Simultaneous separation and determination (in serum) of phenytoin and carbamazepine and their deuterated analogues by high-performance liquid chromatography--ultraviolet detection for tracer studies.

The use of stable isotope-labeled tracer compounds is the safest and most effective method to perform many steady state pharmacokinetic and drug interaction studies. We describe a method by which the heavily deuterated 2H10 analogues of carbamazepine (2H10 CBZ) and phenytoin (2H10 PHT) can be chromatographically separated by high-performance liquid chromatography from unlabeled CBZ and PHT. All compounds are quantitated against an internal standard (IS) (10,11-dihydrocarbamazepine) and measured using conventional UV detection rather than mass spectrometry. Baseline resolution of extracted serum containing 2H10 CBZ, CBZ, 2H10 PHT, PHT and IS is achieved on a heated (55 degrees C) 25 cm x 4.6 mm BioAnalytical Systems Phase II 5 microns ODS column with an isocratic mobile phase consisting of water-acetonitrile-tetrahydrofuran (80:16:4, v/v/v) at 1.2 ml/min. Eluting compounds were monitored at a UV wavelength of 214 nm. Calculated resolution of 2H10 CBZ from CBZ and of 2H10 PHT from PHT were 1.3. Serum standard curves were linear (R greater than or equal to 0.999) over a range of 0.5-14 micrograms/ml for 2H10 CBZ, 0.5-20 micrograms/ml for CBZ, 0.5-20 micrograms/ml for 2H10 PHT, and 0.5-30 micrograms/ml for PHT. Within-day percent relative standard deviations (precision) were less than 6% in all cases.

Carbamazepine metabolism in humans: effect of concurrent anticonvulsant therapy.

Free and total carbamazepine (CBZ) and carbamazepine-epoxide (CBZ-EP) plasma levels were obtained on 113 patients with epilepsy (18-61 years old) controlled on either monotherapy or coadministration with either phenobarbital (PB), phenytoin (PHT), valproic acid (VPA), or all three. A subset of patients were administered tetradeuterium labeled CBZ to evaluate the effects of autoinduction and coadministration of VPA on the kinetics of CBZ and its metabolite CBZ-EP. Polytherapy had variable effect on free and total CBZ plasma levels compared to monotherapy. Coadministered PHT (co-PHT), or all three anticonvulsants together (PHT, PB, and VPA: co-AEDs) decreased free and total CBZ plasma levels. No change was noted for coadministered VPA (co-VPA). Compared to monotherapy the free and total CBZ-EP levels increased with co-VPA, less with coadministered PB (co-PB), and no change with co-PHT or co-AEDs. Protein binding of CBZ and CBZ-EP was not affected by any antiepileptic drugs studied. The free and total CBZ-EP/CBZ ratio was tripled with co-VPA or co-AED's, and doubled with co-PHT or co-PB. Isotope labeling did not demonstrate any differences in half-life (t1/2), plasma clearance (Cl), or volume of distribution (Vd). Compared to naive controls, monotherapy and co-VPA decreased CBZ t1/2 by 50%, and more than doubled the CBZ Cl without a significant change in the Vd. Autoinduction is one explanation for these changes with chronic CBZ therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

Improved delivery through biological membranes. XXXL: Solubilization and stabilization of an estradiol chemical delivery system by modified beta-cyclodextrins.

A dihydropyridine in equilibrium pyridinium salt chemical delivery system (CDS) for estradiol (E2CDS) was complexed with various modified beta-cyclodextrins including hydroxyethyl-beta-cyclodextrin (HECD), hydroxypropyl-beta-cyclodextrin (HPCD), and heptakis(2,6-di-O-methyl)-beta-cyclodextrin (DMCD). Complex formation with all of these cyclodextrins resulted in dramatic increases in the water solubility of E2CDS. Studies on the complex of E2CDS and HPCD (E2CDS-CD) indicated that the encapsulated estrogen was approximately four times more stable than the unmanipulated CDS, producing an estimated half-life of degradation of 4 years compared with 1.2 years for the uncomplexed drug at room temperature. The complexation of E2CDS and HPCD also stabilized the dihydronicotinate in solutions containing potassium ferricyanide. This formulation was shown to be equivalent to E2CDS in dimethyl sulfoxide in delivering the oxidized, estradiol precursor (E2Q+) to the brain, and also produced similar biological responses; these included decreased luteinizing hormone (LH) secretion and a decrease in the rate of weight gain in castrated female rats.

A dihydropyridine conjugate which generates high and sustained levels of the corresponding pyridinium salt in the brain does not exhibit neurotoxicity in cynomolgus monkeys.

Many drugs can be selectively delivered to the brain by using a dihydropyridine in equilibrium pyridinium salt chemical delivery system (CDS). The interaction of these systems with central dopaminergic function was examined in this communication. Castrate female Sprague-Dawley rats when treated with a CDS for estradiol (i.e. 3-hydroxy-17 beta-[( (1-methyl-1,4-dihydropyridin-3-yl)carbonyl]oxy) estra-1,3,5(10)-triene or E2CDS) exhibit sustained and profound suppression of serum levels of leuteinizing hormone (LH). Treatment of rats with pargyline (80 mg/kg) prior to E2CDS (2 mg/kg) did not mitigate the biological effectiveness of this estrogen indicating at least indirectly that monoamine oxidate (MAO) was not involved in the CDS activation. In a more direct examination, cynomolgus monkeys treated with various repeated doses of E2CDS (cumulative doses of 0.2-40.0 mg/kg) demonstrated neither impaired motor function nor depleted striatal dopamine concentrations. The latter parameter was measured using liquid chromatographic-electrochemical analysis. These experiments support the contention that the CDS is not neurotoxic and further strengthens the strict structure-activity requirements for MPTP-induced neurotoxicity.

Neuropharmacology of zonisamide, a new antiepileptic drug.

Zonisamide readily crosses the blood-brain barrier and is readily absorbed after oral administration with a Tmax of about 3 hr. The half-life of ZNA in epileptic patients is about 28 hr. Zonisamide has a broader therapeutic range than other antiepileptic drugs. Neurotoxic, hemapoietic, renal, and liver effects have been minimal in patients participating in controlled clinical studies. It is effective in several experimental models of epilepsy and in initial clinical trials has been shown to be effective in generalized tonic-clonic, simple, and complex partial seizures.

Pharmaceutical and clinical analysis by tandem mass spectrometry.

Tandem mass spectrometry (MS/MS) is a promising technique for trace determination of compounds in complex mixtures. The application of triple-quadrupole MS/MS to clinical and pharmacological studies has been investigated with major emphasis on rapid screening and determination of drugs and biomolecules in physiological fluids and tissues. These techniques have been applied to a range of problems, including the determination of chlorinated compounds in humans, subpicogram analysis of neurochemicals and the detection of illegal drugs in racing animals. A new technique for determining the structures of all the metabolites of a particular drug has also been developed. It is possible to identify in a single sample all molecular ions which contain substructures characteristic of the parent drug. The structure of each metabolite can then be determined by obtaining the MS/MS spectrum of the molecular ion.

Twice-daily dosing of valproate with divalproex.

Equivalent doses of enteric-coated divalproex were substituted for valproic acid in 15 epileptic patients who were on a three- or four-times-a-day dosing schedule. After 2 wk the dosing regimen was changed to twice-daily divalproex dosing, and plasma levels were determined during the 12-hr period after the morning dose. Peak absorption was reached at 4 hr; extended plateaus were noted thereafter. The mean fluctuation between low and high plasma values for the group was 46%, with a decrease of less than 50% of peak levels in 9 of the 15 patients at a sampling time just before the second dose. Breakthrough seizures did not occur as a result of twice-daily dosing.

Pharmacokinetics of primidone elimination by uremic patients.

The hemodialyzability of primidone was investigated in four patients on long-term hemodialysis. Primidone, 500 or 250 mg, was given orally 2 hours before hemodialysis. Blood and dialyzate samples were collected periodically during the 4-hour dialysis and measured by gas-liquid chromatography and high-performance liquid chromatography for primidone. Dialysis clearance calculated by the instantaneous dialyzate method averaged 97.7 ml/min, which is considerably greater than the metabolic clearance of 30 ml/min for the drug. The extraction efficiency of the hollow-fiber dialyzers averaged 40.2 pr cent for plasma samples. A mean of 31.7 per cent of the administered dose of primidone was removed during hemodialysis. The half-life was 5.1 hours in our patients during hemodialysis, a nearly two-thirds reduction of the 13.9-hour half-life calculated in uremic patients. Because of the reduction in elimination half-life, greater dialysis clearance than metabolic clearance, high extraction efficiency, and significant drug removal during dialysis, we conclude that primidone is dialyzable.

In vivo uptake of valproic acid into brain.

The pharmacokinetics of valproic acid (VPA) penetration into the central nervous system of cats were studied. VPA levels in cortical gray matter and plasma were measured at timed intervals after rapid intravenous drug infusion. Brain uptake of the drug was maximal at 1 min postinfusion and decayed rapidly with a mean elimination half-life of 41 min. After a rapid distribution phase, plasma VPA levels remained stable for 90 min. The brain:plasma ratio was maximal at 1 min and also declined rapidly. The volume of distribution was 0.125 1/kg. The small volume of distribution, low brain:plasma ratios and rapid clearance from brain indicate that VPA is not significantly bound in cerebral cortex after a single dose.

Hemodialysis clearance of ethosuximide in patients with chronic renal disease.

Clearance of ethosuximide by hemodialysis was studied. Four patients with chronic renal disease supported by hemodialysis were given ethosuximide 500 mg four hours before dialysis. Samples of arterial and venous blood and dialysate were collected before and during the four-hour dialysis procedure. Ethosuximide concentration was measured by gas-liquid chromatography. The extraction efficiency for the dialysis systems used in this study ranged from 61.1 to 100%, and dialysis clearance was from 122.3 to 156.3 ml/min. Recovery of ethosuximide from the dialysate was 38.8 to 52.4% of the administered dose. Hemodialysis reduced the elimination half-life of ethosuximide from a presumable value of 55 hours to an average of 3.5 hours. The authors concluded that ethosuximide is dialyzable. Hemodialysis patients concurrently receiving ethosuximide may require a supplemental dose or an altered ethosuximide dosing schedule. Because hemodialysis quickly clears ethosuximide, it may be useful in treating ethosuximide overdosage.

Interactions of valproic acid with phenytoin.

The interaction of phenytoin and valproic acid was studied in four adults. We studied serial changes in total phenytoin concentrations, protein binding, urinary hydroxyphenylphenylhydantoin (HPPH) excretion, and half-life. In all four patients valproic acid caused an increase in the free fraction of phenytoin. Total phenytoin plasma concentrations decreased transiently in three patients and remained low throughout the study period in one patient. HPPH excretion increased transiently and then decreased, corresponding to changes in total phenytoin plasma concentrations. Biologic half-life transiently decreased in three patients (not statistically significant) and subsequently increased significantly in all four patients. The data suggest that valproic acid displaced phenytoin from protein-binding sites in all four patients and subsequently inhibited phenytoin metabolism in three patients.

Hemodialysis clearance and total body elimination of carbamazepine during chronic hemodialysis.

The effect of the artificial kidney on the removal of carbamazepine was studied in four uremic patients undergoing chronic hemodialysis. Each patient received 500 mg of carbamazepine PO 8 hr prior to hemodialysis. Predialysis and postdialysis blood samples as well as dialysate samples were collected at various time intervals during a 4-hr hemodialysis. Samples were analyzed for carbamazepine content by HPLC. Dialysis clearance calculated from dialysate measurements averaged 53.6 +/- 10.0 mL/min which is double the reported mean plasma clearance of 27.5 mL/min. Since drug clearance by the dialyzer is twice the endogenous plasma clearance, we conclude that carbamazepine is dialyzable. Despite the significant dialysis clearance, a dosage regimen adjustment may not be necessary because of the long elimination half-life of carbamazepine of 35 hr compared to the short length of the usual hemodialysis treatment of 3-5 hr.

Valproic acid and plasma levels of phenobarbital.

During concurrent administration of phenobarbital and valproic acid, phenobarbital plasma concentrations often increase. This often requires a reduction of phenobarbital dosage. In normal cats and patients with epilepsy, we found no evidence of decreased renal excretion of phenobarbital. Metabolic studies in four patients revealed a decrease in the conversion of phenobarbital to hydroxyphenylphenobarbital and decreased urinary ratios of hydroxyphenylphenobarbital to phenobarbital. These data suggest that phenobarbital metabolism is inhibited by therapeutic plasma levels of valproic acid.

Simultaneous determination of the anticonvulsants, cinromide (3-bromo-n-ethylcinnamamide), 3-bromocinnamamide, and carbamazepine in plasma by high-performance liquid chromatography.

A high-performance liquid chromatographic method is described for monitoring plasma concentrations of cinromide (3-bromo-N-ethylcinnamamide) and its de-ethylated metabolite. Carbamazepine levels can be easily measured by the same technique. The N-isopropyl analogue of cinromide is used as internal standard, and all compounds are easily separated on a reversed-phase column operated at 55 degrees with a small-diameter pre-column maintained at the same temperature. The extraction is rapid and generally applicable to plasma and urine samples that are to be analyzed by reversed-phase chromatography. Short- and long-term reproducibility studies show less than 4% relative standard deviation for replicate determinations for all drugs. Limits of quantitation are 10-20 ng/ml with an internal standard concentration of 3 micrograms/ml. Another metabolite of cinromide, 3-bromocinnamic acid, which may have some anticonvulsant effect, can be analyzed simultaneously by buffering the mobile phase and adding an ion-pairing reagent.

Steady-state kinetics of valproic acid in epileptic patients.

Pharmacokinetic evaluation and prediction were carried out in 20 epileptic patients. Using conventional pharmacokinetic techniques and a one-compartment model, predicted and observed valproic acid plasma concentrations were compared. Valproic acid assay was performed by gas-liquid chromatography. There was good agreement between predicted and observed plasma concentrations. Most patients had predicted half-lives (t1/2s) of 6 to 8 hr, independent of the plasma concentration of valproic acid. Five patients had predicted t1/2s of 12 hr. The correlation between dose and plasma level was poor. Most patients had valproic acid plasma levels between 55 and 100 microgram/ml. Administration of valproic acid three times a day with determination of individual plasma concentrations offers a reliable method of monitoring. Constant levels are maintained in individual patients, but there is substantial intersubject variation.

Gas--liquid chromatographic determination of carbamazepine and phenylethylmalonamide in plasma after reaction with dimethylformamide dimethylacetal.

A previously published procedure for the gas chromatographic analysis of carbamazepine has been modified and expanded to allow simultaneous determination of phenylethylmalonamide, a metabolite of primidone. Internal standards that closely resemble each compound are used, and derivatives are made by reaction with dimethylformamide dimethylacetal. This change of internal standard for carbamazepine and the use of a commercial, pretested column-packing material eliminate the major pitfalls of the original method.