Metabolizers Of Debrisoquine Research Articles

Cytochrome-P450-dependent hydroxylation in cluster headache.

Two isozymes of the cytochrome-P450-dependent drug oxidizing system exhibit polymorphism. Five to 10% of a Caucasian population are deficient in debrisoquine-hydroxylase activity and about 3% in mephenytoin-hydroxylase activity (poor metabolizers). We tested the hypothesis of a possible over-representation of poor metabolizers among patients with cluster headache. The individual metabolic capacity was determined in 30 cluster headache patients after administration of a test dose of 10 mg of debrisoquine and 100 mg of mephenytoin. Two patients (6.7%) were poor metabolizers of debrisoquine and one (3.3%) a poor metabolizer of mephenytoin. This was no different from the rate of poor metabolizers, 7.1% and 3.3% respectively, in a reference panel of healthy Swedish volunteers.

Haloperidol disposition is dependent on debrisoquine hydroxylation phenotype.

To investigate the importance of genetic factors for the regulation of haloperidol metabolism, we studied the disposition of a single oral dose of this drug in a panel of six extensive (EM) and six poor (PM) metabolizers of debrisoquine. PM eliminated haloperidol significantly slower than EM, the plasma half-life being longer (mean 29.4 +/- S.D. 4.2 and 16.3 +/- 6.4 h; p less than 0.01) and the clearance lower (1.16 +/- 0.36 and 2.49 +/- 1.31 L/h/kg; p less than 0.05). A 4-mg dose of haloperidol was given to the first three PM, but all three developed side effects, and a 2-mg dose had to be given to the next three PM subjects. All EM received 4 mg haloperidol. The disposition of haloperidol is thus associated with the genetically determined capacity to hydroxylate debrisoquine. PM of debrisoquine (7% of Caucasian populations) might, therefore, on common doses of haloperidol, achieve high plasma concentrations and thereby have an increased risk of side effects. At the other extreme, very rapid metabolizers may need increased doses of haloperidol.

Evidence for a dissociation in the control of sparteine, debrisoquine and metoprolol metabolism in Nigerians.

The 0-8 hour urinary distributions of the metabolic ratios of sparteine (100 mg), debrisoquine (10 mg) and metoprolol (100 mg) were measured in 165 healthy, unrelated, black Nigerian medical students. There was a weak correlation (rs = 0.51, p < 0.001; n = 82) between the metoprolol/alpha-hydroxymetoprolol (M/HM) and the sparteine/total (2- + 5-) dehydrosparteine (S/DHS) ratios. No significant correlations were found between the debrisoquine/4-hydroxydebrisoquine (D/HD) and M/HM ratios (rs = 0.16, n = 33) and between the D/HD and S/DHS ratios (rs = 0.31, n = 38). Both visual inspection and kernel density analysis of the data suggested the presence of two phenotypic groups for sparteine oxidation, with 4% of the population studied being putative poor metabolizers. In contrast biomodality was not apparent in the distribution of the log10M/HM and log10D/HD ratios. These findings provide evidence for a dissociation in the control of metoprolol, sparteine and debrisoquine oxidation in Nigerians and highlight the difficulties in the interpretation of data from pharmacogenetic studies in different ethnic groups.

Analysis of the CYP2D6 gene in relation to debrisoquin and desipramine hydroxylation in a Swedish population.

The molecular basis of polymorphic debrisoquin hydroxylation was studied in 223 Swedish white subjects, 187 extensive metabolizers and 36 poor metabolizers phenotyped with debrisoquin and desipramine. Restriction fragment length polymorphism (RFLP) analysis of the CYP2D6 gene revealed that 52% of unrelated poor metabolizers were homozygous for Xba I 29 kb fragment, and only 8% had two mutant alleles detected with RFLP. Allele-specific polymerase chain reaction (PCR)-based DNA amplification, however, revealed that all but one of the poor metabolizers had two mutant alleles of the CYP2D6A or CYP2D6B type or both. Extensive metabolizers who were heterozygous for wild-type and CYP2D6B genes had metabolic ratios for debrisoquin and desipramine that were higher than those of subjects who were homozygous for the wild-type gene. The 16 + 9 kb Xba I RFLP pattern was associated with the poor metabolizer phenotype and CYP2D6B mutations. Three extremely rapid metabolizers of debrisoquin had a 44 kb Xba I fragment that did not carry either CYP2D6A or CYP2D6B mutations. In conclusion, in the Swedish population studied, allele-specific PCR amplification allowed prediction of the debrisoquin hydroxylation phenotype with 99% accuracy.

In vitro oxidation of oxicam NSAIDs by a human liver cytochrome P450

The nature of the enzyme(s) catalyzing the major metabolic pathway (5′-hydroxylation) of oxicam NSAIDs was investigated in subcellular preparations of human liver tissue. Microsomal, but not cytosolic, fractions catalyzed the 5′-hydroxylation of tenoxicam. This reaction required NADPH and was inhibited by various nonselective P450 inhibitors (CO, SKF-525A, ketoconazole), but not by the peroxidase inhibitor NaN 3. Tenoxicam 5′-hydroxylation exhibited simple Michaelis-Menten kinetics compatible with catalysis by a single enzyme, but it strongly inhibited its own oxidation at concentrations higher than 100–150 μM. Piroxicam competitively inhibited tenoxicam 5′-hydroxylation and, conversely, tenoxicam competitively inhibited piroxicam 5′-hydroxylation. Tenoxicam 5′-hydroxylation kinetics were similar in microsomes from one poor and five extensive metabolizers of debrisoquin (CYP2D6). Dextromethorphan (CYP2D6 prototype substrate) and midazolam (CYP3A prototype substrate) had no influence on tenoxicam 5′-hydroxylation, whereas mephenytoin, tolbutamide and sulfaphenazole (K i ≈ 0.1 μM) inhibited it. This indicates that the 5′-hydroxylation of both piroxicam and tenoxicam is predominantly catalyzed by at least one cytochrome P450 isozyme of the CYP2C subfamily.

Debrisoquine oxidation in an Italian population: A study in healthy subjects and in schizophrenic patients

The debrisoquine oxidation phenotype was assessed in 137 healthy Italian volunteers and in 41 drug-free schizophrenic patients. A bimodal distribution of the urinary debrisoquine/4-hydroxydebrisoquine metabolic ratio was observed in healthy volunteers. Ten subjects were identified as poor metabolizers, yielding a frequency of 7.3% which is similar to that reported in other European countries. The prevalence of the poor metabolizer phenotype was 9.8% among schizophrenic patients. This indicates that there is no association between polymorphic drug oxidation and schizophrenic disorder. Treatment with chlorpromazine (100 or 150 mg daily) significantly increased the debrisoquine metabolic ratio in nine patients (P less than 0.01). These results confirm that neuroleptics of the phenothiazine class inhibit the oxidative metabolism of debrisoquine.

Debrisoquine polymorphism

Markedly decreased cytochrome P450-mediated metabolism of debrisoquine, sparteine, and more than two dozen additional commonly prescribed drugs is an autosomal recessive trait that has been associated with several RFLP patterns involving the CYP2D6 gene. In Caucasians there are at least six variant alleles known to be correlated with the 'poor metabolizer' (PM) phenotype. We examined debrisoquine and sparteine metabolism and CYP2D6 RFLP patterns in 22 Ngawbé Guaymí Indians of Panama. We studied a two-generation family, a three-generation family, and three other unrelated PM individuals. Digestion of all 22 DNA samples with Xba I or Hind III did not produce the same varying CYP2D6 RFLP patterns as those commonly seen in at least two-thirds of all Northern European Caucasians and Chinese so far screened. In contrast, we found a single heretofore undescribed Bam HI polymorphism that was correlated with the PM phenotype among all Ngawbé Guaymí individuals examined. It is possible that this novel RFLP might represent a recent founder effect that has occurred in this unadmixed Amerindian tribe within the past 20,000-30,000 years.

In vitro forecasting of drugs which may interfere with the biotransformation of midazolam

The biotransformation of midazolam is mediated by a cytochrome P-450 isozyme (P-450 IIIA) whose activity is highly variable. The kinetics of the 1'- and 4-hydroxylation of midazolam, the major routes of midazolam oxidation, by human liver microsomes have been examined to characterize further the cytochrome isozyme(s) catalysing these reactions, and to screen for drugs that might interfere with them. In hepatic microsomal preparation from two kidney donors (extensive and poor metabolisers of debrisoquine) KM values for 1'-hydroxylation were 4.2 and 6.1 microM (extensive and poor metabolisers, respectively), and for the 4-hydroxylation they were 14.7 and 18.1 microM, respectively. The corresponding Vmax values were 25.8 and 29.8 and 17.0 and 18.1 nmol.mg P-1.h-1. Both reactions appeared to be catalysed by the same or by coregulated isozymes. Midazolam hydroxylations in vitro are inhibited by many drugs, including nifedipine and other dihydropyridine-type calcium channel blockers, ergot alkaloids, cyclosporine, erythromycin and phenothiazine-type neuroleptics. A clinical case report illustrates the consequence of such a drug-drug interference with hepatic biotransformation; midazolam-induced sleep in a patient lasted for 6 days (t1/2 = 25 h).

Extremely Long Plasma Half-Life of Amitriptyline in a Woman with the Cytochrome P450IID6 29/29-Kilobase Wild-Type Allele—A Slowly Reversible Interaction with Fluoxetine

A 61-year-old woman, a nonsmoker, was admitted to the hospital because of endogenous depression. No concomitant disease, especially kidney or liver dysfunction, was diagnosed. After 9 days of treatment with 125 mg of amitriptyline (AMI) daily, she developed signs of a severe anticholinergic syndrome. Plasma concentrations of AMI (510 ng/ml) and nortriptyline (NOR; 320 ng/ml) were very high and the half-life of AMI was about 120 h. The debrisoquine metabolic ratio was 0.55 and 0.79 on two occasions, which shows that she had no deficiency of cytochrome P450IID6. This result was confirmed with a dextromethorphan test, analysis of restriction fragment length polymorphisms (29/29-kb fragments), and genotyping with allele-specific polymerase chain reaction (homozygous 29 kb wild-type alleles). Patients with high plasma levels of tricyclic antidepressants are usually poor metabolizers of debrisoquine. Before the administration of AMI, our patient was pretreated with fluoxetine. A slowly reversible interaction with fluoxetine or an extremely long-lasting metabolite may be responsible for the long plasma half-life of AMI.

Clinical Implications of the Competitive Inhibition of the Debrisoquin-Metabolizing Isozyme by Quinidine

Approximately 10% of western populations are genetically deficient in the enzyme that metabolizes the antihypertensive drug debrisoquin. These "poor metabolizers" process many common medications in an aberrant fashion, resulting in a variety of untoward consequences including exaggerated drug effect, subtherapeutic drug concentrations, or complex drug interactions. A variety of medications, including neuroleptics, antidepressants, beta-blockers, and certain antiarrhythmics, are subject to the influence of this metabolic polymorphism. Quinidine administration changes persons to poor metabolizers of debrisoquin for the duration of therapy. Thus, the use of quinidine with any of the other drugs metabolized by this isozyme may be expected to result in a drug interaction in which a person's response will mimic that of a poor metabolizer. Because no test is commonly available to determine directly the debrisoquin metabolic phenotype, clinicians should be alert to unusual drug reactions in patients receiving quinidine concurrently with the other medications.

Clinical implications of the competitive inhibition of the debrisoquin-metabolizing isozyme by quinidine

Approximately 10% of western populations are genetically deficient in the enzyme that metabolizes the antihypertensive drug debrisoquin. These "poor metabolizers" process many common medications in an aberrant fashion, resulting in a variety of untoward consequences including exaggerated drug effect, subtherapeutic drug concentrations, or complex drug interactions. A variety of medications, including neuroleptics, antidepressants, beta-blockers, and certain antiarrhythmics, are subject to the influence of this metabolic polymorphism. Quinidine administration changes persons to poor metabolizers of debrisoquin for the duration of therapy. Thus, the use of quinidine with any of the other drugs metabolized by this isozyme may be expected to result in a drug interaction in which a person's response will mimic that of a poor metabolizer. Because no test is commonly available to determine directly the debrisoquin metabolic phenotype, clinicians should be alert to unusual drug reactions in patients receiving quinidine concurrently with the other medications.

The metabolism of mexiletine in relation to the debrisoquine/sparteine‐ type polymorphism of drug oxidation.

1. The relationship between the metabolism of the antiarrhythmic drug mexiletine and the debrisoquine/sparteine-type polymorphism was studied in vitro, using microsomes from six human livers, and in vivo, in nine healthy drug-free volunteers with wide variation in their ability to hydroxylate debrisoquine. 2. There was a strong and similar correlation between the formation rate of both major mexiletine metabolites, p-hydroxymexiletine (PHM) and hydroxymethylmexiletine (HMM), and the high affinity component of dextromethorphan O-demethylase activity in human liver microsomes (rs = 0.94; P less than 0.01). 3. There were marked interindividual differences in the amounts of PHM and HMM excreted in the urine over 48 h after a single 200 mg oral dose of mexiletine hydrochloride. Recoveries of both metabolites were correlated inversely with the debrisoquine/4-hydroxydebrisoquine (D/HD) urinary metabolic ratio (rs = -0.83; P = 0.006 and rs = -0.85; P = 0.004, respectively) and were lower in poor metabolisers of debrisoquine (PM) than in extensive metabolisers (EM). Moreover, PM had the highest values of mexiletine/PHM and mexiletine/HMM urinary ratios. In addition, there was a strong correlation between these two indices of mexiletine hydroxylation and the D/HD metabolic ratios (rs = 0.92; P = 0.001 and rs = 0.90; P = 0.001, respectively). 4. After mexiletine pretreatment, the values for D/HD ratio were significantly increased in EM while corresponding values in PM were similar. 5. These findings are in accordance with previous in vitro data suggesting that PHM and HMM formation is predominantly catalyzed by the genetically variable human liver cytochrome P450IID6 isoenzyme responsible for the debrisoquine/sparteine-type polymorphism of drug oxidation.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic and metabolic criteria for the assignment of debrisoquine 4-hydroxylation (cytochrome P4502D6) phenotypes

A randomly selected population of 73 volunteers, together with 22 previously established poor metabolisers of debrisoquine, were phenotyped for their ability to 4-hydroxylate debrisoquine and were also analysed for a number of mutations in the CYP2D6 gene. Genotyping was performed using both restriction fragment length polymorphism with the restriction enzyme Xba I, together with two separate polymerase chain reaction assays. Together, these assays detected 98% of mutant alleles in the poor metaboliser group which corresponded to positive identification of 95% of this group. The most common mutant allele detected as the 29B which comprised 75% of total alleles in the poor metaboliser group, whereas the 29A had a frequency of 0.11. Two other allelic variants, which were detectable by restriction fragment length polymorphism analysis occurred at frequencies of 0.07 and 0.05. In the volunteer group, 2.7% of subjects were genotypically poor metabolisers, 35.6% heterozygous extensive metabolisers and 61.7% homozygous extensive metabolisers, on the basis of the genotyping assays used. A good correlation between debrisoquine metabolic ratio and genotype was obtained particularly for subjects genotyped as homozygous extensive metabolisers.

Identification of genetic differences in drug metabolism: Prediction of individual risk of toxicity or cancer

These two reports describe recombinant DNA tests that can identify individuals having a defect in the cytochrome P450IID6 (CYP2D6)-mediated oxidative metabolism of debrisoquine and more than two dozen other drugs that are commonly prescribed. The poor metabolizer (PM), representing 5% to 10% of the northern European white population is homozygous for an autosomal recessive trait. Compared with the extensive metabolizer (EM) phenotype, the PM individual is more prone to toxicity caused by some of these drugs. Curiously, the PM phenotype appears to be associated with a lower risk of lung and bladder cancer. Gough and coworkers propose that the primary gene defect associated with the CYP2D6 polymorphism appears to be a G to A transition mutation affecting the splice junction of intron 3/exon 4 of the CYP2D6 gene. They offer a polymerase chain reaction (PCR) amplification method for diagnosing three variant alleles and claim to have predicted the phenotype of all normal and 34 of 42 (81%) PM individuals. Based on the known frequencies of those variant alleles detectable by this method, however, the assay would be expected to predict less than 60% of PM patients. Heim and Meyer, on the other hand, have combined allelespecific PCR with restriction fragment length polymorphism (RFLP) patterns to identify more than 95% of all mutant alleles, suggesting that their assay would accurately predict more than 90% of all PM individuals. Using this latter assay or a similar test with further improvements, especially in combination with family studies, it should soon be possible for the physician to determine the CYP2D6 phenotype of the patient, thereby avoiding toxic drug overdoses.

Identification of genetic differences in drug metabolism: Prediction of individual risk of toxicity or cancer Gough AC, Miles JS, Spurr NK, Moss JE, Gaedigk A, Eichelbaum M, Wolf CR. Identification of the primary gene defect at the cytochrome P450 CYP2D locus. Nature 1990;347:773–776. Heim M, Meyer UA. Genotyping of poor metabolisers of debrisoquine by allele-specific PCR amplification. Lancet 1990;336:529–532

Identification of genetic differences in drug metabolism: Prediction of individual risk of toxicity or cancer Gough AC, Miles JS, Spurr NK, Moss JE, Gaedigk A, Eichelbaum M, Wolf CR. Identification of the primary gene defect at the cytochrome P450 CYP2D locus. Nature 1990;347:773–776. Heim M, Meyer UA. Genotyping of poor metabolisers of debrisoquine by allele-specific PCR amplification. Lancet 1990;336:529–532

Disposition of the neuroleptic zuclopenthixol cosegregates with the polymorphic hydroxylation of debrisoquine in humans

The pharmacokinetics of a single oral dose of the neuroleptic drug zuclopenthixol (10 or 6 mg) was studied in 6 extensive and 6 poor metabolizers of debrisoquine. The peak plasma concentrations of zuclopenthixol did not differ between the phenotypes, whereas the plasma elimination half-life was significantly longer in poor than in extensive metabolizers (29.9 +/- 6.6 vs 17.6 +/- 6.9 h). Accordingly, the total oral plasma clearance was lower in poor than in extensive metabolizers (0.78 +/- 0.27 vs 2.12 +/- 0.65 1/h/kg). Ten of the volunteers had previously participated in a similar study in which the kinetics of perphenazine, another neuroleptic drug, were studied in poor and in extensive metabolizers of debrisoquine. There was a significant correlation between the oral clearance of perphenazine and that of zuclopenthixol among these 10 subjects. The study indicates that the disposition of zuclopenthixol, as well as that of perphenazine, is related to the genetically determined capacity to hydroxylate debrisoquine. The significance of this polymorphism for the clinical use of neuroleptics is discussed.

Impact of environmental and genetic factors on codeine analgesia

The polymorphic cytochrome P-450 DB1 (P-450 IID6) is responsible for the O-demethylation of codeine to morphine by human liver microsomes. The influence of P-450 DB1 variable activity on the bioactivation of codeine in vivo to morphine and on its analgesic effect was investigated in phenotyped healthy volunteers--7 extensive [EM] and 1 poor [PM] metabolizer of debrisoquine. After pretreatment with oral placebo or quinidine sulphate 50 mg, codeine phosphate 100 mg or placebo were administered orally according to a double-blind randomized crossover design. In EM subjects the plasma morphine Cmax was 17.9 nmol/l, whereas virtually no morphine was detectable after quinidine pretreatment (1.5 nmol/l), and in the PM subject (0.60 nmol/l). In EM codeine significantly increased subjective (VAS) and objective (R-III reflex) pain thresholds in response to selective transcutaneous nerve stimulation, whereas no significant analgesia was detected after placebo, or after codeine with quinidine pretreatment, or in the PM. In PM of genetic origin, or due to environmental alteration of the phenotypic expression (i.e. drug interaction), codeine is not activated into morphine and is an inefficient analgesic.

Acute Effects of 1‐Methyl‐4‐Phenyl‐1, 2, 3, 6‐Tetrahydropyridine in a Model of Rat Designated a Poor Metabolizer of Debrisoquine

The relationship between oxidative polymorphisms and the cause of Parkinson's disease is controversial. The drug 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which induces parkinsonism in humans and in some animal models, is metabolized by cytochrome P450 db1 isozyme (the same enzymatic system implicated in 4-hydroxylation of debrisoquine). In this study, we treated females of three rat species, which differ in their ability to hydroxylate debrisoquine, with MPTP (three doses of 30 mg/kg s.c. at 12-h intervals), and we measured their motor activity and brain monoamine levels. Female dark-adapted rats (poor metabolizers of debrisoquine) showed a more pronounced and more maintained reduction of their motor activity after treatment with MPTP. MPTP-treated, dark-adapted rats also had a depletion of noradrenaline in the diencephalon and a depletion of dopamine and serotonine and their respective metabolites in the limbic system when compared with the other two species. These results suggest that oxidative polymorphism of debrisoquine plays a role in the acute effects of MPTP.

Genetic analysis of the interethnic difference between Chinese and Caucasians in the polymorphic metabolism of debrisoquine and codeine

The Far Eastern and Caucasian populations are strikingly different with respect to the debrisoquine/sparteine hydroxylation polymorphism. The number of poor metabolizers, as defined for Caucasians, is very low among Chinese and Japanese. We investigated the molecular basis for this difference by analysis of the CYP2D6 gene in 115 Chinese subjects, combined with phenotypic classification of codeine and debrisoquine metabolism. A correlation between the rates of metabolism of these two drugs and genotype, as analyzed by RFLP using XbaI, was observed among the Chinese. A high frequency (37%) of alleles indicative of gene insertions (reflected by XbaI 44kb fragments) was recorded in the Chinese, but was not associated with the poor metabolizer phenotype, as it is in Caucasians. PCR amplification of part of the CYP2D6 gene with mutation specific primers for CYP2D6A (29A) and CYP2D6B (29B) allelic variants revealed that the XbaI 44kb fragment in Chinese apparently contains a functional CYP2D6 gene, in contrast to the situation among Caucasians. The results provide a molecular explanation of the interethnic difference in the metabolism of drugs affected by the debrisoquine hydroxylation polymorphism.

Cytochrome P450IID subfamily in non-human primates: Catalytical and immunological characterization

Interindividual variations of debrisoquine metabolism was recently identified in non-human primates tested in vivo. The catalytical and immunological characterization of cytochrome P450IID subfamily was undertaken in hepatic microsomes from extensive metabolizer primates. The NADPH/ O 2 mediated metabolism of debrisoquine, dextromethorphan and bufuralol was similar to the kinetics reported in humans. The CuOOH mediated metabolism of bufuralol suggested that at least two enzymes are responsible for bufuralol 1'-hydroxylation. Eleven compounds were tested for their capacity to modify P450IID function vitro. Eight competitive inhibitors of P450IID6 in man were all and exclusively competitive inhibitors of P450IID subfamily in non-human primates. Quinidine, which is the strongest competitive inhibitor in man, exhibited the higher inhibitory potency in monkey ( K i = 0.75 μM). Anti-LKM antibody against P450IID subfamily cross-reacted with two proteins of 49 and 47 kDa, and sera containing anti-LKM antibody against these two proteins inhibited dextrorphan formation in vitro. These data provide evidence for catalytical and immunological similarities between human and monkey microsomes and indicate that the primate system could be a model for enzymatic studies of P450IID.