Phenytoin Hydroxylation Research Articles

Biotransformation of phenytoin in the electrochemically-driven CYP2C19 system

The possibility of the detection of atypical kinetic profiles of drug biotransformation using electrochemical systems based on immobilized cytochromes P450 with phenytoin hydroxylation by cytochrome P450 2C19 (CYP2C19) as an example was evaluated for the first time. For this purpose, we developed an electrochemical system, where one of the electrodes was modified by didodecyldimethylammonium bromide (DDAB) and was used as an electron donor for reduction of heme iron ion of the immobilized CYP2C19 and initiation of the catalytic reaction, while the second electrode was not modified and served for an electrochemical quantitation of 4-hydroxyphenytoin, which is a metabolite of antiepileptic drug phenytoin, by its oxidation peak. It was revealed that the dependence of the rate of 4-hydroxyphenytoin formation on phenytoin concentration is described by the equation for two enzymes or two binding sites indicating the existing of high- and low-affinity forms of the enzyme. The atypical kinetics and the kinetic parameters of CYP2C19-mediated phenytoin hydroxylation in the electrochemical system correlate to the same characteristics obtained by other authors in an alternative enzymatic system. Our results demonstrate the possibility of electrochemical systems based on cytochromes P450 to be applied for the detection of atypical kinetic profiles of drug metabolism.

Potential herb-drug interaction risk of thymoquinone and phenytoin

Thymoquinone is a main bioactive compound of Nigella sativa L. (N.sativa), which has been used for clinical studies in the treatment of seizures due to its beneficial neuroprotective activity and antiepileptic effects. It has been evidenced that thymoquinone may inhibit the activity of cytochrome P450 2C9 (CYP2C9). However, little is known about the effect of thymoquinone or N.sativa on the pharmacokinetic behavior of phenytoin, a second-line drug widely used in the management of status epilepticus. In this study, we systematically investigated the risk of the potential pharmacokinetic drug interaction between thymoquinone and phenytoin. The inhibitory effect of thymoquinone on phenytoin hydroxylation activity by CYP2C9 was determined using UPLC-MS/MS by measuring the formation rates for p-hydroxyphenytoin (p-HPPH). The potential for drug-interaction between thymoquinone and phenytoin was quantitatively predicted by using in vitro-in vivo extrapolation (IVIVE). Our data demonstrated that thymoquinone displayed effective inhibition against phenytoin hydroxylation activity. Enzyme kinetic studies showed that thymoquinone exerted a competitive inhibition against phenytoin hydroxylation with a Ki value of 4.45 ± 0.51 μM. The quantitative prediction from IVIVE suggested that the co-administration of thymoquinone (>18 mg/day) or thymoquinone-containing herbs (N.sativa > 1 g/day or N.sativa oil >1 g/day) might result in a clinically significant herb-drug interactions. Additional caution should be taken when thymoquinone or thymoquinone-containing herbs are co-administered with phenytoin, which may induce unexpected potential herb-drug interactions via the inhibition of CYP2C9.

Risk prediction of drug-drug interaction potential of phenytoin and miconazole topical formulations

The drug-drug interaction (DDI) risk of phenytoin with several topical formulations of miconazole is still unclear. The present investigation conducted in vitro-in vivo extrapolation to predict the potential risks. Our data indicated that miconazole potently inhibited phenytoin hydroxylation in both pooled human liver microsomes (HLMs) and recombinant cytochrome P450 2C9 (CYP2C9) with the Ki values of 125 ± 7 nM and 30 ± 2 nM, respectively. Quantitative prediction of DDI risk suggests that, beside intravenous administration or swallowed tablet, combination of phenytoin and miconazole high dose oral gel or buccal tablet may also result in a clinically significant increase of phenytoin AUC (>53%) by the inhibition of miconazole against phenytoin hydroxylation, consequently a higher frequency of adverse events, while the coadministration of miconazole vaginal formulation and phenytoin will be safe.

Drug-induced gingival overgrowth: the genetic dimension.

Background:Currently, the etiology of drug-induced gingival overgrowth is not entirely understood but is clearly multifactorial. Phenytoin, one of the common drugs implicated in gingival enlargement, is metabolized mainly by cytochrome P450 (CYP)2C9 and partly by CYP2C19. The CYP2C9 and CYP2C19 genes are polymorphically expressed and most of the variants result in decreased metabolism of the respective substrates.Aims:The present study was undertaken to investigate the influence of the CYP2C9*2 and *3 variant genotypes on phenytoin hydroxylation in subjects diagnosed with epilepsy from South India, thus establishing the genetic polymorphisms leading to its defective hydroxylation process.Materials and Methods:Fifteen epileptic subjects, age 9 to 60 years were included in the study. Among the study subjects, 8 were males and 7 were females. Genomic DNA was extracted from patients’ blood using Phenol-chloroform method and genotyping was done for CYP2C9 using customized TaqMan genotyping assays on a real time thermocycler, by allelic discrimination method. The genetic polymorphisms *1, *2 and *3 on CYP2C9 were selected based on their function and respective allele frequencies in Asian subcontinent among the Asian populations.Results:CYP2C9*1*2 and CYP2C9*3/*3 were identified with equal frequency in the study population. There were seven subjects with CYP2C9*1/*2 genotype (heterozygous mutant), one subject with CYP2C9*1/*1 (wild type) and seven study subjects with CYP2C9*3/*3 (homozygous mutant).Conclusion:The results obtained in the present study will be helpful in the medical prescription purposes of phenytoin, and a more personalized patient approach with its administration can be advocated.

CYP2C9 Amino Acid Residues Influencing Phenytoin Turnover and Metabolite Regio- and Stereochemistry

Phenytoin has been an effective anticonvulsant agent for over 60 years, although its clinical use is complicated by nonlinear pharmacokinetics, a narrow therapeutic index, and metabolically based drug-drug interactions. Although it is well established that CYP2C9 is the major cytochrome P450 enzyme controlling metabolic elimination of phenytoin through its oxidative conversion to (S)-5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH), nothing is known about the amino acid binding determinants within the CYP2C9 active site that promote metabolism and maintain the tight stereocontrol of hydroxy metabolite formation. This knowledge gap was addressed here through the construction of nine active site mutants at amino acid positions Phe100, Arg108, Phe114, Leu208, and Phe476 and in vitro analysis of the steady-state kinetics and stereochemistry of p-HPPH formation. The F100L and F114W mutants exhibited 4- to 5-fold increases in catalytic efficiency, whereas the F100W, F114L, F476L, and F476W mutants lost >90% of their phenytoin hydroxylation capacity. This pattern of effects differs substantially from that found previously for (S)-warfarin and (S)-flurbiprofen metabolism, suggesting that these three ligands bind within discrete locations in the CYP2C9 active site. Only the F114L, F476L, and L208V mutants altered phenytoin's orientation during catalytic turnover. The L208V mutant also uniquely demonstrated enhanced 6-hydroxylation of (S)-warfarin. These latter data provide the first experimental evidence for a role of the F-G loop region in dictating the catalytic orientation of substrates within the CYP2C9 active site.

The “Albumin Effect” and in Vitro-in Vivo Extrapolation: Sequestration of Long-Chain Unsaturated Fatty Acids Enhances Phenytoin Hydroxylation by Human Liver Microsomal and Recombinant Cytochrome P450 2C9

This study characterized the mechanism by which bovine serum albumin (BSA) reduces the K(m) for phenytoin (PHY) hydroxylation and the implications of the "albumin effect" for in vitro-in vivo extrapolation of kinetic data for CYP2C9 substrates. BSA and essentially fatty acid-free human serum albumin (HSA-FAF) reduced the K(m) values for PHY hydroxylation (based on unbound substrate concentration) by human liver microsomes (HLMs) and recombinant CYP2C9 by approximately 75%, with only a minor effect on V(max). In contrast, crude human serum albumin increased the K(m) with both enzyme sources. Mass spectrometric analysis of incubations containing HLMs was consistent with the hypothesis that BSA sequesters long-chain unsaturated acids (arachidonic, linoleic, oleic) released from membranes. A mixture of arachidonic, linoleic and oleic acids, at a concentration corresponding to 1/20 of the content of HLMs, doubled the K(m) for PHY hydroxylation by CYP2C9, without affecting V(max). This effect was reversed by addition of BSA to incubations. K(i) values for arachidonic acid inhibition of human liver microsomal- and CYP2C9-catalyzed PHY hydroxylation were 3.8 and 1.6 microM, respectively. Similar effects were observed with heptadecanoic acid, the most abundant long-chain unsaturated acid present in Escherichia coli membranes. Extrapolation of intrinsic clearance (CL(int)) values for each enzyme source determined in the presence of BSA and HSA-FAF accurately predicted the known CL(int) for PHY hydroxylation in vivo. The results indicate that previously determined in vitro K(m) values for CYP2C9 substrates are almost certainly overestimates, and accurate in vitro-in vivo extrapolation of kinetic data for CYP2C9 substrates is achievable.

CYP2C-catalyzed delta(9)-tetrahydrocannabinol metabolism: Kinetics, pharmacogenetics and interaction with phenytoin

delta(9) -Tetrahydrocannabinol (Δ 9-THC), the primary psychoactive constituent of marijuana, is subject to first pass hepatic metabolism primarily by hydroxylation to yield active and inactive oxygenated products. The primary metabolite is formed via oxidation of the allylic methyl group to yield 11-hydroxy-Δ 9-THC, which is oxidized further to 11-nor-9-carboxy-Δ 9-THC. The hydroxylation is thought to be mediated by CYP2C9. The present study was designed to address the kinetics and pharmacogenetics of CYP2C-mediated metabolism of (Δ 9)-THC by studying its metabolism in human liver microsomes and expressed enzymes. Expressed CYP2C9.1 exhibited high affinity for the hydroxylation of Δ 9-THC (apparent K m, 2 μM), similar to that observed in human liver microsomes (apparent K m, 0.8 μM). In contrast, the calculated intrinsic clearance (apparent V m/ K m) for CYP2C9.2 and CYP2C9.3 was approximately 30% that of the wild type, CYP2C9.1. Given the high affinity of CYP2C9 for the hydroxylation of Δ 9-THC, we evaluated the potential for an interaction between Δ 9-THC, 11-hydroxy-Δ 9-THC, or 11-nor-9-carboxy-Δ 9-THC and the CYP2C9 substrate, phenytoin. Surprisingly, Δ 9-THC increased the rate of phenytoin hydroxylation in human liver microsomes and expressed CYP2C9 enzyme. Similar increases in rate were observed with co-incubation of 11-hydroxy-Δ 9-THC and 11-nor-9-carboxy-Δ 9-THC with phenytoin. These in vitro data suggest the potential for an interaction from the concomitant administration of Δ 9-THC and phenytoin that could result in decreased phenytoin concentrations in vivo.

Effects of cysteine on the pharmacokinetics of intravenous phenytoin in rats with protein–calorie malnutrition

The effects of cysteine on the pharmacokinetics of phenytoin and one of its metabolites, 5-( p-hydroxyphenyl)-5-phenylhydantoin ( pHPPH) were investigated after intravenous administration of phenytoin, 25 mg/kg, to control rats (4-week fed on 23% casein diet) and rats with PCM (protein–calorie malnutrition, 4-week fed on 5% casein diet) and PCMC (PCM with oral cysteine supplementation, 250 mg/kg, twice daily starting from the fourth week). In rats with PCM and PCMC, the phenytoin hydroxylation (to form pHPPH) activities were significantly smaller (164, 103 and 95.3 pmol/min per mg protein for the control rats, and rats with PCM and PCMC, respectively) than that in control rats. In rats with PCMC, the intrinsic clearance of phenytoin, CL int was significantly slower than those in control rats and rats with PCM (0.175, 0.131 and 0.044 ml/min). The above data suggested that the formation of pHPPH could be reduced in rats with PCM and PCMC. This was supported by significantly smaller 24-h urinary excretion of pHPPH (54.7, 35.6 and 32.5% of intravenous dose of phenytoin) in rats with PCM and PCMC than that in control rats. In rats with PCM, the maximum velocity (0.344, 0.203 and 0.196 μg/min), apparent volume of distribution in central compartment (44.4, 65.4 and 72.2 ml/kg) of phenytoin, and total area under the plasma concentration–time curve from time zero to time infinity (609, 714 and 1210 μg min/ml), renal clearance (20.5, 13.4 and 4.67 ml/min per kg) and 24-h urinary excretion (54.7, 35.6 and 32.5% of intravenous dose of phenytoin) of pHPPH were not returned to control levels by cysteine supplementation (rats with PCMC). This could be mainly due to the fact that the phenytoin hydroxylation activity in rats with PCMC was not returned to control level.

Inhibition of phenytoin hydroxylation in human liver microsomes by several selective serotonin re-uptake inhibitors

Several case reports have indicated that the selective serotonin re-uptake inhibitor (SSRI) fluoxetine increases phenytoin blood levels when given concurrently. The mechanism of this drug–drug interaction has been attributed to inhibition of CYP2C9-catalyzed hydroxylation of phenytoin to its major oxidative metabolite in humans, para-hydroxyphenyl phenyl hydantoin (HPPH). With a bank of human liver microsomes (HLM), four SSRIs (fluoxetine, norfluoxetine, sertraline, and paroxetine) were tested for inhibition of HPPH formation. Initially, the K m and V max values of phenytoin hydroxylation to HPPH were determined in the individual HLM samples. The average K m ( n=8) was 9.7±2.9 μM. The V max varied fivefold, with an average value of 113±53 pmol HPPH/min/nmol CYP450. All of the SSRIs inhibited HPPH formation; resulting Ki values were 31.1±10.1 μM (fluoxetine) ( n=5), 51.1±9.4 μM (norfluoxetine) ( n=3), 52.2±21.5 μM (sertraline) ( n=3), and 80.0±7.2 μM (paroxetine) ( n=3). Sulfaphenazole (10 μM), utilized as a positive control for inhibition of HPPH formation, inhibited phenytoin hydroxylation (>95%) in all HLM samples. Diclofenac hydroxylation to 4′-OH diclofenac, a specific marker for CYP2C9 activity, was determined in HLM1–HLM6 and was highly correlated with HPPH formation in HLM1–HLM6, indicating that phenytoin hydroxylation in human liver microsomes is largely due to CYP2C9. This work presents direct evidence that the effect of fluoxetine on phenytoin blood levels may be explained by inhibition of CYP2C9-catalyzed phenytoin hydroxylation. In light of typical SSRI blood levels observed in patients, this study also suggests that the risk of a SSRI–phenytoin interaction is highest with fluoxetine and norfluoxetine, and less likely with sertraline and paroxetine.

Human CYP2C-mediated stereoselective phenytoin hydroxylation in Japanese: difference in chiral preference of CYP2C9 and CYP2C19

Regio- and stereoselective hydroxylation of phenytoin was determined in liver microsomes of nine extensive (EM) and three poor metabolizers (PM) of mephenytoin. Hydroxyphenytoins (HPPH) were isolated and quantified after separation into four regio- and stereoisomers. The total rates of microsomal phenytoin 4′- hydroxylation were approximately 3-fold higher than those of 3′-hydroxylation, and not significantly different in EM and PM. Formation of 4′-( R)-HPPH was 4.4-fold higher in EM than in PM, whereas no clear differences between EM and PM were detected in the formation of 4′-( S)-, 3′- ( R)-, and 3′- ( S)-HPPH. Cytochrome P450 (CYP)2C9, expressed in a fission yeast, Schizosaccharomyces pombe, catalyzed the formation of 4′-( R)- and 4′-( S)-HPPH stereoselectively, as observed with EM, in which predominantly 4′- ( S)-HPPH was formed. Recombinant CYP2C19 was more stereoselective for 4′-( R)-HPPH formation. These results, in addition to inhibition experiments with anti-human CYP2C antibody, indicate that phenytoin hydroxylation is mainly catalyzed by CYP2C9. Furthermore, CYP2C19 showed limited contribution to phenytoin 4′-hydroxylation with a different chiral preference from CYP2C9.

Hydroxylation of Phenytoin (PHT) and the Cytochrome P450 (CYP) 2C Subfamily

Purpose: Four members of the CYP2C subfamily, CYP2C8, CYP2C9, CYP2C18, and CYP2C19, have been identified in humans, and a number of allelic variants of the CYP2C9, CYP2C18. and CYP2C19 genes associated with metabolic polymorphisms have been reported (Pharmacogenetics) 1994;4:285–99). CYP2C9 is a major enzyme responsible for the formation of 5–(4‐hydroxyphenyl)‐5‐phenylhydantoin (p‐HPPH), a major hydroxylation metabolite of PHT. However, we recently reported that CYP2C19 contributes to the stereoselective hydroxylation of PHT (Br J Clin Pharmacol 1997;43:431–5). To clarify the relation between hydroxylation of PHT and the CYP2C subfamily, we examined their stereoselective para‐hydroxylation properties by using cDNAs expressing CYP2C8, 9, 18, or 19. In addition, the allelic linkage among members of the CYP2C subfamily was evaluated. Methods: PHT (200 μM) and microsomes from yeast expressing human CYP2C8, 9, 18, or 19 (200 pmol of P450/ml) were incubated in a reaction buffer (0.1M Kpi [pH 7.41 and 0.1 mM EDTA 2 Na) with the addition of a NADPH‐generating system at 37°C for 60 min. The PHT metabolites, (R)‐ and (S)‐pHPPH, were analyzed by reverse‐phase high‐performance liquid chromatography. Blood samples were obtained from 175 adult Japanese patients with epilepsy who were treated at the Department of Neuropsychiatry of Kyushu University Hospital, and genomic DNA was isolated from peripheral lymphocytes with an extraction kit. The CYP2C19*1 (wild‐type) gene and two mutant alleles, CYP2C19*2 (G to A in exon 5) and CYP2C*3 (G to A in exon 4), and the CYP2C18wt (wild‐type) gene and two mutant alleles, CYP2C18ml (T to A in exon 2) and CYP2C18m2 (T to C in 5′‐flanking region), were identified by the polymerase chain reaction (PCR) and restriction fragment‐length polymorphism (RFLP) analysis by using specific primers and restriction enzymes. Results: The mean formation rates of (R)‐pHPPH by CYP2C19 and by CYP2C18 were 0.37 and 3.05 pmol/h/pmol P450, respectively. The rates of (S)‐pHPPH formation by CYP2C19, by CYP2C18, and by CYP2C9 were 0.69, 2.84, and 0.68 pmol/h/pmo1 P450, respectively. (R)‐ and (S)‐pHPPH formation by CYP2C8 and (R)‐pHPPH formation by CYP2C9 were not detected; stereoselective hydroxylation was suspected for CYP2C9. The genotyping results for CYP2C18 were completely consistent with those for CYP2C19: CYP2C19*1/*1 (n = 75; 42.98). *2/*2 (n = 11; 6.3%), and *3/*3 (n = 4; 2.3%) homozygotes and *1/*2 (n = 54; 30.9%), *1/*3 (n = 25; 14.38). and *2/*3 (n = 6; 3.4%) heterozygotes were homozygous for CYP2C18wt/wt/m2/m2 and m1/m1 and heterozygous for wt/m2, wt/ml and m2/m1, respectively. Conclusions: Not only CYP2C9 but also CYP2C18 and CYP2C19 contributed to the formation of p‐HPPH. The stereoselective differences in the formation of p‐HPPH among the CYP2C subfamily were elucidated. Because the CYP2C18 and CYP2C19 gene mutations were linked, the poor metabolizer (PM) phenotype of CYP2C19 might also be the PM phenotype of CYP2C18. Evidence collected in vivo and in vitro suggested that CYP2C9 might become a key enzyme when PHT is given to patients with the PM phenotype of CYP2C19.

Phenytoin Hydroxylation in a Healthy Caucasian Population: Bimodal Distribution of Hydroxyphenytoin Urinary Excretion

AbstractThe phenytoin hydroxylation index was studied in 122 unrelated Caucasian volunteers. Following a 100 mg oral dose of phenytoin, phenytoin and hydroxyphenytoin were measured in urine from 0‐32 hr after administration. As phenytoin was not found in all urine collections, a phenytoin hydroxylation index was expressed as follows: Phenytoin hydroxylation index=amount of phenytoin administcrcd/0‐32 hr urinary output of hydroxyphenytoin. Phenytoin hydroxylation index values appear to be bimodally distributed, 92% of the population showing a mean (±S.E.M.) value of 0.639±0.099 and 8% a mean (±S.E.M.) value of 1.001 ±0.180 (log10 values). These results are in favour of the existence of a phenytoin genetic polymorphism. Since misuse of urinary metabolite excretion data in drug metabolism studies is a well‐known phenomenon, our data emphazise the need for future population studies on phenytoin pharmacokinetics as well as on CYP2C9 genotyping before concluding about existence of a phenytoin genetic polymorphism.

Teratogenic evaluation of dry hydrollzed mud in fourth generation wistar rats

Regio- and stereoselective hydroxylation of phenytoin was determined in liver microsomes of nine extensive (EM) and three poor metabolizers (PM) of mephenytoin. Hydroxyphenytoins (HPPH) were isolated and quantified after separation into four regio- and stereoisomers. The total rates of microsomal phenytoin 4′- hydroxylation were approximately 3-fold higher than those of 3′-hydroxylation, and not significantly different in EM and PM. Formation of 4′-(R)-HPPH was 4.4-fold higher in EM than in PM, whereas no clear differences between EM and PM were detected in the formation of 4′-(S)-, 3′- (R)-, and 3′- (S)-HPPH. Cytochrome P450 (CYP)2C9, expressed in a fission yeast, Schizosaccharomyces pombe, catalyzed the formation of 4′-(R)- and 4′-(S)-HPPH stereoselectively, as observed with EM, in which predominantly 4′- (S)-HPPH was formed. Recombinant CYP2C19 was more stereoselective for 4′-(R)-HPPH formation. These results, in addition to inhibition experiments with anti-human CYP2C antibody, indicate that phenytoin hydroxylation is mainly catalyzed by CYP2C9. Furthermore, CYP2C19 showed limited contribution to phenytoin 4′-hydroxylation with a different chiral preference from CYP2C9.

Trichoroacetic Acid: A Possible Biological Marker of Environmental Exposure to Trichloroethylene and Tetrachloroethylene

Regio- and stereoselective hydroxylation of phenytoin was determined in liver microsomes of nine extensive (EM) and three poor metabolizers (PM) of mephenytoin. Hydroxyphenytoins (HPPH) were isolated and quantified after separation into four regio- and stereoisomers. The total rates of microsomal phenytoin 4′- hydroxylation were approximately 3-fold higher than those of 3′-hydroxylation, and not significantly different in EM and PM. Formation of 4′-(R)-HPPH was 4.4-fold higher in EM than in PM, whereas no clear differences between EM and PM were detected in the formation of 4′-(S)-, 3′- (R)-, and 3′- (S)-HPPH. Cytochrome P450 (CYP)2C9, expressed in a fission yeast, Schizosaccharomyces pombe, catalyzed the formation of 4′-(R)- and 4′-(S)-HPPH stereoselectively, as observed with EM, in which predominantly 4′- (S)-HPPH was formed. Recombinant CYP2C19 was more stereoselective for 4′-(R)-HPPH formation. These results, in addition to inhibition experiments with anti-human CYP2C antibody, indicate that phenytoin hydroxylation is mainly catalyzed by CYP2C9. Furthermore, CYP2C19 showed limited contribution to phenytoin 4′-hydroxylation with a different chiral preference from CYP2C9.

Metabolism of phenytoin and covalent binding of reactive intermediates in activated human neutrophils

Activation of neutrophils by phorbol-12-myristate-13-acetate (PMA) causes rapid production of superoxide radical (O 2 −), leading to the formation of additional reactive oxygen species, including hydrogen peroxide (H 2O 2), hypochlorous acid (HOCl), and possibly hydroxyl radical (·OH). These reactive oxygen species have been associated with the oxidation of some drugs. We investigated the metabolism of phenytoin (5,5-diphenylhydantoin) and the covalent binding of reactive intermediates to cellular macromolecules in activated neutrophils. In incubations with 100 μM phenytoin, PMA-stimulated neutrophils from six human subjects produced p-, m-, and o-isomers of 5-(hydroxyphenyl)-5-phenylhydantoin (HPPH) in a ratio of 1.0:2.1:2.8, respectively, as well as unidentified polar products. Analysis of cell pellets demonstrated that phenytoin was bioactivated to reactive intermediates that bound irreversibly to macromolecules in neutrophils. Glutathione, catalase, Superoxide dismutase, azide, and indomethacin all diminished the metabolism of phenytoin and the covalent binding of its reactive intermediates. The iron-inactivating chelators desferrioxamine and diethylenetriaminepentaacetic acid had little or no effect on the metabolism of phenytoin by neutrophils, demonstrating that adventitious iron was not contributing via Fenton chemistry. In an ·OH-generating system containing H 2O 2 and Fe 2+ chelated with ADP, phenytoin was oxidized rapidly to unidentified polar products and to p-, m-, and o-HPPH (ratio 1.0:1.7:1.5, respectively). Reagent HOCl and human myeloperoxidase (MPO), in the presence of Cl − and H 2O 2, both formed the reactive dichlorophenytoin but no HPPH. However, no chlorinated phenytoin was detected in activated neutrophils, possibly because of its high reactivity. These findings, which demonstrated that activated neutrophils biotransform phenytoin in vitro to hydroxylated products and reactive intermediates that bind irreversibly to tissue macromolecules, are consistent with phenytoin hydroxylation by ·OH generated by a transition metal-independent process, chlorination by HOCl generated by MPO, and possibly cooxidation by neutrophil hydroperoxidases. Neutrophils activated in vivo may similarly convert phenytoin to reactive intermediates, which could contribute to some of the previously unexplained adverse effects of the drug.

Analysis of mephenytoin, 4-hydroxymephenytoin and 4-hydroxyphenytoin enantiomers in human urine by cyclodextrin micellar electrokinetic capillary chromatography: simple determination of a hydroxylation polymorphism in man.

Using cyclodextrin micellar electrokinetic capillary chromatography (CD-MECC), baseline separation of mephenytoin, 4-hydroxymephenytoin and 4-hydroxyphenytoin enantiomers in urine was effected with beta-cyclodextrin. After single-dose administration of 100 mg of racemic mephenytoin, the 0-8 h urine was collected, and enzymatically hydrolyzed urine specimens were applied. For extensive metabolizers, a single peak for 4-hydroxymephenytoin was detected corresponding to the S-enantiomer. This peak was either very small or undetectable in samples of poor metabolizers. Typically, mephenytoin could not be detected in these samples. However, application of undeglucuronidated extracts revealed the presence of free S-4-hydroxymephenytoin and R,S-mephenytoin and thus permitted phenotyping via both the urinary S:R enantiomeric ratio of mephenytoin and the hydroxylated metabolite. Application of enzymatically hydrolyzed and extracted urines after phenytoin administration (100 mg; 0-8 h urine collection) revealed the presence of S-4-hydroxyphenytoin. Thus, CD- MECC is shown to be a simple and attractive approach for (i) the confirmation of the stereoselectivity of the aromatic hydroxylation of mephenytoin and phenytoin, (ii) the simple and rapid differentiation between extensive and poor metabolizers for mephenytoin, and (iii) assessment of compliance.

Site-directed mutation studies of human liver cytochrome P-450 isoenzymes in the CYP2C subfamily

Evidence from human studies in vivo and in vitro strongly suggests that the methylhydroxylation of tolbutamide and the 4-hydroxylation of phenytoin, the major pathways in the elimination of these two drugs, are catalysed by the same cytochrome P-450 isoenzyme(s). In the present study we used site-directed mutagenesis and cDNA expression in COS cells to characterize in detail the kinetics of tolbutamide and phenytoin hydroxylations by seven CYP2C proteins (2C8, 2C9 and variants, and 2C10) in order to define the effects of small changes in amino acid sequences and the likely proteins responsible in the metabolism of these two drugs in man. Tolbutamide was hydroxylated to varying extents by all expressed cytochrome P-450 isoenzymes, although activity was much lower for the expressed 2C8 protein. While the apparent Km values for the 2C9/10 isoenzymes (71.6-131.7 microM) were comparable with the range of apparent Km values previously observed in human liver microsomes, the apparent Km for 2C8 (650.5 microM) was appreciably higher. The 2C8 enzyme also showed quite different sulphaphenazole inhibition characteristics. The 4-hydroxylation of phenytoin was also more efficiently catalysed by the 2C9/10 enzymes. These enzymes showed similarities in kinetics of phenytoin hydroxylation and sulphaphenazole inhibition compared with human liver phenytoin hydroxylase. Also of interest was the observation that, among the 2C9 variants, small differences in amino acid composition could appreciably affect both tolbutamide and phenytoin hydroxylations. The amino acid substitution Cys-144-->Arg increased both the rates of tolbutamide and phenytoin hydroxylations, while the Leu-359-->Ile change had a greater effect on phenytoin hydroxylation. We conclude that: (1) although 2C8 and 2C9/10 proteins metabolize tolbutamide. only 2C9/10 proteins play a major role in human liver; (2) 2C9/10 proteins also appear to be chiefly responsible for phenytoin hydroxylation; and (3) subtle differences in the amino acid composition of these 2C9/10 proteins can affect the functional specificities towards both tolbutamide and phenytoin.

Two-dimensional high-performance liquid chromatographic method to assay p-hydroxyphenylphenylhydantoin enantiomers in biological fluids and stereoselectivity of enzyme induction in phenytoin metabolism

A two-dimensional high-performance liquid chromatographic method was developed to assay the enantiomers of a major phenytoin metabolite, p-hydroxyphenylphenylhydantoin (p-HPPH). Racemic p-HPPH was first separated from phenytoin and other interfering peaks by a reversed-phase column and monitored by an ultraviolet detector. At the retention time of p-HPPH, the racemic p-HPPH peak was automatically transferred to a chiral ligand-exchange column to separate R-p-HPPH and S-p-HPPH by a time-programmed column-switching valve. The ratio of enantiomers was measured by a second ultraviolet detector. The method can be used to assay R- and S-p-HPPH enantiomers with reasonable sensitivity and reproducibility. By using this method, the stereoselectivity of enzyme induction and inhibition of phenytoin metabolism was investigated. Male rats were treated with phenobarbital, 3-methylcholanthrene, acetone, Aroclor 1254, pregnenolone-16α-carbonitrile, dexamethasone and isosafrole. Microsomes were prepared from the rat liver and phenytoin hydroxylation was measured. Pretreatment with phenobarbital, pregnenolone-16α-carbonitrile or acetone induced phenytoin metabolism non-stereoselectively. Pretreatment with dexamethasone decreased R-p-HPPH formation without affecting the formation of S-p-HPPH. Liver microsomes from female rats showed a higher S-p-HPPH formation, whereas R-p-HPPH formation remained the same. Various inhibitors were added to inhibit phenytoin metabolism by control microsomes. Sulphaphenazole, ketoconazole, 4,4-di( p-methoxyphenyl)hydantoin, cimetidine and diazepam inhibited the formation of R- and S-p-HPPH. Quinidine, tolbutamide and mephenytoin showed no significant inhibitory activity. None of these inhibitors showed stereoselectivity.

Relationship between mephenytoin oxidation polymorphism and phenytoin, methylphenytoin and phenobarbitone hydroxylation assessed in a phenotyped panel of healthy subjects.

1. In a phenotyped panel of healthy subjects correlations were studied between the oxidation of mephenytoin, phenytoin, methylphenytoin and phenobarbitone, with respect to the formation of their 4-hydroxy metabolites (OH-). 2. On different occasions phenotyped extensive metabolizers (EM; n = 16) and poor metabolizers (PM; n = 4) of mephenytoin received phenytoin (100 mg), methylphenytoin (100 mg) and phenobarbitone (50 mg) and urine was collected up to 24 h. The excreted 4-hydroxy metabolites of all compounds were measured by h.p.l.c. 3. Urinary recovery of OH-phenytoin was 31.0 +/- 11.7%, of OH-methylphenytoin 3.4 +/- 2.7% and of OH-phenobarbitone 1.4 +/- 1.2%. No correlation was found between the recovery of OH-mephenytoin and OH-phenytoin. A subject who produced virtually no OH-phenytoin was an EM of mephenytoin, confirming a dissociation of mephenytoin polymorphism and phenytoin hydroxylation. 4. The correlation coefficient for OH-mephenytoin and OH-methylphenytoin recovery was 0.71 (Spearman rank, P = 0.002). The PMs of mephenytoin excreted the least amount of OH-methylphenytoin, suggesting a cosegregation of the 4-hydroxylation pathways. No correlation was found between the urinary recovery of OH-phenobarbitone and that of the other 4-hydroxy metabolites.

Stereoselectivity of hepatic phenytoin hydroxylation: An in vivo probe for the heterogeneity of the cyt P-450 system in various drug hydroxylation phenotypes in man

Stereoselectivity of hepatic phenytoin hydroxylation: An in vivo probe for the heterogeneity of the cyt P-450 system in various drug hydroxylation phenotypes in man