- Email: [email protected]
Inhibition of experimentally-induced gastric ulcers in the rat by a new sulfated glycopeptide
EUROPEAN JOURNAL OF PHARMACOLOGY 15 (1971) 119-126. NORTH-HOLLANDPUBLISHINGCOMPANY
INHIBITION OF EXPERIMENTALLY-INDUCED GASTRIC ULCERS I N T H E R A T BY A N E W S U L F A T E D G L Y C O P E P T I D E G. PRINO, S. PAGLIALUNGA, G. NARDI and A. LIETTI Crinos Biological Research Laboratories, Villa Guardia (Como], Italy
Received 16 November 1970
Accepted 12 March 1971
G. PRINO, S. PAGLIALUNGA,G. NARDI and A. LIETTI, Inhibition o f experimentally-induced gastric ulcers in the rat by a new sulfated glycopeptide, European J. Pharmacol. 15 (1971) 119-126. A sulfated glycopeptide (GLPS) obtained by sulfonation of a glycopeptide derived from pig duodenum was tested as an inhibitor of experimental gastric ulcers in rat. GLPS, given orally, (15-190 mg/kg) protected the rats against gastric ulceration induced by pyloric ligation or by administration of hypertonic glucose to fasting rats; after oral administration (10-25 mg/kg) it was active against hydrocortisone and restraint-induced ulcers. A protective effect of GLPS against Shay ulcers was also demonstrated after intraperitoneal or intravenous administration. A dose-response relationship was always found except for restraint ulcers. In pyloric-figated rats GLPS revealed a strong anti-peptic activity and an inhibitory effect on gastric secretion when given orally. The latter effect was also seen after intravenous administration. On the basis of its anti-peptic and anti-secretory activity, an explanation of the protective effect of GLPS against gastric ulceration is presented. Glycopeptide sulfate
Gastric ulcer protection
1. INTRODUCTION Glycosylglycanic and giycoproteic compounds have been isolated from gastric juice, duodenal juice and gastric mucosa, and identified (De Graef et al., 1968; Dische et al., 1959; H~,kinen, Hartiala and Terho, 1965; Martin et al., 1968a, 1969; Schrager, 1964). Their physiological role and alterations in their metabolism in pathological or experimental conditions have also been investigated (Familiar, 1969; Lambert, Andre" and Martin, 1969; Hfikkinen, 1960, H~ikkinen and Hartiala, 1966; H~3cinen, Johansson and Pantio, 1968; K0walewski and Strutz, 1959; Martin et al., 1968a, 1968b; Menguy and Masters, 1963; Menguy and Desbaillets, 1968). These studies allowed many workers to formulate the hypothesis that an alteration of the 'mucous barrier' is a major factor in the pathogenesis of gastric ulcer. Many data indicate that alterations in the
Peptic inhibition
Gastric secretion
production of certain components of mucus may account for the decrease in its protective capacity. The importance of sulfate groups bound to macromolecular components and modifications in the composition of carbohydrates in the gastric mucus was particularly stressed (Menguy and DesbaiUets, 1968). According to some authors, sulfated polysaccharides and sulfated glycoproteins secreted in gastric juice have a gastro-protective action since their biosynthesis was decreased and their composition altered in animals with gastric ulcers (Hiikkinen, 1960; H~ddnen and Hartiala, 1966; H~ckinen, Johansson and Pantio, 1968; Kowalewski and Strutz, 1959; Lambert, Andre" and Martin, 1969). A peculiar property of these macromolecules is their anti-pepsin effect (Babkin and Komarov, 1932; Levey and Sheinfeld, 1954; Martin et al., 1968b) which was also described for mucus preparations (Babkin and Komarov, 1932; Placer, Roubal and V6k~[6, 1958).
120
G.Prino et al., A sulfated glycopeptide and rat gastric ulcers
Thus gastric mucosal extracts and sulfated polysaccharides of animal or vegetable origin have now become widely used in the therapy of peptic ulcer. During our studies on the chemical composition of swine duodenum a glycoprotein with a molecular weight of 81,000, containing hexoses, hexosamines, sialic acid and a polypeptidic fraction has been isolated (Bertellini et al., 1971a). This substance, calledGLP, has been shown to posses anti-ulcer and anti-inflammatory activity in laboratory animals. These pharmacological properties were potentiated by polysulfation of this glycopeptide (Prino et al., 1970). The glycopeptide sulfate, (GLPS), contained 14% sulfur and retained the same homogeneity and characteristics of the original glycopeptide (Bertellini et al., 1971b). Pharmacological investigations revealed that GLPS had greater anti-ulcer activity than did GLP and manifested anti-peptic property. The anti-peptic activity was particularly evident in gastric juice (Prino et al., 1970). Its clinical efficiency was also proved (Butti, Imbimbo and Brembilla, 1970). The aim of this study was to evaluate the action of glycopeptide sulfate on several types of experimental ulcers in the rat and to examine the protective role of this substance.
2. MATERIALS AND METHODS Sulfated glycopeptide, sodium salt, (GLPS), was prepared in our chemical laboratories according to Bertellini et al. (1971a,b). The material used in this study contained: total sugar 14.6%, sialic acid 2.6%, N-acetyl-hexosamines 24%, total amino acids 9.4%; sulfur 14%. In this study, the GLPS activity was tested on ulcers experimentally produced in the rumen (pyloric ligation and glucose ulcers) or in the ventricle (hydrocortisone and restraint ulcers)of rat stomach. We did not employ a single scoring system to evaluate the degree of the gastric lesions as each type of experimental ulceration revealed peculiar features in its development and aspecL Perforated ulcers were obtained only with the pyloric ligation technique. After pyloric ligation, the ulcers appeared in the rumen as single necrotic lesions of different sizes. The 'glucose ulcers' were localized in the rumen and were gathered in one or more groups. The lesions were round or oval, surrounded by a whitish swollen
border and presented a necrotic-fibrinous degeneration on the bottom of the 'craters'. Hydrocortisone and restraint ulcers were present exclusively in the glandular portion of the rat stomach (ventricle) as linear or oval haemorrhagic superficial lesions. 2.1. eyloric ligation ulcer
A modification of Shay's method (Shay et al., 1945) was used. Male Sprague-Dawley rats, weighing 190-195 g, were fasted for 72 hr before ligation but had free access to water. They were kept in single cages provided with a wire net bottom to avoid coprophagy. After the fasting period, only animals weighing 140-150 g were used. GLPS was administered at 15, 24, 38.4, 51.44, 98.3 or 157.28 mg/kg orally, at 0.75-1.5mg/kg intravenously or at 12.5-25 mg/kg intraperitoneally following pylorus ligation. The animals which were treated intravenously or intraperitoneally received 10 ml/kg of distilled water by gastric tube immediately following pyloric ligation. The rats were killed 8 hr later and their stomachs removed and opened along the greater curvature. For the evaluation of ulcers, the following scores were used: 0.5 for haemorrhages or erosions or for each small ulcer (< 3 mm) 1 for each marked ulcer (> 3 mm) 5 for perforated ulcer (or as maximum score). Animals which died during the experiment but which did not have perforated ulcers were not included in the evaluation. 2.2. Gastric secretion The volume, pH and total acidity of rat gastric juice were determined at 60 min, 90 min and 180 min after pyloric ligation in rats. Total acidity was measured by titrating undiluted gastric juice with 0.01 N NaOH using phenolphthalein as indicator. Peptic activity was determined in undiluted gastric juice of pyloric ligated rats according to the following procedure: 0.1 ml of centrifuged gastric juice (5000g for 10 min) was added to 1 ml of bovine albumin (0.5% w/v in 0.01 N HC1, pH 2) and incubated for 20 min at 37°C. A duplicate background control tube (gastric juice blank) in which the 1 ml albumin was replaced with 1 ml of 0.01 N HC1 was run simultaneously. The hydrolysis was stopped by adding 2 ml of 10% tri-
G.Prino et al., A sulfated glyeopeptide and rat gastric ulcers chloroacetic acid. All tubes were heated in boiling water for 5 min, cooled and faltered, through Whatman no. 42 filter paper. Two ml of fdtrate were added to 0.8ml of 2.5N NaOH and 0.2ml of Folin-Ciocalteu reagent: the volume was brought to 20 ml with distilled water and the absorbancy was measured at 700 nm. 2.3. Glucose ulcer in fasting rats Following Grandjean's (1948) method, male Sprague-Dawley rats weighing 190-200 g were kept in single cages provided with a wire bottom net to avoid coprophagy and fed only with 20% glucose solution ad libitum. GLPS at several doses, was dissolved in 30 ml of glucose solution, which the animals drank in about 18 hr; for the rest of the day the animals received only glucose solution. On the 8th day from the beginning of the experiment the rats were killed with ether and the stomachs removed and opened. The ulcers were manifested as single lesions or as small groups. The adopted evaluation score was the following: 1 when only one group of ulcers was present 2 for two or more groups of ulcers with some isolated ulcers 3 when lesions covered more than 50% of the surface of the rumen 4 as maximum score. 2.4. Hydrocortisone ulcer The technique of Robert and Nezamis (1958) was used. The rats were sacrificed 7 hr after the 4th treatment to avoid an increase in mortality. The animals were treated once a day for four days with hydrocortisone acetate, 10 mg/kg s.c., and with GLPS at various doses, orally (in saline, 5 ml/kg). The animals were fasted during the test but given water ad libitum. The score for ulcer evaluation was the following: 0.5. haemorrhages and/or erosions 1. marked erosions or one small ulcer 1.5. two small ulcers or one marked ulcer 2. three small ulcers or one marked ulcer plus one small ulcer 2.5. four small ulcers or one marked ulcer plus two small ulcers or two marked ulcers. According to this plan a maximum score of 5 was standardized, which corresponds to one or more
121
marked ulcers plus many small lesions ( > 10). 2.5. Restrain t ulcers Male rats of Sprague-Dawley strain weighing 180 g were employed. The ulcers were produced using the technique of Rossi and Bonfils (1956). The animals were restrained for 24 hr. GLPS, was given orally (in saline, 5 ml/kg) once a day for three days before the experiment; the fourth dose was given immediately before immobilization. The degree of ulceration was scored as described for hydrocortisone ulcers. 2.6. Experimental design and statistical evaluation The range of test doses was chosen on the basis of preliminary experiments. Shay ulcer experiments, with GLPS given per os, were planned according to a Graeco-latin square distribution (3 operators, 7 groups, 7 replications). Simpler designs at randomized blocks with 2 operators and 3 or more replications (7 in the case ofhydrocortisone, restraint and glucose ulcers) were used for the other tests. The data were submitted to analysis of variance and the difference versus control groups evaluated with Dunnett's (1955) or Student's t tests.
3. RESULTS 3.1. Pyloric ligation ulcers GLPS, administered per os to pyloric ligated rats, reduced ulcer severity. Even a dose of 15 mg/kg produced 17% inhibition. GLPS activity was proportional to the dose employed and a linear doseresponse relationship was observed (fig. 1). Ulcer 5
4
~3 =2
i
15
38.4
i
98.3 GLPS mg/kg
Fig. 1. Inhibitory effect of the oral administration of GLPS on pyloric iigation ulcer in rats. 21 rats/group.
122
G.Prino et al., A sulfated glycopeptide and rat gastric ulcers
severity was inhibited b y 73% with the highest dose used in our experiments (157 mg/kg). Ulcers i n d u c e d b y pyloric ligation were also inhibited w h e n GPLS was given intraperitoneaUy or intravenously (table 1). A n intraperitoneal injection o f GLPS, 25 mg/kg, resulted in an i n h i b i t i o n of 70.5%. Gastric ulceration was reduced b y 56.6% after the intravenous a d m i n i s t r a t i o n of GLPS, 1.5 mg/kg.
Table 1 Effect of intraperitoneally and intravenously administered GLPS on gastric ulceration in Shay rats. Mean of 20 rats per group.
3.2. Gastric secretion Gastric juice peptic activity was d e t e r m i n e d at the eighth h o u r after pyloric ligation with and w i t h o u t oral t r e a t m e n t with GLPS. Using those doses which were effective in reducing the severity of lesions, a net decrease of peptic activity was observed (table 2). Doses of 98.3 and 157.28 mg/kg inhibited gastric proteases b y 44.2% and 68% respectively. The effect o f oral GLPS on rat gastric acid secretion was observed one hour after pyloric ligation. GLPS, 100 mg/kg, given to t w e n t y rats, reduced gastric juice v o l u m e from 3.77 -+ 0.20 ml (saline group) to 2.79 +-0.23 ml ( m i n u s 26%). Total acid o u t p u t was reduced from 271.7 + 21/,teq I-1" to 151.3 + 22/aeq H ÷ ( m i n u s 44.3%); peptic activity was completely abolished. GLPS, 1.mg/kg, intravenously administered reduced secretory volume b y 43% after 90 m i n o f pyloric ligation. A comparable r e d u c t i o n of total acid o u t p u t (36%) and of pepsin o u t p u t (44%) was present. After 3 hr of ligation this effect o n volume was no longer evident, while the peptic activity
Ulcer score Treatment (mg/kg) Mean _+S.E.
% Inhibition
p vs. controls
Saline GLPS 12.5
i.p. i.p.
4.10 ± 0.26 2.78 ± 0.40
32.2
< 0.01
Saline GLPS 25
i.p. i.p.
3.62 ± 0.37 1.07 ± 0.36
70.5
< 0.001
Saline GLPS 0.75 GLPS 1.5
i.v. i.v. i.v.
3.80 ± 0.48 3.07 ± 0.50 1.65 ± 0.48
19.1 56.6
< 0.05 < 0.01
Table 2 In vivo effect of GLPS on rat gastric peptic activity. Mean of 12 rats/group.
Treatment (mg/kg, p.o.)
Peptic activity /~moles L-tyrosine/hr * Mean ± S.E. '
Distilled water (2 ml/kg)
1.90 ± 0.05
GLPS
1.06 ± 0.11 0.61 ± 0.08
98.3 157.2
% Inhibition
p vs. controls
-
-
44.21 67.89
< 0.01 < 0.01
* Amount released per 1 hr of incubation by 0.2 ml gastric juice.
Table 3 Effect of intravenously administered GLPS on gastric secretion at 90 and 180 min after pyloric ligation in rats. Mean of 12 rats per group. Gastric juice volume (ml)
Total acidity output (~eq H*)
Peptic activity output (/amoles L-tyrosine)
90 min
180 min
90 min
90 min
180 min
Saline (2 ml/kg)
1.95 ± 0.21
3.23 -+ 0.35
185.04 ± 24.07 385.59 -+56.38
13.78 ± 1.92
29.39 ± 4.11
GLPS
1.93 ± 0.25 (1)
3.10 ± 0.41 (4)
190.21 ± 33.43 382.93 ± 56.17 (+2.8) (0.7)
13.42 ± 1.84 (2.6)
25.04 ± 4.78 ( 14.8)
1.11 ± 0.25 * (43)
2.62 ± 0.34 (18.9)
117.19 ± 29.84 334.02 ± 55.2 (36.7) (13.4)
7.67 ± 2.01 * (44.3)
19.71 ± 3.65 (32.9)
Treatment (mg/kg)
0.5
* p < 0.05 (Student t). In brackets: percentage inhibition.
180 min
G.Prino et al., A sulfated glycopeptide and rat gastric ulcers Table 4 Effect of GLPS on glucose ulcer in fasting rats. GLPS was dissolved in 20% glucose solution. Mean of 28 rats per group. Treatment (mg/kg/day)
Ulcer score Mean ± S.E.
Controls
2.69 ± 0.20
GLPS
84.37 126.56 189.84
2.04 ± 0.22 1.61 ± 0.21 1.43 ± 0.18
% Inhibition 24.16 40.15 46.84
p vs. controls < 0.05 < 0.01 < 0.01
output was still 33% (not statistically significant) lower than controls (table 3). However, after intravenous GLPS, a consistent specific inhibition of pepsin secretion could not be ascertained. In fact, in experiments where the pyloric ligation was prolonged to 6 hr we found no differences in volume, total acidity output and peptic output between control and GLPS (0.5-1 mg/kg) treated rats. 3.3. Glucose ulcers Stomach lesions produced in rats by drinking a 20% glucose solution ad libitum were reduced by oral administration of GLPS dissolved in 20% glucose solution (table 4). Under these experimental conditions, a decrease of ulcer severity was observed at a dose of 84 mg/kg; a 40% ulcer score inhibition was obtained with 126 mg/kg. 3.4. Hydrocortisone and restraint ulcers GLPS was also tested for its protective action against ulcerative lesions induced in the glandular portion of the stomach by the subcutaneous administration of hydrocortisone and by the restraint teeh-
Table 5 Effect of orally administered GLPS on hydrocortisone ulcer in rats. Mean of 28 rats per group. Treatment (mg]kg/day)
Ulcer score Mean ± S.E.
Saline (5 ml/kg)
2.35 ± 0.32
-
-
GLPS
1.44 ± 0.30 0.96 + 0.27 1.05 ± 0.28
38.6 59.3 55.2
< 0.05 < 0.01 < 0.01
11.11 16.67 25.0
% Inh~ition
p vs. controls
123
nique. In hydrocortisone ulcers, GLPS administered once a day for 4 days was active even at low doses; 11 mg/kg gave a 38% inhibition, higher doses were more effective (table 5). In restraint-induced ulcer, the oral administration of GLPS 25 mg/kg/day protected the animals from ulceration by 60% (the ulcer score was reduced from 2.08 -+0.39 to 0.83 -+ 0.28). In this test we did not find a dose-response relationship; doses lower than 25mg/kg/day, were ineffective while higher doses did not increase inhibition.
4. DISCUSSION A non-sulfated glycopeptide (GLP), isolated from hog duodenum, revealed interesting anti-inflammatory and anti-ulcer activities in laboratory animals (Bertellini et al. 1971a). The observation that this non-sulfated macromolecule had anti-ulcer activity prompted us to prepare its polysulfated derivative in order to obtain a substance more active on the gastrointestinal tract (Bertellini et al., 1971b; Prino et al., 1970). From various published works, it appears that sulfate groups are peculiar to polysaccharides and mucoproteins with anti-ulcer and anti-peptic properties (Anderson, 1961; Cook, Eich and Cammarata, 1963; Komarov, 1936; Levey and Sheinfeld, 1954; Placer, Roubal and V6k~, 1958). The polysulfonated derivative of GLP (GLPS) showed, in contrast to GLP, an evident in rive and in vitro antipeptic activity, in laboratory animals and in humans. By using a synthetic dipeptide (N-acetyt-L-phenylalanyl-L-diiodo tyrosine) as a peptic substrate it was found that GLPS inactivated gastric proteases directly (by precipitating them) (Prino et al., 1970). The results presented in this paper, confirm the anti-peptic properties of GLPS, and show that it inhibits some ulcers induced experimentally in rats. The activity of GLPS was studied on ulcers produced in rat fore-stomach (pyloric ligation and feeding hypertonic glucose solution to fasting rats) and on ulcers of the stomach glandular portion (hydrocortisone and restraint ulcer). In pyloric ligation ulcers, the volume and the peptic activity of the gastric juice secreted are considered the main ulcerogenic factors (Sun, and Chen, 1963; Vocac, Alphin and Bolton, 1969). In this experimental ulceration, 15-20 mg/kg of GLPS, adminis-
124
G.Prino et al., A sulfated glycopep tide and rat gastric ulcers
tered orally, gave a statistically significant protection against the formation of lesions. A linear doseresponse relationship was observed with the regression y = 7.337-2.81 log x. GLPS was also active after intravenous and intraperitoneal administration against ulcers formed by pyloric ligation. The direct anti-peptic activity manifested by GLPS after oral administration provides a good explanation of its protective role against Shay ulcers. This main activity was accompanied by a short-lived effect (within the first hour of pyloric ligation) on gastric secretion. This effect could be explained if one assumes gastrointestinal absorption of this sulfated glycopeptide. In fact, preliminary experiments made in our laboratories showed that a measurable amount of metachromatic material (about 2% of the GLPS in the incubation medium) was found on the serosal side of the everted stomach preparation. Besides, a metachromatic reaction was given by rat urines after oral administration of GLPS at the dose of 50 mg/rat. In addition Eagleton, Watt and Marcus (1969) demonstrated the presence of metachromatic material in rat urines after oral administration of another sulfated macromolecule: degraded carrageenin. After intravenous treatment, GLPS, 1 mg/kg, at 90 min of pyloric ligation, inhibited gastric secretory volume and gave the same reduction of total acid and pepsin output. In contrast to the results obtained after oral administration, specific inhibition of gastric juice peptic activity could not be obtained. Therefore, while the GLPS activity against pyloric ligation ulcers, after oral administration, seems linked to its presence in the gastric contents, the latter assumption cannot be substantiated after intravenous treatment. In this case the protection exerted against pyloric ligation lesions could be partially explained by the anti-secretory activity of GLPS. Inhibition of gastric secretion by GLPS might be explained by considering its possible interaction with endogenous histamine or its activation of diamine oxidase, as has been shown for other sulfated macromolecules (Kobayashi, Kupelian and Maudsley, 1969; Sanyal and West, 1959). Further, screening tests showed that GLPS (5-10mg/kg) did not modify either cholinergic (evoked by acetylcholine or vagal stimulation) or adrenergic blood pressure response (evoked by noradrenaline, carotid occlusion, or tyramine); in vitro, GLPS (2.5 X 10 -4 g/ml) also proved
to be ineffective on acetylcholine- and histamineinduced contractions of the guinea pig ileum and on serotonin-induced contractions of the rat fundus. It is known that hypertonic solutions reduce gastric motility (Vantrappen et al., 1963) and remove parietal mucus (washing effect, Kowalewski and Schier, 1970). In this condition the unprotected gastric wall could be attacked by pepsin and this may explain the ulceration due to the administration of hypertonic glucose solution. However, other elements are present in the pathogenesis of this experimental ulcer, such as the state of fasting or the lack of many nutritional factors. It is likely that GLPS, mixed with glucose, inhibits this experimental ulcer through its anti-peptic and gastro-protective action. It is more difficult to explain how GLPS acts on ulcerations induced in the glandular portion of rat stomach, although it cannot be excluded that GLPS's protective effect occurs through its anti-peptic and anti-secretory properties. Increased gastric acid secretion could not be demonstrated in rats after steroid administration at ulcerogenic doses (Myhre, 1963) and it is still doubtful if hypersecretion occurs in restrained rats (Bonfils, Ferrier and Caulin, 1966). Nevertheless, a decrease of mucins and a reduced biosynthesis of sulfated mucopolysaccharides and mucoproteins was pointed out by a number of workers (Kowalewski and Strutz, 1959; Lambert, Andr6 and Martin, 1969; Menguy and Masters, 1963)which may be expected to result in an altered structure of the gastric mucosa. The gastric mucosa might then be subjected to the damaging action of acid pepsin even if gastric secretion is normal. GLPS could act by inhibiting peptic activity, but its protective role against these experimental ulcers is still open to further investigations.
ACKNOWLEDGEMENT We gratefully acknowledgethe technical assistance of Miss C. Giarmuzzi.
REFERENCES Anderson, W., 1961, The anti-peptic activity of sulfated polysaccharides, J. Pharm. Pharmacol. 13, 139.
G.Prino et al., A sulfated glycopeptide and rat gastric ulcers Babkin, P.B. and S.A. Komarov, 1932, The influence of gastric mucus on peptic digestion, Can. Med. Assoc. J. 27, 463. Bertellini, G., A. Butti, B. Piantanida, G. Prino, A. Riva, A. Rossi and S. Rossi, 1971a, Studies on glycoproteins of animal origin and their derivatives. I. Isolation of a sialoglycopeptide from hog duodenum; preliminary chemical and pharmacological studies, Arzneimittel-Forsch. 21, 244. _ Bertellini, G., A. Butti, B. Piantanida, G. Prino, A. Riva, A. Rossi and S. Rossi, 1971b, Studies on glycoproteins of animal origin and their derivatives. II. Synthesis of the polysulfuric ester of a natural sialoglycopeptide, Arzneimittel-Forsch. 21,247. Bonfils, S., J.P. Ferrier and Ch. Caulin, 1966, L'ulc~re de contrainte du rat blanc. M~thode de pathologie exp6rimentale et test pharmacologique, Rev. Franc. Etudes Clin. Biol. 11,343. Brodie, D.A., R.W. Marshall and O.M. Moreno, 1962, Effect of restraint on gastric acidity in the rat, Am. J. Physiol. 202, 812. Brodie, D.A., 1966, The mechanism of gastric hyperacidity produced by pylorus ligation in the rat, Am. J. Digest, Diseases New Series 2,231. Butti, A., B. Imbimbo and E. Brembilla, 1970, Clinical evaluation of an anti-ulcer sulfated glycopeptide (GLPS). A report on controlled multicenter trials, 4th World Congress of Gastroenterology, Copenhagen, July 1970. Advance Abstracts, eds. P. Riis, P. Anthonisen and H. Baden p. 308. Cook, D.L., S. Elch and P.S. Cammarata, 1963, Comparative pharmacology and chemistry of synthetic sulfated polysaccharides, Arch. Intern. Pharmacodyn. 144, 1. De Graef, J., A. Gerard, R. Lev and G.B.J. Glass, 1968, Secretion of acid and neutral mucosubstances in the dog stomach, in: The Physiology of Gastric Secretion, eds. L.S. Semb and J. Myren (Universitetsforlaget, Oslo) p. 124. Dische, Z., P. di Sant'Agnese, C. Pallavicini and J. Youlos, 1959, Composition of mucoid fraction from duodenal fluid of children and of adults, Arch. Biochem. 84, 205. Dunnett, C.W., 1955, A multiple comparison procedure for comparing several treatments with a control, Am. Statist. Assoc. J., 50, 1096. Eagleton, G.B., J. Watt and R. Marcus, 1969, Effect of subcutaneously administered degraded carrageenan on the production of histamine-induced gastric and duodenal ulceration, J. Pharm. Pharmacol. 21,123. Familiar, R.G., 1969, Sulphate-containing gastric mucus components and peptic ulcer: a possible relationship, J. Am. Geriat. Soc. 17,680. Grandjean, E., 1948, L'action d'un extrait stomaco-intestinal sur l'ulc~re exl~rimental du rat, Rev. Med. Suisse Rom. 68, 442. Ha'kkinen, I.P.T., 1960, Histamine induced changes in the sulphate metabolism of the duodenum and prepyloric region of the stomach: an experimental study with special reference to ulcer formation, Aeta Physiol. Scand. 51, Suppl. 177, 1.
125
H~ikkinen, I., K. Hartiala and T. Tetho, 1965, The fractionation and characterization of the acid polysaccharides in human gastric juice, Acta Chem. Scand. 19,797. H~kkinen, I. and K. Hartiala, 1966, The effect of cinchophen on the acid polysaccharides of the gastric and duodenal wall in dog, Acta Physiol. Scand. 66,326. H~ikkinen, I., R. Johansson and M. Pantio, 1968, An immunological and histoimmunological study of gastric sulphoglycoproteins in healthy and aspirin-treated dogs, Gut 9", 712. Kobayashi, Y., J. Kupelian and D.V. Maudsley, 1969, Release of diamine oxidase by heparin in the rat, Biochem. Pharmacol. 18, 1585. Komarov, S.A., 1936, The influence of mucoitin sulfuric acid on peptic digestion, Am. J. Digest. Diseases 3, 164. Kowalewski, K. and W.A. Strutz, 1959, Uptake of radiosulfate by the gastric tissue and gastric secretion in cortisone treated Shay rats, Acta Endocrinol. 31,107. Kowalewski, K. and J.F. Schier, 1970, Experimental peptic ulcer in dogs following the induced deficiency of gastric 'mucous barrier ~.Biochemical and histopathological study. 4th World Congress of Gastroenterology, Copenhagen, July 1970. Advance Abstracts, eds. P. Riis, P. Anthonisen and H. Baden, p. 378. Lambert, R., C. Andrd and F. Martin, 1969, Incorporation of radiosulfate in the gastric mucosa of the rat subjected to restraint, Gastroenterology 56,200. Levey, S. and. S. Sheinfeld, 1954, The inhibition of the proteolytic action of pepsin by sulfate-containing polysaccharides, Gastroenterology 27, 625. Martin, F., R. Lambert, M. Martin, J.-L. Vuez et A. B6rard, 1968a, Les glycoprot~ines sulfat6es de la s6cr~tion gastrique humaine, Biol. Gastroenterol. Suppl. 1 Arch. Franc. Maladies Appl. Digestif 57, 5. Martin, F., J.-L. Vuez, A B~rard, C. Andr~ et R. Lambert, 1968b, Inhibition de la prot6olyse peptique par une glycopr6t~ine sulfat~e gastrique humaine, Compt. Rend. Soc. Biol. 162, 1368. Martin, F., R. Mathian, A. B~rard and R. Lambert, 1969, Sulfated glycoproteins in human salivary and gastric secretions, Digestion 2, 103. Menguy, R. and J.-F. Masters, 1963, Effect of cortisone on mucoprotein secretion by gastric antrum of dogs: pathogenesis of steriod ulcer, Surgery 54, 19. Menguy, R. and L. Desbaillets, 1968, The gastric mucous barrier: Influence of protein-bound carbohydrate in mucus on the rate of proteolysis of gastric mucus, Ann. Surg. 168, 475. Myhre, E., 1963, The effect of Corticotrophin and Cortisone on the healing of gastric ulcer, in: Pathophysiology of Peptic Ulcer, ed.. S.C. Skoryna (McGill University Press, Canada) pp. 233-243. Placer, Z., Z. Roubal and V. V6k~E, 1958, The action of gastric mucus and some sulfate compounds on the development of experimental gastric ulcers in rats, Rev. Czech. Med. 4, 111. Prino, G., S. Paglialunga, A. Lietti, G. Nardi and R. Niada, 1970, Anti-ulcer and anti-peptic activity of a new glyco-
126
G.Prino et al., A sulfated glycopeptide and rat gastric ulcers
peptide sulfate (GLPS). Chemical and Pharmacological Study. 4th World Congress of Gastroenterology, Copenhagen, July 1970. Advance Abstracts, eds. P. Riis, P. Anthonisen and H. Baden, p. 307. Robert, A., J.E. Nezamis, 1958, Ulcerogenic property of steroids, Proc. Soc. Exptl. Biol. Med. 99,443. Rossi, G., S. Bonfils, F. Lieffogh, A. Lambling, 1956, Technique nouvelle pour produire des ulcerations gastriques chez le rat blanc: l'ulc~re de contrainte, Compt. Rend. Soc. Biol. 150, 1224. Sanyal, R.K. and G.B. West, 1959, The histamine-heparin complex, J. Pharm. Pharmacol. 11,548. Schrager, J., 1964, Mucopolysaccharides of the gastric secretion, Nature 201, 1220. Shay, H., S.A. Komarov, S.S. Fels, D. Meranze, M. Gruen-
stein and H.A. Siplet, 1945, A simple method for the uniform production of gastric ulceration in the rat, Gastroenterology 5, 43. Sun, D.C.H., and J.K. Chen, 1963, Experimental ulcer production in the pylorus ligated rat, in: Pathophysiology of Peptic Ulcer, ed. S.C. Skoryna (McGill University Press, Canada) pp. 141-152. Vantrappen, G., S. Vandenbroucke, Verbeke and T. Hellemans, 1963, Gastric motility, in: Pathophysiology of Peptic Ulcer, ed. S.C. Skoryna (McGiU University Press, Canada) pp. 115-131. Vocac, J.A., R.S. Alphin and P.J. Bolton, 1969, Role of HC1, pepsin and intragastric pressure in gastric ulceration of the perfused rat stomach in situ. Annual Meeting American Gastroenterological Association, Washington, May 1969. Abstract. Gastroenterology 56, 1266.
INHIBITION OF EXPERIMENTALLY-INDUCED GASTRIC ULCERS I N T H E R A T BY A N E W S U L F A T E D G L Y C O P E P T I D E G. PRINO, S. PAGLIALUNGA, G. NARDI and A. LIETTI Crinos Biological Research Laboratories, Villa Guardia (Como], Italy
Received 16 November 1970
Accepted 12 March 1971
G. PRINO, S. PAGLIALUNGA,G. NARDI and A. LIETTI, Inhibition o f experimentally-induced gastric ulcers in the rat by a new sulfated glycopeptide, European J. Pharmacol. 15 (1971) 119-126. A sulfated glycopeptide (GLPS) obtained by sulfonation of a glycopeptide derived from pig duodenum was tested as an inhibitor of experimental gastric ulcers in rat. GLPS, given orally, (15-190 mg/kg) protected the rats against gastric ulceration induced by pyloric ligation or by administration of hypertonic glucose to fasting rats; after oral administration (10-25 mg/kg) it was active against hydrocortisone and restraint-induced ulcers. A protective effect of GLPS against Shay ulcers was also demonstrated after intraperitoneal or intravenous administration. A dose-response relationship was always found except for restraint ulcers. In pyloric-figated rats GLPS revealed a strong anti-peptic activity and an inhibitory effect on gastric secretion when given orally. The latter effect was also seen after intravenous administration. On the basis of its anti-peptic and anti-secretory activity, an explanation of the protective effect of GLPS against gastric ulceration is presented. Glycopeptide sulfate
Gastric ulcer protection
1. INTRODUCTION Glycosylglycanic and giycoproteic compounds have been isolated from gastric juice, duodenal juice and gastric mucosa, and identified (De Graef et al., 1968; Dische et al., 1959; H~,kinen, Hartiala and Terho, 1965; Martin et al., 1968a, 1969; Schrager, 1964). Their physiological role and alterations in their metabolism in pathological or experimental conditions have also been investigated (Familiar, 1969; Lambert, Andre" and Martin, 1969; Hfikkinen, 1960, H~ikkinen and Hartiala, 1966; H~3cinen, Johansson and Pantio, 1968; K0walewski and Strutz, 1959; Martin et al., 1968a, 1968b; Menguy and Masters, 1963; Menguy and Desbaillets, 1968). These studies allowed many workers to formulate the hypothesis that an alteration of the 'mucous barrier' is a major factor in the pathogenesis of gastric ulcer. Many data indicate that alterations in the
Peptic inhibition
Gastric secretion
production of certain components of mucus may account for the decrease in its protective capacity. The importance of sulfate groups bound to macromolecular components and modifications in the composition of carbohydrates in the gastric mucus was particularly stressed (Menguy and DesbaiUets, 1968). According to some authors, sulfated polysaccharides and sulfated glycoproteins secreted in gastric juice have a gastro-protective action since their biosynthesis was decreased and their composition altered in animals with gastric ulcers (Hiikkinen, 1960; H~ddnen and Hartiala, 1966; H~ckinen, Johansson and Pantio, 1968; Kowalewski and Strutz, 1959; Lambert, Andre" and Martin, 1969). A peculiar property of these macromolecules is their anti-pepsin effect (Babkin and Komarov, 1932; Levey and Sheinfeld, 1954; Martin et al., 1968b) which was also described for mucus preparations (Babkin and Komarov, 1932; Placer, Roubal and V6k~[6, 1958).
120
G.Prino et al., A sulfated glycopeptide and rat gastric ulcers
Thus gastric mucosal extracts and sulfated polysaccharides of animal or vegetable origin have now become widely used in the therapy of peptic ulcer. During our studies on the chemical composition of swine duodenum a glycoprotein with a molecular weight of 81,000, containing hexoses, hexosamines, sialic acid and a polypeptidic fraction has been isolated (Bertellini et al., 1971a). This substance, calledGLP, has been shown to posses anti-ulcer and anti-inflammatory activity in laboratory animals. These pharmacological properties were potentiated by polysulfation of this glycopeptide (Prino et al., 1970). The glycopeptide sulfate, (GLPS), contained 14% sulfur and retained the same homogeneity and characteristics of the original glycopeptide (Bertellini et al., 1971b). Pharmacological investigations revealed that GLPS had greater anti-ulcer activity than did GLP and manifested anti-peptic property. The anti-peptic activity was particularly evident in gastric juice (Prino et al., 1970). Its clinical efficiency was also proved (Butti, Imbimbo and Brembilla, 1970). The aim of this study was to evaluate the action of glycopeptide sulfate on several types of experimental ulcers in the rat and to examine the protective role of this substance.
2. MATERIALS AND METHODS Sulfated glycopeptide, sodium salt, (GLPS), was prepared in our chemical laboratories according to Bertellini et al. (1971a,b). The material used in this study contained: total sugar 14.6%, sialic acid 2.6%, N-acetyl-hexosamines 24%, total amino acids 9.4%; sulfur 14%. In this study, the GLPS activity was tested on ulcers experimentally produced in the rumen (pyloric ligation and glucose ulcers) or in the ventricle (hydrocortisone and restraint ulcers)of rat stomach. We did not employ a single scoring system to evaluate the degree of the gastric lesions as each type of experimental ulceration revealed peculiar features in its development and aspecL Perforated ulcers were obtained only with the pyloric ligation technique. After pyloric ligation, the ulcers appeared in the rumen as single necrotic lesions of different sizes. The 'glucose ulcers' were localized in the rumen and were gathered in one or more groups. The lesions were round or oval, surrounded by a whitish swollen
border and presented a necrotic-fibrinous degeneration on the bottom of the 'craters'. Hydrocortisone and restraint ulcers were present exclusively in the glandular portion of the rat stomach (ventricle) as linear or oval haemorrhagic superficial lesions. 2.1. eyloric ligation ulcer
A modification of Shay's method (Shay et al., 1945) was used. Male Sprague-Dawley rats, weighing 190-195 g, were fasted for 72 hr before ligation but had free access to water. They were kept in single cages provided with a wire net bottom to avoid coprophagy. After the fasting period, only animals weighing 140-150 g were used. GLPS was administered at 15, 24, 38.4, 51.44, 98.3 or 157.28 mg/kg orally, at 0.75-1.5mg/kg intravenously or at 12.5-25 mg/kg intraperitoneally following pylorus ligation. The animals which were treated intravenously or intraperitoneally received 10 ml/kg of distilled water by gastric tube immediately following pyloric ligation. The rats were killed 8 hr later and their stomachs removed and opened along the greater curvature. For the evaluation of ulcers, the following scores were used: 0.5 for haemorrhages or erosions or for each small ulcer (< 3 mm) 1 for each marked ulcer (> 3 mm) 5 for perforated ulcer (or as maximum score). Animals which died during the experiment but which did not have perforated ulcers were not included in the evaluation. 2.2. Gastric secretion The volume, pH and total acidity of rat gastric juice were determined at 60 min, 90 min and 180 min after pyloric ligation in rats. Total acidity was measured by titrating undiluted gastric juice with 0.01 N NaOH using phenolphthalein as indicator. Peptic activity was determined in undiluted gastric juice of pyloric ligated rats according to the following procedure: 0.1 ml of centrifuged gastric juice (5000g for 10 min) was added to 1 ml of bovine albumin (0.5% w/v in 0.01 N HC1, pH 2) and incubated for 20 min at 37°C. A duplicate background control tube (gastric juice blank) in which the 1 ml albumin was replaced with 1 ml of 0.01 N HC1 was run simultaneously. The hydrolysis was stopped by adding 2 ml of 10% tri-
G.Prino et al., A sulfated glyeopeptide and rat gastric ulcers chloroacetic acid. All tubes were heated in boiling water for 5 min, cooled and faltered, through Whatman no. 42 filter paper. Two ml of fdtrate were added to 0.8ml of 2.5N NaOH and 0.2ml of Folin-Ciocalteu reagent: the volume was brought to 20 ml with distilled water and the absorbancy was measured at 700 nm. 2.3. Glucose ulcer in fasting rats Following Grandjean's (1948) method, male Sprague-Dawley rats weighing 190-200 g were kept in single cages provided with a wire bottom net to avoid coprophagy and fed only with 20% glucose solution ad libitum. GLPS at several doses, was dissolved in 30 ml of glucose solution, which the animals drank in about 18 hr; for the rest of the day the animals received only glucose solution. On the 8th day from the beginning of the experiment the rats were killed with ether and the stomachs removed and opened. The ulcers were manifested as single lesions or as small groups. The adopted evaluation score was the following: 1 when only one group of ulcers was present 2 for two or more groups of ulcers with some isolated ulcers 3 when lesions covered more than 50% of the surface of the rumen 4 as maximum score. 2.4. Hydrocortisone ulcer The technique of Robert and Nezamis (1958) was used. The rats were sacrificed 7 hr after the 4th treatment to avoid an increase in mortality. The animals were treated once a day for four days with hydrocortisone acetate, 10 mg/kg s.c., and with GLPS at various doses, orally (in saline, 5 ml/kg). The animals were fasted during the test but given water ad libitum. The score for ulcer evaluation was the following: 0.5. haemorrhages and/or erosions 1. marked erosions or one small ulcer 1.5. two small ulcers or one marked ulcer 2. three small ulcers or one marked ulcer plus one small ulcer 2.5. four small ulcers or one marked ulcer plus two small ulcers or two marked ulcers. According to this plan a maximum score of 5 was standardized, which corresponds to one or more
121
marked ulcers plus many small lesions ( > 10). 2.5. Restrain t ulcers Male rats of Sprague-Dawley strain weighing 180 g were employed. The ulcers were produced using the technique of Rossi and Bonfils (1956). The animals were restrained for 24 hr. GLPS, was given orally (in saline, 5 ml/kg) once a day for three days before the experiment; the fourth dose was given immediately before immobilization. The degree of ulceration was scored as described for hydrocortisone ulcers. 2.6. Experimental design and statistical evaluation The range of test doses was chosen on the basis of preliminary experiments. Shay ulcer experiments, with GLPS given per os, were planned according to a Graeco-latin square distribution (3 operators, 7 groups, 7 replications). Simpler designs at randomized blocks with 2 operators and 3 or more replications (7 in the case ofhydrocortisone, restraint and glucose ulcers) were used for the other tests. The data were submitted to analysis of variance and the difference versus control groups evaluated with Dunnett's (1955) or Student's t tests.
3. RESULTS 3.1. Pyloric ligation ulcers GLPS, administered per os to pyloric ligated rats, reduced ulcer severity. Even a dose of 15 mg/kg produced 17% inhibition. GLPS activity was proportional to the dose employed and a linear doseresponse relationship was observed (fig. 1). Ulcer 5
4
~3 =2
i
15
38.4
i
98.3 GLPS mg/kg
Fig. 1. Inhibitory effect of the oral administration of GLPS on pyloric iigation ulcer in rats. 21 rats/group.
122
G.Prino et al., A sulfated glycopeptide and rat gastric ulcers
severity was inhibited b y 73% with the highest dose used in our experiments (157 mg/kg). Ulcers i n d u c e d b y pyloric ligation were also inhibited w h e n GPLS was given intraperitoneaUy or intravenously (table 1). A n intraperitoneal injection o f GLPS, 25 mg/kg, resulted in an i n h i b i t i o n of 70.5%. Gastric ulceration was reduced b y 56.6% after the intravenous a d m i n i s t r a t i o n of GLPS, 1.5 mg/kg.
Table 1 Effect of intraperitoneally and intravenously administered GLPS on gastric ulceration in Shay rats. Mean of 20 rats per group.
3.2. Gastric secretion Gastric juice peptic activity was d e t e r m i n e d at the eighth h o u r after pyloric ligation with and w i t h o u t oral t r e a t m e n t with GLPS. Using those doses which were effective in reducing the severity of lesions, a net decrease of peptic activity was observed (table 2). Doses of 98.3 and 157.28 mg/kg inhibited gastric proteases b y 44.2% and 68% respectively. The effect o f oral GLPS on rat gastric acid secretion was observed one hour after pyloric ligation. GLPS, 100 mg/kg, given to t w e n t y rats, reduced gastric juice v o l u m e from 3.77 -+ 0.20 ml (saline group) to 2.79 +-0.23 ml ( m i n u s 26%). Total acid o u t p u t was reduced from 271.7 + 21/,teq I-1" to 151.3 + 22/aeq H ÷ ( m i n u s 44.3%); peptic activity was completely abolished. GLPS, 1.mg/kg, intravenously administered reduced secretory volume b y 43% after 90 m i n o f pyloric ligation. A comparable r e d u c t i o n of total acid o u t p u t (36%) and of pepsin o u t p u t (44%) was present. After 3 hr of ligation this effect o n volume was no longer evident, while the peptic activity
Ulcer score Treatment (mg/kg) Mean _+S.E.
% Inhibition
p vs. controls
Saline GLPS 12.5
i.p. i.p.
4.10 ± 0.26 2.78 ± 0.40
32.2
< 0.01
Saline GLPS 25
i.p. i.p.
3.62 ± 0.37 1.07 ± 0.36
70.5
< 0.001
Saline GLPS 0.75 GLPS 1.5
i.v. i.v. i.v.
3.80 ± 0.48 3.07 ± 0.50 1.65 ± 0.48
19.1 56.6
< 0.05 < 0.01
Table 2 In vivo effect of GLPS on rat gastric peptic activity. Mean of 12 rats/group.
Treatment (mg/kg, p.o.)
Peptic activity /~moles L-tyrosine/hr * Mean ± S.E. '
Distilled water (2 ml/kg)
1.90 ± 0.05
GLPS
1.06 ± 0.11 0.61 ± 0.08
98.3 157.2
% Inhibition
p vs. controls
-
-
44.21 67.89
< 0.01 < 0.01
* Amount released per 1 hr of incubation by 0.2 ml gastric juice.
Table 3 Effect of intravenously administered GLPS on gastric secretion at 90 and 180 min after pyloric ligation in rats. Mean of 12 rats per group. Gastric juice volume (ml)
Total acidity output (~eq H*)
Peptic activity output (/amoles L-tyrosine)
90 min
180 min
90 min
90 min
180 min
Saline (2 ml/kg)
1.95 ± 0.21
3.23 -+ 0.35
185.04 ± 24.07 385.59 -+56.38
13.78 ± 1.92
29.39 ± 4.11
GLPS
1.93 ± 0.25 (1)
3.10 ± 0.41 (4)
190.21 ± 33.43 382.93 ± 56.17 (+2.8) (0.7)
13.42 ± 1.84 (2.6)
25.04 ± 4.78 ( 14.8)
1.11 ± 0.25 * (43)
2.62 ± 0.34 (18.9)
117.19 ± 29.84 334.02 ± 55.2 (36.7) (13.4)
7.67 ± 2.01 * (44.3)
19.71 ± 3.65 (32.9)
Treatment (mg/kg)
0.5
* p < 0.05 (Student t). In brackets: percentage inhibition.
180 min
G.Prino et al., A sulfated glycopeptide and rat gastric ulcers Table 4 Effect of GLPS on glucose ulcer in fasting rats. GLPS was dissolved in 20% glucose solution. Mean of 28 rats per group. Treatment (mg/kg/day)
Ulcer score Mean ± S.E.
Controls
2.69 ± 0.20
GLPS
84.37 126.56 189.84
2.04 ± 0.22 1.61 ± 0.21 1.43 ± 0.18
% Inhibition 24.16 40.15 46.84
p vs. controls < 0.05 < 0.01 < 0.01
output was still 33% (not statistically significant) lower than controls (table 3). However, after intravenous GLPS, a consistent specific inhibition of pepsin secretion could not be ascertained. In fact, in experiments where the pyloric ligation was prolonged to 6 hr we found no differences in volume, total acidity output and peptic output between control and GLPS (0.5-1 mg/kg) treated rats. 3.3. Glucose ulcers Stomach lesions produced in rats by drinking a 20% glucose solution ad libitum were reduced by oral administration of GLPS dissolved in 20% glucose solution (table 4). Under these experimental conditions, a decrease of ulcer severity was observed at a dose of 84 mg/kg; a 40% ulcer score inhibition was obtained with 126 mg/kg. 3.4. Hydrocortisone and restraint ulcers GLPS was also tested for its protective action against ulcerative lesions induced in the glandular portion of the stomach by the subcutaneous administration of hydrocortisone and by the restraint teeh-
Table 5 Effect of orally administered GLPS on hydrocortisone ulcer in rats. Mean of 28 rats per group. Treatment (mg]kg/day)
Ulcer score Mean ± S.E.
Saline (5 ml/kg)
2.35 ± 0.32
-
-
GLPS
1.44 ± 0.30 0.96 + 0.27 1.05 ± 0.28
38.6 59.3 55.2
< 0.05 < 0.01 < 0.01
11.11 16.67 25.0
% Inh~ition
p vs. controls
123
nique. In hydrocortisone ulcers, GLPS administered once a day for 4 days was active even at low doses; 11 mg/kg gave a 38% inhibition, higher doses were more effective (table 5). In restraint-induced ulcer, the oral administration of GLPS 25 mg/kg/day protected the animals from ulceration by 60% (the ulcer score was reduced from 2.08 -+0.39 to 0.83 -+ 0.28). In this test we did not find a dose-response relationship; doses lower than 25mg/kg/day, were ineffective while higher doses did not increase inhibition.
4. DISCUSSION A non-sulfated glycopeptide (GLP), isolated from hog duodenum, revealed interesting anti-inflammatory and anti-ulcer activities in laboratory animals (Bertellini et al. 1971a). The observation that this non-sulfated macromolecule had anti-ulcer activity prompted us to prepare its polysulfated derivative in order to obtain a substance more active on the gastrointestinal tract (Bertellini et al., 1971b; Prino et al., 1970). From various published works, it appears that sulfate groups are peculiar to polysaccharides and mucoproteins with anti-ulcer and anti-peptic properties (Anderson, 1961; Cook, Eich and Cammarata, 1963; Komarov, 1936; Levey and Sheinfeld, 1954; Placer, Roubal and V6k~, 1958). The polysulfonated derivative of GLP (GLPS) showed, in contrast to GLP, an evident in rive and in vitro antipeptic activity, in laboratory animals and in humans. By using a synthetic dipeptide (N-acetyt-L-phenylalanyl-L-diiodo tyrosine) as a peptic substrate it was found that GLPS inactivated gastric proteases directly (by precipitating them) (Prino et al., 1970). The results presented in this paper, confirm the anti-peptic properties of GLPS, and show that it inhibits some ulcers induced experimentally in rats. The activity of GLPS was studied on ulcers produced in rat fore-stomach (pyloric ligation and feeding hypertonic glucose solution to fasting rats) and on ulcers of the stomach glandular portion (hydrocortisone and restraint ulcer). In pyloric ligation ulcers, the volume and the peptic activity of the gastric juice secreted are considered the main ulcerogenic factors (Sun, and Chen, 1963; Vocac, Alphin and Bolton, 1969). In this experimental ulceration, 15-20 mg/kg of GLPS, adminis-
124
G.Prino et al., A sulfated glycopep tide and rat gastric ulcers
tered orally, gave a statistically significant protection against the formation of lesions. A linear doseresponse relationship was observed with the regression y = 7.337-2.81 log x. GLPS was also active after intravenous and intraperitoneal administration against ulcers formed by pyloric ligation. The direct anti-peptic activity manifested by GLPS after oral administration provides a good explanation of its protective role against Shay ulcers. This main activity was accompanied by a short-lived effect (within the first hour of pyloric ligation) on gastric secretion. This effect could be explained if one assumes gastrointestinal absorption of this sulfated glycopeptide. In fact, preliminary experiments made in our laboratories showed that a measurable amount of metachromatic material (about 2% of the GLPS in the incubation medium) was found on the serosal side of the everted stomach preparation. Besides, a metachromatic reaction was given by rat urines after oral administration of GLPS at the dose of 50 mg/rat. In addition Eagleton, Watt and Marcus (1969) demonstrated the presence of metachromatic material in rat urines after oral administration of another sulfated macromolecule: degraded carrageenin. After intravenous treatment, GLPS, 1 mg/kg, at 90 min of pyloric ligation, inhibited gastric secretory volume and gave the same reduction of total acid and pepsin output. In contrast to the results obtained after oral administration, specific inhibition of gastric juice peptic activity could not be obtained. Therefore, while the GLPS activity against pyloric ligation ulcers, after oral administration, seems linked to its presence in the gastric contents, the latter assumption cannot be substantiated after intravenous treatment. In this case the protection exerted against pyloric ligation lesions could be partially explained by the anti-secretory activity of GLPS. Inhibition of gastric secretion by GLPS might be explained by considering its possible interaction with endogenous histamine or its activation of diamine oxidase, as has been shown for other sulfated macromolecules (Kobayashi, Kupelian and Maudsley, 1969; Sanyal and West, 1959). Further, screening tests showed that GLPS (5-10mg/kg) did not modify either cholinergic (evoked by acetylcholine or vagal stimulation) or adrenergic blood pressure response (evoked by noradrenaline, carotid occlusion, or tyramine); in vitro, GLPS (2.5 X 10 -4 g/ml) also proved
to be ineffective on acetylcholine- and histamineinduced contractions of the guinea pig ileum and on serotonin-induced contractions of the rat fundus. It is known that hypertonic solutions reduce gastric motility (Vantrappen et al., 1963) and remove parietal mucus (washing effect, Kowalewski and Schier, 1970). In this condition the unprotected gastric wall could be attacked by pepsin and this may explain the ulceration due to the administration of hypertonic glucose solution. However, other elements are present in the pathogenesis of this experimental ulcer, such as the state of fasting or the lack of many nutritional factors. It is likely that GLPS, mixed with glucose, inhibits this experimental ulcer through its anti-peptic and gastro-protective action. It is more difficult to explain how GLPS acts on ulcerations induced in the glandular portion of rat stomach, although it cannot be excluded that GLPS's protective effect occurs through its anti-peptic and anti-secretory properties. Increased gastric acid secretion could not be demonstrated in rats after steroid administration at ulcerogenic doses (Myhre, 1963) and it is still doubtful if hypersecretion occurs in restrained rats (Bonfils, Ferrier and Caulin, 1966). Nevertheless, a decrease of mucins and a reduced biosynthesis of sulfated mucopolysaccharides and mucoproteins was pointed out by a number of workers (Kowalewski and Strutz, 1959; Lambert, Andr6 and Martin, 1969; Menguy and Masters, 1963)which may be expected to result in an altered structure of the gastric mucosa. The gastric mucosa might then be subjected to the damaging action of acid pepsin even if gastric secretion is normal. GLPS could act by inhibiting peptic activity, but its protective role against these experimental ulcers is still open to further investigations.
ACKNOWLEDGEMENT We gratefully acknowledgethe technical assistance of Miss C. Giarmuzzi.
REFERENCES Anderson, W., 1961, The anti-peptic activity of sulfated polysaccharides, J. Pharm. Pharmacol. 13, 139.
G.Prino et al., A sulfated glycopeptide and rat gastric ulcers Babkin, P.B. and S.A. Komarov, 1932, The influence of gastric mucus on peptic digestion, Can. Med. Assoc. J. 27, 463. Bertellini, G., A. Butti, B. Piantanida, G. Prino, A. Riva, A. Rossi and S. Rossi, 1971a, Studies on glycoproteins of animal origin and their derivatives. I. Isolation of a sialoglycopeptide from hog duodenum; preliminary chemical and pharmacological studies, Arzneimittel-Forsch. 21, 244. _ Bertellini, G., A. Butti, B. Piantanida, G. Prino, A. Riva, A. Rossi and S. Rossi, 1971b, Studies on glycoproteins of animal origin and their derivatives. II. Synthesis of the polysulfuric ester of a natural sialoglycopeptide, Arzneimittel-Forsch. 21,247. Bonfils, S., J.P. Ferrier and Ch. Caulin, 1966, L'ulc~re de contrainte du rat blanc. M~thode de pathologie exp6rimentale et test pharmacologique, Rev. Franc. Etudes Clin. Biol. 11,343. Brodie, D.A., R.W. Marshall and O.M. Moreno, 1962, Effect of restraint on gastric acidity in the rat, Am. J. Physiol. 202, 812. Brodie, D.A., 1966, The mechanism of gastric hyperacidity produced by pylorus ligation in the rat, Am. J. Digest, Diseases New Series 2,231. Butti, A., B. Imbimbo and E. Brembilla, 1970, Clinical evaluation of an anti-ulcer sulfated glycopeptide (GLPS). A report on controlled multicenter trials, 4th World Congress of Gastroenterology, Copenhagen, July 1970. Advance Abstracts, eds. P. Riis, P. Anthonisen and H. Baden p. 308. Cook, D.L., S. Elch and P.S. Cammarata, 1963, Comparative pharmacology and chemistry of synthetic sulfated polysaccharides, Arch. Intern. Pharmacodyn. 144, 1. De Graef, J., A. Gerard, R. Lev and G.B.J. Glass, 1968, Secretion of acid and neutral mucosubstances in the dog stomach, in: The Physiology of Gastric Secretion, eds. L.S. Semb and J. Myren (Universitetsforlaget, Oslo) p. 124. Dische, Z., P. di Sant'Agnese, C. Pallavicini and J. Youlos, 1959, Composition of mucoid fraction from duodenal fluid of children and of adults, Arch. Biochem. 84, 205. Dunnett, C.W., 1955, A multiple comparison procedure for comparing several treatments with a control, Am. Statist. Assoc. J., 50, 1096. Eagleton, G.B., J. Watt and R. Marcus, 1969, Effect of subcutaneously administered degraded carrageenan on the production of histamine-induced gastric and duodenal ulceration, J. Pharm. Pharmacol. 21,123. Familiar, R.G., 1969, Sulphate-containing gastric mucus components and peptic ulcer: a possible relationship, J. Am. Geriat. Soc. 17,680. Grandjean, E., 1948, L'action d'un extrait stomaco-intestinal sur l'ulc~re exl~rimental du rat, Rev. Med. Suisse Rom. 68, 442. Ha'kkinen, I.P.T., 1960, Histamine induced changes in the sulphate metabolism of the duodenum and prepyloric region of the stomach: an experimental study with special reference to ulcer formation, Aeta Physiol. Scand. 51, Suppl. 177, 1.
125
H~ikkinen, I., K. Hartiala and T. Tetho, 1965, The fractionation and characterization of the acid polysaccharides in human gastric juice, Acta Chem. Scand. 19,797. H~kkinen, I. and K. Hartiala, 1966, The effect of cinchophen on the acid polysaccharides of the gastric and duodenal wall in dog, Acta Physiol. Scand. 66,326. H~ikkinen, I., R. Johansson and M. Pantio, 1968, An immunological and histoimmunological study of gastric sulphoglycoproteins in healthy and aspirin-treated dogs, Gut 9", 712. Kobayashi, Y., J. Kupelian and D.V. Maudsley, 1969, Release of diamine oxidase by heparin in the rat, Biochem. Pharmacol. 18, 1585. Komarov, S.A., 1936, The influence of mucoitin sulfuric acid on peptic digestion, Am. J. Digest. Diseases 3, 164. Kowalewski, K. and W.A. Strutz, 1959, Uptake of radiosulfate by the gastric tissue and gastric secretion in cortisone treated Shay rats, Acta Endocrinol. 31,107. Kowalewski, K. and J.F. Schier, 1970, Experimental peptic ulcer in dogs following the induced deficiency of gastric 'mucous barrier ~.Biochemical and histopathological study. 4th World Congress of Gastroenterology, Copenhagen, July 1970. Advance Abstracts, eds. P. Riis, P. Anthonisen and H. Baden, p. 378. Lambert, R., C. Andrd and F. Martin, 1969, Incorporation of radiosulfate in the gastric mucosa of the rat subjected to restraint, Gastroenterology 56,200. Levey, S. and. S. Sheinfeld, 1954, The inhibition of the proteolytic action of pepsin by sulfate-containing polysaccharides, Gastroenterology 27, 625. Martin, F., R. Lambert, M. Martin, J.-L. Vuez et A. B6rard, 1968a, Les glycoprot~ines sulfat6es de la s6cr~tion gastrique humaine, Biol. Gastroenterol. Suppl. 1 Arch. Franc. Maladies Appl. Digestif 57, 5. Martin, F., J.-L. Vuez, A B~rard, C. Andr~ et R. Lambert, 1968b, Inhibition de la prot6olyse peptique par une glycopr6t~ine sulfat~e gastrique humaine, Compt. Rend. Soc. Biol. 162, 1368. Martin, F., R. Mathian, A. B~rard and R. Lambert, 1969, Sulfated glycoproteins in human salivary and gastric secretions, Digestion 2, 103. Menguy, R. and J.-F. Masters, 1963, Effect of cortisone on mucoprotein secretion by gastric antrum of dogs: pathogenesis of steriod ulcer, Surgery 54, 19. Menguy, R. and L. Desbaillets, 1968, The gastric mucous barrier: Influence of protein-bound carbohydrate in mucus on the rate of proteolysis of gastric mucus, Ann. Surg. 168, 475. Myhre, E., 1963, The effect of Corticotrophin and Cortisone on the healing of gastric ulcer, in: Pathophysiology of Peptic Ulcer, ed.. S.C. Skoryna (McGill University Press, Canada) pp. 233-243. Placer, Z., Z. Roubal and V. V6k~E, 1958, The action of gastric mucus and some sulfate compounds on the development of experimental gastric ulcers in rats, Rev. Czech. Med. 4, 111. Prino, G., S. Paglialunga, A. Lietti, G. Nardi and R. Niada, 1970, Anti-ulcer and anti-peptic activity of a new glyco-
126
G.Prino et al., A sulfated glycopeptide and rat gastric ulcers
peptide sulfate (GLPS). Chemical and Pharmacological Study. 4th World Congress of Gastroenterology, Copenhagen, July 1970. Advance Abstracts, eds. P. Riis, P. Anthonisen and H. Baden, p. 307. Robert, A., J.E. Nezamis, 1958, Ulcerogenic property of steroids, Proc. Soc. Exptl. Biol. Med. 99,443. Rossi, G., S. Bonfils, F. Lieffogh, A. Lambling, 1956, Technique nouvelle pour produire des ulcerations gastriques chez le rat blanc: l'ulc~re de contrainte, Compt. Rend. Soc. Biol. 150, 1224. Sanyal, R.K. and G.B. West, 1959, The histamine-heparin complex, J. Pharm. Pharmacol. 11,548. Schrager, J., 1964, Mucopolysaccharides of the gastric secretion, Nature 201, 1220. Shay, H., S.A. Komarov, S.S. Fels, D. Meranze, M. Gruen-
stein and H.A. Siplet, 1945, A simple method for the uniform production of gastric ulceration in the rat, Gastroenterology 5, 43. Sun, D.C.H., and J.K. Chen, 1963, Experimental ulcer production in the pylorus ligated rat, in: Pathophysiology of Peptic Ulcer, ed. S.C. Skoryna (McGill University Press, Canada) pp. 141-152. Vantrappen, G., S. Vandenbroucke, Verbeke and T. Hellemans, 1963, Gastric motility, in: Pathophysiology of Peptic Ulcer, ed. S.C. Skoryna (McGiU University Press, Canada) pp. 115-131. Vocac, J.A., R.S. Alphin and P.J. Bolton, 1969, Role of HC1, pepsin and intragastric pressure in gastric ulceration of the perfused rat stomach in situ. Annual Meeting American Gastroenterological Association, Washington, May 1969. Abstract. Gastroenterology 56, 1266.