Inhibition of 17ß-estradiol activation by CYP1A1: genotype- and regioselective inhibition by St. John's Wort and several natural polyphenols.

Several epidemiological studies associate certain CYP1A1 genotypes, alone or in combination, with an increased risk of estrogen-related cancers. Previously we demonstrated that metabolic activation of estrogens by CYP1A1 is a genotype-dependent reaction with the CYP1A1.2 (Ile462Val) variant being the most efficient catalyst (Kisselev et al.). To answer the question whether genotype-dependent inhibition of activation of estrogens by CYP1A1 could also contribute, we studied the inhibition of hydroxylation activity of the most common allelic variants of human CYP1A1 towards 17ß-estradiol. We expressed and purified CYP1A1.1 (wild-type), CYP1A1.2 (Ile462Val), and CYP1A1.4 (Thr461Asn) and performed inhibition assays by natural polyphenols of our diet and drugs of NADPH-dependent estradiol hydroxylation in reconstituted CYP1A1 systems. From the polyphenols studied, a St. John's Wort (Hypericum perforatum) extract, some of its main single constituents hypericin, pseudohypericin, and quercetin, as well as the flavonols kaempferol, myricetin and the phytoestrogens resveratrol and tetramethyl-stilbene exhibited strong inhibition. For the St. John's Wort extract and its single constituents hypericin, pseudohypericin, and quercetin, inhibition exhibited a remarkable dependency on the CYP1A1 genotype. Whereas (wild-type) CYP1A1.1 was most inhibited by the whole crude extract, the variant CYP1A1.2 (Ile462Val) was significantly stronger inhibited by the constituents in its pure form: IC50 values for 2-hydroxylation was more than two times lower compared with the wild-type enzyme and the variant CYP1A1.4 (Thr461Asn). Besides this, the inhibition exhibited a remarkable regioselectivity. The data suggest that risk of estrogen-mediated diseases might be not only influenced by CYP1A1 genotype-dependent activation but also its inhibition by natural polyphenols of our diet and drugs.

Moxifloxacin does not alter ciclosporin pharmacokinetics in transplant patients: a multiple-dose, uncontrolled, single-centre study.

BACKGROUND: Moxifloxacin has a broad antibacterial spectrum and rapid bactericidal activity, and is thus a good option for the treatment of bacterial infections in patients who have undergone organ or bone marrow transplantation. Transplant patients also receive immunosuppressant therapy such as ciclosporin. OBJECTIVE: The primary objective of this study was to assess the steady-state pharmacokinetics of ciclosporin with and without concomitant treatment with moxifloxacin in transplant recipients. A secondary objective was to determine the safety and tolerability of the combined treatment. METHODS: Patients (n = 9) with stable graft function after bone marrow or renal transplantation and who were already receiving ciclosporin therapy were enrolled into the study. The patients were given ciclosporin (Sandimmun Optoral) capsules twice daily (total daily dosage 150-380 mg/day) throughout the study period. Moxifloxacin (Avolox) tablets 400 mg once daily were given on days 2-8 inclusive. The primary outcome measure was the change in ciclosporin pharmacokinetics on coadministration with moxifloxacin. Secondary outcomes were the steady-state pharmacokinetics of moxifloxacin and ciclosporin plus its metabolites in patients receiving moxifloxacin and ciclosporin concomitantly. Moxifloxacin pharmacokinetic parameters in the presence of ciclosporin were compared with previously published pharmacokinetic data for moxifloxacin in healthy individuals. RESULTS: No significant changes occurred in the concentration-time curves of ciclosporin and its metabolites following combination therapy with moxifloxacin. The geometric means of whole blood concentrations of ciclosporin and ciclosporin plus its metabolites on day 1 were similar to those on day 8 following combined administration of ciclosporin and moxifloxacin for 7 days. The ratio of combination treatment to monotherapy for ciclosporin was 1.01 (90% CI 0.91, 1.11) for the area under the blood concentration-time curve from time zero to 12 hours at steady state (AUC(12,ss)) and 0.96 (90% CI 0.88, 1.04) for the maximum steady-state blood drug concentration (C(max,ss)). For ciclosporin plus its metabolites the ratio was 1.07 (90% CI 0.99, 1.17) for AUC(12,ss) and 1.03 (90% CI 0.98, 1.09) for C(max,ss). The pharmacokinetic parameters for moxifloxacin were unaffected by the presence of ciclosporin. CONCLUSIONS: Concomitant administration of moxifloxacin does not alter the pharmacokinetic parameters of ciclosporin or ciclosporin plus its metabolites in immunosuppressed patients. Therefore, no dose adjustments or additional drug monitoring are required when ciclosporin is coadministered with moxifloxacin.

Endothelin-1 gene variant Lys198Asn and plasma endothelin level in obstructive sleep apnea.

Obstructive sleep apnea (OSA) is a recognized risk factor for cardiovascular disorders. Thus, an association between endothelin-1 (EDN1) and OSA can be assumed. We investigated a cohort of 364 consecutive patients (age 57 +/- 10 years) with mild to severe OSA for the EDN1 variant Lys198Asn (G/T) and endothelin plasma levels and compared them with 57 controls. The Lys198Asn genotype was significantly associated with the apnea/hypopnea index (AHI) with a median of 30/h of sleep for GG, 27/h for GT and 59/h for TT genotype (p < 0.05). Further stratification of patients into 2 groups by body mass index (BMI) revealed a strong association between AHI and Lys198Asn polymorphism in 191 obese patients (p = 0.005), whereas in 173 nonobese patients, we observed no association. A substantial effect by BMI on OSA severity was seen with multiple linear regression (p < 0.001). However, this effect was modified by the Lys198Asn polymorphism and by gender: the AHI increase per unit of BMI was more pronounced in males than in females, and about 1.3 times greater in homozygous carriers of the mutant allele than in other carrier groups. EDN1 plasma levels of untreated OSA patients and of patients treated with nasal continuous positive airway pressure were not elevated compared with controls. Our results indicate that the Lys198Asn polymorphism is associated with the severity of OSA in obese subjects. The EDN1 plasma level cannot be used as a marker for OSA or its severity.

Vitamin D receptor poly(A) microsatellite and colorectal cancer risk in Caucasians.

BACKGROUND: Colorectal cancer incidence and prognosis are influenced by vitamin D intake and expression of the vitamin D receptor (VDR). Polymorphisms of the VDR are linked to several diseases. This study was aimed to investigate whether variants of the VDR poly(A) microsatellite are associated with colorectal cancer incidence. MATERIALS AND METHODS: The poly(A) polymorphism was analyzed in a series of 255 colorectal cancer patients and 255 controls of Caucasian origin (case-control study) by a combination of GeneScan and sequencing. RESULTS: There was a distinct separation between long and short alleles. We found 19, 20, 21, and 22 A-repeats for the long variant and 14 and 15 A-repeats for the short variant. Frequencies of long and short alleles did not differ between cases and controls, nor did frequencies of any single variant. CONCLUSION: Our findings do not support an association between VDR poly(A) variants and the incidence of colorectal cancer.

St John's wort extract (Ze 117) does not alter the pharmacokinetics of a low-dose oral contraceptive.

PURPOSE: St John's wort (Hypericum perforatum) is an herbal remedy that is widely used in the treatment of depression. Recent clinical data have demonstrated that St John's wort extracts interfere with the action of various drugs and possibly also with combined oral contraceptives. Therefore, we investigated the effects of a St John's wort extract (Ze 117) with low hyperforin content on the pharmacokinetics of ethinylestradiol and 3-ketodesogestrel. METHOD: Sixteen healthy female volunteers, who had taken a low-dose oral contraceptive (Lovelle contains 0.02 mg ethinylestradiol + 0.15 mg desogestrel) for at least 3 months, participated in the study. Pharmacokinetic data (AUC, C(max), t(max)) were determined the day before (reference) and after (test) a 14-day period of Ze 117 intake (250 mg twice daily). RESULTS: Before the co-administration of Ze 117 on day 7, the geometric mean (geometric coefficient of variation) for the AUC(0-24) of ethinylestradiol was 152.53 pg.h/ml (87.39%) and after co-administration on day 21 it was 196.57 pg.h/ml (78.14%). The respective values for ketodesogestrel were 36.37 pg.h/ml (34.18%) and 41.12 pg.h/ml (34.36%). The mean of individual ratios (reference-to-test) of log-transformed AUC values (90% confidence interval) were 0.951 (0.915-0.986) for ethinylestradiol and 0.968 (0.944-0.992) for ketodesogestrel indicating a small gain [corrected] in bioavilability, but bioequivalence nevertheless. CONCLUSION: These results indicate that the recommended dose of the hypericum extract Ze117, which has a low hyperforin content, does not interact with the pharmacokinetics of the hormonal components of the low-dose oral contraceptive.

Effect of tolterodine on sleep structure modulated by CYP2D6 genotype.

OBJECTIVE: Tolterodine, a drug for the treatment of overactive bladder symptoms, has a limited entry into the brain, which makes cognitive side effects seldom. However, some case reports have described central-nervous side effects such as sleepiness. The aim of this retrospective analysis was to investigate whether tolterodine-related effects on sleep stage parameters could be explained by different CYP2D6 metabolizer characteristics of subjects. METHODS: Data were taken from two randomized, double-blind, placebo-controlled studies conducted in a cross-over design. Forty-eight volunteers underwent 4 two-night attended polysomnographic studies. Subjective quality of sleep and cognitive function were assessed. A single dose of 4 mg tolterodine or placebo was administered before sleep. Forty-four volunteers gave informed consent for genotyping. We found 19 extensive metabolizers (EM), 20 intermediate metabolizers (IM), 4 poor metabolizers (PM) and 1 ultrarapid metabolizer. There were no significant differences between the groups regarding demographic data. RESULTS: Rapid eye movement (REM) sleep duration as a percentage of total sleep time showed significant reduction (p=0.019) in the group carrying one or more deficient alleles (IM+PM). No significant difference was found with two active alleles of CYP2D6 in the EM group. REM latencies under tolterodine displayed a tendency towards prolongation, which was irrespective of the metabolizer status. Subjective sleep parameters did not show statistically significant changes after tolterodine. Cognitive skills were not affected. CONCLUSION: Our retrospective analysis reveals that a decrease of REM sleep under tolterodine is found only in individuals carrying one or two deficient CYP2D6 alleles.

An anomalous haplotype distribution of the arrestin domain-containing 4 gene (ARRDC4) haplotypes in Caucasians.

Little was known about the sequence variability of the human Arrestin domain-containing 4 gene (ARRDC4). We sequenced its DNA from exon 2 to exon 8 in a sample of 92 Russians. Seven variants were identified; one of them has not been described yet. It causes an amino acid change from Thr to Met. Identified variants were genotyped in the complete sample of 253 unrelated men and women to analyze haplotype distribution. Fifteen haplotypes were inferred. Nine haplotypes had estimated frequencies > 1%. Ninety-five percent of all haplotypes were determined by five haplotype-tagging single nucleotide polymorphisms. Haplotypes form two clades. The two most common haplotypes cover 76% of all haplotypes. The certainty of the haplotype reconstruction does not depend on the haplotype-inferring algorithms, but is a result of the anomalous haplotype distribution of ARRDC4, which makes this gene a suitable candidate gene for haplotype association studies. Interestingly, there is a great evolutionary distance between the two most common haplotypes, which could suggest a more complicated coalescent process with either past gene flow, selections, or bottlenecks.

Association of CYP1A1 polymorphisms with differential metabolic activation of 17beta-estradiol and estrone.

Several epidemiologic studies associate certain CYP1A1 genotypes, alone or in combination, with an increased risk of estrogen-related cancers. To answer the question of whether genotype-dependent activation of estrogens by CYP1A1 could be the underlying mechanism, we studied the hydroxylation activity of the most common allelic variants of human CYP1A1 towards both endogenously occurring estrogens, 17beta-estradiol (E2) and estrone (E1). We expressed and purified CYP1A1.1 (wild-type), CYP1A1.2 (Ile(462)Val), and CYP1A1.4 (Thr(461)Asn) and did enzymatic assays of NADPH-dependent estrogen hydroxylation in reconstituted CYP1A1 systems. All CYP1A1 variants catalyzed the formation of 2-, 4-, 6alpha-, and 15alpha-hydroxylated estrogen metabolites from E2 and E1, yet with varying catalytic efficiency and distinct regiospecificity. Whereas the variant CYP1A1.2 (Ile(462)Val) had a significant higher catalytic activity for all hydroxylation sites and both substrates, it was most pronounced for 2-hydroxylation. Catalytic efficiencies for the formation of the major metabolites, 2-OH-E2 and 2-OH-E1, by CYP1A1.2 were 5.7- and 12-fold higher, respectively, compared with the wild-type enzyme. The catalytic efficiencies for hydroxylations catalyzed by CYP1A1.4 were roughly comparable with those of the wild-type enzyme. Enzyme kinetics showed that the superior activity of CYP1A1.2 (Ile(462)Val) is mainly caused by a higher V(max), whereas K(m) values of all variants were similar. The data suggest that risk of estrogen-induced cancers and cardiovascular diseases might be-at least partially-determined by the CYP1A1 genotype.

Influence of the SLCO1B1*1b and *5 haplotypes on pravastatin's cholesterol lowering capabilities and basal sterol serum levels.

We previously showed that variant SLCO1B1 haplotype *1b (A388G) accelerates and that *5 (T521C) delays hepatocellular uptake of the HMG-CoA reductase inhibitor pravastatin [Mwinyi et al. (2004): Clin Pharmacol Ther 75:415-421]. In the present study we checked for differential effects of variant SLCO1B1 haplotypes on hepatocellular cholesterol synthesis. We analyzed the serum levels of cholesterol, lathosterol, and campesterol in healthy white males which had been grouped on the basis of their SLCO1B1 haplotype: *1a (n=10), *1b (n=10), and *5 (n=8). The subjects received a single oral dose of 40 mg pravastatin. Cholesterol and lathosterol levels were lower in all subjects following pravastatin intake for up to 24. Median levels 6 h post-dosing of lathosterol decreased in each SLCO1B1 haplotype group in the rank order of *1b (-0.11 mg dl(-1); min-max: -0.20 to -0.04; p=0.005) > *1a (-0.09 mg dl(-1); min-max: -0.22 to -0.05; p=0.005) > *5 (-0.07 mg dl(-1); min-max: -0.17 to -0.05; p=0.012). Lathosterol median-change values were significantly greater in haplotype *1b than in haplotype *5 individuals (p=0.041, non-adjusted), which was congruent with the extent of mean changes in lathosterol-to-cholesterol ratios, although the latter did not reach statistical significance. Post-treatment serum levels of campesterol were not affected by SLCO1B1 haplotype. Interestingly, sterol basal serum levels tended to be highest in *1b carriers, followed by those in *1a and *5 individuals, with significant differences in lathosterol concentrations between the *1b and *5 (p=0.041, non-adjusted) haplotype group. Our findings suggest an association of SLCO1B1*1b and *5 haplotypes to pravastatin's inhibition of the hepatocellular HMG-CoA reductase. Furthermore, SLCO1B1 haplotypes seem to play a role in basal cholesterol homeostasis.

Systematic analysis of sequence variability of the endothelin-1 gene: a prerequisite for association studies.

We analyzed allele frequencies and pairwise linkage disequilibria of 13 variants in the EDN1 gene of 298 young males, the majority of German ancestry. Our analysis comprises all common variants in the five exons and flanking intronic regions, as well as known polymorphisms in the promoter sequence. In addition to previously analyzed polymorphisms, our haplotype reconstruction included five recently described variants and was done by using three different algorithms to allow inference of result stability. More than 30 haplotypes were predicted. All haplotypes with frequencies > or = 1% were inferred by all three methods and can be described by seven haplotype tagging single-nucleotide polymorphisms (htSNPs), reducing the genotyping load to 65%. Three of these haplotypes with frequencies of about 11%, 9%, and 4% had been mistaken for one haplotype in the previous analysis, which included only six polymorphisms, some of them not being htSNPs. Systematic analysis of sequence variability and comprehensive haplotype analysis of the EDN1 gene determined a substantial part of its genetic variability for further association studies and helped to reduce the genotyping load for common phenotypes.

St. John's wort extracts and some of their constituents potently inhibit ultimate carcinogen formation from benzo[a]pyrene-7,8-dihydrodiol by human CYP1A1.

Commercially available St. John's wort (Hypericum perforatum) preparations and some of their main constituents (hypericin, pseudohypericin, hyperforin, rutin, and quercetin) were examined for their potential to inhibit carcinogen activation by human cytochrome P450 1A1 (CYP1A1). We used a reconstituted system consisting of purified human CYP1A1, purified human NADPH-cytochrome P450 reductase, and dilaurylphosphatidylcholine as lipid component. St. John's wort extracts potently inhibited CYP1A1-catalyzed (+/-)-trans-7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene (7,8-diol-B[a]P) epoxidation, the terminal reaction leading to the ultimate carcinogenic product (+/-)-B[a]P-r-7,t-8-dihydrodiol-t-9,10-epoxide (diolepoxide 2). All constituents, except rutin, were shown to possess strong inhibitory potencies toward diolepoxide 2 formation from 7,8-diol-B[a]P, with IC(50) values of 0.5 microM (hypericin), 1.2 microM (hyperforin), 1.5 microM (quercetin), and 8 microM (pseudohypericin), respectively. Preincubation experiments revealed that their action was not mechanism based. Inhibition kinetics studies showed the anthrodianthrone compound hypericin to be a noncompetitive inhibitor, with a K(i) value of 0.6 microM, and the phloroglucinol hyperforin to be a competitive inhibitor, with a K(i) value of 1.1 microM. When the effects on NADPH-P450 reductase activity were investigated, all constituents of St. John's wort studied turned out to be rather ineffective inhibitors; quercetin was the only exception, with an IC(50) value of approximately 20 microM. These in vitro data indicate that St. John's wort extracts and some of their constituents potently inhibit the major human procarcinogen-activating enzyme CYP1A1.

Variations in the 5-hydroxytryptamine type 3B receptor gene as predictors of the efficacy of antiemetic treatment in cancer patients.

PURPOSE: Serotonin (5-hydroxytryptamine type 3 [5-HT3]) receptor antagonists have substantially reduced but not eliminated nausea and vomiting in patients undergoing cancer chemotherapy. They act through specific binding to the 5-HT3A, 5-HT3B receptor complex. The 5-HT3B subunit seems to be most important for its functionality. We hypothesized that patients with genetic variations in the 5-HT3B receptor gene might respond differently to antiemetic treatment. PATIENTS AND METHODS: We included 242 cancer patients on their first day of chemotherapy. Nausea and vomiting were documented before and twice during the chemotherapy using standardized interviews and visual analog scales. We sequenced the entire 5-HT3B receptor gene, including the 5' flanking region and at least a 20-base pair intronic sequence of each intron-exon splice site of all patients. RESULTS: Approximately 30% of all patients suffered from nausea or vomiting. Sequencing of the 5-HT3B receptor gene revealed 13 polymorphisms: two of them were amino acid exchanges (Tyr129Ser, Ala223Thr) and two were deletion variants. In both observation periods, patients homozygous for the -100_-102delAAG deletion variant of the promotor region experienced vomiting more frequently than did all the other patients. CONCLUSION: A more efficient antiemetic treatment with 5-HT3 receptor antagonists might be possible on a pharmacogenetic basis. However, only a small fraction of the therapeutic failure is explained by the -AAG deletion variant of the 5-HT3B receptor gene. Additional clinical and biochemical studies are needed to confirm the association.

Patient-tailored antiemetic treatment with 5-hydroxytryptamine type 3 receptor antagonists according to cytochrome P-450 2D6 genotypes.

PURPOSE: The use of serotonin 5-hydroxytryptamine type 3 receptor antagonists has substantially reduced, but not eliminated, nausea and vomiting in cancer chemotherapy. This study sought to investigate whether efficacy of antiemetic treatment with ondansetron and tropisetron depends on cytochrome P-450 2D6 (CYP2D6) genotype, hypothesizing that the rapid and particularly the ultrarapid metabolizers of these drugs are at risk of being undertreated. PATIENTS AND METHODS: Included in the study were 270 cancer patients receiving their first day of chemotherapy. Nausea and vomiting were documented using standardized interviews. The intensity of nausea was measured with visual analog scales before and twice during the chemotherapy. The relationship between the CYP2D6 genotypes and the tropisetron serum concentrations 3 and 6 hours after drug administration was analyzed in a subgroup of 42 patients. CYP2D6 genotyping was carried out by polymerase chain reaction-restriction fragment length polymorphism analysis. RESULTS: Genetically defined poor metabolizers had higher serum concentrations of tropisetron than all other patients (P <.03). Approximately 30% of all patients receiving chemotherapy experienced nausea and vomiting. Genetically defined ultrarapid meta-bolizers of CYP2D6 substrates had higher frequency of vomiting within the first 4 hours (P <.001) and within the period 5 to 24 hours (P <.03) after treatment than all the other patients; the tendency for nausea was similar. This difference was more pronounced in patients treated with tropisetron than in those treated with ondansetron. CONCLUSION: Antiemetic treatment with tropisetron or ondansetron could be improved by adjustment for the CYP2D6 genotype; approximately 50 subjects would have to be genotyped to protect one patient from severe emesis.

Pharmacokinetics of levetiracetam in patients with moderate to severe liver cirrhosis (Child-Pugh classes A, B, and C): characterization by dynamic liver function tests.

BACKGROUND: Levetiracetam is an antiepileptic drug approved for use as adjunctive therapy in adults with partial-onset seizures. We sought to investigate possible changes in the pharmacokinetics of levetiracetam and its metabolite ucb L057 in patients with liver cirrhosis, who may require dose adjustments. METHODS: A single dose of levetiracetam was administered to 5 healthy subjects and to patients with Child-Pugh class A (n = 5), B (n = 6), or C (n = 5) alcohol-induced cirrhosis. The pharmacokinetics of levetiracetam and ucb L057 was measured and correlated with biochemical liver function parameters, with creatinine clearance, and with kinetics of caffeine, lidocaine, and d -sorbitol as probes for specific liver functions. RESULTS: Dynamic liver function tests revealed a deterioration of liver function. The pharmacokinetics of levetiracetam and its metabolite did not differ between healthy subjects and those with class A or B cirrhosis. However, in patients with class C cirrhosis, levetiracetam total clearance was reduced by 57% (90% confidence interval [CI], 43%-67%; P < .001). The geometric mean ratio of the area under the plasma concentration-time curve for levetiracetam, Child-Pugh class C versus control, was 2.41 (90% CI, 1.80-3.23), and the geometric mean of the half-life ratio was 2.27 (90% CI, 1.74-2.97). This was explained by the deterioration of renal function in patients with severe hepatic disease. CONCLUSIONS: In pharmacokinetic studies of hepatic impairment, including all classes of cirrhosis may be more revealing than including only selected classes of liver failure. No dose adjustment of levetiracetam is necessary in patients with mild to moderate liver impairment; however, patients with severe cirrhosis should initially receive only half of the commonly recommended dose.

CYP1A1 genotype-selective inhibition of benzo[a]pyrene activation by quercetin.

Epidemiological studies suggest that food rich in quercetin and naringin may protect against certain types of lung cancer, and that genotype dependent inhibition of cytochrome P450 1A1 (CYP1A1)-mediated bioactivation of procarcinogens could be the underlying mechanism. We studied the inhibitory effects of quercetin and naringin on the terminal bioactivation step of benzo[a]pyrene (B[a]P), a member of the major class of lung carcinogens. This reaction (epoxidation of (+/-)-trans-7,8-dihydro-7,8-dihydroxy-B[a]P to the ultimate carcinogenic product, (+/-)-B[a]P-r-7,t-8-dihydrodiol-t-9,10-epoxide (diolepoxide 2)) was examined using three of the most common allelic variants of human CYP1A1, namely wild-type CYP1A1.1, CYP1A1.2, and CYP1A1.4. Quercetin potently inhibited diolepoxide 2 formation by all CYP1A1 types with IC(50) values between 1.6 and 7.0 microM. The differences between the wild-type enzyme and the variants were statistically highly significant (P < 0.01). Enzyme kinetics revealed quercetin as a mixed-type inhibitor of CYP1A1.1, CYP1A1.2, and CYP1A1.4 with K(i) values of 2.0, 6.4, and 9.3 microM, respectively. Naringin inhibited diolepoxide 2 formation only slightly. Our data support the hypothesis that quercetin may have a stronger chemopreventive effect in individuals carrying wild-type compared with variant CYP1A1 genes. Future studies should consider the influence of P450 polymorphisms on both procarcinogen activation and its inhibition to facilitate the development of genotype-specific chemoprevention regimes.

Effect of genetic polymorphisms in cytochrome p450 (CYP) 2C9 and CYP2C8 on the pharmacokinetics of oral antidiabetic drugs: clinical relevance.

Type 2 diabetes mellitus affects up to 8% of the adult population in Western countries. Treatment of this disease with oral antidiabetic drugs is characterised by considerable interindividual variability in pharmacokinetics, clinical efficacy and adverse effects. Genetic factors are known to contribute to individual differences in bioavailability, drug transport, metabolism and drug action. Only scarce data exist on the clinical implications of this genetic variability on adverse drug effects or clinical outcomes in patients taking oral antidiabetics. The polymorphic enzyme cytochrome P450 (CYP) 2C9 is the main enzyme catalysing the biotransformation of sulphonylureas. Total oral clearance of all studied sulphonylureas (tolbutamide, glibenclamide [glyburide], glimepiride, glipizide) was only about 20% in persons with the CYP2C9*3/*3 genotype compared with carriers of the wild-type genotype CYP2C9*1/*1, and clearance in the heterozygous carriers was between 50% and 80% of that of the wild-type genotypes. For reasons not completely known, the resulting differences in drug effects were much less pronounced. Nevertheless, CYP2C9 genotype-based dose adjustments may reduce the incidence of adverse effects. The magnitude of how doses might be adjusted can be derived from pharmacokinetic studies. The meglitinide-class drug nateglinide is metabolised by CYP2C9. According to the pharmacokinetic data, moderate dose adjustments based on CYP2C9 genotypes may help in reducing interindividual variability in the antihyperglycaemic effects of nateglinide. Repaglinide is metabolised by CYP2C8 and, according to clinical studies, CYP2C8*3 carriers had higher clearance than carriers of the wild-type genotypes; however, this was not consistent with in vitro data and therefore further studies are needed. CYP2C8*3 is closely linked with CYP2C9*2. CYP2C8 and CYP3A4 are the main enzymes catalysing biotransformation of the thiazolidinediones troglitazone and pioglitazone, whereas rosiglitazone is metabolised by CYP2C9 and CYP2C8. The biguanide metformin is not significantly metabolised but polymorphisms in the organic cation transporter (OCT) 1 and OCT2 may determine its pharmacokinetic variability. In conclusion, pharmacogenetic variability plays an important role in the pharmacokinetics of oral antidiabetic drugs; however, to date, the impact of this variability on clinical outcomes in patients is mostly unknown and prospective studies on the medical benefit of CYP genotyping are required.

Quantification of pravastatin in human plasma and urine after solid phase extraction using high performance liquid chromatography with ultraviolet detection.

A high performance liquid chromatography (HPLC) method for the estimation of pravastatin in human plasma and urine samples has been developed. The preparation of the samples was performed by automated solid phase extraction using clonazepam as internal standard. The compounds were separated by isocratic reversed-phase HPLC (C(18)) and detected at 239 nm. The method was linear up to concentrations of 200 ng/ml in plasma and 2000 ng/ml in urine. The intra-assay variability for pravastatin in plasma ranged from 0.9% to 3.5% and from 2.5% to 5.3% in urine. The inter-assay variability ranged from 9.1% to 10.2% in plasma and from 3.9% to 7.5% in urine. The validated limits of quantification were 1.9 ng/ml for plasma and 125 ng/ml for urine estimation. These method characteristics allowed the determination of the pharmacokinetic parameters of pravastatin after administration of therapeutic doses.

Implications of pharmacogenetics for individualizing drug treatment and for study design.

Adverse drug reactions and ineffective drug treatment are responsible for a large health care burden. Considerable variability in drug response makes the prediction of the individual reaction difficult. Pharmacogenetics can help to individualize drug treatment in accordance with the genetic make-up of the patient. Drug response is best understood as a complex interplay between pharmacokinetics, pharmacodynamics, and other disease-associated factors. There are a large number of genetic variants in the enzymes of phase I and phase II drug metabolism, in drug transporters, and drug targets, all of which account for differences in drug response. The polymorphisms in the cytochrome P450 enzyme system have been investigated most extensively. Genotype-based dose adjustment which should ensure "bioequivalent" drug concentrations in all patients has been derived from pharmacokinetic parameters, but this approach will have to be verified in prospective studies. Drug transport has recently been recognized as a further crucial determinant in pharmacokinetics. The effect of genetics on disease susceptibility and drug treatment has been studied quite extensively; however, hardly any of this progress is at present reflected in routine health care. The integration of pharmacogenetic factors in clinical trials requires novel considerations for study design and data interpretation. It is to be hoped that the new science bioinformatics will (a) help us identify the contribution of genetics to disease and treatment response and will (b) create data-processing devices which help the physician in the face of the enormously expanding scientific knowledge in selecting the best individually adapted treatment for the patient.

Interaction of CA repeat polymorphism of the endothelial nitric oxide synthase and hyperhomocysteinemia in acute coronary syndromes: evidence of gender-specific differences.

We have recently shown that high CA repeat copy numbers (> or = 34 repeats) in intron 13 of the endothelial nitric oxide (eNOS) gene are associated with excess risk of coronary artery disease. Hyperhomocysteinemia interacts by several mechanisms with the NO system, thereby favoring endothelial dysfunction. Since hyperhomocysteinemia evidently promotes prothrombotic activation, we investigated a possible interaction among hyperhomocysteinemia, the eNOS CA repeat polymorphism, and acute coronary syndromes. The median value of homocysteine in our study population was 9.4 micromol/l. We accordingly determined the relative risk of acute coronary syndromes for homocysteine values higher than 9.4 micromol/l and 9.4 micromol/l or lower in the entire coronary artery disease group, and at different CA repeat cutoff values (34, 35, 36, 37, 38 CA repeats). For the entire coronary artery disease group ( n=1000), homocysteine levels higher than 9.4 micromol/l were not significantly associated with acute coronary syndromes. Although the CA repeat copy numbers were not associated with acute coronary syndromes in the overall group, the relative risk among women with homocysteine higher than 9.4 micromol/l for developing acute coronary syndromes increased nonsignificantly from 0.98 at cutoff 34 CA repeats to 1.68 at 35 CA repeats and significantly to 4.89 at 36 CA repeats, 11.20 at 37 CA repeats, and 18.32 at 38 CA repeats. This effect modification was not observed in men. These data suggest gender-specific gene-environment interaction between the CA repeat eNOS polymorphism and homocysteine in acute coronary syndromes.

Trimipramine pharmacokinetics after intravenous and oral administration in carriers of CYP2D6 genotypes predicting poor, extensive and ultrahigh activity.

OBJECTIVE: The tricyclic antidepressant trimipramine is one of the drugs with the most pronounced differences in pharmacokinetics caused by the CYP2D6 genetic polymorphism. However, the effect of CYP2D6 genotype on steady state kinetics and on bioavailability has not been studied so far. In addition, we were interested in trimipramine pharmacokinetics in genetically defined ultra rapid metabolizers. METHODS: We studied intravenous and multiple dose oral application of 50 mg trimipramine in five, seven, and three healthy volunteers with CYP2D6 genotypes predicting deficient, highly active and ultrarapid metabolism. The latter group included carriers of one wild-type and one duplication allele. Trimipramine and desmethyltrimipramine concentrations were measured by HPLC over a time interval of 72 h after intravenous and after one oral application. RESULTS: Both bioavailability and systemic clearance significantly depended on CYP2D6 genotype with a linear gene dose relationship. Mean bioavailability was 44, 16 and 12% in carriers of zero, two and three active genes of CYP2D6, respectively, and the corresponding data for systemic clearance were 12.0, 24.2, and 30.3 l/h. Consequently, the mean total oral clearances were 27.3, 151, and 253 l/h in poor, extensive and ultrarapid metabolizers. CONCLUSIONS: High bioavailability combined with low systemic clearance of trimipramine in poor metabolizers of CYP2D6 substrates results in a very high exposure to trimipramine with the risk of adverse drug reactions. On the other hand, the extremely high systemic and presystemic elimination may result in sub-therapeutic drug concentrations in carriers of CYP2D6 gene duplications with a high risk of poor therapeutic response.