An insight into an intriguing oxidative biotransformation pathway of 5-O-caffeoylquinic acid by a gut bacterium.

Microbiota is known to play a pivotal role in generating bioavailable and bioactive low-molecular-weight metabolites from dietary polyphenols. 5-O-caffeoylquinic acid (5-CQA), one of the main polyphenols found in human diet, was submitted to a resting cell biotransformation study using three gut bacteria species Lactobacillus reuteri, Bacteroides fragilis and Bifidobacterium longum. These bacteria were selected according to their belonging to the main phyla found in human gut microbiota. Our study highlighted the ability of only one of the strains studied, L. reuteri, to bioconverse 5-CQA into various metabolites due to the expression of the cinnamoyl esterase enzyme as the first step. Interestingly, one known natural compound, esculetin, was described for the first time as a 5-CQA-derived metabolite after conversion by a gut bacterium, the other metabolites had already been reported. This evidence highlighted an interesting oxidative pathway occurring in vivo by intestinal microbiota leading to esculetin. This molecule was also identified after electrochemical and enzymatic oxidations of caffeic acid. The oxidation capacity of L. reuteri led to less diverse metabolites in comparison to those obtained either electrochemically and enzymatically where dimers and trimers were reported. Thus, esculetin may have interesting and benefical biological effects on gut microbiota, which should be further evaluated. Novel synbiotics could be formulated from the association of L. reuteri with 5-CQA.

Chemometrics approach for optimization of capillary electrophoretic conditions for the separation of insulin analogues.

Six insulin analogues with high degree of chemical similarity were separated using Capillary Electrophoresis (CE). In order to improve the method performance, optimization and development the Design of Experiment (DoE) approach has been used and this strategy provided related statistical models. This methodology delivered the information regarding the influence of main factors in the method development and explained the interaction of relevant factors in terms of shortening the analysis time. The response surface methodology (RSM) design employing Central Composite Face Centered (CCF) Design analyzed the effect of the most influencing factors, including background electrolyte pH and concentration, applied voltage and temperature. This study demonstrated that the approach of analyzing the influence of each parameter in the migration behavior of analyzed peptides was capable to assess the best electrophoretic conditions for the separation of insulin analogues.

Application of the capillary zone electrophoresis (CZE) and capillary gel electrophoresis (CGE) for the separation of human insulin, insulin lispro and their degradation products.

Two capillary electrophoresis (CE) methods have been developed for the separation of charge and mass variants of human insulin and its recombinant analogue lispro. Since the capillary zone (CZE) and Capillary gel electrophoresis (CGE) are based on different principles of separation, they can be used to detect different impurities of insulin and its analogues. Application of CZE enabled a separation of compounds with different m/z ratio, therefore CZE is a suitable method for the separation of deamidation products of insulin. After the optimization, this method is validated according ICH requirements. CGE method was used for the separation of higher molecular weight transformation products. Experimental data have shown that CZE and CGE are simple, fast and robust methods which could be used as a routine analysis for quality control of insulin formulations.

Prebiotics as excipients for enhancement of stability and functionality of Bifidobacterium longum ssp. infantis with potential application as symbiotics in food and pharmaceuticals.

Objective: Formulations containing probiotics are promoted due to health benefits. During lyophilization and subsequent storage in the gastrointestinal tract, bacteria are exposed to stress conditions that can lead to impairment and loss of viability. Methods: The suitability of various excipients for enhancing the stability and functionality of Bifidobacterium longum subsp. infantis during storage as freeze-dried powder and through exposure to acid and bile was investigated. Cells were lyophilized in the presence of sucrose, trehalose, lactose, cellobiose and fructooligosaccharide (FOS) and stored at 4 °C or 25 °C. The effect of diverse protectants on the persistence after exposure to acid and bile environment was examined through determination of the colony forming units, the ß-glucosidase and ß-galactosidase activity and the membrane integrity changes. Results: Cells freeze-dried in the presence of cryoprotectants had comparable survivability during storage at 4 °C whereas the survival rate at 25 °C of cells protected by cellobiose and FOS was higher than for those protected with sucrose and trehalose. Furthermore, the respective excipients used as cryoprotectants enhanced the stability of cells exposed to simulated gastric and small intestinal medium. Stabilization may be achieved through different mechanism of action such as protecting the membrane integrity and as metabolizable substrates. Overall, prebiotic and thus metabolizable protectants including cellobiose and FOS were superior to other protectants used. Conclusion: In symbiotic formulas with B. infantis, these sugars might serve as prebiotics and stabilizers of this probiotic strain during lyophilization, storage and in gastrointestinal conditions simultaneously, potentially increasing its health-promoting effects.

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.

Charge heterogeneity study of a Fc-fusion protein, abatacept, using two-dimensional gel electrophoresis.

Medicinal products obtained by recombinant DNA technology are complex molecules and demonstrate a high degree of molecular heterogeneity. Charge heterogeneity and isoform pattern of this class of medicines, are parameters important for their quality, safety, and efficacy. In this study we report the application of two-dimensional gel electrophoresis (2-D electrophoresis) for the quality assessment, identification, charge heterogeneity and isoform pattern study of recombinant protein, CTLA4-Ig (abatacept), which has been selected as an example of the drug class, known as Fc-fusion proteins. In order to achieve an efficient separation of this complex analyte,2-D electrophoresis was optimized employing different experimental conditions regarding the selection of an immobilized pH gradient (IPG), sample pretreatment, presentation and detection procedure. Experimental datadocumented that 2-D electrophoresis is a suitable method for the assessment of identity, purity, structural integrity, isoform pattern and to monitor charge heterogeneity and post-translational glycosylation of the Fc-fusion protein, abatacept.