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Measurement of the absolute bioavailability of phenytoin (PHT) derived from test doses of phenytoin prodrug (PPD) at therapeutic PHT serum concentrations is complicated by two problems: 1) the area under the serum concentration versus time curve (AUC) produced by a given size of test dose will vary directly with background PHT serum concentration due to the nonlinear pharmacokinetic properties of PHT; 2) PPD is more water soluble than PHT, making renal excretion of PPD more likely. The authors describe a double-stable isotope method that obviates these two problems. Using only six subjects, the authors were able to demonstrate bioequivalence of PHT derived from intravenous PPD with intravenous PHT by current FDA standards for AUC ratio of test/reference formulation (90% confidence intervals between 0.80 and 1.20; ratio > or = 0.80 in > or = 80% of subjects; statistical power to detect a difference of 0.20 with a probability of 0.80).

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.

The bioavailability of phenytoin from ACC-9653 versus intravenously administered sodium phenytoin was determined using a crossover design for intravenous and intramuscular administration of ACC-9653 to healthy volunteers. Absolute bioavailability of phenytoin derived from ACC-9653 in each subject was calculated as the ratio of the phenytoin area under the plasma concentration time curve for time 0 to infinity [AUC(0-inf)] after ACC-9653 divided by the phenytoin AUC(0-inf) after intravenous sodium phenytoin. The mean absolute bioavailability of ACC-9653 was 0.992 after intravenous administration and 1.012 after intramuscular administration. These data establish that the bioavailability of ACC-9653 is complete following intravenous or intramuscular administration in single-dose volunteer studies. The absolute bioavailability of phenytoin derived from ACC-9653 in subjects with therapeutic plasma phenytoin concentrations is being studied in patients given simultaneous infusions of stable isotope-labeled tracer doses of ACC-0653 and sodium phenytoin.