Volume 33, Issue 3

Background The aim was to study seven polymorphic markers of genes encoding proteins involved in the absorption, metabolism and pharmacokinetics of clopidogrel among patients with an acute coronary syndrome (ACS), who have undergone percutaneous coronary intervention (PCI). Methods Eighty-one ACS and PCI patients older than 18 years and treated with dual antiplatelet therapy were enrolled in the study. Platelet function testing and ABCB1 , CYP2C19 , CYP3A5 and CYP4F2 genotyping were performed. The predictive role of categorical variables, such as genotypes (carriers and non-carriers of polymorphism), on platelet reactivity (platelet reactivity units [PRU] platelet inhibition [PI]) was assessed by logistic regression (for categorical outcomes) and linear regression (for continuous outcomes) analysis. A p-value<0.05 was considered significant. The allele frequencies were estimated by gene counting, and Hardy-Weinberg equilibrium was tested using the chi-square test. Results Regarding clopidogrel response, 62 patients (76.5%) were clopidogrel responders and 19 were non-responders (23.5%). Mean PRU value and the percentage of platelet inhibition were 170.0±50.9 PRU and 28.6±19.9%, respectively. The effects of the CYP2C19*2 polymorphisms on PRU (166.0±50.8 vs. 190.7±48.2, p<0.038) and PI (30.6±20.0 vs. 18.1±16.3, p<0.013) were observed, and the rates of high platelet reactivity (HPR) were lower in CYP2C19*1/*1 than those in CYP2C19*1/*2+CYP2C19*2/*2 (16.2% vs. 53.8% p<0.0067). In comparison, no significant difference in PRU value and PI was observed at <5 days between the rest of polymorphisms (p>0.05). Based on the logistic regression analysis, CYP2C19*2 (OR: 4.365, CI: 1.25–17.67, p=0.022) was an independent predictor of HPR at <5 days, as was the stent diameter (OR: 0.219, CI: 0.002–0.229, p=0.049). The remaining polymorphisms had no influence. Conclusions The reactivity of the on-clopidogrel platelet in the early phase of ACS is influenced primarily by the CYP2C19 polymorphisms. We believe that the findings of the present study could supply additional evidence regarding the clinical appropriateness of the CYP2C19 genetic testing for designing suitable antiplatelet therapy in the early phase of ACS.

Background In recent years, the use of tramadol as a probe drug for human cytochrome p450 2D6 (CYP2D6) has been investigated. The objective of this study was to assess the recovery of rat CYP2D1 enzymatic activity after mechanism-based inhibition induced by a single dose of ecstasy (MDMA, 3,4-methylenedioxymethamphetamine) and evaluation of the tramadol ability as a probe drug. CYP2D1 is orthologous in rats to human CYP2D6 and was employed in the current study. Methods A total of 16 male rats were selected and divided into control and treatment groups. The control group did not receive MDMA, while rats in the treatment group received a single dose of MDMA (1 mg/kg) and were subsequently divided into groups that were tested at 1 h, 10 days or 30 days post-administration. The rats were subjected to liver perfusion with Krebs-Heinslet buffer containing tramadol for 60 min and the tramadol and M1 levels were determined by HPLC-fluorescence. Results The enzymatic activity of CYP2D1 for the 1-h group decreased significantly when compared with the control group (p<0.05). Moreover, enzymatic activity increased non-significantly in the 10- and 30-day groups in comparison with the control group. The concentration and AUC 0−60 of tramadol increased in the 1-h and 10-day groups when compared with the control group but decreased in the 30-day group; however, none of these changes was statistically significant (p>0.05). The M1 metabolic ratio in the 1-h group decreased significantly when compared with the control group (p<0.05). The M1 metabolic ratio of the 10-day group increased and of the 30-day group decreased, but neither of these changes were significant. Conclusions Regardless of the genotype, the enzymatic activity of rat CYP2D1 recovered by 10 days post-administration of MDMA. It appears that tramadol, irrespective of its stereoselectivity, is not able to appraise rat hepatic CYP2D1 activity. It can be extrapolated that tramadol is a not suitable probe drug for human hepatic CYP2D1 because CYP2D1 in rats is orthologous to human CYP2D6. Further animal and human studies are required to confirm this hypothesis.

Background: Central nervous system (CNS) hemangioblastoma (HB) is the most common tumor in the von Hippel Lindau (VHL) disorder, the hereditary tumor syndrome caused by the biallelic mutations of the VHL gene. The disrupted VHL and Elongin protein interaction on hypoxia-inducible factor-1α (HIF-1α) induces a set of hypoxia-inducible genes, resulting in an unchecked endothelial cell proliferation that then leads to hemangioblastoma formation. However, recent studies have shown that disruptive germline mutations of VHL need not result in hemangioblastoma, though it can cause other manifestations of the VHL syndrome. Similarly, sporadic hemangioblastoma can occur rarely without a somatic biallelic VHL mutation. The VHL protein was earlier found to be associated with the deposition of matrix fibronectin (FN) protein in the renal extracellular matrix. Methods: The present study was designed to investigate the deposition of the matrix FN protein in VHL-associated hemangioblastoma. Results: Seven HB tumor samples from the VHL syndrome had lower expressions of tissue FN compared to the control cerebellum samples or the control blood vessel sample. On comparing the VHL and FN protein expressions in a timed endothelial tube assay, the VHL protein expression was absent during the initial phase of tube formation but started expressing after 6 h. The levels of matrix form of FN gradually increased along with the VHL expression during the maturation of tube formation. Tube formation was found to be enhanced with extraneously added soluble FN and inhibited by matrix FN. Similarly, tube formation was inhibited by a modified tripeptide (RGD) inhibitor of integrin (-αVβ3), namely, Cyclo-Ala-Arg-Gly-Asp-3-aminomethylbenzoyl. Conclusions: Our study implicates that the extracellular deposition and matrix formation of FN is important for vascular endothelial proliferation, and that its absence has roles in the development of hemangioblastoma in the VHL syndrome.

Background Calcium drugs are analogs of endogenous substances. The baseline concentration of endogenous substances in the body and homeostasis mechanisms of regulation make the bioavailability and bioequivalence (BE) evaluation of these drugs very difficult. The aim of the study was to assess the pharmacokinetic parameters of calcium in the studies of comparative bioavailability combined with the assessment of homeostatic regulation mechanisms. Methods The study included 42 healthy male volunteers. We studied the composition of 1000 and 1200 mg of calcium, and 800 and 1000 IU of cholecalciferol. Blood sampling was carried out at 8 a.m. and 0.5, 1, 2, 3, 4, 6, 8 h after administering the drugs. Calcium pharmacokinetic parameters were measured: the area under the curve (AUC) “concentration-time”, and maximum concentration (Cmax) in volunteers. The excretion of calcium in urine in terms of creatinine clearance and parathyroid hormone (PTH) levels was assessed. The maximum excretion of calcium was converted to creatinine (calcium/creatinine). Enzyme-linked immunosorbent assay (ELISA) was used to determine the PTH and the o-cresolphthalein method to quantitatively measure the total calcium serum and urine. Results Results indicated that both the AUC and the excretion of calcium are comparable, and the differences are not statistically significant, p>0.05 (the ratio of calcium and creatinine in the urine maintained within physiological levels). The comparable dynamics of PTH concentration is an indirect measure of the amount of absorbed calcium. Conclusions The study demonstrates that, along with the standard way of calculating the area under the curve “concentration-time” (total calcium in serum), it is important to assess the calcium excretion in urine in terms of creatinine excretion, as well as focusing on the changes in the PTH levels in volunteers, which could be considered as a marker of the calcium level in the blood.

Background Cytochrome P450 2D6 ( CYP2D6 ) allelic distribution exhibits differences amongst worldwide populations. There is lack of data from Mumbai, Western India, on the major CYP2D6 alleles *2, *3, *4, *5, *10 and *41, and gene multiplication alleles. Hence, the present study was undertaken to determine the distribution of these clinically relevant CYP2D6 alleles. Methods Fifty-two healthy individuals were screened using TaqMan SNP genotyping and copy number variation (CNV) assays by real-time polymerase chain reaction. Results The allele frequencies of CYP2D6 *2, *3, *4, *5, *10 and *41 alleles were observed to be 30.8%, 0%, 11.5%, 3.9%, 19.2% and 17.3%, respectively. The frequency of CYP2D6 gene one copy, two copies, three copies and four copies were observed to be 7.7%, 76.9%, 13.5% and 1.9%, respectively. The predicted phenotype frequency was observed to be 78.9%, 3.9% and 9.6% for extensive, intermediate, and ultrarapid metabolizers, respectively, whereas poor metabolizers were not detected. Conclusions CYP2D6 allele frequencies showed heterogeneous distribution in the present study as compared to worldwide populations. High frequency of CYP2D6 *41 allele, gene duplication alleles and UMs was observed. The scarcity and/or lack of data from the Indian population on these alleles further substantiates the need for screening of CYP2D6 genotyping.

Background Simvastatin is a widely used drug for dyslipidemia treatment, and the best therapeutic effects are achieved at night time. Simvastatin administration has been associated with the development of myopathy. Some polymorphisms in genes that are involved in the metabolism and transport of simvastatin seem to have an important role in the development of simvastatin-associated myopathy. The administration of 40 mg of simvastatin to 19 hyperlipidemic subjects with regular sleep rhythms (RR) and shift workers (SW) with predominant evening rhythms and sleep-wake disturbances, uncovered two SW volunteers who developed myopathy. Case presentation We report two SWs who developed simvastatin-dependent myopathy after three weeks of treatment. The SLCO1B1 521T>C major risk allele was observed in one myopathy-affected and one unaffected subjects, the second affected patient shared polymorphisms with the unaffected subjects. Conclusions The lack of consonance in the studied genotypes between SWs affected with simvastatin-associated myopathy can be related to circadian misalignment. Hence, other genes with circadian behavior and induced by simvastatin should be evaluated in future studies.