ABSTRACT
PURPOSE: To evaluate the potential pharmacokinetic interactions between topiramate (TPM) and phenytoin (PHT) in patients with epilepsy by studying their pharmacokinetics (PK) after monotherapy and concomitant TPM/PHT treatment. METHODS: Twelve patients with epilepsy stabilized on PHT monotherapy were enrolled in this study, with 10 and seven patients completing the phases with 400 and 800 mg TPM daily doses, respectively. TPM was added at escalating doses, and after stabilization at the highest tolerated TPM dose, PHT doses were tapered. Serial blood and urine samples were collected for PK analysis during the monotherapy phase or the lowest PHT dose after taper and the concomitant TPM/PHT phase. Potential metabolic interaction between PHT and TPM also was studied in vitro in human liver microsomal preparations. RESULTS: In nine of the 12 patients, PHT plasma concentrations remained stable, with a mean (+/-SD) area under the curve (AUC) ratio (combination therapy/monotherapy) of 1.13 +/- 0.17 (range, 0.89-1.23). Three patients had AUC ratios of 1.25, 1.39, and 1.55, respectively, and with the addition of TPM (800, 400, and 400 mg daily, respectively), their peak PHT plasma concentrations increased from 15 to 21 mg/L, 28 to 36 mg/L, and 27 to 41 mg/L, respectively. Human liver microsomal studies with S-mephenytoin showed that TPM partially inhibited CYP2C19 at very high concentrations of 300 microM (11% inhibition) and 900 microM (29% inhibition). Such high plasma concentrations would correspond to doses in humans that are 5 to 15 times higher than the recommended dose (200-400 mg). TPM clearance was approximately twofold higher during concomitant TPM/PHT therapy CONCLUSIONS: This study provides evidence that the addition of TPM to PHT generally does not cause clinically significant PK interaction. PHT induces the metabolism of TPM, causing increased TPM clearance, which may require TPM dose adjustments when PHT therapy is added or is discontinued. TPM may affect PHT concentrations in a few patients because of inhibition by TPM of the CYP2C19-mediated minor metabolic pathway of PHT.
Subject(s)
Anticonvulsants/pharmacokinetics , Aryl Hydrocarbon Hydroxylases , Epilepsy/drug therapy , Fructose/pharmacokinetics , Phenytoin/pharmacokinetics , Adolescent , Adult , Anticonvulsants/therapeutic use , Cytochrome P-450 CYP2C19 , Cytochrome P-450 Enzyme System/drug effects , Cytochrome P-450 Enzyme System/metabolism , Dose-Response Relationship, Drug , Drug Administration Schedule , Drug Interactions , Drug Therapy, Combination , Epilepsy/metabolism , Female , Fructose/analogs & derivatives , Fructose/therapeutic use , Humans , Male , Middle Aged , Mixed Function Oxygenases/drug effects , Mixed Function Oxygenases/metabolism , Phenytoin/therapeutic use , TopiramateABSTRACT
OBJECTIVE: Several reports indicate that fluvoxamine decreases the clearance of cytochrome P4501A2 (CYP1A2) substrates. This study compared in vitro and in vivo inhibition potencies of fluvoxamine toward CYP1A2 with an approach based on inhibition constants (K(i)) determined in vitro and in vivo. METHODS: In vitro inhibition constant values were determined with human liver microsomes and complementary deoxyribonucleic acid-expressed CYP1A2 (supersomes). Fluvoxamine in vivo inhibition constants (K(i)iv) for CYP1A2 were obtained from an investigation of single-dose theophylline (250 mg) disposition in 9 healthy volunteers receiving steady-state (9 days) fluvoxamine at 3 doses (0, 25, or 75 mg/d) in a randomized crossover design. RESULTS: In vitro K(i) values based on total inhibitor concentrations were 177 +/- 56 nmol/L, 121 +/- 21 nmol/L, and 52 +/- 13 nmol/L in human liver microsomes with 1 mg/ml protein and 0.5 mg/ml protein and in supersomes with 0.3 mg/ml protein, respectively. The corresponding in vitro K(i) values based on unbound fluvoxamine concentrations were 35 nmol/L, 36 nmol/L, and 36 nmol/L. The ratio of 1-methyluric acid formation clearances (control/inhibited) in 8 subjects was positively correlated with fluvoxamine concentration (r (2) = 0.87; P <.001) with an intercept near 1. Mean values for K(i)iv based on total and unbound plasma concentrations at steady state were 25.3 nmol/L (range, 14-39 nmol/L) and 3.6 nmol/L (range, 2.4-5.9 nmol/L), respectively. CONCLUSION: Comparison of in vitro and in vivo K(i) values based on unbound fluvoxamine concentrations suggests that fluvoxamine inhibition potency is approximately 10 times greater in vivo than in vitro.
Subject(s)
Cytochrome P-450 CYP1A2 Inhibitors , Enzyme Inhibitors/pharmacology , Fluvoxamine/pharmacology , Theophylline/pharmacokinetics , Adult , Cross-Over Studies , Drug Interactions , Fluvoxamine/metabolism , Humans , In Vitro TechniquesABSTRACT
The active site topography of rabbit CYP4B1 has been studied relative to CYP2B1 and CYP102 using a variety of aromatic probe substrates. Oxidation of the prochiral substrate cumene by CYP4B1, but not CYP2B1 or CYP102, resulted in the formation of the thermodynamically disfavored omega-hydroxy metabolite, 2-phenyl-1-propanol, with product stereoselectivity for the (S)-enantiomer. Reaction of CYP4B1, CYP2B1, and CYP102 with phenyldiazene produced spectroscopically observable sigma-complexes for each enzyme. Subsequent oxidation of the CYP2B1 and CYP102 complexes followed by LC/ESI--MS analysis yielded heme pyrrole migration patterns similar to those in previous literature reports. Upon identical treatment, no migration products were detected for CYP4B1. Intramolecular deuterium isotope effects for the benzylic hydroxylation of o-xylene-alpha-(2)H(3), p-xylene-alpha-(2)H(3), 2-(2)H(3),6-dimethylnaphthalene, and 4-(2)H(3),4'-dimethylbiphenyl were determined for CYP4B1 and CYP2B1 to further map their active site dimensions. These probes permit assessment of the ease of equilibration, within P450 active sites, of oxidizable methyl groups located between 3 and 10 A apart [Iyer et al. (1997) Biochemistry 36, 7136--7143]. Isotope effects for the CYP4B1-mediated benzylic hydroxylation of o- and p-xylenes were fully expressed (k(H)/k(D) = 9.7 and 6.8, respectively), whereas deuterium isotope effects for the naphthyl and biphenyl derivatives were both substantially masked (k(H)/k(D) approximately equal to 1). In contrast, significant suppression of the deuterium isotope effects for CYP2B1 occurred only with the biphenyl substrate. Therefore, rapid equilibration between two methyl groups more than 6 A apart is impeded within the active site of CYP4B1, whereas for CYP2B1, equilibration is facile for methyl groups distanced by more than 8 A. Collectively, all data are consistent with the conclusion that the active site of CYP4B1 is considerably restricted relative to CYP2B1.
Subject(s)
Aryl Hydrocarbon Hydroxylases , Benzene Derivatives/metabolism , Biphenyl Compounds/metabolism , Cytochrome P-450 Enzyme System/metabolism , Imines/metabolism , Naphthalenes/metabolism , Xylenes/metabolism , Animals , Binding Sites , Deuterium/metabolism , Hydroxylation , Iron , Ligands , Propanols/metabolism , Rabbits , Substrate SpecificityABSTRACT
Human liver microsomes catalyze the oxidation of sulfinpyrazone sulfide (SPZS) to a variable mixture of sulfinpyrazone (SPZ) enantiomers and two minor phenolic metabolites. In one, the thiophenyl ring is hydroxylated, whereas in the second an N-phenyl ring is hydroxylated. SPZ is further oxidized to sulfinpyrazone sulfone (SPZO) and a minor polar metabolite that also has an N-phenyl ring hydroxylated. Determination of the metabolism of SPZ and SPZS under modified incubation conditions of prior heat treatment, higher pH, and the presence of detergent indicated that the formation of SPZ was cytochrome P450 (P450)- but not flavin monooxygenase-dependent. Specific P450 inhibitors (sulfaphenazole, quinidine sulfate, coumarin, diethyldithiocarbamic acid, troleandomycin, and furafylline) and specific cDNA-expressed P450s were used to identify the major isoforms responsible for the oxidation of SPZS to SPZ and SPZ to SPZO. Both P450 2C9 and P450 3A4 were responsible for the oxidation of SPZS to SPZ, whereas P450 3A4 alone catalyzed the further oxidation of SPZ to SPZO. SPZS was found to be metabolized by P450 2C9 to SPZ with a high degree of enantiomeric selectivity (9:1) and a K(m) comparable with its previously determined K(i) for inhibition of the P450 2C9-dependent 7-hydroxylation of (S)-warfarin (WARF). In contrast, the P450 3A4-catalyzed oxidation of SPZS to SPZ proceeded with the same enantioselectivity but to a much lesser degree (58:42). These results provide evidence that the metabolism of both (S)-WARF and SPZS is mediated by a common enzyme, P450 2C9, which is central to understanding the WARF-SPZ interaction and SPZS-mediated drug interactions in general.
Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Microsomes, Liver/enzymology , Sulfinpyrazone/metabolism , Chromatography, High Pressure Liquid , Cytochrome P-450 Enzyme System/genetics , DNA, Complementary , Humans , Oxidation-Reduction , Spectrometry, Mass, Electrospray Ionization , Sulfinpyrazone/pharmacokineticsABSTRACT
A ligand-based model is reported that predicts the Ki values for cytochrome P450 2C9 (CYP2C9) inhibitors. This CoMFA model was used to predict the affinity of 14 structurally diverse compounds not in the training set and appears to be robust. The mean error of the predictions is 6 microM. The experimentally measured Ki values of the 14 compounds range from 0.1 to 48 microM. Leave-one-out cross-validated partial least-squares gives a q2 value of between 0.6 and 0.8 for the various models which indicates internal consistency. Random assignment of biological data to structure leads to negative q2 values. These models are useful in that they establish a pharmacophore for binding to CYP2C9 that can be tested with site-directed mutagenesis. These models can also be used to screen for potential drug interactions and to design compounds that will not bind to this enzyme with high affinity.
Subject(s)
Aryl Hydrocarbon Hydroxylases , Cytochrome P-450 Enzyme System/chemistry , Enzyme Inhibitors/chemistry , Steroid 16-alpha-Hydroxylase , Steroid Hydroxylases/chemistry , Binding Sites , Cytochrome P-450 Enzyme System/metabolism , Drug Interactions , Ligands , Models, Biological , Models, Molecular , Protein Binding , Reproducibility of Results , Steroid Hydroxylases/metabolism , Structure-Activity Relationship , Sulfaphenazole/chemistry , Sulfaphenazole/metabolism , Sulfonamides/chemistry , Sulfonamides/metabolism , Warfarin/chemistry , Warfarin/metabolismABSTRACT
Mechanism-based inactivation of human liver P450 3A4 by L-754,394, a Merck compound synthesized as a potential HIV protease inhibitor, was investigated using recombinant P450 3A4. Enzyme inactivation was characterized by a small partition ratio (3.4 or 4.3 +/- 0.4), i.e., the total number of metabolic events undergone by the inhibitor divided by the number of enzyme inactivating events, lack of reversibility upon extensive dialysis, no decrease in the characteristic 450-nm species relative to control, and covalent modification of the apoprotein. The major and minor products formed during the inactivation of P450 3A4 were the monohydroxylated and the dihydrodiol metabolites of L-754,394, respectively. L-754,394 that had been adducted to P450 3A4 was hydrolyzed under the conditions used for SDS-PAGE, Ni(2+) affinity chromatography, and proteolytic digestion. In addition, the modification was not stable to the acidic conditions of HPLC separation and CNBr digestion. The labile nature of the peptide adduct and the nonstoichiometric binding of the inactivating species to P450 3A4 precluded the direct identification of a covalently modified amino acid residue or the peptide to which it was attached. However, Tricine SDS-PAGE in combination with MALDI-TOF-MS and homology modeling, allowed I257-M317 to be tentatively identified as an active site peptide, while prior knowledge of the stability of N-, O-, and S-linked conjugates of activated furans implicates Glu307 as the active site amino acid that is labeled by L-754, 394.
Subject(s)
Cytochrome P-450 Enzyme Inhibitors , Indans/pharmacology , Mixed Function Oxygenases/antagonists & inhibitors , Piperazines/pharmacology , Binding Sites , Catalysis , Cell Membrane/metabolism , Chromatography, High Pressure Liquid , Computer Simulation , Cyanogen Bromide/metabolism , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/chemistry , Cytochrome P-450 Enzyme System/isolation & purification , Cytochrome P-450 Enzyme System/metabolism , Dialysis , Escherichia coli/cytology , HIV Protease Inhibitors/chemistry , HIV Protease Inhibitors/metabolism , HIV Protease Inhibitors/pharmacology , Humans , Hydroxylation , Indans/chemistry , Indans/metabolism , Microsomes, Liver/enzymology , Mixed Function Oxygenases/chemistry , Mixed Function Oxygenases/isolation & purification , Mixed Function Oxygenases/metabolism , Models, Chemical , Models, Molecular , NADP/metabolism , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Piperazines/chemistry , Piperazines/metabolism , Protein Binding , Protein Structure, Secondary , Recombinant Fusion Proteins/antagonists & inhibitors , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Trypsin/metabolismABSTRACT
Nevirapine (NVP), a non-nucleoside inhibitor of HIV-1 reverse transcriptase, is concomitantly administered to patients with a variety of medications. To assess the potential for its involvement in drug interactions, cytochrome P-450 (CYP) reaction phenotyping of NVP to its four oxidative metabolites, 2-, 3-, 8-, and 12-hydroxyNVP, was performed. The NVP metabolite formation rates by characterized human hepatic microsomes were best correlated with probe activities for either CYP3A4 (2- and 12-hydroxyNVP) or CYP2B6 (3-and 8-hydroxyNVP). In studies with cDNA-expressed human hepatic CYPs, 2- and 3-hydroxyNVP were exclusively formed by CYP3A and CYP2B6, respectively. Multiple cDNA-expressed CYPs produced 8- and 12-hydroxyNVP, although they were produced predominantly by CYP2D6 and CYP3A4, respectively. Antibody to CYP3A4 inhibited the rates of 2-, 8-, and 12-hydroxyNVP formation by human hepatic microsomes, whereas antibody to CYP2B6 inhibited the formation of 3- and 8-hydroxyNVP. Studies using the CYP3A4 inhibitors ketoconazole, troleandomycin, and erythromycin suggested a role for CYP3A4 in the formation of 2-, 8-, and 12-hydroxyNVP. These inhibitors were less effective or ineffective against the biotransformation of NVP to 3-hydroxyNVP. Quinidine very weakly inhibited only 8-hydroxyNVP formation. NVP itself was an inhibitor of only CYP3A4 at concentrations that were well above those of therapeutic relevance (K(i) = 270 microM). Collectively, these data indicate that NVP is principally metabolized by CYP3A4 and CYP2B6 and that it has little potential to be involved in inhibitory drug interactions.
Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Microsomes, Liver/metabolism , Nevirapine/pharmacokinetics , Reverse Transcriptase Inhibitors/pharmacokinetics , Antibodies/pharmacology , Biotransformation/drug effects , Cytochrome P-450 Enzyme Inhibitors , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/immunology , DNA, Complementary/genetics , Drug Interactions , HIV Reverse Transcriptase/drug effects , HIV Reverse Transcriptase/metabolism , Humans , In Vitro Techniques , Ketoconazole/pharmacology , Microsomes, Liver/enzymology , Nevirapine/metabolism , Nevirapine/pharmacology , Phenotype , Recombinant Proteins/metabolism , Reverse Transcriptase Inhibitors/metabolism , Reverse Transcriptase Inhibitors/pharmacology , Troleandomycin/pharmacologyABSTRACT
Possible reasons for the observed differences in metabolic behavior and drug interaction liability between the structurally similar oral anticoagulants warfarin and phenprocoumon were explored. Incubating (S)-phenprocoumon with human liver microsomes and cDNA-expressed CYP2C9 and determining its metabolism both in the absence and presence of the CYP2C9 inhibitor, sulfaphenazole, confirmed that phenprocoumon is a substrate for CYP2C9. Comparing the metabolic behavior of (S)- and (R)-warfarin, (S)- and (R)-phenprocoumon, and fixed structural mimics of the various tautomeric forms [(S)- and (R)-4-methoxyphenprocoumon, (S)- and (R)-2-methoxyphenprocoumon, (S)- and (R)-4-methoxywarfarin, (S)- and (R)-2-methoxywarfarin, and 9(S)- and 9(R)-cyclocoumarol] available to these two drugs with expressed CYP2C9 provides compelling evidence indicating that the ring closed form of (S)-warfarin and the ring opened anionic form of (S)-phenprocoumon are the major and specific structural forms of the two drugs that interact with the active site of CYP2C9. The conclusion that (S)-warfarin and (S)-phenprocoumon interact with CYP2C9 in very different structural states provides a clear basis for the significant differences observed in their metabolic profiles. Moreover, in accord with a previously established CoMFA model these results are consistent with the hypothesis that the active site of CYP2C9 possesses at least two major substrate binding sites, a pi-stacking site for aromatic rings and an ionic binding site for organic anions. An additional electrostatic binding site also appears to contribute to the orientation of coumarin analogs in the CYP2C9 active site by interacting with the C2-carbonyl group of the coumarin nucleus.
Subject(s)
Anticoagulants/chemistry , Anticoagulants/metabolism , Aryl Hydrocarbon Hydroxylases , Cytochrome P-450 Enzyme System/metabolism , Phenprocoumon/chemistry , Phenprocoumon/metabolism , Steroid 16-alpha-Hydroxylase , Steroid Hydroxylases/metabolism , Warfarin/chemistry , Warfarin/metabolism , 4-Hydroxycoumarins/chemistry , 4-Hydroxycoumarins/metabolism , Anticoagulants/pharmacology , Catalytic Domain , Cytochrome P-450 CYP2C9 , Cytochrome P-450 Enzyme Inhibitors , Cytochrome P-450 Enzyme System/chemistry , Drug Interactions , Humans , In Vitro Techniques , Kinetics , Microsomes, Liver/metabolism , Models, Molecular , Phenprocoumon/pharmacology , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Stereoisomerism , Steroid Hydroxylases/antagonists & inhibitors , Steroid Hydroxylases/chemistry , Substrate Specificity , Sulfaphenazole/pharmacology , Warfarin/pharmacologyABSTRACT
Previous modeling efforts have suggested that coumarin ligand binding to CYP2C9 is dictated by electrostatic and pi-stacking interactions with complementary amino acids of the protein. In this study, analysis of a combined CoMFA-homology model for the enzyme identified F110 and F114 as potential hydrophobic, aromatic active-site residues which could pi-stack with the nonmetabolized C-9 phenyl ring of the warfarin enantiomers. To test this hypothesis, we introduced mutations at key residues located in the putative loop region between the B' and C helices of CYP2C9. The F110L, F110Y, V113L, and F114L mutants, but not the F114Y mutant, expressed readily, and the purified proteins were each active in the metabolism of lauric acid. The V113L mutant metabolized neither (R)- nor (S)-warfarin, and the F114L mutant alone displayed altered metabolite profiles for the warfarin enantiomers. Therefore, the effect of the F110L and F114L mutants on the interaction of CYP2C9 with several of its substrates as well as the potent inhibitor sulfaphenazole was chosen for examination in further detail. For each substrate examined, the F110L mutant exhibited modest changes in its kinetic parameters and product profiles. However, the F114L mutant altered the metabolite ratios for the warfarin enantiomers such that significant metabolism occurred for the first time on the putative C-9 phenyl anchor, at the 4'-position of (R)- and (S)-warfarin. In addition, the Vmax for (S)-warfarin 7-hydroxylation decreased 4-fold and the Km was increased 13-fold by the F114L mutation, whereas kinetic parameters for lauric acid metabolism, a substrate which cannot interact with the enzyme by a pi-stacking mechanism, were not markedly affected by this mutation. Finally, the F114L mutant effected a greater than 100-fold increase in the Ki for inhibition of CYP2C9 activity by sulfaphenazole. These data support a role for B'-C helix loop residues F114 and V113 in the hydrophobic binding of warfarin to CYP2C9, and are consistent with pi-stacking to F114 for certain aromatic ligands.
Subject(s)
Aryl Hydrocarbon Hydroxylases , Cytochrome P-450 Enzyme System/chemistry , Cytochrome P-450 Enzyme System/metabolism , Steroid 16-alpha-Hydroxylase , Steroid Hydroxylases/chemistry , Steroid Hydroxylases/metabolism , Warfarin/metabolism , Arachidonic Acid/metabolism , Binding Sites/genetics , Blotting, Western , Catalysis , Cytochrome P-450 Enzyme Inhibitors , Cytochrome P-450 Enzyme System/genetics , Diclofenac/metabolism , Kinetics , Lauric Acids/metabolism , Leucine/genetics , Mutagenesis, Site-Directed , Phenylalanine/genetics , Protein Structure, Secondary , Static Electricity , Steroid Hydroxylases/antagonists & inhibitors , Steroid Hydroxylases/genetics , Substrate Specificity/drug effects , Substrate Specificity/genetics , Sulfaphenazole/pharmacologyABSTRACT
A general scheme for the purification of baculovirus-expressed cytochrome P450s (P450s) from the crude insect cell pastes has been designed which renders the P450s suitable for analysis by high-performance liquid chromatography (HPLC) electrospray ionization mass spectrometry (ESI-MS). An HPLC/ESI-MS procedure has been developed to analyze small amounts of intact purified P450 (P450s cam-HT, 1A1, 1A2, 2A6, 2B1, 2C9, 2C9 C175R, 3A4, 3A4-HT) and rat NADPH cytochrome P450 reductase (P450 reductase). The experimentally determined and predicted (based on the amino acid sequences) molecular masses (MMs) of the various proteins had identical rank orders. For each individual protein, the difference between the experimentally determined (+/-SD, based on experiments performed on at least 3 different days) and predicted MMs ranged from 0.002 to 0.035%. Each experimentally determined MM had a standard deviation of less than 0.09% (based on the charge state distribution). Application of this HPLC/ESI-MS technique made the detection of the covalent modification to P450 2C9 following mechanism-based inactivation by tienilic acid possible. In the absence of glutathione, three P450 2C9 species were detected that produced ESI mass spectra corresponding to native P450 2C9 and both a monoadduct and a diadduct of tienilic acid to P450 2C9. In the presence of glutathione, only native P450 2C9 and the monoadduct were detected. Based on the observed mass shifts for the P450 2C9/tienilic acid adducts, a mechanism for the inactivation of P450 2C9 by tienilic acid is proposed.
Subject(s)
Aryl Hydrocarbon Hydroxylases , Cytochrome P-450 Enzyme System/chemistry , Steroid 16-alpha-Hydroxylase , Steroid Hydroxylases/chemistry , Ticrynafen/chemistry , Animals , Baculoviridae/genetics , Chromatography, High Pressure Liquid , Cytochrome P-450 Enzyme Inhibitors , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/isolation & purification , Electron Spin Resonance Spectroscopy , Electrons , Enzyme Activation , Genetic Vectors , Humans , Rats , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Steroid Hydroxylases/antagonists & inhibitors , Steroid Hydroxylases/genetics , Steroid Hydroxylases/isolation & purificationABSTRACT
Of several furanocoumarins [5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP), 5-hydroxypsoralen (5-OH-P), 8-hydroxypsoralen (8-OH-P), 4',5'-dihydro-8-MOP (DH-8-MOP), and psoralen (P)] tested as mechanism-based inactivators (MBIs) of purified reconstituted cytochrome P450 (P450) 2B1, 8-MOP was found to be the most potent (KI, kinact, and partition ratio of 2.9 microM, 0.34 min-1, and 1.3, respectively). The inactivation was not prevented by reactive oxygen species scavengers or nucleophilic trapping agents and proceeded with a decrease in P450 spectral content. Liquid chromatography (LC) separation of the reconstituted enzyme mixture, followed by liquid scintillation counting, indicated that [14C]-8-MOP binding was specific to the apoprotein of P450 2B1 with a binding stoichiometry of 0.7:1. The major metabolites formed by P450 2B1 from the furanocoumarins that were MBIs were characterized by LC electrospray ionization tandem mass spectrometry (ESI-MS/MS) as dihydro diols. Results from H218O incorporation experiments supported initial oxidation of 8-MOP and P to an epoxide which can react with some nucleophilic active site residue and inactivate the enzyme or partition to a dihydro diol metabolite by hydrolytic ring opening. On the other hand, 5-MOP was converted to an epoxide or gamma-keto enal intermediate prior to inactivation or dihydro diol formation. Comparison of the ESI mass spectra of P450 2B1 and furanocoumarin exposed P450 2B1, indicated a mass difference consistent with the covalent addition of a furanoepoxide to P450 2B1.
Subject(s)
Coumarins/pharmacology , Cytochrome P-450 CYP2B1/antagonists & inhibitors , Cytochrome P-450 CYP2B1/metabolism , Methoxsalen/pharmacology , 5-Methoxypsoralen , Animals , Binding Sites , Chromatography, Liquid , Coumarins/metabolism , Enzyme Activation/drug effects , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Ficusin/metabolism , Male , Mass Spectrometry , Methoxsalen/analogs & derivatives , Methoxsalen/metabolism , Microsomes, Liver/drug effects , Microsomes, Liver/enzymology , NADP/physiology , Rats , Rats, Sprague-DawleyABSTRACT
Several furanocoumarins were tested for their ability to inhibit human P450 2A6 activity. The metabolites and conjugates formed from these furanocoumarins after incubation with reconstituted purified P450 2A6 in the absence and presence of exogenous nucleophiles were characterized by UV and LC/ESI-MS/MS analysis. The results suggest initial oxidation to form a furanoepoxide followed by hydrolytic attack, or attack of exogenous nucleophiles, to form dihydrofuranocoumarin products. Initial epoxidation is confirmed by the finding that a single 18O atom is incorporated into the 8-methoxypsoralen (8-MOP) and psoralen (P) dihydrodiol metabolites when the incubations are performed in the presence of H218O. In contrast, 19% of the dihydrodiol formed from 5-methoxypsoralen (5-MOP) involves incorporation of two 18O atoms, implicating a gamma-ketoenal intermediate in the formation of this metabolite. Thus, the structure of the reactive intermediate(s) formed is dictated by the intrinsic electronic properties of the parent compound. After exposure to [14C]-8-MOP and [14C]-5-MOP, SDS-PAGE and HPLC experiments, followed by radiometric detection, indicated that both P450 2A6 and P450 reductase were covalently modified in the purified system. In contrast, only P450 2A6 was covalently modified in a lymphablastoid cell line (GENTEST). With the purified system, partition ratios were higher (1.5-3.9X), and the ability to scavenge reactive intermediates with exogenous nucleophiles was greater. These results suggest that relative to the cell system, more reactive intermediates can escape, rather than bind to, the active site of purified reconstituted P450 2A6.
Subject(s)
Aryl Hydrocarbon Hydroxylases , Coumarins/pharmacology , Cytochrome P-450 Enzyme Inhibitors , Mixed Function Oxygenases/antagonists & inhibitors , 5-Methoxypsoralen , Binding Sites , Carbon Radioisotopes , Coumarins/metabolism , Cytochrome P-450 CYP2A6 , Cytochrome P-450 Enzyme System/metabolism , Deuterium Oxide/metabolism , Enzyme Activation/drug effects , Humans , Methoxsalen/analogs & derivatives , Methoxsalen/metabolism , Methoxsalen/pharmacology , Microsomes, Liver/enzymology , Mixed Function Oxygenases/metabolism , Oxygen IsotopesABSTRACT
(R)-(+)-Menthofuran is a potent, mechanism-based inactivator of human liver cytochrome P450 (CYP or P450) 2A6. Menthofuran caused a time- and concentration-dependent loss of CYP2A6 activity. The inactivation of CYP2A6 was characterized by a Ki of 2.5 microM and a kinact of 0.22 min-1 for human liver microsomes and a Ki of 0.84 microM and a kinact of 0.25 min-1 for purified expressed CYP2A6. Addition of various nucleophiles, a chelator of iron, or scavengers of reactive oxygen species or extensive dialysis failed to protect CYP2A6 from inactivation. An antibody to metallothionein conjugates of a suspected reactive metabolite of menthofuran was used to detect reactive menthofuran metabolite adducts with CYP2A6. These adducts were formed only in the presence of NADPH-P450 reductase and NADPH. Glutathione, methoxylamine, and semicarbazide did not prevent adduction of reactive menthofuran metabolites to CYP2A6, however. The menthofuran metabolite formation/CYP2A6 inactivation partition ratio was determined to be 3.5 +/- 0.6 nmol/nmol of P450. Menthofuran was unable to inactivate CYP1A2, CYP2D6, CYP2E1, or CYP3A4 in a time- and concentration-dependent manner.
Subject(s)
Aryl Hydrocarbon Hydroxylases , Cytochrome P-450 Enzyme Inhibitors , Enzyme Inhibitors/pharmacology , Mixed Function Oxygenases/antagonists & inhibitors , Monoterpenes , Terpenes/pharmacology , Animals , Cytochrome P-450 CYP2A6 , Humans , NADP/pharmacology , RabbitsABSTRACT
Acetaminophen (APAP), a widely used analgesic and antipyretic agent, is bioactivated by cytochromes P450 to cause severe hepatotoxicity. APAP is oxidized by two pathways to form a toxic intermediate, N-acetyl-p-benzoquinone imine (NAPQI), and a nontoxic catechol metabolite, 3-hydroxy-APAP (3-OH-APAP). We investigated the role of P450 2E1 and 2A6 in APAP oxidation by using baculovirus-expressed and highly purified forms of human P450 2E1 and 2A6. An electrochemical HPLC assay was developed to quantify both oxidative metabolites simultaneously. For the first time, it was demonstrated that human P450 2E1 selectively oxidized APAP to NAPQI (assayed as its glutathione conjugate, GS-APAP), whereas human P450 2A6 selectively oxidized APAP to 3-OH-APAP. At 1 mM APAP, the relative ratio for the formation of GS-APAP vs 3-OH-APAP with human P450 2E1 was approximately 6:1, whereas the ratio with human P450 2A6 was 1:3. Apparent Km and Vmax values for the formation of GS-APAP by human P450 2E1 were 1.3 mM and 6.9 nmol/min/nmol of P450, respectively, whereas they were 4.6 mM and 7.9 nmol/min/nmol of P450 for P450 2A6. Apparent Km and Vmax values for the formation of 3-OH-APAP by human P450 2E1 were 4.0 mM and 2.5 nmol/min/nmol of P450, respectively, whereas they were 2.2 mM and 14.2 nmol/min/nmol of P450, respectively, for P450 2A6. Thus, although at toxic doses of APAP P450 2E1 is the more efficient catalyst for the formation of the toxic metabolite NAPQI, P450 2A6 also can contribute significantly to NAPQI production.
Subject(s)
Acetaminophen/metabolism , Analgesics, Non-Narcotic/metabolism , Aryl Hydrocarbon Hydroxylases , Cytochrome P-450 CYP2E1/metabolism , Cytochrome P-450 Enzyme System/metabolism , Mixed Function Oxygenases/metabolism , Baculoviridae/enzymology , Cytochrome P-450 CYP2A6 , HumansABSTRACT
OBJECTIVE: The spectrum of cytochrome P450 inhibition of stiripentol, a new anticonvulsant, was characterized in vitro and in vivo. METHODS: Stiripentol was incubated in vitro with (R)-warfarin, coumarin, (S)-warfarin, (S)-mephenytoin, bufuralol, p-nitrophenol, and carbamazepine as probes for CYPs 1A2, 2A6, 2C9, 2C19, 2D6, 2E1, and 3A4, respectively. Caffeine demethylation and the 6 beta-hydroxycortisol/cortisol ratio were monitored in vivo before and after 14 days of treatment with stiripentol as measures of CYP1A2 and CYP3A4 activity, and dextromethorphan O- and N-demethylation were used to measure CYP2D6 and CYP3A4 activity, respectively. In vivo inhibition constants for CYP3A4 were calculated with use of data that previously documented the interaction between stripentol and carbamazepine. RESULTS: In vitro, stiripentol inhibited CYPs 1A2, 2C9, 2C19, 2D6, and 3A4, with inhibition constant values at or slightly higher than therapeutic (total) concentrations of stiripentol, but it did not inhibit CYPs 2A6 and 2E1 even at tenfold therapeutic concentrations. In vivo inhibition of caffeine demethylation and dextromethorphan N-demethylation were consistent with inhibition of CYP1A2 and CYP3A4, respectively. The 6 beta-hydroxycortisol/cortisol ratio did not provide a reliable index of CYP3A4 inhibition. Inhibition of CYP2D6-mediated O-demethylation was not observed in vivo. With use of carbamazepine, in vivo inhibition constants for CYP3A4 ranged between 12 and 35 mumol/L, whereas the corresponding in vitro value was 80 mumol/L. CONCLUSIONS: Stiripentol appears to inhibit several CYP450 enzymes in vitro and in vivo. In vivo inhibition constants show that stiripentol inhibition of CYP3A4 is linearly related to plasma concentration in patients with epilepsy.
Subject(s)
Anticonvulsants/pharmacology , Cytochrome P-450 Enzyme Inhibitors , Dioxolanes/pharmacology , Adult , Anticonvulsants/chemistry , Caffeine , Carbon Dioxide/analysis , Carbon Isotopes , Cytochrome P-450 CYP3A , Dextromethorphan , Dioxolanes/chemistry , Epilepsy/drug therapy , Epilepsy/enzymology , Humans , Hydrocortisone , In Vitro Techniques , Mixed Function Oxygenases/antagonists & inhibitors , Reference Values , Time FactorsABSTRACT
Cytochrome P4502C9 (CYP2C9) is largely responsible for terminating the anticoagulant effect of racemic warfarin via hydroxylation of the pharmacologically more potent S-enantiomer to inactive metabolites. Mutations in the CYP2C9 gene result in the expression of three allelic variants, CYP2C9*1, CYP2C9*2 and CYP2C9*3. Both CYP2C9*2 and CYP2C9*3 exhibit altered catalytic properties in vitro relative to the wild-type enzyme. In the present study, a patient was genotyped who had proven unusually sensitive to warfarin therapy and could tolerate no more than 0.5 mg of the racemic drug/day. PCR-amplification of exons 3 and 7 of the CYP2C9 gene, followed by restriction digest or sequence analysis, showed that this individual was homozygous for CYP2C9*3. In addition, patient plasma warfarin enantiomer ratios and urinary 7-hydroxywarfarin enantiomer ratios were determined by chiral-phase high performance liquid chromotography in order to investigate whether either parameter might be of diagnostic value in place of a genotypic test. Control patients receiving 4-8 mg warfarin/day exhibited plasma S:R ratios of 0.50 +/- 0.25:1, whereas the patient on very low-dose warfarin exhibited an S:R ratio of 3.9:1. In contrast, the urinary 7-hydroxywarfarin S:R ratio of 4:1 showed the same stereoselectivity as that reported for control patients. Therefore, expression of CYP2C9*3 is associated with diminished clearance of S-warfarin and a dangerously exacerbated therapeutic response to normal doses of the racemic drug. Analysis of the plasma S:R warfarin ratio may serve as a useful alternative test to genotyping for this genetic defect.
Subject(s)
Anticoagulants/pharmacology , Aryl Hydrocarbon Hydroxylases , Cytochrome P-450 Enzyme System/genetics , Steroid 16-alpha-Hydroxylase , Steroid Hydroxylases/genetics , Warfarin/pharmacology , Anticoagulants/pharmacokinetics , Cytochrome P-450 CYP2C9 , Cytochrome P-450 Enzyme System/metabolism , Heterozygote , Homozygote , Humans , Male , Middle Aged , Phenotype , Stereoisomerism , Steroid Hydroxylases/metabolism , Warfarin/pharmacokineticsABSTRACT
Intramolecular isotope effects associated with the benzylic hydroxylation of a series of selectively deuterated isomeric xylenes and 4,4'-dimethylbiphenyl as catalyzed by various rat liver microsomal preparations and CYP2B1 were determined. Substrate analogs in which each methyl group contained either one (d2 substrates) or two (d4 substrates) deuterium atoms were used to determine the intrinsic isotope effect for the reaction. Specific values of the individual primary (P) and secondary isotope effects (S) were determined. P ranged from a low of 5.32 +/- 0.48 to a high of 7.57 +/- 0.42 depending upon the specific cytochrome P450 preparation used for catalysis. S had an average value of 1.03. The d3 substrates allowed exploration of the effect of distance on the magnitude of the observed isotope effect. The results indicate that the distance of 6.62 A that separates the carbon atoms of the para methyl groups of p-xylene is insufficient to suppress (mask) the intrinsic isotope effect for benzylic hydroxylation by all of the enzyme preparations examined. Conversely, a distance of 11.05 A, the minimal separation between the carbon atoms of the para methyl groups of p,p'-dimethylbiphenyl, is large enough to almost completely mask the intrinsic isotope effect for benzylic hydroxylation by the same set of enzymes.
Subject(s)
Benzene/metabolism , Biphenyl Compounds/metabolism , Cytochrome P-450 Enzyme System/metabolism , Xylenes/metabolism , Animals , Cytochrome P-450 CYP2B1/metabolism , Deuterium , Hydrogen Bonding , Hydroxylation , Isomerism , Kinetics , Male , Methylation , Microsomes, Liver/enzymology , Models, Chemical , Rats , Rats, Sprague-DawleyABSTRACT
The P450 2A6 catalyzed 7-hydroxylation of coumarin proceeded with a mean Km of 0.40 (+/-0.13) microM and Vmax of 6.34 nmol/nmol P450/min (36-fold variation) in microsomal preparations from a panel of 12 human livers. Substrate depletion was avoided during the kinetic determinations. 8-Methoxypsoralen (8-MOP) is a potent mechanism-based inactivator of human liver P450 2A6 and reconstituted purified recombinant P450 2A6 based on the following evidence: 1) 8-MOP causes time, concentration, and NADPH-dependent loss of P450 2A6 activity that is not reversed by potassium ferricyanide or extensive dialysis, 2) loss of P450 2A6 activity is associated with a loss of spectrally observable P450, 3) addition of nucleophiles or reactive oxygen scavengers to the incubations does not prevent inactivation of P450 2A6, and 4) 8-MOP-dependent P450 2A6 inactivation is inhibited (concentration dependent) by the addition of a competitive inhibitor (pilocarpine). Inactivation is selective for P450 2A6 at low concentrations of 8-MOP (2.5 microM) after short incubation time periods (3 min) and was characterized by a KI of 0.8 and 1.9 microM in a reconstituted and microsomal system, respectively, and a kinact of 1 min-1 and 2 min-1 in a reconstituted and microsomal system, respectively. A substrate depletion partition ratio of 21 was calculated for the inactivation of recombinant P450 2A6. Potency and selectivity suggest that 8-MOP could be a useful tool in vitro for evaluating P450 2A6 activity in various enzyme preparations.
Subject(s)
Aryl Hydrocarbon Hydroxylases , Cytochrome P-450 Enzyme Inhibitors , Cytochrome P-450 Enzyme System/metabolism , Methoxsalen/pharmacology , Microsomes, Liver/enzymology , Mixed Function Oxygenases/antagonists & inhibitors , Mixed Function Oxygenases/metabolism , Steroid 16-alpha-Hydroxylase , Adolescent , Adult , Child , Coumarins/metabolism , Cytochrome P-450 CYP1A2 , Cytochrome P-450 CYP1A2 Inhibitors , Cytochrome P-450 CYP2A6 , Cytochrome P-450 CYP2C19 , Cytochrome P-450 CYP2D6 Inhibitors , Cytochrome P-450 CYP2E1 Inhibitors , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/genetics , Cytochromes b5/metabolism , Enzyme Activation/drug effects , Female , Humans , Kinetics , Male , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Middle Aged , Mixed Function Oxygenases/genetics , NADP/metabolism , NADPH-Ferrihemoprotein Reductase/metabolism , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Spectrophotometry , Steroid Hydroxylases/antagonists & inhibitors , Time FactorsABSTRACT
Azelastine, an antihistamine with additional pharmacologic properties, was evaluated for a possible influence on pharmacokinetic and electrocardiographic parameters due to its coadministration with CYP3A4 inhibitor ketoconazole (200 mg every 12 hrs). Twelve volunteers entered this three-period, open-label study. Electrocardiographic parameters (PR, QRS and QTc intervals and U-wave morphology) were monitored after 14 days of azelastine HCl (4.4 mg every 12 hrs), after 7 days of either azelastine/ketoconazole or azelastine/placebo, and after a 21-day washout period, which was then followed by a 7-day administration of ketoconazole alone. None of the treatments resulted in meaningful alterations of electrocardiographic variables. Pharmacokinetic parameters could not be estimated because ketoconazole metabolites interfered with azelastine assay procedures. In vitro tests with human liver microsomes were used to characterize azelastine's inhibition spectrum. Azelastine did not inhibit CYP3A4 activity but it did inhibit CYP2D6 and CYP2C19 activity with Ki values exceeding maximum plasma concentration by 120 to 800-fold. Therefore, in vitro tests and the absence of electrocardiographic effects suggests azelastine can be safely administered with CYP3A4 inhibitors.
Subject(s)
Antifungal Agents/pharmacology , Electrocardiography/drug effects , Heart Rate/drug effects , Histamine H1 Antagonists/pharmacology , Ketoconazole/pharmacology , Phthalazines/pharmacology , Adult , Antifungal Agents/metabolism , Antifungal Agents/pharmacokinetics , Area Under Curve , Cytochrome P-450 Enzyme System/metabolism , Double-Blind Method , Histamine H1 Antagonists/metabolism , Histamine H1 Antagonists/pharmacokinetics , Humans , Ketoconazole/metabolism , Ketoconazole/pharmacokinetics , Male , Phthalazines/metabolism , Phthalazines/pharmacokineticsABSTRACT
The purpose of the present studies was to define the role of the I359L allelic variant of CYP2C9 in the metabolism of the low therapeutic index anticoagulant warfarin, by performing in vitro kinetic studies with the two enantiomers of the drug. To obtain sufficient quantities of these variants to perform kinetic studies at physiologically relevant substrate concentrations, methodology was established for the high-level expression, purification, and structural characterization of wild-type CYP2C9 and CYP2C9V1 using the baculovirus system. Both forms were expressed at levels up to 250 nmol/liter and purified in 50-55% yield to specific contents of 13-14 nmol holoenzyme/mg protein. The purified preparations were characterized by Edman degradation and electrospray-mass spectrometry. Both forms of the enzyme metabolized the pharmacologically more potent (S)-enantiomer of warfarin with the same regioselectivity; however, CYP2C9V1 exhibited a fivefold lower Vmax and a fivefold higher Km compared to the wild-type enzyme for this substrate. Neither form of the enzyme formed significant quantities of the (R)-warfarin phenols. Additional studies performed with prochiral arylalkyl sulfides provided confirmation of the low turnover rates catalyzed by CYP2C9V1 and demonstrated further that sulfoxide product stereochemistry did not differ significantly between the two variants. Therefore, decreased catalytic efficiency rather than a gross alteration in substrate orientation appears to be the consequence of this putative active-site mutation. The greatly decreased catalytic efficiency of the I359L variant suggests that leucine homozygotes would eliminate (S)-warfarin, and probably many other CYP2C9 substrates, at much slower rates in vivo than individuals expressing the wild-type enzyme.