Genetic heterogeneity of histamine H2-receptors in the mouse vas deferens.

The ability of histamine to inhibit the overall contractile ('twitch') response of the isolated vas deferens of the mouse to electrical field stimulation (64 V pulse, 1 ms pulse width, frequency 0.2 Hz) was studied in nine inbred mouse strains. The strains were also characterized in terms of the potency of the histamine H2-receptor antagonist cimetidine in its inhibition of histamine-mediated effects. An apparently bimodal inter-strain variation (8-10 fold) in both characteristics was encountered, with three strains (SWR, A2G and C57BL/10ScSn) relatively sensitive (S) to both agonist and antagonist actions, and six (C3H, A, C57/BL6, DBA/2, Balb/C and 129/Sv) relatively insensitive (IS). These strain differences were independent of extracellular calcium concentration in the range 1.25-5 mM, and also independent of the frequency of tissue stimulation over the range 0.2-6.4 Hz. Representative S (SWR and A2G) and IS (DBA/2 and C3H) mouse vasa were also characterized in terms of their sensitivity to the agonist actions of dimaprit and the antagonist actions of tiotidine. In the S strain tissues, dimaprit produced 50% inhibition of the twitch response at 4.6-1.8 microM (mean +/- s.d.) and was able to elicit complete inhibition of the twitch response at concentrations greater than 100 microM, whereas 48.7 +/- 11.9 microM dimaprit was required to produce 50% inhibition of the twitch response in tissues from IS mice. In addition, the agonist actions of dimaprit were incomplete in the latter tissues, the drug eliciting no more than 75% inhibition of the twitch response at concentrations in the range 300-1000 microM. Tiotidine produced competitive antagonism of the actions of both histamine and dimaprit, the strain differences being of the same magnitude as those observed for cimetidine. 4 Mating of a representative S (SWR) and IS (129/Sv) strain produced F, mice with intermediate histamine and cimetidine sensitivities relative to the parental strains. A backcross of male F1 to female IS mice produced progeny displaying a range of histamine and cimetidine sensitivities representative of those seen in tissues from F1 and IS parental animals, however, the data were not bimodal. Thus, the backcross data provided no evidence to support single gene inheritance of histamine sensitivity and might suggest that more than one gene is responsible for these differences between S and IS mice.

The contribution of genetically determined oxidation status to inter-individual variation in phenacetin disposition.

The oxidative O-de-ethylation and aromatic 2-hydroxylation of phenacetin have been investigated in panels of extensive (EM, n = 13) and poor (PM, n = 10) metabolizers of debrisoquine. The EM group excreted in the urine significantly more paracetamol (EM: 40.8 +/- 14.9% dose/0-8 h; PM: 29.2 +/- 8.7% dose/0-8 h, 2P less than 0.05) and significantly less 2-hydroxylated metabolites (EM: 4.7 +/- 2.3% dose/0-8 h; PM: 9.7 +/- 3.5% dose/0-8 h, 2P less than 0.005) than the PM group. Apparent first-order rate constants, calculated from pooled phenotype data, for overall elimination of phenacetin (k) and formation of paracetamol (kml) were higher in the EM group (EM: k = 0.191 +/- 0.151 h-1; kml = 0.091 +/- 0.025 h-1; PM: k = 0.098 +/- 0.035 h-1, 2P less than 0.05, kml = 0.052 +/- 0.019 h-1, 2P less than 0.05) than the PM group. The apparent first-order rate constant for 2-hydroxylation displayed no significant inter-phenotype differences. Correlation analysis demonstrated that genetically determined oxidation status accounted for approximately 50% of the inter-individual variability in phenacetin disposition encountered in this study.

The metabolism of fenclofenac in the horse.

14C-Fenclofenac (2-(2'-4'-dichlorophenoxy)-phenylacetic acid) was administered orally to horses, and urinary metabolites investigated by chromatography. Fenclofenac was rapidly absorbed and eliminated, with a plasma half-life (t1/2) of 2.3 h, with 83.2 and 85.8% of the dose being recovered in the urine in 0-24 h. The major urinary metabolite was the ester glucuronide (58.8, 70.0% dose), and evidence is presented that this metabolite undergoes a structural rearrangement to give beta-glucuronidase-resistant isomers. The other 14C-labelled components in horse urine were unchanged fenclofenac (13.1, 11.5% dose), and two minor metabolites, one of which was identified as a monohydroxy fenclofenac. This study is the first to show an ester glucuronide to be the major metabolite of a non-steroidal anti-inflammatory drug in the horse.

Genetically determined oxidation capacity and the disposition of debrisoquine.

1 The disposition in urine of debrisoquine and its hydroxylated metabolites has been studied in subjects of the 'extensive metabolizer' (EM; n = 5) and 'poor metabolizer' (PM; n = 5) phenotypes. The 4-hydroxylation of debrisoquine by PM subjects following a 10 mg oral dose was capacity-limited and displayed significant dose-dependency over a range of 1-20 mg. In contrast, the EM subjects' ability to perform this metabolic oxidation did not deviate from first-order kinetics over a dose range of 10-40 mg. 2 The disposition of debrisoquine in plasma following a 10 mg oral dose has been studied in EM (n = 4) and PM (n = 3) subjects. Whilst PM subjects displayed significantly higher plasma levels of debrisoquine at all time points following 1 h post-dosing, and higher values for areas under the plasma concentration-time curve (EM: 105.6 +/- 7.0 ng ml-1 h; PM: 371.4 +/- 22.4 ng ml-1 h, 2P less than 0.0001), neither debrisoquine plasma half-life (EM: 3.0 +/- 0.5 h; PM: 3.3 +/- 0.4 h) nor renal clearance of the drug (EM: 152.8 +/- 30.3 ml min-1; PM: 137 +/- 4.5 ml min-1) displayed significant inter-phenotype differences. 3 The results of these investigations show that the phenotyping of individuals for debrisoquine oxidation status by means of a 'metabolic ratio' derived from a single 0-8 h urine sample has a sound kinetic basis. The kinetic differences between the two phenotypes would strongly suggest that the metabolic defect manifested in PM subjects is one of pre-systemic elimination capacity.

Influence of DH/DL alleles regulating debrisoquine oxidation on phenytoin hydroxylation.

Eleven subjects of previously determined debrisoquine oxidation phenotype status (extensive metabolizer [EM], n = 5; poor metabolizer [PM], n = 6) were studied for their ability to perform the aromatic 4-hydroxylation of phenytoin. The PM subjects studied were found to be slower metabolizers of phenytoin than EM subjects in terms of the metabolite formation rate constant (kfHPPH: EM, 0.030 +/- .007 hr-1; PM, 0.016 +/- 0.003 hr-1, 2p less than 0.001) and cumulative excretion of 4-hydroxyphenytoin (48 hr after dosing: EM, 52.8 +/- 10.7%; PM, 36.9 +/- 7.0%, 2p less than 0.01). It is concluded that the metabolic oxidation of phenytoin is influenced by the same DH and DL alleles, acting at the same locus, that regulate the hydroxylation of debrisoquine and that impaired metabolism of phenytoin may be expected to occur in about 9% of the population, being transmitted as an autosomal-recessive trait. It is suggested that debrisoquine oxidation phenotyping may have predictive value in guiding phenytoin dosage, particularly in those with impaired oxidation.

Some observations on the oxidation phenotype status of Nigerian patients presenting with cancer.

The hypothesis is being explored that there may be an association between genetically determined oxidation status and propensity to develop carcinoma in response to environmental chemical carcinogens. For this purpose, the genetic structure of a normal, healthy Nigerian population with respect to oxidation status, has been compared with that found for a group of 59 Nigerian patients presenting with carcinoma of the liver and gastrointestinal tract. Genetically determined oxidation status was assessed by measuring the extent of oxidation of a probe drug, debrisoquine, to its major metabolite, 4-hydroxydebrisoquine. The cancer group contained a disproportionately large number of individuals who were extensive oxidizers compared to the controls (2 P = 0.0045). The findings support the view that genetically determined oxidation status may be an important host factor in influencing responsiveness to chemical carcinogens that require oxidative metabolic activation.

Influence of the genetically controlled deficiency in debrisoquine hydroxylation on antipyrine metabolite formation.

The influence of the genetically controlled deficiency in debrisoquine hydroxylation on antipyrine metabolite formation was studied by giving 500 mg antipyrine to 14 extensive and 10 poor metabolizers of debrisoquine. The pharmacokinetics of antipyrine were determined on the basis of the saliva concentration time curve and the cumulative urinary excretion of 4-hydroxyantipyrine, norantipyrine, 3-hydroxymethyl-antipyrine, and 3-carboxyantipyrine was measured for 32 h following drug administration. Antipyrine elimination half-life, volume of distribution, and total clearance were almost equal for the two groups. Significant differences in the excretion of antipyrine metabolites were not observed, except for 3-hydroxymethyl-antipyrine which was excreted in poor metabolizers about 30% less than in extensive metabolizers (p less than 0.01). However, this difference only reached borderline significance (p less than 0.1) when clearance values for production of this metabolite were calculated. It is concluded that different species of the drug-oxidizing enzymes (cytochrome P-450 system) are involved in the metabolism of debrisoquine and antipyrine. Possibly the enzyme responsible for hydroxylating debrisoquine is partly involved in the formation of 3-hydroxymethyl-antipyrine.

A study of the debrisoquine hydroxylation polymorphism in a Nigerian population.

1. The metabolic oxidation of debrisoquine has been studied in a group of 123 Nigerian volunteers. 2. All subjects excreted unchanged drug together with five oxidation products, namely, 4-, 5-, 6-, 7- and 8-hydroxy-debrisoquine. 3. The 4-hydroxylation reaction exhibits polymorphism; ten subjects were defective in their ability to effect this reaction. 4. The incidence (q) of the allele governing impaired 4-hydroxylation (DL) among Nigerians was calculated as being 0.28 (95% confidence limit of 0.20-0.37). 5. An association was demonstrated between the ability to effect 4-hydroxylation and 6- and 7-hydroxylation of debrisoquine, suggesting that the alleles controlling alicyclic oxidation also influence aromatic hydroxylation.

A family and population study of the genetic polymorphism of debrisoquine oxidation in a white British population.

A population survey of 258 unrelated white British subjects showed a polymorphism for the 4-oxidation of debrisoquine. "Extensive metabolisers" (EM) and "poor metabolisers" (PM) are recognisable, 8.9% of the population being PM. Nine pedigrees ascertained through PM probands show that the PM phenotype is an autosomal Mendelian recessive character. The EM phenotype is dominant and the degree of dominance has been estimated at 30%. PM subjects are more prone to hypotension during debrisoquine therapy. The alleles controlling this polymorphism appear to control the oxidation of other drugs.

Toxicological implications of polymorphic drug metabolism.

The occurrence of genetic polymorphisms of drug metabolism means that populations contain subgroups (phenotypes) that differ sharply in their abilities to effect a number of metabolic reactions. Because of this, major interphenotype differences occur in responsiveness to drugs and toxic substances. The well established genetic polymorphisms of acetylation and hydrolysis illustrate the important association that exists between phenotype and propensity to develop toxic and exaggerated responses to some substances. Recently, for metabolic oxidation, a new genetic polymorphism of drug metabolism has been described and it promises to provide a better understanding of inter-individual variability in the metabolic handling of, and responsiveness to, drugs and toxic substances. The following effects of the polymorphism are described here: (a) its influence in determining variable presystemic metabolism and hence systemic drug availability; (b) its role in determining alternative toxic pathways of metabolism in individuals who have a genetically determined impairment of oxidative capacity and (c) its influence on the development of agranulocytosis associated with metiamide administration.

Debrisoquin hydroxylation polymorphism among Ghanaians and Caucasians.

The alicyclic and aromatic hydroxylation of debrisoquin was studied in Ghanaians. As in a previously studied Caucasian population, the alicyclic 4-hydroxylation of debrisoquin in Ghanaians was polymorphic. Three phenotypes were observed: homozygous extensive metabolizers (58%), heterozygous extensive metabolizers (36%), and homozygous poor metabolizers (6%). In contrast, British Caucasians are primarily monomorphic extensive metabolizers (92%) and homozygous poor metabolizers comprise 8% of the population. Urinary recovery of the drug and its hydroxylated metabolites was significantly less in the Ghanaian subjects. In both Ghanaian and British populations, aromatic hydroxylation producing 5-, 6-, 7-, and 8-hydroxydebrisoquin was shown to parallel the alicyclic 4-hydroxylation of debrisoquin, and thus to be controlled by the same gene locus. Debrisoquin is advocated as a tool for uncovering polymorphism in drug oxidation and its interethnic variations.

The pharmacokinetics of tritiated ethinylestradiol in the rabbit.

The disappearance of ethinylestradiol from the blood of rabbits has been studied, following the intravenous administration of this steroid. The disappearance followed two exponentials, the first having a half life (t1/2) of 5.5 min and the second, apparently terminal exponential was also rapid (t1/2-69 min). The plasma clearance was 150 ml/min which suggests almost total clearance of this steroid during a single passage through the liver. Bile contained a significant concentration of EE conjugates and thus this steroid could undergo enterophepatic recirculations. A large oral dose of unlabelled EE, given prior to intravenous administration of tritiated EE, considerably altered the pharmacokinetics of the latter by saturating both phase one metabolism (changes of the steroid nucleus) and the secretion of conjugates into bile. It was not clear whether phase two metabolism (conjugation) was also saturated.

Polymorphism of carbon oxidation of drugs and clinical implications.

Eight volunteers previously phenotyped for their ability to hydroxylate debrisoquine (four extensive metabolisers (EM), four poor metabolisers (PM) were investigated for their metabolic handling of guanoxan and phenacetin. All three drugs are oxidised at carbon centres. Oxidative dealkylation of phenacetin was determined by measuring the rate of formation of paracetamol. The EM subjects excreted mostly metabolites of guanoxan (mean 29% of dose), whereas the PM group excreted large amounts of unchanged drug (48% of dose). The rate of formation of paracetamol was noticeably slower in the PM group, and, when analysed by minimum estimates of apparent first-order rate constants, the difference between the two phenotypes was significant. Thus the hydroxylation defect shown for debrisoquine metabolism carries over to the oxidative metabolism of phenacetin and guanoxan. Some 5% of the population are genetically defective hydroxylators of drugs. Thus methods for evaluating the metabolism of new drugs in respect of usage and side effects need to be revised.