MEP1A allele for meprin A metalloprotease is a susceptibility gene for inflammatory bowel disease.

The MEP1A gene, located on human chromosome 6p (mouse chromosome 17) in a susceptibility region for inflammatory bowel disease (IBD), encodes the alpha-subunit of metalloproteinase meprin A, which is expressed in the intestinal epithelium. This study shows a genetic association of MEP1A with IBD in a cohort of ulcerative colitis (UC) patients. There were four single-nucleotide polymorphisms in the coding region (P=0.0012-0.04), and one in the 3'-untranslated region (P=2 x 10(-7)) that displayed associations with UC. Moreover, meprin-alpha mRNA was decreased in inflamed mucosa of IBD patients. Meprin-alpha knockout mice exhibited a more severe intestinal injury and inflammation than their wild-type counterparts following oral administration of dextran sulfate sodium. Collectively, the data implicate MEP1A as a UC susceptibility gene and indicate that decreased meprin-alpha expression is associated with intestinal inflammation in IBD patients and in a mouse experimental model of IBD.

Modulatory effects of plasma and colonic milieu of patients with ulcerative colitis on neutrophil reactive oxygen species production in presence of a novel antioxidant, rebamipide.

Rebamipide protects gastrointestinal mucosal integrity against reactive oxygen species (ROS). The effect of rebamipide on the capability of PMNs to produce ROS in the presence of plasma and rectal dialysates (RD) of control and ulcerative colitis (UC) subjects was evaluated. We recruited six healthy volunteers for obtaining PMNs, control plasma, and control RD and six patients with inactive UC for obtaining plasma and RD. PMNs were activated using fMLP, and ROS was measured by fluorescent microplate assay (DCFD). Rebamipide significantly inhibited the neutrophil respiratory burst by 45%. Plasma from both control subjects and UC patients significantly blunted the fMLP-induced respiratory burst. However, the plasma of the UC patients was significantly less inhibitory than the plasma of control subjects. RD from control subjects significantly blunted the fMLP-induced respiratory burst while, RD from patients with UC did not. Rebamipide maintained its antioxidant effects in the presence of plasma or RD obtained from both controls and UC patients. In conclusion, partial loss of the inhibitory effects of plasma and RD in patients with UC may contribute to oxidative-induced tissue damage in UC and rebamipide antioxidant properties were not hampered by the biological milieu of patients with UC.

Caffeic acid phenethyl ester, an inhibitor of nuclear factor-kappaB, attenuates bacterial peptidoglycan polysaccharide-induced colitis in rats.

Caffeic acid phenethyl ester (CAPE) is an anti-inflammatory component of propolis (honeybee resin). CAPE is reportedly a specific inhibitor of nuclear factor-kappaB (NF-kappaB). The aims of our study were 1) to evaluate the effect of CAPE on cytokine production, NF-kappaB, and apoptosis in two cell lines; 2) to assess the effect of CAPE on NF-kappaB in rats with peptidoglycan-polysaccharide (PG-PS)-induced colitis; and 3) to evaluate the efficacy of CAPE against this colitis. In vitro experiments used rat macrophage (NR8383) and colonic epithelial cell (SW620) lines. NF-kappaB was evaluated by electrophoretic mobility shift assay. Cytokines and apoptosis were measured by enzyme-linked immunosorbent assay. Colitis was induced by intramural injections of PG-PS into the distal colon. CAPE (30 mg/kg) or vehicle was administered once daily to rats by intraperitoneal injection, for 1 week. Various macroscopic and biochemical indices were measured on day 21. CAPE (30 microg/ml) significantly inhibited NF-kappaB and TNF-alpha production in the macrophage cell line. In macrophages, CAPE significantly increased DNA fragmentation. CAPE exhibited generally similar effects in the colonic epithelial cell line. CAPE treatment reduced the mean level of colonic NF-kappaB in rats. CAPE also induced a significant reduction in gross colonic injury. Moreover, colonic cytokine levels (TNF-alpha and IL-1beta) were significantly reduced in CAPE-treated rats. In summary, CAPE inhibits NF-kappaB, causes a reduction of pro-inflammatory cytokine production, and induces apoptosis in macrophages. These mechanisms likely contributed to the attenuation of PG-PS-induced colitis by CAPE.

In vitro and in vivo effects of gliotoxin, a fungal metabolite: efficacy against dextran sodium sulfate-induced colitis in rats.

Gliotoxin is a fungal metabolite that has immunosuppressive properties. First, we determined if gliotoxin could inhibit cytokine production from macrophage and colonic epithelial cell lines, as well as whether it inhibited nuclear factor-kappa B in these same cell types. Second, we evaluated whether gliotoxin could reduce dextran sodium sulfate-induced colitis in rats. A disease activity index, myeloperoxidase activity, and cytokine levels were evaluated on either day 7 or 21. In both cell lines, gliotoxin dose dependently inhibited cytokine production and nuclear factor-kappa B. On day 21, gliotoxin significantly reduced disease activity (diarrhea and bloody stools) in rats. On day 7, gliotoxin treatment significantly improved various indices of colitis, including colonic cytokine levels. Decreased food consumption and weight gain was evident with a larger dose of gliotoxin. In summary, gliotoxin, a nuclear factor-kappa B inhibitor, effectively reduced dextran sodium sulfate-induced colitis in rats. However, gliotoxin exhibited a narrow therapeutic to toxicity ratio in these rats.

Effect of naproxen on the hamster gastric antrum: ulceration, adaptation and efficacy of anti-ulcer drugs.

BACKGROUND: Various animal models of non-steroidal anti-inflammatory drug (NSAID)-induced gastric ulceration exist. These models have limitations, which make them less relevant to the human situation. AIM: : To develop a more simple and more relevant model of NSAID-induced gastric ulceration and adaptation. METHODS: Gastric ulceration was evaluated following the orogastric administration of naproxen (80 mg/kg b.d.) to hamsters. The effects of misoprostol and famotidine on gastric acid secretion and ulceration were also determined. Gastric adaptation was evaluated by proliferating cell nuclear antigen (PCNA) immunohistochemistry, in hamsters given naproxen for 3 weeks. Antral resistance to acute injury by NSAIDs and ethanol was also determined in these animals. RESULTS: Naproxen caused primarily gastric antral ulceration, which decreased from day 3 to day 21. This gastric adaptation was accompanied by an increase in PCNA positive cells, particularly on days 7 and 14. The adapted gastric antral mucosa was resistant to acute damage by various agents. Misoprostol (1 or 100 microg/kg) prevented antral ulceration, without affecting gastric acid secretion. Despite decreasing acid output by> 90%, famotidine (30 mg/kg) failed to prevent ulceration. CONCLUSION: The administration of naproxen (80 mg/kg b.d.) to hamsters is a simple, reliable and relevant method for evaluating NSAID-induced gastric antral ulceration and adaptation.

Indomethacin-induced gastric antral damage in hamsters: are neutrophils involved?

BACKGROUND: A direct role for neutrophils in the pathophysiology of indomethacin-induced gastric damage is controversial. Therefore, such damage was evaluated in hamsters. METHODS: Gastric antral damage was evaluated 4 h after the oro-gastric administration of indomethacin (30 mg/kg). Prior to indomethacin, hamsters were treated with various pharmacological agents: rebamipide, methotrexate or anti-neutrophil serum (ANS). The number of circulating neutrophils was determined from Wright-Giemsa stained blood smears. Myeloperoxidase (MPO) activity was measured as a marker of gastric antral neutrophil infiltration. RESULTS: Indomethacin caused primarily gastric antral damage. By histology, this damage did not penetrate the muscularis mucosa. A significant increase in gastric antral MPO activity was also found in indomethacin-treated hamsters. Rebamipide decreased macroscopic gastric antral damage in a dose-related fashion. Methotrexate treatment reduced the circulating blood neutrophil number by 38-44%, but did not affect gastric damage. ANS treatment resulted in near complete neutropenia, and also in a substantial reduction (84%) in gastric antral MPO activity. However, gastric antral damage was not significantly altered by ANS. CONCLUSIONS: Neutrophils are not directly involved in the pathophysiology of indomethacin-induced damage to the hamster gastric antrum.

RP 73870, a gastrin/cholecystokinin-B receptor antagonist with potent anti-ulcer activity in the rat.

RP 73870, the racemic potassium salt of (([N-(methoxy-3-phenyl)-N-(N-methyl-N-phenyl-carbamoylmethyl)- carbamoylmethyl]-3-ureido)-3-phenyl)-2-ethylsulfonate-(RS) is a potent, reversible antagonist of both gastrin and cholecystokinin-B receptors in guinea pig and rat tissues. This compound is a potent inhibitor of pentagastrin-stimulated gastric acid secretion in the perfused rat stomach. RP 73870 also inhibits basal gastric acid secretion in the rat, although at doses higher than that required for inhibition of pentagastrin-stimulated gastric acid secretion. RP 73870 is a potent inhibitor of aspirin-induced gastric damage in the rat. In the prevention of aspirin-induced gastric damage, RP 73870, given p.o., was 10-fold less potent than when given i.v. RP 73870 was as potent as a H2 receptor antagonist or proton pump inhibitor in the prevention of cysteamine-induced duodenal ulcers in the rat. Relative to other gastrin/cholecystokinin-B antagonists, RP 73870 demonstrates greater affinity to gastrin binding sites, and possesses a unique spectrum of in vivo biological activities appropriate for an anti-ulcer indication.

The gastrin/cholecystokinin-B receptor antagonist L-365,260 reduces basal acid secretion and prevents gastrointestinal damage induced by aspirin, ethanol and cysteamine in the rat.

L-365,260, a nonpeptide antagonist of gastrin/CCK-B receptors, was evaluated in receptor binding, antisecretory and gastrointestinal damage assays. L-365,260 binds potently and stereo-selectively to gastrin and CCK-B sites in guinea pig tissue. In contrast, L-365,260 binds to the isolated canine parietal cell gastrin receptor weakly, and without stereoselectivity. In the pylorus-ligated rat, low doses of L-365,260, given i.v., attenuated pentagastrin-stimulated acid secretion, whereas higher doses were required to inhibit both histamine-stimulated and basal acid secretion. In an aspirin-induced gastric damage model, L-365,260 was 2.4-fold less potent than the standard histamine H2 antagonist cimetidine in preventing gastric damage when given i.v., and was 8.3-fold less potent than cimetidine when given p.o. Moreover, the ED50 value for L-365,260, given i.v., in prevention of aspirin-induced gastric damage (11.5 mg/kg) agreed well with its ED50 value for inhibition of basal acid secretion (12.6 mg/kg). At doses as great as 100 mg/kg p.o., neither L-365,260 nor cimetidine had an effect on ethanol-induced gastric damage. L-365,260, although p.o. less bioavailable relative to cimetidine in the aspirin gastric damage model, was as potent as cimetidine in the prevention of cysteamine-induced duodenal ulcers in the rat. We conclude that the gastrin/CCK-B receptor antagonist L-365,260, at doses supramaximal for the inhibition of pentagastrin-stimulated secretory responses in vivo, inhibits gastrointestinal damage in models of peptic ulcer disease by an antisecretory mechanism of action.

Development of high-affinity 5-HT3 receptor antagonists. 1. Initial structure-activity relationship of novel benzamides.

This report describes the development of novel benzamides which are orally active, highly potent, specific antagonists of 5-HT3 receptors. Described in this first report are the structure-activity relationships that led to novel structures with improved potency and selectivity. From this series of compounds, (S)-28 was identified and selected for further evaluation as a 5-HT3 receptor antagonist. Compared with 5-HT3 antagonists such as GR 38032F, BRL 43694, and metoclopramide, (S)-28 was most active in (a) inhibiting binding to 5-HT3 receptor binding sites in rat entorhinal cortex with an Ki value of 0.19 nM and (b) blocking cisplatin-induced emesis in the ferret with an ED50 value determined to be 9 micrograms/kg po.

Acidic fibroblast growth factor accelerates the healing of acetic-acid-induced gastric ulcers in rats.

Acidic fibroblast growth factor (aFGF) was evaluated for the healing of acetic-acid-induced gastric ulcers in rats. The effect of aFGF on angiogenesis in the gastric ulcer bed was determined by the carmine dye infusion method, while its effect on gastric acid secretion was assessed in chronic gastric fistula rats. Oral treatment with aFGF, in the presence of heparin, reduced (ED50 value = 30.2 micrograms/kg/day) the acetic-acid-induced gastric ulcer area, when assessed 1 week later. aFGF was about 1,333-fold more potent than famotidine for healing such ulcers. At a dose of 200 micrograms/kg/day, aFGF increased the carmine density 3-fold and correspondingly reduced (80%) the gastric ulcer area. Thus, the ulcer healing effect of this agent involves angiogenesis in the gastric ulcer bed. This effect of aFGF appears to be unrelated to an inhibition of gastric acid secretion, as it was ineffective in chronic gastric fistula rats. In summary, oral aFGF significantly accelerates the healing of experimental gastric ulcers in rats. It may be a potent and effective agent for the treatment of peptic ulcers in humans.

Gastroprotective and ulcer healing profile of the mast cell stabilizer quazolast in rats.

Quazolast, a mast cell stabilizer, was evaluated for efficacy against acid independent (alcohol, HCl), or dependent (aspirin, indomethacin) gastric damage in rats. Its gastroprotective profile was compared to that of ranitidine. In addition, the antisecretory and gastric ulcer (acetic acid induced) healing capabilities of these agents were examined. Quazolast, in direct contrast to ranitidine, protected the rat gastric mucosa from acid-independent, but not acid-dependent gastric damage. Quazolast lacked antisecretory activity in rats; however, it did heal acetic acid induced gastric ulcers in this species. On day 15 after acetic acid injection, quazolast significantly healed such ulcers, while ranitidine did not. Although the exact mechanisms of gastroprotection and ulcer healing action for quazolast remain to be determined, it may be an effective agent for the treatment of gastric ulcers.

Protection by acidified Maalox against indomethacin-induced gastric antral ulceration in hamsters.

The protective capability of Maalox against indomethacin-induced gastric damage was evaluated in hamsters. The effect of acidification on the gastroprotection provided by Maalox against such damage was also determined. Maalox was ineffective against indomethacin-induced gastric antral ulceration in hamsters. Acidification of this antacid to pHs of 1.5-3.5 resulted in significant (80-90%) gastroprotection against indomethacin. Macroscopic and histologic evidence of binding by acidified Maalox to the hamster antral mucosa was clearly evident. In summary, no correlation exists between acid neutralization and the gastroprotective capability of Maalox against indomethacin in hamsters. The gastroprotection by acidified Maalox against antral ulceration in this species corresponds well with the reported presence of its hexaaquoaluminum cation moiety at a pH below 4. Such gastroprotection may involve binding of this cation to the hamster pyloric antrum thereby protecting the antral mucosa against indomethacin-induced ulceration.

RG 12915: a potent 5-hydroxytryptamine-3 antagonist that is an orally effective inhibitor of cytotoxic drug-induced emesis in the ferret and dog.

RG 12915 [4-[N-(1-azabicyclo[2.2.2.]octan-3-(S)-yl)]2-chloro-cis 5a-(S)-9a-(S)-5a,6,7,8,9,9a-hexahydrobenzofurancarboxamide hydrochloride] is a potent and effective agent against cisplatin-induced emesis in the ferret after i.v. or p.o. administration. This agent (p.o.) is also highly protective against cisplatin-induced emesis in the dog, as well as cyclophosphamide/doxorubicin-induced emesis in the ferret. When administered either p.o. or i.v., RG 12915 has a lower ED50 value (0.004 mg/kg) than GR 38032F, BRL 43694 and metoclopramide for attenuating cisplatin-induced emetic episodes in the ferret. It also has a long duration of action against cisplatin-induced emesis in the ferret. In contrast to metoclopramide, RG 12915 lacks significant antidopaminergic activity both in vitro [( 3H]spiroperidol displacement), as well as in vivo (apomorphine-induced emesis). In radioligand binding assays, RG 12915 is a potent and selective displacer of binding of 5-hydroxytryptamine (5-HT)3 binding sites (IC50 value = 0.16 nM), whereas failing to displace binding of ligands for the alpha-1, alpha-2 and beta adrenergic, 5-HT1 or 5-HT2 or cholinergic-muscarinic sites with IC50 values less than 1 microM. At a p.o. dose (1 mg/kg) in which RG 12915 is highly protective against cisplatin-induced emesis in the dog, RG 12915 has no significant gastroprokinetic activity in the same species. In summary, RG 12915 is a potent and p.o. effective agent against cytotoxic drug-induced emesis in animal models. The antiemetic potency of RG 12915 against cisplatin is unrelated to antidopaminergic or gastroprokinetic activity, but may be related to its affinity for 5-HT3 binding sites.

Antiinflammatory effects of various drugs on acetic acid induced colitis in the rat.

The efficacy of various drugs used to treat ulcerative colitis, (sulfasalazine, 5-aminosalicylate, hydrocortisone) was investigated in a model of acetic acid-induced colitis in the rat. Subsequently, we tested the ability of antioxidant/5-lipoxygenase inhibitors (gossypol and nordihydroguiaretic acid [NDGA]) and a cyclooxygenase inhibitor (indomethacin) to attenuate the macroscopic colonic damage and/or neutrophil influx (myeloperoxidase activity [MPO]) associated with this model of colitis. Oral pretreatment with either sulfasalazine, gossypol, or NDGA significantly decreased colonic MPO activity induced by acetic acid. Intrarectal administration of such drugs resulted in an even larger reduction of the colonic inflammation, with gossypol being the most potent compound. Oral or intrarectal administration of corticosteroids (dexamethasone, hydrocortisone) also attenuated the parameters of acetic acid induced colitis. In contrast, pretreatment with indomethacin was ineffective, or when administered daily after colitis induction, indomethacin actually increased colonic neutrophil influx significantly. Our data suggest that both the route of drug administration and dosing regimen employed affect the antiinflammatory potency and/or efficacy of compounds on colitis induced by acetic acid in the rat. Drugs which were effective against this colitis may act by scavenging of oxygen derived free radicals.

Prostaglandin-mediated trophic effects on the rat duodenum: the role of polyamines.

Ornithine decarboxylase (ODC) is the initial rate-limiting enzyme in polyamine biosynthesis. The relationship between ODC and polyamines and the trophic effect of prostaglandin in the rat gastrointestinal tract are unknown. In these studies we determined whether inhibition of ODC activity and subsequent polyamine biosynthesis with the specific enzyme inhibitor difluoromethyornithine (DFMO) would attenuate prostaglandin-mediated trophic effects in the rat duodenum. Significant increases in duodenal mucosal wet weight, RNA, DNA, and protein were found following treatment with 16,16-dimethyl-PGE2 (1 mg/kg) for 1 week. This trophic response was significantly reduced (P less than 0.01) in the duodenum of rats treated concomitantly with DFMO. In addition, this prostenoid significantly increased (4X) duodenal ODC levels, 2 hr following acute administration. These results suggest that polyamines are required for the prostaglandin-stimulated growth of the rat duodenal mucosa.

Relationship between ornithine decarboxylase activity and gastric damage.

Hypertonic NaCl increases the activity of gastric mucosal ornithine decarboxylase (ODC). Intragastric administration of concentrated NaCl solution also induces ulcers in the glandular gastric mucosa. The relationship between ODC activity and gastric mucosal damage and the significance of ODC increases in hypertonic NaCl-treated rats are unknown. Rats were fasted 24 h before being given 1.0 ml of 3.4 M NaCl, 120 mM aspirin in 100 mM HCl or 50% ethanol intragastrically. The oxyntic gland mucosa was removed and assayed for ODC and in some experiments DNA, RNA, and protein content. DNA, RNA, and protein content were decreased by 3.4 M NaCl, and these decreases were much greater if ODC was inhibited by pretreatment with alpha-difluoromethylornithine (DFMO). Both aspirin and 3.4 M NaCl induced ODC activity 6 h later. However, DFMO increased the lesion index only in NaCl-treated rats. Although ethanol produced damage, it had no effect on ODC levels, and DFMO did not alter the severity of ethanol lesions. When different concentrations (0.4, 0.8, 1.6, 2.5, and 3.4 M) of NaCl were administered, ODC activities were increased 6 h later in rats receiving 1.6, 2.5, and 3.4 M NaCl but not lower concentrations. Gross lesions appeared in response to the 2.5 M dose and increased with increasing NaCl concentration. However, microscopic damage of the gastric mucosa occurred at all the concentrations tested. These data show that 1) ODC activation is not necessarily produced by damage, 2) in the case of NaCl, increasing damage increases ODC, and 3) ODC appears to have a role in the prevention of a recovery to damage caused by NaCl.

Effect of epidermal growth factor on polyamine-synthesizing enzymes in rat enterocytes.

A number of peptides having trophic activity on gastrointestinal mucosa and growth factors are known to induce small intestinal ornithine decarboxylase (ODC) activity. The effect of peptides on ODC and S-adenosylmethionine (SAMDC) activities (key enzymes in polyamine biosynthesis) in isolated enterocytes is unknown. Male Sprague-Dawley rats were fasted for 72 h and injected intraperitoneally with epidermal growth factor (EGF), pentagastrin, or glucagon, or intragastrically with EGF. A similar volume of water served as a control. Villus tip, midvillus, and crypt cell fractions were collected and identified. ODC and SAMDC activities were determined in these cells 4 h after peptide injection. EGF given intraperitoneally, but not intragastrically, stimulated ODC activity along the cryptvillus column. Pentagastrin and glucagon did not induce polyamine biosynthetic enzyme activity. ODC and SAMDC activities in intestinal mucosal scrapings from fasted animals also were increased 2-4 h after intraperitoneal EGF treatment. It is possible that EGF binding at the serosal surface of the crypt enterocyte and subsequent ODC induction is important in initiating the cellular proliferation that is known to occur after treatment with this peptide.

Effect of refeeding on polyamine biosynthesis in isolated enterocytes.

Ornithine decarboxylase (ODC) activity has been found to be preferentially associated with small intestinal villus cells rather than crypt cells in the rat. In the present study, ODC, S-adenosylmethionine decarboxylase (SAMDC), and polyamines were measured in isolated enterocytes to determine which cell populations increased polyamine biosynthetic activity after refeeding. Two hours following refeeding, significant increases in ODC were observed in villus tip (10 times) and midvillus (20 times) enterocytes. No increase in ODC activity was found in isolated crypt cells. A similar pattern was observed for SAMDC. Enzyme activity increased in villus tip (2 times) and midvillus (27 times) cells but not in crypt enterocytes. Putrescine contents were increased following refeeding in midvillus enterocytes (P less than 0.05) and in crypt cells (P less than 0.05). The accumulation of putrescine in midvillus cells occurs via ODC-induced biosynthesis, whereas in crypt enterocytes it may be due to putrescine uptake. The lack of induction of ODC and SAMDC in crypt enterocytes following acute refeeding suggests these enzymes are apparently not involved in the initiation of cell proliferation known to occur under this condition.