Transcriptomic Signatures of Experimental Alkaloid Consumption in a Poison Frog.

In the anuran family Dendrobatidae, aposematic species obtain their toxic or unpalatable alkaloids from dietary sources, a process known as sequestering. To understand how toxicity evolved in this family, it is paramount to elucidate the pathways of alkaloid processing (absorption, metabolism, and sequestering). Here, we used an exploratory skin gene expression experiment in which captive-bred dendrobatids were fed alkaloids. Most of these experiments were performed with Dendrobates tinctorius, but some trials were performed with D. auratus, D. leucomelas and Allobates femoralis to explore whether other dendrobatids would show similar patterns of gene expression. We found a consistent pattern of up-regulation of genes related to muscle and mitochondrial processes, probably due to the lack of mutations related to alkaloid resistance in these species. Considering conserved pathways of drug metabolism in vertebrates, we hypothesize alkaloid degradation is a physiological mechanism of resistance, which was evidenced by a strong upregulation of the immune system in D. tinctorius, and of complement C2 across the four species sampled. Probably related to this strong immune response, we found several skin keratins downregulated, which might be linked to a reduction of the cornified layer of the epidermis. Although not conclusive, our results offer candidate genes and testable hypotheses to elucidate alkaloid processing in poison frogs.

[Derivatives of lupinin and epilupinin as ligands of various cholinesterases].

Literature data have been summarized on interaction of cholinesterases of some mammals and arthropods with a group of isomer derivatives of alkaloid lupini and its epimer epilupinin. As substrates of cholinesterases of several mammals there are studied 8 acetates containing in their molecules the chinolysidin bicycle with different structure of N-alkyl radical, which showed certain elements of specificity of action. For 2 isomer esters that are derivatives of the protonated base of the lupinin and epilupinin structures, differences in their substrate characteristics were revealed. The polyenzyme analysis if anticholinesterase efficiency was performed for 30 organophosphorus inhibitors that are dialkoxyphosphorus derivatives of lupinin and epilupinin; as a result, quite a few peculiarities of their action depending on their structure were revealed. Several tested compounds turned out to act as specific inhibitors of cholinesterases of some mammals and arthropods.

Estimation of the interindividual variability of cytochrome 2D6 activity from urinary metabolic ratios in the literature.

Cytochrome P450 2D6 (CYP2D6) is an enzyme with a large interindividual variability in its metabolic activity due to genetic polymorphisms. In the present study, both its intrinsic metabolic activity (CL(int,CYP2D6,app)) relative to extensive metabolizers (EM) and its variability were estimated by analyzing the urinary metabolic ratios (MR) based on the well-stirred model. Sparteine and debrisoquine were considered to be appropriate probes for our methodology, whereas dextromethorphan was not appropriate since the formation of its metabolite of interest is not described by the well-stirred model. From the analysis of MRs of sparteine and debrisoquine for Caucasian subjects in the literature, CL(int,CYP2D6,app) for intermediate metabolizers (IM) was estimated to be approximately 15% of that for EM. The coefficient of variability (CV) of CL(int,CYP2D6,app) was estimated to be approximately 60% for both EM and IM and 100% for the combined population of ultrarapid metabolizer, EM and IM [i.e., the non-poor metabolizer (non-PM) population]. Simulation of exposure in the non-PM population showed that the CV of exposure was 140% for dextromethorphan and 71% for metoprolol, which reflected the reported values of 110% and 53% for dextromethorphan and metoprolol, respectively. The present study should be useful for predicting the interindividual variability in exposure to investigational drugs that are metabolized by CYP2D6.

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.

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.

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.

Analysis of the CYP2D6 gene mutations and their consequences for enzyme function in a West African population.

The data on differences in the metabolic handling of the CYP2D6 probe drugs sparteine and debrisoquine, and the relationship between phenotype and genotype and gene frequencies for several mutant CYP2D6 alleles in African populations are limited and sometimes controversial. Therefore, in a West African population (Ghana), we investigated (i) the phenotype for sparteine debrisoquine by phenotyping 201 individuals with both drugs and (iii) the genotype for CYP2D6 (n = 326) and debrisoquine (n = 201) oxidation, (ii) the coregulatory control of sparteine and alleles *3 and *4 in 133 individuals and for the alleles *1, *2, *3, *4, *5, *6, *7, *8, *9, *10, *14, *16, *17, *2b, *2xN, *2bxN in 193 individuals. Of the 326 individuals phenotyped with sparteine, eight had a metabolic ratio (MR)sp > 20 corresponding to a poor metabolizer frequency of 2.5% [95% (confidence interval) CI = 1.06-4.77]. The prevalence of the poor metabolizer phenotype for debrisoquine oxidation was 3% (95% CI = 1.1-6.39) with six of the 201 individuals having a MR greater than 12.6. The distribution of the MR of sparteine was trimodal whereas MR of debrisoquine was unimodally distributed with a pronounced kurtosis. In individuals phenotyped with both drugs, there was a significant correlation between the MRs (r(s) = 0.63, P < 0.001). The CYP2D6 alleles *1, *2 and *17 were the most common functional alleles occurring with frequencies of 43.7, 10.6 and 27.7%, respectively. The three other observed functional alleles *2xN, *10 and *20 had much lower frequencies (1.6%, 3.1% and 0.3%, respectively). Of the eight non-functional alleles, only *4 (6.3%) and *5 (6.0%) could be found. The allele *5 occurred with the same frequency as in Caucasian populations (4.1%) but the *4 allele had a much lower frequency (Caucasians 19.5%). One individual with *1/*1 was a poor metabolizer for sparteine and debrisoquine indicating the existence of as yet unknown non-functional alleles in this West African population. Although the prevalence of poor metabolizers and the number of heterozygotes for non-functional alleles was much lower in Ghanaians, the median MRsp of 0.7 was significantly higher in this population compared with a median MRsp of 0.4 in Caucasians, indicating a lower metabolic clearance for CYP2D6 substrates in the West Africans. The lower metabolic activity in Ghanaians could not be explained solely by the high frequency of the *17 allele, which is associated with an impairment of CYP2D6 enzyme function. In addition, a higher median MRsp of 0.5 corresponding to metabolic clearance of 346 ml/min was observed among extensive metabolizers with the genotype *1/*1. Thus, compared with the median of MRsp = 0.28 (CLmet 573 ml/min) in Caucasians homozygous for *1, the metabolic clearance of sparteine was 40% lower on average in respective Ghanaians.

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.

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.

Prediction of CYP2D6-mediated polymorphic drug metabolism (sparteine type) based on in vitro investigations.

Discovery of genetic polymorphism in drug metabolism has contributed a great deal to understanding the variability in dose-concentration relationships introduced by genetic factors, thereby elucidating the mechanisms responsible for unexpected drug reactions. This knowledge should find its way into clinical practice in order to make therapy more efficient and safe. Moreover, genetic factors in drug metabolism should be taken into account during drug development. Therefore, in vitro methods for identifying the metabolic pattern of new compounds during early stages of drug development should be improved. This review summarizes in vitro methods available to identify genetic polymorphism in drug oxidation, in particular the CYP2D6-related polymorphism.

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).

Genetically determined sparteine oxidation polymorphism in a Polish population.

The genetic oxidation polymorphism was determined in 160 healthy Polish volunteers from the south-west of Poland (Wroclaw region), using sparteine as a model drug. The results of a Polish population study revealed a bimodal distribution of the sparteine metabolic ratio and showed the existence of two oxidation phenotypes designated as extensive and poor metabolizers. The frequency of poor metabolizers in our study (8.8%) compares well with most results of poor oxidation metabolizers in Caucasian populations.

Debrisoquine hydroxylase and Parkinson's disease.

The relationship between the genotypes of Xba I and Bam H I restriction fragment length polymorphisms (RFLPs) at a gene for debrisoquine hydroxylase (CYP2D6) and phenotypes of the metabolic function of debrisoquine/sparteine, the EM and the PM in a healthy Japanese population was investigated. The genotypes of Xba I 11.5 kb and Xba I 44 kb-Bam H I 2.3 kb- were responsible for the Japanese PM. Genotype of Xba I RFLP at CYP2D6 locus was analyzed in 43 healthy individuals and 51 patients with IDP. The relative risk of IDP was 2.15 times more for individuals with the Xba I 44 kb allele compared to those without the allele (chi 2 = 4.149, d.f. = 1, p < 0.05) and it was 6.32 times greater for the Xba I 44 kb homozygotes than the Xba I 29 kb homozygotes (chi 2 = 4.935, d.f. = 1, p < 0.05). These data suggest that the PM for debrisoquine/sparteine hydroxylase might be one of the genetic factors making humans susceptible to IDP acquisition.

[The polymorphism of pachycarpine oxidation]. / Polimorfizm okisleniia pakhikarpina.

The ratio of urinary excretory pachycarpine to its oxidized metabolites, 2- and 5-dehydropachycarpines (metabolic ratio) was determined in a selective group of 81 unrelated cardiac patients from a Moscow Caucasian population given pachycarpine in a dose of 25 g. The metabolic ratio distribution was shown to be bimodal. Ninety five per cent of the patients had the metabolic ratio lower than 28 while 4 (5%) patients higher than 70. In 25 patients of the group, the pachycarpine metabolic ratio was evaluated after quinidine, 50 mg. Twenty-two patients with a relatively low metabolic ratio showed a dramatic (several times) increase, while in 3 patients with the prior metabolic ratio higher than 70, the effect of quinidine was insignificant. The findings suggest that pachycarpine oxidation is genetically polymorphic and similar to the polymorphism of sparteine/debrisoquine oxidation. Pachycarpine may be used as a marker in phenotyping the population.

Differential effects of quinidine on the disposition of nifedipine, sparteine, and mephenytoin in humans.

The effects of quinidine on oxidative routes of drug metabolism mediated by different forms of cytochrome P450 were investigated in 10 healthy subjects. Each subject was studied on three different occasions and separately received oral administration of (1) a "cocktail" of nifedipine (5 mg), sparteine sulfate (90 mg), and mephenytoin (100 mg), (2) quinidine sulfate (200 mg), and (3) quinidine sulfate followed by the "cocktail" 1 hour later. Quinidine pretreatment significantly inhibited the aromatization of nifedipine to its major first-pass pyridine metabolite (M-0) and prolonged the elimination half-life of the calcium channel antagonist, both by about 40%. More marked inhibition of metabolism was observed with sparteine, and the formation of dehydrosparteine was abolished. A significant correlation was found between the 0-8-hour urinary ratio and the plasma concentration ratio of sparteine to dehydrosparteine obtained 4 hours after drug administration. No quinidine-induced changes were observed in the 4-hydroxylation of mephenytoin. The interaction between quinidine and nifedipine supports the involvement of a common P450 (P450IIIA4) in the metabolism of the two drugs.