Can the genotype or phenotype of two polymorphic drug metabolising cytochrome P450-enzymes identify oral lichenoid drug eruptions?

BACKGROUND: Lichenoid drug eruptions (LDE) in the oral cavity are adverse drug reactions (ADR) that are impossible to differentiate from oral lichen planus (OLP) as no phenotypic criteria exist. Impaired function of polymorphic cytochrome 450-enzymes (CYPs) may cause increased plasma concentration of some drugs resulting in ADR/LDE. In an earlier study we did not find more patients with OLP (OLPs) with impaired CYP-genotype. OBJECTIVES: To test if more OLPs have an impaired CYP-phenotype than to be expected from the CYP-genotype and to find clinical criteria characterising oral LDE. METHODS: One hundred and twenty OLPs were genotyped for the most common polymorphisms of CYP2D6 and CYP2C19 that result in impaired function. One hundred and ten did a phenotype test of both enzymes. The exposure to drugs and polypharmacy and the CYP metabolism of the drugs were evaluated. The OLP manifestations were registered. RESULTS: The only difference in OLP manifestations was that patients with a CYP2D6 genotype with less than two fully functional alleles presented more asymmetrical OLP distribution in particular in non-medicated patients (P < 0.05). No more OLPs than expected from the genotype had a phenotype with reduced function. However, the established phenotypic categories could not differentiate between the genotypes with two or one fully functional allele. Nevertheless, among the patients with a phenotype with normal function the patients with only one functional allele had a statistically significant higher metabolic ratio compared to patients with two fully functional alleles (P < 0.05). CONCLUSION: It was not possible to identify LDE by impaired function of polymorphic CYPs.

Pharmacokinetics of therapeutic doses of tropisetron in healthy volunteers.

AIMS: To establish the bioavailability of tropisetron (5 mg) administered orally as capsule compared with 2 mg given intravenously. METHODS: Using a randomized crossover design, 18 healthy volunteers received a single oral dose of tropisetron (5 mg) and an intravenous bolus of tropisetron (2 mg) separated by a wash-out period of 1 week. Plasma concentrations of tropisetron were determined by h.p.l.c. and the pharmacokinetic parameters were estimated. RESULTS: The mean pharmacokinetic parameters for 5 mg tropisetron given orally were Cmax 3.46 ng ml(-1), t(max) 2.6 h, t(1/2) 5.7 h and AUC(0,infinity) 32.9 ng ml(-1) h. After intravenous administration initial plasma concentration was 15.1 ng ml(-1), t(1/2) 5.6 h, AUC(0,infinity) 20.7 ng ml(-1) h, V 678 l and CL 1800 ml min(-1). An inverse correlation was demonstrated between CYP2D6 activity, measured by the sparteine metabolic ratio, and the bioavailability (mean 0.60, range 0.27-0.99) of oral tropisetron. CONCLUSIONS: Tropisetron exhibits a wide range of oral bioavailability at therapeutic doses, which is mainly determined by CYP2D6 activity.

Influence of CYP2D6 genotype and medication on the sparteine metabolic ratio of psychiatric patients.

OBJECTIVE: To investigate the influence of CYP2D6 genotype and medication on the reliability of phenotyping in a naturalistic setting of psychiatric inpatients. METHODS: The phenotype of 160 psychiatric inpatients was estimated by taking the urinary metabolic ratio (MR) of the concentrations of sparteine to 2- and 5-dehydrosparteine. Genotyping identified CYP2D6*1, *3, *4, *5 and *6 alleles as well as duplication of the CYP2D6 gene. All subjects underwent detailed drug history including drug dose and therapeutic drug monitoring to control compliance and abuse of other psychotropic drugs. These data were compared with those of 195 unmedicated healthy Germans. RESULTS: The cumulative distribution of the MR in patients showed a significant shift to higher MR when compared with that of healthy subjects (P < or = 0.001). Patients medicated either with selective serotonin reuptake inhibitors (SSRIs, P < or = 0.001), antipsychotic drugs (P= 0.002) or other drugs known to be substrates or inhibitors of CYP2D6 (P < or = 0.001) showed a significantly higher mean MR than unmedicated patients. However, there was no significant effect of tricyclic antidepressants on the MR. Healthy subjects with CYP2D6 deficiency were separated by a MR of greater than 20 from those who expressed functional CYP2D6. Seven patients carrying at least one functional CYP2D6 allele revealed a MR of greater than 20, indicating the occurrence of phenocopying. CONCLUSION: The results of phenotyping may be falsified by drugs known to be substrates or inhibitors of CYP2D6; thus, this method is not sufficiently reliable. However, since we observed the phenomenon of phenocopying only in patients treated with a SSRI such as fluoxetine, fluvoxamine or paroxetine, we conclude that sparteine phenotyping of medicated patients detects CYP2D6 deficiency correctly, provided that patients treated with these SSRIs are excluded.

Absorption and disposition of levocetirizine, the eutomer of cetirizine, administered alone or as cetirizine to healthy volunteers.

The primary objective of the present study was to compare the absorption and disposition of levocetirizine, the eutomer of cetirizine, when administered alone (10 mg) or in presence of the distomer. An additional objective was also to investigate the configurational stability of levocetirizine in vivo in humans. The study was performed in a randomized, two-way cross-over, single-dose design with a wash-out phase of 7 days between the two periods. A total of 12 healthy male and 12 healthy female volunteers were included in the study. Bioequivalence can be concluded from the analysis of the pharmacokinetic parameters of levocetirizine when administered alone or as the racemate cetirizine. No chiral inversion occurs in humans when levocetirizine is administered, i.e. there is no formation of the distomer. When comparing the pharmacokinetic characteristics of levocetirizine and the distomer, the apparent volume of distribution of the eutomer is significantly smaller than that of the distomer (0.41 and 0.60 L/kg, respectively). For an H1-antagonist a small distribution volume can be considered as a positive aspect, both in terms of efficacy and safety. Moreover the non-renal clearance of levocetirizine is also significantly lower than that of the distomer (9.70 and 28.70 mL/min, respectively), which constitutes an additional positive aspect particularly as far as metabolism-based drug interactions are concerned. The information collected in the present study on the pharmacokinetics of levocetirizine and the distomer provide additional reasons for eliminating the distomer and developing levocetirizine as an improvement on cetirizine.

Phenotype of cytochrome P450 CYP2D6 in patients with familial adenomatous polyposis.

This paper describes an attempt to establish the distribution of the oxidative phenotype of sparteine in patients with familial adenomatous polyposis (FAP). The oxidative polymorphism of sparteine was determined in 30 patients with FAP. One hundred and twenty-six normal subjects were examined as a control group. Subjects with urinary metabolic ratios (MR) greater than 20 (the metabolic ratio of sparteine/dehydrosparteines excreted in urine) were defined as poor metabolizers of sparteine. None of the patients were classified as poor metabolizers of sparteine, although 5 control subjects were. No significant differences were found in the distribution of frequencies between patients and control subjects. However, there was a higher metabolic ratio (mean 1.58 +/- 1.13) in 5 patients with malignant changes in large bowel adenomas compared with other FAP patients without malignant changes (mean MR 0.89 +/- 0.66).

In-vivo indices of enzyme activity: the effect of renal impairment on the assessment of CYP2D6 activity.

Urinary drug:metabolite ratios and urinary recoveries of metabolites, have been used to assess specific enzyme activity non-invasively in vivo. These indices are potentially confounded by the effect of renal function. A recent study of the effects of renal impairment has found discrepancies between different indices used to mark CYP2D6 activity based on sparteine and dextromethorphan urinary recoveries. We have re-examined these experimental data from a theoretical viewpoint. The results suggest that the dependence of fractional urinary recovery of metabolites on renal function varies with the importance of different elimination routes. Therefore, no consistent behaviour of this index is expected when markers with different pharmacokinetics are used. However, when collecting the urine until full recovery of drug and metabolite, drug:metabolite ratios show the same degree of dependence on renal function regardless of the marker. The application of the analysis to the experimental data indicates that CYP2D6 activity is compromised in parallel with deterioration of renal function.

[Clinical significance of oxidation and acetylation genetic polymorphism in patients with Parkinson's disease]. / Kliniczne znaczenie polimorfizmu oksydacji sparteiny i acetylacji sulfadimidyny u chorych na chorobe Parkinsona.

The relationship between genetically determined polymorphic oxidation and acetylation and susceptibility to some disease has aroused much interest. The aim of our study was to evaluate whether patients with Parkinson's disease differ from healthy persons in their ability to oxidize sparteine and acetylate sulfadimidine as model drugs. Oxidation and acetylation phenotypes were estimated in 50 patients with Parkinson's disease. The control group consisted of 160 healthy volunteers for comparison of oxidation phenotype and 60 healthy volunteers for comparison of acetylation phenotype. The phenotyping of oxidation revealed two distinct populations among 50 patients with Parkinson's disease: 47 persons (94%) were extensive metabolizers of sparteine and 3 persons (6%) were poor metabolizers. In 160 healthy persons, 146 persons (91.2%) were extensive metabolizers of sparteine and 14 persons (8.8%) were poor metabolizers. The difference between frequency distribution of PMs and EMs in healthy persons and in patients with Parkinson's disease was not statistically significant. The phenotyping of acetylation showed among 50 patients with Parkinson's disease 38 persons (76%) slow acetylators and 12 persons (24%) rapid acetylators. In 60 healthy volunteers the phenotype of slow acetylation was observed in 29 persons (48.3%) and rapid acetylation in 31 persons (51.7%). The prevalence of slow acetylators among patients with Parkinson's disease in comparison to healthy volunteers was statistically significant (chi 2 = 8.7677/p < 0.003). The results of our study may suggest that the slow acetylation phenotype is associated with increased risk of the development of Parkinson's disease.

Codeine in post-operative pain. Study of the influence of sparteine phenotype and serum concentrations of morphine and morphine-6-glucuronide.

OBJECTIVE: Within the past decade, human experimental pain studies have supported the 50-year-old hypothesis that codeine is a prodrug, which has to be converted to morphine to exert an analgesic effect. This study aimed at evaluating the impact of sparteine phenotype and serum concentrations of morphine on the efficacy of codeine in post-operative pain. METHODS: Eighty-one patients with a pain rating of 3 or more on a 0-10 numerical rating scale 0.5 h after surgery were included in the study. The patients were given an oral dose of 100 mg codeine and rated pain with the numerical rating scale 0.5 h and 1 h after medication. Blood for determination of serum concentration of codeine and its metabolites was collected 1 h after medication, and a 12-h urine sample after administration of 100 mg sparteine was used to determine the sparteine phenotype. RESULTS: Eight patients were poor metabolizers and 66 were extensive metabolizers of sparteine, while the urine samples for the remaining seven patients were lost. In 22 patients, including the eight poor metabolizers, the serum concentrations of both morphine and morphine-6-glucuronide (M6G) were below the limit of determination of the assay, i.e. 1.5 nmol x l(-1) and 2 nmol x l(-1), respectively. A sum of the concentration of these two substances below 10 nmol x l(-1) was found in an additional eight patients. The sum of differences between pre- and post-operative pain ratings did not differ between the two phenotypes (P = 0.60), whereas the 30 patients with serum concentrations of morphine plus M6G below 10 nmol x l(-1) had a marginally significant lower sum than the 51 patients with higher levels of these substances (median 1.5 vs 2.5, P = 0.058). CONCLUSION: A low serum concentration of morphine and M6G seems to be common in patients treated with codeine for post-operative pain, and low concentrations of these active substances may be related to decreased efficacy of codeine.

Comparison of three CYP2D6 probe substrates and genotype in Ghanaians, Chinese and Caucasians.

The ability to metabolize CYP2D6 substrates sparteine, debrisoquine, and dextromethorphan was studied in healthy Caucasian (n = 20), Ghanaian (n = 21), and Chinese (n = 22) CYP2D6 extensive metabolizers. Genotype analysis for the CYP2D6*1, *3, *4, *5, *9, *10, and *17 alleles was performed. Interethnic differences in the disposition of the probe drugs were found among the extensive metabolizers; extensive metabolizer status was confirmed by phenotype and genotype analysis. The mean metabolic rate was lower for Caucasians than for Ghanaians for sparteine (P < 0.02) and for both Ghanaians and Chinese for debrisoquine (P < 0.02). Correlation comparisons resulted in lower pairwise correlation coefficients in Ghanaians compared with Chinese and Caucasians for every combination of probe substrates. In addition, in Chinese and Caucasians, metabolic rates for each pair of probe drugs were significantly correlated (P < 0.002), but in Ghanaians the dextromethorphan metabolic rates were not correlated to either sparteine or debrisoquine (P < 0.05). Even when only those with a CYP2D6*1/*1 genotype were included in the correlation calculations, the Ghanaians had very low correlation coefficients (r(s) - 0.02-0.2, n = 9); much lower than those found in Caucasian (r(s) 0.78-0.92, n = 14) or Chinese (r(s) 0.54-0.96, n = 7) individuals. Quinidine had significantly less affect on sparteine metabolic rates in Ghanaians than both Caucasians and Chinese (P < 0.02). In addition, five of the 21 Ghanaian individuals had dextromethorphan metabolic ratios which were unaffected by quinidine. These individuals also had differences in urinary recovery of dextromethorphan and its metabolites when compared to the other Ghanaian individuals. These results confirm the large ethnic differences in probe drug metabolism and quinidine sensitivity among these ethnic groups. They also suggest that the Ghanaians have an additional unidentified allele(s) with altered substrate specificity and quinidine sensitivity which is currently genotyped as CYP2D6*1.

A new CYP2D6 allele with a nine base insertion in exon 9 in a Japanese population associated with poor metabolizer phenotype.

The CYP2D6 gene of a Japanese sparteine poor metabolizer (PM, proband) showing a urinary sparteine metabolic ratio of 31.6 was analysed, and a heterozygous CYP2D6(D), a deletional type, was found by restriction fragment length polymorphism analysis with Xba I enzyme. The PM did not have any other previously described mutations in the CYP2D6 gene causing the loss of catalytic activity of the CYP2D6 enzyme. Thus, a possible new allele(s) responsible for the PM phenotype was analysed. The results indicated that the PM possessed a new 9-base insertion in exon 9, designated CYP2D6(J9). The CYP2D6(J9) and CYP2D6(D) alleles were clarified to be inherited from the mother [2D6(W)/2D6(J9)] and the father [2D6(W)/2D6(D)], respectively. The 9-base insertion caused a large increase in the apparent K(m) value for bufuralol 1'-hydroxylation as examined by expression of the enzyme protein in yeast. Four of 300 Japanese carried a heterozygous CYP2D6(J9) allele (0.7%, 4/600 chromosomes) as determined by a polymerase chain reaction analysis.

Metabolic ratios of four probes of CYP2D6 in Turkish subjects: a cross-over study.

The relationships among the metabolic ratios for the standard probe drugs of CYP2D6 activity, such as debrisoquine, sparteine, metoprolol and dextromethorphan, were studied in 32 Turkish subjects. All subjects were randomly selected according to their phenotypes from a group of 111 Turkish subjects whose oxidation status had been tested for debrisoquine previously. All subjects were given a 10 mg debrisoquine tablet, a 100 mg sparteine tablet, a 100 mg. metoprolol tablet and a 20 mg dextromethorphan capsule orally with a wash-out period of at least 1 week between each probe administration. Metabolic ratios were calculated as percentage of dose excreted as parent drug/percentage of dose excreted as its hydroxymetabolite of parent drug in 0-8 h urine. Three poor metabolisers (PM) of debrisoquine were identified. They were also PMs of the other test probes and no misclassification by the 4 phenotyping methods was observed. All six correlations among the metabolic ratios of the 4 probe drugs assessed by Spearman's rank test were highly significant (P < 0.001). The present findings indicate that the oxidative metabolism of debrisoquine, sparteine, metoprolol and dextromethorphan is catalysed by the same cytochrome P450 in the Turkish subjects.

Use of site-specified tritium labelling to confirm the formation of 17-oxosparteine as a minor urinary metabolite of sparteine in man.

1. The synthesis of [17,17-3H2]-sparteine and its oral administration has enabled the specific identification of 17-oxosparteine as a minor urinary metabolite (approximately 1% dose) in two healthy male volunteers.

Assessment of individual CYP2D6 activity in extensive metabolizers with renal failure: comparison of sparteine and dextromethorphan.

OBJECTIVES: To examine whether the variability of CYP2D6 activity in patients with chronic renal failure can be assessed, particularly among subjects with the extensive metabolizer phenotype, by use of standard in vivo indexes of CYP2D6 activity derived from oral administration of dextromethorphan and sparteine. METHODS: A single 100 mg oral dose of sparteine and a single 40 mg oral dose of dextromethorphan were administered on two occasions to 12 patients with chronic renal failure (creatinine clearance ranging from 20 to 70 ml/min) and 12 age- and sex-matched healthy subjects. Sparteine clearances, sparteine metabolic ratio, and urinary recovery of dextrorphan were calculated. Patients and healthy control subjects were not selected on the basis of their CYP2D6 phenotypes. RESULTS: Chronic renal failure was associated with a decrease in sparteine partial metabolic clearance to dehydrosparteine (median of 322 ml/min and range of 62 to 670 ml/min in patients with renal failure versus median of 635 ml/min and range of 77 to 1276 ml/min in normal subjects; p < 0.02). Sparteine apparent oral clearance (p < 0.03) and renal clearance (p < 0.001) decreased in patients with renal failure. However, sparteine metabolic ratio was not significantly altered in patients with renal failure and showed that all patients were extensive metabolizers of sparteine. Although fractional urinary excretion of dextrorphan decreased in patients with renal failure (median, 24.4%; range, 9.7% to 55.9%) compared with control (median, 47.5%; range, 24.1% to 72.1%) (p = 0.02), it also showed that all subjects were extensive metabolizers of dextromethorphan. The amount of dextromethorphan excreted in urine correlated with creatinine clearance independently from CYP2D6 activity measured as sparteine partial metabolic clearance. However, it did not correlate with sparteine metabolic ratio or with fractional urinary excretion of dehydrosparteine. CONCLUSION: Assessment of CYP2D6 activity by use of dextromethorphan and sparteine is possible in extensive metabolizer patients with chronic renal failure. However, in these subjects, dextromethorphan and sparteine do not reflect CYP2D6 activity in the same way.

Sparteine metabolism in a Nigerian population.

The oxidative metabolism of sparteine has been investigated in a Nigerian population. The distribution of metabolic capacities was shown to be skewed with two subjects (2/97, 2.1%) being relatively deficient in their ability to produce the dehydrometabolites. These observations afford evidence that sparteine oxidation is under polymorphic control in Nigerians.

Oxidative phenotype polymorphism (P450 2D6) and metabolism of toluene.

Oxidative phenotype P-450 2D6 was examined using sparteine test in 3 groups of persons to determine if there is a coincidence in the defect of the oxidative biotransformation of sparteine and impaired oxidation of toluene, which could explain interindividual differences in the amounts of hippuric acid in the urine in exposed persons. The following groups of persons were examined: 30 rotogravure printers exposed to toluene vapors at concentrations of 8-307 ppm; 20 workers, 2 months after the cessation of the long-term exposure to toluene at concentrations of 104-1,170 ppm; 48 healthy volunteers with no exposure to toluene. Among the 98 persons 5 poor metabolizers (PMs) of sparteine were found, none in the group of printers exposed to toluene. In the experimental exposure chamber 5 PMs and 6 extensive metabolizers (EMs) were exposed to toluene concentration of 245 ppm for 5 hours. Hippuric acid and o-cresol in the urine, and toluene both in blood and in alveolar air were measured. However, no significant differences were found in either of these parameters between the PM and EM groups. Thus, the sparteine test does not appear to be applicable in the identification of persons with higher risk arising from toluene exposure.

Mechanism of cytochrome P450 2D6-catalyzed sparteine metabolism in humans.

Two different reaction mechanisms for the formation of the two human enamine-structured sparteine metabolites by cytochrome P450 2D6 have been discussed in the literature. These mechanisms are either initial one-electron oxidation of N1 of sparteine followed by deprotonation of the aminium radical cation, resulting in the formation of different carbon radicals and oxygen rebound of the carbon radicals, or oxidation of the carbon atoms adjacent to N1 by the enzyme, directly producing the respective carbon radicals. With a spectrum of deuterium-labeled isotopomers of sparteine, stereoselectivity and kinetic isotope effects of human sparteine metabolism were investigated by in vitro and in vivo experiments and were compared with chemical oxidation of 17-oxosparteine. These experiments revealed that the major human sparteine metabolite 2,3-didehydrosparteine is formed via highly stereoselective abstraction of the 2 beta-hydrogen atom; the deuterium label was completely retained during metabolism when 2R-[2H]sparteine was used as substrate. Chemical oxidation of 17-oxosparteine by Ce4+, as a model for one-electron oxidation of N1 of a sparteine-like structure, resulted in the sole formation of the 5,6-unsaturated enamine, and no 2,3-unsaturated enamine, structurally equivalent to the human major metabolite, was found. An unequivocal discrimination between the two possible reaction mechanisms was not possible by simple interpretation of the magnitude of the kinetic deuterium isotope effects. However, results of competitive and noncompetitive experiments revealed the presence of a nondissociative enzymatic mechanism for the formation of the two sparteine metabolites, i.e., the sparteine molecule that is bound to the substrate binding site of cytochrome P450 2D6 performs orientational changes without dissociating from the activated enzyme/substrate complex before the product-determining first irreversible reaction step. These results agree with the hypothesis that sparteine metabolism proceeds by direct carbon oxidation. Because electron transfer from amines to P450 may occur over some distance, the possibility of a sequential electron-proton transfer reaction during sparteine metabolism cannot be ruled out completely as an alternative reaction mechanism for sparteine metabolism.

Imipramine metabolism in relation to the sparteine and mephenytoin oxidation polymorphisms--a population study.

1. Sparteine and mephenytoin phenotyping tests were carried out in 327 healthy Danish subjects. Two weeks later each subject took 25 mg imipramine followed by urine collection for 24 h. The urinary content of imipramine, desipramine, 2-hydroxy-imipramine and 2-hydroxy-desipramine was assayed by h.p.l.c. 2. The medians of the hydroxylation ratios (i.e. 2-hydroxy-metabolite over parent compound) were 6 to 14 times higher in 300 extensive metabolizers of sparteine (EMs) as compared with 27 poor metabolizers (PMs), but none of the ratios separated the two phenotypes completely. 3. There were 324 EM of mephenytoin (EMM) and three PM (PMM) in the sample. The demethylation ratios between desipramine, 2-hydroxy-desipramine and their corresponding tertiary amines showed statistically significant correlations with the mephenytoin S/R isomer ratio (Spearman's rs: -0.20 and -0.27, P < 0.05). 4. The demethylation ratios were higher in 80 smokers than in 245 non-smokers. This indicates that CYP1A2, which is induced by cigarette smoking, also catalyzes the N-demethylation of imipramine. 5. CYP2D6 genotyping was carried out by PCR in 325 of the subjects, and the D6-wt allele was amplified in 298 EMs, meaning that they were genotyped correctly. One PMs was D6-wt/D6-B, another PMs had the genotype D6-wt/ and hence both were misclassified as EMs. The remaining 25 PMs were D6-A/D6-B (n = 5), D6-B/ (n = 18) or D6-D/D6-D (no PCR amplification, n = 2).(ABSTRACT TRUNCATED AT 250 WORDS)

S-mephenytoin, sparteine and debrisoquine oxidation: genetic polymorphisms in a Turkish population.

A mephenytoin test was carried out in 106 unrelated healthy Turkish volunteers. Racemic mephenytoin was coadministered with either debrisoquine or sparteine. The S/R mephenytoin ratio ranged from < 0.1 to 0.73 in 105 subjects, accordingly phenotyped as extensive metabolisers. One subject had an S/R mephenytoin ratio of 1.02, showing that he was a poor metaboliser of mephenytoin (0.94%, confidence interval 0.25% and 13.65%). In 48 subjects, the metabolic ratios of debrisoquine and sparteine were correlated significantly (rs = 0.61, P < 0.001).

Rapid determination of sparteine and its metabolites in urine.

A method is presented for the isolation, separation and determination of sparteine and its metabolites in urine. The isolation is based on rapid extraction with dichloromethane and pentane in a glass separator. For the separation and determination, capillary gas chromatography with nitrogen-phosphorus detection was used. The recovery of the method ranged from 81.6% to 94.8%, and the limit of determination varied between 0.2 and 0.5 microgram ml-1. For quantification, 17-ethylsparteine was used as the internal standard.