Martin Kunes

Preclinical electrogastrography in experimental pigs

Preclinical electrogastrography in experimental pigs Surface electrogastrography (EGG) is a non-invasive means of recording gastric myoelectric activity or slow waves from cutaneous leads placed over the stomach. This paper provides a comprehensive review of preclinical EGG. Our group recently set up and worked out the methods for EGG in experimental pigs. We gained our initial experience in the use of EGG in assessment of porcine gastric myoelectric activity after volume challenge and after intragastric administration of itopride and erythromycin. The mean dominant frequency in pigs is comparable with that found in humans. EGG in experimental pigs is feasible. Experimental EGG is an important basis for further preclinical projects in pharmacology and toxicology.

Bacteriocinogeny in experimental pigs treated with indomethacin and Escherichia coli Nissle

AIM To evaluate bacteriocinogeny in short-term high-dose indomethacin administration with or without probiotic Escherichia coli Nissle 1917 (EcN) in experimental pigs. METHODS Twenty-four pigs entered the study: Group A (controls), Group B (probiotics alone), Group C (indomethacin alone) and Group D (probiotics and indomethacin). EcN (3.5×10(10) bacteria/d for 14 d) and/or indomethacin (15 mg/kg per day for 10 d) were administrated orally. Anal smears before and smears from the small and large intestine were taken from all animals. Bacteriocin production was determined with 6 different indicator strains; all strains were polymerase chain reaction tested for the presence of 29 individual bacteriocin-encoding determinants. RESULTS The general microbiota profile was rather uniform in all animals but there was a broad diversity in coliform bacteria (parallel genotypes A, B1, B2 and D found). In total, 637 bacterial strains were tested, mostly Escherichia coli (E. coli). There was a higher incidence of non-E. coli strains among samples taken from the jejunum and ileum compared to that of the colon and rectum indicating predominance of E. coli strains in the large intestine. Bacteriocinogeny was found in 24/77 (31%) before and in 155/560 (28%) isolated bacteria at the end of the study. Altogether, 13 individual bacteriocin types (out of 29 tested) were identified among investigated strains. Incidence of four E. coli genotypes was equally distributed in all groups of E. coli strains, with majority of genotype A (ranging from 81% to 88%). The following types of bacteriocins were most commonly revealed: colicins Ia/Ib (44%), microcin V (18%), colicin E1 (16%) and microcin H47 (6%). There was a difference in bacteriocinogeny between control group A (52/149, 35%) and groups with treatment at the end of the study: B: 31/122 (25%, P=0.120); C: 43/155 (28%, P=0.222); D: 29/134 (22%, P=0.020). There was a significantly lower prevalence of colicin Ib, microcins H47 and V (probiotics group, P<0.001), colicin E1 and microcin H47 (indomethacin group, P<0.001) and microcins H47 and V (probiotics and indomethacin group, P=0.025) compared to controls. Escherichia fergusonii (E. fergusonii) was identified in 6 animals (6/11 isolates from the rectum). One strain was non-colicinogenic, while all other strains of E. fergusonii solely produced colicin E1. All animals started and remained methanogenic despite the fact that EcN is a substantial hydrogen producer. There was an increase in breath methane (after the treatment) in 5/6 pigs from the indomethacin group (C). CONCLUSION EcN did not exert long-term liveability in the porcine intestine. All experimental pigs remained methanogenic. Indomethacin and EcN administered together might produce the worst impact on bacteriocinogeny.

High-performance liquid chromatographic determination of tiapride and its phase I metabolite in blood plasma using tandem UV photodiode-array and fluorescence detection.

New bioanalytical SPE-HPLC-PDA-FL method for the determination of the neuroleptic drug tiapride and its N-desethyl metabolite was developed, validated and applied to xenobiochemical and pharmacokinetic studies in humans and animals. The sample preparation process involved solid-phase extraction of diluted plasma spiked with sulpiride (an internal standard) using SPE cartridges DSC-PH Supelco, USA. Chromatographic separation of the extracts was performed on a Discovery HS F5 250 mm × 4 mm (Supelco) column containing pentafluorophenylpropylsilyl silica gel. Mobile phase (acetonitrile-0.01 M phosphate buffer pH=3, flow rate 1 ml min(-1)) in the gradient mode was employed in the HPLC analysis. Tandem UV photodiode-array→fluorescence detection was used for the determination of the analytes. Low concentrations of tiapride and N-desethyl tiapride were determined using a more selective fluorescence detector (λ(exc.)/λ(emiss.)=232 nm/334 nm), high concentrations (500-6000 pmol ml(-1)) using a UV PDA detector at 212 nm with a linear response. Each HPLC run lasted 15 min. Lower limits of quantification (LLOQ) for tiapride (N-desethyl tiapride) were found to be 8.24 pmol ml(-1) (10.11 pmol ml(-1)). The recoveries of tiapride ranged from 89.3 to 94.3%, 81.7 to 86.8% for internal standard sulpiride and 90.9 to 91.8% for N-desethyl tiapride.

The effect of general anaesthesia on gastric myoelectric activity in experimental pigs

BackgroundSurface electrogastrography (EGG) is a non-invasive method for clinical assessment of gastric myoelectrical activity. Different forms of general anaesthesia might have various effects on porcine EGG. The aim of this study was to evaluate the impact of different anaesthetic agents on EGG in experimental pigs.MethodsFour 15-minute EGG intervals were recorded and analysed. A baseline EGG recording was started 20 minutes after intramuscular injection of ketamine and azaperone (periods A and B). Four different regimens of general anaesthesia followed immediately after the baseline EGG (5 pigs in each experimental group): thiopental, isoflurane, nitrous oxide and isoflurane plus nitrous oxide. EGG recordings followed for the next 30 minutes under general anaesthesia (periods C and D). The dominant frequencies of slow waves were compared between the baseline intervals A and B and periods C and D under general anaesthesia.ResultsThe mean dominant frequency was within the normal range (2.3 – 3.5 cycles per minute) in all animals in all regimens. Thiopental general anaesthesia did not influence any change of the dominant frequency of slow waves. Nitrous oxide general anaesthesia increased the dominant frequency of slow waves in a statistically significant manner (baseline: 2.93 ± 0.53 and 3.01 ± 0.53; under general anaesthesia: 3.25 ± 0.34 and 3.29 ± 0.38 cycles per minute; p < 0.001, p = 0.003, p < 0.001, p < 0.001). Nitrous oxide together with isoflurane induced a statistically significant decrease of dominant frequency in the last 15-minute interval (2.66 ± 0.55 cycles per minute) compared to the baseline recording (2.81 ± 0.49; p = 0.030).ConclusionsAll changes of porcine gastric myoelectric activity assessed by the dominant frequency of slow waves during EGG remained within the normal range although some of them achieved statistical significance. Thus all tested agents used for general anaesthesia can be recommended in preclinical studies with porcine models focused on gastric myoelectric activity without any risk of compromising the results. Thiopental seems to be the most suitable as it did not cause any changes at all.

Morphometric analysis of the porcine gastrointestinal tract in a 10-day high-dose indomethacin administration with or without probiotic bacteria Escherichia coli Nissle 1917

Background: Nonsteroidal anti-inflammatory drugs may cause severe injury to all parts of the gastrointestinal tract. It has been hypothesised that probiotic bacteria might reduce this adverse effect. The aim of this study was to perform a morphometric evaluation of the gastrointestinal tract in experimental pigs treated using a 10-day high-dose of indomethacin with or without Escherichia coli Nissle 1917 (EcN). Methods: Twenty-four healthy mature pigs were included: Group A (controls; 6 animals), Group B (EcN; n = 6), Group C (indomethacin; n = 6) and Group D (EcN & indomethacin; n = 6). EcN (3.5 × 1010 live bacteria/day for 14 days) and/or indomethacin (15 mg/kg/day for 10 days) were administered. Specimens of the stomach, small and large bowel were routinely processed for microscopic examination. The height of glandular mucosa, height and width of interfoveolar spaces and villi and basement size of epithelial cells were evaluated. Results: Different effects of indomethacin and EcN on particular parts of the gastrointestinal tract were shown. The indomethacin and probiotics group demonstrated a significantly lower height of cryptal mucosa and colonocytes and widening of the basement size of colonocytes compared to controls (p = 0.004; p < 0.001; p = 0.025). The height of cryptal mucosa was significantly higher in the EcN group compared to controls (p = 0.001). Conclusions: Indomethacin alone induced marked adaptation of the gastric mucosa. EcN alone provided a significant favourable trophic effect on the colonic mucosa. However, indomethacin and probiotics administered together comprise the worst impact on all porcine stomach, small and large bowel.

Transintestinal transport mechanisms of 5-aminosalicylic acid (in situ rat intestine perfusion, Caco-2 cells) and Biopharmaceutics Classification System.

The aim of the study was 1) to estimate permeability of 5-aminosalicylic acid (5-ASA), 2) to categorize 5-ASA according to BCS (Biopharmaceutics Classification System), and 3) to contribute to determination of 5-ASA transintestinal transport and biotransformation mechanisms. The in situ rat intestine perfusion was used as an initial method to study 5-ASA transport. The amount of 5-ASA (released from tablet) transferred into portal circulation reached 5.79 ± 0.24%. During this transport, the intestinal formation of 5-ASA main metabolite (N-ac-5-ASA) occurred. N-ac-5-ASA was found in perfusate both from intestinal lumen and from v. portae. In in vitro Caco-2 monolayers, transport of 5-ASA (10-1000 µmol/l) was studied in apical-basolateral and basolateral-apical direction (iso-pH 7.4 conditions). The transport of total 5-ASA (parent drug plus intracellularly formed N-ac-5-ASA) was linear with time, concentration- and direction-dependent. Higher basolateral-apical (secretory) transport was mainly caused by higher transport of the metabolite (suggesting metabolite efflux transport). Transport of 5-ASA (only parent drug) was saturable (transepithelial carrier-mediated) at low doses, dominated by passive, paracellular process in higher doses which was confirmed by increased 5-ASA transport using Ca2+-free transport medium. The estimated low 5-ASA permeability and its low solubility enable to classify 5-ASA as BCS class IV.

Imaging and Evaluating Method as Part of Endoscopical Diagnostic Approaches

Novel imaging diagnostic method - wireless capsule enteroscopy was used in experimental gastroenterology studies in pigs. We monitored the disintegration of two types of drug forms in gastrointestinal tract for next optimalisation of pharmaceutical technology process. In parallel, the plasma time profiles of the active component were detected. In second study, scanning of intestinal mucous lesions was done as diagnostic tool. The disintegration of tablets was perceptible in the duodenal-jejunal segment (until the 90th min) and culminated in the proximal jejunum (at the 3rd hour). The peak of maximal concentration was reached between the 3rd and 6th hour. The most common lesions induced by non-steroidal anti-inflammatory drugs were red spots and erosions. Sensitivity and specificity of capsule endoscopy were more than 80% and 95%, respectively. Wireless capsule endoscopy is a highly accurate non-invasive method for evaluation of experimental NSAID-induced enteropathy and evaluation of tablet disintegration proces in the intestine.

TOXCON 2013 - 18th Interdisciplinary Czech-Slovak Toxicology Meeting (Meeting Report)

Traditional toxicology conference TOXCON 2013 (Figure 1) was held in Hradec Kralove (Czech Republic) from 19th to 21st June, 2013. The toxicologists from a variety of institutions, government, academic and industry from the Czech republic and abroad have the great opportunity to meet each other at this meeting annually, alternately in the Czech Republic and the Slovak Republic. The conference was organized by the Czech Society for Experimental and Clinical Pharmacology and Toxicology CLSJEP (CSEKFT) and Slovak Toxicology Society (SETOX).

The Importance of Wireless Capsule Endoscopy for Research into the Intestinal Absorption Window of 5-Aminosalicylic Acid in Experimental Pigs

BACKGROUND Absorption windows in particular segments of the small intestine can contribute to the development of orally administered drug formulations and can limit the bioavailability of released compounds. OBJECTIVE The aim of this study was to evaluate use of wireless capsule enteroscopy regarding the disintegration kinetic process of tablets in the small intestine and its comparison with the levels of the model drug (5- aminosalicylic acid; 5-ASA), and its majority metabolite (N-acetyl-5-aminosalicylic acid; N-acetyl-5-ASA) in blood plasma. METHODS Tablets were endoscopically introduced into the duodenum and their disintegration was monitored using wireless capsule enteroscopy in anaesthetised pigs. In parallel, blood plasma time profiles of the model drug (5-ASA) released from tablets and its metabolite (N-acetyl-5-ASA) were detected. RESULTS The disintegration of tablets was evident in the proximal jejunum (until the 90-minute mark) and culminated at the 3rd hour. The maximum plasmatic concentration of 5-ASA was reached at the 3rd hour and in the case of its metabolite (N-acetyl-5-ASA) at the 4th hour. CONCLUSION The study demonstrated the advantage of combination of wireless capsule enteroscopy and bioanalytical determination of pharmacokinetic parameters in an animal experiment to localise the disintegration site of solid dosage form and following kinetics of intestinal absorption of the released active agent.

Probiotics: Preclinical Testing for Verification of Their Gastrointestinal Effectiveness

Abstract Currently, no studies exist addressing the issue of influence of probiotics on pharmacokinetics of concurrent conventional drug administration. In the present chapter we document some findings from our own preclinical experimental studies. We describe methodology and results from studies evaluating the influence of probiotics (especially of Escherichia coli Nissle 1917) on absorption and biotransformation processes of model drugs and describe the prophylactic effects reducing gastrointestinal injury (experimentally induced with indomethacin and dextran sodium sulfate).