Increased mucosal TNF-α production in Crohn's disease can be modulated locally by probiotics

idiopathic CD may stem from deficiencies in neutrophil and/or monocyte function, rhu GMCSF (Sargramostim, Leukine) stimulates anti-microbial function of both cell types. Aims: The aim of this open labeled therapeutic trial was to investigate the safety and efficacy of rhu GM-CSF in the treatment of active CD. Methods: Eleven patients with active CD were treated with daily subcutaneous injections of 4, 6, or 8 micrograms of GM-CSF for eight weeks. Efficacy was assessed by Crohn's disease activity index (CDAI) and Inflammatory Bowel Disease Questionnaire (IBDQ) quality of life score at week O, 1, 2, 4, 6, 8. Results: Starting CDAIs (week O) ranged between 228 and 471with a mean of 345. The mean CDAI decreased to 257, 216,176, and 148 at weeks 2, 4, 6, and 8 respectively. Nine patients (9/1 f) responded having a decrease in CDAI of >100 points and six patients (6/11) achieved remission (CDAI <150). One patient had closure of a longstanding rectovaginal fistula. Two patients were retreated after experiencing increasing diseaseactivity 3 and 6 weeks after the initial treatment course. Both again responded with a decrease in CDAI of nearly 200 points within 3 weeks and both achieved remission by 8 weeks. Decreases in CDAI were matched by significant improvements in the IBDQ. Adverse events were minimal and included injection site reactions and transient bone pain. No patient worsened. Treatment resulted in a dose dependentincrease in absolute neutrophil count and variable changes in the absolute monocyte count. At higher doses, patients responded more rapidly. Conclusion: These results demonstrate the safety and efficacy of GM-CSFadministration to patients with active CD. The useof colony stimulating factors to modulate the innate immune system offers an alternate paradigm for the treatment of CO. Further exploration of possible pathophysiologic defects in the innate arm of the immune response in CD is warranted. 1438 Collageuous Colitis Remission with Melholrnxate Lybus C. Hillman, Christopher Ashton, Louise Chiragakis, Graham Kaye, Anthony C. Clarke, Medical Specialists Assoc, Garran, ACT Australia

model, SB-265610 decreasedacute and chronic inflammation and preserved mucosal integrity. Thus, SB-265610 may have therapeutic potential in the treatment of inflammatory bowel disease. 1440 Succmmful Treatment of Active Crehn's Disease with Oral Dehydroepiandresterone (DHEA}: An Open Controlled Pilot Trial Tilo Andus, Frank Klebl, Gerhard Rogler, Nicole Bregenzer, Juergen Schoelmerich, Rainer H. Straub, Dept of Int Med, Univ of Reoensburg, Regensburg Germany BACKGROUND/OBJECTIVES:Dehydroepiandrosterone(DHEA) has been shown to be effective for the treatment of lupus erythercatosus. DHEAinhibits nuclear factor KB and interleukin-6 via PPARe. We previously showed that patients with inflammatory bowel diseasehavesignificantly lower serum concentrations of dehydroepiandrosteronesulfate (DHEAS)compared to controls. Furthermore, we found that 7 of 12 patients treated with DHEAfor refractory ulcerative colitis responded to DHEA therapy (Gut 2000; 47 Suppi III A 241). METHODS: Six patients with active Cmhn's disease (CDAI: 237-+54, mean-+SD; range: 161-309) were treated for 56 days with 1 x 200 mg DHEA orally (Audor Pharma, Regensburg, Germany). Prior to the study 4 patients had been treated with corticosteroids, 3 with mesalamine, 2 with azathioprine, all without success. All these drugs remained stable during the trial. RESULTS:Serum concentrations of DHEAS raised significantly from 0.76-+0.85 to 11.8-+2mg/1. Five of the 6 patients went into remission COAl: 90_+65). One of these had a relapse (COAl 172) on day 56. The other 4 patients remained in remission during the study period of 8 weeks. One patients did not improve and stopped medication. C reactive protein decreasedfrom 18.5-+.5 to 6.7-+1.5 mg/I. No severe adverse events occurred with the exception of an unrelated fracture of the femur in one patienL CONCLUSION(S):Treatment of active Cmhn's disease with DHEA was save and effective in our pilot trial. A larger prospective randomised contolied trial is warranted.

Background We have previously reported the dramatic improvement of diarrhoea in a patient with collagenous colitis treated with methntrexate for inflammatory arthritis. This prompted a study to determine the effect of methotrexate on collagenous colitis in patients resistant to other therapeutic modalities. Methods Eleven patients with refractory, histologically proven collagenous colitis were identified from a patient databaseand offered methotrexate. Previous failed therapy included Ioperamide, sulphaealazine, olseiazine, mesalazine and high dose prednisolone. Methotrexate was initiated at a dose of 7.5mg in all but one patient (5mg) and titrated up till a clinical response occurred. Hematological and biochemical indices were monitored. Three patients had follow up colonoscopy and biopsies alter clinical remission was documented. Results Seven females and four males with refractory collagenous colitis, aged 37 to 88 years (mean 64.7), were treated with methotrexate. Comorbidity included celiac disease (1), inflammatory polyarthritis (2) and osteoarthritis (3). The dose of methutrexate ranged from 5 to 20 g per week (median 7.5). Patients were followed for 2 to 28 months (median 11). A clinical response was seen by the third week in all patients except the patient started on 5rag per week who withdrew from the trial after 6 weeks due to lack of improvement. Clinical remission occurred in 7 patients at 7.5 mg per week and in 2 patients at 10 mg per week. One patient with inflammatory polyarthritis received20 mg per week although the colitis was controlled at a lower dose. One patient developed mouth ulcers which were not sufficiently severe to cease therapy. No adverse hematological or biochemical events occurred. One patient ceased methotrexate after sustained clinical remission and relapsed with severe watery diarrhoea 2-3 weeks later. Reintroduction of methotrexate resulted in complete clinical response. Three patients were able to discontinue methotrexate without relapse and 2 had complete resolution of the histological changes including the collagenous layer at rebiopsy. A further patient had marked reduction in the thickness of the collagen layer at rebiopsy. Conclusions • this open trial showed low dose methotrexate to be a safe, effective therapy for collagenous colitis • the response to methotrexate is rapid, occurring within 3 weeks • controlled studies to confirm these findings appear to be justified

1441

Thalidomide inhibits inflammatory And Angiogenic Activation Of Human Intestinal Microvascular Endothelial Ceils (HIMEC). Daniel J. Stein, Parvaneh Rafiee, Angle Taras, Thomas H. Lamirand, Pamela J. Fisher, Hitoshi Ogawa, Gordon L Telford, Mary F. Otterson, Christopher P. Johnson, David G= 8inion, Medical Coil of Wisconsin, Milwaukee, Wl

Backgroundand AJms:The immunomodulator thalidomide (~N-phthalimido-glutarimide; MW 258) is a transcriptional inhibitor of TNF-e, but is also known to exert effects on human blood vessels which may underlie its teratooenicity during fetal development. Thalidomide has been sucessfully used in the treatment of refractory Crohn's disease (CO) chronic intestinal inflammation, but the therapeutic mechanism is not defined. Because endothelial activation and growth (neovascularization) are important components of chronic inflammation, we examined the effect of thalidomide on primary cultures of HIMEC, the relevant endothelial cell population in IBD. We performed experiments to determine the effect of thalidomide on HIMECactivation, leukocyte interaction and growth (angiogenesis). Methods: Primary cultures of HIMEC from control (n = 3) and CD (n = 3) pts were used in all experiments. HIMEC were pretreated with thalidomide (0.1 - 10 mg/ml; Celgene, Inc.) prior to activation with TNF-~ LPS or the selective endothelial growth factor, VEGF. A low shear stress flow adhesion assay with whole blood was used to assess HIMEC inflammatory activation, and a Wrioht's stain was used to identify leukocytes which remained adherent to the endothelial monolayer. Expression of pro-inflammatory cell adhesion molecules (CAM; E-selectin, ICAM-1, VCAM1) was assessed using whole cell ELISA, and iNOS expression was examined using Western blotting. HIMEC growth across a leading edge was quantified by cell enumeration following VEGF stimulation. Results: Thalidomide blocked adhesion of whole blood leukocytes by 50% (p = <0.05) in control and CD HIMEC, inhibiting binding of all classes of leukocytes (i.e. mononuclear cells, neutrophils). Increasing doses of thalidomide blocked TNF-oELPSactivated CAM expression in a dose dependent manner. In marked contrast, thalidomide did not effect iNOS expression, which plays a key role in the downregulation of HIMEC activation following TNF-a/LPS. VEGF-induced HIMEC growth was significantly inhibited by thalidomide between 24-72h, again demonstrating a dose response. Conclusions: Thalidomide exerted a potent inhibitory effect on HIMEC inflammatory and angiogenic activation. These data suggest that both anti-inflammatory as well as anti-angiooenic effects of thalidomide may play a role in the therapeutic effect of this compound in the treatment of chronic inflammation in CD.

1439 SB-265610, an IL-8 Antagonist, is a Potent Inhibitor of PMN Chemutaxis and is Effective in the Rabbit Immune Complex Model ol Colitis. John R. White, Judithann M Lee, SmithKline Beecham, King of Prussia, PA; Ann Flanigan, MCP Hahnemann Univ, Philadelphia, PA; Katherine L. Widdowson, Henry M. Sarau, SmithKline Beecham, King of Prussia, PA; Domenico Coppola, Univ of South F)orida, Tampa, FL; Sreekant Murthy, MCP Hahnemann Univ, Philadelphia, PA

1442

Background: Interieukin-8(IL-8) and the closely related CXC chemokines GRO~, /3 and ~/ are potent neutrophil chemoattractants which are proposed to be major mediators of the inflammatory response. The CXCchemokines activate neutrophils by binding to the cell surface receptors, CXCR1 (IL-8 selective) and CXCR2(activated by GRO(z,/3, -y and ENA-78. Methods and Results: Here we describe an orally active CXCR2antagonist which concentration-dependently inhibited binding of human ~2Sl-lL-8to hCXCR2-membranes,ICso= 39 -+ 11 nM This compound potently blocked GRO(~mediated Ca2÷ mobilization in human neutrophils (IC5o = 5 nM). In particular, SB-265610 inhibited human and rabbit PMN chemofaxis induced by both IL-8 and GRO~. This indicated that chemotaxis is mediated via CXCR2. We further investigated the therapeutic potential of SB-265610 in a rabbit immune complex model of colitis as described by Cominelli, F. et.a/. (JCI 1990 86, p972). Vehicle, SB-265610 (25 mg/ kg, p.o., b.i.d.) or 5-ASA (50 mg/kg, p.o., b.i.d.) was administered one day prior to induction of colitis. Individual therapies were administered for 7 days and animals were euthanized on day 8. At necropsy, SB-265610 treated animals showed no apparent gross lesions in the colon (score = O) as opposed to the vehicle and 5-ASA treated groups, which had scores of 0.83 -+ 0.41 and 2.33 -+ 1.6, respectively. Histological evaluation of the SB-265610 treated group showed a 29% improvement in chronic inflammation (monocytes and T-cell infiltration) and a 40% improvement in acute inflammation (PMN infiltration) compared to the vehicle treated group. The 5-ASA animal group showed a 30% improvement in chronic inflammation and a 43% improvement in acute inflammation. In the SB-265610, treated group, none of the animals developed ulcers were as in the 5-ASA and the vehicle treated groups, ulcers were present. The SB-265610 group had intact superficial epithelium and showed only modest shortening in the crypt architecture (<1/3 shortening of crypt). Based on these data, in this

Increased Mucosal TNF-u Produofionin Crohn's Disease Can Be Modulated Locally by Pmbioliss. Natalia 8orruel, Francesc Casellas, Maria Antolin, Monica Carol, Felipe De Lara, Eloy Espin, Francisco Guarner, Juan R. Malagelada, Hosp Gen Vail d'Rebron, Barcelona Spain BACKGROUND:InCrohn's disease (CD), TNF-~ appears to play a key role in the pathogenesis of altered mucosal immune function. Indeed, monoclonal anti-TNF therapy has shown a clinical, endoscopic and histological benefit in various clinical trials. We hypothesized that since pmbiotics may interact with immune cells of the gut mucosa and regulate cytokine production, they could exert modulatory effects on mucosal TNF-~ production. METHODS:We assessed the effect of selected lactobacilli strains on TNF-a release on explants of intestinal mucosa obtained at surgery from 8 patients with CD(6M/2F;agerange 19-45 years)undergoing ileal resection for stricture. Ileal specimens from patients undergoing right hemi-colectomy for cecal cancer served as normal controls(n = 4). Mucosal explants (20-30 mg)from inflamed and non-inflamed areas were immediately cultured at 37°C with 5%C02 in RPM11640. Thirtysix mucosal explants from each specimen were cultured with 106 viable bacteria/mL of nonpathogenic Ecoli, L.casei DN114001, L.bu/garicus and L.jansenii. Each study contained blank wells with no bacteria. After 24 hours culture, TNF-a concentration in supernatants was measured by ELISA. Tissue viability was always confirmed by LDH release. RESULTS:Tissue viability was not challenged by co-culture with bacteria. TNF-~ is expressed in Table as pg/ mL (*= p
A-278

mucosa as comparedwith non-inflamed CD and controls. Incubation with lactobacilli significantly reducedTNF-~ releasefrom inflamedmucosa.CONCLUSION:Selectedlactobacillistrains may exert a pronounced antiinflammatory effect on the affected intestinal mucosa of patients with Crohn's disease. TNF-a (pg/mL)

Blank E.coli L.cesei L.bulgaricus L.jensenii

InflamedCD

Non-inflamedCD

Control

122_+36 278+140 25.8_+7.2* 41.3_+15.5* 69.3-+22 *

27.4_+3.3 26.2-+4.7 24.5_+3.8 22.5_+3.8 24.~4.9

8.1_+2.2 10~2.6 9.~2.9 5.7_+1.2 5.7_+0.9

1443 Clarithromycin Treatment In Patients With Chronic Treatment-Resiatant Ponchitis Fernando Ri77ello,Paolo Gionchetti, Cristina Amadini, Alessandro Ventud, Oept of Int Med and Gastroenterol, Univ of Bologna, Bologna Italy; Federica Ugolini, Surg Unit, Univ of Bologna, Bologna Italy; Karen M. Lammers, Rossella Romagnoli, Dept of Int Med and Gastroenterol, Univ of Bologna, Bologna Italy; Gill)erto Poggioli, Surg Unit, Univ of Bologna, Bologna Italy; Massimo Campieri, Dept of Int Med and Gastroenterol, Univ of Bologna, Bologna Italy

pathogenesisof UC. The aim of the present study was to evaluatethe efficacy and safety of monoclonal, humanized,anti human interleukin-2 receptor (CD25) antibodies, in refractory ulcerative colitis in an open-label pilot study. Patients and methods. Up to present 5 patients with refractory UC were included in the study and received 1 mg/kg of ZenapaxtV at week 0 with a second infusion at week 4, The clinical and endoscopicatRachmilewitzseverity score, a histological score and IBDQ were monitored to evaluate efficacy at week 0,1, 2 and 8. Circulating blood lymphocytes were followed throughout the study. Immuno-histochemistry for CD25 was performed on all biopsies. Results. Zenapaxtreatment was well tolerated in all patients. The clinical activity index improved in 4 out of 5 patients at 2 weeks after the first infusion. However,at this time point there was no change in the endoscopical index and in the histological severity score. CD25 positivity in the muocsal biopsies diminshed over time. At week 2 no change in the total number or in the CD4/CD8 ratio of circulating lymphocytes was observed. All patients have not yet reachedthe 4 week time point but the improvement appearsto be persistent. Conclusion. The preliminary results of this pilot study indicate that anti IL-2 receptor antibodies, Zenapax, may be an efficacious new biological treatment for refractory UC and confirm the role of IL-2 receptor activation in the pathogenesisof UC. 1446 A Colon Epithelial Protein(s) Induces Oral Tolerance in a Dose Dependent Manner in the Trinitrobenzene Sulfonic Acid (TNBS) Model of Colitis in Rats Arunansu Dasgupta,Jorge Giraldo, Peter S. Amenta, Kiron M. Das, UMDNJ-RobertWood Johnson Medical Sch, New Brunswick, NJ

Backgroun¢. Pouchitis is the major long-term complication after ileal pouch-analanastomosis for ulcerative colitis. Broad-spectrum antibiotics (mainly metronidazoleor ciprofloxacin) are the mainstayof treatment, however, 15% of patients with pouchitis havea chronic, treatmentresistant disease.Clarithromycin is a broad-spectrumantibiotic that can stimulate macrophage proliferation, phagocytosis, chemotaxis and cytocidal activity. Aim: To determine the safety and efficacy of the treatment with oral clarithromycin 500mg tablets b.i.d, in patients with chronic trsatment-resistant pouchitis. Patients and method~. Ten patients with treatmentresistant chronic pouchitis [previous failed treatment was metronidazole1g/daily for 1 month (n = 8) and ciprofloxacin 1g/daily for 1 month (n = 2)] were treated orally with clarithromycin 500mg b.i.d, for 30 days. Diagnosis of pouchitis and response to treatment were evaluated with the Pouchitis Disease Activity Index (PDAI), wich includes clinical, endoscopic and histological criteria. Results: Eight of 10 patients(80%) went into remission with a significant decrease of mean total PDAI score from 11.2 (range 8-16) to 2.8 (range 0-12) (p< 0.002). The improvementwas registered in all clinical, endoscopic and histological portions of PDAL Clarithromycin was well tolerated and no serious side-effects were reported during the treatment. Conclusion~. Clarithromycin 1g/daily was effective and safe in patients with active chronic, treatment-resistantpouchitis, suggestingthe need, in these patients, of an antibiotic with a wide antibacterial spectrum.

Rectal (PR) administration of TNBS produces chronic colitis in the distal colon in rodents. This is a reproducibleimmune mediatedexperimentalmodel of chronic colonic inflammation. We have earlier demonstrated that colon, but not small intestinal extract, is involved in the induction of oral tolerance in this model in rats, suggesting the organ specificity of this oral tolerance. Using a colon epithelial cal~extract iCE) of a human colon cancer cell line, LS1BO, we examined the production of oral tolerance at various protein concentrations. Seventeen rats were fed with CE (Gr A-125/~g in=5), Gr B-250/~g in=6) & Gr C-500,u,gin=6)) each time in the drinking water on alternating days, 5 times, over 10 days, followed by the introduction of TNBS 30mg/rat PR. Gr O (n = 6) consisted of TNBS given PR without feeding CE; Gr E in=6) were the control animals without TNBS-PR. Two weeks after, the animals were sacrificed, blood collected, colon (proximal (PC) and distal (DC)) and small intestine (SI) processed separately.Diseasewas localizedin the distal colon and was scored on a 0-5 scale.Tissuewas examinedfor histology and intestinal extractswere usedfor myeloperoxidase (MPO, unif/mg Pr) assay. Serum was used for estimation of gamma interferon. Resu~: Mean+_SEMvalues are shown in the table. There were no significant differences in various parameters in the PC and SI specimens among the groups (A-D). Conclusion: These results demonstrate that LS180 colon epithelial cell extract is capable of inducing oral tolerance in the TNBS model and it is dose-dependent.Further studies are neededto identify the specific epithelial antigen involved in this oral tolerization. Table

1444

Groups

Fistula Healing in MAP Positive Crnhn's Disease Patients Following Rifabutin and Macrolide Antibiotic Therapy Ira Shefran, Lynn J. Kugler, Javier E. Sandoval, Univ of Cent Florida, Orlando, FL

A B C D E

Background:Prior studies haveshown the beneficialrole of Ritabutin and MacrolideAntibiotic Therapy (RMAT) in the treatment of intractable, fistulizing, Crohn's disease. Methods: We identified a subset of 10 MAP positive patients (3 malesand 7 females) ages 41-20 presenting acute lower GI Crohn s with fistulization. Thesepatientsfailed to respondto prior corticosteroid and anti-inflammatory therapy and presented a mean Crohn's Disease Activity Index (CDAI) score of 269 (250 - 300). The distribution of fistula was scrotal 1 (10%), scrotal-anal 1 (10%), vaginal-anal 1 (10%), perianal5 (50%), ileocecal 1(10%) and colonic 1 (10%). These patients were treated with RMAT consisting of 250 mgm 1 po bid Clarithromycin and 150 mgm 1 po bid RifabuUn. Antibiotic therapy was complemented with 200 mgm po bid of probioticcontaining equal amounts of Lactobacillusacidophilusand Lactobacillusrhamnosus. Results: Patients treated with RMAT had complete closure of fistuli in all cases within an average time of 32 weeks (8 - 60) of treatment and presenting a mean of 126.5 points in their final CDAI score. All patients reached a state of remission classically defined by a CDAI score below 150 points with a minimum differential of 100 points from initial to final score. Subsequentfollow up showed a recurrence of fistuli within an averagetime o1 8 months (213) in 4 (40%) patients who chose to discontinue RMAT. Conclusion: This subset of MAP positive patients may represent a particularly responsive group of Crohn's patients whose disease is remarkably responsive to antimycobactedal therapy but who require effective alternativetherapies for the maintenanceof remission in their Crohn's disease.Double blind, placebo controlled trials are required to further examinethe effectivenessof antibiotics in the management of fistulizing Cronh's disease.

DC.wt(g)

MPO-DC

IFN.,y

Mean Disease Score

3+1 4_+2 2-+1a 7_+1 1_+0

77_+6 103~20 34_+7b 76_+9 11_+7

27_+2 28_+2 17_+4c 4Z~5 16_+1

5 4 2e 5 0

a p 3 months with > 1 unsuccessful taper attempt). Patients were stratified by baseline prednisone dose (10-19 or 20-40 mg/d) and randomized to 1 of 3 treatment groups: placebo, or ISiS 2302 (2 mg/kg/d IV 3 times a week) for 2 OR 4 weeks, with infusion courses in both months 1 and 3. Forced steroid withdrawal was completed, if tolerated, by the end of week 9. Patients in remission at month 6 were followed for 1 year to assess remission duration. Completeclinical remission, defined as CDAI <150 at the end of week 14 and off steroids, was the primary study endpoint. Plasma half-life was 1.11 + 0.2 hours with an average maximum concentration of 14.8 + 3 pg/ml. Using population pharmacokinetic(PK) analysis,the calculatedconcentration of drug in plasma over time (AUC) was used as an index of a patient s total exposureto drug, Genderand weight were associated with AUC variability. Although all patients receiveda 2 mg/kg ISIS 2302 dose, heavier people and females had the highest AUCs. The remission rate was similar for 2 or 4 week ISIS 2302 (20.2%) and placebo (18.8%). However, the remission rate for ISIS 2302 treated patients (N = 9) in the highest AUC subgroup (AUC>65ug*hr/ml) was 55.6% (p = 0.025 vs placebo). Other measuresof clinical responseconsistentlydemonstratedimprovementfor the high AUC subgroup. High AUC patients median CDAI score decreased by 136 (+112.5) at week 14 compared to a 52.2 (+107.2) for placebo (p=0.027). The Inflammatory Bowel Disease Quotient (IBDQ) improved 42.9 (+31) in the high AUC subgroup versus 15.1 (+32) for placebo (p=0.027). Duration of remission of 4/5 high AUC responderswas > 1 year (1 not followed past Week 14). The incidence of adverse reactions was low. Vasodilatation was

1445 The Use of Humanized, Anti Interleukin-2 ReceptorAntibodies (Zenapax~, Roche) in Refractory Ulcerative Colitis. Gert Van Assche, Dept of Gastroenterology,Univ of Leuven, Leuven Belgium; lgnace Dalle, Dept of Pathology, Univ of Leuven, Leuven Belgium; Maja Noman, Isolde Aerden, Caroline Swijsen, Dept of Gastroenterology,Univ of Leuven, Leuven Belgium; Bart Maes, Dept of Nephrology, Univ of Leuven, Leuven Belgium; Karel Geboes, Dept of Pathology, Univ of Leuven, Leuven Belgium; Jan Ceuppens, Dept of Immunology, Univ of Leuven, Leuven Belgium; Paul Rutgeerts, Dept of Gastroenterology,Leuven Belgium Introduction. Although the etiology of ulcerative colitis (UC) has not yet been elucidated, immunosuppressivetherapy has proven to be valuable in the treatment of refractory disease. Although non-specific immunosuppressivetreatment with azathioprineis widely acceptedas maintenance therapy, the efficacy of acute treatment with cyclosporine in severe flares has suggested the importance of interleukin-2 ilL-2) mediated T-lymphocyte activation in the

A-279