- Email: [email protected]
Randomized trial of tinzaparin, a low molecular weight heparin (LMWH), versus placebo in the treatment of mild to moderately active ulcerative colitis
DNA-microarray chips. Changes in mRNA levels were confirmed by LightCycler RT-PCR quantification. Promoters were analyzed by transfection into SaOS-2 cells cotransfected with p53 and p63 expressing plasmids followed by luciferase assays. Mutants of the promoters were created to elucidate the mechanism of transcriptional regulation. Results: p53 downregulates transcription of cdc25C and cydin B. Repression by p53 is not controlled by a p53-consensus binding-site in the cdc25C promoter but by CCAAT-boxes binding the transcription factor NF-Y. Down-regntation of cyclin B2 is mediated by at least two mechanisms which are both independent of p53 sites in its promoter. In contrast, no splice variant of p63 is able to change transcription of these two genes. Two out o| six p63 splice variants tested can activate cyclin-dependent kinase inhibitor p21WAFI/CIP1, FAS/Apo-1 and GADD45 transcription. Two other p63 forms can up- or down-regulate Bax transcription. Only one of the p63 isoforms can upregnlate Mdm2 expression. The differences in regulation point at important functional differences of p63 versus p53 in G2 checkpoint control and regulation of programmed cell death. Conclusion: The tumor suppressor p53 and its homologue p63 differentially regulate cell cycle and apoptosis genes in cotorectal adenocarcinorm cells. This is likely the molecular basis for the difference in tumor suppressor function of p63 and p53.
54O
Randomized Trial of Tinzaparin, a Low Molecular Weight Heparin (LMWH), Versus Placebo in The Treatment of Mild to Moderately Active Ulcerative Colitis Stuart Bloom, S. Kiilerich, M. R. Lassen, C. O'Morain, A. Forbes, S. Orm Background and aims: Heparin may have anti-inflammatory and immunomodulatory activity in the treatment of inflammatory bowel disease. Study Design: A prospective, double-blind, randomized, placebo-controUed, multi-centre study comparing tinaaparin 175 anti-Xa [U/kg/day (innohep *, LEO Pharraa) subcutaneously (sc) for 14 days followed by tinzaparin 4500 anti-Xa 1U/day sc for 28 days with placebo (isotonic sodium chloride solution) in pre-filled syringes, administered sc once daily for up to 42 days. The primary outcome measure was a change in colitis activity from baseline to the end of study treatment assessed by the sum of scores of stool frequency, rectal bleeding, sigmoidoscopy and histology. Secondary outcome measures included individual assessments of the above parameters and changes in laboratory parameters. Patients were clinically assessed at weekly intervals for 6 weeks and within 1 week of completing treatment. Sigmoidoscopy with rectal biopsy was done between 1 and 4 days pre treatment and between 2 and 7 days after completing treatment. Results: 100 patients with a flare of ulcerative colitis (up to 6 bloody stools/day, no fever, no tachycardia or systemic disturbances) were randomized. 48 received tinaaparin and 52 received placebo. Primary outcome data are shown in the table. The mean percentage change in colitis activity was not significantly different between the two groups (p = 0.84 by analysis of variance adjusting for country). The mean difference between tinzaparin and placebo groups (95% confidence interval) was: 2.2 (-19.2 to 23.5). There was no difference between tinzaparin and placebo in any secondary outcome measure. There was only one major bleed (rectal), occurring in a patient receiving placebo. Conclusion: This is the largest study to date of heparin in ulcerative colitis. The results show conclusively no benefit of LMWH over placebo in mild to moderately active ulcerative colitis.
538 lntraepithelial Lymphocyte-derived Keratinocyte Growth Factor Expression DuringIntestinal Adaptation Hua Yang, Barbara E. Wildhaber, Daniel H. Teitelbaum Purpose: Keratiuocyte growth factor (KGF) is important for intestinal epithelial (EC) growth. In the mocosa KGF is expressed by T-cell receptor (TCR) "yS+ IEL, and EC express KGF receptors (KGFR). The functional contribution of mucosal KGF has not been fully explored. We previously showed that IEL KGF mRNA was up-regulated in a short bowel syndrome (SBS)mouse model. We hypothesized that this increased expression would have impact in intestinal adaptation in SBS. This study examined the role of IEL KGF in the SBS model, and the expression of mucosal KGF and KGFR along the crypt-villus axis. Methods: Adult mice underwent a 55% mid-small bowel resection (SBS) or transsection without resection (control). After 7 days small bowel IEL and EC were harvested. ~,B+ TCR IEL were isolated using flow cytometry. IEL KGF and EC KGFR expression were studied using real-time PCR. [EL KGF protein expression utilized immunoblotting. EC proliferation was determined by 8rdU incorporation. To assess the location of IEL KGF and EC KGFR mRNA expression (crypt, lower third (LV), and upper third of villus (UV)), laser capture microdissection was performed. Results are expressed as mean-+SD, ANOVA was used for statistics, P
SI~ 'Pr
Percentage change In colitis activity at end of b l a ~
Intention-to.treat analysis sea
Percentage change In colitis acUvlr Unzaparln ( n ~ ) Median 0 Mean -10.0 SD 44.7 Minimum -100 Ihximm 75 *No~: A nega0ve percentage change indicates disease improvement.
placebo (n:52) -9 -13.0 61.1
-100 150
541 Seal Oil Compared to Soy Oil in Patients with Joint Pain and Inflammatory Bowel Disease (IBD) Gulen Arslan, Tormod Bjorkkjaer, Linn Anne Brunborg, P,agna Lind, Merete Valen, Johan Bran, Livar Froyhnd, Arnold Berstad Background: Marine polyunsaturated fatty acids like eicosapentaenoic acid (EPA, 20:5 n-3) and docosahexaenoic acid (DHA, 22:6 n-3) have anti-inflammatory effects, partly due to their modulating effects on prostaglandin synthesis. In a previous pilot study, seal oil (rich in n-3 fatty acids) administration reduced joint pain in patients with IBD. Fatty acid composition and n-3/n-6 ratio in blood and intestinal mucosa were changed. In this study we wanted to compare the effects of seal oil and soy oil (rich in n-6 fatty acids). Material and methods: Twenty IBD-patients with joint pain were randomly allocated to two groups, one received placebo, the other seal oil treatment. A thin nasojejunal feeding tube was placed by means of fluoroscopy. The patients themselves administered 30 ml seal oil (total 4.4g EPA and DHA) or 30 ml soy oil daily (10 ml 3 times daily) through the feeding tube for 10 days. IBD score, joint pain index, blodlipids and fatty acid composition were determined before and after treatment. Results: Both 1BD-score (p = 0.02) and joint pain index (duration of morning stiffness p=O.01; number of tender joints p=0.036; doctors evaluation of joint pain p=0.OOI; subjective assessment of pain last week p=0.O02) were sigmficantly decreased after seal oil treatment. Soy oil had no effect on IBD-score or joint pain, but reduced significantly totabcholesterol (p = 0.008) and LDL-cholesterol levels (p = 0.04) in serum. N-3/n-6 ratio in serum was significantly (p = 0.00t) increased after seal oil treatment. Concfusion: Seal oil treatment has beneficial effects on joint pain and IBD-seore in patients with IBD. Soy oil reduces total- and LDL-cholesterol levels, but not joint pain.
Total1" Cqfpt LV UV To~t Crypt LV W 885,~9 205~64 145,50 70~23 1351,298 723,169 499~167 426q(~ 1429~300" 594+107" 202,26* 86+27 4261~588* 1513:129" 980~71" 548q14 r Iocor~ol.TNunterofcopies~the gene~ KGForKGFRpern~er ~ co,oresof~ .
539 Mahieenter, Randomized, Double-blind, Placebo-controlled Trial of Deligoparin (Ultra Low Molecular Weight Heparin) for Active Ulcerative Colitis Joshua Korzenik, Philip Miner Jr., David Stanton, Kim lsaacs, Efien Zimmerman, Dennis Ihff, Willem De Villiers, Robert Venuti Background: The use of heparin or low molecular weight heparin (LMWH) for the treatment of active ulcerative colitis (UC) has been evaluated in several small clinical trials, with varying outcomes. Deligoparin is an uhra-LMWH with an average molecular weight of approximately 3000 dahons. Methods: In this study, 138 patients (Pts) with active UC were randomized to receive placebo (PLA; n=46), 75rag deligoparin (D75; n = 4 7 ) or 125mg deligoparin (0125; n=45) once daily by subcutaneous injection for six weeks. Efficacy was assessed using a total colitis activity index (TCA1) score based on severity of rectal bleeding, stool frequency (SF), endoscopic findings and a physician's global assessment, each graded 0 to 3 points. The primary efficacy end point was the distribution of Pts among four response categones: remission (TCAI = 0, or 1 for SF only), improvement (TCAI 1 -> 3), no change, and worsening (TCAI [up arrow] -> 2). Concomitant medications included anunosallcylates (88% of Pts), steroids (17%) and/or azathioprine/6-MP (17%). ResuIts: Mean TCAl scores at study entry ranged from 8.1 (D75) to 8.6 (PLA). Mean % increases in activated partial thromboplastin time (2.7% PLA, 16.7% D75, 26.8% D125) four hours after the first dose of study medication were consistent with anticipated peak detigoparin plasma levels and its in mroratio of anti-activated Factor X to anti-activated Factor II activity of approximately 13. Early withdrawals (21 Pts) were for adverse events (7% PLA, 2% D75, 2% 0125), treatment failure (2%, 6%, 13%), withdrawal of consent (2%, 4%, 2%) and a positive C d~fficile(0%, 0%, 4%). In the intent-to-treat analysis, no significant difference in response was observed between PLA (41% in remission or improved) and either of the D75 (36%) and D125 (42%) treatment groups. Mean decreases in TCAI score of 2.2 (PLA), 2.4 (D75) and 2.4 (D125) points were also not significantly different. Analyses by disease severity, of component scores and using other definitions of improvement (TCAI I > 4; TCAl ~ ::" 5) also failed to show a trend in favor of active treatment. No serious adverse event related to a sigmlicant increase in rectal bleeding or other hemorrhage was reported. No Pt developed heparin-induced thrombocytopenia. Conclusions: Dellgoparm, while well tolerated, was ineffective for the treatment of active UC when added to standard therapy.
542 Multicenter Randomized Double Blind Controlled Trial for Ulcerative Colitis Therapy with Leukocytapheresis Koji Sawada, Kazuo Kusugam, Yasuo Suzuki, Tadao Bamba, Akihiro Munakata, Toshifumi Hibi, Yoshihiro Fukuda, Takashi Shimoyama Background: In results of a muhicenter trial with randomized assignment of 80 active-stage ulcerative colitis (UC) patients, leukocytapberesis (LCAP) group with Cellsorba leukocyte removal column added to on-going conventional drug therapy showed a significantly higher effectiveness and lower adverse effects incidence compared with high dose of prednisolone (PSL, 30 to 80 rag/day) added to on-going conventional drug therapy group. Objective: In order to make a more objective assessment of LCAP as an intensive therapy of UC, effectiveness and safety were investigated by a double blind controlled study with randomized assignment of active-stage UC patients. Methods: Ulcerative colitis patients with more than 10 of Lichtiger's cfinical activity index (L-CAI) were entered in the trial. Assignment was performed by a controller on the basis of L-CAI, sex, and age to obtain two groups with matching patient backgrounds. Procedure such as LCAP or sham perfusion added weekly for five weeks to the patients who were treated by the on-going drug therapy such as 5-aminosalicylic acid (5-ASA) with 0.5 mg/kg/day of steroid. Nafamostat mesilate was used as an anticoagulant
A-67
AGA
Abstracts
54O
Randomized Trial of Tinzaparin, a Low Molecular Weight Heparin (LMWH), Versus Placebo in The Treatment of Mild to Moderately Active Ulcerative Colitis Stuart Bloom, S. Kiilerich, M. R. Lassen, C. O'Morain, A. Forbes, S. Orm Background and aims: Heparin may have anti-inflammatory and immunomodulatory activity in the treatment of inflammatory bowel disease. Study Design: A prospective, double-blind, randomized, placebo-controUed, multi-centre study comparing tinaaparin 175 anti-Xa [U/kg/day (innohep *, LEO Pharraa) subcutaneously (sc) for 14 days followed by tinzaparin 4500 anti-Xa 1U/day sc for 28 days with placebo (isotonic sodium chloride solution) in pre-filled syringes, administered sc once daily for up to 42 days. The primary outcome measure was a change in colitis activity from baseline to the end of study treatment assessed by the sum of scores of stool frequency, rectal bleeding, sigmoidoscopy and histology. Secondary outcome measures included individual assessments of the above parameters and changes in laboratory parameters. Patients were clinically assessed at weekly intervals for 6 weeks and within 1 week of completing treatment. Sigmoidoscopy with rectal biopsy was done between 1 and 4 days pre treatment and between 2 and 7 days after completing treatment. Results: 100 patients with a flare of ulcerative colitis (up to 6 bloody stools/day, no fever, no tachycardia or systemic disturbances) were randomized. 48 received tinaaparin and 52 received placebo. Primary outcome data are shown in the table. The mean percentage change in colitis activity was not significantly different between the two groups (p = 0.84 by analysis of variance adjusting for country). The mean difference between tinzaparin and placebo groups (95% confidence interval) was: 2.2 (-19.2 to 23.5). There was no difference between tinzaparin and placebo in any secondary outcome measure. There was only one major bleed (rectal), occurring in a patient receiving placebo. Conclusion: This is the largest study to date of heparin in ulcerative colitis. The results show conclusively no benefit of LMWH over placebo in mild to moderately active ulcerative colitis.
538 lntraepithelial Lymphocyte-derived Keratinocyte Growth Factor Expression DuringIntestinal Adaptation Hua Yang, Barbara E. Wildhaber, Daniel H. Teitelbaum Purpose: Keratiuocyte growth factor (KGF) is important for intestinal epithelial (EC) growth. In the mocosa KGF is expressed by T-cell receptor (TCR) "yS+ IEL, and EC express KGF receptors (KGFR). The functional contribution of mucosal KGF has not been fully explored. We previously showed that IEL KGF mRNA was up-regulated in a short bowel syndrome (SBS)mouse model. We hypothesized that this increased expression would have impact in intestinal adaptation in SBS. This study examined the role of IEL KGF in the SBS model, and the expression of mucosal KGF and KGFR along the crypt-villus axis. Methods: Adult mice underwent a 55% mid-small bowel resection (SBS) or transsection without resection (control). After 7 days small bowel IEL and EC were harvested. ~,B+ TCR IEL were isolated using flow cytometry. IEL KGF and EC KGFR expression were studied using real-time PCR. [EL KGF protein expression utilized immunoblotting. EC proliferation was determined by 8rdU incorporation. To assess the location of IEL KGF and EC KGFR mRNA expression (crypt, lower third (LV), and upper third of villus (UV)), laser capture microdissection was performed. Results are expressed as mean-+SD, ANOVA was used for statistics, P
SI~ 'Pr
Percentage change In colitis activity at end of b l a ~
Intention-to.treat analysis sea
Percentage change In colitis acUvlr Unzaparln ( n ~ ) Median 0 Mean -10.0 SD 44.7 Minimum -100 Ihximm 75 *No~: A nega0ve percentage change indicates disease improvement.
placebo (n:52) -9 -13.0 61.1
-100 150
541 Seal Oil Compared to Soy Oil in Patients with Joint Pain and Inflammatory Bowel Disease (IBD) Gulen Arslan, Tormod Bjorkkjaer, Linn Anne Brunborg, P,agna Lind, Merete Valen, Johan Bran, Livar Froyhnd, Arnold Berstad Background: Marine polyunsaturated fatty acids like eicosapentaenoic acid (EPA, 20:5 n-3) and docosahexaenoic acid (DHA, 22:6 n-3) have anti-inflammatory effects, partly due to their modulating effects on prostaglandin synthesis. In a previous pilot study, seal oil (rich in n-3 fatty acids) administration reduced joint pain in patients with IBD. Fatty acid composition and n-3/n-6 ratio in blood and intestinal mucosa were changed. In this study we wanted to compare the effects of seal oil and soy oil (rich in n-6 fatty acids). Material and methods: Twenty IBD-patients with joint pain were randomly allocated to two groups, one received placebo, the other seal oil treatment. A thin nasojejunal feeding tube was placed by means of fluoroscopy. The patients themselves administered 30 ml seal oil (total 4.4g EPA and DHA) or 30 ml soy oil daily (10 ml 3 times daily) through the feeding tube for 10 days. IBD score, joint pain index, blodlipids and fatty acid composition were determined before and after treatment. Results: Both 1BD-score (p = 0.02) and joint pain index (duration of morning stiffness p=O.01; number of tender joints p=0.036; doctors evaluation of joint pain p=0.OOI; subjective assessment of pain last week p=0.O02) were sigmficantly decreased after seal oil treatment. Soy oil had no effect on IBD-score or joint pain, but reduced significantly totabcholesterol (p = 0.008) and LDL-cholesterol levels (p = 0.04) in serum. N-3/n-6 ratio in serum was significantly (p = 0.00t) increased after seal oil treatment. Concfusion: Seal oil treatment has beneficial effects on joint pain and IBD-seore in patients with IBD. Soy oil reduces total- and LDL-cholesterol levels, but not joint pain.
Total1" Cqfpt LV UV To~t Crypt LV W 885,~9 205~64 145,50 70~23 1351,298 723,169 499~167 426q(~ 1429~300" 594+107" 202,26* 86+27 4261~588* 1513:129" 980~71" 548q14 r Iocor~ol.TNunterofcopies~the gene~ KGForKGFRpern~er ~ co,oresof~ .
539 Mahieenter, Randomized, Double-blind, Placebo-controlled Trial of Deligoparin (Ultra Low Molecular Weight Heparin) for Active Ulcerative Colitis Joshua Korzenik, Philip Miner Jr., David Stanton, Kim lsaacs, Efien Zimmerman, Dennis Ihff, Willem De Villiers, Robert Venuti Background: The use of heparin or low molecular weight heparin (LMWH) for the treatment of active ulcerative colitis (UC) has been evaluated in several small clinical trials, with varying outcomes. Deligoparin is an uhra-LMWH with an average molecular weight of approximately 3000 dahons. Methods: In this study, 138 patients (Pts) with active UC were randomized to receive placebo (PLA; n=46), 75rag deligoparin (D75; n = 4 7 ) or 125mg deligoparin (0125; n=45) once daily by subcutaneous injection for six weeks. Efficacy was assessed using a total colitis activity index (TCA1) score based on severity of rectal bleeding, stool frequency (SF), endoscopic findings and a physician's global assessment, each graded 0 to 3 points. The primary efficacy end point was the distribution of Pts among four response categones: remission (TCAI = 0, or 1 for SF only), improvement (TCAI 1 -> 3), no change, and worsening (TCAI [up arrow] -> 2). Concomitant medications included anunosallcylates (88% of Pts), steroids (17%) and/or azathioprine/6-MP (17%). ResuIts: Mean TCAl scores at study entry ranged from 8.1 (D75) to 8.6 (PLA). Mean % increases in activated partial thromboplastin time (2.7% PLA, 16.7% D75, 26.8% D125) four hours after the first dose of study medication were consistent with anticipated peak detigoparin plasma levels and its in mroratio of anti-activated Factor X to anti-activated Factor II activity of approximately 13. Early withdrawals (21 Pts) were for adverse events (7% PLA, 2% D75, 2% 0125), treatment failure (2%, 6%, 13%), withdrawal of consent (2%, 4%, 2%) and a positive C d~fficile(0%, 0%, 4%). In the intent-to-treat analysis, no significant difference in response was observed between PLA (41% in remission or improved) and either of the D75 (36%) and D125 (42%) treatment groups. Mean decreases in TCAI score of 2.2 (PLA), 2.4 (D75) and 2.4 (D125) points were also not significantly different. Analyses by disease severity, of component scores and using other definitions of improvement (TCAI I > 4; TCAl ~ ::" 5) also failed to show a trend in favor of active treatment. No serious adverse event related to a sigmlicant increase in rectal bleeding or other hemorrhage was reported. No Pt developed heparin-induced thrombocytopenia. Conclusions: Dellgoparm, while well tolerated, was ineffective for the treatment of active UC when added to standard therapy.
542 Multicenter Randomized Double Blind Controlled Trial for Ulcerative Colitis Therapy with Leukocytapheresis Koji Sawada, Kazuo Kusugam, Yasuo Suzuki, Tadao Bamba, Akihiro Munakata, Toshifumi Hibi, Yoshihiro Fukuda, Takashi Shimoyama Background: In results of a muhicenter trial with randomized assignment of 80 active-stage ulcerative colitis (UC) patients, leukocytapberesis (LCAP) group with Cellsorba leukocyte removal column added to on-going conventional drug therapy showed a significantly higher effectiveness and lower adverse effects incidence compared with high dose of prednisolone (PSL, 30 to 80 rag/day) added to on-going conventional drug therapy group. Objective: In order to make a more objective assessment of LCAP as an intensive therapy of UC, effectiveness and safety were investigated by a double blind controlled study with randomized assignment of active-stage UC patients. Methods: Ulcerative colitis patients with more than 10 of Lichtiger's cfinical activity index (L-CAI) were entered in the trial. Assignment was performed by a controller on the basis of L-CAI, sex, and age to obtain two groups with matching patient backgrounds. Procedure such as LCAP or sham perfusion added weekly for five weeks to the patients who were treated by the on-going drug therapy such as 5-aminosalicylic acid (5-ASA) with 0.5 mg/kg/day of steroid. Nafamostat mesilate was used as an anticoagulant
A-67
AGA
Abstracts