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.

The effect of probiotic Escherichia coli strain Nissle 1917 lipopolysaccharide on the 5-aminosalicylic acid transepithelial transport across Caco-2 cell monolayers.

The object of this study was to investigate the effect of probiotic Escherichia coli strain Nissle 1917 (EcN) (i) EcN lipopolysaccharide (EcN LPS) and (ii) bacteria-free supernatant of EcN suspension (EcN supernatant) on in vitro transepithelial transport of mesalazine (5-aminosalicylic acid, 5-ASA), the most commonly prescribed anti-inflammatory drug in inflammatory bowel disease (IBD). Effect of co-administered EcN LPS (100 µg/ml) or EcN supernatant (50 µg/ml) on the 5-ASA transport (300 µmol/l) was studied using the Caco-2 monolayer (a human colon carcinoma cell line) as a model of human intestinal absorption. Permeability characteristics for absorptive and secretory transport of parent drug and its intracellularly-formed metabolite were determined. The quantification of 5-ASA and its main metabolite N-acetyl-5-amino-salicylic acid (N-Ac-5-ASA) was performed by high performance liquid chromatography. Obtained results suggest that neither EcN LPS nor EcN supernatant had effect on the total 5-ASA transport (secretory flux greater than absorptive flux) and on the transport of intracellularly formed N-Ac-5-ASA (preferentially transported in the secretory direction). The percent cumulative transport of the total 5-ASA alone or in combination with EcN LPS or EcN supernatant did not exceed 1%.

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.

Caco-2 cells, biopharmaceutics classification system (BCS) and biowaiver.

Almost all orally administered drugs are absorbed across the intestinal mucosa. The Caco-2 monolayers are used as an in vitro model to predict drug absorption in humans and to explore mechanism of drug absorption. The Caco-2 cells are derived from a human colon adenocarcinoma and spontaneously differentiate to form confluent monolayer of polarized cells structurally and functionally resembling the small intestinal epithelium. For studying drug permeability, Caco-2 cells are seeded onto the Transwell inserts with semipermeable membrane and grown to late confluence (21 days). After determination of cell viability, the integrity of monolayer is checked by phenol red permeability and by 14C-mannitol permeability. The transport from apical to basolateral (AP-BL) and basolateral to apical (BL-AP) is studied by adding the diluted drug on the apical or basolateral side and withdrawing the samples from the opposite compartment, respectively, for HPLC analysis or liquid scintillation spectrometry. Ca2+ -free transport medium is used to determine paracellular component of the drug transport. On the basis of permeability and solubility, drugs can be categorized into four classes of Biopharmaceutics Classification System (BCS). For certain drugs, the BCS-based biowaiver approach can be used which enables to reduce in vivo bioequivalence studies.

Caco-2 cell monolayer integrity and effect of probiotic Escherichia coli Nissle 1917 components.

OBJECTIVES: Different probiotic strains used in clinical trials have shown prophylactic properties in different inflammatory diseases of the gastrointestinal tract. This study was aimed to investigate the influence of Escherichia coli strain Nissle 1917 (EcN) components on the integrity of the Caco-2 cell monolayer (human adenocarcinoma cell line). METHODS: The effect of supernatant of EcN suspension and lipopolysaccharide (LPS) isolated from EcN (in concentrations from 0.001 to 1 000 µg/ml) on paracellular transport of 14C-mannitol marker through epithelial cell monolayer was estimated. RESULTS: Both LPS and EcN supernatant exerted almost the same effect; whereas no effect was shown using high concentrations (100 and 1 000 µg/ml), low concentrations (0.001, 0.1 and 1 µg/ml) significantly decreased permeability of 14C-mannitol. Concentration (0.001 µg/ml) decreased 14C-mannitol permeability approximately about 20% (LPS) and 30% (EcN supernatant). To elucidate the observed changes in monolayer permeability ("tighter monolayer") induced by concentrations of LPS or supernatant, media able to open epithelial intercellular junctions were used. The effects of Ca2+-free transport medium and of medium containing 5, 10, 20, 50, and 100% of Ca2+ on the 14C-mannitol transport in the presence of the lowest (0.001 µg/ml) and high (100 µg/ml) concentrations of LPS were studied. Using Ca2+-free medium both concentrations of LPS significantly decreased apparent permeability coefficient (Papp) of 14C-mannitol indicating that changes of 14C-mannitol permeability are independent of dimensions of paracellular spaces. CONCLUSION: The decrease of 14C-mannitol permeability caused by EcN LPS indicates the ability of components of probiotic EcN strain to restore disrupted epithelial barrier.

Wireless video capsule enteroscopy in preclinical studies: methodical design of its applicability in experimental pigs.

The aim of this project was to develop a methodology to introduce wireless video capsule endoscopy in preclinical research. Five mature female pigs (Sus scrofa domestica) were selected for the study. Capsule endoscopes (the EndoCapsule system; Olympus) were introduced into the duodenum endoscopically in each of the animals. The life span of batteries (i.e., total time of endoscopy recording) was 487-540 min (median 492 min). The capsule endoscope reached the cecum during enteroscopy once (after 7 h 57 min), in the remaining cases, endoscopy recordings terminated in the distal or terminal ileum. All capsule enteroscopies found a normal pattern of the small intestine. The intestinal lumen is narrower, transverse folds are sparse or even absent, villi are wider but less prominent in pigs compared to humans. Capsule endoscopy in experimental pigs will be helpful for future trials on injury of different drugs and xenobiotics to the small bowel.

Transepithelial transport of ambroxol hydrochloride across human intestinal Caco-2 cell monolayers.

This study aimed i) to characterize the transepithelial transport of the mucolytic agent ambroxol hydrochloride across the intestinal barrier, ii) to classify the ambroxol according to Biopharmaceutics Classification System (BCS) and iii) to predict ambroxol absorption in humans. Transport of ambroxol (100, 300 and 1000 micromol/l) was studied in a human colon carcinoma cell line Caco-2 in apical to basolateral and basolateral to apical direction, under iso-pH 7.4 and pH-gradient (6 vs. 7.4) conditions. The relative contribution of the paracellular route was estimated using Ca2+-free transport medium. Ambroxol samples from receiver compartments were analysed by HPLC with UV detection (242 nm). Results showed that ambroxol transport is linear with time, pH-dependent and direction-independent, displays non-saturable (first-order) kinetics. Thus, the transport seems to be transcellular mediated by passive diffusion. Estimated high solubility and high permeability (P(app) = 45 x 10(-6) cm/s) of ambroxol rank it among well absorbed compounds and class I of BCS. It can be expected that the oral dose fraction of ambroxol absorbed in human intestine is high.

Caco-2 cells and Biopharmaceutics Classification System (BCS) for prediction of transepithelial transport of xenobiotics (model drug: caffeine).

OBJECTIVES: The Caco-2 cell monolayer model is widely used as a standard screening tool for studying the mechanisms of cellular drug transport. Caffeine was chosen as a model drug and is supposed to be class I of the Biopharmaceutics Classification System (BCS). Our study was conducted 1) to characterize the mechanisms of caffeine transport across the intestinal barrier, 2) to classify caffeine according to BCS, 3) to predict drugs intestinal absorption in humans. METHODS: Caffeine transport (0.1, 0.3, 1 and 10 mmol/l) was studied in Caco-2 cell monolayer in apical to basolateral (AP-BL) and basolateral to apical (BL-AP) direction, under iso-pH 7.4 and pH-gradient (6/7.4) conditions. The relative contribution of the paracellular route was estimated using Ca2+- free transport medium (opening tight junctions). RESULTS: The caffeine transport was linear with time, transport direction and pH independent, displaying non-saturable (first-order) kinetics, with high permeability coefficient (Papp): in AP-BL direction Papp = 46.3-53.5 x 10-6 cm/s; in BL-AP direction Papp = 45.6-49.4 x 10-6 cm/s. Thus, the transport seems to be transcellular mediated by passive diffusion. Using Ca2+- free transport medium tight junctions were opened (confirmed by increased Papp of mannitol) but the caffeine Papp was not changed. Thus, the paracellular route is only a minor way of caffeine transport. CONCLUSION: High solubility and high permeability of caffeine rank it among class I of BCS and well absorbed compounds.

Different solutions used for submucosal injection influenced early healing of gastric endoscopic mucosal resection in a preclinical study in experimental pigs.

BACKGROUND: We hypothesised that different solutions for submucosal injection may influence early healing of endoscopic mucosal resection (EMR). The aim of this study was to evaluate histological and immunological changes after EMR in experimental pigs. MATERIALS AND METHODS: Two parallel EMRs on the anterior and posterior wall of the gastric body were performed by means of the cap technique in 21 female pigs. A glycerol-based solution (anterior EMR) and hydroxypropyl methylcellulose solution (posterior EMR) were applied for submucosal injection. The animals were sacrificed 7 days later, and tissue sections of all EMRs were stained using combined trichrome. Computer image analysis was used for objective evaluation of elastic and collagen fibres content. Two-colour indirect immunophenotyping of blood and gastric samples were performed using mouse anti-pig monoclonal antibodies. RESULTS: The values of collagen fibre content 7 days after EMR were significantly higher in lesions after the use of solution A in comparison with solution B (2.10 +/- 0.25% versus 1.57 +/- 0.25%, p = 0.009). Concordant results were found in elastic fibres (3.23 +/- 0.49% versus 2.93 +/- 0.61%, p = 0.018). No systemic changes in major leukocyte subpopulations were found. In gastric tissue, lymphocyte subsets exhibited only minor changes. CD4(+) T-lymphocytes were increased in the healing tissue after EMR using solution A (17.08 +/- 9.24% versus 9.76 +/- 7.97%, p = 0.011). Significant increase of SWC3(+) leukocytes was observed after EMR using solution B (47.70 +/- 25.41% versus 18.70 +/- 12.16%, p = 0.001). CONCLUSIONS: The use of glycerol-based solution for submucosal injection was associated with more pronounced histological signs of early healing of EMRs compared with hydroxypropyl methylcellulose.

High-performance liquid chromatographic method with UV photodiode-array, fluorescence and mass spectrometric detection for simultaneous determination of galantamine and its phase I metabolites in biological samples.

Galantamine, an alkaloid isolated from the bulbs and flowers of Caucasian snowdrop (Galanthus woronowii, Amaryllidaceae) and related species, is employed in human medicine for the treatment of various neuromuscular and neurodegenerative diseases. After the administration, the products of oxidative biotransformation (O-desmethyl-galantamine, N-desmethyl-galantamine, galantamine-N-oxide) and chiral conversion (epigalantamine) are formed in various concentrations from parent compound. For the identification and determination of galantamine and its phase I metabolites in blood plasma and tissues, a new bioanalytical method based on a reversed-phase high-performance liquid chromatography with UV photodiode-array, fluorescence and mass spectrometric detection was developed, validated and applied to pharmacokinetic and biotransformation studies. Sample preparation included a homogenization of the rat tissues (liver, brain, hypophysis) in a phosphate buffer 0.05 mol/L pH 7.4. Plasma samples and tissue homogenates were purified using a mixed-mode solid-phase extraction (Waters Oasis MCX cartridges). Galantamine, its above-mentioned metabolites and the internal standard codeine were separated on a Discovery HS F5 column (Supelco, 150 mmx4.6 mm I.D., 5 microm) at flow rate of 1 mL/min using a linear gradient elution. UV photodiode-array and mass spectrometric detection were employed for the identification of individual galantamine metabolites in various biomatrices, the fluorescence detection (lambdaexcit=280 nm/lambdaemiss=310 nm) was chosen for the quantification of galantamine and its metabolites. The developed method was applicable in liver tissue in the range from 0.50 to 63.47 nmol/g of galantamine, from 0.32 to 41.42 nmol/g of O-desmethyl-galantamine, from 0.54 to 69.40 nmol/g of N-desmethyl-galantamine and from 0.70 to 89.03 nmol/g of epigalantamine. Limit of detection was found to be 0.04 nmol/g for galantamine, 0.19 nmol/g for O-desmethyl-galantamine, and 0.07 nmol/g for N-desmethyl-galantamine and epigalantamine.

New insights into gene positional clustering and its properties supported by large-scale analysis of various differentiation pathways.

To understand how genes are distributed on chromosomes we bring new insights into gene positional clustering and its properties. We have made a large-scale analysis of three types of differentiation and we observed that genes that subsequently enter into different cell processes are positionally clustered on chromosomes. Genes from the clusters are transcribed subsequently with respect to time kinetics and also to position. This means that the genes related to a cellular process are clustered together, independent of the period of time during which they are active and important for the process. Our results also demonstrate not only that there are general regions of increased or decreased levels of gene expression, but also that, in fact, in some chromosome regions we can find clustering of genes related to specific cell processes. The results provided in this paper also support the theory of "transcription factories" and show that transcription of genes from the clusters is managed by softer epigenetic mechanisms.

Effect of galantamine on acetylcholinesterase and butyrylcholinesterase activities in the presence of L-carnitine in rat selected brain and peripheral tissues.

OBJECTIVES: The alkaloid galantamine (GAL), which exhibits a combined anticholinesterase and direct parasympathomimetic mechanism of action, is employed in conjunction with therapeutic interventions in the stimulation of central cholinergic transfer in cognitive diseases. We attempted to achieve pharmacologically-induced enhancement of the parasympathomimetic activity of GAL in the key areas of rat brain, using an interactive combination of the alkaloid with the transmembrane enhancer L-carnitine (CAR). METHODS: We investigated activities of acetylcholinesterase (AChE) in brain areas (frontal cortex, basal ganglia, septum and hippocampus) and the hypophysis, and that of butyrylcholinesterase (BuChE) in plasma and liver. RESULTS: Following administration of the highest of the GAL doses used (2.5; 5; 10 mg/kg i.m.), AChE activity decreased mainly in the frontal cortex, hippocampus and hypophysis. In the interaction of GAL and CAR, AChE inhibition was stronger but without any statistical significance. The peripheral inhibition of BuChE was found to be dose-dependent. Premedication by CAR led to a slight change in the values of the activities monitored. CONCLUSIONS: CAR in terms of positive modulation of GAL targeting to the central nervous system had no statistically significant effect.

Evaluation of CD34+ - and Lin- -selected cells from peripheral blood stem cell grafts of patients with lymphoma during differentiation in culture ex vivo using a cDNA microarray technique.

OBJECTIVE: Hematopoietic stem cells (enriched in fraction of CD34+ cells) have the ability to regenerate hematopoiesis in all of its lineages, and this potential is clinically used in transplanting bone marrow or peripheral blood stem cells. Our objective was to assemble a suitable method for evaluating gene expression in enriched populations of hematopoietic stem cells. We compared biologic properties of cells cultured ex vivo obtained using two different ways of immunomagnetic separation (positive selection of CD34+ cells and negative selection of Lin- cells) by means of a cDNA microarray technique. METHODS: CD34+ and Lin- cells were enriched from peripheral blood stem cell (PBSCs) grafts of patients with non-Hodgkin's lymphoma. Isolated cells were in the presence of cytokine PBSCs, Flt-3 ligand, interleukin-3, interleukin-6, and granulocyte colony-stimulating factor. At days 0, 4, 6, 8, 10, 12, and 14 cells were harvested and analyzed by cDNA microarrays. Total cell expansion, CD34+, colony-forming unit for granulocyte-macrophage and megakaryocytes expansion, vitality, and phenotype of cells were also analyzed. RESULTS: cDNA microarray analysis of cultured hematopoietic cells proved equivalence of the two enrichment methods for PBSC samples and helped us characterize differentiating cells cultured ex vivo. CONCLUSION: Our methodologic approach is helpful in characterizing cultured hematopoietic cells cultured ex vivo, but it is also suitable for more general purposes. Equivalence of CD34+ and Lin- selection methods from PBSC samples proved by cDNA microarray may have an implication for graft manipulation in an experimental setting of hematopoietic transplantation. Total cell expansion and colony formation and phenotype from CD34+ selected and from Lin- samples were comparable.

Galantamine antiacetylcholinesterase activity in rat brain influenced by L-carnitine.

Galantamine (GAL) is a selective, competitive and reversible acetylcholinesterase (AChE) inhibitor, which increases the activity of the cholinergic system and hence gives rise to an improvement of cognitive functions in patients suffering from dementia of Alzheimer type. L-Carnitine (CAR) is a natural component of the mammalian tissue and is known to increase penetration of some chemical compounds/groups across biological membranes. The aim of this study was to evaluate the influence of pretreatment with CAR on AChE inhibition caused by GAL in selected brain parts in rat (basal ganglia, septum, frontal cortex, hippocampus) and in hypophysis, which does not lay beyond the blood-brain-barrier. During the first stage of the study, GAL was administered i.m. in different doses ranging from 2.5 to 10 mg/kg. The highest degree of AChE dose dependent inhibition was observed in hypophysis, while that in CNS was lower and became apparent in frontal cortex and hippocampus only after the administration of the dose of 10 mg/kg i.m. In the second stage, CAR was administered daily during 3 consecutive days at a dose of 250 mg/kg p.o. prior to the administration of GAL (10 mg/kg i.m.). Pretreatment with CAR enhanced trend of AChE inhibition in all selected brain parts comparing with single GAL administration, however, significant difference was not observed. Comparing these results with control group, statistical significance was found in frontal cortex, hippocampus and hypophysis.

Intestinal single-pass in situ perfusion technique in rat: the influence of L-carnitine on absorption of 7-methoxytacrine.

7-Methoxytacrine (7-MEOTA) is an acetylcholine-esterase inhibitor that is potentially useful in the therapy of some neurodegenerative disorders. L-carnitine (CRT) is a naturally occuring compound that is known to increase penetration of some compounds through biological barriers. Aim of this study was how CRT influenced transintestinal absorption transport 7-MEOTA in rat using single-pass intestinal in situ perfusion method. The rate of absorption of 7-MEOTA during luminal perfusion with single 7-MEOTA was compared with rate of absorption during simultaneous perfusion with 7-MEOTA and CRT and with absorption rate after the premedication with CRT for period of three days before beginning of perfusion. The methodical system was the perfusion of mesenterial bed (from arteria mesenterica superior to vena portae) and intestinal luminal perfusion (from duodenum to ileum). The lower transintestinal absorption in the course of simultaneously administration of CRT than just in case of perfusion with single 7-MEOTA has been found. On the contrary a significantly higher absorption of 7-MEOTA has been noted in group of rats premedicated with CRT for three consecutive days. The interpretation suggested that molecules of CRT incorporated into the metabolism of intestinal cells facilitated transport of 7-MEOTA (as a representative substance which is at least partly transferred by carrier mechanism). In case of simultaneous luminal perfusion with CRT and 7-MEOTA competitive over-saturation of carrier systems is probably.

Cyclosporine A inhibits acetylcholinesterase activity in selected parts of the rat brain.

Cyclosporine A (CsA) is the major immunosuppressive drug used for organ and neural transplantation and the therapy of selected autoimmune diseases. We investigated the effect of CsA on the activity of acetylcholinesterase (AChE) in the frontal cortex, hippocampus, septum, and basal ganglia. AChE was determined spectrophotometrically with acetylthiocholine as substrate and 5,5-bis-2-nitrobenzoic acid as chromogen. CsA was administered in single doses of 20 or 45 mg/kg perorally; in the case of the higher dose we also performed a repeated administration of CsA in three consecutive doses separated by 24 h intervals. Both lower and higher doses of CsA decreased AChE activity in the frontal cortex and hippocampus to practically the same extent. On the contrary, AChE activity was more diminished in the case of the higher dose of CsA used in the septum and basal ganglia. Repeated administration of the higher dose of CsA did not lead, with the exception of the hippocampus, to a further decrease in AChE activity in the brain structures observed. These findings contribute to rare evidence concerning the interaction of CsA and the cholinergic system in the brain.

Minipig as a model for drug metabolism in man: comparison of in vitro and in vivo metabolism of propafenone.

To prove the suitability of minipigs as experimental animal in modeling of the drug metabolism and pharmacokine-tics in man, propafenone metabolism in vitro at the microsomal level as well as propafenone pharmacokinetics in the minipig was studied. The results were compared with those obtained for humans. It can be concluded that whereas the microsomal in vitro system of minipig may be a good model for drug metabolism in the man, the pharmacokinetics in the whole organism is more complex reflecting differences in substrate specificities of many enzymatic and transport systems. In this particular case, it has been documented that the glucuronidation of propafenone principal metabolite (5-hydroxypropafenone) is more efficient in the minipig.