Porcine Gastric Mucosa Research Articles

On the mucoadhesive properties of synthetic and natural polyampholytes

HypothesisThe mucoadhesive characteristics of amphoteric polymers (also known as polyampholytes) can vary and are influenced by factors such as the solution's pH and its relative position against their isoelectric point (pHIEP). Whilst the literature contains numerous reports on mucoadhesive properties of either cationic or anionic polymers, very little is known about these characteristics for polyampholytes ExperimentsHere, two amphoteric polymers were synthesized by reaction of linear polyethylene imine (l-PEI) with succinic or phthalic anhydride and their mucoadhesive properties were compared to bovine serum albumin (BSA), selected as a natural polyampholyte. Interactions between these polymers and porcine gastric mucin were studied using turbidimetric titration and isothermal titration calorimetry across a wide range of pHs. Model tablets were designed, coated with these polymers and tested to evaluate their adhesion to porcine gastric mucosa at different pHs. Moreover, a retention study using fluorescein isothiocyanate (FITC)-labelled polyampholytes deposited onto mucosal surfaces was also conducted FindingsAll these studies indicated the importance of solution pH and its relative position against pHIEP in the mucoadhesive properties of polyampholytes. Both synthetic and natural polyampholytes exhibited strong interactions with mucin and good mucoadhesive properties at pH < pHIEP.

Investigation of a biopolymer-based pH-responsive and sustained release raft-gel-forming tablet of famotidine: In-vitro, ex-vivo, bioavailability and anti-ulcer evaluation in New Zealand albino rabbit

Famotidine (FAM), a histamine H2 receptor antagonist, routinely used for gastric ulcers. However, the poor permeability, short biological half-life and degradation in acidic gastric environment, limits its bioavailability and reduces the expression of potential bioactivity. The present study investigated sodium alginate as a pH responsive and raft-gel-forming biopolymer along with slowly releasing ingredients to achieve stomach site-specific, stable and sustained release FAM tablet (FAMRFT) for the management of gastric ulcer. The Box Behnken design was adopted to optimize raft strength and sustained release profile. The optimized FAMRFT was evaluated in terms of in-vitro, ex-vivo and in-vivo in suitable animal models. In-vitro release study showed that FAMRFT can effectively and continuously sustained-release with diffusion mechanism. Ex-vivo permeation study in porcine gastric mucosa showed two-fold increase in permeation parameters. In-vivo studies in New Zealand albino rabbits, the X-ray scintigraphy confirmed the formation of raft-gel in gastric and buoyancy, improved 3-fold bioavailability as compared to pure FAM and commercial tablet, respectively. Further, the improved gastro protective effects were reflected in gastric volume, total acid output, pH, ulcer score, ulcer index, ulcer protection and biomarkers level such as malondialdehyde (MDA), total glutathione (GSH) and nitric oxide (NO) in aspirin-induced gastric ulcers in albino rabbit. Moreover, histopathological study revealed the absence of epithelial erosion, submucosal edema and leucocytes infiltration in gastric mucosa. The results indicate that sodium alginate-based pH responsive raft-gel may be a new formulation for the application of FAM for the treatment of gastric ulcer.

Detection of Helicobacter suis bacteria in pigs of different age groups

Currently, the pathogenesis of gastric ulcer in pigs remains largely unexplored. The origin of this pathology is most often associated with the type and the technologies of feeding, stresses and disorders of homeostasis of the animal body. The possible involvement of bacteria of the genus Helicobacter in the development of chronic gastritis and gastric ulcer disease in pigs was suggested by the researchers relatively recently. The article comprises the results of investigations aimed at detection of Helicobacter suis bacteria and the contamination degree of porcine gastric mucosa in pigs of different age groups. The stomachs, obtained from suckling pigs, fattening pigs and sows in the slaughterhouse of the Mari El Republic, were examined. The study determined the dependence of pathomorphological changes in the gastric mucosa of pigs on the detection of H. suis in microscopic and biochemical tests as well as in PCR. Thus, no pathomorphological changes in the gastric mucosa of suckling pigs were detected. Severe hyperkeratosis, erosions, and ulcers were found on the stomach mucosa of fattening pigs and sows that were infected with H. suis bacteria. Sows also had ulcerative lesions in the non-glandular region of esophagus. In the biomaterial of suckling piglets the DNA of H. suis bacteria was found only in the pyloric region of the stomach, while in fattening pigs, the DNA of these bacteria was most often isolated from the fundal region, and in sows – from the fundal and cardial regions. This indicates a shift in colonization by helicobacters of the mucous membrane of the stomach from the pyloric to the cardiac sectionincreased with animal age. The obtained research data provide the additional evidence of the etiological role of H. suis in the pathogenesis of gastric ulcer in pigs.

Fluorescence detection of deep intramucosal cancer excited by green light for photodynamic diagnosis using protoporphyrin IX induced by 5-aminolevulinic acid: an ex vivo study.

.Significance: The diagnostic depth of photodynamic diagnosis (PDD) for gastric cancer with protoporphyrin IX (PpIX) is limited, which leads to missing intramucosal cancers in screening and surgery.Aim: The reason is that the excitation light, whose wavelength is determined by the highest absorption peak of PpIX (), is strongly attenuated by mucosal tissues. We investigated an excitation wavelength that can extend the diagnostic depth of PpIX fluorescence at the mucosal subsurface.Approach: By calculating the depth-dependent intensity of the excitation light in porcine gastric mucosa for each wavelength, relationships among the wavelength, fluorophore depth, and fluorescence intensity were assessed and fluorescence images of PpIX pellets located at different fluorophore depths were compared experimentally by changing the excitation wavelength.Results: The numerical calculation showed that a 505-nm excitation light provided the highest fluorescence intensities at a fluorophore depth deeper than 1.1 mm. In the fluorescence observation, the fluorescence intensities at fluorophore depths of 0 and 1.0 mm at 405 nm were and units, whereas those at 505 nm were and units, respectively.Conclusion: The experimental results suggest that the diagnosis depth of PDD with PpIX for intramucosal cancer can be extended by 505-nm excitation light.

Hydrolytic degree and antioxidant activity of purified casein characterised by different haplotypes (αs1‐, β‐ and k‐casein) after enzymatic hydrolysis with pepsin and enzymatic extract from Pleurotus eryngii

SummaryThe aim of this study was to estimate the hydrolytic degree and antioxidant activity of purified casein characterised by different haplotypes (αs1‐, β‐ and k‐casein) after in vitro digestion with two different enzymatic systems: pepsin from porcine gastric mucosa (EP) and a crude enzymatic extract from the edible mushroom Pleurotus eryngii. The used enzymes showed a different mode of casein catalysis with a consequent production of peptides of different antioxidant activity. The CN haplotype significantly influenced peptides production; in fact, the amino acid substitutions caused by genetic polymorphisms at the αs1‐, β‐ and k‐CN loci influenced the sites of enzymatic cleavage and therefore the produced peptides. The above is evidenced by the different antioxidant activity found in the hydrolysates depending on the used enzymatic system, the CN haplotype, and the CN haplotype × enzymatic treatment interaction. The findings of this study are a perspective for the production of specific foods that exert a biological effect in addition to the nutritional one.

ChemInform Abstract: Enzyme‐Catalyzed Asymmetric Domino Thia‐Michael/Aldol Condensation Using Pepsin.

The novel catalytic promiscuity of pepsin from porcine gastric mucosa for the asymmetric catalysis of the domino thia-Michael/aldol condensation reaction in MeCN and buffer was discovered for the first time. Broad substrate specificity was tested, and a series of corresponding products were obtained with enantioselectivities of up to 84% ee. This specific catalysis was demonstrated by using recombinant pepsin and control experiments with denatured and inhibited pepsin. The reaction was also shown to occur in the active site by site-directed mutagenesis (the Asp32Ala mutant of pepsin), and a possible mechanism was proposed.

ChemInform Abstract: Enzyme‐Catalyzed Asymmetric Domino Aza‐Michael/Aldol Reaction for the Synthesis of 1,2‐Dihydroquinolines Using Pepsin from Porcine Gastric Mucosa.

Abstract An unprecedented enzyme-catalyzed asymmetric domino aza-Michael/aldol reaction of 2-aminobenzaldehyde and α,β-unsaturated aldehydes is achieved. Pepsin from porcine gastric mucosa provided mild and efficient access to diverse substituted 1,2-dihydroquinolines in yields of 38%–97% with 6%–24% enantiomeric excess (ee). This work not only provides a novel method for the synthesis of dihydroquinoline derivatives, but also promotes the development of enzyme catalytic promiscuity.

Esomeprazole immediate release tablets: Gastric mucosa ex vivo permeation, absorption and antisecretory activity in conscious rats

The aim of this work was to study the esomeprazole activity on the control of gastric secretion after administration of a novel immediate release tablet. The ex vivo permeation of esomeprazole across porcine gastric mucosa from immediate release tablets, containing sodium carbonate or magnesium oxide as alkalinizing agents, was firstly assessed. Pharmacokinetics and pharmacodynamics studies in conscious rats following the administration of immediate release tablets with sodium carbonate, in comparison with delayed-release tablets having the same formula, were also conducted. The results showed an important effect of sodium carbonate and magnesium oxide on the drug release, on the ex vivo trans-mucosal transport and the stability in acid environment. In particular, the presence of sodium carbonate in esomeprazole tablet formulation provided the maximum increase of the drug in vitro transport across the mucosa. Then, the absorption and the antisecretory activity of this proton pump inhibitor orally administered in rats as immediate release tablets containing Na2CO3, was superior but not significantly different compared to delayed-release tablets having the same formula. In the adopted animal model, an activity of esomeprazole from immediate release alkaline formulation was seen also in presence of partial gastric absorption allowing inhibition of proton pumps reached via systemic circulation. This esomeprazole immediate release formulation could be used for the on-demand treatment of acid-related disorders such as gastro-esophageal reflux disease.

Enzyme-Catalyzed Asymmetric Domino Thia-Michael/Aldol Condensation Using Pepsin.

The novel catalytic promiscuity of pepsin from porcine gastric mucosa for the asymmetric catalysis of the domino thia-Michael/aldol condensation reaction in MeCN and buffer was discovered for the first time. Broad substrate specificity was tested, and a series of corresponding products were obtained with enantioselectivities of up to 84% ee. This specific catalysis was demonstrated by using recombinant pepsin and control experiments with denatured and inhibited pepsin. The reaction was also shown to occur in the active site by site-directed mutagenesis (the Asp32Ala mutant of pepsin), and a possible mechanism was proposed.

Enzyme-catalyzed asymmetric domino aza-Michael/aldol reaction for the synthesis of 1,2-dihydroquinolines using pepsin from porcine gastric mucosa

Abstract An unprecedented enzyme-catalyzed asymmetric domino aza-Michael/aldol reaction of 2-aminobenzaldehyde and α,β-unsaturated aldehydes is achieved. Pepsin from porcine gastric mucosa provided mild and efficient access to diverse substituted 1,2-dihydroquinolines in yields of 38%–97% with 6%–24% enantiomeric excess (ee). This work not only provides a novel method for the synthesis of dihydroquinoline derivatives, but also promotes the development of enzyme catalytic promiscuity.

Enzyme-catalyzed domino reaction: efficient construction of spirocyclic oxindole skeleton using porcine pepsin

Pepsin from porcine gastric mucosa was used as a catalyst in the domino Knoevenagel/Michael/Michael reaction for the synthesis of spirooxindoles.

Design and Evaluation of Floating Multi Unit Mini Tablets (MUMTS) Muco Adhesive Drug Delivery System of Famotidine to Treat Upper Gastro Intestinal Ulcers

Ulcer is one of the most common evaluated diseases caused by many reasons like, H. pylori bacteria infection, over secretion of acid levels in the stomach, irregular formation of mucosa and some of the NSAIDs treatment. Famotidine is a H2 receptor antagonist, used to reduce the gastric acid secretion in the stomach. The aim of the present investigation was the development and evaluation of famotidine multi-unit tablet encapsulated system to treat upper gastro intestinal ulcers. The drug release was controlled with matrix polymers like POLYOXWSR 1105 and HPMC K4M. The tablets were prepared by direct compression technique. The prepared tablets were evaluated for physico chemical parameters like weight variation, hardness, friability, buoyancy studies, drug content, in vitro dissolution and mucoadhesive property in porcine gastric mucosa. Further, the intra gastric behaviour of optimized formulation was evaluated in human volunteers under fasting conditions. All the parameters of prepared tablets were within the acceptable limits as per USP. The optimized formulation exhibited floating lag time of 10 ± 2 sec with total floating time of 12 h and in vitro release profile of 99.14 ± 3.21% in 12 h. The mucoadhesion time was found to be visually >12 h in ph 6.8 phosphate buffer. In vivo radiographic imaging studies revealed that the mean residence time of single mini tablet in stomach was found to be 8h in healthy human volunteers.

POZylation: a new approach to enhance nanoparticle diffusion through mucosal barriers.

The increasing use of nanoparticles in the pharmaceutical industry is generating concomitant interest in developing nanomaterials that can rapidly penetrate into, and permeate through, biological membranes to facilitate drug delivery and improve the bioavailability of active pharmaceutical ingredients. Here, we demonstrate that the permeation of thiolated silica nanoparticles through porcine gastric mucosa can be significantly enhanced by their functionalization with either 5 kDa poly(2-ethyl-2-oxazoline) or poly(ethylene glycol). Nanoparticle diffusion was assessed using two independent techniques; Nanoparticle Tracking Analysis, and fluorescence microscopy. Our results show that poly(2-ethyl-2-oxazoline) and poly(ethylene glycol) have comparable abilities to enhance diffusion of silica nanoparticles in mucin dispersions and through the gastric mucosa. These findings provide a new strategy in the design of nanomedicines, by surface modification or nanoparticle core construction, for enhanced transmucosal drug delivery.

Effect of entrapped α-tocopherol on mucoadhesivity and evaluation of the release, degradation, and swelling characteristics of zein–chitosan composite electrospun fibers

A gastro-mucoadhesive matrix of ultrafine fibers was formed from composite polymers of zein, poly(ethylene oxide), and chitosan at a ratio of 87.5/10/2.5 via electrospinning. Alpha-tocopherol (α-TOC), a hydrophobic compound, was embedded at 20wt% into the fiber matrix to investigate its effects on fiber morphology and mucoadhesivity. α-TOC release characteristics were examined in simulated gastric fluid (SGF) at pH 1.2 with pepsin and SGF at pH 2 without pepsin at 37±0.5°C. Gastro-mucoadhesivity was tested in porcine gastric mucosa. Fiber morphology was characterized by scanning electron microscopy (SEM). α-TOC did not affect fiber morphology, but significantly enhanced mucoadhesivity (p<0.05). α-TOC release in SGF at pH 1.2 with pepsin was triggered by erosion, and α-TOC release in SGF at pH 2 without pepsin was triggered by swelling, and driven by diffusion. SEM micrographs revealed that the fibers lost their fibrous structure and later fused, minimizing the fibrous film release surface area.

Amoxycillin Trihydrate Floating-Bioadhesive Drug Delivery System for Eradication of Helicobacter pylori: Preparation, In Vitro and Ex Vivo Evaluation

The aim of the present investigation was to formulate and evaluate floating-bioadhesive tablets of amoxicillin trihydrate (ATFBT), to provide local action in the treatment of Helicobacter pylori (H. pylori) by prolonging the gastric residence time. The FBT were prepared by direct compression method using hydroxy propyl methyl cellulose (HPMC K4M) / chitosan (CH), carbopol (CP974P) / polymethacrylic acid (PMA) as release retarding agent / bioadhesive respectively, sodium bicarbonate (NaHCO3) as a gas-former. The prepared tablets were evaluated for their physical characters such as drug content, in vitro buoyancy, swelling index, drug release and stability. Further, the bioadhesive strength (BS) was determined using porcine gastric mucosa. DSC studies indicate the compatibility of the drug and excipients used in the formulation. The floating lag time, total floating time, bioadhesive strength and swelling index of the optimized formulation (F7) was 32 ± 2.7 sec, more than 12 h, 1.86 ± 0.14 N and more than 3.5 respectively. Formulation (F7) was also found to be physically stable when stored at 40oC / 75% RH for 3 months. The drug release profiles of all the formulations were fitted into various kinetic models. The optimized formulation followed the Peppas model (r2 > 0.99) with a non-Fickian diffusion mechanism (n = 0.625). From the results, a matrix floatingbioadhesive tablet incorporating an insoluble active substance such as amoxicillin trihydrate was developed for the successful eradication of H. pylori.

Gastro protective properties of the novel prostone SPI-8811 against acid-injured porcine mucosa

To evaluate the protective properties of novel prostone ClC-2 agonist SPI-8811 in porcine model of gastric acid injury. Porcine gastric mucosa was mounted in Ussing chambers and injured by bathing mucosal tissues in an HCl Ringer's solution (pH = 1.5) with or without SP1-8811 (1 μmol/L), cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor (inhibitor 172, 10 μmol/L, apical) and ClC-2 inhibitor ZnCl₂, 300 μmol/L, apical), on the apical surface of tissues. Transepithelial resistance and mucosal-to-serosal ³H-mannitol fluxes were measured over a 90-min period. Tissues were analyzed by morph metric techniques, Immunofluorescence and by western blots. Compared with control tissues, acid exposure decreased transepithelial electrical resistance (TER) and increased ³H-mannitol flux. Pretreatment of gastric mucosa with SPI-8811 was protective against acid-induced decreases in TER (TER, 50 Ω.cm² vs 100 Ω.cm²) and abolished increases in flux (³H-mannitol flux, 0.10 μmol/L.cm² vs 0.04 μmol/L.cm²). Evidence of histological damage in the presence of acid was markedly attenuated by SPI-0811. Immunofluorescence and western analysis for occludin revealed enhanced localization to the region of the tight junction (TJ) after treatment with SPI-8811. Pretreatment with the ClC-2 inhibitor ZnCl₂, but not the selective CFTR inhibitor 172, attenuated SPI-8811-mediated mucosal protection, suggesting a role for ClC-2. Prostone may serve both protective and reparative roles in injured tissues. ClC-2 agonist SPI-8811 stimulated enhancement of mucosal barrier function by protecting TJ protein occludin in porcine gastric mucosa and thus protected the gastric acid injury in porcine stomach.

A large scale enzyme screen in the search for new methods of silicon–oxygen bond formation

Biotransformations make use of biological systems to catalyze or promote specific chemical reactions. Transformations that utilize enzymes as “greener” and milder catalysts compared to traditional reaction conditions are of particular interest. Recently, organosilicon compounds have begun to be explored as non-natural enzymatic substrates for biotransformations. The aims of this study were to screen readily available (approximately eighty) enzymes for their ability to catalyze in vitro siloxane bond formation under mild reaction conditions using a model monoalkoxysilane as the substrate and to make a preliminary evaluation of potential factors that might lead to activity or inactivity of a particular enzyme. Several new hydrolase enzymes were observed to catalyze the formation of the condensation product when compared to peptide controls, or buffer solutions at the same pH, as judged from quantitative analyses by gas chromatography. Aspergillus ficuum phytase, Aspergillus niger phytase, chicken egg white lysozyme, porcine gastric mucosa pepsin, and Rhizopus oryzae lipase all catalyzed the condensation of silanols in aqueous media. Factors involved in determining the activity of an enzyme towards silanol condensation appear to include: the presence of imidazole and hydroxyl functions in the active site; solvent; the presence of water; the surface properties of the enzyme; possible covalent inhibition; and steric factors in the substrate.

Impact of diets' physical form (particle size; meal/pelleted) on the stomach content (dry matter content, pH, chloride concentration) of pigs

Pars nonglandularis (PN) is the main affected area of gastric ulcers in pigs. Several factors are discussed to affect integrity of porcine gastric mucosa, but special emphasis is given to physical form of the feed. A total of 26 pigs (bw: 36.4 ± 10.5 kg) were housed individually and test diets were fed ad libitum for 3 days. The four diets used in this study were identical in botanical and chemical composition, but differed in grinding intensity (fine/coarse) and physical form (pelleted/meal). On day 3, animals were sacrificed 6 hours after free access to feed (after an overnight fast). The stomach was withdrawn in toto and frozen immediately at − 80 °C. A sagittal cut was made and samples of the frozen gastric content were taken from 15 standardized localisations to determine dry matter content, pH and chloride (Cl) concentration using standard methods. Physical form of diets noticeably affected quality and composition of stomach content. Marked effects were found in the PN. The finely ground and pelleted diet (FP) resulted in a more liquid chyme and a significantly higher Cl-concentration at PN in comparison to the results observed after feeding the unpelleted diets (CM/FM). There was no distinct pH gradient between the regions after feeding FP. However, the coarsely ground meal (CM) caused highest pH values at PN and low pH values at fundus region. Cl-concentrations at PN were markedly lower when pigs were fed the unpelleted diets (CM/FM). In pigs fed FP a significantly higher Cl-secretion rate of the upper gastrointestinal tract was found. Therefore this study shows clearly effects of grinding intensity and physical form of the diet on composition and quality of gastric chyme within the different regions of porcine stomach. The relatively high pH at PN after feeding the FP indicates that pH presumably is not the key factor for development of gastric ulcers in pigs at PN.

Gastro‐protective Properties of Cobiprostone against Acid‐induced Mucosal Injury in Porcine Gastric Mucosa

Activation of ClC‐2 Cl− channels is an important mechanism of intestinal mucosal barrier repair. Lubiprostone, a prostone known to activate epithelial ClC‐2 Cl− channels, has been shown to hasten repair of ischemic‐injured intestinal mucosa. The objective of the present study was to evaluate the gastro protective properties of the novel prostone, cobiprostone, in a porcine model of gastric acid injury. We hypothesized that cobiprostone would ameliorate acid injury associated with maintenance of tight junctions and activation of chloride channels. Porcine gastric mucosa was mounted in Ussing chambers and injured by bathing mucosal tissues in an HCl Ringer's solution (pH=1.5). Transepithelial resistance and mucosal‐to‐serosal 3H‐mannitol flux was measured over a 90 minute period. Compared with control tissues, acid exposure decreased TER and increased 3H‐mannitol flux. Pretreatment of gastric mucosa with cobiprostone was protective against acid‐induced decreases in TER and abolished increases in flux .Histological damage were attenuated in cobiprostone treated gastric mucosa exposed to acid. Immunofluorescence and western analysis for occludin revealed enhanced localization of to the tight junction after treatment. Pretreatment with the ClC‐2 inhibitor ZnCl2, and the CFTR inhibitor DASU attenuated cobiprostone mediated mucosal protection, indicating a critical role for Cl− channels.The Research is funded by Sucampo Pharmaceuticals, Inc. Bethesda, MD

Formulation development of oral controlled release tablets of hydralazine: Optimization of drug release and bioadhesive characteristics

The current study involves development of oral bioadhesive hydrophilic matrices of hydralazine hydrochloride, and optimization of their in vitro drug release profile and ex vivo bioadhesion against porcine gastric mucosa. A 32 central composite design was employed to systematically optimize the drug delivery formulations containing two polymers, viz., carbomer and hydroxypropyl methyl cellulose. Response surface plots were drawn and optimum formulations were selected by brute force searches. Validation of the formulation optimization study indicated a very high degree of prognostic ability. The study successfully undertook the development of an optimized once-a-day formulation of hydralazine with excellent bioadhesive and controlled release characteristics.