In vivo inhibitory effect of lanreotide (BIM 23014), a new somatostatin analog, on prostaglandin- and cholera toxin-stimulated intestinal fluid in the rat

Peptides, Vol. 14, pp. 297-301. 1993

0196-9781/93 $6.00 + .00 Copyright © 1993 PergamonPress Ltd.

Printed in the USA.

In Vivo Inhibitory Effect of Lanreotide (BIM 23014), a New Somatostatin Analog, on Prostaglandin- and Cholera Toxin-Stimulated Intestinal Fluid in the Rat ALAIN BOTELLA,* FRANt~OISE VABRE,* JEAN FIORAMONTI,* F R A N f f O I S T H O M A S I " A N D L I O N E L B U E N O *l

*Department o f Pharmacology, I N R A , BP3, F-31931 Toulouse, France and - ? I P S E N - B I O T E C H Laboratory, F- 75 73 7 Paris, France R e c e i v e d 1 J u n e 1992 BOTELLA, A., F. VABRE, J. FIORAMONTI, F. THOMAS AND L. BUENO. In vivo inhibitoo, effect oflanreotide (BIM 23014), a new somatostatin analog, on prostaglandin- and cholera toxin-stimulated intestinal fluid in the rat. PEPTIDES 14(2) 297-301, 1993--The antisecretory action of subcutaneously (SC) administered somatostatin(1-14), octreotide, and lanreotide on jejunal net flux of water under basal, prostagtandin El (PGEI)- and cholera toxin (CT)-stimulated secretory conditions was determined in vivo on isolated intestinal loop in anesthetized rats. Both PGEI and CT induced intestinal hypersecretion in the rats. This secretory effect was not affected by SC administration of saline. Lanreotide ( l, 10, and 100 ug/kg) reduced the maximal PGE l-induced secretion, while 200 ~tg/kg had no effect. Similarly, octreotide (l and 10 #g/kg) and somatostatin(1-14) (0. l and l ~,g/kg) reduced the increase of net water flux induced by PGE I. However, higher doses of octreotide ( 100 and 200 ~,g/kg) and somatostatin(l-14) (l 0 and 100 ug/kg) had no effect on PGE l-induced secretion. Lanreotide, octreotide, and somatostatin(114) (l and l0/~g/kg) abolished the maximal secretion induced by cholera toxin. However, 100 ~g/kg of lanreotide, octreotide, and somatostatin(l-14) had no effect on cholera toxin-induced secretion. The present study shows that lanreotide, octreotide, and somatostatin(l-14) reduce the secretion induced by PGEI and abolish that induced by CT. These effects were obtained with doses of less than 100 #g/kg of the products, higher doses being ineffective. The higher efficacy against CT-induced hypersecretion as compared to PGEl-induced hypersecretion suggests a direct antisecretory effect at the enterocyte level and indicates the usefulness of these products as antidiarrheal agents in nonhormonally mediated diarrhea. Lanreotide

Octreotide

Somatostatin( I- 14)

Prostaglandin E 1

ISOLATED initially from hypothalamic tissues and shown to be an inhibitor of growth hormone release (8), somatostatin, a tetradecapeptide, has been found to exist in the stomach, pancreas, and intestine in N-terminally extended form (31). Somatostatin inhibits secretion of gastric acid and pepsin (4,5), pancreatic bicarbonate and protein output (21 ), biliary secretion (27), and the release of several gastrointestinal hormones (6,25,34). These actions suggest that this endogenous peptide may play a role in the control of digestive functions. Although somatostatin does not alter net basal jejunal water and electrolyte fluxes in h u m a n s and other species (2,13,14,26), in vitro it inhibits small intestine secretion stimulated by prostaglandin El (PGEI) (14), fatty acids (1), theophyUine (14,24), glucagon (2), and vasoactive intestinal peptide (l 5) in animal species. Indeed, somatostatin has been shown to reduce diarrhea

Cholera toxin

Jejunal hypersecretion

in patients with carcinoid syndrome (13) and pancreatic cholera (36). Two major problems that limit the therapeutic antidiarrheric effectiveness of somatostatin are its short plasma half-life (i.e., its short duration of action after systemic administration) and its marked diversity of actions (i.e., its lack of selectivity for a given target cell or tissue). Thus, structural analogues of somatostatin have been synthesized in an effort to overcome these problems. One of these analogues, lanreotide [BIM-23014; 3-( 2-naphthyl)-D-Ala-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2], is derived from a potent cyclic octapeptide. Lanreotide has been used with some success in the treatment of diarrhea, as well as for inhibiting growth hormone release (28,29). However, up to now, the mechanism by which somatostatin and its analogs inhibit small intestinal secretion has not been fully elucidated.

Requests for reprints should be addressed to Dr. L. Bueno.

297

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BOTELLA ET AL.

The aims of our study were to i) determine the antisecretory action of subcutaneously administered lanreotide in vivo on intestinal fluid under basal and stimulated secretory conditions in rat, and ii) compare its effects with those of somatostatin and one of its analogues, octreotide (SMS 201-995) (3). We have chosen two secretagogues, prostaglandin E 1 and cholera toxin, which are able to reverse intestinal net water flux by two different intracellular pathways (22).

Cholera toxin. Thirteen groups of six rat were used. After a 120-min equilibration period, cholera toxin (Sigma) was added to the perfusate for 1 h and infused at a rate of 25 ug/kg/h. The medium was perfused at a rate of 12 ml/h. At the end of cholera toxin infusion, the animals received 0.4 ml of saline alone (control) or containing either somatostatin( 1-14), octreotide, or lanreotide at concentrations from 0.1 to 200 ag/kg. The outflow of the segment was collected every 15 min over a 5-h infusion period.

METHOD RESULTS

Animals and Surgical Procedures In vivo transport studies using the isolated loop technique were performed as previously described (33). Male Wistar rats, weighing between 300-400 g, were deprived of food 18-24 h prior to anesthesia and surgery. The animals were anesthetized with urethane (3 g/kg SC) and a midline laparotomy was performed to expose the small intestine. A 15-cm segment of proximal jejunum 3 cm from the ligament of Treitz was ligated at its extremities and cannulated for intraluminal perfusion. Care was taken to ensure neural and vascular integrity in each segment. Cannulated segments were rinsed, replaced in the abdominal cavity, and the laparotomy incision was closed. Rats were placed on heating pads in order to maintain their body temperature around 37°C throughout each experiment.

Gut Perfusion After clearing and elimination of contents with warm saline, the intestinal loops were infused with a Ringer's buffer solution containing (in mmol/I): Na ÷ 142.6; K ÷ 5.0; CI- 123.8; Mg2+ 1.2; Ca 2÷ 1.3; HCO3- 25.0; HPOa 2- 17.0; H2PO4- 0.3; glucose 5.0. The solution also contained 5/~Ci/l of []4C] polyethylene glycol (mol.wt. 4000) as a nonabsorbed dilution marker of water flux and 5 g/l of unlabeled polyethylene glycol 4000 as a carrier. The jejunal segment was infused at a constant rate and the perfusate coming out of the loop was collected during consecutive period of 15 min over 2 to 5 h. ~'C activity in collected samples was determined by liquid scintillometry and net water flux for each 15-min interval was calculated by the following formula: (1 DPMs/DPMx) × P/L = net water flux (,l/cm × h), where DPMs and DPMx are ~4Cactivity in buffer solution and perfusate sample, respectively, P is the rate of perfusion, and L the length of intestinal segment (in cm). Water flux occurring over four successive 15-min intervals was averaged to obtain a mean net flux of water over l-h periods. Positive values represent net absorption; negative values indicate a net secretion of water. Values (net water absorption) are expressed as mean + SE for n rats and were analyzed using unpaired Student t-test. The criterion for statistical significance between controls and treated animals was set at p < 0.05 and p < 0.01.

Experimental Procedure PGE1. Sixteen groups of six rats were used. After a 120-min equilibration period, PGE 1 (Sigma, St. Louis, MO) was added to the perfusate (12 ml/h) for 1 h at a constant rate of I ~g/kg/ h. Five minutes before the start ofPGE1 perfusion, the animals received subcutaneously (SC) 0.4 ml of saline alone (control) or containing either somatostatin( 1-14) (Sigma), octreotide (SMS 201995) (Sandoz, Basle, Switzerland), or lanreotide (BIM 23014) (IPSEN/BIOTECH, France) at concentrations from 0.01 to 200 /~g/kg. The outflow of the infused jejunal segment was collected every 15 min over 3 h (after their administration).

Secretion Induced by PGEI After the 120-min of equilibration period, the net flux of water measured over the last 30 min was 62 + 10,1/cm/h. This value was not significantly modified by SC administration of saline. Injected alone, lanreotide at a high dosage (500 ~tg/kg) did not significantly modify the values of absorption compared with the preinjection period. A significant (p < 0.01) reduction in water absorption occurred 30 min after the start of PGEI infusion and was maximal 75 min after the start of perfusion, corresponding to a net secretion of -232 _+ 28 , i / c m / h (Fig. l ). This secretion of water induced by PGE l lasted I 15 min after the start of PGE l infusion. Administered SC at a dose of 0.1 #g/kg, lanreotide failed to modulate the PGE l-induced secretion; the net water flux measured 75 min after the start of perfusion was - 2 4 6 + 17 gl/cm/ h. At doses of 1, 10, and 100 #g/kg, lanreotide significantly (p < 0.01) reduced by 32, 32, and 28%, respectively, the PGElinduced secretion measured 75 min after the start of the PGE 1 infusion (Fig. 2). However, administered at higher dose (200 #g/ kg), lanreotide had no effect on PGEl-induced secretion. Similarly, octreotide and somatostatin( l - 14) at doses o f 0.01 and 0.1 ,g/kg failed to modulate the PGE l-induced secretion (Table 1). When administered SC at higher doses (1 and i0 , g / kg), octreotide significantly reduced (p < 0.01) the increase in net water flux induced by PGE 1 by 15 and 17%, respectively. Somatostatin(1-14) at 0. l and 1 #g/kg also reduced the PGElinduced secretion by 28 and 14%, respectively. Octreotide administered at 100 and 200 #g/kg and somatostatin( l - 14) at l 0 and 100 #g/kg had no effect on PGE l-induced secretion.

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FIG. 1. Influence of lanreotide ( 1 .gkg) on the time-related changes in net water flux induced by PGE1 infusion in isolated jejunal loop of anesthetized rats. The jejunal segment was infused with a solution conrainingPGE 1during 60 rain ( l ~g/kg/h)at a rate of 12 ml/h. The secretory effect of PGE 1 was maximal 15 min after the end of this infusion. At the start of the secretagogue infusion lanreotide was injected. Values were determinedfrom samplescollectedat 15-rainintervalsand expressed as the means (±SEM) for six rats.

LANREOTIDE AND INTESTINAL ABSORPTION

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FIG. 2. Comparative influence of SC administration of lanreotide, octreotide, and somatostatin(I-14) on water jejunal secretion induced by PGEI. The peptides were administered at doses ranging from 0.01 to 200/~g/kg. Results were expressed as the percentage of maximal secretion (75 min after the start of infusion) induced by PGEI. Values are means (+_SEM) for six rats in each group.

FIG. 3. Influence of lanreotide (1 gg/kg) on the time-related changes in net water flux induced by cholera toxin infusion in isolated jejunal loop of anesthetized rats. The jejunal segment was infused with a solution containing cholera toxin during 60 min (25 ug/kg/h) at a rate of 12 ml/ h. The secretory effect of CT was maximal 5 h after the end of this infusion. At the end of the secretagogue infusion somatuline was injected. Values were determined from the samples collected at 15-min intervals during 1 h and expressed as the means (_+SEM) in six rats.

Secretion Induced by Cholera Toxin T h e SC a d m i n i s t r a t i o n of saline (vehicle) did not influence the net water flux. T h e kinetic o f net water m o v e m e n t following cholera toxin administration was different from that obtained after P G E 1. The m a x i m a l secretion has o b t a i n e d d u r i n g the fifth h o u r after the end of cholera toxin infusion, with a net secretion of - 2 8 _+ 6 ~ l / c m / h (Fig. 3). W h e n administered SC at a dose o f 0.1 ~tg/kg, lanreotide failed to reduce the cholera toxin-induced secretion. T h e net water flux m e a s u r e d 5 h after the e n d o f infusion was - 3 3 + 3 ~d/cm/h. In contrast, higher doses ( 1 a n d 10 ~tg/kg) o f l a n r e o t i d e significantly (p > 0.01) reduced the cholera toxin-induced secretion by 94 a n d 83%, respectively, w h e n m e a s u r e d 5 h after the e n d o f cholera toxin infusion (Fig. 4). However, w h e n administered at higher dose (i.e., 100 #g/kg), lanreotide had n o effect on cholera toxin-induced secretion. Similarly, octreotide a n d somatostatin( 1-14), at a dose of 0.1 ~g/kg SC, failed to reduce the cholera toxin-induced secretion (Table 1). At higher doses (1 a n d 10 #g/kg), octreotide reduced by 91 a n d 95%, respectively (p < 0.01 ), the increase o f net water

flux induced by cholera toxin. S o m a t o s t a t i n ( l - 1 4 ) , at doses of 1 and 10 ~g/kg, also reduced the cholera toxin-induced secretion by 95 a n d 85%, respectively. Neither o f the peptides had any effect o n cholera toxin-induced secretion, w h e n administered a dose of 100 ~tg/kg. DISCUSSION T h e results of the present study show that a new stable somatostatin analog, lanreotide, has a n antisecretory effect o n jej u n a l water hypersecretion induced by b o t h P G E I a n d C T in the rat. However, while lanreotide blocks the hypersecretion induced by CT, it only partially reduces the water secretion induced by P G E I . O u r experiments also d e m o n s t r a t e that its potency does not differ significantly from that o f octreotide or somatos t a t i n ( l - 1 4 ) when all these products were administered at the same doses. These findings are in agreement with previous in vivo data obtained in rats, showing that somatostatin infused intravenously inhibits P G E l - i n d u c e d hypersecretion (14,15).

TABLE 1 INHIBITORY ACTIONS OF SOMATOSTATIN(I-14), OCTREOTIDE, AND LANREOTIDE ON PROSTAGLANDIN El- AND

CHOLERA TOXIN-INDUCED CHANGES IN NET JEJUNAL WATER FLUX Doses (gg/kg)

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Values are mean _+ SEM for six rats expressed in ul/cm/h. In PGEI experiment, these values were determined 75 min after the start of perfusion of PGE1 and in CT experiment, 5 h after the end of cholera toxin perfusion. * p < 0.01. t p > 0.01.

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FIG. 4. Comparative influence of SC administration of lanreotide, octreotide, and somatostatin(I-14) on water jejunal secretion induced by cholera toxin. The peptides were administered at differentdoses ranging from 0.1 to 100 ug/kg. Results were expressed as the percentage of maximal secretion induced by cholera toxin. Values are means (__SEM) for six rats.

However, our results differ from previous in vitro (20) and in vivo (18) studies, which found that octreotide did not reduce cholera toxin-stimulated net water flux in rats. These previous studies showed that octreotide did not alter cholera toxin-stimulated intestinal secretion and concluded that despite a previously identified intestinal antisecretory and proabsortive effect of octreotide in vitro (35), this effect was not present in vivo and thus limited the use of this product as an antidiarrheal agent in nonhormonally mediated diarrhea. These opposite effects obtained with this somatostatin analog on CT-induced secretion may be explained by the different concentrations of agent employed, as indicated by the bell-shaped dose-effect relationships observed in our study (no effect at 100 ug/kg). Indeed, in their studies, Fedorack et al. (18) had subcutaneously injected octreotide at 100 or 1000 ug/kg/day. Our results indicate that octreotide injected at I00/zg/kg had no effect on cholera toxin-induced secretion. Moreover, our results are in agreement with other in vitro studies suggesting an antisecretory effect of octreotide on the intestinal epithelium (35). The lack of antisecretory effects of high doses of lanreotide and other analogues is difficult to explain. However, previous study have shown that somatostatin and octreotide, injected centrally, reduce jejunal absorption of water in dogs (32). Consequently, it could be hypothetized that at higher doses, this centrally mediated effect may occur and counterbalance a peripheral antisecretory action.

Regarding the secretory mechanism of action of PGEI and CT, many studies have suggested that these two agents induce intestinal hypersecretion by different intracellular pathways in enterocytes, both pathways resulting in increase of intracellular cAMP (7,22,37). More recent studies have shown that cholera toxin transfers ADP-ribose to the stimulatory GTP-binding protein Gs, which is linked to adenylate cyclase (17,19). The consequence of this covalent modification of G~ is a reduced GTPase activity, resulting in a stabilization of the active GTP-bound state and thus prolonged adenylate cyclase activation (12). Elevation of intracellular cAMP in intestinal epithelial cells leads to increased fluid secretion (30). The secretory effect of PGEI is associated with an increase of cAMP mucosa cells via an activation ofadenylate cyclase complex (22,23), but the mechanism of PGE seems to be more complex. In vitro (10,16) and in vivo (9) studies have demonstrated two sites of action of PGE depending on concentrations used. Administered at lower doses, PGE act mainly by eliciting local secretory nervous reflexes and it is proposed that PGE does not exert its effect through cAMP. At higher concentrations, PGE acts directly on receptors located on enterocytes, eliciting an increase in tissue cAMP (38). In our study, the concentration of PGE 1 used corresponds to low or physiologic dose employed by the authors previously noted. The utilization of this low dose of PGE 1 strongly suggests the case of a fluid secretion mediated by a nervous reflex. These differences in intracellular pathways between PGE 1- and CT-induced secretion are supported by the type of kinetics obtained with both secretagogues in our study. After addition of CT, the net water flux decreased gradually and the secretory rate was the highest 5 h after the end of CT perfusion. In contrast, addition ofPGE 1 induced a more potent and rapid, but less longer-lasting decrease in net water flux. However, the involvement of these two mechanisms together (neural and direct) could not be completely omitted. This could explain the weaker action of somatostatin and its analogs on prostaglandin-mediated diarrea. Indeed, somatostatin, octreotide, and lanreotide are able to reduce the secretory effect induced by PGEI and cholera toxin, indicating that these compounds inhibit cAMP-stimulated adenylate cyclase activity. However, the more potent antisecretory effect obtained by somatostatin and its analogs in CT-induced secretion suggests that these peptides preferably act on the intracellular pathway triggered by this secretagogue by reversing ADP ribosylation. Finally, our observations suggest that octreotide and lanreotide may be used in the treatment of nonhormonaUy mediated watery diarrhea. However, the usefulness of these analogs in prostaglandin-mediated diarrheal diseases could be suggested but seems to be less convincing. ACKNOWLEDGEMENTS The authors thank INRA and IPSEN-BIOTECH for their financial support.

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LANREOTIDE AND INTESTINAL ABSORPTION

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22. Kimberg, D. V.; Field, M.; Gershon, E.; Henderson, A. Effects of prostaglandins and cholera enterotoxin on intestinal mucosal cyclic AMP accumulation. J. Clin. Invest. 53:941-949; 1974. 23. Kimberg, D. V.; Field, M.; Henderson, A.; Gershon, E. Stimulation of intestinal mucosal adenylate cyclase by cholera enterotoxin and prostaglandins. J. Clin. Invest. 50:1218-1230; 1971. 24. Knoblock, S. F.; Dierner, M.; Rummel, W. Antisecretory effects of somatostatin and vasopressin in the rat colon descendens in vitro. Regul. Pept. 25:75-79; 1989. 25. Konturek, S.; Tasler, J. J.; Obtulowicz, W.; Coy, D. H.; Schully, A. V. Effect of growth hormone release inhibiting hormones on hormones stimulating exocrine pancreatic secretion. J. Clin. Invest. 58: 1-6; 1976. 26. Krejs, G. J.; Browne, R.; Raskin, P. Effect of intravenous somatostatin on jejunal absorption of glucose, amino acids, water and electrolytes. Gastroenterology 78:1554-1558; 1980. 27. Meyer, W. C.; Hanks, J. B.; Jones, R. S. Inhibition of basal and meal-stimulated choleresis by somatostatin. Surgery 86:301-306; 1979. 28. Moreau, S.; Murphy, W. A.; Coy, D. H. Comparison ofsomatuline (BIM-23014) and somatostatin on endocrine and exocrine activities in the rat. Drug Dev. Res. 22:79-93; 1991. 29. Murphy, W. A.: Lance, V. A.; Moreau, S.; Moreau, J. P.; Coy, D. H. Inhibition of rat prostate tumor growth by an octapeptide analog of somatostatin. Life Sci. 40:2515-2522; 1987. 30. Powell, D. W. Intestinal conductances and perm-selectivity changes with theophylline and choleragen. Am. J. Physiol. 227:1436-1444; 1974. 31. Pradayrol, L.; Jomvall, J.; Mutt, V.; Ribet, A. N-terminally extended somatostatin the primary structure of somatostatin 28. FEBS Lett. 109:55-58; 1980. 32. Primi, M. P.; Bueno, L. Influence of centrally administered somatostatin and two related peptides on intestinal absorption of water and electrolytes in conscious dogs. Peptides 8:619-623; 1987. 33. Quito, F. L.; Brown, D. R. [D-ala2, metS]-enkephalinamide: CNSmediated inhibition of prostaglandin-stimulated intestinal fluid and ion transport in the rat. Peptides 8:1029-1033; 1987. 34. Raptis, S.; Dollinger, H. C.; Von Berger, L.; Schleger, W.; Schroder, K. E.; Pfeiffer, E. F. Effects of somatostatin on gastric secretion and gastrin release in man. Digestion 13:259-263; 1975. 35. Robert, W. G.; Fedorack, R. N.; Chang, E. B. In vitro effects of the long acting somatostatin analogue SMS 201-995 on electrolyte transport by the rabbit ileum. Gastroenterology 94:1343-1350; 1988. 36. Ruskon& A.; Rent, E.; Chayvialle, J. A. Effect of somatostatin on diarrhea and on small intestinal water and electrolyte transport in a patient with pancreatic cholera. Dig. Dis. Sci. 27:459-466; 1982. 37. Sharp, G. W.; Hynie, S. Stimulation of intestinal mucosal adenyl cyclase by cholera toxin. Nature 229:266-269; 1971. 38. Smith, G.; Warhurst, G.; Martin, L.; Turnbergh, L. Evidence that PGE2 stimulates intestinal epithelial cell adenylate cyclase by a receptor-mediated mechanism. Dig. Dis. Sci. 32:71-75; 1987.