CYP2C9
CYP2C9 (substrate)

In order to produce 5-phenyl-5-ethylhydantoin (nirvanol), (R)-mephenytoin can be N-demethylated by the cytochrome P450 (CYP) isoform CYP2C9[1].
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[1].

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(R)-Mephenytoin can be N-demethylated by the cytochrome P450 (CYP) isoform CYP2C9 to form the active metabolite 5-ethyl-5-phenylhydantoin (nirvanol). This reaction is used as a probe to assess CYP2C9 activity in vitro. The ratio of (S)-mephenytoin to (R)-mephenytoin metabolism distinguishes between CYP2C19 (S-enantiomer substrate) and CYP2C9 (R-enantiomer substrate). (R)-Mephenytoin is also a substrate of CYP2B6. In vitro, the compound has been used to study CYP2C9-mediated drug metabolism and drug-drug interactions. No direct anticonvulsant activity is attributed to the R-enantiomer alone; the anticonvulsant activity is associated with the S-enantiomer (which is a CYP2C19 substrate) and its metabolites.

No specific in vivo data for (R)-Mephenytoin as a therapeutic agent; however, as a substrate of CYP2C9, its metabolism can be studied in vivo. Administration of racemic mephenytoin (the parent drug) has been used clinically and in research to phenotype CYP2C19 (S-mephenytoin) and CYP2C9 (R-mephenytoin) activity in humans. Following oral administration, the ratio of (S)-mephenytoin to (R)-mephenytoin in urine is used to determine CYP2C19 and CYP2C9 metabolic phenotypes. The compound itself is not used therapeutically as a single enantiomer; the racemate was used as an anticonvulsant but has been largely replaced by newer drugs.

Previous biochemical studies have suggested that tolbutamide and mephenytoin are metabolized by the same cytochrome P450 enzyme. Conversely, clinical studies indicate the involvement of different P450 forms in tolbutamide and mephenytoin metabolism. Our objective was to elucidate further those P450 enzymes responsible for hydroxylation of these two drugs. We studied both tolbutamide and (S)-mephenytoin hydroxylation in microsomes from 38 different normal adult human livers, and found large variability in the rates of metabolism for both reactions (1.75-47.4 nmol/mg/hr for hydroxytolbutamide formation and 0.1-7.2 nmol/mg/hr for 4-hydroxymephenytoin formation). No significant correlation was found between the two activities. However, both reactions shared common inhibitors in vitro, including inhibition by antikidney-liver-microsome autoantibodies (Meier and Meyer, Biochemistry 26: 8466-8474, 1987) and by teniposide. Two human liver cDNAs for P450s of the CYP2C subfamily designated IIC8 and IIC9 (S. Kimura, J. Pastewka, H. V. Gelboin and F. J. Gonzalez, Nucl. Acids Res. 15: 10053-10054, 1987), were functionally expressed in human HepG2 and TK- cells using a vaccinia virus vector. Interestingly, tolbutamide was hydroxylated by both expressed P450s. Only IIC9 catalyzed the 4-hydroxylation of (R)-mephenytoin and neither enzyme metabolized (S)-mephenytoin. We conclude that tolbutamide and (R)-mephenytoin are both metabolized by the same P450 enzyme, IIC9, and that tolbutamide is hydroxylated by an additional highly related enzyme, IIC8, contributing to the lack of correlation of the two hydroxylase activities among human liver microsomes and indicating the absence of a monogenically controlled polymorphism for tolbutamide[2].

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Recombinant human CYP2C9 is expressed in insect cells or E. coli and reconstituted with NADPH-cytochrome P450 reductase and cytochrome b5. The enzyme activity assay uses (R)-Mephenytoin (50-500 uM) as a substrate. The reaction is incubated at 37degC for 30-60 minutes in a buffer containing potassium phosphate (pH 7.4), MgCl2, and an NADPH-regenerating system (glucose-6-phosphate, glucose-6-phosphate dehydrogenase, NADP+). The reaction is stopped by adding acetonitrile or methanol. The metabolite, 5-ethyl-5-phenylhydantoin (nirvanol), is quantified by HPLC-UV or LC-MS/MS. Enzyme kinetic parameters (Km, Vmax) are determined. The assay can be used to screen for CYP2C9 inhibitors or to assess the effect of genetic polymorphisms on enzyme activity.

Primary human hepatocytes or CYP2C9-overexpressing cell lines (e.g., HEK293-CYP2C9) are cultured in appropriate media. Cells are treated with (R)-Mephenytoin (50-500 uM) for 1-24 hours. The metabolite (nirvanol) in the culture supernatant is quantified by LC-MS/MS. CYP2C9 enzyme activity is expressed as the metabolite formation rate. Cell viability is assessed by MTT or LDH assays. The assay can be used to study the effects of CYP2C9 inducers or inhibitors on (R)-Mephenytoin metabolism. The ratio of (S)-mephenytoin to (R)-mephenytoin metabolism can be assessed in the same cell system by adding both enantiomers.

No specific in vivo animal study for (R)-Mephenytoin alone. In human clinical pharmacology studies, racemic mephenytoin (100 mg orally) is administered to human subjects. Urine is collected for 0-8 hours or 0-24 hours. The concentrations of (S)-mephenytoin, (R)-mephenytoin, and their metabolites (nirvanol) in urine are measured by HPLC or LC-MS/MS. The ratio of (S)-mephenytoin to (R)-mephenytoin (S/R ratio) or the metabolic ratio of (S)-mephenytoin to its 4'-hydroxylated metabolite is used as a phenotypic measure of CYP2C19 activity, while (R)-mephenytoin clearance is used as a measure of CYP2C9 activity. In animal models, (R)-Mephenytoin can be administered to rats or mice (typically 10-50 mg/kg orally or intraperitoneally) to study CYP2C9 (or rat CYP2C isoforms) metabolism, but such studies are less common.

Mephenytoin (racemate) is absorbed after oral administration and undergoes extensive hepatic metabolism. The (R)-enantiomer is metabolized primarily by CYP2C9 to nirvanol, which is then further metabolized. The (S)-enantiomer is metabolized by CYP2C19 to 4'-hydroxymephenytoin. The half-life of mephenytoin in humans is approximately 2-4 hours for the (S)-enantiomer and longer for the (R)-enantiomer due to slower metabolism. The pharmacokinetic parameters of (R)-Mephenytoin alone are not specifically reported. Protein binding is high (>90%). Excretion is primarily in urine as metabolites.

Mephenytoin (racemate) was used as an anticonvulsant but is no longer widely used due to its adverse effect profile, including dose-dependent CNS depression (drowsiness, ataxia, nystagmus, dizziness), gastrointestinal disturbances, and rare but serious hypersensitivity reactions (rash, fever, hepatotoxicity, blood dyscrasias, Stevens-Johnson syndrome). The (R)-enantiomer is not approved as a single agent. As a research tool, (R)-Mephenytoin is used at low concentrations in vitro or in low doses in vivo for phenotyping studies and is generally considered safe for research purposes when handled appropriately.

[1]. Assay of mephenytoin metabolism in human liver microsomes by high-performance liquid chromatography. Anal Biochem. 1985 Dec;151(2):286-91.

[2]. Tolbutamide and mephenytoin hydroxylation by human cytochrome P450s in the CYP2C subfamily. J Pharmacol Exp Ther. 1990 Jan;252(1):442-7.

(R)-Mephenytoin (CAS: 71140-51-7) is the (R) enantiomer of the anticonvulsant mephenytoin. It is a substrate of CYP2C9 and is N-demethylated by CYP2C9 to form 5-ethyl-5-phenylhydantoin (nirvanol). It is a valuable research tool for studying CYP2C9-mediated drug metabolism, pharmacogenetics, and drug-drug interactions. The compound is not approved for clinical use as a single enantiomer. The racemate was used as an anticonvulsant but has been largely replaced by newer agents. Mephenytoin is also known as 3-methyl-5,5-phenylethylhydantoin. The compound is for research use only. Synonyms: (-)-Mephenytoin. Molecular formula: C12H14N2O2. Molecular weight: 218.25. References: (R)-Mephenytoin is described as a CYP2C9 substrate and is used in CYP phenotyping studies.

C12H14N2O2

218.25

218.106

71140-51-7

Mephenytoin;50-12-4;(S)-Mephenytoin;70989-04-7

119127

Typically exists as solid at room temperature

1.154g/cm3

137-138ºC

1.541

1.74

1

2

2

16

310

1

CCC1(C(=O)N(C(=O)N1)C)C2=CC=CC=C2

GMHKMTDVRCWUDX-GFCCVEGCSA-N

InChI=1S/C12H14N2O2/c1-3-12(9-7-5-4-6-8-9)10(15)14(2)11(16)13-12/h4-8H,3H2,1-2H3,(H,13,16)/t12-/m1/s1

(5R)-5-ethyl-3-methyl-5-phenylimidazolidine-2,4-dione

(R)-Mephenytoin; 71140-51-7; (R)-(-)-Mephenytoin; R-Mephenytoin; (-)-Mephenytoin; (5r)-5-ethyl-3-methyl-5-phenylimidazolidine-2,4-dione; Mephenytoin, (-)-; (R)-5-Ethyl-3-methyl-5-phenylimidazolidine-2,4-dione;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 220 mg/mL (1008.02 mM)

Solubility in Formulation 1: ≥ 5.5 mg/mL (25.20 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 55.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 2: ≥ 5.5 mg/mL (25.20 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 55.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)