Jordan L. Holtzman

The role of catalytic superoxide formation in the O2 inhibition of nitroreductase.

Abstract Many nitroreductases are strongly inhibited by oxygen. The first intermediate of nitroreductase activity, the nitroaromatic anion free radical, cannot be detected in aerobic microsomal incubations. Even though the nitro compounds are unchanged, both nitrofurantoin and p -nitrobenzoate profoundly increase the NADPH-supported oxygen uptake. This catalytic oxygen consumption is partially reversed by superoxide dismutase, suggesting that superoxide anion free radical is being formed by the rapid air oxidation of the nitroaromatic anion radical.

The pharmacokinetics of daunomycin in man

Through the use of fluorescence assay methods, the plasma levels, urinary excretion, and tissue levels of daunomycin and its fluorescent metabolites were determined in patients treated with this drug for disseminated solid malignancies. The patients received daunomycin in a single rapid intravenous dose at 80 mg. per square meter and 120 mg. per square meter. Fluorescence studies showed the short plasma half‐life to be 0.75 hours, the long plasma half‐life to be 55 hours, the apparent volume of distribution to be ab out 1,000 L., and the relative volume of distribution to be 580 L. per square meter. Drug and fluorescent metabolites in urine averaged 13 to 14 per cent of the total dosage in 7 days. Tritiated daunomycin was used, but isotopic assay produced values different from those with fluorescence assay. These were interpreted to be the result of tritium exchange or metabolism of the daunomycin. Daunomycin fluorescence was detected in autopsy specimens 16 and 19 hours after administration, with the highest levels in kidney, spleen, liver, and lung. The fluorescent species extracted from both plasma and urine consisted predominantly of daunomycin (D1) and daunomycin metabolite (D2), although small amounts of the aglycones were detected. It is proposed that, because of the long half‐life of daunomycin, the current treatment schedule be revised to a single intravenous dose.

Ethanol induction of acetaminophen toxicity and metabolism.

Chronic consumption of moderate amounts of ethanol (10% solution in the drinking water) dramatically enhances the toxicity of acetaminophen in CF-1 mice as demonstrated by a significant decrease in the LD50. The increased toxicity appears to result from an acceleration in the microsomal biotransformation of acetaminophen to a reactive intermediate. Accelerated acetaminophen metabolism in ethanol-treated animals, in turn, causes more rapid depletion of glutathione. These studies suggest a mechanism to account for the toxic synergism between chronic alcohol consumption and acute acetaminophen intake.

Interaction of flecainide with digoxin and propranolol

The effect of flecainide, 200 mg twice daily for 5 days, on steady-state plasma digoxin levels was determined in 15 healthy male subjects who received 0.25 mg of digoxin per day. The predose mean plasma digoxin level before flecainide administration on days 9 and 10 was 0.46 ng/ml, compared with 0.57 ng/ml on day 13 (p less than 0.05) and 0.49 ng/ml on day 15 (difference not significant [NS]) when flecainide was given concurrently with digoxin. The 6-hour postdose mean level for days 9 and 10 was 0.58 ng/ml, compared with mean levels of 0.62 ng/ml on day 13 (NS) and 0.65 ng/ml on day 15 (p less than 0.05). On average, predose and 6-hour postdose digoxin levels increased by 24 +/- 35% and 13 +/- 19%, respectively, during co-administration. A significant prolongation of the electrocardiographic PR interval in 6 of 15 subjects was noted on the combined drug dosage. This reverted to normal after cessation of drug administration. Vital signs showed no significant clinical change during the course of the study. Ten other healthy male subjects were given propranolol, 80 mg 3 times daily, or flecainide, 200 mg twice daily, alone or in combination. Effects on vital signs, exercise heart rate, electrocardiographic interval, M-mode indexes of ventricular function and plasma drug levels were monitored to determine effects of the study drugs when given separately or concurrently. Both drugs caused a decrease in blood pressure, with the systolic pressure affected more than the diastolic; the effects of propranolol and flecainide were additive.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetic studies on the metabolism of ethylmorphine by isolated hepatocytes from adult rats

Hepatocytes of adult rats were isolated by infusion of a hyaluronidase collagenase mixture. High yields of cells excluding trypan blue were obtained. These cells, in Hanks buffer containing rat serum and 0.1% glucose, N-demethylate [3H-CH3-N]ethylmorphine. The formaldehyde initially formed is further metabolized to tritiated water. Fifteen per cent of the original metabolic activity was observed after 21 hr at 37° in 5% CO2-air, and cumulative metabolism is linear for up to 90 min under these conditions. The Km for the N-demethylation of [3H-CH3-N]ethylmorphine is 50 μM, 20 per cent of the value observed for this reaction by musomal preparations. An active transport of the substrate into the cell is postulated to account for this difference.

Effect of influenza vaccine on warfarin anticoagulation

Recent studies have indicated that viral infections, influenza vaccination, or drugs that increase interferon synthesis all decrease hepatic drug metabolism. We report a case in which influenza vaccination was temporally related to an increased anticoagulant effect of warfarin. A prospective study evaluating the effect of influenza vaccination on the prothrombin time of eight patients anticoagulated over the long term showed that there was prolongation of prothrombin time of 40%. In a second study, the effect of influenza vaccination on warfarin t½ was determined in healthy subjects. No significant effect on warfarin metabolism was observed after vaccination. We conclude that influenza vaccination is associated with increased anticoagulant response in some patients receiving anticoagulants over a long term. This effect appears to be related to some step in the coagulation pathway and not to decreased warfarin metabolism and a subsequent rise in serum concentration.

Studies on the effect of chronic consumption of moderate amounts of ethanol on male rat hepatic microsomal drug-metabolizing activity☆

Weanling, male Sprague-Dawley rats given 10% ethanol in the drinking water and food ad lib. for up to 8 weeks consumed 17% of their calories as ethanol. The alanine aminotransferase (ALT), aspartate aminotransferase (AST), and liver histology by light microscopy were unaffected by this treatment. Similarly, hepatic microsomal NADPH-cytochrome c reductase, ethylmorphine N-demethylase and benzphetamine N-demethylase activities were also not affected by ethanol consumption. On the other hand, cytochrome P-450 content, aniline hydroxylase activity and acetaminophen metabolism as measured by both the cysteine conjugate and the [3H]acetaminophen covalently-bound to microsomal protein were increased significantly by ethanol consumption. The maximal effect was seen by 6 weeks. The 2- to 3-fold increase in aniline and acetaminophen metabolism, the absence of liver damage, and the similarity in weight gains and caloric intakes for controls and treated animals suggest that the rat on 10% ethanol in the drinking water is a reasonable model for studies of the effect of moderate alcohol consumption on specific biochemical pathways.

The formation of an azo anion free radical metabolite during the microsomal azo reduction of sulfonazo III

Abstract An ESR spectrum is observed during the anaerobic incubation of the diazonaphthol dye sulfonazo III, with rat hepatic microsomes and NADPH. This spectrum is characterized by a partially resolved 17-line hyperfine pattern and g = 2.0043, as is consistent with the spectrum of an azo anion free radical, [R-N-N-R′] • . Oxygen, which strongly inhibits microsomal azoreductase, destroys the ESR signal. The oxidation of the azo anion radical metabolite by oxygen to the parent azo dye may account for the oxygen inhibition of microsomal azoreductase.

The temperature dependence of the components of the hepatic microsomal mixed-function oxidases.

Abstract The activation energies of ethylmorphine N -demethylase (16.8 kcal/mole) and aniline hydroxylase (17.6 kcal/mole) did not greatly differ. The endogenous NADPH oxidase (16.8 kcal/mole) and the oxidase in the presence of ethylmorphine (15.7 kcal/mole) and aniline (14.7 kcal/mole) are only slightly different. On the other hand, the activation energy for NADPH-cytochrome c reductase is only 8.53 kcal/mole, suggesting that the reduction of the flavin in this enzyme does not significantly contribute to the rate of the overall reaction. The addition of ethylmorphine reduces the NADPH-cytochrome P-450 reductase activation energy from 12.5 kcal/mole to 10.8 kcal/mole. This energy is slightly increased by the presence of aniline (14.7 kcal/mole). Since NADPH-cytochrome P-450 reductase in the presence of ethylmorphine is presumed to be the rate-limiting step in the N -demethylase, the large difference between the energy of activation of this reaction and the energy of activation of ethylmorphine N -demethylase would suggest the participation of two reactions of similar rate in the control of the overall rate of the oxygenase reaction.

Metabolism of phenytoin by the gingiva of normal humans: The possible role of reactive metabolities of phenytoin in the initiation of gingival hyperplasia

Gingival hyperplasia is a well‐known complication of therapy with cyclosporine, calcium channel blockers, and phenytoin. It is characterized by the presence of inflammation and a marked fibrotic response. The mechanism of this adverse reaction is unknown. We propose that it may be initiated by the metabolic activation of these drugs to form reactive metabolites. These then cause cellular injury and lead to the gingival hyperplasia. To evaluate this hypothesis we examined phenytoin metabolism and the cytochrome P450 contents of gingival tissues from 10 patients undergoing surgery for various periodontal conditions. We found that microsomes obtained from the gingiva show significant phenytoin hydroxylase activity as determined by the production of 5‐(4′‐hydroxyphenyl)‐5‐phenylhydantoin (HPPH) (range, 12.8 pmol HPPH/min · mg microsomal protein to 276.9 pmol HPPH/min · mg microsomal protein; rat control, 133.7 ± 11.5 pmol HPPH/min · mg microsomal protein). We also found that CYP1A1, CYP1A2, CYP2C9, CYP2E1, and CYP3A4 were present in these microsomes. We detected no CYP2B6 or CYP2D6. We believe that these data support our hypothesis that the proliferative inflammation observed with drugs such as phenytoin, nifedipine, and cyclosporine may be initiated by the formation of reactive metabolites and that the formation of these metabolites may be catalyzed by one or more CYPs found in the gingiva. These metabolites may then cause cellular injury and induce a reactive inflammatory response, followed by fibroblastic proliferation. This proliferation leads to the excess collagen deposition observed with gingival hyperplasia.