522 SMALL INTESTINE Gut: first published as 10.1136/gut.51.4.522 on 1 October 2002. Downloaded from Antidiarrhoeal properties of a novel sigma ligand (JO 2871) on toxigenic diarrhoea in mice: mechanisms of action V Theodorou, M Chovet, H Eutamene, H Fargeau, M Dassaud, M Toulouse, C Bihoreau, F J Roman, L Bueno ............................................................................................................................. Gut 2002;51:522–528 Background and aims: Sigma ligands display antisecretory activity against various secretagogues, suggesting antidiarrhoeal properties. In this study, we evaluated: (i) the antidiarrhoeal effect of JO 2871, a high affinity sigma ligand, in three models of toxigenic diarrhoea in mice; and (ii) the site and mechanism of action of this compound. Methods: Faeces were collected after toxin or vehicle administration in male DBA2 or NMRI mice. Diarrhoea was determined by cumulative stool weight (mg) over a 120 minute period. Diarrhoea was induced by intravenous administration of Salmonella enteriditis lipopolysaccharide (LPS), or oral administration of Escherichia coli heat stable (E coli-sta) or Clostridium difficile toxins. Two sigma lig- ands, igmesine and JO 2871, were administered either orally or intravenously, 60 and 30 minutes before the toxins, respectively. JO 2871 was also given orally 30 minutes after E coli-sta. In addition, JO 2871 was administered intracerebroventricularly five minutes before LPS and E coli-sta. BMY 14802 (1000 µg/kg orally), a sigma receptor antagonist, or cyclosomatostatin (CSS 1 µg/kg intrave- See end of article for nously), a somatostatin antagonist, were given five minutes prior to JO 2871 in LPS, E coli-sta, and authors’ affiliations C difficile toxin treated mice. Gastric emptying and intestinal transit were evaluated after oral JO 2871 ....................... and BMY 14802 and intravenous CSS. Correspondence to: Results: Stool weight measured 120 minutes after administration of the toxins was significantly Dr V Theodorou, increased. Oral JO 2871 and igmesine dose dependently inhibited toxigenic diarrhoea in all models. Neurogastroenterology ED50 values obtained using JO 2871 (1–20 µg/kg) were more than 40 times lower than those obtained and Nutrition Unit, INRA, with igmesine. Oral JO 2871 given after E coli-sta also inhibited diarrhoea in a dose dependent man- 180 chemin de http://gut.bmj.com/ Tournefeuille, BP 3, 31931 ner (ED50 50 µg/kg). Both sigma ligands were active by the intravenous route on LPS and E coli-sta Toulouse cedex, France; induced stool weight increases. JO 2871 administered intracerebroventricularly failed to block this [email protected] effect at any dose tested. Both BMY 14802 and CSS reversed the antidiarrhoeal effect of oral JO Accepted for publication 2871. JO 2871, BMY 14802, and CSS did not affect transit parameters. 5 March 2002 Conclusions: JO 2871 exerts a potent oral antidiarrhoeal effect, acting peripherally through sigma ....................... sites and somatostatin release. on September 26, 2021 by guest. Protected copyright. iarrhoea is one of the most common maladies facing of various origins is commonly treated with orally active anti- medicine. Intestinal pathogenic bacteria such as diarrhoeal drugs, such as opiates. However, opiate drugs, such Escherichia coli (E coli), Salmonella enteriditis (S enteriditis), as loperamide, have more pronounced effects on gut motility D 8 and Clostridium difficile (C difficile) cause diarrhoeal diseases by than on epithelial functions. Other drugs such as octreotide, producing highly potent enterotoxins. Binding of these a peptidergic analogue of somatostatin, must be administered enterotoxins to the intestinal epithelium results in activation subcutaneously or intravenously in severe cases, but this pep- of cyclic nucleotide second messengers leading to intestinal tide exhibits side effects such as steatorrhoea and gall bladder water secretion. In contrast, intestinal water secretion stasis.9 There is still a need for a specific antisecretory agent depends on neural regulation, including intrinsic and extrin- which improves the severity of diarrhoeal illness without any sic components. A large number of studies have indicated a related gastrointestinal motor effects or other side effects. regulatory effect of the enteric nervous system in intestinal σ σ 1 Receptors are a subtype of receptor known to bind secretion induced by intraluminal secretagogues. Thus intra- diverse classes of pharmacological agents with high affinity. mural nervous reflexes have been shown to be responsible for 1 These agents include neurosteroids, antipsychotics, and a major part of secretion elicited by luminal cholera toxin, dextrorotatory benzomorphans.10 11 σ Receptors are currently heat stable E coli enterotoxin (E coli-sta),2 bile salt,3 and secre- 1 4 thought to be involved in various biological processes, such as tion induced by noxious stimulation of the intestinal serosa. 12–15 σ learning, memory, and analgesia. The 1 receptor has been Extrinsic innervation is also involved in this regulation. cloned16 17 and its amino acid sequence does not resemble that Indeed, several peptides injected centrally have been shown to of any mammalian protein. The sequence of σ receptors con- suppress toxins and prostaglandin E induced diarrhoea.56 1 2 tains an endoplasmic reticulum retention signal close to the N Further capsaicin sensitive afferents are involved in E coli-sta induced diarrhoea.7 Secretion of water and electrolytes from the intestine results in hypovolaemia and decreased circula- ............................................................. tion to vital organs which causes high morbidity and Abbreviations: E coli-sta, Escherichia coli heat stable toxin; CSS, mortality, especially in children and in the elderly. To date, oral cyclosomatostatin; LPS, lipopolysaccharide; ED50, efficacy dose 50; IC50, rehydratation salts are widely used for the treatment of diar- inhibitor concentration 50; TTX, tetrodotoxin; GE, gastric emptying; GC, rhoea (cholera). In addition, acute but not profuse diarrhoea geometric centre; VIP, vasoactive intestinal polypeptide. www.gutjnl.com Antidiarrhoeal properties of JO 2871 on toxigenic diarrhoea in mice 523 terminus, a binding domain for steroids, and one putative Incubation was stopped by washing the membranes on transmembrane region. GF/B filters previously soaked in the incubation buffer with Sigma sites are widely distributed in the organism. They 0.5% polyethyleneimine added. Washing was performed with Gut: first published as 10.1136/gut.51.4.522 on 1 October 2002. Downloaded from have been characterised in the central nervous system, liver, 3×2 ml of the incubation buffer maintained at 4°C. endocrine organs, lymphocytes, and also in the digestive tract. Filters were transferred to scintillation picovials (Packard For example, using binding studies, Roman and colleagues18 Meridien, USA) and added to 5 ml of Emulsifier Scintillator provided evidence for the presence of σ receptors in the Plus (Packard). Counting of scintillation was perormed in a myenteric plexus of guinea pig small intestine. Moreover, Tri-carb sprectometer (Packard). autoradiographic studies19 revealed a dense distribution of σ sites in the mucosa and the submucosal plexus of guinea pig Calculations gastrointestinal tract. Inhibitor concentration 50 (IC50) values were determined by Igmesine, a potent and selective sigma ligand,20 was used as non-linear regression from the inhibition values of specific a probe to demonstrate the effects of sigma receptor activation binding using GraphPad Prism software. Ki values were 28 on modulation of intestinal secretion. Indeed, igmesine can calculated from IC50 values according to Cheng and Prussoff. exert a proabsorptive effect on jejunal mucosa in vitro through The Hill slope corresponding to each curve was calculated by a neuronal pathway.21 Similarly, igmesine reverses the vasoac- the software. tive intestinal polypeptide (VIP) induced increase in short cir- cuit current22 and inhibits prostaglandin induced intestinal In vivo experimental design secretion in humans.23 Recently, we have shown that igmesine Faecal output measurement displays antidiarrhoeal activity in S enteriditis lipopolysaccha- Animals ride (LPS), E coli-sta enterotoxin, and C difficile toxin induced Male DBA2 mice (20–25 g body weight; Janvier, Le Genest St diarrhoea in mice.24 In addition, Turvill and colleagues25 Isle, France) or NMRI mice (30–35 g body weight; Harlan, showed that igmesine inhibited cholera toxin and E coli-sta Gannat, France) were individually housed in propylene cages induced jejunal secretion in rats. However, as stimulated and kept in a temperature controlled room (21 (1)°C). They intestinal water secretion can be centrally mediated56 and were allowed free access to water and fed ad libitum with igmesine can act centrally to modulate visceral functions,26 27 laboratory pellets (UAR Epinay-sur-Orge, France). we have evaluated the site of action (central v peripheral) of Faeces were collected and weighted every 30 minutes over a JO 2871 (a follow up compound of igmesine) in this study. 120 minute period after administration of S enteriditis LPS, E Hence the aims of the present study were: (i) to evaluate the coli-sta, or C difficile toxins A and B. Faecal output was potent antidiarrhoeal effect of JO 2871 ((E)-3-(1-cyclo- determined by measuring cumulative stool weight (mg) over propylmethyl-2-azinanyl)-1-(3,4-dichlorophenyl)-1-propene a 120 minute