Early adventures in drug metabolism: 4. Diphenylhydantoin (phenytoin).

This series comes to a close with a discussion of problems related to phenytoin metabolites, their discovery and role in the toxicity of this drug. More particularly, it looks at the influence of the p-hydroxyphenyl metabolite of phenytoin (HPPH) on the metabolic disposition of the parent compound, and the relationship of the dihydrodiol metabolite to the putative presence of arene oxides. The question of pro-drugs is examined, including factors that may influence any decision to market the drug. The role of organizational committees and the good that can be accomplished outside the confines of the laboratory are also discussed.

Early adventures in drug metabolism. 3. Chloramphenicol.

The saga of our early experiences with drug metabolism studies in the Parke-Davis Research Laboratories continues with a brief resume of the behavior of chloramphenicol under unusual clinical conditions and a consideration of the toxic effects of this drug and its metabolites in laboratory animals and in humans. The dose-related reversible effects of chloramphenicol upon the ferrokinetic pattern and cellular respiration are clearly separated from its nonreversible effects on the bone marrow cells of susceptible individuals. A number of possible research approaches are suggested for the exploration of these problems.

Early adventures in drug metabolism: 2. The absorption of drugs.

This paper deals with our experiences in drug metabolism in the Parke-Davis Research Laboratories at a time when good analytical procedures were just starting to make their appearance and pharmacokinetics was an undiscovered territory. Some of our groping efforts to understand the factors influencing absorption are described. These included formulation differences in generic products and their effect upon absorption. Compounds such as p-aminosalicylic acid, chloramphenicol, phenytoin and acedapsone are used as examples. Our intent is to show how our knowledge of drug metabolism developed during this period, and to describe some areas that still need additional investigation.

Early adventures in drug metabolism: 1. Role of the Bratton-Marshall reagent.

The Bratton-Marshall reagent is one of the real land-marks in the development of drug metabolism and pharmacokinetics, coming at a time when highly sensitive and specific analytical procedures were desperately needed for the measurement of drug concentrations in the body. Examples of its applications are taken from early work in the mid-40's and 50's in the Parke-Davis Research Laboratories, extending from primary aromatic amines (e.g., sulfonamides), to p-nitrophenyl compounds that must first be reduced to amines (e.g., chloramphenicol), and to phenyl derivatives that must be nitrated on a microgram scale and then reduced to aryl amines (e.g., phenytoin). The development and use of separation techniques such as liquid/liquid counter-current partition and paper chromatography is described. Emphasis is placed upon continued, progressive improvement in the basic assay procedures over long periods of time.

Discovery of phenytoin.

Numerous letters and reports located in the Parke, Davis and Smithsonian files add to the story of Merritt's and Putnam's discovery of the anticonvulsant (AC) properties of phenytoin. The major events preceding this work were the fortuitous discovery of phenobarbital as an AC agent, structure/hypnotic activity studies with barbiturates and hydantoins in the early 1920s by A. W. Dox in the Parke, Davis laboratories, and the development of AC assay techniques in animals, by a number of laboratories. Phenytoin was the first item on the list of compounds sent to Putnam by Dox and W. G. Bywater in April 1936. It was found to have AC properties in animals late in 1936, but no public reports were issued until the following year. Clinical efficacy was established in 1937, but no public reports were issued until 1938. Dilantin sodium capsules were prepared by Parke, Davis & Co. and were ready for marketing the same year.

Bioavailability of calcium valproate in normal men compared with the free acid and sodium salt.

Calcium valproate has been formulated into tablets containing the equivalent of 250 mg valproic acid (VPA). The bioequivalence of this preparation (Valontin, Parke-Davis) has been studied in 12 normal adult males in parallel with other products containing the free acid (Depakene capsules, Abbott) and the sodium salt (Depakene syrup, Abbott). Each subject received a single 500-mg oral dose of each product at 2-week intervals in a randomized three-way crossover study. Assays were carried out for VPA in blood and urine specimens which were collected over extended time periods. Peak plasma levels were attained on the average within 30 min with the syrup, 1.13 h with the capsules, and 1.38 h with the tablets. There were no significant differences in the peak plasma levels attained with the three products, or in the plasma half-lives of VPA and areas under the time-concentration curves. The plasma level curves appeared to be biexponential, with terminal half-lives that averaged 16.6 h. One subject showed a remarkably long half-life (28-38 h) after each of the three doses, indicating the possibility of genetic differences between individuals in the disposition of VPA. The urinary excretion of VPA in most subjects ran parallel to the plasma levels and could not be detected 96 h after dosing; however, the subject with the long plasma half-life continued to excrete VPA in his urine for at least 2 additional days. The mean recovery of VPA in 6-day urine represented 12-14% of the dose and ranged from 5.5 to 27.9% in different individuals. There were no significant differences between the three formulations in the pattern of urinary excretion.

Phenytoin metabolism in subjects with long and short plasma half-lives.

Normal adult men with long and short phenytoin plasma half-lives were given 300-mg oral doses of phenytoin once daily for 15 days. Plasma levels of phenytoin (DPH) and its major metabolite (p-HPPH) were measured during the period of drug administration and for 5 days thereafter. Average steady-state plasma levels of DPH rose to 13.4 micrograms/ml in the long half-life group, compared with 3.6 micrograms/ml in the short half-life group. HPPH levels in the long half-life group were about one half of those observed in the short half-life group. The DPH/HPPH ratios in plasma specimens showed excellent correlation with the plasma half-lives of DPH and average steady-state levels, suggesting that this ratio could provide guidance in the selection of optimum dosage regimens for problem patients.

Bioavailability of norethindrone in human subjects.

A competitive protein binding assay for norethindrone was developed to measure plasma levels in human subjects. The plasma levels were considerably higher in women than in men, especially at low dose levels. The plasma levels were directly related to the dose in men; but greater variations in the plasma levels were observed in women. The plasma half-life was about 5 h in both sexes with single oral doses of 5 to 20 mg. A comparative bioavailability study with norethindrone from 2 different manufacturers, formulated in the same manner, showed no significant differences in absorption characteristics and provided sufficient data for pharmacokinetic analysis.

Simplified fluorometric assay for diphenylhydantoin in plasma.

The fluorometric benzophenone procedure for diphenylhydantoin has been simplified by eliminating the need for preliminary extraction of plasma with an organic solvent. Assays are done directly on 0.2 ml of plasma by treating it with alkaline permanganate to form benzophenone, extracting the benzophenone with heptane, and then shaking the heptane layer with sulfuric acid and measuring the fluorescence of the acid layer. The assay is highly reproducible, and adequately sensitive to detect 1 mug of the drug per milliliter of plasma. Fluorometric and gas-liquid chromatographic assays of 154 plasmas gave results that were not significantly different, even in the presence of phenobarbital, other barbiturates and anticonvulsant drugs, or of various tranquilizers and other commonly used drugs. The assay is rapid, the unit cost per assay low.

Effect of an adenosine deaminase inhibitor on the uptake and metabolism of arabinosyl adenine (Vidarabine) by intact human erythrocytes.

Tritium-labeled vidarabine was incubated with fresh citrated human blood in the absence and presence of an adenosine deaminase inhibitor, co-vidarabine was rapidly deaminated to form ara-Hx with minimal incorporation into the erythrocytes. Ara-HxMP was identified as the major component in the erythrocytic nucleotide pool, together with small amounts of IMP, adenosine nucleotides and traces of arabinosyl nucleotides. Addition of the inhibitor completely protected vidarabine from enzymatic deamination and resulted in much greater accumulation of vidarabine 5'-mono-, di-, and triphosphates in the erythrocytes.

The comparative metabolism of zolazepam in rat, dog and monkey.

The metabolic disposition.of zolazepam, a pyrazolodiazepinone, was studied in male and female Spartan rats, Beagle dogs and Rhesus monkeys. Six principal urinary metabolites were characterized by gas chromatography and combined gas chromatography-mass spectrometry. The major metabolite in male and female rats was produced by N-demethylation and hydroxylation. In addition, female rats, but not the male demethylated zolazepam at the 1-position. Male and female dogs also demethylated zolazepam in the 1-position and hydroxylated in a position other than C-6, producing a metabolite peculiar to the dog. In marked contrast, the major metabolite in the monkey involved demethylation without subsequent hydroxylation.