Scandinavian Journal of Gastroenterology
ISSN: 0036-5521 (Print) 1502-7708 (Online) Journal homepage: http://www.tandfonline.com/loi/igas20
Effect of Drugs on Colonic Eicosanoid Accumulation in Active Ulcerative Colitis R. Eliakim, F. Karmeli, M. Chorev, E. Okon & D. Rachmilewitz To cite this article: R. Eliakim, F. Karmeli, M. Chorev, E. Okon & D. Rachmilewitz (1992) Effect of Drugs on Colonic Eicosanoid Accumulation in Active Ulcerative Colitis, Scandinavian Journal of Gastroenterology, 27:11, 968-972, DOI: 10.3109/00365529209000172 To link to this article: http://dx.doi.org/10.3109/00365529209000172
Published online: 08 Jul 2009.
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Date: 19 April 2016, At: 23:57
Effect of Drugs on Colonic Eicosanoid Accumulation in Active Ulcerative Colitis R. ELIAKIM, F. KARMELI, M. CHOREV, E. OKON & D. RACHMILEWITZ Dept. of Medicine, Hadassah University Hospital-Mount Scopus, Hebrew University-Hadassah Medical School, Jerusalem, Israel
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Eliakim R, Karmeli F, Chorev M, Okon F, Rachmilewitz D. Effect of drugs on colonic eicosanoid accumulation in active ulcerative colitis. Scand J Gastroenterol 1992;27:96%972. The effect of immunosuppressive drugs, 4-aminosalicylicacid (4-ASA), acetyl 5-aminosalicylic acid ( S ASA), and ketotifen on human colonic eicosanoid accumulation was evaluated in view of enhanced accumulation in patients with active ulcerative colitis. Azathioprine (100 pg/ml), cyclosporin (100 pg/ ml), and methotrexate (100 wg/ml) significantly inhibited, by 25-35%, prostaglandin E, (PGE,) accumulation by organ-cultured colonic mucosa of ulcerative colitis patients. Methotrexate was the only immunosuppressive drug that inhibited leukotriene B4 (LTB4) accumulation (50%), whereas azathioprine inhibited the accumulation of leukotriene C4 (LTC,) (25%). 5-ASA and its metabolite, acetyl 5-ASA, inhibited by 20-70% PGE,, LTB,, and LTC4 accumulation in the culture, supporting the contention that acetyl 5-ASA is as active as 5-ASA in these respects. 4-ASA had no effect on any of the eicosanoids. Ketotifen, a mast cell stabilizer, significantly inhibited the accumulation of PGE,, LTBJ, and LTC4by 3340%. These results suggest a potential, new, unrecognized mode by which the immunomodulators induce part of their therapeutic effects in inflammatory bowel disease and support the contention that acetyl 5-ASA is as active as 5-ASA. The results obtained also indicate that ketotifen, used effectively in the prevention of bronchial asthma, inhibits the accumulation of colonic eicosanoids and, thus, may be of value in the treatment of inflammatory bowel disease. Key words: Acetyl 5-aminosalicylic acid; 4-aminosalicylic acid; 5-aminosalicylic acid; azathioprine; chloroquine; cyclosporin; ketotifen; methotrexate; ulcerative colitis
R. Eliakim, M. D . , Dept. of Medicine, Hadamah Unioersity Hospital, Mount Scopus, P.O. Box 24035, Jerusalem 91240, Israel
The inflamed colonic mucosa in ulcerative colitis is infiltrated by equally prominent components of ‘active’ and ‘chronic’ leukocyte populations (1-3). The presence of neutrophils and macrophages suggests the involvement of soluble chemotactic and other mediators, causing migration of inflammatory cells into the mucosa (4). We and others have shown increased colonic accumulation of mediators-that is, prostaglandins (PG), leukotriene B4 (LTB4), plateletactivating factor (PAF), and interleukin 1 (IL-1)-by the inflamed colonic mucosa in patients with active ulcerative colitis (5-9). The therapeutic effects of the drugs currently used for the treatment of inflammatory bowel disease, such as corticosteroids, sulfasalazine, and its active moiety, 5aminosalicylic acid (5-ASA), are ascribed in part to their induced inhibition of the colonic accumulation of inflammatory mediators ( 5 , & l l ) . All these drugs have been shown to inhibit the generation of colonic eicosanoids, PAF, and IL-1 (5,8-11). 4-Aminosalicylic acid (4-ASA) and the immunomodulators azathioprine, cyclosporin, and methotrexate have also been used successfully in inflammatory bowel disease (1218). The therapeutic efficacy in ulcerative colitis of disodium
cromoglycate, a mast cell stabilizer, remains controversial, whereas that of chloroquine is unknown or preliminary (1922). Ketotifen, a potent eosinophil and mast cell stabilizer, has been shown to be effective in the prevention of gastric and colonic mucosal damage in the rat (23,24). The effects of these drugs on colonic eicosanoid accumulation in ulcerative colitis has not yet been established. The aim of the present study was to evaluate the effects of immunosuppressive drugs, ketotifen, chloroquine, 4-ASA, and acetyl 5-ASA, the possible active metabolite of 5-ASA, on eicosanoid accumulation by cultured colonic mucosa in patients with active ulcerative colitis. MATERIALS A N D METHODS Colonic biopsy specimens were obtained with an Olympus CF-1OL colonoscope 8-10cm from the anus from 25 untreated patients with active ulcerative colitis (mean age, 40.8 years (range, 20-81 years); mean duration of disease, 4.3 years; and disease extent s 6 0 cm in 65%) and from 11 subjects without any signs or symptoms of gastrointestinal disease (mean age, 50.5 years; range, 18-81 years). No
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Effect of Drugs on Colonic Eicosanoids
complications ensued. The study was approved by the Committee on Research Involving Human Subjects of the Hadassah University Hospital. In all ulcerative colitis patients the disease was confirmed histologically. Biopsy specimens were obtained with biopsy forceps, washed in 0.15 M NaCI, weighed, and cultured within 10 min after excision. Organ culture was performed as previously described by us (5). In brief, tissue specimens were cultured (37"C, 5% C 0 2 , 95% air) for 24 h. The tissue was placed on a metal grid over the central well of the culture disk (Falcon) and cultured in 0.8 ml RPMI 1640 (Bio-Lab, Israel), containing penicillin (100 U/ml) and streptomycin (100 pg/ml). After 24 h of incubation specimens and media were transferred to separate tubes. All drug studies were performed on specimens obtained from ulcerative colitis patients. Specimens obtained from the same subject were cultured in medium containing either 5-ASA (100 to 500 pg/ml) (Sigma, Israel), acetyl5-ASA (100 to 500pg/ml) (kindly provided by Dr. Chorev, Pharmacy School, Hebrew University, Jerusalem), 4-ASA (50 to 500 pg/ml) (Sigma, Israel), azathioprine (25 to 100 pg/ml) (Pharmachemie, The Netherlands), cyclosporin (1to 100 pg/ ml) (Sandoz, Switzerland), methotrexate (1 to 100 pg/ml) (Pharmachemie), ketotifen (1 to 100 pg/ml) (Sandoz), chloroquine (25 to lOOpg/ml) (Sigma). Stock solutions of the drugs were as follows: azathioprine, chloroquine, and ketotifen, 10 mg/ml H 2 0 ;methotrexate, 100 mg/ml H 2 0 ;cyclosporin, 100 mg/ml ethanol; and 5-ASA and acetyl 5-ASA, 100 mg/ml dimethyl sulfoxide. Working solutions were prepared in RPMI, final pH of 7.5, identical to that of RPMI alone. In these series of experiments one biopsy specimen obtained from each subject was cultured in drug-free medium and served as control. At the end of the culture period the tissue and organ culture medium were kept at -20°C for up to a month before prostaglandin or leukotriene determination. To test whether there were drug toxicity effects on the specimens, they were assessed both histologically and for lactic dehydrogenase accumulation in the medium. The tissue was homogenized in 1.0 ml ice-cold 50 mM Tris-HCI buffer, pH 7.0, containing 0.02 M ethylenediaminetetraacetic acid (EDTA). The homogenate was extracted twice with two volumes of ether, and both the aqueous phase and the incubation media were then assayed for PGE2, LTC4, and LTB4 concentration by means of a radioimmunoassay, as previously described (25, 26). Statistical methods used were the t test for paired (drug studies) and unpaired (comparison between control and ulcerative colitis) data; p values were two- or one-tailed, as stated in the tables. RESULTS Eicosanoid accumulation in the medium of organ-cultured colonic mucosa of patients with active ulcerative colitis was significantly higher than the respective accumulation in cul-
969
Table I. Eicosanoid accumulation (pg/mg/24 h) by cultured colonic mucosa
PGEi LTB LTC,
,
Ulcerative colitis
Normal subjects
1546 f 182* 46 t 9* 244 f 17*
305 f 41 20 f 4 180 ? 22
Biopsy specimens obtained from patients with active ulcerative colitis and normal controls were cultured for 24 h in medium containing RPMI, penicillin (100 U/ml), and streptomycin (100 pg/ml), as described in Methods. At the end of the culture period the tissue and organ culture medium were kept at -2O"C, processed as described in Methods, and assayed for prostaglandin E Z (PGE,), leukotriene B4 (LTB,), and leukotriene C4 (LTC,). Results are mean t SE of 25 subjects with ulcerative colitis and 11 controls. * Denotes significantly different from normal controls ( p < 0.05).
tures of colonic mucosa obtained from normal controls (Table I). In active ulcerative colitis patients PGE2 generation was five times higher than in controls. LTB4 generation was double, whereas LTC4 generation was only 25% higher. The ratio of PGE2 levels in tissue and medium was 35:65 in both ulcerative colitis patients and controls, and the ratios for LTB4 and LTC4 was 17 : 83. The effect of the cytotoxic immunomodulators used in inflammatory bowel disease-azathioprine, cyclosporin, and methotrexate-on colonic eicosanoids was tested. The addition of each of these (100 pg/ml) drugs to the culture medium of rectal biopsy specimens obtained from patients with active ulcerative colitis significantly inhibited PGE2 accumulation by 25% to 35% (Table 11). Azathioprine, when administered in a lower dose of 50 pg/ml, also significantly inhibited colonic PGE? accumulation (35%, n = 6), whereas lower doses of cyclosporin and methotrexate had no effect. Only methotrexate (100 pg/ml) inhibited LTB4 accumulation (by 5 0 % , p < 0.02), whereas azathioprine (100 pg/ml) was the only drug to inhibit LTC4 accumulation (by 25%, p < 0.02) (Table 11). The effect of 4-ASA, 5-ASA, and its metabolite, acetyl 5-ASA, on eicosanoid accumulation was also evaluated. As previously reported, the addition of 5-ASA (500 pg/ml) to the culture medium of colonic specimens from patients with active ulcerative colitis significantly inhibited PGEz and LTC4 accumulation by 70% and 30%, respectively (Table 11). 5-ASA (250 pg/ml) significantly decreased PGEz and LTC4 accumulation by 47% and 46%, respectively ( n = 6, p < 0.05). Lower dose of 5-ASA (100 pg/ml) decreased PGE2 and LTC4, but not to statistically significant levels. The addition of the metabolite, acetyl 5-ASA (500 pg/ml), to the culture medium effectively and significantly inhibited the accumulation of PGE2, LTB4 and LTC4 by 20% to 65% (Table 11). At a dose of 100 and 250pg/ml, it decreased LTB4 by 50% and LTC4 by 20% ( n = 6, p < 0.05). When 4-ASA (100 pg/ml) was added to the culture medium, there was no effect on PGE2 or LTB4 accumulation (Table 11). The effect of ketotifen (Zaditenm), a mast cell stabilizer,
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R . Eliakim et al.
Table 11. Effect of drugs o n eicosanoid accumulation by cultured colonic mucosa
Azathioprine Methotrexate Cyclosporin 5-ASA
Acetyl 5-ASA
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4-ASA Ketotifcn
Chloroquine
25 (5)t 50 (6) 100 ( 5 ) 1 (6) 10 (6) 100 (7) 1 (4) 10 (4) 100 (7) 100 ( 6 ) 250 ( 6 ) 500 ( 6 ) 100 (8) 250 (7) 500 (8) 50 (8) 100 (8) 0.1 (4) 1 (4) 10 (3) 100 ( 6 8 ) 25 ( 5 ) 50 (5) 75 (3) 100 (9)
62 t 14 66 t 9* 6 9 + 16 102 t 10 Rc)? 1 1 65 t 11* 90 t 20 119+-33 76 t 9% 85 t 16 58 t 8** 25 t 5** 103 -t 11 9 8 2 16 33 t 7** 135 2 34 133 t 23 118 t 14 111 - + 7 78 t 15 54 + 9* 57 t 15* 63 t 10* Not tested 53 t 7**
7 4 t 13 106 t 27 97 -t 13 105 t 20 7 2 2 13 52 2 lo** 118 t 36 95 rt 33 107 t 14 92 t 28 97 t 32 85 -t 16 62 t 14** 40 2 7** 65 t 11** 97 2 25 7 2 t 14 1002 16 7 9 t 13 94 t 23 43 t 8* 90 t 24 86 t 29 92 2 20 75 t 15
6 5 t 13 77 t 12 75 t 7* 177 t 25 125 t 37 8 9 t 17 Not tested 113 t 27 130 2 29 81 t 24 55 2 14* 54 t 7** 99 2 12 81 t 12 74 7** Not tested Not tested 159 2 45 109 t 13 Not tested 68 2 7' 150 t 35 1 0 4 2 18 116239 9 2 2 10
*
Biopsy specimens obtained from patients with active ulcerative colitis were cultured for 24 h in RPMI medium with or without drugs, as described in Methods. Results are given as percentage drug-free medium with mean 2 SE. Prostaglandin E2 (PGE2), leukotriene B, (LTBJ, and leukotriene C, (LTC,) (pg/mg/24 h) accumulations in drug-free medium were considered 100%. 5-ASA = 5-arninosalicylic acid; 4-ASA = 4-acetylsalicylic acid. * Denotes statistically different from drug-free medium, p < 0.05 ( t test for paired data, one-tailed). * * t Test for paired data, two-tailed. t No. of experiments.
shown to prevent mucosal damage and eicosanoid generation in two rat models of inflammatory bowel disease, and of chloroquine o n human colonic eicosanoid accumulation was tested. The addition of ketotifen (100 pg/ml) to the culture medium of rectal specimens from patients with active ulcerative colitis significantly inhibited the accumulation of PGE2, LTC,, and LTB4 by 33% to 60% (Table 11). Chloroquine, at the dose of 100kg/ml, significantly inhibited PGE2 accumulation by 40% (Table 11). It had no effect on LTB, or LTC4accumulation. Lower doses (10 pg/ml) had no effect on eicosanoid generation. Histologic examination of biopsy specimens after their culture in the presence of the various drugs in the doses tested and examination of lactic dehydrogenase levels in culture medium did not disclose any toxic effects of the drugs on the tissue. DISCUSSION Substantial evidence has accumulated in support of the role of eicosanoids in the pathogenesis of inflammatory bowel disease, although their specific contribution remains unclear (5-7, 10, 11, 27, 28). In the present study high levels of eicosanoids were found in the medium of cultured colonic mucosa of patients with active ulcerative colitis. These
mediators play a role in the recruitment of leukocytes to the diseased area and the amplification of the inflammatory process via activation of oxygen free radicals and proteases from neutrophils, causing more tissue damage. Other effects include a change in vascular permeability, causing vasoconstriction and stimulation of smooth-muscle contraction (5-9, 29, 30). An orally active inhibitor of leukotriene synthesis has been shown to accelerate healing in a rat model of colitis, adding further evidence for the role of these mediators in colitis (28,31). Azathioprine is used for the treatment of steroid-dependent patients with refractory Crohn's disease or for treating fistula. Its role in ulcerative colitis remains unclear, and it is used in patients with refractory disease who wish to avoid colectomy (12,13, 18). Cyclosporin and methotrexate are two promising drugs used so far in small trials for the treatment of inflammatory bowel disease. Cyclosporin, a polypeptide derived from the fungus Tolypocadium inflaturn selectively inhibits activation of helper T cells and interleukin-2, -3, -4, and -5 production (14,32). Methotrexate, a folic acid antagonist, has anti-metabolic and anti-inflammatory properties (15,33). Bernard et al. (34) have shown that methotrexate decreases ileal PGEz content in the rat. In the present study all three drugs significantly decreased colonic PGE2 accumulation; azathioprine also decreased
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Effect of Drugs on Colonic Eicosanoids
LTC4, and methotrexate decreased LTB4 accumulation, thus shedding some light onto a potential additional mechanism whereby these drugs may be beneficial in the treatment of inflammatory bowel disease. 5-ASA is the therapeutically active moiety of sulfasalazine. 5-ASA is subsequently acetylated in the gut wall to N-acetyl-5-ASA (35,37), which was considered to be therapeutically inert (38). Acetylation is an irreversible process (39), and the metabolite is present in the plasma shortly after both oral and rectal administration (40,41). The halflife of 5-ASA is short (s1.5 h), whereas that of N-acetyl 5ASA is longer (
ISSN: 0036-5521 (Print) 1502-7708 (Online) Journal homepage: http://www.tandfonline.com/loi/igas20
Effect of Drugs on Colonic Eicosanoid Accumulation in Active Ulcerative Colitis R. Eliakim, F. Karmeli, M. Chorev, E. Okon & D. Rachmilewitz To cite this article: R. Eliakim, F. Karmeli, M. Chorev, E. Okon & D. Rachmilewitz (1992) Effect of Drugs on Colonic Eicosanoid Accumulation in Active Ulcerative Colitis, Scandinavian Journal of Gastroenterology, 27:11, 968-972, DOI: 10.3109/00365529209000172 To link to this article: http://dx.doi.org/10.3109/00365529209000172
Published online: 08 Jul 2009.
Submit your article to this journal
Article views: 9
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=igas20 Download by: [RMIT University Library]
Date: 19 April 2016, At: 23:57
Effect of Drugs on Colonic Eicosanoid Accumulation in Active Ulcerative Colitis R. ELIAKIM, F. KARMELI, M. CHOREV, E. OKON & D. RACHMILEWITZ Dept. of Medicine, Hadassah University Hospital-Mount Scopus, Hebrew University-Hadassah Medical School, Jerusalem, Israel
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Eliakim R, Karmeli F, Chorev M, Okon F, Rachmilewitz D. Effect of drugs on colonic eicosanoid accumulation in active ulcerative colitis. Scand J Gastroenterol 1992;27:96%972. The effect of immunosuppressive drugs, 4-aminosalicylicacid (4-ASA), acetyl 5-aminosalicylic acid ( S ASA), and ketotifen on human colonic eicosanoid accumulation was evaluated in view of enhanced accumulation in patients with active ulcerative colitis. Azathioprine (100 pg/ml), cyclosporin (100 pg/ ml), and methotrexate (100 wg/ml) significantly inhibited, by 25-35%, prostaglandin E, (PGE,) accumulation by organ-cultured colonic mucosa of ulcerative colitis patients. Methotrexate was the only immunosuppressive drug that inhibited leukotriene B4 (LTB4) accumulation (50%), whereas azathioprine inhibited the accumulation of leukotriene C4 (LTC,) (25%). 5-ASA and its metabolite, acetyl 5-ASA, inhibited by 20-70% PGE,, LTB,, and LTC4 accumulation in the culture, supporting the contention that acetyl 5-ASA is as active as 5-ASA in these respects. 4-ASA had no effect on any of the eicosanoids. Ketotifen, a mast cell stabilizer, significantly inhibited the accumulation of PGE,, LTBJ, and LTC4by 3340%. These results suggest a potential, new, unrecognized mode by which the immunomodulators induce part of their therapeutic effects in inflammatory bowel disease and support the contention that acetyl 5-ASA is as active as 5-ASA. The results obtained also indicate that ketotifen, used effectively in the prevention of bronchial asthma, inhibits the accumulation of colonic eicosanoids and, thus, may be of value in the treatment of inflammatory bowel disease. Key words: Acetyl 5-aminosalicylic acid; 4-aminosalicylic acid; 5-aminosalicylic acid; azathioprine; chloroquine; cyclosporin; ketotifen; methotrexate; ulcerative colitis
R. Eliakim, M. D . , Dept. of Medicine, Hadamah Unioersity Hospital, Mount Scopus, P.O. Box 24035, Jerusalem 91240, Israel
The inflamed colonic mucosa in ulcerative colitis is infiltrated by equally prominent components of ‘active’ and ‘chronic’ leukocyte populations (1-3). The presence of neutrophils and macrophages suggests the involvement of soluble chemotactic and other mediators, causing migration of inflammatory cells into the mucosa (4). We and others have shown increased colonic accumulation of mediators-that is, prostaglandins (PG), leukotriene B4 (LTB4), plateletactivating factor (PAF), and interleukin 1 (IL-1)-by the inflamed colonic mucosa in patients with active ulcerative colitis (5-9). The therapeutic effects of the drugs currently used for the treatment of inflammatory bowel disease, such as corticosteroids, sulfasalazine, and its active moiety, 5aminosalicylic acid (5-ASA), are ascribed in part to their induced inhibition of the colonic accumulation of inflammatory mediators ( 5 , & l l ) . All these drugs have been shown to inhibit the generation of colonic eicosanoids, PAF, and IL-1 (5,8-11). 4-Aminosalicylic acid (4-ASA) and the immunomodulators azathioprine, cyclosporin, and methotrexate have also been used successfully in inflammatory bowel disease (1218). The therapeutic efficacy in ulcerative colitis of disodium
cromoglycate, a mast cell stabilizer, remains controversial, whereas that of chloroquine is unknown or preliminary (1922). Ketotifen, a potent eosinophil and mast cell stabilizer, has been shown to be effective in the prevention of gastric and colonic mucosal damage in the rat (23,24). The effects of these drugs on colonic eicosanoid accumulation in ulcerative colitis has not yet been established. The aim of the present study was to evaluate the effects of immunosuppressive drugs, ketotifen, chloroquine, 4-ASA, and acetyl 5-ASA, the possible active metabolite of 5-ASA, on eicosanoid accumulation by cultured colonic mucosa in patients with active ulcerative colitis. MATERIALS A N D METHODS Colonic biopsy specimens were obtained with an Olympus CF-1OL colonoscope 8-10cm from the anus from 25 untreated patients with active ulcerative colitis (mean age, 40.8 years (range, 20-81 years); mean duration of disease, 4.3 years; and disease extent s 6 0 cm in 65%) and from 11 subjects without any signs or symptoms of gastrointestinal disease (mean age, 50.5 years; range, 18-81 years). No
Downloaded by [RMIT University Library] at 23:57 19 April 2016
Effect of Drugs on Colonic Eicosanoids
complications ensued. The study was approved by the Committee on Research Involving Human Subjects of the Hadassah University Hospital. In all ulcerative colitis patients the disease was confirmed histologically. Biopsy specimens were obtained with biopsy forceps, washed in 0.15 M NaCI, weighed, and cultured within 10 min after excision. Organ culture was performed as previously described by us (5). In brief, tissue specimens were cultured (37"C, 5% C 0 2 , 95% air) for 24 h. The tissue was placed on a metal grid over the central well of the culture disk (Falcon) and cultured in 0.8 ml RPMI 1640 (Bio-Lab, Israel), containing penicillin (100 U/ml) and streptomycin (100 pg/ml). After 24 h of incubation specimens and media were transferred to separate tubes. All drug studies were performed on specimens obtained from ulcerative colitis patients. Specimens obtained from the same subject were cultured in medium containing either 5-ASA (100 to 500 pg/ml) (Sigma, Israel), acetyl5-ASA (100 to 500pg/ml) (kindly provided by Dr. Chorev, Pharmacy School, Hebrew University, Jerusalem), 4-ASA (50 to 500 pg/ml) (Sigma, Israel), azathioprine (25 to 100 pg/ml) (Pharmachemie, The Netherlands), cyclosporin (1to 100 pg/ ml) (Sandoz, Switzerland), methotrexate (1 to 100 pg/ml) (Pharmachemie), ketotifen (1 to 100 pg/ml) (Sandoz), chloroquine (25 to lOOpg/ml) (Sigma). Stock solutions of the drugs were as follows: azathioprine, chloroquine, and ketotifen, 10 mg/ml H 2 0 ;methotrexate, 100 mg/ml H 2 0 ;cyclosporin, 100 mg/ml ethanol; and 5-ASA and acetyl 5-ASA, 100 mg/ml dimethyl sulfoxide. Working solutions were prepared in RPMI, final pH of 7.5, identical to that of RPMI alone. In these series of experiments one biopsy specimen obtained from each subject was cultured in drug-free medium and served as control. At the end of the culture period the tissue and organ culture medium were kept at -20°C for up to a month before prostaglandin or leukotriene determination. To test whether there were drug toxicity effects on the specimens, they were assessed both histologically and for lactic dehydrogenase accumulation in the medium. The tissue was homogenized in 1.0 ml ice-cold 50 mM Tris-HCI buffer, pH 7.0, containing 0.02 M ethylenediaminetetraacetic acid (EDTA). The homogenate was extracted twice with two volumes of ether, and both the aqueous phase and the incubation media were then assayed for PGE2, LTC4, and LTB4 concentration by means of a radioimmunoassay, as previously described (25, 26). Statistical methods used were the t test for paired (drug studies) and unpaired (comparison between control and ulcerative colitis) data; p values were two- or one-tailed, as stated in the tables. RESULTS Eicosanoid accumulation in the medium of organ-cultured colonic mucosa of patients with active ulcerative colitis was significantly higher than the respective accumulation in cul-
969
Table I. Eicosanoid accumulation (pg/mg/24 h) by cultured colonic mucosa
PGEi LTB LTC,
,
Ulcerative colitis
Normal subjects
1546 f 182* 46 t 9* 244 f 17*
305 f 41 20 f 4 180 ? 22
Biopsy specimens obtained from patients with active ulcerative colitis and normal controls were cultured for 24 h in medium containing RPMI, penicillin (100 U/ml), and streptomycin (100 pg/ml), as described in Methods. At the end of the culture period the tissue and organ culture medium were kept at -2O"C, processed as described in Methods, and assayed for prostaglandin E Z (PGE,), leukotriene B4 (LTB,), and leukotriene C4 (LTC,). Results are mean t SE of 25 subjects with ulcerative colitis and 11 controls. * Denotes significantly different from normal controls ( p < 0.05).
tures of colonic mucosa obtained from normal controls (Table I). In active ulcerative colitis patients PGE2 generation was five times higher than in controls. LTB4 generation was double, whereas LTC4 generation was only 25% higher. The ratio of PGE2 levels in tissue and medium was 35:65 in both ulcerative colitis patients and controls, and the ratios for LTB4 and LTC4 was 17 : 83. The effect of the cytotoxic immunomodulators used in inflammatory bowel disease-azathioprine, cyclosporin, and methotrexate-on colonic eicosanoids was tested. The addition of each of these (100 pg/ml) drugs to the culture medium of rectal biopsy specimens obtained from patients with active ulcerative colitis significantly inhibited PGE2 accumulation by 25% to 35% (Table 11). Azathioprine, when administered in a lower dose of 50 pg/ml, also significantly inhibited colonic PGE? accumulation (35%, n = 6), whereas lower doses of cyclosporin and methotrexate had no effect. Only methotrexate (100 pg/ml) inhibited LTB4 accumulation (by 5 0 % , p < 0.02), whereas azathioprine (100 pg/ml) was the only drug to inhibit LTC4 accumulation (by 25%, p < 0.02) (Table 11). The effect of 4-ASA, 5-ASA, and its metabolite, acetyl 5-ASA, on eicosanoid accumulation was also evaluated. As previously reported, the addition of 5-ASA (500 pg/ml) to the culture medium of colonic specimens from patients with active ulcerative colitis significantly inhibited PGEz and LTC4 accumulation by 70% and 30%, respectively (Table 11). 5-ASA (250 pg/ml) significantly decreased PGEz and LTC4 accumulation by 47% and 46%, respectively ( n = 6, p < 0.05). Lower dose of 5-ASA (100 pg/ml) decreased PGE2 and LTC4, but not to statistically significant levels. The addition of the metabolite, acetyl 5-ASA (500 pg/ml), to the culture medium effectively and significantly inhibited the accumulation of PGE2, LTB4 and LTC4 by 20% to 65% (Table 11). At a dose of 100 and 250pg/ml, it decreased LTB4 by 50% and LTC4 by 20% ( n = 6, p < 0.05). When 4-ASA (100 pg/ml) was added to the culture medium, there was no effect on PGE2 or LTB4 accumulation (Table 11). The effect of ketotifen (Zaditenm), a mast cell stabilizer,
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R . Eliakim et al.
Table 11. Effect of drugs o n eicosanoid accumulation by cultured colonic mucosa
Azathioprine Methotrexate Cyclosporin 5-ASA
Acetyl 5-ASA
Downloaded by [RMIT University Library] at 23:57 19 April 2016
4-ASA Ketotifcn
Chloroquine
25 (5)t 50 (6) 100 ( 5 ) 1 (6) 10 (6) 100 (7) 1 (4) 10 (4) 100 (7) 100 ( 6 ) 250 ( 6 ) 500 ( 6 ) 100 (8) 250 (7) 500 (8) 50 (8) 100 (8) 0.1 (4) 1 (4) 10 (3) 100 ( 6 8 ) 25 ( 5 ) 50 (5) 75 (3) 100 (9)
62 t 14 66 t 9* 6 9 + 16 102 t 10 Rc)? 1 1 65 t 11* 90 t 20 119+-33 76 t 9% 85 t 16 58 t 8** 25 t 5** 103 -t 11 9 8 2 16 33 t 7** 135 2 34 133 t 23 118 t 14 111 - + 7 78 t 15 54 + 9* 57 t 15* 63 t 10* Not tested 53 t 7**
7 4 t 13 106 t 27 97 -t 13 105 t 20 7 2 2 13 52 2 lo** 118 t 36 95 rt 33 107 t 14 92 t 28 97 t 32 85 -t 16 62 t 14** 40 2 7** 65 t 11** 97 2 25 7 2 t 14 1002 16 7 9 t 13 94 t 23 43 t 8* 90 t 24 86 t 29 92 2 20 75 t 15
6 5 t 13 77 t 12 75 t 7* 177 t 25 125 t 37 8 9 t 17 Not tested 113 t 27 130 2 29 81 t 24 55 2 14* 54 t 7** 99 2 12 81 t 12 74 7** Not tested Not tested 159 2 45 109 t 13 Not tested 68 2 7' 150 t 35 1 0 4 2 18 116239 9 2 2 10
*
Biopsy specimens obtained from patients with active ulcerative colitis were cultured for 24 h in RPMI medium with or without drugs, as described in Methods. Results are given as percentage drug-free medium with mean 2 SE. Prostaglandin E2 (PGE2), leukotriene B, (LTBJ, and leukotriene C, (LTC,) (pg/mg/24 h) accumulations in drug-free medium were considered 100%. 5-ASA = 5-arninosalicylic acid; 4-ASA = 4-acetylsalicylic acid. * Denotes statistically different from drug-free medium, p < 0.05 ( t test for paired data, one-tailed). * * t Test for paired data, two-tailed. t No. of experiments.
shown to prevent mucosal damage and eicosanoid generation in two rat models of inflammatory bowel disease, and of chloroquine o n human colonic eicosanoid accumulation was tested. The addition of ketotifen (100 pg/ml) to the culture medium of rectal specimens from patients with active ulcerative colitis significantly inhibited the accumulation of PGE2, LTC,, and LTB4 by 33% to 60% (Table 11). Chloroquine, at the dose of 100kg/ml, significantly inhibited PGE2 accumulation by 40% (Table 11). It had no effect on LTB, or LTC4accumulation. Lower doses (10 pg/ml) had no effect on eicosanoid generation. Histologic examination of biopsy specimens after their culture in the presence of the various drugs in the doses tested and examination of lactic dehydrogenase levels in culture medium did not disclose any toxic effects of the drugs on the tissue. DISCUSSION Substantial evidence has accumulated in support of the role of eicosanoids in the pathogenesis of inflammatory bowel disease, although their specific contribution remains unclear (5-7, 10, 11, 27, 28). In the present study high levels of eicosanoids were found in the medium of cultured colonic mucosa of patients with active ulcerative colitis. These
mediators play a role in the recruitment of leukocytes to the diseased area and the amplification of the inflammatory process via activation of oxygen free radicals and proteases from neutrophils, causing more tissue damage. Other effects include a change in vascular permeability, causing vasoconstriction and stimulation of smooth-muscle contraction (5-9, 29, 30). An orally active inhibitor of leukotriene synthesis has been shown to accelerate healing in a rat model of colitis, adding further evidence for the role of these mediators in colitis (28,31). Azathioprine is used for the treatment of steroid-dependent patients with refractory Crohn's disease or for treating fistula. Its role in ulcerative colitis remains unclear, and it is used in patients with refractory disease who wish to avoid colectomy (12,13, 18). Cyclosporin and methotrexate are two promising drugs used so far in small trials for the treatment of inflammatory bowel disease. Cyclosporin, a polypeptide derived from the fungus Tolypocadium inflaturn selectively inhibits activation of helper T cells and interleukin-2, -3, -4, and -5 production (14,32). Methotrexate, a folic acid antagonist, has anti-metabolic and anti-inflammatory properties (15,33). Bernard et al. (34) have shown that methotrexate decreases ileal PGEz content in the rat. In the present study all three drugs significantly decreased colonic PGE2 accumulation; azathioprine also decreased
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Effect of Drugs on Colonic Eicosanoids
LTC4, and methotrexate decreased LTB4 accumulation, thus shedding some light onto a potential additional mechanism whereby these drugs may be beneficial in the treatment of inflammatory bowel disease. 5-ASA is the therapeutically active moiety of sulfasalazine. 5-ASA is subsequently acetylated in the gut wall to N-acetyl-5-ASA (35,37), which was considered to be therapeutically inert (38). Acetylation is an irreversible process (39), and the metabolite is present in the plasma shortly after both oral and rectal administration (40,41). The halflife of 5-ASA is short (s1.5 h), whereas that of N-acetyl 5ASA is longer (
