Distribution of the most Common Genetic Variants Associated with a Variable Drug Response in the Population of the Republic of Macedonia.

Genetic variation in the regulation, expression and activity of genes coding for Phase I, Phase II drug metabolizing enzymes (DMEs) and drug targets, can be defining factors for the variability in both the effectiveness and occurrence of drug therapy side effects. Information regarding the geographic structure and multi-ethnic distribution of clinically relevant genetic variations is becoming increasingly useful for improving drug therapy and explaining inter-individual and inter-ethnic differences in drug response. This study summarizes our current knowledge about the frequency distribution of the most common allelic variants in three broad gene categories: the Phase I oxidation-cytochrome P450 (CYP450) family (CYP2C9, CYP2C19, CYP3A5, CYP2D6); the Phase II conjugation (GSTT1, SULT1A1; UGT1A1) and drug target (TYMS-TSER, MTHFR and VKORC1) in the population of the Republic of Macedonia and compares the information obtained with data published for other indigenous European populations. Our findings define the population of the Republic of Macedonia as an ethnic group with a highly polymorphic genetic profile. These results add to the evidence regarding the distribution of clinically important variant alleles in DME and drug target genes in populations of European ancestry.

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.

Characterization of the most common CYP2C9 and CYP2C19 allelic variants in the population from the Republic of Macedonia.

The aim of this study was to evaluate the most common CYP2C9 and CYP2C19 polymorphisms in the population of Macedonia and compare them with the global geographic data reported from different ethnic populations. In total, 184 healthy volunteers from the general population were included. Genotypes for the CYP2C9 (*2 [rs1799853] and *3[rs1057910]) and CYP2C19 (*2-[rs4244285] and *17 [rs12248560]) polymorphisms were detected by Real-Time PCR using TaqMan SNP genotyping assay. The CYP2C9 wildtype allele (*1) was the most frequent (78.8%) and the non-functional alleles *2 and *3 had a frequency of 13.9% and 7, 3%, respectively. Seven subjects (2.97%) were poor metabolites (PMs) for CYP2C9 because of the *2/*2 and *3/*3 genotype. For CYP2C19, the frequencies of the*1 (wild-type) and the non-functional alleles (*2 and *17) were 65.4%, 14.4% and 20.1%, respectively. The *2/*2 genotype, corresponded to the predicted frequency of 2.7% for the CYP2C19 PM phenotype. The total of 59 out of 184 subjects (32.0%) was determined as UMs because of the *1/*17 and *17/*17 genotypes. The compound heterozygote (*2/*17), which is associated with a difficult-to-predict phenotype, was detected in 8 subjects (4.34%). The CYP2C9 and CYP2C19 are polymorphic in the population of the Republic of Macedonia. The frequencies of the most common CYP2C9 and CYP2C19 allelic variants are similar to those reported for Caucasians of European descendant, but differ from those of North America Caucasians. Our results suggest that the genetically determined capacity of CYP2C9 and CYP2C19 has to be taken into account in order to improve the individual risk / benefit ratio of the drug therapy in Macedonia.

Perindopril treatment in streptozotocin induced diabetic nephropaty.

Diabetic nephropathy (DN) is one of the most common causes of terminal stadium damage to the kidneys. The angiotensin-converting enzyme (ACE) represents a significant risk factor for the progression of DN. ACE inhibitors are medications of particular interest knowing the role of angiotensin II in the development of DN. This study aimed to examine the effects of ACE inhibitor treatment perindopril (PER), administered to rats with streptozotocin (STZ) induced DN, that developed albuminuria, renal hypertrophy and mild glomerulussclerosis. DN was induced by a STZ (60 mg/kg ip) single injection to normotensive Wistar rats. The administration of STZ caused diabetes mellitus (DM) with symptoms and signs of DN including poor general condition, body-weight loss, kidney weight increase as well as increased values of BUN and serum creatinine, accompanied by increased diuresis as well as distinct albuminuria. The majority of these symptoms were manifested 4 weeks after, and even more distinctly 8 and 12 weeks after administering STZ. The perindopril treatment (6 mg/kg BW), starting 4 weeks after administering STZ, resulted in a significant improvement of all symptoms and signs of DN, significantly lowering the values of BUN and serum creatinine, albuminuria and diuresis. The histopathological examination of the renal samples at 8 and 12 weeks after the beginning of the study have shown that perindopril significantly lowers the progression of glomerulopathy, and significantly improves the glomerulosclerotic index, as well as the progression of renal histological abnormalities induced with STZ. Thus perindopril treatment ameliorates STZ-induced nephropathic changes in DM rats.

Erythropoietin reduces cumulative nephrotoxicity from cisplatin and enhances renal tubular cell proliferation.

Cisplatin, a heavy metal complex, is one of the most active drugs used in the treatment of several human malignancies. However, high-dose therapy with cisplatin is limited by its cumulative nephrotoxicity. The main objectives of this study were to determine the role of recombinant human erythropoietin (Epoetin alfa) in the prevention of nephrotoxicity induced experimentally in Wistar rats by long-term administration of cisplatin (2 mg/kg/b.w./week) over eight weeks, and an evaluation of its effect on renal tubular cell proliferation. The animals were randomly assigned into three groups, each including 25 rats. Group 1 (CP) received only cisplatin (2 mg/kg/b.w./week), group 2 (CP+EPO) received cisplatin (2 mg/kg/b.w./week) and epoetin alfa (150 IE/kg/b.w./three times a week), and group 3 (control group) received only saline. During the study, the following tests for the assessment of the renal function and renal damages were performed: determination of concentration of serum creatinine and BUN and determination of total protein quantity in 24-hour urine samples. At the end of the study, the abdomen was opened and both kidneys of the rats were removed and sent for histological and morphometric analysis. Ki-67 was used as a tool to determine a proliferative index. The results obtained have shown that epoetin alfa significantly reduced the functional renal failures and renal damages, and increased toleration of high doses of cisplatin. At the same time, our results with regard to tubular proliferative index have confirmed that one of the possible mechanisms by which erythropoietin accomplishes its renoprotective effect is stimulation of tubular cell proliferation and regeneration.