Ivan Barisic

Inhibition of methyldigoxin-induced arrhythmias by pentadecapeptide BPC 157: A relation with NO-system

Pentadecapeptide BPC 157 (GEPPPGKPADDAGLV, MW 1419) reversed congestive heart failure and various arrhythmias, influenced the NO-system and showed no proarrhythmic effect. In therapy analogy, we challenged rats with digitalis, to show attenuation by BPC 157 and the relation between the NO-system and digitalis toxicity. (i). BPC 157 prophylactic effect. Development of cumulative intravenous digitalis toxicity, BPC 157 (50 microg, 10 microg, 10 ng/kg applied intravenously immediately before a methyldigoxin increment regimen (2.0/1.5/1.5/1.0 mg/kg at 15 min-intervals, total dose 6.0 mg/kg/45 min)) reduced the number of ventricular premature beats, prolonged the time before onset of ventricular tachycardia, reduced ventricular tachycardia and AV-block duration (microg-regimes) or reduced mainly the AV-block duration (ng-regimen). (ii). BPC 157 therapy. Advanced methyldigoxin toxicity (6.0 mg/kg i.v. bolus). BPC 157 applied at the 20th second of the grade 3 AV-block shortened AV-blocks, mitigated a further digitalis toxicity course. Ventricular tachycardias were either avoided (50 microg), or markedly reduced (10 microg, 10 ng). Fatal outcome was either avoided (50 microg), reduced (10 microg), or only delayed (10 ng) (iii) BPC 157, L-NAME, l-arginine, L-NAME+l-arginine application. L-NAME-application (5 mg/kg i.p.) aggravated methyldigoxin-arrhythmias. l-arginine (200 mg/kg i.p.) alone had no effect but blunted L-NAME-exaggeration (L-NAME+l-arginine). In this respect, BPC 157 (50 microg/kg i.p.) was prophylactically and therapeutically more effective: the antagonism of L-NAME with BPC 157 produced an effect similar to BPC 157 alone. In conclusion, digitalis-induced arrhythmias in rats could be prevented and counteracted by pentadecapeptide BPC 157, mainly through an interaction with the NO-system.

Stable gastric pentadecapeptide BPC 157-NO-system relation.

We reviewed stable gastric pentadecapeptide BPC 157-NO-system-relation, its close participation in Moncadas (maintained vascular integrity, platelets control) homeostatic healing response of NO-system to injury. Namely, BPC 157s particular healing effect also affects all events after vascular integrity loss (dependent on circumstances, it reduces either thrombosis (abdominal aorta anastomosis) or bleeding/thrombocytopenia (amputation, heparin, warfarin, aspirin)) and in a series of different injurious models, acute and chronic, BPC 157 consistently advances healing after severe injuries in various tissues spontaneously unable to heal; stimulates egr-1 and naB2 genes; exhibits high safety (LD1 not achieved)). Hypothesis, that BPC 157 (since formed constitutively in the gastric mucosa, stable in human gastric juice, along with significance of NO-synthase and the basal formation of NO in stomach mucosa, greater than that seen in other tissues) exhibits a general, effective competing both with L-arginine analogues (i. e., L-NAME) and L-arginine, and that this has some physiologic importance (NO-generation), later, practically supports its beneficial effects illustrating BPC 157 and NOsystem mutual (with L-NAME/L-arginine; alone and together) relations in (i) gastric mucosa and mucosal protection, following alcohol lesions, in cytoprotection course, NO-generation, and blood pressure regulation; (ii) alcohol acute/chronic intoxication, and withdrawal; (iii) cardiovascular disturbances, chronic heart failure, pulmonary hypertension, and arrhythmias; (iv) disturbances after hypokalemia and hyperkalemia, and potassium-cell membrane dysfunction; and finally, in (v) complex healing failure, proved by the fistulas healing, colocutaneous and esophagocutaneous. However, how this advantage of modulating NO-system (i. e., particular effect on eNOS gene), may be practically translated into an enhanced clinical performance remains to be determined.

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.

Mortal Furosemide-Hypokalemia-Disturbances in Rats NO-System Related Shorten Survival by L-NAME. Therapy Benefit with BPC 157 Peptide More Than With L-Arginine

Background: We focused on NO-system-relations (worsening/amelioration) of furosemide (100 mg/kg intraperitoneally)-diuresis-hypokalemia mortal course in rats and beneficial effect of BPC 157 therapy. Methods: Electrocardiographically 90-150 min post-furosemide application duration of PR, RR, QRS, QT intervals, P, R, S, T waves and its amplitude as well were analysed along with appearance of AV block, ventricular premature beats, ventricular tachycardia. Clinically, skeletal muscle myoclonal activity and lethality at 150 min were also analysed. Results: All NO-system-related agents (alone and/or combined, before/after furosemide) not changed hypokalemia and all averted to some extent furosemide-forced diuresis. NOS-blocker, L-NAME (5 mg/kg intraperitoneally) accelerated mortality, aggravated cardiac and extra-cardiac manifestations, thereby, NO-systemrelated. Prevented hypokalemia-mortality was with NO-precursor L-arginine (100 mg/kg intraperitoneally) and stable gastric pentadecapeptide BPC 157 (10 ug, 10 ng/kg intraperitoneally/intragastrically). Specifically, BPC 157 showed most complete benefit. i. BPC 157 given 15 min before furosemide. All BPC 157 regimens maintained sinus rhythm, had no ventricular premature beats, ventricular tachycardia, AV block, no prolongation of intervals and waves without reduction of amplitude. ii. BPC 157 given 90 min after furosemide (with hypokalemia, 3rd grade AV block and/ or ventricular tachycardia being present). Within 5-10 minutes, BPC 157 regimens normalized P, R, S, T waves, PR, RR, QRS, QT interval duration, R, S, T wave amplitude, total AV block and terminated ventricular tachycardia. Likewise, BPC 157 eliminated skeletal muscle myoclonus. Conclusion: L-NAME/L-arginine was mutual counteraction while BPC 157 completely eliminated L-NAME (arrhythmias, myoclonus, mortality), without an additive benefit when combined with L-arginine. Thus, we showed potentially effective therapeutic interventions for acute hypokalemia.

Stable gastric pentadecapeptide BPC 157 and bupivacaine

Bupivacaine toxicity following accidental overdose still lacks therapeutic solution. However, there are major arguments for testing BPC 157 against bupivacaine toxicity in vivo in rats, in particular, and then finally, in vitro. These are: the lack of any known BPC 157 toxicity, a lifesaving effect via the mitigation of arrhythmias in rats underwent hyperkalemia or digitalis toxicity, the elimination of hyperkalemia and arrhythmias in rats underwent succinylcholine toxicity and finally, the reduction of potassium-induced depolarization in vitro (in HEK293 cells) in severe hyperkalemia. Most importantly, BPC 157 successfully prevents and counteracts bupivacaine cardiotoxicity; BPC 157 is effective even against the worst outcomes such as a severely prolonged QRS complex. Here, rats injected with bupivacaine (100mg/kg IP) exhibited bradycardia, AV-block, ventricular ectopies, ventricular tachycardia, T-wave elevation and asystole. All of the fatalities had developed T-wave elevation, high-degree AV-block, respiratory arrest and asystole. These were largely counteracted by BPC 157 administration (50µg/kg, 10µg/kg, 10ng/kg, or 10pg/kg IP) given 30min before or 1min after the bupivacaine injection. When BPC 157 was given 6min after bupivacaine administration, and after the development of prolonged QRS intervals (20ms), the fatal outcome was markedly postponed. Additionally, the effect of bupivacaine on cell membrane depolarization was explored by measuring membrane voltages (Vm) in HEK293 cells. Bupivacaine (1mM) alone caused depolarization of the cells, while in combination with BPC 157 (1µm), the bupivacaine-induced depolarization was inhibited. Together, these findings suggest that the stable gastric pentadecapeptide BPC 157 should be a potential antidote for bupivacaine cardiotoxicity.

Rat inferior caval vein (ICV) ligature and particular new insights with the stable gastric pentadecapeptide BPC 157

Rat inferior caval vein (ICV) ligation (up to the right ovarian vein (ROV)) commonly represents a recapitulation of Virchow: with ligation leading to vessel injury, stasis, thrombosis and hemodynamic changes. We revealed that BPC 157s therapy collectively attenuated or counteracted all these events and the full syndrome. METHODS We applied BPC 157 (10 μg, 10 ng/kg) as an early regimen or as a delayed therapy. Assessment includes gross assessment by microcamera; microscopy, venography, bleeding, blood pressure, ECG, thermography, MDA and NO-level in plasma and ICV, and gene expression. RESULTS Direct vein injury, thrombosis, thrombocytopenia, prolonged bleeding were all counteracted. Also, rapid presentation of collaterals and redistribution of otherwise trapped blood volume (bypassing through the left ovarian vein (LOV) and other veins), with venous hypertension, arterial hypotension and tachycardia counteraction were shown. BPC 157-rats presented raised plasma NO-values, but normal MDA-values; in ICV tissue reverted low NO-values and counteracted increased MDA-levels. Altered expression of EGR, NOS, SRF, VEGFR and KRAS in ICV, ROV and LOV revealed increased or decreased levels, while some genes continuously remained unchanged. CONCLUSION As a new insight, BPC 157 application largely attenuated or even completely eliminated all consequences of ICV ligation in rats.

BPC 157 and Standard Angiogenic Growth Factors. Gastrointestinal Tract Healing, Lessons from Tendon, Ligament, Muscle and Bone Healing

Commonly, the angiogenic growth factors signify healing. However, gastrointestinal ulceration is still poorly understood particularly with respect to a general pharmacological/pathophysiological role of various angiogenic growth factors implemented in growth factors wound healing concept. Thereby, we focused on the stable gastric pentadecapeptide BPC 157, a peptide given always alone vs. standard peptidergic angiogenic growth factors (EGF, FGF, VEGF), and numerous carriers. Further, we reviewed how the gastrointestinal tract healing could be generally perceived (i) in terms of angiogenic growth factors, and/or (ii) through the healing of extragastrointestinal tissues healing, such as tendon, ligament, muscle and bone, and vice versa. Respected were the beneficial effects obtained with free peptides or peptides with different carriers; EGF, FGF, VEGF, and BPC 157, their presentation along with injuries, and a healing commonality, providing their implementation in both gastrointestinal ulcer healing and tendon, ligament, muscle and bone healing. Only BPC 157 was consistently effective in all of the models of acute/chronic injury of esophagus, stomach, duodenum and lower gastrointestinal tract, intraperitoneally, per-orally or locally. Unlike bFGF-, EGF-, VEGF-gastrointestinal tract studies demonstrating improved healing, most of the studies on tendon, muscle and bone injuries provide evidence of their (increased) presentation along with the various procedures used to produce beneficial effects, compared to fewer studies in vitro, while in vivo healing has a limited number of studies, commonly limited to local application, diverse healing evidence with diverse carriers and delivery systems. Contrary to this, BPC 157 - using same regimens like in gastrointestinal healing studies - improves tendon, ligament and bone healing, accurately implementing its own angiogenic effect in the healing. Thus, we claim that just BPC 157 represents in practice a pharmacological and pathophysiological role of various peptidergic growth factors.

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.

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.