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Maintenance treatment with balsalazide provides more effective control of nocturnal symptoms of ulcerative colitis (UC) than delayed-release mesalazine
Immunology, Microbiology, and Inflammatory Disorders A987
April 1998 factors binding to multiple cis-acting elements critical for regulating IFN-T expression in the gut. This work was supported by USPHS Grant DK43211. G4044
EFFECT OF VACCINATION AGAINST HELICOBACTER PYLORI INFECTION AND ITS IMMUNE RESPONSES IN EXPERIMENTAL MOUSE MODEL. T.Got0h 1, A.Nishizono 2, J.Ikewaki 2, M.Kimoto 1 K.Mifune 2, T.Fujioka 1, M.NasuL 1Second Department of Internal Medicine and 3Department of Microbiology, Oita Medical University, Oita; Japan. Introduction: It has been reported that chronic active gastritis caused by Helicobacter pylori (H.pylori) is concerned with immunological abnormalities, especially with an inbalance of helper T (TH) cells consisting of TH1 and TH2 ceils. The aim of this study is to investigate the relationship between the effect of vaccination and the variety of TH cell responses in C57BL/6 mouse model. Materials and Methods: Six-week-old female C57BL/6 mice were used in the study and H.pylori (Sydney strain) was kindly provided by Dr.A.Lee. Mice were divided into six groups; group 1 to 3: oral vaccination of cholera toxin (5 lag), with a dose of 2 mg (groupl), 200 lag (group 2) and 2 lag (group 3) of sonicated H.pylori, respectively, group 4: intraperitoneal (ip) vaccination with sonicated H.pylori (100 lag), group 5: ordinary infection without vaccination, group 6: normal control. One week after the last vaccination, blood and fecal samples were collected to evaluate the H.pylorispecific antibody response by ELISA. Except for group 5 and 6, mice were challenged with three times inoculations with 108 cfu H.pylori organisms and were sacrificed at six weeks after the last challenge. The stomach of each mouse was removed for bacterial examination of H.pylori, histological examination and the determination of cytokine expression by RT-PCR. Results: Before the challenge, TH2 responses were dominant in orally vaccinated mice, while TH1 responses were dominant in ip-immunized mice. In orally vacccinated groups 1 and 2, colonization of H.pylori was completely inhibited. However, marked infiltration of inflammatory cells were observed in corpus region at the early phase after challenge. On the contrary, in ip-vaccinated group mice (group 4), H.pylori was well colonized in all subjects but the inflammation in gastric tissues was mild. Conclusions" It was suggested that dominant TH2 responses by oral immunization could be protective against H.pylori infection. Of interest was that induction of TH1 response (group 4) is able to give rise to quiescence of active gastritis rather than eradication of organisms. • G4045
GLUCOCORTICOID RECEPTOR CHARACTERISTICS, NUCLEAR TRANSLOCATION & MOLECULAR EFFECTS IN HUMAN INTESTINAL SMOOTH MUSCLE (HISM) CELLS. M. Graham A. Willey, Y. Zhu, J. Schreher, R. Witorsch, H. Sugerman. Laboratory of Tissue Repair, Depts. Pediatrics, Surgery & Physiology, MCV/VCU, Richmond VA. Glucocorticoids are potent regulators of collagen and collagenase expression in HISM cells, inducing the former and repressing the latter (Graham et al. Gastroenterology 1995, 1997). The molecular mechanisms of these actions are currently under investigation. The action of glucocorticoids is mediated via the glucocorticoid receptor (GR), a cytoplasmic protein that exerts its effect by translocating to the cell nucleus and then binding to glucocorticoid response elements (GRE'S) on the gene promoter. The purpose of this study was to characterise the GR and the pharmacodynamics of nuclear translocatinn in HISM cells. Cnmpetetive binding analysis with [3H]-dexamethasone demonstrated 50% inhibition of binding at 10-8 M cold dex. This effect was 2 orders of magnitude greater than that of cold testosterone. Westem blot analysis of GR demonstrated a single band of MW 94 kD that was 80-90% cytoplasmic. Treatment with dexamethasone resulted in translocation of 85% and 90% of GR to the nucleus at 10-s and 10-7 M Dex. This effect was partially simulated, and partially inhibited, by the specific glucocorticoid competetive analogue RU38486. HISM cells from a patient with Crohn's disease showed an increased sensitivity at 10-9 M dex. Co-transfection of HISM cells with GR and a collagenase reporter plasmid pcCAT demonstrated inhibition of collagenase transcriptional activity at 10-s M dex. That effect was blocked by c-jun cotransfection. These studies characterise, for the first time, the glucocorticoid receptor in human intestinal smooth muscle cells. They demonstrate specific binding, nuclear translocation, and molecular effect at physiologic concentrations of glucocorticoid, suggesting that the biologic effect on these cells can be evoked under conditions of stress and at relatively low pharmacologic doses. The inhibition, by c-jun, of the glucocorticoid effect suggests that this oncoprotein is involved in the regulation of GR action in HISM cells, probably by modulating the binding of GR to the collagenase promoter. Supported by NIH grant DK34151
• G4046
MAINTENANCE TREATMENT WITH BALSALAZIDE PROVIDES MORE EFFECTIVE CONTROL OF NOCTURNAL SYMPTOMS OF ULCERATIVE COLITIS (UC) THAN DELAYED-RELEASE MESALAZINE. JRB Green, JA Gibson, GD Kerr, ET Swarbrick, CD HoldsworttdAJ Lobo, JP Crowe, KJ Schofield, MD Taylor and the ABACUS Investigator group. Gastroenterology units in Stoke, Stafford, Shrewsbury, Wolverhampton, Sheffield, Dublin and Astra Pharmaceuticals Ltd., Kings Langley, U.K. Aim:- This study compared balsalazide (Colazide ®) and mesalazine (Asacol ®) in terms of efficacy and tolerability in the maintenance treatment of inactive UC over 12 months. Methods:- Ninety-nine patients with asymptomatic, sigmoidoscopicallyinactive (grade 0-1) UC were randomised, to receive double-blind, balsalazide 1.5 g b.i.d. (equivalent to 1.04 g mesalazine) (n=49) or mesalazine 0.4 g o.m. plus 0.8 g o.n. (n=46) for 12 months. The study was performed at 33 centres (UK and Ireland). Symptom and laboratory assessments were performed at entry and after 3, 6, 9 and 12 months. Results:- The two groups were well matched at entry with respect to patient characteristics and disease history. Diary card data were collected for 3 months. Analysis showed that baisalazide patients experienced more asymptomatic nights (90% vs 77%, p=0.0011) and days (58% vs 50%, n.s.). By 3 months, balsalazide patients were experiencing significantly better night-time symptom relief.
Symt~tom (nights~week)
Balsalazide
Mesalazine
F value
Completely s:cmptom-free 6.40 -+ 1.71 4.71 -+ 2.84 0.0006 Blood with stools 0.05 -+0.22 0.56 -+ 1.42 0.0241 Blood on toilet paper 0.05-+0.22 0.71-+1.35 0.0054 Mucus with stools 0.0183 0.07 -+0.46 0.68 -+ 1.77 Sleep disturbed - toilet visit 0.31 -+ 1.32 0.95 -+ 1.90 0.0072 Sleep disturbed - symptoms 0.25 -+ 1.16 1.00 -+ 2.09 0.0090 Mean + SD presented, analysed using Wilcoxon rank sum test. Fewer balsalazide patients relapsed within 3 months (10% vs 28%; p=0.0354). The remission rate at 12 months was 58% in both groups. Fiftyseven percent of patients failed to complete the study (29 balsalazide, 27 mesalazine), the main reason being treatment failure (16 balsalazide, 18 mesalazine). Logistic regression identified symptom severity at entry as being a prognostic factor associated with relapse by 3 (p=0.0256) and 12 (p=0.0114) months, regardless of study treatment. Similar proportions of patients reported adverse events (61% balsalazide vs 65% mesalazine). Conclusion:- Both balsalazide and mesalazine are effective in maintaining remission and are equally well tolerated. However, balsalazide controls nocturnal UC symptoms more effectively. This research was funded by Astra Pharmaceuticals Ltd, Kings Langley, U.K. G4047
ANTI-INFLAM1VIATORY EFFECTS OF A NOVEL ANTIOXIDANT IN RATS. B. Greenwood-Van Meerveld 1 K.R. Tyler 1, L.J. Peters 1, W. Flitter 2 and W. Garland 2. iOklahoma Foundation for Digestive Research Basic Science Labs, VA Medical Center, Oklahoma City, OK and 2Centaur Pharmaceuticals Inc., Sunnyvale, CA. Recent evidence suggests that the release of free radicals is important in the pathogenesis of intestinal inflammation. Nitrone-related therapeutics (NRTs) represent a new class of small molecules that may be effective in the treatment of inflammatory bowel disease (IBD) because they trap free radicals and reduce oxidative stress. The objective of this study was to determine the efficacy and potency of CPI-1189, a novel NRT, in both the prevention and healing of colonic inflammation in a rat model. Methods: In our experiments we induced a severe distal colitis via an intra-rectal enema of trinitrobenzene sulphonic acid (TNBS) [50 mg/kg dissolved in 50% ethanol]. Inflammation was assessed by gross morphological appearance using a grading scale (0-5) and through measurement of myeloperoxidase (MPO) activity, an enzymatic marker of inflammation intensity. Tissue wet weight was also measured. Results: We found that in methylcellulose vehicle-treated rats the TNBS enema produced distal colonic damage, stimulated MPO levels and increased the wet weight of the colonic segment. In treated animals, 10-90 mg/kg of CPI-1189 given by oral garage 1 hr. before the TNBS enema provided dosedependent protection from the colonic damage induced by TNBS. The lowest dose of CPI-1189 (10mg/kg) provided 19% protection whereas the greatest protection (46%, P<0.01) was observed with 30 mg/kg p.o. of CPI-1189. Furthermore, these findings were confirmed biochemically, through decreases in MPO activity and reductions in tissue wet weight. Greater protection (70%) was seen when CPI-1189 (30 mg/kg) was administered intravenously 1 hr. pre-TNBS enema which is consistent with the 50% oral bioavailability of CPI-1189. The protective effects of intra-colonic administration of CPI-1189 (30 mg/kg 1 hr. pre-TNBS enema) resembled those seen following oral administration. In the healing experiments, CPI-1189 (30 mg/kg p.o.) was administered 3 times at 4, 8 and 24 hr. after the TNBS enema. In these experiments we observed a significant inhibition (48%, P<0.01) of the colonic inflammatory response induced by TNBS. Condusion: CPI-1189 is effective in both preventing and healing severe colonic inflammatory response induced by TNBS in rats. These observations suggest that CPI-1189 may be a useful therapeutic agent in the treatment of idiopathic inflammatorybowel disease in man. This research was funded by Centaur Pharmaceuticals Inc. Sunnyvale, CA.
April 1998 factors binding to multiple cis-acting elements critical for regulating IFN-T expression in the gut. This work was supported by USPHS Grant DK43211. G4044
EFFECT OF VACCINATION AGAINST HELICOBACTER PYLORI INFECTION AND ITS IMMUNE RESPONSES IN EXPERIMENTAL MOUSE MODEL. T.Got0h 1, A.Nishizono 2, J.Ikewaki 2, M.Kimoto 1 K.Mifune 2, T.Fujioka 1, M.NasuL 1Second Department of Internal Medicine and 3Department of Microbiology, Oita Medical University, Oita; Japan. Introduction: It has been reported that chronic active gastritis caused by Helicobacter pylori (H.pylori) is concerned with immunological abnormalities, especially with an inbalance of helper T (TH) cells consisting of TH1 and TH2 ceils. The aim of this study is to investigate the relationship between the effect of vaccination and the variety of TH cell responses in C57BL/6 mouse model. Materials and Methods: Six-week-old female C57BL/6 mice were used in the study and H.pylori (Sydney strain) was kindly provided by Dr.A.Lee. Mice were divided into six groups; group 1 to 3: oral vaccination of cholera toxin (5 lag), with a dose of 2 mg (groupl), 200 lag (group 2) and 2 lag (group 3) of sonicated H.pylori, respectively, group 4: intraperitoneal (ip) vaccination with sonicated H.pylori (100 lag), group 5: ordinary infection without vaccination, group 6: normal control. One week after the last vaccination, blood and fecal samples were collected to evaluate the H.pylorispecific antibody response by ELISA. Except for group 5 and 6, mice were challenged with three times inoculations with 108 cfu H.pylori organisms and were sacrificed at six weeks after the last challenge. The stomach of each mouse was removed for bacterial examination of H.pylori, histological examination and the determination of cytokine expression by RT-PCR. Results: Before the challenge, TH2 responses were dominant in orally vaccinated mice, while TH1 responses were dominant in ip-immunized mice. In orally vacccinated groups 1 and 2, colonization of H.pylori was completely inhibited. However, marked infiltration of inflammatory cells were observed in corpus region at the early phase after challenge. On the contrary, in ip-vaccinated group mice (group 4), H.pylori was well colonized in all subjects but the inflammation in gastric tissues was mild. Conclusions" It was suggested that dominant TH2 responses by oral immunization could be protective against H.pylori infection. Of interest was that induction of TH1 response (group 4) is able to give rise to quiescence of active gastritis rather than eradication of organisms. • G4045
GLUCOCORTICOID RECEPTOR CHARACTERISTICS, NUCLEAR TRANSLOCATION & MOLECULAR EFFECTS IN HUMAN INTESTINAL SMOOTH MUSCLE (HISM) CELLS. M. Graham A. Willey, Y. Zhu, J. Schreher, R. Witorsch, H. Sugerman. Laboratory of Tissue Repair, Depts. Pediatrics, Surgery & Physiology, MCV/VCU, Richmond VA. Glucocorticoids are potent regulators of collagen and collagenase expression in HISM cells, inducing the former and repressing the latter (Graham et al. Gastroenterology 1995, 1997). The molecular mechanisms of these actions are currently under investigation. The action of glucocorticoids is mediated via the glucocorticoid receptor (GR), a cytoplasmic protein that exerts its effect by translocating to the cell nucleus and then binding to glucocorticoid response elements (GRE'S) on the gene promoter. The purpose of this study was to characterise the GR and the pharmacodynamics of nuclear translocatinn in HISM cells. Cnmpetetive binding analysis with [3H]-dexamethasone demonstrated 50% inhibition of binding at 10-8 M cold dex. This effect was 2 orders of magnitude greater than that of cold testosterone. Westem blot analysis of GR demonstrated a single band of MW 94 kD that was 80-90% cytoplasmic. Treatment with dexamethasone resulted in translocation of 85% and 90% of GR to the nucleus at 10-s and 10-7 M Dex. This effect was partially simulated, and partially inhibited, by the specific glucocorticoid competetive analogue RU38486. HISM cells from a patient with Crohn's disease showed an increased sensitivity at 10-9 M dex. Co-transfection of HISM cells with GR and a collagenase reporter plasmid pcCAT demonstrated inhibition of collagenase transcriptional activity at 10-s M dex. That effect was blocked by c-jun cotransfection. These studies characterise, for the first time, the glucocorticoid receptor in human intestinal smooth muscle cells. They demonstrate specific binding, nuclear translocation, and molecular effect at physiologic concentrations of glucocorticoid, suggesting that the biologic effect on these cells can be evoked under conditions of stress and at relatively low pharmacologic doses. The inhibition, by c-jun, of the glucocorticoid effect suggests that this oncoprotein is involved in the regulation of GR action in HISM cells, probably by modulating the binding of GR to the collagenase promoter. Supported by NIH grant DK34151
• G4046
MAINTENANCE TREATMENT WITH BALSALAZIDE PROVIDES MORE EFFECTIVE CONTROL OF NOCTURNAL SYMPTOMS OF ULCERATIVE COLITIS (UC) THAN DELAYED-RELEASE MESALAZINE. JRB Green, JA Gibson, GD Kerr, ET Swarbrick, CD HoldsworttdAJ Lobo, JP Crowe, KJ Schofield, MD Taylor and the ABACUS Investigator group. Gastroenterology units in Stoke, Stafford, Shrewsbury, Wolverhampton, Sheffield, Dublin and Astra Pharmaceuticals Ltd., Kings Langley, U.K. Aim:- This study compared balsalazide (Colazide ®) and mesalazine (Asacol ®) in terms of efficacy and tolerability in the maintenance treatment of inactive UC over 12 months. Methods:- Ninety-nine patients with asymptomatic, sigmoidoscopicallyinactive (grade 0-1) UC were randomised, to receive double-blind, balsalazide 1.5 g b.i.d. (equivalent to 1.04 g mesalazine) (n=49) or mesalazine 0.4 g o.m. plus 0.8 g o.n. (n=46) for 12 months. The study was performed at 33 centres (UK and Ireland). Symptom and laboratory assessments were performed at entry and after 3, 6, 9 and 12 months. Results:- The two groups were well matched at entry with respect to patient characteristics and disease history. Diary card data were collected for 3 months. Analysis showed that baisalazide patients experienced more asymptomatic nights (90% vs 77%, p=0.0011) and days (58% vs 50%, n.s.). By 3 months, balsalazide patients were experiencing significantly better night-time symptom relief.
Symt~tom (nights~week)
Balsalazide
Mesalazine
F value
Completely s:cmptom-free 6.40 -+ 1.71 4.71 -+ 2.84 0.0006 Blood with stools 0.05 -+0.22 0.56 -+ 1.42 0.0241 Blood on toilet paper 0.05-+0.22 0.71-+1.35 0.0054 Mucus with stools 0.0183 0.07 -+0.46 0.68 -+ 1.77 Sleep disturbed - toilet visit 0.31 -+ 1.32 0.95 -+ 1.90 0.0072 Sleep disturbed - symptoms 0.25 -+ 1.16 1.00 -+ 2.09 0.0090 Mean + SD presented, analysed using Wilcoxon rank sum test. Fewer balsalazide patients relapsed within 3 months (10% vs 28%; p=0.0354). The remission rate at 12 months was 58% in both groups. Fiftyseven percent of patients failed to complete the study (29 balsalazide, 27 mesalazine), the main reason being treatment failure (16 balsalazide, 18 mesalazine). Logistic regression identified symptom severity at entry as being a prognostic factor associated with relapse by 3 (p=0.0256) and 12 (p=0.0114) months, regardless of study treatment. Similar proportions of patients reported adverse events (61% balsalazide vs 65% mesalazine). Conclusion:- Both balsalazide and mesalazine are effective in maintaining remission and are equally well tolerated. However, balsalazide controls nocturnal UC symptoms more effectively. This research was funded by Astra Pharmaceuticals Ltd, Kings Langley, U.K. G4047
ANTI-INFLAM1VIATORY EFFECTS OF A NOVEL ANTIOXIDANT IN RATS. B. Greenwood-Van Meerveld 1 K.R. Tyler 1, L.J. Peters 1, W. Flitter 2 and W. Garland 2. iOklahoma Foundation for Digestive Research Basic Science Labs, VA Medical Center, Oklahoma City, OK and 2Centaur Pharmaceuticals Inc., Sunnyvale, CA. Recent evidence suggests that the release of free radicals is important in the pathogenesis of intestinal inflammation. Nitrone-related therapeutics (NRTs) represent a new class of small molecules that may be effective in the treatment of inflammatory bowel disease (IBD) because they trap free radicals and reduce oxidative stress. The objective of this study was to determine the efficacy and potency of CPI-1189, a novel NRT, in both the prevention and healing of colonic inflammation in a rat model. Methods: In our experiments we induced a severe distal colitis via an intra-rectal enema of trinitrobenzene sulphonic acid (TNBS) [50 mg/kg dissolved in 50% ethanol]. Inflammation was assessed by gross morphological appearance using a grading scale (0-5) and through measurement of myeloperoxidase (MPO) activity, an enzymatic marker of inflammation intensity. Tissue wet weight was also measured. Results: We found that in methylcellulose vehicle-treated rats the TNBS enema produced distal colonic damage, stimulated MPO levels and increased the wet weight of the colonic segment. In treated animals, 10-90 mg/kg of CPI-1189 given by oral garage 1 hr. before the TNBS enema provided dosedependent protection from the colonic damage induced by TNBS. The lowest dose of CPI-1189 (10mg/kg) provided 19% protection whereas the greatest protection (46%, P<0.01) was observed with 30 mg/kg p.o. of CPI-1189. Furthermore, these findings were confirmed biochemically, through decreases in MPO activity and reductions in tissue wet weight. Greater protection (70%) was seen when CPI-1189 (30 mg/kg) was administered intravenously 1 hr. pre-TNBS enema which is consistent with the 50% oral bioavailability of CPI-1189. The protective effects of intra-colonic administration of CPI-1189 (30 mg/kg 1 hr. pre-TNBS enema) resembled those seen following oral administration. In the healing experiments, CPI-1189 (30 mg/kg p.o.) was administered 3 times at 4, 8 and 24 hr. after the TNBS enema. In these experiments we observed a significant inhibition (48%, P<0.01) of the colonic inflammatory response induced by TNBS. Condusion: CPI-1189 is effective in both preventing and healing severe colonic inflammatory response induced by TNBS in rats. These observations suggest that CPI-1189 may be a useful therapeutic agent in the treatment of idiopathic inflammatorybowel disease in man. This research was funded by Centaur Pharmaceuticals Inc. Sunnyvale, CA.