Current Medical Research and Opinion (1996), 13, No. 7, 417-425
| |
The effect of 5-aminosalicylate and para-aminosalicylate
on the synthesis of prostaglandin E2 and
leukotriene B4 in isolated colonic mucosal cells
|
Christoph Schmidt, M.D.,
Thomas Fels, M.D., Bernhard Baumeister, M.D. and Hans Vetter, M.D.
Medical Poliklinik, Accepted: 19th December 1995 |
![]() |
Both 5-ASA and PAS did not alter the PGE2 production but, compared with PAS and the control, 5-ASA decreased the LTB4 synthesis in a dose-related fashion. As a result, the LTB4 PGE2 ratio was significantly diminished by 10-4 mol/l 5-ASA. These findings are consistent with those of other authors, indicating that 5-ASA, at least in part, modulates the colonic eicosanoid synthesis. In contrast, PAS did not influence the mucosal production of PGE2 and LTB4 and therefore must exert some other biochemical action in order to explain its therapeutic effects in the treatment of Crohn's disease or ulcerative colitis.
Crohn's disease and ulcerative colitis are both characterized by diarrhoea, ulceration and inflammatory infiltration of intestinal mucosa. Eicosanoid mediators including prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) play an important role in this inflammatory process and their synthesis is increased in the intestinal mucosa of patients with chronic inflammatory bowel disease.12, 16 Both steroids and sulfasalazine are effective in decreasing elevated levels of PGE2 and LTB4 in these diseases
The purpose of this study is to evaluate whether 5-ASA and PAS have the same effects on colonic synthesis as steroids and sulfasalazine in decreasing the mucosal production of eicosanoids
Eight biopsies were taken by colonoscopy from the distal end of the descending colon, placed in physiological saline, transported to the laboratory, and weighed. The biopsies were similar in size and had an average weight of 5 mg. Additional specimens were obtained for histological examination to exclude any pathological findings.
To isolate mucosal cells from the biopsies, the specimens were incubated in medium A (0.5 m M NaH2PO4, 20 mM NaHCO3, 1 mM Na2HPO4, 70 mM NaCl, 5 mM KCl, 11 mM glucose, 50 mM HEPES, 2 mM EDTA and 20 g/l bovine serum albumin, pH 7.4) for 30 min, taken out, and then added to medium B (analogous to medium A, but including 1 mM MgCl2, 1 mM CaCl2, 10 g/l bovine serum albumin, 0.25 mg/ml pronase, and without EDTA) for a further 60 min. The suspension was centrifuged to sediment the isolated cells and passed through a 60 mm nylon filter to remove mucus and remaining cell clumps. Then the cells were resuspended with medium C (analogous to medium B, but with 1 g/l bovine serum albumin) and the concentration of the suspension was determined by cytometry under light microscopy. To identify dead cells, trypan blue was added, which stained less than 6% of cells in each assay. The suspension was diluted to a concentration of 500 000 cells/ml with medium C, and transferred into vials.
Concurrently, 5-aminosalicylate and para-aminosalicylate were diluted with distilled water to a concentration of 10-4 or 10-6 mol/l. Then 3 sets of vials were set up: 5-ASA, PAS and a control (distilled water). They were incubated in a water bath at 37°C for 0 (to determine the basal PGE2 concentration of the incubation medium), 10, 20, 30 or 45 min, respectively. The calcium ionophore A 23187 (dissolved in DMSO, final concentration 10 µmol/l) was added for the last 15 min to the 45 min vials. At the end of each incubation time, the suspensions were recentrifuged (12 000 U/min), and the supernatant was carefully decanted, frozen and stored for later determination of PGE2 and LTB4 by radioimmunoassay (125I PGE2 RIA and 3H LTB4 RIA by NEN/DuPont, Dreieich, Germany). Light microscopy showed no evidence of remnant cells in the supernatant.
For statistical analysis, the Wilcoxon test for pairs and the Mann-Whitney U-test for independent samples were performed.
The leukotriene B4 concentration was below the detection limit of the radioimmunoassay until stimulated by the calcium ionophore. The LTB4 accumulation after 45 min incubation is shown in Figure 3. Compared with PAS and the control, vials with 5-ASA reveal a diminished LTB4 synthesis when incubated with 10-4 mol/l 5-ASA as opposed to 10-6 mol/l. Because of this dose-related effect of 5-ASA, the LTB4/PGE2 ratio decreases significantly ( p < 0.05) when 5ASA was used in a higher concentration (10-4 mol/l). PAS did not change this ratio.
As shown in Figures 1 and 2, the PGE2 concentration in all suspensions rose significantly at the beginning of the incubation. Following this, there was only a moderate further increase between minutes 10 and 30. This suggested a reduced capacity of the isolated cells to produce prostaglandins, caused either by an increased mucosal catabolism or, more likely, by the shortage of substrate, as neither arachidonic acid nor any other predecessor of PGE2 were added to the medium. The stimulation with the calcium ionophore A 23187, however, led to another marked rise of PGE2 at minute 45. This drug increased the permeability of cell membranes to calcium. As the arachidonate metabolism is integrated with Ca2+ mobilization, calcium ionophores allow an energy- and carrier-independent influx of Ca2+ across impermeable membranes and thus stimulate the synthesis of eicosanoids to their maximum. The fact that consecutive measurements of PGE2 concentrations showed increasing values, even though separate vials were used for different incubation times, underlined the reliability of our measurements. Leukotriene B4 became detectable only after stimulation with the calcium ionophore between minutes 30 and 45.
The aim of this study was to find out whether 5-ASA and PAS exert their therapeutic action on patients with inflammatory bowel disease by altering the PGE2 and LTB4 synthesis of colonic mucosa. 5-ASA and PAS are chemically related to sulfasalazine which has, in addition to steroids, been used for the treatment of Crohn's disease and ulcerative colitis for decades. 5-ASA was shown to be the pharmacologically active component of sulfasalazine, whose sulfapyridine moiety is responsible for most of its side-effects. PAS was used in the treatment of tuberculosis before it was proved to be as effective as 5-ASA for chronic inflammatory bowel disease. Para-aminosalicylate is chemically characterized as 4-aminosalicylate and is thus closely related to 5-ASA.
Although steroids and sulfasalazine have been used for a long time in the therapy of Crohn's disease and ulcerative colitis, it is not known how these drugs influence the pathophysiology of these diseases. There is some evidence that the inflammatory process is, at least in part, influenced and caused by prostaglandins and leukotrienes. These eicosanoids are found to be significantly increased in active disease and both PGE2 and LTB4 are decreased by steroids or sulfasalazine.18 The rate of PGE2 synthesis correlates well with disease activity3 and is also a reliable predictor of the response to drug treatment. Patients with increased PGE2 levels show a substantial risk of relapse.4
Prostaglandin E2 acts synergistically with other mediators of inflammation,13 causes central hyperthermia and sensitizes local receptors to pain.7 PGE2 is a potent vasodilator, increases vascular permeability causing local oedema, and mediates the secretory diarrhoea observed in Crohn's disease and ulcerative colitis. Furthermore, PGE2 regulates the immune response by enhancing it at low concentrations, but suppressing it at higher concentrations.9 In addition, PGE2 promotes the cytoprotective activity in intestinal mucosa.2 Therefore prostaglandin E2 does not only contribute to negative aspects of the disease, but may also activate healing mechanisms. This may explain why isolated modulation of the PGE2 level does not necessarily improve the course of chronic inflammatory bowel disease. Inhibition of prostaglandin E2 synthesis by indomethacin, for example, does not improve but, in fact, worsens the symptoms of Crohn's disease and ulcerative colitis.8
Leukotriene B4 amplifies and modulates the inflammatory response in active Crohn's disease or ulcerative colitis. LTB4 induces neutrophil aggregation and degranulation, increases microvascular permeability and induces the release of lysosomal enzymes.6, 10
As 5-ASA and PAS are chemically related to sulfapyridine, it was suggested that they exert their pharmacological action in the same way as sulfapyridine (and steroids), which alter the eicosanoid metabolism in intestinal mucosa. The results of our study showed that there were considerable differences between 5ASA and PAS. The leukotriene synthesis was markedly decreased in concentrations of 10-4 mol/l, whereas 10-6 mol/l did not show this effect (Figure 2). This finding underlines the dose-related effect of 5-ASA in altering the leukotriene production of colonic mucosal cells. PAS or the control (distilled water, the diluent for 5-ASA and PAS) did not alter LTB4 synthesis. The prostaglandin synthesis was not affected by 5-ASA (or PAS), as shown in Figures 1 and 2. As a result, 5-ASA decreased the LTB4/PGE2 ratio significantly ( p < 0.05). This could contribute to the therapeutic effect of 5-ASA. By shifting the LTB4/PGE2 ratio towards prostaglandin E2, the inflammatory process in inflammatory bowel disease is diminished. LTB4 is a potent mediator of inflammation, whereas PGE2, as discussed before, exerts a more ambivalent role by worsening, but also ameliorating, the inflammatory process. In addition, as only healthy subjects were examined, the influence of 5-ASA on the eicosanoid metabolism may be underestimated. We feel that the potential number of untreated patients with chronic inflammatory bowel disease would be too small for statistical evaluation. However, it is quite likely that the increased LTB4 and PGE2 synthesis in patients with active disease are much more affected by 5-ASA than in healthy subjects with normal eicosanoid levels. The alteration of mucosal eicosanoid production has been reported by other authors previously.17
No effect of para-aminosalicylate on the eicosanoid synthesis of colonic mucosa could be demonstrated in our study. There was no alteration either in PGE2 or LTB4 production in the cell suspension. Again these findings are consistent with other studies, indicating that PAS does not alter the eicosanoid metabolism.11 However, this result is confusing for two reasons. Firstly, the clinical benefit of PAS in the treatment of Crohn's disease and ulcerative colitis is well established.1, 15 Secondly, the chemical structure of PAS (= 4-ASA) is quite similar to 5-ASA. Nevertheless, our study confirms the striking fact that two chemically closely related drugs, which show the same clinical effect on inflammatory bowel disease, exert their action by different mechanisms. 5Aminosalicylate alters the LTB4/PGE2 ratio in colonic mucosa, but the way para-aminosalicylate works remains unknown.
The enhanced PGE2 and LTB4 production in patients with active Crohn's disease or ulcerative colitis might be the consequence rather than the cause of the inflammatory process in these diseases.14 However, as LTB4 and PGE2 amplify and modulate the signs and symptoms of chronic inflammatory bowel disease, the pharmacological alteration of these mediators of inflammation contributes to the healing process in these patients. The growing understanding of how empirical drugs like steroids and sulfasalazine, as well as 5-ASA and PAS, influence the clinical course of chronic inflammatory bowel disease, will provide a better knowledge of the pathophysiology and thus of newly developed therapeutic means in the treatment of Crohn's disease and ulcerative colitis.
2. Hawkey, C. J., and Rampton, D. S., (1985). Prostaglandins and the gastrointestinal mucosa: are they important in its function, disease, or treatment? Gastroenterology, 89, 1162-1188.
3. Lauritsen, K., Laursen, L. S., Bukhave, K., and Rask-Madsen, J., (1986). Effects of topical 5-aminosalicylic acid and prednisolone on prostaglandin E2 and leukotriene B4 levels determined by equilibrium in vivo dialysis of rectum in relapsing ulcerative colitis. Gastroenterology, 91, 837-844.
4. Lauritsen, K., Laursen, L. S., Bukhave, K., and Rask-Madsen, J., (1988). Use of colonic eicosanoid concentrations as predictors of relapse in ulcerative colitis. Gut, 29, 1316-1321.
5. Lauritsen, K., Laursen, L. A., Bukhave, K., and Rask-Madsen, J., (1986). Effects of topical 5-aminosalicylic acid and prednisolone on prostaglandin E2 and leukotriene B4 levels determined by equilibrium in vivo dialysis of rectum in relapsing ulcerative colitis. Gastroenterology, 91, 837-844.
6. Lobos, E. A., Sharon, P., Stenson, W. F., (1985). Chemotactic activity in inflammatory bowel disease: role of leukotriene B4. Dig. Dis. Sci., 32, 1380.
7. Malmsten, C. L., (1985). Prostaglandins, thromboxanes and leukotrienes in inflammation. Semin. Arthritis Rheum., 15, 29-35.
8. Marcus, A. J., (1985). Eicosanoids as bioregulators in clinical medicine. Am. J. Med., 78, 805-810.
9. Mertin, J., Stackpoole, A., and Shumway, S.J., (1984). Prostaglandins and cell-mediated immunity. Transplantation, 37, 396-402.
10. Naccache, P. H., and Sha'afi, R. I., (1983). Arachidonic acid, leukotriene B4, and neutrophil activation. Ann. N. Y. Acad. Sci., 414, 125-139.
11. Nielsen, O. H., and Ahnfeld-Ronne, I., (1988). 4-Aminosalicylic acid, in contrast to 5aminosalicylic acid, has no effect on arachidonic acid metabolism in human neutrophils, or on free radical 1,1-diphenyl-1-2-picrylhydrazyl. Pharmacol. Toxicol., 62, 223-226.
12. Schmidt, C., Kipnowski, J., and Baumeister, B., (1988). Veränderungen im Eikosanoid-stoffwechsel bei entzündlichen Darmerkrankungen. Klin. Wochenschr., 66 (Suppl 13), 211.
13. Schumert, R., Towner, J., and Zipser, R. D., (1988). Role of eicosanoids in human and experimental colitis. Dig. Dis. Sci., 33 (Suppl 3), 58S-64S.
14. Rask-Madsen, J., Bukhave, K., et al., (1984). Prostaglandins in the gastrointestinal tract. Acta Med. Scand., 685 (Suppl), 30-46.
15. Selby, W. S., Bennett, M. K., and Jewell, D. P., (1984). Topical treatment of distal ulcerative colitis with 4-aminosalicylic acid enemas. Digestion, 29, 231-234.
16. Sharon, P., and Stenson, W. F., (1984). Enhanced synthesis of leukotriene B4 by colonic mucosa in inflammatory bowel disease. Gastroenterology, 86, 453.
17. Sircar, J. C., Schwender, C. F. and Carethers, M. E., (1983). Inhibition of soybean lipogenase by sulfasalazine and 5-aminosalicylate: a possible mode of action in ulcerative colitis. Biochem. Pharmacol., 32, 170-172.
18. Wallace, J. L., Wallace, K., MacNaughton, W. K., Morris, G. P. and Beck, P. J., (1989). Inhibition of leukotriene synthesis markedly accelerates healing in a rat model of inflammatory bowel disease. Gastroenterology, 96, 29-36.