Major Metabolite Of Phenytoin Research Articles

Effect of moderate haemodilution with Fluosol-DA or normal saline on low-dose phenytoin and (+/-)-5-(4-hydroxyphenyl)-5-phenylhydantoin kinetics.

Phenytoin kinetics were determined in the rat following moderate (50%) blood exchange with either Fluosol-DA or normal saline. Rats received an intravenous phenytoin dose (10 mg kg-1) 0.5, 24, 48, or 72 h after exchange and were compared with non-exchanged controls. Phenytoin t 1/2 was not altered by exchange with either fluid. Its Cl and Vd were decreased and AUC increased 24, 48, and 72 h after saline exchange and 24 h after Fluosol-DA exchange. (+/-)-5-(4-Hydroxyphenyl)-5-phenylhydantoin (HPPH), a major metabolite of phenytoin, showed a decreased t1/2 and VHPPH 24, 48, and 72 h after exchange with either fluid; t1/2 was also reduced 0.5 h after Fluosol-DA exchange. The decreased Vd and VHPPH may result from changes in cardiac output secondary to haemodilution, or may represent a redistribution in the microcirculation. Fluosol-DA appears to enhance phenytoin and HPPH metabolism 48 and 72 h after exchange.

Analytic Performance Evaluation of a New Turbidimetric Immunoassay for Phenytoin on the ADVIA 1650® Analyzer

Phenytoin is an anticonvulsant that requires therapeutic drug monitoring. Recently, Bayer HealthCare, Diagnostics Division released a turbidimetric immunoassay of phenytoin on the ADVIA 1650 analyzer. We evaluated the analytic performance of this assay by comparing values obtained in 52 patients receiving Phenytoin using this new assay with the values obtained by using a widely used fluorescence polarization immunoassay (FPIA). The new turbidimetric immunoassay for phenytoin showed the following imprecision with the low, medium, and high controls: total CV of 5.2% (mean 4.81 microg/mL), 3.7% (mean 16.24 microg/mL), and 4.1% (mean 22.65 microg/mL), respectively. The detection limit of the assay was 0.79 microg/mL, and the assay was linear up to a phenytoin concentration of 46.1 microg/mL. The assay showed excellent dilution recovery and recovery of spiked samples (mean recovery 101.4% and 94.4%, respectively). We observed an excellent correlation between the values obtained by the FPIA (x-axis) assay and the new turbidimetric (y-axis) assay (y=1.06 x-0.61, r=0.98, n=52). We also determined the cross-reactivity of 5-(p-hydroxyphenyl)-5-phenylhydantoin (HPPH), a major metabolite of phenytoin, and of oxaprozine, an analogue with a similar chemical structure to phenytoin, in both phenytoin assays. Both assays showed almost no cross-reactivity to oxaprozine and only small (5%-8%) cross-reactivity to HPPH. We also found that the turbidimetric assay was free from interference at least up to 1200 mg/dL of hemolysis, 30 mg/dL of free bilirubin, 34.5 mg/dL of conjugated bilirubin, and 750 mg/dL of triglyceride (Intralipid). When a drug-free serum was followed by a serum sample containing 38.5 microg/mL of phenytoin, no sample probe carryover effect was observed. We conclude that the new turbidimetric assay can be used for routine monitoring of phenytoin in clinical laboratories.

Effect of mild therapeutic hypothermia on phenytoin pharmacokinetics.

The authors examined the effect of mild therapeutic hypothermia on phenytoin pharmacokinetics in 14 patients with brain damage. Each patient was given phenytoin during and after mild therapeutic hypothermia. Plasma concentrations of total phenytoin, unbound phenytoin, and 5-(p-hydroxyphenyl)-5-phenylhydantoin (5-p-HPPH), the major metabolite of phenytoin, were measured. Pharmacokinetic parameters during and after mild therapeutic hypothermia were compared. Plasma concentrations of total and unbound phenytoin were significantly higher during hypothermia than after hypothermia. The area under the plasma concentration-time curve (zero to infinity) was increased by 180% and mean residence time was prolonged by 180% during hypothermia compared with the corresponding values after hypothermia. Moreover, the elimination constant (ke) was decreased by 50% and elimination clearance of phenytoin was decreased by 67% during hypothermia compared with the corresponding values after hypothermia. The plasma concentration of 5-p-HPPH was significantly lower during hypothermia than after hypothermia. These findings suggest that phenytoin metabolism is inhibited by mild therapeutic hypothermia.

The effects of genetic polymorphisms of CYP2C9 and CYP2C19 on phenytoin metabolism in Japanese adult patients with epilepsy: studies in stereoselective hydroxylation and population pharmacokinetics.

The aim of this study was to clarify the effects of genetic polymorphisms of cytochrome P450 (CYP) 2C9 and 2C19 on the metabolism of phenytoin (PHT). In addition, a population pharmacokinetic analysis was performed. The genotype of CYP2C9 (Arg144/Cys, Ile359/Leu) and CYP2C19(*1, *2 or *3) in 134 Japanese adult patients with epilepsy treated with PHT were determined, and their serum concentrations of 5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) enantiomers, being major metabolites of PHT, were measured. A population pharmacokinetic analysis (NONMEM analysis) was performed to evaluate whether genetic polymorphism of CYP2C9/19 affects the clinical use of PHT by using the 336 dose-serum concentration data. The mean maximal elimination rate (Vmax) was 42% lower in the heterozygote for Leu359 allele in CYP2C9, and the mean Michaelis-Menten constants (Km) in the heterozygous extensive metabolizers and the poor metabolizers of CYP2C19 were 22 and 54%, respectively, higher than those without the mutations in CYP2C9/19 genes. (R)- and (S)-p-HPPH/PHT ratios were lower in patients with mutations in CYP2C9 or CYP2C19 gene than those in patients without mutations. Although the hydroxylation capacity of PHT was impaired with mutations of CYP2C9/19, the impairment was greater for CYP2C9. In view of the clinical use of PHT, two important conclusions were derived from this population study. First, the serum PHT concentration in patients with the Leu359 allele in CYP2C9 would increase dramatically even at lower daily doses. Second, the patients with CYP2C19 mutations should be treated carefully at higher daily doses of PHT.

Limited value of the urinary phenytoin metabolic ratio for the assessment of cytochrome P4502C9 activity in vivo

Relationships between the ratio of p-hydroxyphenytoin (p-HPPH), the major metabolite of phenytoin, to unchanged phenytoin excreted in urine (the urinary metabolic ratio or MR) were compared with a number of indices of the metabolic clearances of phenytoin and tolbutamide published previously for seventeen subjects separately administered these known cytochrome P4502C9 (CYP2C9) substrates. Significant correlations (rs = 0.50-0.60, P < 0.05) were observed between the phenytoin MR, derived from either 0-24 or 24-48 h urine collections, and inverse areas under the plasma unbound concentration-time curves (measured over various time intervals) of phenytoin and with plasma unbound tolbutamide clearance. Significant correlations (rs = 0.59-0.74) were also observed between the phenytoin MRs and metabolic unbound clearances for p-hydroxyphenytoin formation. Despite the significant correlations, variability in tolbutamide and phenytoin metabolic clearance parameters tended to account for < 50% of the variability in phenytoin MR. Correlations between the renal clearance of phenytoin and the phenytoin MRs suggest that variability in the renal clearance of unchanged drug limits the usefulness of the phenytoin MR for the investigation of factors influencing CYP2C9 activity in vivo.

Plasma and saliva concentrations of phenytoin and 5-(4-hydroxyphenyl)-5-phenylhydantoin in relation to the incidence and severity of phenytoin-induced gingival overgrowth in epileptic patients.

This study examined the relationships between plasma and saliva concentrations of phenytoin and 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH), the major metabolite of phenytoin in man, and the prevalence and severity of gingival overgrowth. Thirty-six adult epileptic patients who had been receiving phenytoin for greater than 6 months without a recent change in dosage were assessed for signs of periodontal disease and gingival overgrowth. Plasma and saliva samples were analyzed by high performance liquid chromatography for the determination of phenytoin and HPPH concentrations. Seventeen patients demonstrated clinically significant gingival over-growth (responders; overgrowth index > or = 30%). There were significant correlations between the gingival overgrowth index and both the papillary bleeding index (r = 0.495; P < 0.005) and probing depth (r = 0.632; P < 0.005). The plaque index correlated with the papillary bleeding index (r = 0.420; P < 0.05) and the probing depth (r = 0.301; P < 0.005), but not with the gingival overgrowth index. The extent of gingival overgrowth did not correlate significantly with either plasma or saliva concentrations of phenytoin or HPPH. Mean plasma and saliva concentrations of phenytoin and HPPH did not differ significantly between non-responders and responders, nor did the mean plaque index. The mean papillary bleeding index (32.5 +/- 21.2 vs. 63.8 +/- 37.7; P < 0.01) and mean probing depth (12.4 +/- 14.4% vs. 35.9 +/- 25.3%; P < 0.02) were significantly greater in the responders. This study found no evidence of a relationship between phenytoin or HPPH concentrations in plasma or saliva and the extent, or prevalence of phenytoin-induced gingival overgrowth. Further studies with larger populations may be necessary to establish the relationship, if any, between phenytoin or HPPH levels and gingival overgrowth.

Effect of 5-(p-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) enantiomers, major metabolites of phenytoin, on the occurrence of chronic-gingival hyperplasia: in vivo and in vitro study.

The purpose of this study was to assess the possible role of the (R)- and (S)- enantiomers of the phenytoin metabolite p-HPPH in the pathogenesis of gingival hyperplasia (GH). About 98% of circulating p-HPPH is in the (S)-form. There were significant differences between patients with and without GH in (R)-p-HPPH level (0.055 vs 0.042 microgram.ml-1), both enantiomer/racemate level ratios, and R/S enantiomeric ratio (0.0313 vs 0.0232); an increase in serum (R)-p-HPPH level was observed in patients with GH. In separate experiments, the effect of p-HPPH enantiomers on the proliferation of the normal human dermal fibroblast was studied. The in vitro study showed that (R)-p-HPPH selectively stimulated fibroblast growth. The results suggest that the least abundant metabolite, (R)-p-HPPH, is the most toxic with respect to gingival hyperplasia.

Effect of barbiturate therapy on phenytoin pharmacokinetics.

To evaluate the effect of high-dose pentobarbital therapy on phenytoin pharmacokinetics. A prospective, clinical study. The intensive care unit of a university hospital. Ten adult patients with cerebral lesions requiring anticonvulsants and control of intracranial pressure. Each patient received phenytoin sufficient to maintain a plasma concentration at 15 micrograms/mL (60 mumol/L) both before and after barbiturate therapy. Plasma concentrations of total phenytoin, unbound phenytoin, and the major metabolite of phenytoin, 5-(p-hydroxyphenyl)-5-phenylhydantoin, were measured, and pharmacokinetic variables obtained before and after barbiturate therapy were compared. Plasma concentrations of total phenytoin remained within the therapeutic range during the 12-hr period preceding barbiturate therapy. After barbiturate therapy, plasma concentrations of both total and unbound phenytoin were significantly less than those concentrations before barbiturate therapy. For total phenytoin, maximum metabolic velocity was increased by 62% (1.09 +/- 0.62 to 1.77 +/- 0.52 mg/L/hr, 1.20 +/- 0.68 to 1.95 +/- 0.57 nmol/L/sec, p < .05), and area under the plasma concentration-time curve (0 to infinity) and mean residence time were each decreased by 73% (32.5 +/- 20.0 to 8.7 +/- 3.1 min.mg/mL, 2.14 +/- 1.25 to 0.57 +/- 0.19 sec.mmol/L, p < .01, and 135,000 +/- 69,000 to 37,000 +/- 11,000 secs, p < .005, respectively) after barbiturate therapy. The plasma concentration of the principal metabolite of phenytoin, 5-(p-hydroxyphenyl)-5-phenylhydantoin, was significantly increased after barbiturate therapy. Phenytoin metabolism is increased by barbiturate therapy, and supplemental doses of phenytoin and frequent drug monitoring may be required after barbiturate therapy.

Simultaneous determination of p-hydroxylated and dihydrodiol metabolites of phenytoin in urine by high-performance liquid chromatography

Accurate urinary measurements of the two major metabolites of phenytoin, 5-(p-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) and 5-(3,4-dihydroxy-cyclohexa-1,5-dienyl)-5-phenylhydantoin (dihydrodiol, DHD), are necessary for pharmacokinetic and drug-interaction studies of this commonly used antiepileptic drug. We describe a simple, rapid, acid hydrolysis, with liquid-liquid extraction and simultaneous isocratic reversed-phase high-performance liquid chromatography of p-HPPH and 5-(m-hydroxyphenyl)-5-phenylhydantoin (m-HPPH) (hydrolytic end product of DHD). p-HPPH and m-HPPH were quantitated against their separate respective internal standards of alphenal and tolylbarb. The mobile phase consisted of water-dioxane-tetrahydrofuran (80:15:5, v/v/v) at 2 ml/min and at 50 degrees C, with detection at 225 nm. Baseline separation was achieved by use of a 16 cm x 3.9 mm Nova-Pak C18 column and total analysis time of 12 min. p-HPPH and m-HPPH concentrations ranged from 10 to 200 and from 2 to 30 micrograms/ml, respectively, with between-day coefficients of variations of 3.3-4.5% and 2.2-5.1% for controls. All standard curves were linear with r values greater than 0.993. The DHD concentration was determined by multiplying m-HPPH concentrations by 2.3.

Detection of carbon-14 eluting from a capillary gas chromatograph using chemical reaction interface—mass spectrometry

We have applied a chemical reaction interface-mass spectrometric (CRIMS) technique to the detection of 14C-labeled phenytoin and its metabolites in urine following separation by capillary gas chromatography. The presence of 14C was followed by monitoring 14CH4 which is produced by the chemical reaction interface from carbon when hydrogen is used as the reactant gas. Chromatograms showing 14C are obtained by measuring 14CH4 at m/z 18.034 with a resolution of 1800. The chemical reaction interface permits symmetrical, narrow chromatographic peaks to be obtained. Detection limits of 6.3 Bq/ml (1.26 Bq on-column which is equivalent to detection of 187 pg) of a diethylated 14C-labeled metabolite was detected at a signal-to-noise ratio of 3. The analysis was determined to be linear and similar detection limits were obtained following analysis of phenytoin-spiked urine or a phenytoin solution. Radiocarbon detection is not completely selective because nitrogen-containing compounds present at greater than 250 ng on-column are detected as NH4+. However, such interferences are infrequently of analytical importance because the capacity limits of CRIMS are 500-1000 ng. Furthermore, these interferences are readily discovered in a NH3 chromatogram which is the precursor of NH4+. After a 3.3-MBq dose, the major metabolites of phenytoin were detected by CRIMS.

Kinetic equivalence of stable-isotope-labeled and unlabeled phenytoin.

Stable isotope labeling (SIL) of a drug results in a higher molecular weight than that of the unlabeled drug. SIL tracer doses can be quantitated separately from unlabeled drug by gas chromatography-mass spectrometry (GC-MS) without exposing the patient to radiation. The higher molecular weight of SIL drug could cause a higher energy of activation for (and slowing of) metabolic reactions ("isotope effect"). To evaluate possible isotope effect, three dogs and three men were infused with a mixture containing equal amounts of SIL (2-13C-1,3-15N2) and unlabeled phenytoin (PHT). Plasma and urine were collected at regular intervals. Concentrations of SIL and unlabeled PHT and HPPH (the major metabolite of PHT) were determined by GC-MS. Within each subject there was no trend for concentrations of SIL PHT or HPPH to be higher or lower than concentrations of their unlabeled analogs (greater than 0.20 to 0.90). There was no difference in the distribution and elimination half-lifes (t 1/2s), volume of distribution, volume of central compartment, or clearance of the two forms of PHT. Thus, no isotope effect was found.

A mass spectrometric method for the determination of stable isotope labeled phenytoin suitable for pulse dosing studies.

A gas chromatographic mass spectrometric method has been developed for the determination in biological fluids of phenytoin (5,5-diphenylhydantoin), 5-(4-hydroxyphenyl)-5-phenylhydantoin (the major metabolite of phenytoin), and simultaneously, their stable isotope labeled analogs [5,5-diphenyl-2-13C-1,3-15N2-hydantoin and 5-(4-hydroxyphenyl)-5-phenyl-2-13C-1,3-15N2-hydantoin]. Quantification was achieved by an isotopic dilution technique: 5,5-di(pentadeuterophenyl)-hydantoin and 5-(4-hydroxy-3,5-dideuterophenyl)-5-phenyl-2-13C-1,3-15N2-hydantoin were used as internal standards. Molecular ion abundances of the permethylated derivatives were measured using a limited mass range repetitive scanning technique. The method is accurate, selective, reproducible and linear for analysis of 1.0 ml of serum and 0.5 ml of urine samples at the expected concentrations of drug (serum: 0.1-30.0 micrograms ml-1) and metabolite (serum: 0.1-10.0 micrograms ml-1; urine: 5.0-200.0 micrograms ml-1). The pharmacological equivalence of labeled and unlabeled phenytoin is demonstrated for a human volunteer. The results are discussed in the light of the further applications of the method, i.e. determination of the pharmacokinetics of a pulse dose of the labeled drug administered to patients who are taking a steady state dose of the unlabeled drug.

Substrate-labeled fluorescent immunoassay for phenytoin in human serum.

A homogeneous substrate-labeled fluorescent immunoassay has been applied to the measurement of phenytoin concentrations in human serum. We coupled a fluorogenic enzyme substrate, galactosyl-umbelliferone, covalently to a derivative of phenytoin. Under assay conditions, this drug-substrate conjugate was nonfluorescent but became fluorescent upon hydrolysis catalyzed by bacterial beta-galactosidase. When antibody to phenytoin is bound to the drug-substrate conjugate, it is inactive as an enzyme substrate. Addition of phenytoin to competitive-binding reactions relieves the inactivation, and the resulting fluorescence is proportional to the phenytoin concentration. We validated the fluorescent immunoassay by comparing values for phenytoin obtained with this technique to those obtained by gas chromatography and by enzyme immunoassay (EMIT). All three methods correlated well. The major metabolite of phenytoin, 5-(p-hydroxyphenyl)-5-phenylhydantoin, and other drugs at concentrations expected in serum had no effect on the assay. The fluorescent immunoassay is rapid and simple to perform and requires only 2 microL of serum sample per test.

Determination of 5-(3,4-Dihydroxy-1,5-cyclohexadien-1-yl)-5-phenylhydantoin (Dihydrodiol) and quantitative studies of phenytoin metabolism in man.

A routine gas chromatographic assay for urinary 5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH), the major metabolite of phenytoin (PHT) in man, was adapted to allow quantitation of 5-(3,4-dihydroxy-1,5-cyclohexadien-1-yl)-5-phenylhydantoin (Dihydrodiol, DHD) is based on the observation that acid-catalyzed dehydration of DHD quantitatively yields a mixture of p-HPPH and m-HPPH in a reproducible molar ratio of 56:44p-HPPH: m-HPPH and on the assumption that all m-HPPH found in urine after heating with acid has been derived from DHD. The urinary DHD content was verified by a "specific" method in which urine was incubated with beta-glucuronidase and the released phenolic metabolites completely removed by extraction. Subsequent acid-catalyzed dehydration of the remaining DHD yielded p-HPPH and m-HPPH, from the sum of which the original DHD concentration in urine could be calculated. In all of the urine samples from PHT patients examined to date, there was close agreement between the DHD values obtained by the "specific" method and those calculated from m-HPPH, in the simple acid-hydrolysis method. It can be inferred that much the greater part (greater than 90%) of m-HPPH found in human urine after acid treatment has been derived from DHD. All samples of urine after acid treatment has been derived from DHD. All samples of urine from PHT patients examined have shown detectable quantities of DHD. The methods described here may be useful in a survey of PHT patients to reveal unusual patterns of PHT metabolism and to permit recognition of possible associations between such unusual patterns and the occurrence of adverse reactions.

Influence of food on the absorption of phenytoin in man.

The influence of food intake on the absorption of phenytoin was examined in eight healthy volunteers, by study of single-dose kinetics following ingestion of phenytoin 300 mg either with a standardized breakfast or on an empty stomach. Blood samples were collected at regular intervals from 0 to 48 h, and serum concentrations of unmetabolized phenytoin were determined by gas chromatography. Serum concentrations of the major metabolite of phenytoin, 4-hydroxyphenytoin, were measured by mass fragmentography. Concurrent intake of food and phenytoin appeared to accelerate absorption of the drug from the formulation used, and the peak concentrations were significantly higher (mean increase 40%) in the postprandial than in the preprandial state. As reflected by the AUC (area under the curve), the amount of drug absorbed was increased during postprandial conditions, although the difference only reached borderline significance. It is suggested that phenytoin should always be taken in a defined relation to meals.

The major metabolite of phenytoin (Dilantin) induces gingival overgrowth in cats.

The major metabolite of phenytoin (Dilantin) induces gingival overgrowth in cats.