- Email: [email protected]
Thalidomide inhibits inflammatory and angiogenic activation of human intestinal microvascular endothelial cells (HIMEC)
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
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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
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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
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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
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