Dennis C. Mays

Safety, Tolerance and Pharmacokinetics of Intravenous Doses of the Phosphate Ester of 3‐Hydroxymethyl‐5,5‐Diphenylhydantoin: A New Prodrug of Phenytoin

A new prodrug of phenytoin, the disodium phosphate ester of 3‐hydroxymethyl‐5,5‐diphenylhydantoin (ACC‐9653), was administered intravenously over 30 minutes to four different groups of volunteers at doses of 150, 300, 600, and 1200 mg. The prodrug and phenytoin were measured in plasma samples, collected at specified times, by specific high performance liquid chromatography (HPLC) assays. The prodrug, after achieving a maximum concentration at the end of the 30‐minute infusion (Cmax 20, 36, 75, 129 μg/mL) declined rapidly with a half‐life (t1/2) of about 8 minutes. The area under the plasma concentration‐time curve (10, 19, 43, 77 mg ṁ hr/L) was proportional to dose whereas the total clearance, 14 L/hr, was independent of dose. The volume of distribution of the prodrug, a polar, water‐soluble molecule was about 2.6 L, indicating that most of the dose remained in the plasma. The concentration of phenytoin reached 90% of its maximum about 12 minutes after the end of the infusion of ACC‐9653. At the dose of 1200 mg of prodrug, the average peak concentration of phenytoin was about 17 μg/mL, near the upper limit of the therapeutic range. Adverse reactions (lightheadedness, nystagmus, incoordination) were minor and attributed to phenytoin. No significant abnormalities in ECG, Holter monitoring, or EEG were noted after the infusion of ACC‐9653.

Improved method for the determination of aspirin and its metabolites in biological fluids by high-performance liquid chromatography: applications to human and animal studies

An improved method has been developed for the determination of acetylsalicylic acid, salicylic acid, gentisic acid, and salicyluric acid in plasma and urine of rabbits and man. Samples are extracted with dichloromethane containing mephenytoin as an internal standard, the solvent is evaporated under reduced pressure, the residue reconstituted and analyzed by high-performance liquid chromatography. Extraction efficiencies, linearity and assay precision were determined. This method has been applied to human bioavailability studies and the data are presented.

Hydroxylation of salicylate by activated neutrophils

Salicylates are metabolized in vivo to hydroxylated compounds, including 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid (gentisic acid). The present study hypothesized that activated neutrophils represent one pathway for salicylate hydroxylation. Human neutrophils were incubated in medium containing 10 mM salicylate and stimulated with phorbol myristate acetate (PMA) for 1 hr. The cell-free supernatant fractions were analyzed by HPLC. Neutrophils (1 x 10(6) cells) produced 55 +/- 11 ng of gentisic acid. Neutrophils also produced smaller quantities of 2,3-dihydroxybenzoic acid. Antioxidant inhibitor experiments indicated that superoxide dismutase (SOD), heme protein inhibitors, and glutathione blocked gentisic acid formation, whereas catalase, mannitol, and deferoxamine failed to inhibit. Experiments with the reagent hypochlorous acid (HOCl) and the model myeloperoxidase (MPO) enzyme system did not support a role for the MPO pathway in gentisic acid formation. These findings demonstrate that activated neutrophils can hydroxylate salicylate by an unknown pathway. This pathway may contribute to the increased recovery of hydroxylated salicylates in patients with inflammatory disorders.

Inhibition of elimination of caffeine by disulfiram in normal subjects and recovering alcoholics

The kinetics of caffeine elimination were investigated in 10 normal male subjects and in 11 recovering alcoholics before and during disulfiram dosing. In normal subjects the total body clearance of caffeine declined 30% (142 to 99 ml/min) at the maintenance dose of disulfiram, 250 mg/day, and 29% (161 to 114 ml/min) at the loading dose of 500 mg/day. In recovering alcoholics, the total body clearance decreased from 333 to 253 ml/min, a 24% change. The mean caffeine t½increased 39% and 34% in normal subjects after 250 and 500 mg disulfiram, respectively, and 29% in recovering alcoholics. The inhibition of caffeine elimination was moderate in most subjects. However, the clearance of caffeine decreased by ≥50% after disulfiram in three of the 11 recovering alcoholics. These patients may have an increased risk of cardiovascular and cerebral excitation associated with higher concentrations of caffeine, which could complicate withdrawal from alcohol.

Methoxsalen is a potent inhibitor of the metabolism of caffeine in humans

The acute effect of a single oral dose of methoxsalen on the pharmacokinetics of caffeine was investigated in five nonsmoking volunteers with psoriasis. Caffeine, 200 mg orally, was administered to each subject at baseline before treatment with methoxsalen. One week later each subject was given a single oral dose of 1.2 mg/kg methoxsalen 1 hour before administration of another oral dose of 200 mg caffeine. The clearance of caffeine declined markedly from 110 ± 17 ml/min (mean ± SE) in the control study to 34 ± 5 ml/min after methoxsalen. During the period of maximum inhibition the mean elimination half‐life of caffeine increased from 5.6 hours at baseline to 57 hours after administration of methoxsalen. The peak concentration of caffeine and the time to reach the peak concentration of caffeine were not affected by pretreatment with methoxsalen. Thus, methoxsalen, administered acutely, is a potent inhibitor of caffeine metabolism in humans with psoriasis. Results of this investigation suggest that the elimination of concurrently administered drugs may be inhibited in patients receiving methoxsalen. In comparison with other drugs, methoxsalen is the most potent inhibitor of drug metabolism in humans. Other work has shown that inhibition of drug metabolism by methoxsalen is associated with both extensive covalent binding of metabolite(s) of methoxsalen to liver microsomal protein in vitro and in vivo and inactivation of cytochrome P‐450.

Activation of 8-methoxypsoralen by cytochrome P-450. Enzyme kinetics of covalent binding and influence of inhibitors and inducers of drug metabolism.

The kinetics of covalent binding of reactive metabolites of 8-methoxypsoralen (8-MOP) to protein were measured in incubations of liver microsomes of rats pretreated for 3 days with i.p. injections of 80 mg/kg/day of beta-naphthoflavone (BNF), phenobarbital (PB), 8-MOP, or vehicle. Covalent binding of radioactivity derived from [14C]8-MOP (labeled at the metabolically stable 4-position in the coumarin ring) required NADPH, obeyed classical Michaelis-Menten kinetics, and was inducible by both PB and BNF. Plots of V versus V/[S] were linear in liver microsomes of rats pretreated with vehicle, PB, or 8-MOP; respective values for Km were 26, 24 and 13 microM and for Vmax were 0.61, 1.70 and 0.50 nmol bound/min/mg protein. In microsomes of rats pretreated with BNF, high- and low-affinity components of covalent binding were observed with respective values for Km of 4.7 and 117 microM and for Vmax of 0.77 and 1.71 nmol bound/min/mg protein. Addition of glutathione and cysteine to the incubations decreased covalent binding by 33 and 67%, respectively, presumably by trapping reactive electrophilic metabolites. Inhibition of epoxide hydrolase with 1,1,1-trichloropropene-2,3-oxide did not affect covalent binding of reactive metabolites of 8-MOP. SKF-525A was a potent inhibitor of both the metabolism of 8-MOP and covalent binding in microsomes from rats pretreated with PB, but had only a slight effect in microsomes from rats pretreated with BNF. In contrast, alpha-naphthoflavone almost completely inhibited metabolism of 8-MOP and covalent binding in BNF-induced microsomes but had no effect in PB-induced microsomes. Apparent covalent binding was reduced by 39% in incubations with 8-MOP labeled with tritium in the metabolically labile methoxy group. Collectively, these results indicate that 8-MOP is biotransformed by two or more isozymes of cytochrome P-450 to reactive electrophiles capable of binding to tissue macromolecules.

Metabolism of phenytoin and covalent binding of reactive intermediates in activated human neutrophils

ontivation of neutrophils by phorbol-12-myristate-13-acetate (PMA) causes rapid production of superoxide radical (O2-), leading to the formation of additional reactive oxygen species, including hydrogen peroxide (H2O2), hypochlorous acid (HOCl), and possibly hydroxyl radical (.OH). These reactive oxygen species have been associated with the oxidation of some drugs. We investigated the metabolism of phenytoin (5,5-diphenylhydantoin) and the covalent binding of reactive intermediates to cellular macromolecules in activated neutrophils. In incubations with 100 microM phenytoin, PMA-stimulated neutrophils from six human subjects produced p-, m-, and o-isomers of 5-(hydroxyphenyl)-5-phenylhydantoin (HPPH) in a ratio of 1.0:2.1:2.8, respectively, as well as unidentified polar products. Analysis of cell pellets demonstrated that phenytoin was bioactivated to reactive intermediates that bound irreversibly to macromolecules in neutrophils. Glutathione, catalase, superoxide dismutase, azide, and indomethacin all diminished the metabolism of phenytoin and the covalent binding of its reactive intermediates. The iron-inactivating chelators desferrioxamine and diethylenetriaminepentaacetic acid had little or no effect on the metabolism of phenytoin by neutrophils, demonstrating that adventitious iron was not contributing via Fenton chemistry. In an .OH-generating system containing H2O2 and Fe2+ chelated with ADP, phenytoin was oxidized rapidly to unidentified polar products and to p-, m-, and o-HPPH (ratio 1.0:1.7:1.5, respectively). Reagent HOCl and human myeloperoxidase (MPO), in the presence of Cl- and H2O2, both formed the reactive dichlorophenytoin but no HPPH. However, no chlorinated phenytoin was detected in activated neutrophils, possibly because of its high reactivity. These findings, which demonstrated that activated neutrophils biotransform phenytoin in vitro to hydroxylated products and reactive intermediates that bind irreversibly to tissue macromolecules, are consistent with phenytoin hydroxylation by .OH generated by a transition metal-independent process, chlorination by HOCl generated by MPO, and possibly cooxidation by neutrophil hydroperoxidases. Neutrophils activated in vivo may similarly convert phenytoin to reactive intermediates, which could contribute to some of the previously unexplained adverse effects of the drug.

Inhibition and induction of drug metabolism by psoralens: Alterations in duration of sleep induced by hexobarbital and in clearance of caffeine and hexobarbital in mice

1. Hexobarbital (100 mg/kg i.p.) sleeping times in male CD-1 mice pretreated (-1 h) with a single i.p. injection of 150 mumol/kg of psoralen or coumarin analogues were increased, most markedly (6-fold) by linear, methoxy-substituted psoralens. 2. Hexobarbital sleeping times of mice which received three daily injections (231 mumol/kg; 50 mg/kg) of 8-methoxypsoralen (8-MOP) were 44% of controls (corn oil). 3. The whole-body half-life of caffeine (1 mg) in mice was 10.2, 1.2, and 0.37 h following 8-MOP (50 mg/kg per day) x 1, vehicle, and 8-MOP x 3 respectively. 4. The whole-body concentrations of hexobarbital (100 mg/kg dose) in mice 30 min after dosing were 14.3 +/- 0.9, 8.4 +/- 0.3, and 5.2 +/- 0.5 micrograms/ml (1 mouse = 150 ml) following 8-MOP (50 mg/kg per day) x 1, vehicle, and 8-MOP x 3 respectively. 5. It is concluded that, administered acutely, psoralen analogues inhibit hexobarbital metabolism in mice; and 8-MOP administered acutely inhibits the metabolism of caffeine and hexobarbital, but administered repeatedly increases their metabolism.

AMINOBENZOIC ACID COMPOUNDS AS HOCL TRAPS FOR ACTIVATED NEUTROPHILS

This study was designed to develop traps for hypochlorous acid (HOCl) which could be used to detect HOCl in the microenvironment of activated neutrophils. Reagent HOCl was found to react with para-aminobenzoic acid (PABA) in aqueous solution to produce a predominant metabolite detectable by high performance liquid chromatography (HPLC). Mass spectroscopy and nuclear magnetic resonance identified this metabolite as the ring addition product 3-chloro PABA. The related compound para-aminosalicylic acid (PAS) was also metabolized by HOCl to 3-chloro PAS. The formation of the 3-chloro metabolite was specific for reactions involving HOCl, since several other oxidants in chloride buffer failed to produce the metabolite. Human blood neutrophils activated by phorbol myristate acetate or zymosan in the presence of PABA (or PAS) used their HOCl to produce large amounts of the 3-chloro metabolite. The formation of 3-chloro PABA was inhibited by azide, catalase, and taurine, which is consistent with the production of the metabolite by the neutrophil myeloperoxidase (MPO) pathway. The reaction of HOCl with PABA and PAS was relatively slow as shown by competitive reactions with endogenous antioxidants like taurine, methionine, and glutathione. This was confirmed in reactions involving PABA/PAS and reagent HOCl or HOCl generated by the MPO enzyme system. In these in vitro systems, glutathione and serum completely inhibited the formation of the 3-chloro metabolite. In contrast, activated neutrophils metabolized PABA/PAS to the 3-chloro metabolite even in the presence of 1% serum. These findings demonstrate that PABA and PAS are specific trapping agents for HOCl produced by neutrophils in complex biological conditions.

The effects of gallium nitrate on osteopenia induced by ovariectomy and a low-calcium diet in rats.

The effects of gallium nitrate (GN) were evaluated on osteopenia induced by ovariectomy (OVX) and a low-calcium diet (LCD) in Sprague-Dawley rats. Twenty-five rats (300-400 g) were randomized into four groups of 5-7 animals: (I) OVX LCD treated with GN for 22 weeks; (II) OVX LCD treated with GN for 10 weeks; (III) OVX LCD treated with saline; and (IV) sham-operated (SO), normal diet, treated with saline. GN-treated rats received a 30-mg/kg subcutaneous single dose of elemental gallium, followed by 10 mg/kg per week, whereas control animals received an equal volume of saline. All animals were euthanized at 22 weeks. Measurements of bone density and histomorphometry, performed on the proximal portion of the tibia, indicated significant bone loss in all OVX LCD animals. GN-treated rats in group I gained significantly less weight than those in the other groups, and their blood urea nitrogen increased, suggesting a nephrotoxic effect. After discontinuation of GN, rats in group II gained weight at the same rate as those which had received only saline. Bone formation rates in the GN-treated rats were double those of the saline-treated OVX animals and more than 10 times those of SO controls. Although the bone formation rate in GN-treated rats increased, GN had no effect in preventing the loss of bone surface, density and volume induced by OVX LCD. These findings suggest that although GN may enhance osteoblastic activity, this agent alone does not appear effective in the prevention of bone loss induced by OVX LCD.