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Effect of protease inhibitors on experimental peptic ulceration
Effect of Protease Inhibitors on Experimental
Peptic Ulceration CASSIUS M ELLIS, MD, Minneapolis, Minnesota JOHN B LUNSETH, MD, PhD, Minneapolis, Minnesota DEMETRE M NICOLOFF, MD, PhD, Minneapolis, Minnesota
Although considerable research has been carried out on the cause and management of peptic ulcers, the factors that are responsible for their development, healing, and recurrence are still not completely understood. The statement, “no acid-no ulcer,” has been widely accepted by the medical community primarily on the clinical and experimental evidence that a pH of 4.0 or less is necessary for the development of peptic ulceration. However, this does not necessarily mean that acid per se is responsible for the ulceration, but rather that it may only be one of the necessary coexisting factors. Other indirect evidence that control of gastric acidity alone is not of primary importance is the equivocal success of the usual mode of therapy directed at buffering gastric acidity such as the use of antacids and antisecretory drugs. Although much less emphasized in the literature on peptic ulceration, the presence of pepsin, the major acid protease in gastric juice, is believed by some to be just as important as gastric acidity in the development of peptic ulceration [j-3]. Experimental evidence has shown that in the absence of peptic activity, peptic ulceration does not occur even though the pH of the gastric juice may be 2 or less [4,5]. In view of the aforementioned facts, a closer examination of the peptic element in the etiology of peptic ulceration was believed to be warranted. The purpose of this experiment was to determine whether compounds that have the capacity to inhibit protease activity in vitro without changing the pH of the gastric juice would influence the incidence and severity of chronic peptic ulceration produced in a standard ulcerogenic preparation in dogs. Material
and Methods
The ulcerogenic operation of placing the antrum as an antiperistaltic diverticulum on the colon as described by Dragstedt, Oberhelman, and Smith [6] was performed under sterile surgical conditions on seventy dogs. (Figure 1.) Gastrointestinal continuity was re-established by perFrom the Department of Surgery, Veterans Administration and University of Minnesota Hospital, Minneapolis, Minnesota of
Presented at the Tenth Annual Meeting the Alimentary Tract, New York, New
Vol. 119, February 1970
Hospital 55455.
of the Society for Surgery York, July 12 and 13, 1969.
forming gastrojejunostomy. After surgery the dogs were allowed to drink only water and milk for the first three days. Thereafter, they were allowed a standard kennel diet and water ad libitum. All dogs received antibiotics during the first seven postoperative days. On the seventh postoperative day the dogs were randomly placed into one of seven groups of ten. Each group was given one of six medications at the following three-hour intervals every day of the week: 7 AM, 10 AM, 1 PM, 4 PM, 7 PM, and 10 PM. No medications were given between 10 PM and 7 AM. All medications were given in gelatin capsules by trained personnel who made sure the capsules were swallowed. All dogs underwent necropsy at death and surviving dogs were sacrificed at sixty days. At necropsy the number of ulcers, their locations, their size, and whether they had perforated were recorded. The nature and location of the ulceration that occurs in this preparation are shown in Figure 2. There is a great similarity in the gross and histologic appearance of these ulcers and those found in man. The control group received only gelatin capsules. One group received 1 gm of antacids whose composition was aluminum hydroxide and magnesium trisilicate. The remaining five groups of dogs received compounds that, except for cellulose sulfate, showed protease inhibition in vitro with little or no change in gastric juice pH. One group received 3 gm of powdered activated charcoal. The charcoal was placed in the capsule by hand since the addition of mineral oil which is necessary for mechanical encapsulation nearly completely destroyed its protease inhibitory action. Another group received 3 gm of Amberlitea IRC-50 per dose. This is a cation exchange in finely divided form composed of a polymethacrylic matrix with carboxyl side groups. It is of medium porosity with a 5 per cent cross linkage and a moisture capacity of 48 t 5 per cent. It has an exchange capacity of 10 mEq/gm between pH of 5 and 14. Below this pH, it is essentially unionized and in this form serves as an effective hydrogen bonding agent [7]. It has been used as an adsorbent of pepsin and other macromolecular components present in gastric juice [8]. Adsorbent bonding of pepsin from human or canine gastric juice is essentially the same from pH 1 to 3.5. Above pH 4, however, as the resin ionizes, the adsorbed pepsin is released 191. The remaining three compounds used are all sulfated polysaccharides. Attention to this group of compounds has been drawn by their ability to interfere with peptic diges213
Ellis, Lunseth, and Nicoloff 1
Figure 1. Schematic presentation of the ulcerogenic preparation. The antrum is placed on the colon as an antiperistaltic diverticulum. Gastrointestinal continuity is re-established by a gastrojejunostomy. Figure 2. The ulcers are chronic stoma1 ulcers that occur primarily on the jejunal side of the anastomosis in the efferent limb.
tion [IO,II]. Cellulose sulfate* was given in a dose of 3 gm. This substance is a long straight chain synthetic polysaccharide with multiple sulfate side groups. Sulfated amylopectin (SN-263, Depepsin)t, which is a branched polysaccharide with multiple sulfate side groups, was given in a dose of 500 mg. This synthetic polysaccharide is the sodium salt of sulfated potato amylopectin having substantially 1.6 sulfate groups per glucose unit, a molecular weight of 2 x 107 to 8 x IO’, and contains 15 to 16.5 per cent sulphur [2]. Carrageenan was administered in a dose of 1 gm. This substance is composed primarily of sulfated d-galactose residues, linked together to form longchained polymers having a molecular weight of several hundred thousands. The ester sulfate group expressed as sulfate is approximately 32 per cent by weight. Results
The results are given in Table I. In the control group, ulcers developed in eight of the ten dogs. Six of the dogs had perforated ulcers. The average number of ulcers per dog was two with a mean size of 3.25 cm. In the group receiving carrageenan, ulceration developed in only two of the dogs and both ulcers perforated. Each dog had one ulcer with a mean size of 1.5 cm. When SN-263 was given, ulceration developed in three dogs with perforation occurring in two of the dogs. In the group receiving charcoal, ulcers developed in four dogs and perforation occurred in two of the dogs. The decrease in ulceration rate and size in these three groups from the control group is statistically significant (p < 0.05). In the group receiving antacids, five dogs had ulcers and perforation occurred in all of the dogs. The size and numbers of the ulcers were the same as in the control group. In the group receiving cellulose sulfate, eight dogs had ulcers with perforation occurring in seven. The size and number of the ulcers * Supplied fornia. t Supplied Illinois. 214
by the Kelco as Depepsin
Company,
SCS,
San
Diego,
by G.D. Searle & Company,
Cali-
Chicago,
were the same as in the control group. Of surprise and considerable interest was the finding that all ten dogs receiving Amberlite IRC-50 showed severe ulceration and perforation, The average number of ulcers per dog was almost three times that of the control dogs, and all of the dogs in this group died within a two to three week period. Comments The most effective substance for the reduction of ulceration in this experimental model was SN-263 or sulfated amylopectin. It significantly reduced the rate and severity of ulceration in a dose that was one-sixth that of the other protease inhibitors. Carrageenan was also effective, but at a larger dose. The use of sulfated polysaccharides as protease inhibitors and antiulcerogenie agents has been reported by several investigators [I-3,10--121. Cook, Eich, and Cammarata [13] compared the pharmacologic properties and chemical behavior of a number of synthetic sulfated polysaccharides and concluded that the molecule had to be of a certain minimal size to show protease inhibition. In addition, the protease inhibitory activity appeared to be directly related with the sulfate content and the degree of branching of the polysaccharide. The cellulose sulfate used in our experiment was highly sulfated, but had little or no branching. This lack of branching may be the reason for the absence of antiulcerogenic activity. The mode of action of SN-263 and carrageenan is not clear. Bianchi and Cook [14] showed that SN263 had antipeptic activity in vitro and antiulcerogenic activity in Shay rats. They found, as we did, that similar doses of antacids failed to produce inhibition of ulceration under similar conditions. Anderson [IS] has suggested that the antiulcerogenic activity is related to the formation of complexes between the gastric mucus and the polysaccharide with subsequent prevention of diffusion of pepsin into this complex. Houck, Bhayana, and Lee [16] have produced evidence which sugThe American Journal of Surgery
Protease TABLE
I
Effect
of
Protease
Inhibitors
on
Ulcer
Inhibitors
and
Peptic
Ulceration
Formation Percentage
Percentage Experimental
Individual
Group*
of
Dose
Perforation
km)
Mean
Number
Ulcers
per
a0
60
3.25
2
20
1.5
1
mg
30
20
1 .o
1
Charcoal
3 gm
40
20
2.0
1
Antacids
1 gm
50
50
3.0
1.75
3 gm
a0
70
3.0
1
100
3.0
5.50
500
SN-263
Cellulose
sulfate
100
3 gm
Amberlite
dogs per group; dogs surviving
sixty
days
were
3.
4.
5.
6.
7.
8.
9.
10.
11.
Summary
The effects of various protease inhibitors were studied in a canine model in which chronic peptic ulceration similar to that seen in man was produced. The administration of 500 mg of SN-263 and 3 gm of carrageenan at three hour intervals daily significantly reduced the severity and incidence of ulceration. Antacids given in, comparable doses had little or no effect. That the chemical structure of sulfated polysaccharides is important is demonstrated by the lack of antiulcerogenie activity of the cellulose sulfate used in our experiment. No apparent adverse side effects were seen from the administration of SN-263 or carrageenan. These findings would indicate that protease inhibition by these compounds should be considered in the treatment of chronic peptic ulceration in man.
12.
13.
14.
15.
16.
17.
18.
References 1. Sun DCH: Effect of a synthetic
sulfated polysaccharide on gastric peptic activity in human. Ann York Acad Sci 140: 749, 1967. and Drill VA: Pharmacological properties of pep-
(SN-263)
New Cook DL
119. February 1970
of
Dog
sacritlced.
gests that sulfated polysaccharides act as competitive inhibitors of pepsin. However, Anderson and Soman [I71 demonstrated that carrageenan placed in the duodenum of animals in which the continuity of the duodenum and stomach had been interrupted still reduced the incidence of histamine-induced gastric ulceration. Although some studies have suggested that sulfated polysaccharides reduce gastric acidity by inhibiting gastric secretion, most studies have shown that the polysaccharides have no effect on gastric acidity [28, 191. It would appear that the protective effect of this group of compounds is a local one and largely related to their ability to inhibit the protease activity of gastric juice. The increase in the rate of ulceration and perforation seen with the administration of Amberlite IRC50 is striking and of interest. This finding cannot be explained by the amount of hydrogen ions liberated from the compound. At present we have no explanation for this finding and are undertaking additional experiments to determine what factors might be responsible.
Vol.
of Ulcer
20
Carrageenan
2.
Size
3 gm
Control
* Ten
of
Ulceration
Mean
19.
sin inhibitors. Ann New York Acad Sci 140: 724, 1967. Zimmion DS, Miller G, Cox G, and Tesler MA: Specific inhibition of gastric pepsin in the treatment of gastric ulcer. Gastroenterology 56: 19, 1969. Nicoloff DM, Griffen WO, Salmon PA, Peter ET, WangenSteen OH: Local gastric hypothermia in the management of massive gastrointestinal hemorrhage. Surg Gynec & Obst 114: 495, 1962. Wangensteen OH, Salmon PA, Griffen WO, Paterson JRS, and Fattah F: Studies of local gastric cooling as related to peptic ulcer. Ann Surg 150: 346, 1959. Dragstedt LR, Oberhelman AA, and Smith CA: Experimental hyperfunction of the gastric antrum with ulcer formation. Ann Surg 134: 332, 1951. Boardman NK and Partridge SM: Separation of neutral proteins on ion-exchange resins. Biochem J 59: 543, 1955. Richmond V, Tang J, Wolf S, Tricco RE, and Cap&to R: Chromatographic isolation of gastricsin, the proteolytic enzyme from gastric juice with pH optimum 3.2. Biochim et biophys acta 29: 453, 1958. Lunseth JB: The isolation of pepsin from human gastric juice; gross preparatory techniques. PhD thesis, University of Minnesota, 1966. Levey S and Sheinfeld S: The inhibitor of the proteolytic action of pepsin by sulfate-containing polysaccharides. Gastroenterology 27: 625, 1954. Anderson W and Watt J: Inhibition of peptic activity, protection against histamine ulceration in the guinea pig, and combination with gastric mucin by an algal polyanion. J Pharm & Pharmacol 11: 318, 1959. Cayer D and Ruffin JM: Effect of depepsen in the treatment of peptic ulcer. Ann New York Acad Sci 140: 744, 1967. Cook DL, Eich S, and Cammarata PS: Comparative pharmacology and chemistry of synthetic sulfated polysaccharides. Arch internat pharmacodyn 144: 1, 1963. Bianchi RG and Cook DL: Antipeptic and anti-ulcerogenic properties of synthetic sulfated polysaccharide (SN263). Gastroenterology 47: 409, 1964. Anderson W: The effect of a sulfated polysaccharide upon diffusion of pepsin through mucin. J Pharm Pharmacof 13: 122 T, 1961. Houck JC, Bhayana J, and Lee T: The inhibition of pepsin and peptic ulcers. Gastroenterology 39: 196, 1960. Anderson W, and Soman PD: Degraded carrageenan and experimental acute gastric ulceration in the guinea pig. Nature (London) 199: 389, 1963. Anderson W, Marcus R, and Watt J: The effect of a sulfated polysaccharide on the acidity and volume of histamine-stimulated gastric secretion in the guinea pig. J Pharm Pharmacof 14: 119 T, 1962. Turner MD, Miller LL, and Segal HL: Gastric proteases and protease inhibitors. Gastroenterology 53: 967, 1967. 215
Ellis, Lunseth,
and Nicoloff
Discussion HARRY H LEVEEN (Brooklyn, NY): The literature on this subject goes back as far as 1914 when Langenskiold published his classic paper on tissue resistance to proteinases.* The great point that Langenskiold made, which has also been confirmed by Northrup and others, is that when proteins are digested, peptones are formed which combine with the pepsin and inactivate it. Northrup found that the formation of peptone during protein digestion brought digestion to a standstill. Peptic digestion is not a monomolecular reaction-it is a bimolecular reaction, with the products of digestion inactivating the pepsin. Langenskiold showed that a meal of protein or irrigation of the mucosa makes the bowel very resistant to ulceration by pepsin hydrochloric acid for a number of hours. This is a good reason for using proteins which do combine with acid and do form very potent inhibitors. * Scandinav arch physiol 31: I, 1914; reprinted in Am J Digest Dis 7: 35, 1940 (On the resistance of some living tissue to the action of proteinases).
216
Before one experiments with sulfated compounds such as the carrageenins (heparin has heen shown to have such activity), one should compare the activity of ordinary protein, which seems to be about the best protective substance. Drug companies make no money from the sale of protein. and they rarely tell us these facts. CASSIUSM ELLIS (closing) : We believe that this chronic ulcerogenic preparation, which produces ulcers similar to human peptic ulceration, is a better model than the Shay rat for the study of the effect of various drugs on peptic ulcer disease. Antacids were not as effective as SN-263 on an equa1 weight to weight basis. Suifonation of the polysaccharides itself is not the determining factor in protease inhibitor activity as demonstrated by the cellulose sulfate results in our experiments. Pepsin binding by nonadsorbable substances such as Amberlite does not seem to be as effective an antiulcerogenic agent as SN-263. Activated charcoal, given in powder rather than in tablet form, has been used for ages for dyspepsia and appears to be an effective antiulcerogenic agent by its ability to adsorb pepsin and in this way act as a protease inhibitor.
The American Journal of Surgery
Peptic Ulceration CASSIUS M ELLIS, MD, Minneapolis, Minnesota JOHN B LUNSETH, MD, PhD, Minneapolis, Minnesota DEMETRE M NICOLOFF, MD, PhD, Minneapolis, Minnesota
Although considerable research has been carried out on the cause and management of peptic ulcers, the factors that are responsible for their development, healing, and recurrence are still not completely understood. The statement, “no acid-no ulcer,” has been widely accepted by the medical community primarily on the clinical and experimental evidence that a pH of 4.0 or less is necessary for the development of peptic ulceration. However, this does not necessarily mean that acid per se is responsible for the ulceration, but rather that it may only be one of the necessary coexisting factors. Other indirect evidence that control of gastric acidity alone is not of primary importance is the equivocal success of the usual mode of therapy directed at buffering gastric acidity such as the use of antacids and antisecretory drugs. Although much less emphasized in the literature on peptic ulceration, the presence of pepsin, the major acid protease in gastric juice, is believed by some to be just as important as gastric acidity in the development of peptic ulceration [j-3]. Experimental evidence has shown that in the absence of peptic activity, peptic ulceration does not occur even though the pH of the gastric juice may be 2 or less [4,5]. In view of the aforementioned facts, a closer examination of the peptic element in the etiology of peptic ulceration was believed to be warranted. The purpose of this experiment was to determine whether compounds that have the capacity to inhibit protease activity in vitro without changing the pH of the gastric juice would influence the incidence and severity of chronic peptic ulceration produced in a standard ulcerogenic preparation in dogs. Material
and Methods
The ulcerogenic operation of placing the antrum as an antiperistaltic diverticulum on the colon as described by Dragstedt, Oberhelman, and Smith [6] was performed under sterile surgical conditions on seventy dogs. (Figure 1.) Gastrointestinal continuity was re-established by perFrom the Department of Surgery, Veterans Administration and University of Minnesota Hospital, Minneapolis, Minnesota of
Presented at the Tenth Annual Meeting the Alimentary Tract, New York, New
Vol. 119, February 1970
Hospital 55455.
of the Society for Surgery York, July 12 and 13, 1969.
forming gastrojejunostomy. After surgery the dogs were allowed to drink only water and milk for the first three days. Thereafter, they were allowed a standard kennel diet and water ad libitum. All dogs received antibiotics during the first seven postoperative days. On the seventh postoperative day the dogs were randomly placed into one of seven groups of ten. Each group was given one of six medications at the following three-hour intervals every day of the week: 7 AM, 10 AM, 1 PM, 4 PM, 7 PM, and 10 PM. No medications were given between 10 PM and 7 AM. All medications were given in gelatin capsules by trained personnel who made sure the capsules were swallowed. All dogs underwent necropsy at death and surviving dogs were sacrificed at sixty days. At necropsy the number of ulcers, their locations, their size, and whether they had perforated were recorded. The nature and location of the ulceration that occurs in this preparation are shown in Figure 2. There is a great similarity in the gross and histologic appearance of these ulcers and those found in man. The control group received only gelatin capsules. One group received 1 gm of antacids whose composition was aluminum hydroxide and magnesium trisilicate. The remaining five groups of dogs received compounds that, except for cellulose sulfate, showed protease inhibition in vitro with little or no change in gastric juice pH. One group received 3 gm of powdered activated charcoal. The charcoal was placed in the capsule by hand since the addition of mineral oil which is necessary for mechanical encapsulation nearly completely destroyed its protease inhibitory action. Another group received 3 gm of Amberlitea IRC-50 per dose. This is a cation exchange in finely divided form composed of a polymethacrylic matrix with carboxyl side groups. It is of medium porosity with a 5 per cent cross linkage and a moisture capacity of 48 t 5 per cent. It has an exchange capacity of 10 mEq/gm between pH of 5 and 14. Below this pH, it is essentially unionized and in this form serves as an effective hydrogen bonding agent [7]. It has been used as an adsorbent of pepsin and other macromolecular components present in gastric juice [8]. Adsorbent bonding of pepsin from human or canine gastric juice is essentially the same from pH 1 to 3.5. Above pH 4, however, as the resin ionizes, the adsorbed pepsin is released 191. The remaining three compounds used are all sulfated polysaccharides. Attention to this group of compounds has been drawn by their ability to interfere with peptic diges213
Ellis, Lunseth, and Nicoloff 1
Figure 1. Schematic presentation of the ulcerogenic preparation. The antrum is placed on the colon as an antiperistaltic diverticulum. Gastrointestinal continuity is re-established by a gastrojejunostomy. Figure 2. The ulcers are chronic stoma1 ulcers that occur primarily on the jejunal side of the anastomosis in the efferent limb.
tion [IO,II]. Cellulose sulfate* was given in a dose of 3 gm. This substance is a long straight chain synthetic polysaccharide with multiple sulfate side groups. Sulfated amylopectin (SN-263, Depepsin)t, which is a branched polysaccharide with multiple sulfate side groups, was given in a dose of 500 mg. This synthetic polysaccharide is the sodium salt of sulfated potato amylopectin having substantially 1.6 sulfate groups per glucose unit, a molecular weight of 2 x 107 to 8 x IO’, and contains 15 to 16.5 per cent sulphur [2]. Carrageenan was administered in a dose of 1 gm. This substance is composed primarily of sulfated d-galactose residues, linked together to form longchained polymers having a molecular weight of several hundred thousands. The ester sulfate group expressed as sulfate is approximately 32 per cent by weight. Results
The results are given in Table I. In the control group, ulcers developed in eight of the ten dogs. Six of the dogs had perforated ulcers. The average number of ulcers per dog was two with a mean size of 3.25 cm. In the group receiving carrageenan, ulceration developed in only two of the dogs and both ulcers perforated. Each dog had one ulcer with a mean size of 1.5 cm. When SN-263 was given, ulceration developed in three dogs with perforation occurring in two of the dogs. In the group receiving charcoal, ulcers developed in four dogs and perforation occurred in two of the dogs. The decrease in ulceration rate and size in these three groups from the control group is statistically significant (p < 0.05). In the group receiving antacids, five dogs had ulcers and perforation occurred in all of the dogs. The size and numbers of the ulcers were the same as in the control group. In the group receiving cellulose sulfate, eight dogs had ulcers with perforation occurring in seven. The size and number of the ulcers * Supplied fornia. t Supplied Illinois. 214
by the Kelco as Depepsin
Company,
SCS,
San
Diego,
by G.D. Searle & Company,
Cali-
Chicago,
were the same as in the control group. Of surprise and considerable interest was the finding that all ten dogs receiving Amberlite IRC-50 showed severe ulceration and perforation, The average number of ulcers per dog was almost three times that of the control dogs, and all of the dogs in this group died within a two to three week period. Comments The most effective substance for the reduction of ulceration in this experimental model was SN-263 or sulfated amylopectin. It significantly reduced the rate and severity of ulceration in a dose that was one-sixth that of the other protease inhibitors. Carrageenan was also effective, but at a larger dose. The use of sulfated polysaccharides as protease inhibitors and antiulcerogenie agents has been reported by several investigators [I-3,10--121. Cook, Eich, and Cammarata [13] compared the pharmacologic properties and chemical behavior of a number of synthetic sulfated polysaccharides and concluded that the molecule had to be of a certain minimal size to show protease inhibition. In addition, the protease inhibitory activity appeared to be directly related with the sulfate content and the degree of branching of the polysaccharide. The cellulose sulfate used in our experiment was highly sulfated, but had little or no branching. This lack of branching may be the reason for the absence of antiulcerogenic activity. The mode of action of SN-263 and carrageenan is not clear. Bianchi and Cook [14] showed that SN263 had antipeptic activity in vitro and antiulcerogenic activity in Shay rats. They found, as we did, that similar doses of antacids failed to produce inhibition of ulceration under similar conditions. Anderson [IS] has suggested that the antiulcerogenic activity is related to the formation of complexes between the gastric mucus and the polysaccharide with subsequent prevention of diffusion of pepsin into this complex. Houck, Bhayana, and Lee [16] have produced evidence which sugThe American Journal of Surgery
Protease TABLE
I
Effect
of
Protease
Inhibitors
on
Ulcer
Inhibitors
and
Peptic
Ulceration
Formation Percentage
Percentage Experimental
Individual
Group*
of
Dose
Perforation
km)
Mean
Number
Ulcers
per
a0
60
3.25
2
20
1.5
1
mg
30
20
1 .o
1
Charcoal
3 gm
40
20
2.0
1
Antacids
1 gm
50
50
3.0
1.75
3 gm
a0
70
3.0
1
100
3.0
5.50
500
SN-263
Cellulose
sulfate
100
3 gm
Amberlite
dogs per group; dogs surviving
sixty
days
were
3.
4.
5.
6.
7.
8.
9.
10.
11.
Summary
The effects of various protease inhibitors were studied in a canine model in which chronic peptic ulceration similar to that seen in man was produced. The administration of 500 mg of SN-263 and 3 gm of carrageenan at three hour intervals daily significantly reduced the severity and incidence of ulceration. Antacids given in, comparable doses had little or no effect. That the chemical structure of sulfated polysaccharides is important is demonstrated by the lack of antiulcerogenie activity of the cellulose sulfate used in our experiment. No apparent adverse side effects were seen from the administration of SN-263 or carrageenan. These findings would indicate that protease inhibition by these compounds should be considered in the treatment of chronic peptic ulceration in man.
12.
13.
14.
15.
16.
17.
18.
References 1. Sun DCH: Effect of a synthetic
sulfated polysaccharide on gastric peptic activity in human. Ann York Acad Sci 140: 749, 1967. and Drill VA: Pharmacological properties of pep-
(SN-263)
New Cook DL
119. February 1970
of
Dog
sacritlced.
gests that sulfated polysaccharides act as competitive inhibitors of pepsin. However, Anderson and Soman [I71 demonstrated that carrageenan placed in the duodenum of animals in which the continuity of the duodenum and stomach had been interrupted still reduced the incidence of histamine-induced gastric ulceration. Although some studies have suggested that sulfated polysaccharides reduce gastric acidity by inhibiting gastric secretion, most studies have shown that the polysaccharides have no effect on gastric acidity [28, 191. It would appear that the protective effect of this group of compounds is a local one and largely related to their ability to inhibit the protease activity of gastric juice. The increase in the rate of ulceration and perforation seen with the administration of Amberlite IRC50 is striking and of interest. This finding cannot be explained by the amount of hydrogen ions liberated from the compound. At present we have no explanation for this finding and are undertaking additional experiments to determine what factors might be responsible.
Vol.
of Ulcer
20
Carrageenan
2.
Size
3 gm
Control
* Ten
of
Ulceration
Mean
19.
sin inhibitors. Ann New York Acad Sci 140: 724, 1967. Zimmion DS, Miller G, Cox G, and Tesler MA: Specific inhibition of gastric pepsin in the treatment of gastric ulcer. Gastroenterology 56: 19, 1969. Nicoloff DM, Griffen WO, Salmon PA, Peter ET, WangenSteen OH: Local gastric hypothermia in the management of massive gastrointestinal hemorrhage. Surg Gynec & Obst 114: 495, 1962. Wangensteen OH, Salmon PA, Griffen WO, Paterson JRS, and Fattah F: Studies of local gastric cooling as related to peptic ulcer. Ann Surg 150: 346, 1959. Dragstedt LR, Oberhelman AA, and Smith CA: Experimental hyperfunction of the gastric antrum with ulcer formation. Ann Surg 134: 332, 1951. Boardman NK and Partridge SM: Separation of neutral proteins on ion-exchange resins. Biochem J 59: 543, 1955. Richmond V, Tang J, Wolf S, Tricco RE, and Cap&to R: Chromatographic isolation of gastricsin, the proteolytic enzyme from gastric juice with pH optimum 3.2. Biochim et biophys acta 29: 453, 1958. Lunseth JB: The isolation of pepsin from human gastric juice; gross preparatory techniques. PhD thesis, University of Minnesota, 1966. Levey S and Sheinfeld S: The inhibitor of the proteolytic action of pepsin by sulfate-containing polysaccharides. Gastroenterology 27: 625, 1954. Anderson W and Watt J: Inhibition of peptic activity, protection against histamine ulceration in the guinea pig, and combination with gastric mucin by an algal polyanion. J Pharm & Pharmacol 11: 318, 1959. Cayer D and Ruffin JM: Effect of depepsen in the treatment of peptic ulcer. Ann New York Acad Sci 140: 744, 1967. Cook DL, Eich S, and Cammarata PS: Comparative pharmacology and chemistry of synthetic sulfated polysaccharides. Arch internat pharmacodyn 144: 1, 1963. Bianchi RG and Cook DL: Antipeptic and anti-ulcerogenic properties of synthetic sulfated polysaccharide (SN263). Gastroenterology 47: 409, 1964. Anderson W: The effect of a sulfated polysaccharide upon diffusion of pepsin through mucin. J Pharm Pharmacof 13: 122 T, 1961. Houck JC, Bhayana J, and Lee T: The inhibition of pepsin and peptic ulcers. Gastroenterology 39: 196, 1960. Anderson W, and Soman PD: Degraded carrageenan and experimental acute gastric ulceration in the guinea pig. Nature (London) 199: 389, 1963. Anderson W, Marcus R, and Watt J: The effect of a sulfated polysaccharide on the acidity and volume of histamine-stimulated gastric secretion in the guinea pig. J Pharm Pharmacof 14: 119 T, 1962. Turner MD, Miller LL, and Segal HL: Gastric proteases and protease inhibitors. Gastroenterology 53: 967, 1967. 215
Ellis, Lunseth,
and Nicoloff
Discussion HARRY H LEVEEN (Brooklyn, NY): The literature on this subject goes back as far as 1914 when Langenskiold published his classic paper on tissue resistance to proteinases.* The great point that Langenskiold made, which has also been confirmed by Northrup and others, is that when proteins are digested, peptones are formed which combine with the pepsin and inactivate it. Northrup found that the formation of peptone during protein digestion brought digestion to a standstill. Peptic digestion is not a monomolecular reaction-it is a bimolecular reaction, with the products of digestion inactivating the pepsin. Langenskiold showed that a meal of protein or irrigation of the mucosa makes the bowel very resistant to ulceration by pepsin hydrochloric acid for a number of hours. This is a good reason for using proteins which do combine with acid and do form very potent inhibitors. * Scandinav arch physiol 31: I, 1914; reprinted in Am J Digest Dis 7: 35, 1940 (On the resistance of some living tissue to the action of proteinases).
216
Before one experiments with sulfated compounds such as the carrageenins (heparin has heen shown to have such activity), one should compare the activity of ordinary protein, which seems to be about the best protective substance. Drug companies make no money from the sale of protein. and they rarely tell us these facts. CASSIUSM ELLIS (closing) : We believe that this chronic ulcerogenic preparation, which produces ulcers similar to human peptic ulceration, is a better model than the Shay rat for the study of the effect of various drugs on peptic ulcer disease. Antacids were not as effective as SN-263 on an equa1 weight to weight basis. Suifonation of the polysaccharides itself is not the determining factor in protease inhibitor activity as demonstrated by the cellulose sulfate results in our experiments. Pepsin binding by nonadsorbable substances such as Amberlite does not seem to be as effective an antiulcerogenic agent as SN-263. Activated charcoal, given in powder rather than in tablet form, has been used for ages for dyspepsia and appears to be an effective antiulcerogenic agent by its ability to adsorb pepsin and in this way act as a protease inhibitor.
The American Journal of Surgery