Influence of SLCO1B1 polymorphisms on atorvastatin efficacy and safety in Macedonian subjects.

Atorvastatin, as 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, is a widely prescribed medication for the treatment of dyslipidemia. However, despite its clinical efficacy in reducing major cardiovascular events, a wide inter-individual variability in its response exists. Several studies in this area point to the effect of polymorphisms in the solute carrier organic anion transporter 1B1 (SLCO1B1) gene encoding the multiple organic anion-transporting polypeptide 1B1 (OATP1B1) involved in hepatic uptake of atorvastatin. Hence, the aim of this study was to analyze the association between the SLCO1B1 c.388A>G, c.521T>C, c.571T>C, c.597C>T, c.1086C>T, c.1463G>C and c.*439T>G polymorphisms and lipid-lowering effect and safety of atorvastatin. A hundred and fifty six patients with hyperlipidemia IIa and IIb, all of Macedonian origin, were included in the study receiving atorvastatin 20 - 80 mg/day for 3 months. SLCO1B1 single nucleotide polymorphisms (SNPs) were genotyped using the TaqMan allelic discrimination assay. As parameters of atorvastatin response, total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglycerides (TG), apolipoprotein A (ApoAI), apolipoprotein B (ApoB), lipoprotein(a) (Lp(a)), creatine phosphokinase (CPK), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured, using standard laboratory methods, at baseline and after 3 months of treatment. No statistically significant association between the different SLCO1B1 SNPs and atorvastatin response was observed. However, the carriers of c.521CC manifested a lower decrease in plasma levels of TG, TC, LDL-C and Lp(a), with percentage difference being 16%, 7%, 29% and 149%, respectively, compared to the carriers of c.521TT variant. Lower increase in HDL-C (271%) and ApoAI (293%) and higher increase in CPK (69%) in c.521CC carriers were also observed, confirming the lower OATP1B1 activity in carriers of the variant c.521 C allele. Similar results were obtained when a comparison between the percentage of biochemical parameter change was made between *15/*16/*17 heterozygotes and *15/*16/*17 non-carriers. The lack of a statistically significant association between the SLCO1B1 polymorphism and atorvastatin response can be explained dominantly by the low number of individuals homozygous for the rare c.521C variant allele. Despite this limitation, the study offers valuable information on the influence of the genetic determinant SLCO1B1 on atorvastatin response in the Macedonian population.

Effects of single nucleotide polymorphisms and haplotypes of the SLCO1B1 gene on the pharmacokinetic profile of atorvastatin in healthy Macedonian volunteers.

OATP1B1 is an influx transporter known to mediate the uptake of various endogenous compounds and xenobiotics. Several sequence variations have been discovered in the SLCO1B1 gene encoding OATP1B1. The aim of this study was to investigate the effects of SLCO1B1 polymorphisms on the pharmacokinetics of atorvastatin in healthy volunteers of Macedonian origin. Twenty three participants, genotyped for SLCO1B1 c.388A > G, c.521T > C, c.571T > C, c.597C > T, c.1086C > T, c.1463G > C and c.*439T > G polymorphisms using TaqMan allelic discrimination assay, ingested a single 80 mg dose of atorvastatin. The plasma concentrations of atorvastatin were measured for 48 h using Tandem Liquid Chromatography-Mass Spectrometry, LC-MS-MS, and the peak plasma concentration (C(max)), time to peak plasma concentration (T(max)), elimination half-life (t1/2), constant rate of elimination (k(el)), mean residence time (MRT, expo), volume of distribution (Vd/kg), clearance (CL/kg), area under curve AUC(0.48h) and AUC(0-∞), were determined. Our data confirmed that the SLCO1B1 gene is highly polymorphic, with a frequency of the c.521T > C single-nucleotide polymorphism (SNP) being the lowest (app. 15%) and of all other SNPs alleles above 40%. Exceptions were c.1463G > C and c.1086C > T SNPs for which variant alleles were not identified. The strongest correlation was observed between the c.521T > C and c.571T > C SNPs pair. The haplotype analysis revealed 10 different haplotypes, with *1J/*1K/*1L being the dominant, with a frequency of app. 40%. The haplotype *15/*16/*17, containing both variant alleles of the functionally most distinguished SNPs, c.388A > G and c.521T > C, occurred with a frequency of 13%. However, *15/*16/*17 homozygotes were not identified in the study group. In this study, no significant differences in the k(el), t1/2, C(max), T(max), AUC(0-48h), AUC(0-∞), MRT expo, Vd and CL between the carriers of different c.388A > G, c.597C > T and c.*439T > G genotypes were observed. Subject with a variant allele C in the c.521T > C SNP, c.521CC genotype, had markedly higher values for C(max) and AUC(0.48h), 140% and 67%, respectively, in comparison with the carriers of the c.521TT genotype. Also, the carriers of the variant allele C at c.571T > C SNP, c.571 CC genotype, had 55% and 43% lower mean C(max) and AUC(0-48h) in comparison with the carrier of c.571TT. These differences lacked statistical significance due to the size of the sample. In addition, no significant differences in the pharmacokinetic parameters of atorvastatin between the *15/*16/*17 heterozygotes and *15/*16/*17 non-carriers were observed. In conclusion, this extensive analysis of the effect of SLCO1B1 polymorphisms on the pharmacokinetic profile of atorvastatin showed that c.521T > C and c.571T > C SNPs may affect the inter-individual response to atorvastatin. Additional studies, with a large sample size, are needed to confirm this finding.

Frequencies of single-nucleotide polymorphisms and haplotypes of the SLCO1B1 gene in selected populations of the western balkans.

As a membrane influx transporter, organic anion-transporting polypeptide 1B1 (OATP1B1) regulates the cellular uptake of a number of endogenous compounds and drugs. The aim of this study was to characterize the diversity of the solute carrier organic anion transporter family member 1B1 (SLCO1B1) gene encoding this transporter in two ethnic groups populating the Western Balkans. The distribution of SCLO1B1 alleles was determined at seven variant sites (c.388A>G, c.521T>C, c.571T>C, c.597C>T, c.1086C>T, c.1463G>C and c.*439T>G) in 266 Macedonians and 94 Albanians using the TaqMan allelic discrimination assay. No significant difference in the frequencies of the single nucleotide polymorphisms (SNPs) was observed between these populations. The frequency of the c.521T>C SNP was the lowest (<13.7 and 12.2%, respectively), while the frequencies of all other SNP alleles were above 40.0%. Variant alleles of c.1463G>C and c.1086 C>T SNPs were not identified in either ethnic group. The haplotype analysis revealed 20 and 21 different haplotypes in the Macedonian and Albanian population, respectively. The most common haplotype in both ethnic groups, *1J/*1K/*1L, had a frequency of 39.0% and 26.6%, respectively. In both populations, the variant alleles of the functionally significant c.521T>C and c.388A>G SNPs existed in one major haplotype (*15/*16/*17), with a frequency of 8.6 and 2.4% in the Macedonian and Albanian subjects, respectively. In conclusion, sequence variations of the SLCO1B1 gene in the studied populations occur at high frequencies, which are similar to that of the Caucasian population. Further studies are needed to evaluate the clinical significance of these SNPs and/ or the major SLCO1B1 haplotypes they form for a large number of substrates and for susceptibility to certain diseases.

Development and validation of automated SPE-LC-MS/MS method for determination of indapamide in human whole blood and its application to real study samples.

A fast and simple liquid chromatography-electrospray ionization tandem mass spectrometry method for determination of indapamide in human whole blood was developed and validated. The sample extraction of indapamide from human whole blood was achieved using automated solid-phase extraction. Chromatographic separation was performed on Kinetex C18 column (100 × 2.1 mm, 1.7 µm particle size) using acetonitrile and 2 mm ammonium formate in ratio 90:10 (v/v) as a mobile phase. The mass spectrometer was operated in the multiple reaction monitoring mode using positive electrospray ionization for indapamide and the internal standard (zolpidem tartarate). The total run time was 2.5 min. The present method was found to be linear in the concentration range of 1-50 ng/mL with the coefficient of determination 0.9987. The absolute recoveries of indapamide were 90.51-93.90%. The method was validated according the recommendations for validation of bioanalytical methods of European Medicines Agency guideline and was successfully used to analyze human whole blood samples for application in a pharmacokinetic study.

Colon-specific delivery of 5-aminosalicylic acid from chitosan-Ca-alginate microparticles.

Chitosan-Ca-alginate microparticles for colon-specific delivery and controlled release of 5-aminosalicylic acid after peroral administration were prepared using spray drying method followed by ionotropic gelation/polyelectrolyte complexation. Physicochemical characterization pointed to the negatively charged particles with spherical morphology having a mean diameter less than 9 microm. Chitosan was localized dominantly in the particle wall, while for alginate, a homogeneous distribution throughout the particles was observed. (1)H NMR, FTIR, X-ray and DSC studies indicated molecularly dispersed drug within the particles with preserved stability during microencapsulation and in simulated in vivo drug release conditions. In vitro drug release studies carried out in simulated in vivo conditions in respect to pH, enzymatic and salt content confirmed the potential of the particles to release the drug in a controlled manner. The diffusional exponents according to the general exponential release equation indicated anomalous (non-Fickian) transport in 5-ASA release controlled by a polymer relaxation, erosion and degradation. Biodistribution studies of [(131)I]-5-ASA loaded chitosan-Ca-alginate microparticles, carried out within 2 days after peroral administration to Wistar male rats in which TNBS colitis was induced, confirmed the dominant localization of 5-ASA in the colon with low systemic bioavailability.

5-ASA loaded chitosan-Ca-alginate microparticles: Preparation and physicochemical characterization.

The objective of the work was to prepare chitosan-Ca-alginate microparticles that can effectively deliver 5-ASA to the colon after peroral administration. For these requirements, a spray-drying technique was applied to 5-ASA/sodium alginate aqueous solution to obtain spherical particles having a mean diameter less than 10microm. The microparticles formed were cross-linked and coated into solution of CaCl(2) and chitosan to obtain stable microsystem. (1)H NMR and UV-vis spectra of 5-ASA have shown no degradation when working in adequate conditions, such as light protection, freshly prepared solution and use of nitrogen to prevent the oxidative self-coupling of 5-ASA moieties. By imaging with SEM, acceptable spherical morphology was observed, but also flattened, disk-shaped particles of smooth surface and low porosity. CLSM imaging showed dominant localization of chitosan in the particle wall, while for alginate, a homogeneous distribution throughout the particle was observed giving the particles negative charge. In the FTIR spectra of 5-ASA loaded Ca-alginate microparticles the characteristic peaks of 5-ASA were not altered indicating no covalent interaction between the drug and the polymer. DSC and X-ray diffraction studies revealed that 5-ASA was molecularly dispersed within the chitosan-Ca-alginate microparticles during the production process.

Biodegradation and drug release studies of BSA loaded gelatin microspheres.

Certain variations in the process parameters (emulsification time, surfactant concentration) were performed in order to prepare BSA-loaded gelatin microspheres with high loading efficacy and particle size ranging from 1 to 10 microm using a procedure originally employed by Tabata and Ikada. The mathematical modelling of drug release in the presence of collagenase showed a biphasic release pattern, where the rate constant for the initial time release confirmed the influence of the particle size and/or enzymatic degradation rate on drug release rate.

Biodistribution studies of BSA loaded gelatin microspheres after peroral application.

Biodistribution studies of radiolabelled [131I]BSA loaded gelatin microspheres were carried out on BALB/c mice after peroral administration. To two groups, the radiolabelled [131I]BSA gelatin microspheres with different mean particle size, 1.196+/-1.961 and 7.028+/-1.231 microm were administered orally. To the control group, a solution of [131I]BSA was also orally administered. Biodistribution was followed periodically within 15 days as a percent of total radioactivity present in stomach, small intestine with Peyer's patches and mesentery, colon with Peyer's patches, appendix and mesentery, liver, spleen, blood, kidney, lungs and heart. The biodistribution data confirmed that uptake in mice into Peyer's patches and passage to the liver and spleen via the mesentery lymph supply and nodes, increased with decreasing particle size.

Release profile of lidocaine HCl from topical liposomal gel formulation.

Liposomal hydrogel formulations of lidocaine HCl, suitable for topical application, have been prepared and drug release properties in vitro have been evaluated. Liposomes composed of Soya lechitin and cholesterol, with lidocaine HCl, entrapped in the inner water compartment, were prepared by simple hydration method. Topical liposomal gel formulations were prepared by incorporation of liposomes into a structured vehicle (hydrogels of Carbopol 940 in concentration of 1.5, 1.75 and 2%). High percentage of encapsulated drug in liposomes has been obtained (over 70%). Liposomal gel formulations provided prolonged drug release rate. The concentration of gelling agent in a range 1.5-2.0% affected the release rate slightly. In vitro release data showed that release kinetic can be described as diffusion-controlled, while liposomes act as reservoir systems for continuous delivery of drug. Proposed formulations provided stable percentage of entrapped drug and drug release within an examination period of 3 weeks.