active site.

SUBMITTER: Kang P

PROVIDER: S-EPMC2881839 | biostudies-literature | 2008 Mar

REPOSITORIES: biostudies-literature

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CYP3A4-Mediated carbamazepine (CBZ) metabolism: formation of a covalent CBZ-CYP3A4 adduct and alteration of the enzyme kinetic profile.

Kang Ping P Liao Mingxiang M Wester Michael R MR Leeder J Steven JS Pearce Robin E RE Correia Maria Almira MA

Drug metabolism and disposition: the biological fate of chemicals 20071220 3

Carbamazepine (CBZ) is a widely prescribed anticonvulsant whose use is often associated with idiosyncratic hypersensitivity. Sera of CBZ-hypersensitive patients often contain anti-CYP3A antibodies, including those to a CYP3A23 K-helix peptide that is also modified during peroxidative CYP3A4 heme-fragmentation. We explored the possibility that cytochromes P450 (P450s) such as CYP3A4 bioactivate CBZ to reactive metabolite(s) that irreversibly modify the P450 protein. Such CBZ-P450 adducts, if stab ...[more]

PMID: 18096676

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Project description:BACKGROUND: The aim of this study was to investigate the effect of two genetic polymorphisms in the coding regions (exon 3 and exon 4) of the EPHX1 gene, ie, 337T>C and 416A>G, respectively, on the metabolism of carbamazepine (CBZ) 10,11-epoxide (the active metabolite of CBZ) by evaluating the variation in serum CBZ 10,11-epoxide levels 4 hours after administration of the drug. Moreover, we reported the genotype frequencies of the CYP3A4*22 (rs 35599367, C>T) variant and its influence on the metabolism of CBZ. METHODS: The analysis was performed in 50 patients receiving CBZ as monotherapy. DNA was extracted from leukocytes using a commercially available kit. Serum CBZ 10,11-epoxide levels were measured by high-performance liquid chromatography. Allelic discrimination was performed using polymerase chain reaction-restriction fragment length polymorphism. Statistical analysis of the difference in mean values for CBZ 10,11-epoxide levels according to genotype was performed using the Student's t-test with Statistical Package for the Social Sciences version 13 software. RESULTS: Fourteen percent of the study group were CC, 42% were CT, and 44% were TT for the EPHX1 337T>C variant. No GG homozygote was identified for the EPHX1 416A>G variant; 64% were AA and 36% were AG. When we compared serum CBZ 10,11-epoxide levels 4 hours after drug administration, we found no statistically significant difference between the 337 CC, CT, and TT genotypes. Similarly, no difference in serum CBZ 10,11-epoxide levels was found between 416A>G AA and AG. Genotype frequencies for the CYP3A4*22 (rs 35599367 C>T) allelic variant were 94% for CC and 6% for CT, with no statistically significant difference in serum CBZ 10,11-epoxide levels between these genotypes 4 hours after administration of the drug (2.6±1.3 ?g/?L and 2.5±1.2 ?g/?L, respectively). CONCLUSION: Although there is some evidence of involvement of these polymorphisms in enzyme activity in vitro, we found no interference with CBZ metabolism in vivo.

Project description:Drug toxicity is a hurdle to drug development and to clinical translation of basic research. Antiepileptic drugs such as carbamazepine (CBZ) and selective serotonin reuptake inhibitors such as sertraline (SRT) are commonly co-prescribed to patients with epilepsy and comorbid depression. Because SRT may interfere with cytochrome P450 (CYP) enzyme activity and CYPs have been implicated in the conversion of CBZ to reactive cytotoxic metabolites, we investigated in vitro models to determine whether SRT affects the neurotoxic potential of CBZ and the mechanisms involved.Human fetal brain-derived dopaminergic neurons, human brain microvascular endothelial cells (HBMECs), and embryonic kidney (HEK) cells were used to evaluate cytotoxicity of CBZ and SRT individually and in combination. Nitrite and glutathione (GSH) levels were measured with drug exposure. To validate the role of CYP3A4 in causing neurotoxicity, drug metabolism was compared to cell death in HEK CYP3A4 overexpressed and cells pretreated with the CYP3A4 inhibitor ketoconazole.In all cellular systems tested, exposure to CBZ (127 ?M) or SRT (5 ?M) alone caused negligible cytotoxicity. By contrast CBZ, tested at a much lower concentration (17 ?M) in combination with SRT (5 ?M), produced prominent cytotoxicity within 15 min exposure. In neurons and HBMECs, cytotoxicity was associated with increased nitrite levels, suggesting involvement of free radicals as a pathogenetic mechanism. Pretreatment of HBMECs with reduced GSH or with the GSH precursor N-acetyl-L-cysteine prevented cytotoxic response. In HEK cells, the cytotoxic response to the CBZ + SRT combination correlated with the rate of CBZ biotransformation and production of 2-hydroxy CBZ, further suggesting a causative role of reactive metabolites. In the same system, cytotoxicity was potentiated by overexpression of CYP3A4, and prevented by CYP3A4 inhibitor.These results demonstrate an unexpected neurotoxic interaction between CBZ and SRT, apparently related to increased CYP3A4-mediated production of reactive CBZ metabolites. The potential clinical implications of these findings are discussed.

Project description:To investigate the effects of P. ginseng C.A. Mey (P. ginseng) on the metabolism of diester alkaloids and explore the potential mechanism. P. ginseng was administered orally to rats for 7 days, after which liver microsome samples were prepared and then incubated with diester alkaloids. Ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry was used to determinate the concentration of diester alkaloids to calculate the clearance rate. The cocktail method was used to evaluate the effects of oral administration of P. ginseng extracts on the activities of cytochrome P450 (CYP) isoforms in rats through the changes in the pharmacokinetic parameters of the probe drugs. The protein and gene expression of CYP3A2 and pregnane X receptor (PXR) in rats were evaluated by western blotting and quantitative PCR. The specific enzyme inhibitor method and human recombinant enzyme method were used to identify the involvement of sub-CYPs in the metabolism of diester alkaloids in human liver microsomes (HLMs). The clearances of aconitine, mesaconitine, and hypaconitine in the P. ginseng groups were lower than those of the control group. The areas under the curve of midazolam were 2.37 ± 1.05, 4.96 ± 0.51, and 6.23 ± 1.30 mg·L-1·h for the low-, medium-, and high-dose P. ginseng groups, respectively, which were higher than that of the control (2.23 ± 0.64 mg·L-1·h). The clearances of midazolam for the medium- (1.87 ± 0.16 L·h-1·kg-1) and high-dose (1.60 ± 0.34 L·h-1·kg-1) P. ginseng groups were lower than that of the control group (4.66 ± 1.43 L·h-1·kg-1). After exposure to P. ginseng extracts, the gene and protein expression levels of CYP3A4 and PXR were decreased. The hepatic metabolism rates of aconitine, mesaconitine, and hypaconitine in HLMs were decreased to 60.37%, 21.67%, and 10.11%, respectively, when incubated with ketoconazole, a specific inhibitor for CYP3A. The kinetic plots indicated that the KM and V max values of CYP3A4 were 10.08 ± 3.26 μM and 0.12 ± 0.01nmol·mg protein-1·min-1 for aconitine, 131.3 ± 99.75 μM and 0.73 ± 0.44 nmol·mg protein-1·min-1 for mesaconitine, and 17.05 ± 9.70 μM and 0.16 ± 0.04 nmol·mg protein-1·min-1 for hypaconitine, respectively. The in vitro mean intrinsic clearance rates by CYP3A4 were 0.0119, 0.0056, and 0.0091 mL·nmol CYP-1·min-1 for aconitine, mesaconitine, and hypaconitine, respectively. Therefore we implied that P. ginseng inhibited the metabolism of diester alkaloids in vitro and decreased the CYP3A4 enzyme activity as well as the gene and protein expression of CYP3A4 and PXR in vivo. CYP3A4 had a larger effect on diester alkaloid metabolism than the other human CYP isoforms, CYP1A2, CYP2C9, and CYP2E1.

Dataset Information

CYP3A4-Mediated carbamazepine (CBZ) metabolism: formation of a covalent CBZ-CYP3A4 adduct and alteration of the enzyme kinetic profile.

Publications

CYP3A4-Mediated carbamazepine (CBZ) metabolism: formation of a covalent CBZ-CYP3A4 adduct and alteration of the enzyme kinetic profile.

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