George K. Szabo

Bioavailability of ACC-9653 (Phenytoin Prodrug)

Summary: 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(O‐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.

Alternatives to Least Squares Linear Regression Analysis for Computation of Standard Curves for Quantitation by High Performance Liquid Chromatography: Applications to Clinical Pharmacology

Standard curves and validation points for high‐performance liquid chromatography (HPLC) determination of four drugs (carbamazepine and phenytoin at therapeutic drug monitoring concentrations and deuterium labeled carbamazepine and phenytoin at tracer dose concentrations) were computed using standard least squares linear regressions analysis and six alternative regression techniques (weighted 1/x, 1/y, 1/x2, 1/y2 least squares linear, log/log least squares linear, and robust). The coefficient of determination (R2) and the coefficient of prediction (R2pred) values for standard curves and the computed values for validation points did not differ significantly among the seven methods. The lower limit of quantitation (LLQ) values obtained with all six of the alternative regression methods were significantly (P < .01) lower than the LLQ values obtained with least squares linear regression analysis. The lowest LLQ values were obtained with 1/x2 and 1/y2 weighting and were threefold to tenfold less than the values obtained with unweighted least squares linear regression analysis (P < .001). The authors conclude that alternative regression analysis techniques (especially 1/x2 and 1/y2 weighting) offer significant advantages for clinical pharmacology studies when concentration values being measured by HPLC are near the LLQ of the method determined by unweighted least squares linear regression analysis. In other situations, alternative forms of regression analysis had no significant advantages in our study.

Studies with stable isotopes II: Phenobarbital pharmacokinetics during monotherapy.

Six healthy adults receiving no other medications were given tracer doses of 90 mg of stable isotope‐labeled phenobarbital (PB) intravenously before, and four weeks after, and 12 weeks after beginning therapy. Serum samples were collected for 96 hours after each injection, and the concentration of stable isotope‐labeled PB in each sample was determined by gas chromatographic mass spectrometry. The volume of distribution, elimination half‐life, and total clearance of PB did not differ significantly on any of the three occasions measured. Phenobarbital clearance did not correlate significantly with total PB serum concentration. Clearances determined from single‐dose studies before beginning PB therapy accurately predicted steady‐state PB serum concentrations. Therefore, it is not necessary to adjust PB dosage for time‐dependent or dose‐dependent changes in clearance during monotherapy. In addition, clearance or serum concentration determined at one dosing rate directly predicts serum concentration at another dosing rate.

Methsuxirnide for complex partial seizures Efficacy, toxicity, clinical pharmacology, and drug interactions

Methsuximide (MSM; Celontin) was administered for 8 weeks to 26 patients with complex partial seizures (CPS) refractory to phenytoin and carbamazepine and phenobarbital or primidone. A 50% or greater reduction in CPS frequency was obtained in eight patients. MSM therapy was continued chronically in these eight patients, and five continued to have a 50% or greater reduction in CPS frequency after 3 to 34 months of follow-up. Drowsiness, gastrointestinal disturbance, hiccups, irritability, and headache were the common side effects of MSM. No serious toxicity occurred. N-desmethylmethsuximide was the principal substance detected in plasma and had the following pharmacokinetic values: accumulation half-life, 49.7 hours; time to steady state, 10.4 days; elimination half-life, 72.2 hours; therapeutic range of plasma concentration, 10 to 30 mg per liter. Plasma concentrations of phenytoin and phenobarbital derived from primidone rose significantly (p < 0.05) after addition of MSM.

Studies With Stable Isotopes I: Changes in Phenytoin Pharmacokinetics and Biotransformation During Monotherapy

Six patients were given tracer doses of 13C15N2‐phenytoin (PHT) before and four and 12 weeks after beginning monotherapy. The following significant (P < .05) changes occurred during monotherapy: (1) Apparent (from tracer doses) PHT total clearance by linear method decreased; (2) apparent PHT elimination half‐life increased; (3) apparent mean PHT serum concentration per unit dose increased; (4) apparent rate of excretion of p‐hydroxyphenyl‐phenylhydantoin (p‐HPPH) decreased; (5) apparent rate of excretion of PHT dihydrodiol increased; and (6) apparent PHT total clearance and elimination half‐life and apparent p‐HPPH rate of excretion were dose dependent. Phenytoin apparent pharmacokinetic and biotransformation values undergo a typical series of changes after beginning monotherapy at typical dosing rates, because PHTs dose‐dependent pharmacokinetics result in differing apparent vaiues as the serum concentration rises to steady state. Stable iostope methods are particularly suitable for investigating such phenomena.

Variable Absorption of Carbidopa Affects Both Peripheral and Central Levodopa Metabolism

Carbidopa (CD), a competitive inhibitor of aromatic l‐amino acid decarboxylase that does not cross the blood‐brain barrier, is routinely administered with levodopa (LD) to patients with Parkinson disease (PD) to reduce the peripheral decarboxylation of LD to dopamine. Using a stable isotope‐labeled form of LD, the authors examined in 9 PD patients the effects of variable CD absorption on peripheral and central LD metabolism. Subjects were administered orally 50 mg of CD followed in 1 hour by a slow bolus intravenous infusion of 150 mg stable isotope‐labeled LD (ring 1′,2′,3′,4′,5′,6′‐13C). Eight patients underwent a lumbar puncture 6 hours following the infusion. Blood and cerebrospinal fluid (CSF) samples were analyzed for labeled and unlabeled metabolites using a combination of high‐performance liquid chromatography and mass spectrometry. When patients were divided into “slow” and “rapid” CD absorption groups, significantly greater peripheral LD decarboxylation (as measured by area under the curve [AUC]‐labeled serum HVA) was noted in the poor absorbers (p = 0.05, Mann‐Whitney U test). Elimination half‐lives for serum LD did not differ between groups, suggesting a further capacity for decarboxylation inhibition in the “rapid” absorbers. A significant correlation between AUC serum CD and percent‐labeled HVA in CSF was found for all patients (R = 0.786, p = 0.02). “Rapid” as compared to “slow” CD absorbers had significantly more percent‐labeled CSF HVA (60 vs. 49, p = 0.02, Mann‐Whitney U test), indicating greater central‐labeled DA production in the better CD absorbers. The data suggest that peripheral aromatic l‐amino acid decarboxylase activity is not saturated at CD doses used in current practice. The authors believe that future studies to better examine a dose dependence of CD on peripheral LD decarboxylation and LD brain uptake are warranted.

Performance of Human Mass Balance/Metabolite Identification Studies Using Stable Isotope (13C, 15N) Labeling and Continuous‐Flow Isotope‐Ratio Mass Spectrometry as an Alternative to Radioactive Labeling Methods

Stable isotope labeling in therapeutic and subtherapeutic quantities of drug (15N213C‐phenobarbital) can be quantitated in biological matrices (urine) and high performance liquid chromatography (HPLC) peaks from urine using continuous‐flow isotope‐ratio mass spectrometry (CF‐IRMS). Standard curves for 15N213C‐phenobarbital were reproducible and linear (R2 > 0.985) over the ranges of 3–100 μg/mL for whole urine (1SN2 or 13C labeling) and 0.1–8.0 μg/mL for HPLC peaks derived from urine (15N2 labeling). The lower limit of quantitation values for urine drug concentration was 0.46–2.62 μg/mL in whole urine and 0.10–0.70 μg/mL in HPLC peaks. Validation samples quantitated with these standard curves yielded close to expected values. These data suggest stable isotope labeling and CF‐IRMS may be used as an alternative to 14C labeling and radioactivity counting methods in mass balance/metabolite identification and other biomedical studies.

Studies With Stable Isotopes III: Pharmacokinetics of Tracer Doses of Drug

Stable isotope labeled tracer doses of phenytoin (PHT) and phenobarbital (PB) were given intravenously before and four and 12 weeks after beginning monotherapy in two groups of six patients. Phenytoin demonstrated nonlinear pharmacokinetics, while PB demonstrated linear pharmacokinetics. Each of the 36 sets of tracer dose serum concentration versus time data points appeared linear during the elimination phase on semilog plots, and each demonstrated a high degree of linearity using semilog regression analysis (r2 = .977‐.999, P < .001, for PHT; r2 = .791‐.996, P < .005, for PB). We conclude tracer doses administered at steady‐state serum concentration will exhibit linear serum concentration versus time relationships on semilog plots regardless of whether the steady‐state serum concentration is in the linear or the nonlinear portion of a drugs dose versus steady‐state serum concentration relationship. The mechanism and implications of this conclusion are discussed.

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.

Performance of Human Mass Balance Studies with Stable Isotope-Labeled Drug and Continuous Flow-Isotope Ratio Mass Spectrometry: A Progress Report

We propose performing human mass balance studies by administering stable isotope labeled (13C or 15N) drug and quantitating excess (above background) 13C or 15N in urine, serum, and feces by continuous flow‐isotope ratio mass spectrometry (CF‐IRMS). Theoretical calculations and empirical data (dynamic range, linearity, sensitivity, precision, accuracy) are presented to establish that commercially available CF‐IRMS instruments can quantitate stable isotope labeled (one or two 15N or 13C labels) drug concentrations of 1.0 μg/mL or greater in urine, serum (15N), or feces. More than two 13C labels may be necessary to quantitate 1.0 μg/mL of drug in serum. Three volunteers received 650 mg of 15N13C2‐acetaminophen, and urine was collected for 72 hours. Percent of administered label recovered in urine from the three subjects was 97.4, 78.9, and 95.4 for 13C and 90.3, 77.0, and 90.6 for 15N. Fecal recovery of label for one subject was 0.9% (13C2) and 1.1% (15N). Serum pharmacokinetic values obtained by counting 13C or 15N in one subject were as expected for acetaminophen. This method appears to be promising, and further validation is ongoing.