Bing K. Tang

Differences in Metabolism of Sulfonamides Predisposing to Idiosyncratic Toxicity

Individual differences in metabolism of the sulfonamides may predispose patients to idiosyncratic reactions. Sulfonamides are metabolized by N-acetylation (mediated by a genetically polymorphic enzyme) and oxidation to potentially toxic metabolites. We examined 6 patients who had severe reactions to sulfonamides and compared them with 20 controls. Acetylator phenotype was determined with caffeine, a safe in-vivo probe of enzyme activity. All 6 patients were slow acetylators (expected, 55%; p less than 0.05). Detoxification of oxidative metabolites was studied in vitro with a lymphocyte assay evaluating cell death from metabolites generated by a murine hepatic microsomal system. Cells from each patient showed increased toxicity from sulfonamide metabolites but not from the drugs themselves. Cells from parents of 3 patients had intermediate toxicity from sulfonamide metabolites, whereas cells from a sibling of 1 patient had a normal response. Susceptibility to sulfonamide reactions may be due to interaction of metabolic pathways, possibly under genetic control, regulating N-acetylation and specific detoxification of toxic metabolites of the drugs.

Biotransformation of caffeine, paraxanthine, theobromine and theophylline by cDNA-expressed human CYP1A2 and CYP2E1.

Six human cytochrome P450s expressed in HepG2 cells using vaccinia virus cDNA-directed expression, were used to study the biotransformation of caffeine and its metabolites. CYP1A2 alone was responsible for caffeine 3-demethylation and paraxanthine 7-demethylation; in addition, 1A2 catalysed virtually all reactions related to caffeine and its metabolites. The metabolic profile of caffeine biotransformation by CYP1A2 averaged 81.5% for paraxanthine, 10.8% for theobromine and 5.4% for theophylline formation. It remained quite uniform when caffeine concentrations were varied. The most striking finding was that CYP2E1 (the ethanol-inducible form) had major influences upon caffeine metabolism: in particular, it catalysed the formation of theophylline and theobromine from caffeine. Thus, the in vivo metabolite profiling of caffeine may reveal CYP2E1 activities in addition to the previously documented activities of CYP1A2, polymorphic N-acetyltransferase and xanthine oxidase.

Variability in caffeine metabolism

Urinary metabolites excreted after oral caffeine were quantified in a healthy sample (n = 68) from the Toronto population by HPLC analyses. The profile of metabolites, assessed by examining particular metabolite ratios, was found to differ widely among subjects. Ratios denoting cytochrome P‐450–dependent activities were shown to be interethnically variable between Oriental and Caucasian groups, whereas those indicative of xanthine oxidase activity exhibited neither significant interindividual variation nor an ethnic difference. It was also shown that a ratio providing an index of polymorphic N‐acetyltransferase activity holds promise as a simple marker for acetylator status in man.

Hydrolysis of cocaine in human plasma by cholinesterase

Abstract Hydrolysis of cocaine to ecgonine methyl ester in human plasma is mediated by cholinesterase. Cocaine hydrolysis by plasma is blocked by DFP and eserine and partially inhibited by fluoride. Highly purified cholinesterase from human plasma when diluted to the same benzoylcholine hydrolyzing activity as human plasma, shows the same rate of cocaine hydrolysis as human plasma. There was no detectable enzymatic conversion of cocaine to benzoyl ecgonine in plasma.

Polymorphic N-acetylation of a caffeine metabolite

In the course of investigations into variability in the metabolism of caffeine in human populations, urinary levels of 5‐acetylamino‐6‐formylamino‐3‐methyluracil (AFMU), a newly discovered ring‐opened metabolite of caffeine, were found to be both bimodally distributed and interethnically variable in samples (Caucasian: n = 42; Oriental: n = 26) from the Toronto population. To test the premise that the polymorphic N‐acetyltransferase enzyme (E.C.2.3.1.5) could be responsible for the production of AFMU, 20 of the subjects were phenotyped for acetylator status using sulfamethazine (SMZ). Concordance for all subjects between AFMU production and SMZ acetylation strongly suggests that the acetylation polymorphism is involved in the formation of AFMU in man.

Biotransformation of caffeine by microsomes from human liver: Kinetics and inhibition studies☆

The nature of the cytochrome P-450-dependent enzyme reactions giving rise to four primary metabolites of caffeine was investigated using microsomes isolated from livers of human kidney donors. Metabolite formation proceeded at a lower rate than that predicted from in vivo caffeine elimination half-lives, as has been observed in other species using this compound as a substrate in microsomal incubations. Kinetic experiments indicated that the formation of each of the N-demethylated metabolites paraxanthine, theobromine and theophyline was mediated by both a high- and a low-affinity catalytic site over a substrate concentration range from 0.05 mM to 80.0 mM, although only the high-affinity component is likely to be of any importance at normally encountered in vivo caffeine concentrations. 7-Ethoxyresorufin and acetanilide, selective substrates for two polycyclic aromatic hydrocarbon (PAH)-inducible isozymes of cytochrome P-450 in the mouse (P1-450 and P3-450, respectively) were each able to inhibit competitively the formation of caffeine metabolites by human liver microsomes, while caffeine could in turn similarly inhibit the biotransformations of these two compounds. The isozyme-selective P-450 inhibitor alpha-naphthoflavone (ANF) potently inhibited the high-affinity component of caffeine N-demethylations, while 1-phenylimidazole (PI) was a more potent inhibitor of the low-affinity component. The inhibition studies also indicated that the formation of 1,3,7-trimethyluric acid was mediated by both ANF-sensitive and PI-sensitive sites. Taken together, the data support suggestions from in vivo studies that a PAH-inducible isozyme of cytochrome P-450 plays a significant role in the biotransformation of caffeine in man.

Distinction of CYP1A1 and CYP1A2 activity by selective inhibition using fluvoxamine and isosafrole

Ethoxyresorufin O-deethylation (EROD) has been used as a specific probe for CYP1A1 and CYP1A2. Selective inhibition of one of these cytochromes P450 may differentiate their activity in human liver. Four inhibitors were chosen to examine the selective inhibition of EROD activity, using cDNA of CYP1A1 and CYP1A2. The two flavones, alpha-naphthoflavone and apigenin, while differing in potency, inhibited expressed human CYP1A1, CYP1A2, and human liver microsomes to a similar extent. Isosafrole and fluvoxamine were found to inhibit CYP1A2 selectively, with Ki values of 14 and 800 times, respectively, lower than those for CY1A1. A set of equations was developed to estimate both CYP1A1 and CYP1A2 activity. Levels of CYP1A2 in four human liver specimens ranged from 44.4 to 76.7 pmol/mg protein, which significantly correlated with phenacetin O-deethylase activity (r = 0.99; P < 0.001). Low levels of CYP1A1 activity were present in all four investigated livers, ranging from 0.4 to 2.7 pmol/mg protein.

Interindividual variability in the glucuronidation of (S) oxazepam contrasted with that of (R) oxazepam.

Although conjugation with glucuronic acid is a major process for converting many xenobiotics into hydrophilic, excretable metabolites, relatively little has been reported concerning interindividual variability of glucuronidation in human populations. Oxazepam, a therapeutically active metabolite of diazepam, is one of a number of C3-hydroxylated benzodiazepines for which glucuronide conjugation is the predominant pathway of biotransformation. The drug is normally formulated as a racemic mixture of inactive (R) and active (S) enantiomers. In the present study we have investigated the use of oxazepam as a potential probe drug for studying the variability of glucuronide conjugation, and for demonstrating the extent to which genetic factors may be responsible. In preliminary studies we determined oxazepam pharmacokinetics metabolite profiles after administration of racemic (R,S) oxazepam to eleven human volunteers. The (S) glucuronide was preferentially formed and excreted in nine of the eleven subjects. The ratios of (S) to (R) glucuronide metabolites (S/R ratios) were 3.87 +/- 0.79 (mean +/- SD) and 3.52 +/- 0.60 in urine and plasma, respectively. However, both ratios were significantly lower in two subjects (p < 0.01). In these two atypical subjects, the half-life of (R,S) oxazepam was also markedly longer (14.7 and 15.9 h) than in the other subjects (8.1 +/- 3.2 h). A good correlation (rs = 0.90) between the S/R-glucuronide ratio in urine and the plasma clearance of (R,S) oxazepam suggested that a low S/R ratio may be a marker of poor elimination of oxazepam. In further investigations, the drug was administered to 66 additional subjects. The S/R-glucuronide ratio in 8 h pooled urine was bimodally distributed, with 10% of all subjects possessing ratios below an apparent antimode of 1.9. A survey of the in vitro formation of oxazepam glucuronides by microsomes from 37 human livers also showed that 10% of the livers displayed an abnormally high apparent Michaelis constant (Km) for the formation of the (S) glucuronide, but not of the (R) glucuronide. These results suggest that the glucuronidation of the pharmacologically active (S) enantiomer of oxazepam is decreased in a significant percentage (10%) of Caucasian individuals. The observed in vitro differences in apparent kinetics of the S-glucuronidation reaction may reflect defects at the genetic level, leading to structural changes in the isozyme(s) of UDP-glucuronyltransferase that catalyse this reaction.

Induction of P-450 in workers exposed to dioxin.

OBJECTIVES--To examine the effects of occupational exposure to substances contaminated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on cytochrome P-4501A2 activity in a cross sectional medical survey. METHODS--The exposed workers had been employed at two chemical plants > 15 years earlier in the manufacture of 2,4, 5-trichlorophenol and its derivatives. The control group consisted of people with no occupational exposure to phenoxy herbicides and who lived within the communities of the exposed workers. A total of 58 workers and 125 unexposed controls participated in the analysis. Cytochrome P-450 activity was assessed with test that measures caffeine metabolites in the urine. A ratio of metabolites of caffeine (CMR) constituted a measure of P-4501A2 activity. RESULTS--Compared with the control group in multivariate logistic regression, raised non-significant associations were found for three of four categories of TCDD in exposed workers (TCDD < 20 pg/g, odds ratio (OR) 1.7, 95% confidence interval (95% CI) 0.6 to 5.0, TCDD 20-66, OR 0.3, 95% CI 0.0 to 1.7; TCDD 67-147, OR 2.3, 95% CI 0.6 to 8.8; TCDD > or = 148, OR 3.1, 95% CI 0.8 to 12.5). We found a strongly significant association of CMR and urinary cotinine, a measure of smoking, and urinary free ethanol. We found weak non-significant associations between P-4501A2 activity and increased serum TCDD among workers. CONCLUSIONS--The absence of an association between serum TCDD and cytochrome P-4501A2 may be due to the size of the study, insensitivity of the CMR to assess cytochrome P-4501A2 activity, or inadequate levels of exposure, although these were among the highest in human groups tested.

Variation in amobarbital metabolism: Evaluation of a simplified population study

Kinetic constants of amobarbital metabolism were established for 52 subjects on the basis of urinary analysis extending over several days, usually 96 hr. There was no evidence of effect of age or sex on any of the constants. C‐Hydroxylation was induced by cigarette smoking as much as 100%, but glucosidation was not affected. A factor influencing the constants was ethnicity of subjects (Caucasian or Oriental). This study confirms ethnic differences in amobarbital metabolism that were reported after measuring the concentration of metabolites in single samples of urine, that is, urine specimens voided during the postdistributive phase after oral drug intake. It appears that extreme simplification of sampling methods may be contemplated in the design of metabolic investigations of populations.