Werner Kalow

Evaluation of the genetic component of variability in CYP3A4 activity: a repeated drug administration method.

The CYP3A4 enzyme contributes to the disposition of more than 60 therapeutically important drugs and displays marked person-to-person variability of the catalytic function. However, the extent of genetic contribution to variability in CYP3A4 activity remains elusive. Recently, we showed that a comparison of between- (SDb2) and within-person (SDW2) variances provides an estimate of the genetic component of variability in drug disposition. The aim of the present analysis was to assess the genetic control of CYP3A4 activity in vivo. A computerized literature search was conducted covering 1966 to September 1999 to identify studies reporting repeated administration of CYP3A4 substrates. The genetic contribution (rGC) to disposition of each CYP3A4 substrate was obtained by the formula (SDb2-SDW2)/SDb2. The rGC values approaching 1.0, point to overwhelming genetic control, whereas those close to zero suggest that environmental factors dominate. A total of 16 studies with 10 different CYP3A4 substrates were identified (n = 161 subjects). The rGC for hepatic CYP3A4 activity as measured by midazolam plasma clearance or the erythromycin breath test was 0.96 (0.92-0.98) (95% Cl) and 0.89 (0.65-0.98), respectively (P < 0.05). The point estimates of rGC for composite (hepatic + intestinal) CYP3A4 activity measured after oral administration of cyclosporine, ethinylestradiol, ethylmorphine, nifedipine and nitrendipine, ranged from 0.66-0.98 (median: 0.83) (P < 0.05). Cyclosporine data suggested a higher genetic control of CYP3A4 at night than during the day. These data indicate that further molecular genetic investigations are warranted to identify genetic variants at CYP3A4 or elsewhere in the genome which contribute to regulation of CYP3A4 activity.

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.

Malignant hyperthermia: A statistical review

SummaryInformation was collected on 89 patients who responded to general anaesthetics with malignant hyperthermia. The syndrome occurred at the rate of about one in 14,000 anaesthetics among a hospital population of children. The patient mortality was 64 per cent. The finding that males were somewhat more commonly affected than were females does not contradict previous observations of dominant inheritance of the syndrome. About one-third of patients had relatives who were also affected with malignant hyperthermia, although a few patients had had previous uneventful general anaesthetics. The racial origin was varied. A pre-existing muscle or musculoskeletal disease was present more frequently than expected in patients who manifested rigidity.Clinical manifestations followed the administration of a muscle relaxant or a potent inhalational agent, usually halothane. Fever was invariably present within the first one to two hours of the induction. Skeletal muscle rigidity occurred in more than two-thirds of cases. The use of anticholinergic drugs given preoperatively appeared to increase the incidence of rigidity. The use of non-depolarizing relaxants in vain attempts to overcome the rigidity has certainly not improved the chances of survival. The higher the absolute maximum temperature and the longer the duration of anaesthesia, the greater was the mortality rate.It is possible that the cases with and without rigidity represent slightly different disorders. In cases characterized by rigidity there were often tachypnoea, tachycardia, arrhythmias, acute heart failure, late neurological deterioration, hypoxia, respiratory and metabolic acidoses, hyperkalaemia, hypocalcaemia, elevated serum enzymes, impaired blood coagulation, haemo- and myoglobinuria, oliguria, and muscle biopsy abnormalities.Treatment included a wide variety of therapeutic measures. No particular agent could be credited with having improved the survival rate. So far, the most effective treatment was early detection and early cessation of anaesthesia.RésuméOn a recueilli des renseignements chez 89 malades qui ont présenté de ľhypothermie maligne au cours ďune anesthésie générale. Le syndrome est apparu au taux ďun pour 14,000 anesthésies parmi la clientèle ďun hôpital pour enfants. Le taux de mortalité a été de 64 pour cent. La constatation que les sujets de sexe masculin sont le plus fréquemment affectés ne contredit pas les observations précédentes. Environ un tiers des malades avaient vu des membres de leur famille souffrir ďhyperthermie maligne, cependant que quelques uns avaient déjà subi une anesthésie générale sans incidents. Ľorigine raciale était variée. Il y avait une maladie musculaire ou musculosquelettique pré-existante plus souvent qu’on ne s’y attendait chez les malades qui ont manifesté de la rigidité.Les manifestations cliniques se sont produites après ľadministration ďun myorésolutif ou ďun agent inhalatoire puissant, généralement ľhalothane. On a toujours observé de la fièvre au cours de la première ou de la deuxième heure à compter de ľinduction. La rigidité des muscles squelettiques s’est produite dans plus des deux tiers des cas. Ľusage de produits anticholinergiques en médication préopératoire a semblé augmenter la fréquence de la rigidité. Ľusage de résolutifs non dépolarisants, pour tenter en vain de combattre la rigidité, n’a certainement pas augmenté les chances de survie. Plus la température a été élevée et plus ľanesthésie a été longue, plus le taux de mortalité a été élevé. Il est possible que les cas avec ou sans rigidité représentent des pathologies légèrement différentes. Chez les malades présentant de la rigidité, il y avait souvent de la tachypnée, de la tachycardie, des arythmies, une défaillance cardiaque aigue, une détérioration neurologique tardive, de ľhypoxie, de ľacidose respiratoire et métabolique, de ľhypercaliémie, de ľhypocalcémie, une élévation des enzymes aériques, des troubles de la coagulation du sang, de ľhémoglobinurie, de la myoglobinurie, de ľoligurie et des anomalies de la biopsie musculaire.Le traitement comprend une grande variété de mesures thérapeutiques. Aucun agent en particulier ne peut recevoir le crédit ďavoir amélioré le taux de survie. Pour le moment, le traitement le plus efficace a été le diagnostic précoce et la cessation rapide de ľanesthésie.

Pharmacogenetics of drug metabolism

Genetic variation in drug metabolizing capacity deserves attention not only in terms of inter-individual but also in terms of inter-ethnic differences.

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.

The use of caffeine for enzyme assays: A critical appraisal

Clinical Pharmacology and Therapeutics (1993) 53, 503–514; doi:10.1038/clpt.1993.63

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.

Hypothesis : Comparisons of inter- and intra-individual variations can substitute for twin studies in drug research

Twin studies are useful devices to determine the heritability of persistent but variable characteristics that tend to differ among individuals. Drug responses are not persistent affairs; they are temporary characteristics. One therefore may ask whether twin studies are necessary to assess the genetic element in pharmacological responsiveness. To measure the genetic component contributing to their variability, it seems logical to investigate the response variation by repeated drug administration to given individuals, and to compare the variability of the responses within and between individuals. We attempt here to describe a theoretical background of this venture, and to show some results of the exercise. Potential sources of error or uncertainty are discussed.

Cocaine metabolism: Cocaine and norcocaine hydrolysis by liver and serum esterases

The hydrolysis of cocaine and its N‐demethylated product, norcocaine, by esterases was examined in liver and serum. Both liver and serum enzymatically formed ecgonine methyl ester from cocaine. The liver enzyme had a much lower affinity for cocaine than that of serum, indicating that a different form of esterase was present in liver. The liver enzyme had a similar affinity for both norcocaine and cocaine. Likewise, the serum enzyme showed similar affinities for both substrates. The Vmax estimates, however, were consistently higher for norcocaine than cocaine in both liver and serum. Benzoyl ecgonine, a major metabolite of cocaine formed by hydrolysis, was not produced enzymatically in either serum or liver; the rate of spontaneous formation at physiological pH suggests that this metabolite may arise nonenzymatically in the body.

Use of caffeine metabolite ratios to explore CYP1A2 and xanthine oxidase activities

Caffeine was used as a metabolic probe to screen healthy subjects for their activities of two enzymes, deduced to be CYP1A2 (an inducible cytochrome P450) and xanthine oxidase. A longitudinal study revealed modest effects of caffeine dose, ethanol intake, and time‐of‐day on the CYP1A2 index, without any effect on the xanthine oxidase index. The coefficients of intraindividual variation not accounted for were 5.0% for the xanthine oxidase and 17.2% for the CYP1A2 index. In a population study, both indexes showed a log normal distribution, with CYP1A2 values of most subjects covering a 6.3‐fold range but only a 1.7‐fold range with xanthine oxidase. The CYP1A2 index was 33% decreased in women who used oral contraceptives and substantially increased in cigarette smokers. Neither the CYP1A2 nor the xanthine oxidase index differed between volunteers of Chinese and European extraction. Four of 178 subjects showed unexplained low xanthine oxidase values (i.e., values several standard deviations below the mean).