Lidija Berkopić

Stable gastric pentadecapeptide BPC 157 in trials for inflammatory bowel disease (PL-10, PLD-116, PL14736, Pliva, Croatia) heals ileoileal anastomosis in the rat.

PurposeGastric pentadecapeptide BPC 157 (BPC 157), which has been shown to be safe in clinical trials for inflammatory bowel disease (PL-10, PLD-116, PL14736, Pliva, Croatia), may be able to cure intestinal anastomosis dehiscence. This antiulcer peptide shows no toxicity, is limit test negative, and a lethal dose is not achieved. It is stable in human gastric juice. In comparison with other standard treatments it is more effective for ulcers and various wounds, and can be used without a carrier needed for other peptides, both locally and systemically (i.e., perorally, parenterally). We studied the effectiveness of BPC 157 for ileoileal anastomosis healing in rats.MethodsWe assessed ileoileal anastomosis dehiscence macroscopically, histologically, and biomechanically (volume [ml] infused through a syringe-perfusion pump system (1u2009ml/10u2009s), and pressure [mmHg] to leak induction [catheter connected to a chamber and a monitor, at 10u2009cm proximal to anastomosis]), at 1, 2, 3, 4, 5, 6, 7, and 14 days. BPC 157 (10u2009µg, 10u2009ng, 10u2009pg/kg i.p. (or saline [5u2009ml/kg]) was first administered after surgery, while it was last given 24u2009h before either assessment or sacrifice.ResultsThroughout the experiment, both higher doses of BPC 157 were shown to improve all parameters of anastomotic wound healing. The formation of adhesions remained slight, the blood vessels were filled with blood, and a mild intestinal passage obstruction was only temporarily observed. Anastomosis without leakage induces markedly higher volume and pressure values, with a continuous increase toward healthy values. From day 1, edema was markedly attenuated and the number of granulocytes decreased, while from days 4 or 5 necrosis decreased and granulation tissue, reticulin, and collagen formation substantially increased, thus resulting in increased epithelization.ConclusionThis study showed BPC 157 to have a beneficial effect on ileoileal anastomosis healing in the rat.

Mortal hyperkalemia disturbances in rats are NO-system related. The life saving effect of pentadecapeptide BPC 157

We demonstrate the full counteracting ability of stable gastric pentadecapeptide BPC 157 against KCl-overdose (intraperitoneal (i), intragastric (ii), in vitro (iii)), NO-system related. (i) We demonstrated potential (/kg) of: BPC 157 (10ng, 10μg ip, complete counteraction), l-arginine (100mg ip, attenuation) vs. L-NAME (5mg ip, deadly aggravation), given alone and/or combined, before or after intraperitoneal KCl-solution application (9mEq/kg). Therapy was confronted with promptly unrelenting hyperkalemia (>12mmol/L), arrhythmias (and muscular weakness, hypertension, low pressure in lower esophageal and pyloric sphincter) with an ultimate and a regularly inevitable lethal outcome within 30min. Previously, we established BPC 157-NO-system interaction; now, a huge life-saving potential. Given 30min before KCl, all BPC 157 regimens regained sinus rhythm, had less prolongation of QRS, and had no asystolic pause. BPC 157 therapy, given 10min after KCl-application, starts the rescue within 5-10min, completely restoring normal sinus rhythm at 1h. Likewise, other hyperkalemia-disturbances (muscular weakness, hypertension, low sphincteric pressure) were also counteracted. Accordingly with NO-system relation, deadly aggravation by L-NAME: l-arginine brings the values to the control levels while BPC 157 always completely nullified lesions, markedly below those of controls. Combined with l-arginine, BPC 157 exhibited no additive effect. (ii) Intragastric KCl-solution application (27mEq/kg) - (hyperkalemia 7mmol/L): severe stomach mucosal lesions, sphincter failure and peaked T waves were fully counteracted by intragastric BPC 157 (10ng, 10μg) application, given 30min before or 10min after KCl. (iii). In HEK293 cells, hyperkalemic conditions (18.6mM potassium concentrations), BPC 157 directly affects potassium conductance, counteracting the effect on membrane potential and depolarizations caused by hyperkalemic conditions.

Gastric Pentadecapeptide BPC 157 and Short Bowel Syndrome in Rats

The gastric pentadecapeptide BPC 157, which was shown to be safe as an antiulcer peptide in trials for inflammatory bowel disease (PL14736, Pliva), successfully healed intestinal anastomosis and fistula in rat. Therefore, we studied for 4xa0weeks rats with escalating short bowel syndrome and progressive weight loss after small bowel resection from fourth ileal artery cranially of ileocecal valve to 5xa0cm beneath pylorus. BPC 157 (10xa0μg/kg or 10xa0ng/kg) was given perorally, in drinking water (12xa0ml/rat/day) or intraperitoneally (once daily, first application 30xa0min following surgery, last 24xa0h before sacrifice). Postoperatively, features of increasingly exhausted presentation were: weight loss appearing immediately regardless of villus height, twofold increase in crypt depth and fourfold increase in muscle thickness within the first week, jejunal and ileal overdilation, and disturbed jejunum/ileum relation. In contrast, constant weight gain above preoperative values was observed immediately with BPC 157 therapy, both perorally and parenterally, and villus height, crypt depth, and muscle thickness [inner (circular) muscular layer] also increased, at 7, 14, 21, and 28xa0days. Moreover, rats treated with pentadecapeptide BPC 157 showed not different jejunal and ileal diameters, constant jejunum-to-ileum ratio, and increased anastomosis breaking strength. In conclusion, pentadecapeptide BPC 157 could be helpful to cure short bowel syndrome.

Therapy for Unhealed Gastrocutaneous Fistulas in Rats as a Model for Analogous Healing of Persistent Skin Wounds and Persistent Gastric Ulcers: Stable Gastric Pentadecapeptide BPC 157, Atropine, Ranitidine, and Omeprazole

Objective This study focused on unhealed gastrocutaneous fistulas to resolve whether standard drugs that promote healing of gastric ulcers may simultaneously have the same effect on cutaneous wounds, and corticosteroid aggravation, and to demonstrate why peptides such as BPC 157 exhibit a greater healing effect. Therefore, with the fistulas therapy, we challenge the wound/growth factors theory of the analogous nonhealing of wounds and persistent gastric ulcers. Methods The healing rate of gastrocutaneous fistula in rat (2-mm-diameter stomach defect, 3-mm-diameter skin defect) validates macro/microscopically and biomechanically a direct skin wound/stomach ulcer relation, and identifies a potential therapy consisting of: (i) stable gastric pentadecapeptide BPC 157 [in drinking water (10xa0μg/kg) (12xa0ml/rat/day) or intraperitoneally (10xa0μg/kg, 10xa0ng/kg, 10xa0pg/kg)], (ii) atropine (10xa0mg/kg), ranitidine (50xa0mg/kg), and omeprazole (50xa0mg/kg), (iii) 6-alpha-methylprednisolone (1xa0mg/kg) [intraperitoneally, once daily, first application at 30xa0min following surgery; last 24xa0h before sacrifice (at postoperative daysxa01, 2, 3, 7, 14, and 21)]. Results Greater anti-ulcer potential and efficiency in wound healing compared with standard agents favor BPC 157, efficient in inflammatory bowel disease (PL-14736, Pliva), given in drinking water or intraperitoneally. Even after 6-alpha-methylprednisolone aggravation, BPC 157 promptly improves both skin and stomach mucosa healing, and closure of fistulas, with no leakage after up to 20xa0ml water intragastrically. Standard anti-ulcer agents, after a delay, improve firstly skin healing and then stomach mucosal healing, but not fistula leaking and bursting strength (except for atropine). Conclusion We conclude that BPC 157 may resolve analogous nonhealing of wounds and persistent gastric ulcers better than standard agents.

Sa1949 Intragastric Kcl-Overdose Induces Severe Stomach Mucosal Lesions, Sphincters Failure and Peaked T Waves, Fully Counteracted by Intragastric BPC 157 Application, Given Before or After Kcl Intragastric Challenge

Background. Experimental evidence obtained in rats (J Pharmacol Sci. 2006 Nov;102(3):26977, Dig Dis Sci. 1996 Jul;41(7):1518-26) suggests a novel causative and mutually detrimental relation between esophagitis-sphincter failure and acute pancreatitis (Gastroenterology, 2009). Aim. To show that the acute pancreatitis patients have lower pressure in esophageal sphincters compared with healthy subjects. Methods/Results. In 10 patients with acute pancreatitis (6 women and 4 men, mean age 54.5 ± 8.2 years), esophageal manometry was performed immediately after admission to the department. In 8 patients the cause of pancreatitis was biliary in 2 etilic genesis. The majority of patients (7 cases) had mild pancreatitis and the recovery proceeded without any major complications. All patients underwent esophageal manometry according to standard protocol, and the results were compared with values of control healthy subjects (our GI motility lab) (Table 1). The following parameters investigated esophageal motility: the length, pressure, and the ability to relax the upper and lower esophageal sphincter, values of contraction amplitude in the upper, lower and middle esophagus and peristaltic wave velocity along the body of the esophagus. The results of this preliminary study show that patients with acute pancreatitis had significantly lower mean pressure at rest in both the lower and in upper esophageal sphincter compared to a control group of healthy subjects. There were no statistically significant differences in other parameters studied esophageal motor function between compared groups. Table 1

W1339 Safe Anti-Ulcer Peptide, in Trial for Inflammatory Bowel Desease, Stable Gastric Pentadecapeptide BPC 157 (PL14736) Can Cure Rats With Short Bowel Syndrome Complicated With Ulcerative Colitis

Stable gastric pentadecapeptide BPC 157 recovered short bowel sydrome in rats after massive small bowel resection, improved intestinal adaptation, villus height, crypt depth, and muscle thickness (and inner (circular) and/or outer (longitudinal) muscular layer)), induced weight gain in weight of normal healthy rats (Dig Dis Sci, 2008 in press). BPC 157 is a safe anti-ulcer peptide (PL-14736, Pliva) in trial for IBD, and wound therapy, no toxicology reported (Gastroenterology, 2005) that healed intestinal anastomosis and fistulas (Dig Dis Sci, 2008, J Pharm Sci, 2008). BPC 157 also prevented and reversed ulcerative colitis induced by cysteamine enema (J Physiol, 1999). However, BPC 157 was not tested after small bowel massive resection in rats with ulcerative colitis. Small bowel resection. Throughtout a 4 week period we tested rats with escalating bowel syndrome and progressive weight loss that had only 20% of small bowel (Dig Dis Sci, 2008, in press). Ulcerative colitis. Cysteamine enema 400 mg/kg i.r., 1ml/rat. Ulcerative colitis+small bowel resection. Enema was applied at 10 min before surgery. BPC 157 medication. BPC 157 (10 μg, 10ng/kg i.p. or in drinking water) first application 30 min following surgery, last 24 h before sacrifice (7, 14, 21, 28 days). Small bowel resection. The rats had an escalating bowel syndrome and progressive weight loss despite the fourfold muscle thickness increase and twofold villus height and crypt depth increase during the first week. BPC 157 groups recovery showed improved intestinal adaptation, villus height, crypt depth, and muscle thickness (and inner (circular) and/or outer (longitudinal) muscular layer))additionally increased. The rats immediately gained weight, ultimately to the weight of normal healthy rats. Ulcerative colitis. Controls exhibited pertinent ulceration, initial weight loss, and then decreased weight gain. BPC 157 rats immediately gained weight, ultimately to the weight of normal healthy rats, and had consistently less ulcerations. Ulcerative colitis+small bowel resection. Ulcerative colitis significantly aggravated all these parameters. BPC 157 retained the same beneficial effects, decreased ulcerative colitis, improved intestinal adaptation, villus height, crypt depth, and muscle thickness (and inner (circular) and/or outer (longitudinal) muscular layer)) additionally increased, early weight gain, ultimately to the weight of normal healthy rats. In addition to the recovery of the rats with short bowel syndrome or ulcerative colitis, BPC 157 can cure rats with short bowel syndrome complicated with ulcerative colitis.

W1337 Therapy With Gastric Pentadecapeptide BPC 157 (PL14736) and L-NAME in Short Bowel Syndrome and Entero-Enteral Anastomosis Healing in Rats

Gastric pentadecapeptide BPC 157 (BPC 157), safe in trials for inflammatory bowel disease (IBD) (PL14736 Pliva)(Gastroenterology, 2005), could also be useful in IBD complications. It successfully heals the intestinal anastomosis in rats (Surg Today, 2007) and has a therapeutic effect on the short-bowel syndrome that compromises nutritional status and healing process (Dig Dis Sci, 2008). Also, it interacts with the NO-system and counteracts the effects of NOS-inhibition, induced by application of NOS-inhibitor, N-G-nitro-L-arginine-methyl ester (L-NAME) (Eur J Pharmacol, 1997, J Pharm Sci, 2008). Thereby, this study investigates whether the therapeutic effect of the pentadecapeptide BPC 157 on the short-bowel syndrome could be abolished by L-NAME application. Extensive small bowel resection (from the 4th ileal artery cranially of the ileocecal valve to 5 cm underneath the pylorus) and entero-enteral anastomosis were carried out in male rats as described before (Surg Today, 2007, Gastroenterology, 2007) Medication (BPC 157 (10 µg, 10 ng, 10 pg), L-NAME (5 mg) or an equivolume of saline (5 ml) was given once daily, i.p. (/kg), first application 30 min following surgery, last 24 h before sacrifice (at the post-operative day 1, 3, 5, 6, 7, 14). The assessment includes macroscopic presentation (body weight, mass of small intestine (3 cm cranially to 3 cm caudally from anastomosis), diameter measurements (jejunal, ileal and anastomosis diameter), biomechanical presentation (the volume (ml) instilled through a syringe-perfusion pump system (1ml /10 sec)) to the induction of leakage), microscopic presentation (Surg Today, 2007, Dig Dis Sci, 2008). A severe bowel syndrome was observed (severe body weight loss, jejunal diameter increase, a small volume (2-3 ml) before leakage, particularly decreased height of the villi distal to anastomosis). This was completely counteracted by both pentadecapeptide BPC 157 regimens. Weight gain to healthy values along with markedly higher volume values (4-6 ml) was obtained without leakage. Anastomosis healing is improved (edema markedly attenuated, granulocyte number decreased, necrosis attenuated, granulation tissue, reticulin and collagen formation markedly increased, and finally epithelization increased). All measured diameters were similar to diameter of healthy animals, the height of the villi distal to the anastomosis were higher than in controls. BPC 157 therapeutic effect could be not abolished by L-NAME application. Alone, L-NAME application aggravated short bowel syndrome. Short bowel syndrome and entero-enteral anastomosis healing may be a particular target for therapy with BPC 157.

W1851 BPC 157 Reduced Postoperative Adhesion Formation in Rats

Background: Intestinal scarring is a major cause of morbidity in Crohns disease. Current therapies treat inflammation, but do not alter the progression of fibrosis and bowel obstruction. Given the observation that late use of potent anti-inflammatory therapies does not reduce stenosis or obstruction, we hypothesized that intestinal fibrosis becomes self-propagating, despite removal of inflammatory stimuli. Methods: The Salmonella typhimurium murine model of inflammation and fibrosis in which clearance of commensal microbiota by streptomycin, followed by infection with S. typhimurium, produces chronic inflammation, culminating with fibrosis by day 21 post infection was used. The inflammatory stimulus (S. typhimurium) was removed with the oral antibiotic levofloxacin at day 2, 4, or 8 post-infection. Eradication of S.typhimurium was confirmed by stool plating and T-RFLP analysis. Results: By day 2 post-infection, the mouse cecae infected with S.typhimurium developed an inflammatory phenotype, characterized by a shrunken yet heavier cecum, expansion of the cecal submucosa, and induction of inflammatory genes (IL-1b, TNFa, IL-6, IL-17, IL12p40). However, fibrotic gene expression (CTGF, IGF-1, TGFb) was indistinguishable from uninfected controls. In addition, aSMA protein expression was not induced until day 8 postinfection. Early intervention (day 2 & 4) repressed inflammation as determined by gross pathology, histopathology, and repression of inflammatory genes. Fibrotic gene expression was partially repressed by day 2 levofloxacin treatment. However, day 8 levofloxacin treatment did not prevent induction of aSMA protein or pro-fibrotic genes (CTGF, IGF-1, TGFb) by day 8 and which continued to increase after levofloxacin treatment to day 21, remaining significantly higher than uninfected controls or matched S.typhimurium infected mice harvested at day 4 and 8 post-infection (p < 0.03) Conclusions: Early removal of the inflammatory stimulus reduces fibrosis, but fibrosis after initiation continues to propagate in the absence of an inflammatory stimulus. Since many Crohns patients develop complications of fibrosis, understanding the mechanisms of auto-propagation of fibrosis and developing anti-fibrotic therapies is critical to improving outcomes in Crohns disease.