CYP2C19 Gene Profiling as a Tool for Personalized Stress Ulcer Prophylaxis With Proton Pump Inhibitors in Critically Ill Patients - Recommendations Proposal.

To this date, there are no recommendations for personalized stress ulcer prophylaxis (SUP) in critical care that would take the patient's individual genetic predispositions into account. Of drugs used for this purpose, proton pump inhibitors (PPIs) are the first-choice drugs in intensive care unit patients. The degradation of proton pump inhibitors is mediated by cytochrome P450 (CYP) enzymes; in particular, CYP2C19 and, to a lesser extent, CYP3A4 are involved. Expression and metabolic activity of, namely in, CYP2C19 is significantly affected by single nucleotide polymorphisms, the drug metabolization rate varies greatly from ultrarapid to poor and likely influences the optimal dosage. As these CYP2C19 predictive phenotypes via CYP2C19 haplogenotypes (rs12248560/rs4244285) can be relatively easily determined using the current standard equipment of hospital laboratories, we prepared a set of recommendations for personalized PPI-based stress ulcer prophylaxis taking into account the patient's CYP2C19 predictive phenotype determined in this way. These recommendations are valid, in particular, for European, American and African populations, because these populations have the high representations of the CYP2C19*17 allele associated with the overexpression of the CYP2C19 gene and ultrarapid degradation of PPIs. We propose the CYP2C19 gene profiling as a tool for personalized SUP with PPI in critically ill patients.

Successful Mercaptopurine Usage despite Azathioprine-Induced Pancreatitis in Paediatric Crohn's Disease.

BACKGROUND: Azathioprine [AZA] and mercaptopurine [MP] are recommended for maintenance of steroid-free remission in children with Crohn`s disease [CD]. Azathioprine-induced pancreatitis, an idiosyncratic and major side effect, has been considered as an absolute contraindication for the use of a second thiopurine in IBD patients. MATERIALS AND METHODS: We describe two children with CD in whom MP were successfully trialled after a confirmed azathioprine-induced pancreatitis, being well tolerated in both cases. RESULTS: Two boys [13 and 10 years old] started exclusive enteral nutrition after diagnosis of moderate (Pediatric Crohn's Disease Activity Index [wPCDAI] = 45) and mild [wPCDAI = 35] CD. Both developed an acute mild to moderate pancreatitis after 2 and 3 weeks, respectively, of AZA treatment but recovered fully in hospital after AZA withdrawal. They started on MP treatment without any adverse effect. They were tested for the presence of polymorphisms 238G>C, 460G>A, and 719A>G in the TPMT gene and 94C>A and 21>C in the ITPase. Both patients were wild-type for all tested polymorphisms. CONCLUSIONS: Azathioprine-induced acute pancreatitis should not be considered as an absolute contraindication for the use of MP. Further investigation is required to create a better understanding of the mechanism underlying the adverse events and to allow more possibilities for personalised therapy.

Diplacone and mimulone ameliorate dextran sulfate sodium-induced colitis in rats.

Diplacone (1) and mimulone (2), two geranylated flavanones, have previously shown anti-inflammatory and antiradical activity in vitro. The present study aimed to evaluate their activity in vivo on a model of colitis induced in Wistar rats by an oral administration of dextran sulfate sodium (DSS). Diplacone (1) and mimulone (2) were administered at a bolus dose of 25mg/kg by gastric gavage 48 and 24h prior to the induction of colitis by DSS and every 24h on the following days of the experiment. The effect of the treatment was assessed by monitoring the disease activity index (DAI), histopathological examination, evaluation of the weight and length of the colon and by analysis of the levels and activities of cyclooxygenase-2 (COX-2), matrix metalloproteinase-2 (MMP2), superoxide dismutase-2 (SOD2), and catalase (CAT) in the inflamed tissue. Administration of the test compounds prior and after induction of colitis ameliorated the symptoms of colitis (diarrhea, presence of the blood in the stool) and delayed their onset. The ability of compounds 1 and 2 to reduce the levels of COX-2 and to increase the ratio of pro-MMP2/MMP2 activity correlates with the values of the DAI. The lowering of the levels of the antioxidant enzymes SOD2 and CAT reflects the ability of the test compounds to scavenge reactive oxygen species.

Antibacterial efficiency of vermiculite/chlorhexidine nanocomposites and results of the in vivo test of harmlessness of vermiculite.

Clay minerals have been proposed as very useful materials for modulating drug delivery. These are the commonly used materials in pharmaceutical production both as inorganic carriers or active agents. We focused on the development of suitable long-acting material for local treatment of oral infection where clay minerals act as inorganic drug carriers. Organovermiculites with antibacterial activity were prepared by ion exchange reactions using different concentrations of chlorhexidine diacetate. The samples were characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and thermal analysis (TGA). The antibacterial activity was evaluated by finding the minimum inhibitory concentration (MIC). All studied organoclays possessed good antibacterial activity after 24h exposure against Escherichia coli, Enterococcus faecalis and particularly against Staphylococcus aureus. Pseudomonas aeruginosa however proved very resistant as only the sample with the highest concentration of CA that successfully inhibited bacterial growth. Furthermore, clay mineral vermiculite was subjected to in vivo toxicological analysis and its influence on gastrointestinal tract during its oral application was investigated. Tissue samples from buccal mucosa, tongue, esophagus, stomach, terminal duodenum, small intestine, caecum, distal colon and liver were subjected to histological examination, both macroscopically and microscopically. Neither systemic nor local reactions were observed. Therefore the toxicity of vermiculite to a mammal model organism can be excluded.

Frequencies of HLA-DQ2 and HLA-DQ8 haplotypes in Czech and Slovak coeliac patients and the healthy population.

Coeliac disease is an autoimmune disorder with genetic predisposition. The aim was to determine the frequency of HLA-DQ2 and HLA-DQ8 in Czech and Slovak patients and the healthy population. The study included 127 patients and 66 healthy volunteers. HLA-DQ2 was identified in 85.03% patients, and 24.24% healthy individuals (P=0.0001; OR17.7632; CI=8.4347-37.4088). HLA-DQ8 was identified in 11.81% patients and 15.5% healthy individuals. HLA-DQ8 occurred more often in HLA-DQ2-negative patients compared to HLA-DQ2-positive patients (P=0.0494; OR3.5; CI 1.0428-11.7468). At least one of the studied HLA-variants was found more often in patients than in healthy individuals (P=0.0001; OR58.8; CI 7.6856-449.8602).

Three polymorphisms in promoter of protein C gene with endothelial protein c receptor gene and risk of venous thrombosis.

The primary abnormalities that are associated with a risk of venous thrombosis are the deficiencies of protein C. Protein C (PROC), encoded by the PROC gene, acts through its affinity for binding to its transmembrane endothelial cell protein C receptor (EPCR) encoded by the EPCR gene. The objective of the study was to analyze the link between three polymorphisms in the promoter of PROC gene, the polymorphism in the EPCR gene and the occurrence of venous thrombosis. We genotyped 135 individuals - 51 cases with documented venous thrombosis and 84 healthy volunteers without a history of venous thrombosis. The occurrence of the TAA haplotype of PROC gene was significantly more frequent in the controls (N = 48; 57.1%), compared with the patients (N = 18; 35.3%), (P = 0.0206). The healthy individuals were also significantly often carriers of the TAA haplotype and the standard genotype AA of EPCR gene (50 vs. 25.5%) than the patients (P = 0.0066). The frequency of haplotypes CAA and CGT of PROC gene was insignificantly higher in the patients (15.7 and 21.6%, respectively) than in the control group (9.5 and 13.1%). The combination of haplotype CAA/CAA of PROC gene and variant genotype AG of EPCR gene was confirmed with a higher frequency in the group of patients (3.9 vs. 1.2%).This analysis showed that the PROC haplotype associated with a high protein C level (TAA) and the EPCR AA genotype was significantly more frequent in the healthy volunteers (P = 0.0066). Haplotypes associated with a low production of protein C (CAA or CGT) were more frequent in patients with venous thrombosis.

A comparison of the impact of amaranth flour and squalene on plasma cholesterol in mice with diet-induced dyslipidemia.

Amaranth was identified as a possible component of an anti-sclerotic diet. To date, particular substances responsible for this effect have not been exactly specified. Squalene, which is contained in amaranth, could be responsible for this effect. However, there are also other potential substances and the hypolipidemic effect of amaranth can be caused by a synergistic effect of several components. This study investigated and compared the impact of amaranth flour and squalene on the total cholesterol (CHOL(TOT)) and LDL cholesterol (CHOL(LDL)) levels in mice with dyslipidemia induced by a cholesterol- and sugar-rich diet. The experiment included 40 inbred mice (C57Bl/6J SPF). After a 7-days acclimatization period, animals were divided into four groups by random. Individual groups were fed different diets for 49 days: control (group C), high energy diet (group HED), high energy diet with amaranth flour (group HED+A) and high energy diet with squalene (group HED+S). The sugar- and cholesterol-rich diet in HED resulted in the significant increase in the levels of CHOL(TOT) by 125% (P < 0.05) and CHOL(LDL) by 304% (P < 0.05), and at the same time in a decrease of HDL cholesterol (CHOL(HDL)) levels by 58% (P < 0.05) compared to group C. To the contrary, amaranth flour enriched diet in group HED+A led to a decrease of CHOL(TOT) levels by 33% (P < 0.05) and CHOL(LDL) by 37% (P < 0.05), compared to HED. Both, amaranth flour and squalene, had a positive impact on CHOL(HDL) levels. Compared to group HED, there was a 47% increase in HED+A and a 60% increase in HED+S. Results proved the favorable impact of amaranth flour on the levels of total cholesterol CHOL(TOT) and also on CHOL(LDL). Furthermore, the results imply that amaranth flour contains besides squalene other substances, which can actively participate in its hypolipidemic effect.

Polymorphism G-308A in the promoter of the tumor necrosis factor-α gene and its association with the risk of venous thromboembolism.

The main abnormalities associated with the increased risk of venous thrombosis are the inherited deficiencies of antithrombin, protein C, protein S, the point mutations known as factor V Leiden and factor II G20210A. The association of other specific genes with thrombotic risk is less known. G-308A polymorphism in the promoter area of the gene coding for tumor necrosis factor α (TNF-α) is associated with an increased transcription activity of this gene, increased TNF-α production and subsequent predisposition to some illnesses. The aim of this work was to study the link between this polymorphism and predisposition to deep venous thrombosis (DVT). The research determined the frequency of the variant allele -308A in the gene for TNF-α in a group of 67 patients diagnosed with DVT and in a group of 62 healthy volunteers. We confirmed statistically significant link between the occurrence of the variant allele -308A and DVT (P = 0.02). This mutation was associated with a 2.64-fold greater risk of venous thrombosis, 95% confidence interval (1.19-5.87). When excluding heterozygous and homozygous carriers of the Leiden mutation from both groups, the difference between the occurrence of the variant allele -308A in the groups of ill and healthy individuals remained statistically significant (P = 0.04). The statistical significance was also confirmed after the exclusion of patients with mutation in the gene for prothrombin (P = 0.02). The results of this work imply possible association between the variant allele -308A and the development of DVT.

Gene polymorphisms involved in manifestation of leucopenia, digestive intolerance, and pancreatitis in azathioprine-treated patients.

BACKGROUND: Approximately 10-28 % of patients experience adverse drug reactions related to treatment with thiopurines. The most serious reaction is myelosuppression, typically manifested as leucopenia, which occurs in approximately 2-5 % of patients. Other adverse drug reactions that often accompany thiopurine therapy are pancreatitis, hepatotoxicity, allergic reactions, digestive intolerance, arthralgia, febrile conditions, and rash. OBJECTIVE: The objective of this study was to assess the relationship between variant alleles of thiopurine S-methyltransferase (SNPs 238G > C, 460G > A and 719A > G), inosine triphosphate diphosphatase (SNPs 94C > A and IVS2 + 21A > C), and xanthine dehydrogenase (837C > T) and the occurrence of adverse drug reactions to azathioprine therapy. METHODS: Genotype was determined for 188 Caucasians diagnosed with inflammatory bowel disease treated with a standard dose of azathioprine (1.4-2.0 mg/kg/day). Allelic variants were determined by PCR-REA and real-time PCR methods. Results were statistically evaluated by use of Fisher's test and by odds ratio calculation. RESULTS: Variant genotype thiopurine S-methyltransferase predisposes to development of leucopenia (P = 0.003, OR = 5, CI 95 %, 1.8058-13.8444). Although not statistically significant, we observed a trend that suggested correlation between the occurrence of digestive intolerance and the variant genotype inosine triphosphate diphosphatase (P = 0.1102; OR 15.63, CI 95 %, 1.162-210.1094), and between the occurrence of pancreatitis and the variant allele xanthine dehydrogenase 837T (P = 0.1124; OR 12,1, CI 95 %, 1.15-126.37). CONCLUSION: The variant genotype thiopurine S-methyltransferase has been associated with the occurrence of leucopenia. The involvement of polymorphisms in inosine triphosphate diphosphatase and xanthine dehydrogenase genes in the development of digestive intolerance and pancreatitis will require further verification.

Local tissue reaction after the application of topical hemostatic agents in a rat partial nephrectomy model.

Various hemostatics are used for renal surgical procedures. We investigated the hemostatic efficacy of cellulose derivatives on the model of partial nephrectomy in rats focusing on the local reaction of renal parenchyma. A total of 50 Wistar rats were divided into five groups of 10 animals each. Partial nephrectomy of the caudal pole without hilar vascular control was performed. Oxidized cellulose (OC), sodium salt of oxycellulose (OCN), carboxymethyl cellulose (CMC), dialdehyde cellulose (DAC), and gelatin-based hemostatic (C) were applied to the bleeding wounds. The time to hemostasis was monitored. Half of the animals were euthanized after 3 days, the second half 30 days from the experiment start date. The left kidney was excised and subjected to histopathological examination. The biochemical data was subjected to statistical analysis. The time to hemostasis in all groups was significantly less than in the C group (in OC p = 0.0057, OCN p = 0.0039, CMC and DAC p = 0.0001). In the C group, massive hemorrhages and necrosis did occur. In the OC and OCN groups, there were regenerative changes, a receding inflammatory reaction and hemorrhage. DAC caused an immune reaction and massive interstitial hemorrhages with biochemical signs of liver damage. Parenchyma in CMC revealed a reduction of necrosis and interstitial hemorrhages with regenerative processes. The most effective hemostatics were CMC and OC, achieving the best results both in the time to hemostasis, and for histopathological evaluation.

Cardioprotective effects of 44Bu, a newly synthesized compound, in rat heart subjected to ischemia/reperfusion injury.

Excessive intracellular Na+ accumulation followed by Ca2+ overload in cardiac tissue is one of the important mechanisms leading to ischemia/reperfusion injury. In the present study, the cardioprotective effects of 44Bu, 2-hydroxy-3-(butylamino) propyl-4-{(butoxycarbonyl)amino}benzoate hydrochloride, a novel Na+ channel blocker, on ischemia/reperfusion injury were investigated and compared to lidocaine. Isolated rat hearts perfused at the constant flow were exposed to global ischemia for 60 min followed by 30 min of reperfusion. In control hearts, ischemia/reperfusion markedly decreased left ventricular developed pressure and increased left ventricular end-diastolic pressure, and caused lactate dehydrogenase release and infarction. 44Bu (0.1, 0.3 and 1 microM) or lidocaine (1 and 200 microM) was administrated during the last 10 min before ischemia and the first 5 min of the reperfusion period. A significant post-ischemic functional recovery in the same degree was elicited by 0.3 and 1 microM 44Bu or 200 microM lidocaine. These effects of 44Bu and lidocaine closely correlated with the reduction in the infarct size and lactate dehydrogenase release. In contrast, 44Bu (0.1 microM) or lidocaine (1 microM) treatment did not result in a significant recovery in any of the examined parameters. In accordance with functional results, our electrophysiological data demonstrated that 44Bu was a more potent agent than lidocaine in terms of transient Na+ current inhibition. On the other hand, 44Bu did not cause any change in Ca2+ currents and on Na+/H+ exchange activity. These results show that 44Bu, as a novel Na+ channel blocker, has cardioprotective effects against ischemia/reperfusion injury.

Importance of thiopurine S-Methyltransferase gene polymorphisms for prediction of azathioprine toxicity.

OBJECTIVES: Our study aims to find the relationship between metabolic enzyme thiopurine S-methyltransferase (TPMT) gene polymorphisms and clinical output of the therapy with azathioprine. We focused on patients who experienced leucopenia caused by high blood levels of active azathioprine metabolites. DESIGN: Our group consists of 87 patients who have been treated by azathioprine. 21 individuals experienced leucopenia during treatment with standard dose of azathioprine. We have used PCR-REA and "real-time" PCR methods for genotype detection G238C, G460G and A719G substitutions in TPMT gene. RESULTS: We have found statistical association between the presence of non-standard TPMT alleles and adverse event associated with azathioprine treatment - leucopenia (p=0.0033). CONCLUSION: Our results confirm that TPMT genotyping prior to the treatment with azathioprine could predict patients with genetic predisposition for serious leucopenia and seems to be a useful genetic marker for individualisation of the therapy.

Antiarrhythmic effect of newly synthesized compound 44Bu on model of aconitine-induced arrhythmia -- compared to lidocaine.

The antiarrhythmic action of the newly developed compound 44Bu (an original compound that was synthesized at our Faculty of Pharmacy) was tested on a model of aconitine-induced arrhythmia and compared with the effect of lidocaine. Both tested substances were administered either as therapeutic or prophylactic agents. 44Bu was highly effective in reducing the occurrence of ventricular fibrillation from 94% to 8% by therapeutic administration, and to 0% by prophylactic administration. The overall mortality rate was significantly reduced by 44Bu from 100% to 25% in the case of therapeutic administration, and to 0% in the case of prophylactic administration. In contrast, there was not any significant difference between therapeutic and prophylactic administration of lidocaine. The occurrence of ventricular fibrillation dropped from 94% to 50% with therapeutic administration, and to 67% with prophylactic administration of lidocaine. The overall mortality rate was significantly reduced from 100% to 63% and to 67%, respectively. We conclude that the 44Bu compound is a highly effective agent in suppressing aconitine-induced arrhythmias. The antiarrhythmic effect of 44Bu was significantly more evident in comparison with lidocaine, particularly in the case of its prophylactic administration.

Effect of a new ultrashort betalytic agent on aconitine-induced arrhythmia.

The anti-arrhythmic effect was tested on the model of aconitine-induced arrhythmia. The experiment was performed in vivo with 31 male Wistar laboratory rats. Group A was first administered aconitine and, after the onset of the first sinus rhythm disorders, the 44Bu compound was administered. Group B was first administered the 44Bu compound and only after that the aconitine. The control group was administered aconitine and saline as a replacement of the tested compound. In group A, there was a decrease in the ventricular fibrillation occurrence from 100 % to 8 % (p < 0.001) after the administration of the 44Bu compound. In the B group, the onsets of all monitored arrhythmia types were delayed by an average of 15.6 min. Ventricular rhythm occurrence was decreased from 100 to 20 %, as well as ventricular fibrillations, from 100 to 0 % (p < 0.001).