Sparteine Oxidation Research Articles

A Natural Variant of the Heme-Binding Signature (R441C) Resulting in Complete Loss of Function of CYP2D6

A new variant allele CYP2D6*62 (g.4044C>T; R441C) of the drug-metabolizing cytochrome P450 (P450) CYP2D6 was identified in a person with reduced sparteine oxidation phenotype, which was unexpected based on a genetic CYP2D6*1A/*41 background. The recombinantly expressed variant protein had no activity toward propafenone as a result of missing heme incorporation. Sequence alignment revealed that the positively charged R441 residue is part of the heme-binding signature but not strictly conserved among all the P450s. A compilation of described P450 monooxygenase variants revealed that other enzymes can functionally tolerate even nonconservative amino acid changes at the corresponding position (i.e., the invariant cysteine 2). This suggests that heme binding in mammalian P450s depends not only on the integrity of the heme-binding signature but also on other family- and subfamily-specific sequence determinants.

A silent mutation (2939G>A, exon 6; CYP2D6*59) leading to impaired expression and function of CYP2D6

We analyzed CYP2D6 in two individuals characterized by impaired sparteine oxidation (intermediate metabolizer phenotype) and genotype 2D62/4 (1661G>C; 2850C>T; 4180G>C) usually associated with normal function. Full genomic sequencing and haplotype analysis confirmed the previously identified silent mutation 2939G>A in exon 6 (former allele variant 2D62J, now termed 2D659), as well as an additional novel 2291G>A change in intron 4. Transient expression in Huh7 hepatoma cells of the entire CYP2D6 gene of constructs carrying either both or only the 2939G>A change resulted in about three-fold reduced levels of mRNA, immunoreactive 2D6 protein and propafenone hydroxylase activity. These data demonstrate profound effects of a silent mutation on expression and function of CYP2D6, resulting in impaired drug oxidation phenotype. The 2939G>A single nucleotide polymorphism in exon 6 was present heterozygously in two individuals out of 308 (0.65%), corresponding to an allele frequency of 0.3%. Genotyping for this mutation thus improves phenotype-genotype correlation for CYP2D6 and may help to predict adverse drug treatment events.

The effect of selective serotonin re‐uptake inhibitors on cytochrome P4502D6 (CYP2D6) activity in human liver microsomes

Inhibition of human cytochrome P4502D6 (CYP2D6)-catalysed metabolism can lead to clinically significant alterations in pharmacokinetics. Since there is evidence that the selective serotonin reuptake inhibitor (SSRI) class of antidepressant drugs might inhibit CYP2D6, the effects of five SSRIs on human liver microsomal CYP2D6 activity were compared with each other and with three tricyclic antidepressant drugs. On a molar basis, paroxetine was the most potent of the SSRIs at inhibiting the CYP2D6-catalysed oxidation of sparteine (Ki = 0.15 microM), although fluoxetine (0.60 microM) and sertaline (0.70 microM) had Ki values in the same range. Fluvoxamine (8.2 microM) and citalopram (5.1 microM) also inhibited CYP2D6 activity. The major circulating metabolites of paroxetine in man produced negligible inhibition. In contrast, norfluoxetine the active metabolite of fluoxetine, was a potent CYP2D6 inhibitor (0.43 microM). CYP2D6 activity was also diminished by the tricyclic antidepressant drugs clomipramine (2.2 microM), desipramine (2.3 microM) and amitriptyline (4.0 microM). These findings suggest that compounds with SSRI activity are likely to interact with human CYP2D6 in vivo with the potential of causing drug interactions.

Dose‐Dependent Inhibition of the CYP2D6 Catalyzed Oxidation of Sparteine by Mepyramine in Healthy Volunteers

Dose‐Dependent Inhibition of the CYP2D6 Catalyzed Oxidation of Sparteine by Mepyramine in Healthy Volunteers

Elucidation of the genetic basis of the common 'intermediate metabolizer' phenotype for drug oxidation by CYP2D6.

A subgroup of 10-15% of Caucasians are termed phenotypical 'intermediate metabolizers' of drug substrates of CYP2D6 because they have severely impaired yet residual in-vivo function of this cytochrome P450. Genotyping based on the currently known CYP2D6 alleles does not predict this phenotype satisfactorily. A systematic sequencing strategy through 1.6 kb of the CYP2D6 5'-flanking sequence revealed six mutations of which three were exclusively associated with the functional CYP2D6*2 allele (-1496 C to G; -652 C to T; and -590 G to A), two were associated with the nonfunctional *4 and with the functional *10-alleles (-1338 C to T and -912 G to A) and one (-1147 A to G) was seen in all *2, *4 and *10-alleles investigated. The -1496 C to G mutation was found to be polymorphic within CYP2D6*2 alleles. In a family study, the wild-type CYP2D6 *2[-1496 C] and the novel variant [-1496 G] allele co-segregated with lower and higher CYP2D6 in-vivo function, respectively, as shown by phenotyping using sparteine as probe drug. In a representative population sample selected for genotypes comprising one CYP2D6*2 and one non-functional allele, the median urinary metabolic ratio (MRs) for sparteine oxidation was 4.4-fold reduced in individuals with the variant allele (*2[-1496 G], MRs = 0.53, n = 27) compared with individuals lacking the mutation (*2[-1496 C], MRs = 2.33, n = 12; P < 0.0001). The mutation -1496 C to G has an estimated frequency of approximately 20% in the general population and allows establishment of a genotype for the identification of over 60% of intermediate metabolizers in Caucasian populations.

Polymorphic CYP2D6 mediates O-demethylation of the opioid analgesic tramadol.

This study was designed to investigate whether the in vivo metabolism of tramadol was influenced by CYP2D6 polymorphism. The extent of tramadol O- and N-demethylation was calculated by determining the amounts of tramadol and O- and N-desmethyltramadol in 24 h urine after ingestion of a test dose of tramadol. The O- and N-demethylation rates were calculated by dividing the 24-h urinary excretion amount of tramadol by that of O- and N-desmethyltramadol. Volunteers were phenotyped for CYP2D6 polymorphism using sparteine as an in vivo probe. High correlation was found between tramadol-O-demethylation and sparteine oxidation in 71 extensive metabolizers of sparteine (rs = 0.544). The mean metabolic ratio of tramadol O-demethylation was significantly higher in poor metabolizers of sparteine than in extensive metabolizers (4.4 vs 0.8). These in vivo results confirm that tramadol O-demethylation is carried out to a large extent by the polymorphic CYP2D6.

Evaluation of genetically determined sparteine oxidation and sulfadimidine acetylation polymorphism in women with breast cancer

Abstract The relationship between genetically determined polymorphic oxidation and acetylation and susceptibility to neoplastic diseases has aroused much interest. The aim of our study was to evaluate whether women with breast cancer differ from healthy women in their ability to oxidize sparteine and acetylate sulfadimidine as model drugs. The results of our study revealed a predominance of the percentage of extensive metabolizers (EMs) of sparteine (97.8%) among 90 women with breast cancer in comparison to the percentage of EM (93.2%) in 74 healthy women. Among very extensive metabolizers of sparteine (VEM), with metabolic ratio (MR)

Imipramine metabolism in relation to the sparteine oxidation polymorphism--a family study.

The relationship between genetic polymorphism and imipramine metabolism has never been studied in a family study. A sparteine/mephenytoin test was carried out in 31 parents and 20 siblings of 18 Danish poor metabolizers of sparteine (PMs). One week 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 HPLC. There were 10 PMs (20%; 9.8-33%, 95% confidence interval) and 41 extensive metabolizers of sparteine (EMs) among parents and siblings. In 26 of the 28 PMs among probands and relatives, there were concordance between phenotype and genotype: D6-A/D6-D (n = 2), D6-A/D6-B (n = 5), D6-B/(n = 15) or D6-B/D6-D (n = 4). Two PMs were apparently heterozygous (EMs), D6-wt/D6-B. Accordingly, based on the present sample of 28 PMs the specificity of the genotype test was 100% and the sensitivity was 92.9%. Two EMs were homozygous dominant D6-wt/and 39 were heterozygous EMs; D6-wt/D6-D (n = 5), D6-wt/D6-B (n = 27), D6-wt/D6-A (n = 6), D6-wt/D6-wt* (unknown mutation) (n = 1). As previously reported in a population study the hydroxylation ratios (i.e. 2-hydroxymetabolite over parent compound) of imipramine were much lower in PMs than in EMs. This and the pedigrees confirmed the co-segregation of sparteine oxidation, imipramine 2-hydroxylation and the CYP2D6 genotype. None of the hydroxylation ratios could separate EMs and PMs completely, mainly because the 2-hydroxylation of imipramine also depends on P450s other than CYP2D6.

The influence of environmental and genetic factors on CYP2D6, CYP1A2 and UDP-glucuronosyltransferases in man using sparteine, caffeine, and paracetamol as probes

The impact of gender, use of oral contraceptive steroids (OCS), coffee consumption and of smoking on the metabolism of sparteine, caffeine, and paracetamol was studied in 194 randomly selected subjects (98 male and 95 female). Thirty-eight of the male volunteers were cigarette smokers, 40 of the female subjects were smokers and/or users of OCS. The metabolic ratio of sparteine oxidation (MRs) showed a trimodal distribution. 7.7% of the subjects had a MRs > 20 and thus were poor metabolizers (PMs). Within the extensive metabolizer (EM) subjects, a distinct subgroup accounting for 11% was observed with 20 > MRs > 1.2. Six of the 15 phenotypical PMs were heterozygous EMs by genotyping. This indicates the existence of one or several CYP2D6 mutations which cannot be identified by the currently employed genotyping methods. In each subgroup, i.e. smokers/OCS and non-smokers/non-OCS, the cumulative frequency distribution of the heterozygous (wt/B) phenotype caused a shift to higher MRs compared with the wild-type homozygotes (wt/wt). Thus, for the in vivo activity of CYP2D6, genetic determinants prevail over environmental factors. Smoking, use of oral contraceptive steroids, caffeine consumption, or gender had no influence on sparteine metabolism. The distribution of the paracetamol glucuronide/paracetamol metabolic ratio appeared to be unimodal although skewed. Glucuronidation capacity was clearly affected by gender, OCS use and smoking. It was higher in male than in female subjects. Male smokers had the highest, and female non-smokers/non-OCS users the lowest metabolic ratio. CYP1A2 activity, as determined by a caffeine metabolic ratio ((AFMU + 1X + 1U)/1, 7U), was multimodally distributed and was clearly increased in smokers. It was significantly correlated to paracetamol glucoronidation in male heavy smokers (r=0.85), suggesting an element of co-regulation of CYP1A2 and of paracetamol conjugating UDP-glucuronosyltransferase isozymes, including UGTI.6.

Evolutionary pharmacogenetics of CYP2D6 in Ngawbe Guaymi of Panama: allele-specific PCR detection of the CYP2D6B allele and RFLP analysis

Gene cluster CYP2D controls the biosynthesis of enzyme CYP2D6, which is responsible for the polymorphic oxidation of sparteine, debrisoquine and related drugs. This cluster consists of the functional gene D6 and of two pseudogenes, D7 and D8. RFLP Bam HI analysis of CYP2D in 37 unrelated and eight related Ngawbe Guaymi Amerindians of Panama showed a polymorphism characterized by the presence of two alleles: 4.7 + 7.9 and 2.3 + 6.0 (frequencies: 0.63 and 0.37, respectively, n = 35 unrelated subjects). The possible genotypes for these alleles follow the Hardy-Weinberg distribution (chi 2 = 1.76; 0.10 < p < 0.25). All PMs of sparteine or debrisoquine (n = 7) were homozygotes for the second allele, but not all homozygotes (n = 10) were PMs, so there was not an exclusive association between the Bam HI genotype and the observed phenotype. A similar analysis with the endonuclease Xba I proved to be non-informative in relation to phenotype, since all subjects (n = 40) showed only the 29 kb allele. Allele-specific PCR studies of selected subjects indicated the existence of the CYP2D6B allele (freq = 0.17; C.I.95% = 0.085, 0.29; n = 30 unrelated subjects), in addition to the wild-type. The mutant CYP2D6B allele was responsible for the enzyme deficiency present in PMs. Its presence in Amerindians suggests that this allele has a far more ancient evolutionary history than previously thought. The over-all RFLP and PCR analyses point to a diminished genetic diversity for the Ngawbe subjects, consistent with their demographic history and population genetics.

Gas chromatography/mass spectrometry assays for the determination of debrisoquine and sparteine metabolites in microsomal fractions of rat liver

Debrisoquine and sparteine are prototype substrates of a genetic deficiency in cytochrome P450-dependent drug metabolism. Sensitive assays of in vitro oxidation of sparteine and debrisoquine are required for evaluation of this polymorphism. The activities were measured by quantitative analysis of 2-dehydrosparteine and 4-hydroxydebrisoquine production, respectively, using capillary column gas chromatography coupled with mass selective ion detection. With a single extraction, separation of parent drug, metabolite, and a suitable internal standard was readily achievable. Time-dependent production of both metabolites could be detected from as little as 40 μg of microsomal protein. Both activities showed a maximal activity with a 240-min incubation period. The ability to simultaneously quantify the parent drug and its metabolite suggests it would also be useful for evaluation of in vivo metabolism.

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.

The relationship between paroxetine and the sparteine oxidation polymorphism.

The relationship between the selective serotonin reuptake inhibitor paroxetine and the sparteine oxidation polymorphism was investigated in a combined single-dose (30 mg) and steady-state (30 mg/day for 2 weeks) study including a panel of nine extensive metabolizers and eight poor metabolizers of sparteine. The median area under the plasma concentration-time curve (AUC) after the first paroxetine dose was about seven times higher in poor metabolizers than in extensive metabolizers (3910 versus 550 nmol.hr/L), whereas at steady state the median AUCss tau interphenotype difference was only twofold (4410 versus 2550 nmol.hr/L). Plasma half-life and steady-state plasma concentration were significantly longer and higher, respectively, in poor metabolizers than in extensive metabolizers (41 versus 16 hours and 151 versus 81 nmol/L). Paroxetine pharmacokinetics were linear in poor metabolizers and nonlinear only in extensive metabolizers. Sparteine metabolic ratio (MR = 12 hour urinary ratio of sparteine/dehydrosparteine), increased during treatment with paroxetine in subjects who were extensive metabolizers, and after 14 days treatment two extensive metabolizers were phenotyped as poor metabolizers and the remaining extensive metabolizers were changed into extremely slow extensive metabolizers with sparteine MRs of 5.7 to 16.5. The inhibition of sparteine metabolism was rapidly reversed after cessation of paroxetine administration. In the poor metabolizers there were no significant changes in MRs during the study. It is concluded that paroxetine and sparteine metabolism cosegregates, but the interphenotype difference in metabolism was less prominent at steady state than after a single dose, presumably because of saturation of the sparteine oxygenase (CYP2D6) in subjects who were extensive metabolizers. Paroxetine is a potent inhibitor of sparteine oxidation by CYP2D6 in vivo.

Pharmacokinetics of the selective serotonin reuptake inhibitor paroxetine: nonlinearity and relation to the sparteine oxidation polymorphism.

Steady-state plasma concentrations of paroxetine were studied at five or more paroxetine dose levels (10 to 70 mg/day) in each of 13 extensive metabolizers of sparteine and at three or four dose levels (10 to 40 mg/day) in each of three poor metabolizers of sparteine, all treated for diabetic neuropathy symptoms. On a dose of 30 mg/day there was a 25-fold variation in steady-state concentrations (25 to 670 nmol/L). The upper extreme of this variation was made up by the poor metabolizers of sparteine and the lower extreme by some fast extensive metabolizers. Further, within the extensive metabolizer group, steady-state levels showed a significant, positive correlation with sparteine metabolic ratio at all dose levels. On increasing doses, a disproportionate increase in plasma drug levels was observed in the majority of patients. In nearly all extensive metabolizers the concentration-dose data were best described by a pharmacokinetic model assuming elimination by at least two kinetically distinct processes, one a high-affinity saturable process and one a low-affinity linear process. Estimates of clearance at low drug levels of the high-affinity process showed a significant negative correlation with the sparteine metabolic ratio. Clearance of the low-affinity process was not related to the metabolic ratio and was of the same magnitude in extensive and poor metabolizers. The data thus confirmed that the metabolism of paroxetine and sparteine cosegregates and indicated that the enzyme responsible for a high-affinity saturable paroxetine elimination process is identical with CYP2D6, the source of the sparteine oxidation polymorphism.

Sparteine and mephenytoin oxidation: Genetic polymorphisms in East and West Greenland

The oxidation of sparteine and mephenytoin was examined in a group of subjects living in Greenland: 300 in East Greenland and 171 in West Greenland. The distribution of the ratio between the chromatographic peak areas of S- and R-mephenytoin in the urine, the S/R ratio was clearly bimodal in both populations. Thus 9.3% of the East Greenlanders had S/R ratios of 0.9 or more and were phenotyped as poor metabolizers of mephenytoin. In the West Greenlanders, 2.9% of the sample had S/R ratios of 0.90 or more and were accordingly phenotyped as poor metabolizers. The intraethnic difference with regard to the frequency of the mephenytoin poor metabolizer is probably attributable in part to a much higher proportion of admixed Caucasian genes in the West Greenlanders than in the East Greenlanders. In both the East and the West Greenlanders, the sparteine metabolic ratio displayed marked interindividual differences without a clear bimodal distribution. Poor metabolizers arbitrarily defined as subjects with an metabolic ratio of 20 or more made up 3.3% of the East Greenlanders and 2.3% of the West Greenlanders, but the difference between the two groups was not statistically significant.

Oxidation of reduced haloperidol to haloperidol: involvement of human P450IID6 (sparteine/debrisoquine monooxygenase).

1. The conversion of haloperidol (HAL) to reduced haloperidol (RHAL) and then back to HAL has been established in vivo and observed in psychiatric patients. The reduction of HAL to RHAL is known to be catalysed by a ketone reductase, while the nature of oxidation back to HAL is the subject of the present study. 2. We examined the in vitro oxidation of RHAL to HAL in human livers. The activity was microsomal and evidence is presented to suggest that the sparteine/debrisoquine metabolizing isoenzyme P450IID6 contributes to this oxidation. 3. Reciprocal inhibition studies between RHAL and sparteine, a specific substrate for cytochrome P450IID6, indicated that both compounds compete for the same binding site. Quinidine, the most specific inhibitor for this cytochrome P450 potently inhibited the oxidative conversion of reduced haloperidol to haloperidol. A significant correlation (rs = 0.62, P less than 0.01) was found between RHAL oxidation and sparteine oxidation in a study involving 17 human liver samples.

Absence of Polymorphism of Sparteine Oxidation in the South African Venda

1 This study has found no occurrence of poor metabolism of sparteine within a South African Venda population of 97 subjects. 2 On the basis of MR (metabolic ratio) the mean and distribution of the results are very similar to those found in Ghanaians. 3 The distribution is also similar to that for fast metabolizers in Caucasians. 4 It is concluded that different P450 cytochromes are responsible for immediate oxidation of debrisoquine and sparteine, but that both may be activated by the same P450 reductase.

Codeine increases pain thresholds to copper vapor laser stimuli in extensive but not poor metabolizers of sparteine

The analgesic efficacy and kinetics of a single oral dose of 75 mg codeine was investigated in 12 extensive metabolizers and 12 poor metabolizers of sparteine in a double-blind, placebo-controlled crossover study. The cosegregation of the O-demethylation of codeine to morphine with the sparteine oxidation polymorphism was confirmed. Hence morphine could not be detected in the plasma of any of the poor metabolizers, whereas detectable morphine plasma levels were found in 10 of 12 extensive metabolizers. Pain thresholds to laser stimuli were determined before drug intake and 90, 150, and 210 minutes after drug intake. Codeine significantly increased the pricking pain thresholds in the extensive metabolizers (p less than 0.05), whereas there were no significant changes in the poor metabolizers. No change in pain thresholds occurred with placebo in any of the two phenotypes. In the extensive metabolizers there was a significant positive correlation between the increase in pain threshold and plasma concentration of codeine. The study supports the hypothesis that morphine formation is essential for achievement of analgesia during codeine treatment.

LKM-1 ANTIBODY POSITIVE AUTOIMMUNE HEPATITIS IN IDENTICAL TWINS: IN VITRO INHIBITION OF THE TARGET ANTIGEN CYTOCHROME P45O db1 AND IN VIVO PHENOTYPE

A subgroup of autoimmune type chronic active hepatitis (AI-CAH) is associated with LKM-1 autoantibodies. They are directed against Cytochrome P450 dbl and inhibit its function in vitro. We observed LKM-1 positive AI-CAH and colitis in a 13 year old girl. Liver disease responded well to corticosteroids. Father, mother, brother, the patient and her identical twin sister were investigated for HLA class I–III phenotypes, auto-antibodies, in vitro inhibition of P450 dbl, and in vivo phenotype for drug metabolism (sparteine) mediated by this enzyme. Both twins had the autoimmune HLA haplotype A1, B8, DR3, C4A-Q0. While the mother is a homozygous extensive metabolizer (EM) (metabolic ratio 0.33), the father is a homozygous poor metabolizer (MR 65.25) and all children are heterozygote EM (MR 1.01, 0.99 & 1.76). Only the index patient had signs of liver disease and was positive for LKM-1 antibodies, and only her serum inhibited P450 dbl catalysed oxidation of sparteine in vitro up to 90 %.Conclusion: We describe for the first time occurence of LKM-1 positive AI-CAH in a pair of identical twins, who were discordant for the disease. Since both twins are of the EM metabolizer type, i.e. express functionally intact P450 dbl, and share the autoimmune HLA haplotype, we conclude that environmental factors trigger the manifestation of this autoimmune liver disease.

A dose‐effect study of the in vivo inhibitory effect of quinidine on sparteine oxidation in man.

1. Twelve healthy extensive metabolisers of sparteine were sparteine tested daily for 6 days (19.00 h to 07.00 h). A small but statistically significant rise in sparteine metabolic ratio (MR) was observed. 2. Following 100 mg quinidine sulphate given to four of the subjects at 16.00 h, sparteine tests were carried out 19.00 h to 07.00 h on the same day and then daily for 6 days. Quinidine caused an immediate twenty-fold increase in sparteine-MR which then gradually returned to normal over the following 4-6 days. Quinidine concentrations in plasma were measurable only up to 20 h after the quinidine test dose. 3. At weekly intervals, all 12 subjects received single doses of quinidine sulphate of 5, 10, 20, 40 and 80 mg at 16.00 h, each time followed by a sparteine test 19.00 h to 07.00 h on the same day. A clear dose-effect relationship was found with a significant rise in the sparteine-MR even after 5 mg quinidine. After 80 mg quinidine, 8 of 12 subjects became phenotypically poor metabolisers (MR greater than 20).