Scandinavian Journal of Gastroenterology
ISSN: 0036-5521 (Print) 1502-7708 (Online) Journal homepage: http://www.tandfonline.com/loi/igas20
Gastric Protection by Nocloprost against Aspirin Damage in Humans Possible Role of Epidermal Growth Factor S. J. Konturek, J. W. Konturek, N. Kwiecien, W. Obtulowicz, J. Oleksy, Z. Hebzda & I. Amon To cite this article: S. J. Konturek, J. W. Konturek, N. Kwiecien, W. Obtulowicz, J. Oleksy, Z. Hebzda & I. Amon (1991) Gastric Protection by Nocloprost against Aspirin Damage in Humans Possible Role of Epidermal Growth Factor, Scandinavian Journal of Gastroenterology, 26:3, 231-236, DOI: 10.3109/00365529109025036 To link to this article: http://dx.doi.org/10.3109/00365529109025036
Published online: 08 Jul 2009.
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Gastric Protection by Nocloprost against Aspirin Damage in Humans, Possible Role of Epidermal Growth Factor
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S. J. KONTUREK, J. W. KONTUREK, N. KWIECIEN, W. OBTULOWICZ, J. OLEKSY, Z . HEBZDA & I. AMON Institute of Physiology, Academy of Medicine, Cracow, Poland, and Dept. of Pharmacology, Asche AG, Hamburg, Germany Konturek SJ, Konturek JW, Kwiecien N, Obtuiowicz W, Oleksy J, Hebzda Z , Amon I. Gastric protection by nocloprost against aspirin damage in humans. Possible role of epidermal growth factor. Scand J Gastroenterol 1991, 26, 231-236 Ten healthy young male subjects took part in a double-blind, placebo-controlled crossover study to assess the effects of nocloprost on gastric microbleeding and endoscopic mucosal injury induced by the administration of aspirin (2.5 8). In addition, basal and pentagastrin-induced gastric acid and pepsin secretion and salivary and plasma contents of epidermal growth factor (EGF) were measured after placebo plus aspirin or nocloprost plus aspirin treatment in these subjects. Nocloprost (100 pg/ dose) significantly reduced spontaneous gastric microbleeding and almost completely prevented gastric mucosal injury induced by aspirin. Nocloprost failed to affect basal and pentagastrin-stimulated gastric acid and pepsin secretion but increased significantly the salivary outputs and plasma concentrations of EGF. In conclusion, nocloprost is effective in preventing gastric injury by aspirin even at a non-antisecretory dose, and this protection may involve an excessive release of EGF. Key words: Bleeding; cytoprotection; endoscopy; prostaglandins
Prof. S. J . Konturek, M . D . , Institute of Physiology, 31-531 Krakow, ul Grzegorzecka 16, Poland
Gastric mucosal injury by non-steroidal antiinflammatory drugs (NSAID) such as aspirin (ASA) has been ascribed predominantly to a deficiency of prostaglandins (PG) in the gastric mucosa due to the suppression of their mucosal biosynthesis (1,2). Studies in normal volunteers with PGE analogs, primarily misoprostol and enprostil, have shown that these agents, used in antisecretory doses, were highly effective in the protection of gastric mucosa against various NSAID (3,4). Such a protection against certain NSAID has also been obtained by using gastric acid inhibitors such as H2-receptor antagonists (5-7), suggesting that acid suppression may be a critical factor in gastroprotection against NSAIDinduced mucosal damage. Nocloprost is a unique PGE2 analog that has
been shown in experimental animals to display a potent gastroprotective activity against both acidindependent and acid-dependent ulcerogens when administered orally in non-antisecretory doses (8). Since the gastroprotective activity of nocloprost against aspirin damage has not been studied in humans, a double-blind, placebocontrolled crossover study was undertaken to examine the effects of this PG on mucosal damage caused by ASA, as assessed by the examination of gastric microbleeding and by gastric endoscopy. Furthermore, gastric acid and pepsin secretion and the release of epidermal growth factor (EGF), which has been implicated in the maintenance of mucosal integrity (9), have been investigated in these subjects.
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SUBJECTS AND METHODS
Subjects This study was approved by the Institutional Ethical Committee, and informed consent was obtained from each subject. The study was carried out on 10 male volunteers aged 21-25 years (mean age, 22 years) and weighing 67-74 kg (mean weight, 72 kg). All subjects were in good health without any previous or present gastrointestinal disease and with normal laboratory values for blood biochemistry, hematology, and urinalysis. All subjects underwent pre-study assessments, including examination of gastric microbleeding and gastroduodenoscopy to exclude any mucosal abnormalities or gastroduodenal ulcers. All subjects were hospitalized, and 1 week before the trial all medication was withdrawn and alcohol was forbidden. Study design The study was randomized, double-blind, placebo-controlled, and crossover. After the prestudy assessments, each volunteer underwent two treatments, one with nocloprost and another with placebo with 7-10 days of ‘washout’ in between. One tablet (100 pg) of nocloprost (Asche AG, Hamburg, Germany) or placebo was taken on day 1 at 0800 and 2000 h. Thirty minutes after taking the nocloprost or placebo tablet, the subject took two tablets of 0.5 g unbuffered aspirin (Polfa, Poland). On day 2, one nocloprost or one placebo tablet was taken at 0600 h, followed 30 min later by one ASA tablet. Post-treatment assessment was performed 2 h after the last ASA tablet. Assessments Pre- and post-treatment assessments were carried out as described previously ( 5 ) . In brief, venous blood samples were withdrawn to measure plasma salicylate concentration (10) and to determine plasma EGF levels. For this purpose the blood was collected in chilled tubes with heparin (10 U/ml) and the kallikrein inhibitor aprotinin (400 U/ml) (Trasylolm, Polfa, Poland) and centrifuged, and the plasma was frozen until radioimmunoassay of EGF as described (11). Then,
the rate of gastrin microbleeding into the gastric lumen was determined in three consecutive 10min gastric washings (5,12). Gastric endoscopy was performed with an Olympus GIF Q panendoscope to evaluate the mucosal appearance by means of the Lanza score system (13, 14). Grading was from 0 = normal mucosa to 4 = widespread involvement of the stomach with submucosal hemorrhages or superficial ulcerations. After completion of gastric washing and endoscopy, the collection of the saliva and gastric content was started and continued for 120min. During the initial 30-min period, gastric aspiration was collected to determine basal acid output, and ‘then parafilm chewing was carried out for 15 min to stimulate salivary secretion for the determination of EGF output. Finally, saliva and gastric secretion were collected for 45 min during intravenous infusion of pentagastrin ( 2 Fg/kg-h), to assess maximal acid output in these subjects. Nocloprost (supplied in tablets by Dr. R. Wabnitz from Asche AG, Hamburg, Germany), is a 9P-chloro-16,16-dimethyl-prostaglandinE 2 with clathrate of P-cyclodextrin to ensure its water solubility. The results are presented in the figures and the table as individual data and mean values (*SEM). Friedman’s two-way analysis of variance was used, supplemented, when applicable, by the Wilcoxon’s two-sided test. The significance level was set at 5%. RESULTS
Effects of nocloprost on gastric microbleeding and the endoscopic appearance of the gastric mucosa The pre-treatment assessment of gastric microbleeding was about 14.6 ? 3.9 pl/30 min, and in all 10 subjects examined endoscopically the mucosa was normal. After pre-treatment with placebo plus ASA, gastric microbleeding increased in all subjects and reached on average 129 33 p1/30 min. Endoscopy showed a significant increase in the mucosal damage in all subjects tested, and on average the score was 3.2 2 0.3 (Figs. 1 and 2 ) . The endoscopic changes ranged from a single submucosal hemorrhage (1 subject)
*
Gastroprotection by Nocloprost
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to widespread involvement of the mucosa with over 10 hemorrhages and superficial erosions ( 5 subjects). After treatment with nocloprost, ASAinduced gastric microbleeding was reduced in 9 of the 10 subjects and averaged about 64 ? 15 PI/
NOCLOPROST
ASA
Fig. 2. Gastric endoscopic scores (individual data and means ? SEM) in healthy subjects treated with placebo plus ASA or nocloprost plus ASA. Asterisk indicates significant decrease below the placebo value.
30 min. Endoscopy showed a decrease in mucosal lesions in all subjects treated with nocloprost. The mean mucosal damage on average was 0.32 k 0.21, and this score was significantly lower than that in placebo-treated subjects. Basal and maximal pentagastrin-stimulated gastric acid and pepsin secretion in subjects
Table I. Effects of nocloprost plus aspirin or placebo plus aspirin on basal and pentagastrin-stimulated maximal acid and pepsin outputs and salivary secretion and plasma levels of EGF (under basal conditions and after parafilm chewing) and plasma levels of salicylate. Means t SEM of 10 determinations in 10 subjects Treatment Placebo Gastric H' (mmol/30 min) Basal Maximal Gastrin pepsin (mg/30 min) Basal Maximal Salivary EGF (ng/l5 min) Basal Chewing Plasma EGF (pg/ml) Basal Chewing Plasma salicylate (mg%)
+ ASA
Nocloprost
+ ASA
2.84 t 0.76 9.88 t 1.55
1.92 ? 0.57 8.36 t 1.10
21.28 t 4.02 56.51 t 9.64
23.28 t 4.84 59.55 t 9.48
17.50 t 6.54 29.41 t 7.62
48.60 ? 9.22" 54.85 t 12.42*
37.1 t 6.1 51.2 t 12.6 5.84 t 1.26
68.2 ? 14.3* 96.8 t 13.6* 4.88 2 1.36
* Significant ( p < 0.05j increase above the placebo value.
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treated with placebo plus ASA were not significantly different from the values obtained in these subjects after nocloprost plus ASA administration. Plasma concentrations of salicylates were also similar in subjects treated with placebo and nocloprost plus ASA (Table I). EGF outputs in salivary secretion were significantly increased during chewing both in placebo- and in nocloprost-treated subjects. The values of salivary outputs and plasma concentration of EGF were significantly higher in nocloprost-treated subjects than in those treated with placebo plus ASA (Table I). DISCUSSION This study provides evidence that nocloprost applied orally in a dose (1OOpg) that failed to affect basal or maximally stimulated gastric acid or pepsin secretion reduced significantly aspirininduced gastric microbleeding and gastric mucosal injury in humans. The gastrotoxic effect of NSAID has been well recognized ever since Douthwaite & Lintott (15) in 1938 described the first endoscopic observations of gastric mucosal injury in patients taking aspirin. Lanza et al. (13, 14) reported first detailed endoscopic evaluation and scoring system of mucosal injury in normal volunteers taking various NSAID. The mucosal injury by NSAID was assessed by measuring fecal blood loss with the chromium-51 technique or gastric blood loss in gastric washings as proposed by Hunt & Fisher (12). Endoscopic injury scores and the blood loss in subjects taking aspirin were then compared and found, to be closely correlated (16,17). Our present study used both these techniques to examine the alterations in gastric microbleeding and mucosal damage by aspirin and the possible protective activity of nocloprost. We found that aspirin at a total dose of 2.5 g causes severalfold increase in gastric bleeding and endoscopic mucosal damage in all tested subjects. Several mechanisms have been proposed to explain aspirin-induced gastric mucosal damage, but the inhibition of mucosal PG biosynthesis and subsequent decrease of mucosal resistance to noxious agents has been considered to be the
major factor (1,2). Ligumsky et al. (18) confirmed (19) that in rats aspirin causes a marked inhibition of PG btosynthesis (by about 90%) but suggested that the mucosal damage occurred only when the mucosa was additionally exposed to another irritant such as luminal acid. In contrast, Cohen & MacDonald (20) reported that aspirin in humans may induce fundic mucosal injury even without affecting mucosal PG significantly. These observations could suggest that the reduction in PG biosynthesis is neither sufficient (rats) nor even necessary (humans) for the production of gastric mucosal damage by aspirin. More detailed studies (21) confirmed the dependence of ASAinduced mucosal damage on the marked (about 95%) reduction in PG content in fundic mucosa. Our finding that xiocloprost is capable of reducing the extent of mucosal injury caused by aspirin is in keeping with the concept that the deficiency of the protective PG in the mucosa is the major factor in gastrotoxicity by NSAID. Other PGE analogs have also been reported to protect the gastric mucosa against the injury caused by NSAID (13,14), but these analogs were used in gastric inhibitory doses. Since the damaging action of aspirin is related to the rate of its absorption into the mucosal cells and depends on its lipophilic nature at acid pH and the pH of gastric contents (22), the reduction in gastric acid secretion seems to be the crucial factor in the mechanism of gastroprotection by these PG analogs. Nocloprost applied orally at the dose used was completely devoid of gastric inhibitory action both in experimental animals (8) and, as shown in this report, also in humans. Although nocloprost is chemically more stable and, like other PGE analogs, is well absorbed from the gastrointestinal tract, it does not reach the systemic circulation because of the metabolism in the mucosa and first-passage uptake and inactivation by the liver (8,23). This may explain the failure of nocloprost administered orally to affect gastric acid secretion or to provoke any adverse effects such as diarrhea or abortion (23). Thus, after oral administration, nocloprost mainly acts locally on the gastric rnucosa to maintain its protective mechanisms, including stimulation of mucus-alkaline secretion, enhancing mucosal blood flow, and
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1300 mg of aspirin: an endoscopic study. Dig Dis promoting mucosal growth, as other PGE analogs Sci 1986, 31, 137-1418 do (24). 4. Cohen MM, McGready DR, Clark L, Sevilius H. It may be that these and Yet other u ~ k r " J n Protection against aspirin-induced antral and duodenal mucoia with enprostil. Gastroenterology properties of nocloprost are operative in the 1985, 88, 382-386 stomach in providing protection to the mucosa 5. Konturek SJ, Kwiecien N, Obtulowicz W, Polanski against NSAID-induced damage. The results of M, Kopp B, Oteksy J. Comparison of prostaglandin E2 and ranitidine in prevention of gastric bleeding this study suggest the possible implication of by aspirin in man. Gut 1983, 24, 89-93 EGF as a mediator of the favorable influence of 6. Roth SH, Bennett RE, Mitchell CS, Hartmann RJ. nocloprost on human gastric mucosa. EGF was Cimetidine therapy in NSAID gastropathy. Double blind long-term evaluation. Arch Intern Med 1987, reported to show gastroprotective activity against 147, 1798 aspirin damage in rats, and this effect was inde7. Rachmilewitz D, Polak D, Eliskin R, et al. Cimependent of the mucosal generation of PG (19). tidine significantly decreases indomethacin-induced gastro-duodenal mucosal damage. GastroenterNSAID appear, however, to suppress the release 1986, 90, 1596 ology of EGF from the salivary glands (9), and this may 8. Konturek SJ, Brzozowski T, Drozdowicz D, Amon contribute, at least in part, to the damaging action I. Nocloprost, a stable PGE2 analog with unique local gastroprotective and ulcer-healing activity. of these drugs on the gastric mucosa. In the Gastroenterology 1990, 98, A70 present study the administration of nocloprost in 9. Konturek SJ. Role of growth factors in gasaspirin-treated subjects resulted in a significant troduodenal protection and healing of peptic ulcers. Gastroenterology Clin N Am 1990, 19, 41-66 increase in EGF release into the saliva and in Satzman A . A fluorometric method for the esti10. the plasma concentration of this peptide. Further mation of salicylate in blood. J Biol Chem 1948, studies are needed to determine whether the alter174, 399-404 ations in salivary and plasma EGF by nocloprost 11. Konturek JW, Bielanski W, Konturek SJ, Bogdal J, Oteksy J. Distribution and release of epidermal is a specific effect of this PG analog in humans growth factor in man. Gut 1989, 30, 1194-1200 and whether it represents an important factor in 12. Hunt JN, Fisher NA. Aspirin-induced gastric bleeding stops despite rising plasma salicylate. Dig Dis the gastroprotective activity of this agent.
ACKNOWLEDGEMENTS The authors thank Prof. I. Amon and Dr. R. Wabnitz from Clinical Research of Asche AG, Hamburg, Germany, for the help in supplying the experimental agents and assistance in preparing this paper. This study was supported in part by research grants from Polish Academy of Sciences, Warsaw, Poland, CPBP 06.03.3.6and from Asche AG, Hamburg, Germany. REFERENCES 1. Vane JR. Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nature 1971, 231, 232-235 2. Konturek SJ, Obtulowicz W, Sito E, Oieksy J, Wilkon S, Kiec-Dembinska A. Distribution of prostaglandins in gastric and duodenal mucosa of healthy subjects and duodenal ulcer patients: Effects of aspirin and paracetamol. Gut 1981, 22, 283-289 3. Silverstein FE, Kimmey MB, Saunders DR, Levine DS. Gastric protection by misoprostol against
Sci 1980, 25, 135-139 13. Lanza FL, Royer GL, Nelson RS, Chen TT, Seckman CE, Rack MF. The effects of ibuprofen, indomethacin, aspirin, naproxen and placebo on the gastric mucosa of normal volunteers. A gastriscopic and photographic study. Dig Dis Sci 1979,24, 823828 14. Lanza FL, Royer GL, Nelson RS. Endoscopic evaluation of the effects of aspirin, buffered aspirin, and enteric coated aspirin on gastric and duodenal mucosa. N Engl J Med 1980, 303, 136-138 15. Douthwaite AH, Lintott GAM. Gastroscopic observation of the effect of aspirin and certain other substances in the stomach. Lancet 1938, 2, 12221225 16. Loebl DH, Craig RM, Culic D D , Rudolf0 AS, Falk J, Schmidt FR. Gastrointestinal blood loss: effect of aspirin, fenoprofen and acetaminophen in rheumatoid arthritis as determined by sequential gastroscopy and radioactive fecal markers. JAMA 1977, 237, 976-981 17. Chernish SM, Rosenak BD, Brunelle RL, Cabtree R . Comparison of gastrointestinal effects of aspirin and fenoprofen: a double-blind crossover study. Arthritis Rheum 1979, 22, 371-375 18. Ligumsky M, Golanska EM, Hansen DG, Kauffman GL. Aspirin can inhibit gastric mucosal cyclooxygenase without causing lesions in rats. Gastroenterology 1983, 84, 756-761 19. Konturek SJ, Radecki T, Brzozowski T, Gryglewski
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R. Gastric cytoprotection by epidermal growth factor. Role of endogenous prostaglandins and DNA synthesis. Gastroenterology 1981, 81, 438445 20. Cohen MM, MacDonald WC. Mechanism of aspirin injury to human gastroduodenal mucosa. Prostaglandins Leukotrienes Med 1982, 9, 241-255 21. Faust TW, Fedfern JS, Dodolsky I, Lee E, Grundy SM, Feldman M. Effects of aspirin on gastroduodenal mucosal prostaglandin E2 and F content and on gastric rnucosal injury in humans
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Received 2 June 1990 Accepted 17 August 1990
receiving fish oil and olive oil. Gastroenterology 1990, 98, 586591 22. Cooke AR. The role of acid in pathogenesis of aspirin-induced gastrointestinal erosions and hemorrhage. Am J Dig Dis 1973, 18, 225-237 23. Amon I. Nocloprost AH T475. Asche AG, Hamburg, 1989 24. Muller TA. Protective effects of prostaglandins against mucosal damage; current knowledge and proposed mechanisms. Am .IPhysioll984,245,601623
ISSN: 0036-5521 (Print) 1502-7708 (Online) Journal homepage: http://www.tandfonline.com/loi/igas20
Gastric Protection by Nocloprost against Aspirin Damage in Humans Possible Role of Epidermal Growth Factor S. J. Konturek, J. W. Konturek, N. Kwiecien, W. Obtulowicz, J. Oleksy, Z. Hebzda & I. Amon To cite this article: S. J. Konturek, J. W. Konturek, N. Kwiecien, W. Obtulowicz, J. Oleksy, Z. Hebzda & I. Amon (1991) Gastric Protection by Nocloprost against Aspirin Damage in Humans Possible Role of Epidermal Growth Factor, Scandinavian Journal of Gastroenterology, 26:3, 231-236, DOI: 10.3109/00365529109025036 To link to this article: http://dx.doi.org/10.3109/00365529109025036
Published online: 08 Jul 2009.
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Article views: 1
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Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=igas20 Download by: [University of California, San Diego]
Date: 05 April 2016, At: 17:22
Gastric Protection by Nocloprost against Aspirin Damage in Humans, Possible Role of Epidermal Growth Factor
Downloaded by [University of California, San Diego] at 17:22 05 April 2016
S. J. KONTUREK, J. W. KONTUREK, N. KWIECIEN, W. OBTULOWICZ, J. OLEKSY, Z . HEBZDA & I. AMON Institute of Physiology, Academy of Medicine, Cracow, Poland, and Dept. of Pharmacology, Asche AG, Hamburg, Germany Konturek SJ, Konturek JW, Kwiecien N, Obtuiowicz W, Oleksy J, Hebzda Z , Amon I. Gastric protection by nocloprost against aspirin damage in humans. Possible role of epidermal growth factor. Scand J Gastroenterol 1991, 26, 231-236 Ten healthy young male subjects took part in a double-blind, placebo-controlled crossover study to assess the effects of nocloprost on gastric microbleeding and endoscopic mucosal injury induced by the administration of aspirin (2.5 8). In addition, basal and pentagastrin-induced gastric acid and pepsin secretion and salivary and plasma contents of epidermal growth factor (EGF) were measured after placebo plus aspirin or nocloprost plus aspirin treatment in these subjects. Nocloprost (100 pg/ dose) significantly reduced spontaneous gastric microbleeding and almost completely prevented gastric mucosal injury induced by aspirin. Nocloprost failed to affect basal and pentagastrin-stimulated gastric acid and pepsin secretion but increased significantly the salivary outputs and plasma concentrations of EGF. In conclusion, nocloprost is effective in preventing gastric injury by aspirin even at a non-antisecretory dose, and this protection may involve an excessive release of EGF. Key words: Bleeding; cytoprotection; endoscopy; prostaglandins
Prof. S. J . Konturek, M . D . , Institute of Physiology, 31-531 Krakow, ul Grzegorzecka 16, Poland
Gastric mucosal injury by non-steroidal antiinflammatory drugs (NSAID) such as aspirin (ASA) has been ascribed predominantly to a deficiency of prostaglandins (PG) in the gastric mucosa due to the suppression of their mucosal biosynthesis (1,2). Studies in normal volunteers with PGE analogs, primarily misoprostol and enprostil, have shown that these agents, used in antisecretory doses, were highly effective in the protection of gastric mucosa against various NSAID (3,4). Such a protection against certain NSAID has also been obtained by using gastric acid inhibitors such as H2-receptor antagonists (5-7), suggesting that acid suppression may be a critical factor in gastroprotection against NSAIDinduced mucosal damage. Nocloprost is a unique PGE2 analog that has
been shown in experimental animals to display a potent gastroprotective activity against both acidindependent and acid-dependent ulcerogens when administered orally in non-antisecretory doses (8). Since the gastroprotective activity of nocloprost against aspirin damage has not been studied in humans, a double-blind, placebocontrolled crossover study was undertaken to examine the effects of this PG on mucosal damage caused by ASA, as assessed by the examination of gastric microbleeding and by gastric endoscopy. Furthermore, gastric acid and pepsin secretion and the release of epidermal growth factor (EGF), which has been implicated in the maintenance of mucosal integrity (9), have been investigated in these subjects.
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SUBJECTS AND METHODS
Subjects This study was approved by the Institutional Ethical Committee, and informed consent was obtained from each subject. The study was carried out on 10 male volunteers aged 21-25 years (mean age, 22 years) and weighing 67-74 kg (mean weight, 72 kg). All subjects were in good health without any previous or present gastrointestinal disease and with normal laboratory values for blood biochemistry, hematology, and urinalysis. All subjects underwent pre-study assessments, including examination of gastric microbleeding and gastroduodenoscopy to exclude any mucosal abnormalities or gastroduodenal ulcers. All subjects were hospitalized, and 1 week before the trial all medication was withdrawn and alcohol was forbidden. Study design The study was randomized, double-blind, placebo-controlled, and crossover. After the prestudy assessments, each volunteer underwent two treatments, one with nocloprost and another with placebo with 7-10 days of ‘washout’ in between. One tablet (100 pg) of nocloprost (Asche AG, Hamburg, Germany) or placebo was taken on day 1 at 0800 and 2000 h. Thirty minutes after taking the nocloprost or placebo tablet, the subject took two tablets of 0.5 g unbuffered aspirin (Polfa, Poland). On day 2, one nocloprost or one placebo tablet was taken at 0600 h, followed 30 min later by one ASA tablet. Post-treatment assessment was performed 2 h after the last ASA tablet. Assessments Pre- and post-treatment assessments were carried out as described previously ( 5 ) . In brief, venous blood samples were withdrawn to measure plasma salicylate concentration (10) and to determine plasma EGF levels. For this purpose the blood was collected in chilled tubes with heparin (10 U/ml) and the kallikrein inhibitor aprotinin (400 U/ml) (Trasylolm, Polfa, Poland) and centrifuged, and the plasma was frozen until radioimmunoassay of EGF as described (11). Then,
the rate of gastrin microbleeding into the gastric lumen was determined in three consecutive 10min gastric washings (5,12). Gastric endoscopy was performed with an Olympus GIF Q panendoscope to evaluate the mucosal appearance by means of the Lanza score system (13, 14). Grading was from 0 = normal mucosa to 4 = widespread involvement of the stomach with submucosal hemorrhages or superficial ulcerations. After completion of gastric washing and endoscopy, the collection of the saliva and gastric content was started and continued for 120min. During the initial 30-min period, gastric aspiration was collected to determine basal acid output, and ‘then parafilm chewing was carried out for 15 min to stimulate salivary secretion for the determination of EGF output. Finally, saliva and gastric secretion were collected for 45 min during intravenous infusion of pentagastrin ( 2 Fg/kg-h), to assess maximal acid output in these subjects. Nocloprost (supplied in tablets by Dr. R. Wabnitz from Asche AG, Hamburg, Germany), is a 9P-chloro-16,16-dimethyl-prostaglandinE 2 with clathrate of P-cyclodextrin to ensure its water solubility. The results are presented in the figures and the table as individual data and mean values (*SEM). Friedman’s two-way analysis of variance was used, supplemented, when applicable, by the Wilcoxon’s two-sided test. The significance level was set at 5%. RESULTS
Effects of nocloprost on gastric microbleeding and the endoscopic appearance of the gastric mucosa The pre-treatment assessment of gastric microbleeding was about 14.6 ? 3.9 pl/30 min, and in all 10 subjects examined endoscopically the mucosa was normal. After pre-treatment with placebo plus ASA, gastric microbleeding increased in all subjects and reached on average 129 33 p1/30 min. Endoscopy showed a significant increase in the mucosal damage in all subjects tested, and on average the score was 3.2 2 0.3 (Figs. 1 and 2 ) . The endoscopic changes ranged from a single submucosal hemorrhage (1 subject)
*
Gastroprotection by Nocloprost
233
350-
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.-C
. 8
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22 u
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z
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-
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to widespread involvement of the mucosa with over 10 hemorrhages and superficial erosions ( 5 subjects). After treatment with nocloprost, ASAinduced gastric microbleeding was reduced in 9 of the 10 subjects and averaged about 64 ? 15 PI/
NOCLOPROST
ASA
Fig. 2. Gastric endoscopic scores (individual data and means ? SEM) in healthy subjects treated with placebo plus ASA or nocloprost plus ASA. Asterisk indicates significant decrease below the placebo value.
30 min. Endoscopy showed a decrease in mucosal lesions in all subjects treated with nocloprost. The mean mucosal damage on average was 0.32 k 0.21, and this score was significantly lower than that in placebo-treated subjects. Basal and maximal pentagastrin-stimulated gastric acid and pepsin secretion in subjects
Table I. Effects of nocloprost plus aspirin or placebo plus aspirin on basal and pentagastrin-stimulated maximal acid and pepsin outputs and salivary secretion and plasma levels of EGF (under basal conditions and after parafilm chewing) and plasma levels of salicylate. Means t SEM of 10 determinations in 10 subjects Treatment Placebo Gastric H' (mmol/30 min) Basal Maximal Gastrin pepsin (mg/30 min) Basal Maximal Salivary EGF (ng/l5 min) Basal Chewing Plasma EGF (pg/ml) Basal Chewing Plasma salicylate (mg%)
+ ASA
Nocloprost
+ ASA
2.84 t 0.76 9.88 t 1.55
1.92 ? 0.57 8.36 t 1.10
21.28 t 4.02 56.51 t 9.64
23.28 t 4.84 59.55 t 9.48
17.50 t 6.54 29.41 t 7.62
48.60 ? 9.22" 54.85 t 12.42*
37.1 t 6.1 51.2 t 12.6 5.84 t 1.26
68.2 ? 14.3* 96.8 t 13.6* 4.88 2 1.36
* Significant ( p < 0.05j increase above the placebo value.
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treated with placebo plus ASA were not significantly different from the values obtained in these subjects after nocloprost plus ASA administration. Plasma concentrations of salicylates were also similar in subjects treated with placebo and nocloprost plus ASA (Table I). EGF outputs in salivary secretion were significantly increased during chewing both in placebo- and in nocloprost-treated subjects. The values of salivary outputs and plasma concentration of EGF were significantly higher in nocloprost-treated subjects than in those treated with placebo plus ASA (Table I). DISCUSSION This study provides evidence that nocloprost applied orally in a dose (1OOpg) that failed to affect basal or maximally stimulated gastric acid or pepsin secretion reduced significantly aspirininduced gastric microbleeding and gastric mucosal injury in humans. The gastrotoxic effect of NSAID has been well recognized ever since Douthwaite & Lintott (15) in 1938 described the first endoscopic observations of gastric mucosal injury in patients taking aspirin. Lanza et al. (13, 14) reported first detailed endoscopic evaluation and scoring system of mucosal injury in normal volunteers taking various NSAID. The mucosal injury by NSAID was assessed by measuring fecal blood loss with the chromium-51 technique or gastric blood loss in gastric washings as proposed by Hunt & Fisher (12). Endoscopic injury scores and the blood loss in subjects taking aspirin were then compared and found, to be closely correlated (16,17). Our present study used both these techniques to examine the alterations in gastric microbleeding and mucosal damage by aspirin and the possible protective activity of nocloprost. We found that aspirin at a total dose of 2.5 g causes severalfold increase in gastric bleeding and endoscopic mucosal damage in all tested subjects. Several mechanisms have been proposed to explain aspirin-induced gastric mucosal damage, but the inhibition of mucosal PG biosynthesis and subsequent decrease of mucosal resistance to noxious agents has been considered to be the
major factor (1,2). Ligumsky et al. (18) confirmed (19) that in rats aspirin causes a marked inhibition of PG btosynthesis (by about 90%) but suggested that the mucosal damage occurred only when the mucosa was additionally exposed to another irritant such as luminal acid. In contrast, Cohen & MacDonald (20) reported that aspirin in humans may induce fundic mucosal injury even without affecting mucosal PG significantly. These observations could suggest that the reduction in PG biosynthesis is neither sufficient (rats) nor even necessary (humans) for the production of gastric mucosal damage by aspirin. More detailed studies (21) confirmed the dependence of ASAinduced mucosal damage on the marked (about 95%) reduction in PG content in fundic mucosa. Our finding that xiocloprost is capable of reducing the extent of mucosal injury caused by aspirin is in keeping with the concept that the deficiency of the protective PG in the mucosa is the major factor in gastrotoxicity by NSAID. Other PGE analogs have also been reported to protect the gastric mucosa against the injury caused by NSAID (13,14), but these analogs were used in gastric inhibitory doses. Since the damaging action of aspirin is related to the rate of its absorption into the mucosal cells and depends on its lipophilic nature at acid pH and the pH of gastric contents (22), the reduction in gastric acid secretion seems to be the crucial factor in the mechanism of gastroprotection by these PG analogs. Nocloprost applied orally at the dose used was completely devoid of gastric inhibitory action both in experimental animals (8) and, as shown in this report, also in humans. Although nocloprost is chemically more stable and, like other PGE analogs, is well absorbed from the gastrointestinal tract, it does not reach the systemic circulation because of the metabolism in the mucosa and first-passage uptake and inactivation by the liver (8,23). This may explain the failure of nocloprost administered orally to affect gastric acid secretion or to provoke any adverse effects such as diarrhea or abortion (23). Thus, after oral administration, nocloprost mainly acts locally on the gastric rnucosa to maintain its protective mechanisms, including stimulation of mucus-alkaline secretion, enhancing mucosal blood flow, and
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1300 mg of aspirin: an endoscopic study. Dig Dis promoting mucosal growth, as other PGE analogs Sci 1986, 31, 137-1418 do (24). 4. Cohen MM, McGready DR, Clark L, Sevilius H. It may be that these and Yet other u ~ k r " J n Protection against aspirin-induced antral and duodenal mucoia with enprostil. Gastroenterology properties of nocloprost are operative in the 1985, 88, 382-386 stomach in providing protection to the mucosa 5. Konturek SJ, Kwiecien N, Obtulowicz W, Polanski against NSAID-induced damage. The results of M, Kopp B, Oteksy J. Comparison of prostaglandin E2 and ranitidine in prevention of gastric bleeding this study suggest the possible implication of by aspirin in man. Gut 1983, 24, 89-93 EGF as a mediator of the favorable influence of 6. Roth SH, Bennett RE, Mitchell CS, Hartmann RJ. nocloprost on human gastric mucosa. EGF was Cimetidine therapy in NSAID gastropathy. Double blind long-term evaluation. Arch Intern Med 1987, reported to show gastroprotective activity against 147, 1798 aspirin damage in rats, and this effect was inde7. Rachmilewitz D, Polak D, Eliskin R, et al. Cimependent of the mucosal generation of PG (19). tidine significantly decreases indomethacin-induced gastro-duodenal mucosal damage. GastroenterNSAID appear, however, to suppress the release 1986, 90, 1596 ology of EGF from the salivary glands (9), and this may 8. Konturek SJ, Brzozowski T, Drozdowicz D, Amon contribute, at least in part, to the damaging action I. Nocloprost, a stable PGE2 analog with unique local gastroprotective and ulcer-healing activity. of these drugs on the gastric mucosa. In the Gastroenterology 1990, 98, A70 present study the administration of nocloprost in 9. Konturek SJ. Role of growth factors in gasaspirin-treated subjects resulted in a significant troduodenal protection and healing of peptic ulcers. Gastroenterology Clin N Am 1990, 19, 41-66 increase in EGF release into the saliva and in Satzman A . A fluorometric method for the esti10. the plasma concentration of this peptide. Further mation of salicylate in blood. J Biol Chem 1948, studies are needed to determine whether the alter174, 399-404 ations in salivary and plasma EGF by nocloprost 11. Konturek JW, Bielanski W, Konturek SJ, Bogdal J, Oteksy J. Distribution and release of epidermal is a specific effect of this PG analog in humans growth factor in man. Gut 1989, 30, 1194-1200 and whether it represents an important factor in 12. Hunt JN, Fisher NA. Aspirin-induced gastric bleeding stops despite rising plasma salicylate. Dig Dis the gastroprotective activity of this agent.
ACKNOWLEDGEMENTS The authors thank Prof. I. Amon and Dr. R. Wabnitz from Clinical Research of Asche AG, Hamburg, Germany, for the help in supplying the experimental agents and assistance in preparing this paper. This study was supported in part by research grants from Polish Academy of Sciences, Warsaw, Poland, CPBP 06.03.3.6and from Asche AG, Hamburg, Germany. REFERENCES 1. Vane JR. Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nature 1971, 231, 232-235 2. Konturek SJ, Obtulowicz W, Sito E, Oieksy J, Wilkon S, Kiec-Dembinska A. Distribution of prostaglandins in gastric and duodenal mucosa of healthy subjects and duodenal ulcer patients: Effects of aspirin and paracetamol. Gut 1981, 22, 283-289 3. Silverstein FE, Kimmey MB, Saunders DR, Levine DS. Gastric protection by misoprostol against
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Received 2 June 1990 Accepted 17 August 1990
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