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PubMedDate not supplied

Mephenytoin stereoselective elimination in the rat: II. Comparison of mephenytoin stereoselective clearance during chronic intravenous and hepatic portal vein administration.

The stereoselective clearances of R- and S-mephenytoin were determined in rats receiving either an intravenous or hepatic portal vein infusion of racemic mephenytoin. The mean +/- SD intravenous clearances of R- and S-mephenytoin were 1630 +/- 250 ml/hr and 630 +/- 250 ml/hr, respectively. The corresponding portal vein clearances for these enantiomers were 2560 +/- 1230 ml/hr (R-mephenytoin) and 540 +/- 230 ml/hr (S-mephenytoin). In spite of the slightly higher clearance for R-mephenytoin following portal vein administration, the difference between the intravenous and portal vein clearances for R- or S-mephenytoin were not found to be significant. Subsequent computer simulations of the data indicated there was less than a 5% probability that this result could be attributed solely to interanimal variability in drug clearance. The estimated extraction ratio of R-mephenytoin by the liver was modest and suggested mephenytoin may undergo a substantial degree of extrahepatic elimination in the rat.

Animals↗
PubMed1986

Mephenytoin-type polymorphism of drug oxidation: purification and characterization of a human liver cytochrome P-450 isozyme catalyzing microsomal mephenytoin hydroxylation.

A genetic polymorphism causing deficient metabolism of the anticonvulsant drug mephenytoin occurs in 5% of the Caucasian and 23% of the Japanese population. By monitoring the activities of the two major oxidative pathways of mephenytoin metabolism in the column eluates, we have purified from human livers a cytochrome P-450 isozyme, P-450 meph, which exclusively and stereoselectively catalyzes the 4-hydroxylation of (S)-mephenytoin, the major pathway affected by the polymorphism, whereas P-450 meph was virtually devoid of catalytic activity for N-demethylation of mephenytoin, the pathway remaining unaffected by the genetic deficiency. P-450 meph had an apparent Mr of 55 000 and a lambda max in the reduced CO-binding spectrum of 450 nm. Polyclonal rabbit antibodies against purified human P-450 meph almost completely inhibited the 4-hydroxylation of mephenytoin but had little effect on N-demethylation in human liver microsomes. In microsomes of liver biopsies of two subjects characterized in vivo as 'poor metabolizers' of mephenytoin, immunocrossreactive and immunoinhibitable material was observed with similar or identical properties to those of P-450 meph. There was no difference in the extent of the immunochemical reaction between microsomes of in vivo phenotyped poor metabolizers and extensive metabolizers of mephenytoin. These data suggest that P-450 meph is the target of the genetic deficiency and support the concept that a functionally altered variant form of P-450 meph causes this polymorphism.

Animals↗
PubMed1997

An S-mephenytoin cysteine conjugate identified in urine of extensive but not of poor metabolizers of S-mephenytoin.

A conjugate of S-mephenytoin excreted in urine of extensive but not of poor metabolizers of S-mephenytoin has previously been reported. This conjugate, which is easily hydrolysed back to S-mephenytoin, has now been isolated and identified in urine from one extensive metabolizer after a single dose of 100 mg racemic mephenytoin. High performance liquid chromatography purification, followed by gas chromatographic, mass spectrometric and amino acid analyses showed that the isolated compound is a cysteine conjugate of S-mephenytoin. The significant mass spectrometric ions have been confirmed in three additional extensive metabolizers of S-mephenytoin, but were not detectable in urine from three poor metabolizer subjects. The exact structure of the conjugate is unknown, but we suggest that an S-N bond between cysteine and S-mephenytoin is formed via an oxidative radical mechanism catalyzed by CYP2C19.

Chromatography, High Pressure Liquid↗
PubMed1984

Stereoselectivity of the arene epoxide pathway of mephenytoin hydroxylation in man.

Stereoselective metabolism of mephenytoin has been investigated in four normal subjects by comparing urinary recoveries of hydroxylated metabolites after administration of racemic RS-mephenytoin (1.4 mmol/day) and R-mephenytoin (0.7 mmol/day) on separate occasions. Gas chromatography-mass spectrometry was employed to measure the urinary recovery of 3-methyl-5-(4-hydroxyphenyl)-5-ethylhydantoin (4-OH-M) and mephenytoin catechol, methylcatechol, and dihydrodiol metabolites. Following a single oral dose of racemic mephenytoin, 4-OH-M, mephenytoin catechol, and methylcatechol metabolites were identified in urine mainly as conjugates, whereas the dihydrodiol metabolite was recovered mainly in its unconjugated form. Urinary elimination of each metabolite was similar on days 1 and 10 of chronic racemic mephenytoin administration. Following R-mephenytoin administration, urinary recoveries of hydroxylated metabolites were five to 10 times smaller than after administration of the racemic drug. This implies substrate-stereoselective hydroxylation of the S-enantiomer of mephenytoin. In one subject with a genetic deficiency of aromatic mephenytoin hydroxylation deficiency, the excretion of each hydroxylated mephenytoin metabolite after RS-mephenytoin administration was decreased to 5-15% of the values found in the four extensively hydroxylating study volunteers. The impaired formation of hydroxylated mephenytoin metabolites in genetic hydroxylation deficiency, in conjunction with stereoselective hydroxylation of S-mephenytoin via an extensive NIH shift in normal man, is consistent with the hypothesis that the formation of the S-mephenytoin arene oxide is under genetic control and represents the initial enzymatic reaction of stereoselective aromatic mephenytoin hydroxylation. The formation of this potentially reactive metabolite of S-mephenytoin may have implications in mephenytoin-induced toxicity.

Ethers, Cyclic↗
PubMed1995

Comparison of chloroguanide and mephenytoin for the in vivo assessment of genetically determined CYP2C19 activity in humans.

OBJECTIVES: The main objective of this study was to examine the relations between chloroguanide (proguanil) and mephenytoin metabolic ratios to determine whether or not chloroguanide could replace mephenytoin as a probe for the indirect in vivo measurement of CYP2C19 activity. An additional objective was to examine the interactions between chloroguanide, omeprazole, and mephenytoin, which are three substrates of CYP2C19. METHODS: Twenty healthy volunteers received 200 mg chloroguanide orally on three separate occasions in an open, randomized-sequence crossover design: once alone, once 2 hours before the oral administration of 100 mg mephenytoin, and once after oral administration for 7 days of 40 mg/day omeprazole. During one additional period, 100 mg mephenytoin was administered orally. The chloroguanide to cycloguanil ratio was determined in plasma 4 hours after drug administration; it was determined in urine collected over 4, 8, and 24 hours. The mephenytoin hydroxylation index was also measured in urine. RESULTS: All subjects were extensive metabolizers of chloroguanide and mephenytoin. We found no correlation between the mephenytoin hydroxylation index and the chloroguanide to cycloguanil ratio in any of the urine samples collected or in plasma. In the presence of chloroguanide, mephenytoin hydroxylation index increased from a baseline value of 1.2 +/- 0.2 to 1.7 +/- 1.0 (p < 0.05). In the presence of omeprazole, the chloroguanide to cycloguanil metabolic ratio in 24-hour urine increased from 2.2 +/- 1.0 to 5.6 +/- 3.2 (p < 0.001). CONCLUSION: Chloroguanide inhibits the CYP2C19-dependent 4'-hydroxylation of mephenytoin. The bioactivation of chloroguanide to cycloguanil is inhibited by the CYP2C19 substrate omeprazole. However, the chloroguanide to cycloguanil metabolic ratio does not reflect the same array of S-mephenytoin hydroxylase activities found in extensive metabolizers as that show by the mephenytoin hydroxylation index.

Adult↗
PubMed1990

Polymorphism in hydroxylation of mephenytoin and hexobarbital stereoisomers in relation to hepatic P-450 human-2.

Stereoselective 4'-hydroxylations of R-(-)-mephenytoin and S-(+)-mephenytoin and 3'-hydroxylation of R-(-)-hexobarbital and S-(+)-hexobarbital were determined in liver microsomes of 14 Japanese subjects who were extensive metabolizers of mephenytoin and in five Japanese subjects who were poor metabolizers of mephenytoin. Content of P-450 human-2 assessed by Western blots was correlated to microsomal S-(+)-mephenytoin 4'-hydroxylation, R-(-)-hexobarbital 3' alpha-hydroxylation, and S-(+)-hexobarbital 3' beta-hydroxylation, and was less correlated to R-(-)mephenytoin 4'-hydroxylation, R-(-)-hexobarbital 3' beta-hydroxylation, and S-(+)-hexobarbital 3' alpha-hydroxylation. Antibodies raised against P-450 human-2 inhibited microsomal S-(+)-mephenytoin 4'-hydroxylation efficiently but was less efficient on R-(-)-mephenytoin 4'-hydroxylation in extensive metabolizers and on 4'-hydroxylation of mephenytoin enantiomers in poor metabolizers. The antibodies also inhibited R-(-)-hexobarbital 3' alpha-hydroxylation and S-(+)-hexobarbital 3' beta-hydroxylation but did not effectively inhibit the hydroxylation of the two other optical isomers of hexobarbital in extensive metabolizers and of four stereoisomers in poor metabolizers. These findings indicate the close relationship between polymorphic mephenytoin 4'-hydroxylation and two stereospecific hexobarbital hydroxylations, and they suggest that P-450 human-2 is a typical S-(+)-mephenytoin 4'-hydroxylase and a major hexobarbital 3'-hydroxylase in the livers of extensive metabolizers. The findings were further supported by the experiments that used P-450 human-2 complementary dexoyribonucleic acid-derived protein in yeast microsomes.

Cytochrome P-450 Enzyme System↗
PubMed1984

Pharmacogenetics of mephenytoin: a new drug hydroxylation polymorphism in man.

Inherited deficiency in mephenytoin hydroxylation was observed in a family study. It is important that the propositus was of the extensive metabolizer phenotype for the genetically controlled hydroxylation of debrisoquine. Thus, a genetic polymorphism of drug hydroxylation was suspected for mephenytoin. A population study of mephenytoin hydroxylation, combined with identification of extensive and poor debrisoquine hydroxylation phenotypes, was carried out in 221 unrelated normal volunteers. Twelve of them (5%) exhibited defective aromatic hydroxylation of mephenytoin, and 23 (10%) could be identified as poor metabolizers of debrisoquine. Amongst these 35 subjects with a drug hydroxylation deficiency, 3 (or 0.5%; 1 female, 2 males) displayed both defects simultaneously. A panel study of 10 extensive and 10 poor metabolizers of mephenytoin showed that the ability to perform aromatic hydroxylation of the demethylated mephenytoin metabolite nirvanol (5-phenyl-5-ethylhydantoin) was co-inherited with the mephenytoin hydroxylation polymorphism. Family studies suggested that poor metabolizer phenotypes of nirvanol and mephenytoin were most likely to have the homozygous genotype for an autosomal recessive allele of deficient aromatic drug hydroxylation. Intra-subject comparison of the debrisoquine and mephenytoin hydroxylation phenotypes in these subjects indicated that deficiency in the two drug hydroxylations occurred independently. Consequently, the co-inheritance of extensive and poor hydroxylation of mephenytoin and nirvanol, respectively, represents a new drug hydroxylation polymorphism in man.

Adult↗
PubMed1985

Mephenytoin hydroxylation polymorphism: characterization of the enzymatic deficiency in liver microsomes of poor metabolizers phenotyped in vivo.

The rate of 4-hydroxylation and of N-demethylation of S- and R-mephenytoin was determined in liver microsomes of 13 extensive (EM) and two poor (PM) metabolizers of mephenytoin. Detailed kinetic studies were performed in microsomes of eight EMs and the two PMs. Microsomal mephenytoin metabolism in PMs was characterized by an increased Km (150.6 and 180.6 vs. a mean [+/- SD] 37.8 +/- 9.6 mumol/L S-mephenytoin in 8 EMs), a decreased maximum rate of metabolism for S-mephenytoin hydroxylation (0.76 and 0.69 vs 4.85 +/- 1.65 nmol 4-hydroxymephenytoin per milligram protein per hour), and loss of stereoselectivity for the hydroxylation of the R- and S-enantiomers of mephenytoin (R/S ratio: 1.10 and 0.76 vs. 0.11 +/- 0.04 in 13 EMs). The formation of 4-OH-mephenytoin from R-mephenytoin and the demethylation reaction remained unaffected. These results support our hypothesis that the mephenytoin polymorphism is caused by a partial or complete absence or inactivity of a cytochrome P-450 isozyme with high affinity for S-mephenytoin.

Adult↗
PubMed1986

Polymorphic metabolism of mephenytoin in man: pharmacokinetic interaction with a co-regulated substrate, mephobarbital.

The simultaneous dosing of two drugs with co-regulated genetic polymorphisms determined by a single cytochrome P-450 isozyme could result in competitive inhibition of metabolism. We investigated this hypothesis in vivo by studying the interaction of mephobarbital and mephenytoin in eight normal subjects with wide variability in S-mephenytoin 4-hydroxylation. Each received oral racemic mephenytoin (100 mg) alone and, on a separate occasion, 1 hour after oral racemic mephobarbital (200 mg). After mephenytoin dosing alone, the 8-hour urinary enantiomeric (R/S) ratio indicated one poor (PM), one intermediate (IM), and six extensive (EM) metabolizers. Total intrinsic clearance of S-mephenytoin varied more than 100-fold, whereas the range for R-mephenytoin was only twofold. The urinary R/S ratio correlated (r = 0.92) with the enantiomeric ratio of the plasma AUCs over the same period, indicating no stereoselectivity in renal clearance. When mephenytoin was taken in the presence of mephobarbital, peak levels and AUC of S-mephenytoin increased while those of the R-enantiomer remained unchanged. Accordingly, the R/S ratios in both plasma and urine were reduced, with the change rank order-related to the control value of the total intrinsic clearance of S-mephenytoin (i.e., greatest in the most extensive EM). Thus the urinary R/S ratio can be used as a measure of the enantiomeric ratio of the plasma concentrations over the same time period of collection. Moreover, this ratio may be used to detect drug interactions that involve the cytochrome P-450 isozyme(s) responsible for the polymorphic 4-hydroxylation of mephenytoin.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Oral↗
PubMed1993

Species differences in stereoselective metabolism of mephenytoin by cytochrome P450 (CYP2C and CYP3A).

Stereoselective involvement of hepatic cytochrome P450 in the metabolism of mephenytoin was investigated in vitro by using livers of five different experimental animal species and humans. The rates of microsomal 4'-hydroxylation were 2 to 6 times higher with the R-enantiomer than the S-enantiomer in rabbits, dogs and rats, whereas the rates of the 4'-hydroxylation in female mice were not different between R- and S-enantiomers. Preferential S-mephenytoin 4'-hydroxylation was observed in monkeys as similar to that in humans. The rates of microsomal mephenytoin N-demethylation were approximately 2 times higher with the R-enantiomer than the S-enantiomer in male rats and both sexes of dogs. Antibodies raised against CYP2C11 (anti-CYP2C) clearly inhibited microsomal 4'-hydroxylation of S-mephenytoin and N-demethylation of R-mephenytoin in rats, monkeys and humans. Antibodies raised against CYP3A2 (anti-CYP3A) clearly inhibited microsomal 4'-hydroxylation of R-mephenytoin, but marginally S-mephenytoin, in rats. Anti-CYP3A, however, showed no clear inhibition on microsomal 4'-hydroxylation and N-demethylation of both enantiomers in monkeys and humans, except for slight inhibition of R-mephenytoin 4'-hydroxylation in male monkeys. The results suggest that stereoselective involvement of rat CYP3A and scant involvement of human CYP3A in R-mephenytoin 4'-hydroxylation are major determinants of the species differences between rats and humans in stereoselective mephenytoin 4'-hydroxylation.

Adult↗
PubMed1996

Catalytic role of cytochrome P4502B6 in the N-demethylation of S-mephenytoin.

In vitro methods were used to identify the cytochrome P450 (CYP) enzyme(s) involved in S-mephenytoin N-demethylation. S-Mephenytoin (200 microM) was incubated with human liver microsomes, and nirvanol formation was quantitated by reversed-phase HPLC. S-Mephenytoin N-demethylase activity in a panel of human liver microsomes ranged 35-fold from 9 to 319 pmol/min/mg protein and correlated strongly with microsomal CYP2B6 activity (r = 0.91). Additional correlations were found with microsomal CYP2A6 and CYP3A4 activity (r = 0.88 and 0.74, respectively). Microsomes prepared from human beta-lymphoblastoid cells transformed with individual P450 cDNAs were assayed for S-mephenytoin N-demethylase activity. Of 11 P450 isoforms (P450s 1A1, 1A2, 2A6, 2B6, 2E1, 2D6, 2C8, 2C9, 2C19, 3A4, and 3A5) tested, only CYP2B6 catalyzed the N-demethylation of S-mephenytoin with an apparent K(m) of 564 microM. Experiments with P450 form-selective chemical inhibitors, competitive substrates, and anti-P450 antibodies were also performed. Troleandomycin, a mechanism-based CYP3A selective inhibitor, and coumarin, a substrate for CYP2A6 and therefore a potential competitive inhibitor, failed to inhibit human liver microsomal S-mephenytoin N-demethylation. In contrast, orphenadrine, an inhibitor of CYP2B forms, produced a 51 +/- 4% decrease in S-mephenytoin N-demethylase activity in human liver microsomes and a 45% decrease in recombinant microsomes expressing CYP2B6. Also, both CYP2B6-marker 7-ethoxytrifluoromethylcoumarin O-deethylase and S-mephenytoin N-demethylase activities were inhibited by approximately 65% by 5 mg anti-CYP2B1 IgG/mg microsomal protein. Finally, polyclonal antibody inhibitory to CYP3A1 failed to inhibit S-mephenytoin N-demethylase activity. Taken together, these studies indicate that the N-demethylation of S-mephenytoin by human liver microsomes is catalyzed primarily by CYP2B6.

Antibodies, Monoclonal↗
PubMed1984

Mephenytoin hydroxylation deficiency: kinetics after repeated doses.

Deficient aromatic hydroxylation of S-mephenytoin was observed in an index subject during a kinetic study of stereoselective metabolism of mephenytoin. A genetic basis for this defect was suggested by decreased urinary recovery of 3-methyl-5-(4-hydroxyphenyl)-5-ethylhydantoin (4-OH-M) in the 24 hr after oral racemic mephenytoin in two brothers of the propositus. The parents and a third brother had urinary recoveries of 4-OH-M of the same order as in a group of 20 normal subjects. The kinetic implications of this defect were studied in the index subject and compared with four normal subjects after a single oral dose of differentially radiolabeled pseudoracemic mephenytoin (5 microCi of 14C-S-mephenytoin, 45 microCi of H3-R-mephenytoin, and 11.5 mumol/kg of both S- and R-mephenytoin) followed by single oral doses of 1.4 mmol of unlabeled racemic mephenytoin daily the next 4 days. In normal subjects, there was substrate stereoselective metabolism with the S-enantiomer rapidly excreted as 4-OH-M and the R-enantiomer slowly excreted as 5-phenyl-5-ethylhydantoin (PEH). Stereoselective metabolism persisted during repeated dosing. In the hydroxylation-deficient subject, there was no evidence of stereoselective metabolism, recovery of 4-OH-M was low, and both enantiomers were slowly excreted, predominantly as PEH. Plasma PEH concentrations and urinary PEH excretion rates were approximately twice that in normal subjects. Thus a genetic deficiency in ability to hydroxylate S-mephenytoin results in the S-enantiomer metabolization by the alternate route of demethylation to PEH that cumulates, thereby, in comparison to the normal, effectively doubling the dose of total hydantoin.

Adult↗
PubMed1992

Relation between chloroguanide bioactivation to cycloguanil and the genetically determined metabolism of mephenytoin in humans.

It has been suggested recently that the bioactivation of chloroguanide hydrochloride (proguanil) to its active antimalarial metabolite cycloguanil cosegregates with the genetically determined polymorphism of mephenytoin hydroxylation. We determined the chloroguanide to cycloguanil ratio in urine after oral administration of a single dose of 200 mg proguanil either alone or together with 100 mg racemic mephenytoin or 40 mg dextromethorphan in a randomized crossover study performed in 24 healthy subjects. The mephenytoin hydroxylation index was also determined after administration of 100 mg racemic mephenytoin either alone or together with 200 mg proguanil. Two subjects were poor metabolizers and one subject was an intermediate metabolizer of mephenytoin. These three subjects had chloroguanide to cycloguanil ratios of more than 50. The 21 subjects with the extensive metabolizer phenotype for mephenytoin hydroxylation had chloroguanide to cycloguanil ratios of less than 10. The chloroguanide to cycloguanil ratio was not significantly altered by mephenytoin or dextromethorphan coadministration. The trend toward a correlation between chloroguanide/cycloguanil ratio and log mephenytoin hydroxylation index did not reach statistical significance. Inclusion of the dextromethorphan metabolic ratio into the model did not improve the relationship. These findings confirm that the bioactivation of chloroguanide to cycloguanil cosegregates with the genetically determined activity of the CYP2C family. However, the chloroguanide to cycloguanil ratio and the mephenytoin hydroxylation index do not similarly reflect the variable activity of CYP2C.

Adult↗
PubMed1993

Pharmacokinetics of citalopram in relation to the sparteine and the mephenytoin oxidation polymorphisms.

The relationship between the metabolism of the selective serotonin reuptake inhibitor citalopram and the sparteine and mephenytoin oxidation polymorphisms was studied in 24 healthy male volunteers, constituting panels of extensive metabolizers of sparteine and mephenytoin (n = 10), poor metabolizers of sparteine (n = 8), and poor metabolizers of mephenytoin (n = 6). Each subject was given 40 mg/day citalopram for 10 days and citalopram, and its des- and didesmethylmetabolites were assayed in serum and urine. Using a nonenantioselective analytical method (high-performance liquid chromatography), it was shown that the citalopram elimination partially depends on the mephenytoin oxygenase, since steady-state serum concentration, half-life, and area under the serum concentration/time curve for citalopram were significantly higher in poor metabolizers of mephenytoin than in extensive metabolizers of mephenytoin. Both citalopram total clearance and demethylation clearance (formation of desmethylcitalopram) were significantly lower in poor metabolizers of mephenytoin compared to extensive metabolizers (median 15.2 vs. 27.3 and 2.6 vs. 5.9 L/h, respectively). It was further indicated that the demethylation of desmethylcitalopram to didesmethylcitalopram depends on the sparteine oxygenase CYP2D6. Didesmethylcitalopram could virtually not be detected in any poor metabolizers of sparteine, contrasting measurable serum levels in all sparteine/mephenytoin extensive metabolizers. The demethylation clearance of desmethylcitalopram was significantly lower in sparteine poor metabolizers compared to extensive metabolizers (0.3 vs. 2.4 L/h, respectively). During administration of citalopram, there was a modest increase in sparteine metabolic ratio from median 0.31 to 0.80 in extensive metabolizers of sparteine, whereas the mephenytoin S/R ratio was unaltered during citalopram treatment. Both the sparteine and the mephenytoin oxidation polymorphism thus appear to contribute partially to the total pharmacokinetic variability of citalopram.

Citalopram↗
PubMedDate not supplied

Polymorphism in stereoselective hydroxylations of mephenytoin and hexobarbital by Japanese liver samples in relation to cytochrome P-450 human-2 (IIC9).

1. Stereoselective 4'-hydroxylations of R-(--)-mephenytoin and S-(+)-mephenytoin were determined in liver microsomes of 19 Japanese subjects. 2. The content of P-450 human-2 assessed by Western-blots correlated with microsomal S-(+)-mephenytoin 4'-hydroxylation. Antibody raised against P-450 human-2 effectively inhibited microsomal S-(+)-mephenytoin 4'-hydroxylation, but was less efficient for inhibition of R-(--)-mephenytoin 4'-hydroxylation in extensive metabolizers, and 4'-hydroxylation of both mephenytoin enantiomers in poor metabolizers. 3. Similar results were observed on the stereoselective hydroxylations of R-(--)- and S-(+)-hexobarbital. Clear correlations were observed for the content of P-450 human-2 and microsomal R-(--)-hexobarbital 3'alpha-hydroxylation and S-(+)-hexobarbital 3'beta-hydroxylation. 4. Moreover, yeast microsomes expressing P-450 human-2 cDNA showed high stereoselectivities for hydroxylations of mephenytoin and hexobarbital similar to those observed in human liver. 5. Two other cytochromes P-450(IIC 9/10) expressed in yeast, whose cDNA were synthesized by site-directed mutagenesis from human-2 cDNA, showed no stereoselectivity for the hydroxylations of mephenytoin and hexobarbital, in spite of the modification of only two amino acid substitutions or deletions in the whole sequence. 6. Only a cytochrome derived from P-450 human cDNA corresponding to P-450 human-2 was expressed in human livers, the two cytochromes of the three related IIC9/10 forms were not expressed. 7. These findings indicate that P-450 human-2 is the major cytochrome P-450 responsible for the polymorphisms in stereoselective hydroxylations of mephenytoin and hexobarbital.

Cytochrome P-450 Enzyme System↗
PubMedDate not supplied

The metabolism of 3-methyl-5-ethyl-5-phenylhydantoin (mephenytoin) to 5-ethyl-5-phenylhydantoin (Nirvanol) in mice in relation to anticonvulsant activity.

Mephenytoin (3-methyl-5-ethyl-5-phenylhydantoin) is metabolized to Nirvanol (5-ethyl-5-phenylhydantoin). Both compounds block the hind leg tonic extensor phase of the Maximal Electroshock Seizure (M.E.S.) Test in mice. The M.E.S. ED50 of mephenytoin is 42 mg/kh at 30 min and 35 mg/kg at 2 hr after ip administration. The M.E.S. ED50 for Nirvanol at 30 min and 2 hr was 23 and 30 mg/kg, respectively. Brain and plasma levels of mephenytoin and Nirvanol were determined by gas-liquid chromatography after ip administration of 40 mg of mephenytoin per kg. At 30 min the brain levels of mephenytoin and Nivanol were 19.2 and 8.1 mug/g, respectively. The brain levels of mephenytoin fell to 5.8 mug/g and those of Nirvanol rose to 18.2 mug/g at 2 hr. The total molar concentration of mephenytoin and Nirvanol, however, did not change more than 10% during the 2-hr period. Although the anticonvulsant activity of mephenytoin did not vary greatly during 2 hr after administration, the early activity is due in major part fo mephenytoin and the later activity to Nirvanol.

Animals↗
PubMed1985

Assay of mephenytoin metabolism in human liver microsomes by high-performance liquid chromatography.

The metabolism of mephenytoin to its two major metabolites, 4-OH-mephenytoin (4-OH-M) and 5-phenyl-5-ethylhydantoin (nirvanol) was studied in human liver microsomes by a reversed phase HPLC assay. Because of preferential hydroxylation of S-mephenytoin in vivo, microsomes (5-300 micrograms protein) were incubated separately with S- and R-mephenytoin. After addition of phenobarbital as internal standard, the incubation mixture was extracted with dichloromethane. The residue remaining after evaporation was dissolved in water and injected on a 60 X 4.6-mm reversed-phase column (5 mu-C-18). Elution with acetonitrile/methanol/sodium perchlorate (20 mM, pH 2.5) led to almost baseline separation of mephenytoin, metabolites, and phenobarbital. Quantitation was performed by uv-absorption at 204 nm by the internal standard method. Propylene glycol was found to be the best solvent for mephenytoin, but inhibited the reaction noncompetitively. 4-OH-M and nirvanol could be detected at concentrations in the incubation mixture as low as 40 and 80 nM, respectively. The rates of metabolite formation were linear with time and protein concentration. The reaction was found to be substrate stereoselective. At substrate concentrations below 0.5 mM S-mephenytoin was preferentially hydroxylated to 4-OH-M, while R-mephenytoin was preferentially demethylated to nirvanol at all substrate concentrations tested (25-1600 microM). These data provide a mechanistic explanation for the stereospecific pharmacokinetics in vivo. The dependence of both metabolic relations on NADPH and the inhibition by CO suggest that they are mediated by cytochrome P-450-type monooxygenases.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromatography, High Pressure Liquid↗
PubMed1991

The mephenytoin oxidation polymorphism is partially responsible for the N-demethylation of imipramine.

The metabolism of imipramine in six poor metabolizers of mephenytoin was compared with the metabolism of 16 extensive metabolizers of mephenytoin from an earlier study. Each subject was given single doses of 100 mg imipramine hydrochloride and 100 mg desipramine hydrochloride on separate occasions. Imipramine demethylation clearance was 0.74 L.min-1 (mean; range, 0.31-1.24) in poor metabolizers of mephenytoin compared with 1.43 L.min-1 (mean; range, 0.61-3.81) in extensive metabolizers of mephenytoin (p = 0.01, Mann-Whitney U test). It has previously been shown that the imipramine clearance by way of other pathways and desipramine oral clearance, both largely representing 2-hydroxylation, are considerably lower in poor metabolizers of sparteine than in extensive metabolizers of sparteine. In contrast, five subjects who were poor metabolizers of mephenytoin and extensive metabolizers of sparteine and a control group of 11 subjects who were extensive metabolizers of mephenytoin and sparteine showed no statistically significant difference with regard to these parameters. One subject who was a poor metabolizer of mephenytoin and sparteine had the lowest imipramine oral clearance of all 22 subjects studied. In conclusion, this and an earlier study show that the oxidation of imipramine is mediated by means of two different polymorphic P450 isozymes, 2-hydroxylation by way of the sparteine oxygenase (P450IID6) and demethylation by way of the mephenytoin oxygenase (P450IIC8).

Adult↗