Intercellular Adhesion Molecule-1 Antisense Oligonucleotides Research Articles

Alicaforsen, an Antisense Inhibitor of Intercellular Adhesion Molecule-1, in the Treatment for Left-Sided Ulcerative Colitis and Ulcerative Proctitis

Background: Data on the efficacy of intercellular adhesion molecule-1 antisense oligonucleotide alicaforsen in ulcerative colitis (UC) is inconsistent. Methods: All patients, who had received at least one dose of alicaforsen, were analyzed retrospectively. Alicaforsen’s efficacy was assessed in patients treated for left-sided UC and proctitis by comparing clinical and (if applicable) endoscopic disease activity before/after treatment. Results: Twelve patients were treated for left-sided UC or proctitis. Eleven patients received a 6-week course of a once-daily 240 mg alicaforsen enema formulation. In 1 patient, treatment was discontinued, because it was found to be inefficient. Disease activity measured by the partial Mayo score and 6-point symptom score was significantly reduced after treatment (6.0 vs. 2.4, p = 0.011 and 3.7 vs. 1.4, p = 0.008). Faecal calprotectin showed a trend towards reduction (484.4 vs. 179.5 μg/g, p = 0.063). Clinical improvement was achieved in 10 patients (83.3%). In 7 patients, a relapse occurred (70%). Median duration of clinical improvement was 18.0 weeks (range 1–112). Three patients showed an ongoing improvement of >9 months. No adverse events were reported. Conclusions: A 6-week course of alicaforsen seemed to be safe and efficacious in inducing clinical improvement in patients with left-sided UC and proctitis. Prolonged clinical improvement was observed in many but not all patients.

Intercellular adhesion molecule‐1 (ICAM‐1) in ulcerative colitis: presence, visualization, and significance

The intensive research of recent years has suggested that the cause of ulcerative colitis (UC) involves a genetic predisposition to an uncontrolled or unbalanced immune response to luminal or epithelial antigens or against other external factors. Intercellular adhesion molecule-1 (ICAM-1) is pivotal for the influx of neutrophil granulocytes into colonic mucosa, and gene analyses have found polymorphisms in the gene encoding ICAM-1, indicating that changes in ICAM-1 function may be involved in the pathogenesis of UC. Clinical trials of the ICAM-1 antisense oligonucleotide Alicaforsen, which inhibits the synthesis of ICAM-1, have shown positive results in the treatment of patients with left-sided (distal) UC. In addition to emphasizing the central role of ICAM-1 in active stages of UC, the results provide hope for the development and introduction of a more specific and efficient treatment for UC than those currently available. This review discusses the results from studies on the expression of ICAM-1 in colonic tissue from patients with UC.

Positive Role of CCAAT/Enhancer-Binding Protein Homologous Protein, a Transcription Factor Involved in the Endoplasmic Reticulum Stress Response in the Development of Colitis

Although recent reports suggest that the endoplasmic reticulum (ER) stress response is induced in association with the development of inflammatory bowel disease, its role in the pathogenesis of inflammatory bowel disease remains unclear. The CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) is a transcription factor that is involved in the ER stress response, especially ER stress-induced apoptosis. In this study, we found that experimental colitis was ameliorated in CHOP-null mice, suggesting that CHOP exacerbates the development of colitis. The mRNA expression of Mac-1 (CD11b, a positive regulator of macrophage infiltration), Ero-1alpha, and Caspase-11 (a positive regulator of interleukin-1beta production) in the intestine was induced with the development of colitis, and this induction was suppressed in CHOP-null mice. ERO-1alpha is involved in the production of reactive oxygen species (ROS); an increase in ROS production, which is associated with the development of colitis in the intestine, was suppressed in CHOP-null mice. A greater number of apoptotic cells in the intestinal mucosa of wild-type mice were observed to accompany the development of colitis compared with CHOP-null mice, suggesting that up-regulation of CHOP expression exacerbates the development of colitis. Furthermore, this CHOP activity appears to involve various stimulatory mechanisms, such as macrophage infiltration via the induction of Mac-1, ROS production via the induction of ERO-1alpha, interleukin-1beta production via the induction of Caspase-11, and intestinal mucosal cell apoptosis.

Delivery of Intercellular Adhesion Molecule-1 Antisense Oligonucleotides Using a Topical Hydrogel Tissue Sealant in a Murine Partial Nephrectomy/Ischemia Model

Ischemia/reperfusion injury is a leading cause of renal damage which can be improved with antisense oligonucleotide gene therapy. We have shown that polyethylene glycol (PEG) hydrogel, which also functions as a tissue sealant, is an effective topical delivery vehicle for oligonucleotides in a murine partial nephrectomy model. The objective of this study was to use and evaluate this method against intercellular adhesion molecule-1 (ICAM-1) to prevent tissue damage. Sixty mice underwent left upper pole partial nephrectomy with 45 minutes of warm ischemia, randomized to treatment with 50 microg ICAM-1 antisense oligonucleotides embedded in PEG hydrogel, no therapy, or sham surgery. Kidneys were harvested at 24 hours and 3, 4, and 5 days. The specimens were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) for ICAM-1 messenger ribonucleic acid (mRNA), immunohistochemical staining for ICAM-1 protein, and standard histology. At 24 hours, qRT-PCR and immunohistochemistry data showed a significant reduction in ICAM-1 mRNA and protein expression in the antisense group versus the ischemia group, but no difference at 3 to 5 days. Histologically there was reduced inflammation and necrosis in the cortex at 24 hours. The outer and inner medulla also showed improvement at 3 to 5 days in the antisense group as opposed to the ischemia group. Topical PEG hydrogel delivery of antisense ICAM-1 oligonucleotides demonstrated decreased ICAM-1 mRNA expression, reduced ICAM-1 protein staining, and decreased cellular damage. The application of gene therapy through this novel topical delivery system holds potential for a highly specific, localized method of preventing tissue damage after ischemia/reperfusion injury.

The ICAM‐1 antisense oligonucleotide ISIS‐3082 prevents the development of postoperative ileus in mice

Intestinal manipulation (IM) during abdominal surgery triggers the influx of inflammatory cells, leading to postoperative ileus. Prevention of this local muscle inflammation, using intercellular adhesion molecule-1 (ICAM-1) and leukocyte function-associated antigen-1-specific antibodies, has been shown to shorten postoperative ileus. However, the therapeutic use of antibodies has considerable disadvantages. The aim of the current study was to evaluate the effect of ISIS-3082, a mouse-specific ICAM-1 antisense oligonucleotide, on postoperative ileus in mice. Mice underwent a laparotomy or a laparotomy combined with IM after treatment with ICAM-1 antibodies, 0.1-10 mg kg(-1) ISIS-3082, saline or ISIS-8997 (scrambled control antisense oligonucleotides, 1 and 3 mg kg(-1)). At 24 h after surgery, gastric emptying of a 99mTC labelled semi-liquid meal was determined using scintigraphy. Intestinal inflammation was assessed by myeloperoxidase (MPO) activity in ileal muscle whole mounts. IM significantly reduced gastric emptying compared to laparotomy. Pretreatment with ISIS-3082 (0.1-1 mg kg(-1)) as well as ICAM-1 antibodies (10 mg kg(-1)), but not ISIS-8997 or saline, improved gastric emptying in a dose-dependent manner. This effect diminished with higher doses of ISIS-3082 (3-10 mg kg(-1)). Similarly, ISIS-3082 (0.1-1 mg kg(-1)) and ICAM-1 antibodies, but not ISIS-8997 or higher doses of ISIS-3082 (3-10 mg kg(-1)), reduced manipulation-induced inflammation. Immunohistochemistry showed reduction of ICAM-1 expression with ISIS-3082 only. ISIS-3082 pretreatment prevents postoperative ileus in mice by reduction of manipulation-induced local intestinal muscle inflammation. Our data suggest that targeting ICAM-1 using antisense oligonucleotides may represent a new therapeutic approach to the prevention of postoperative ileus.

Intercellular adhesion molecule-1 (ICAM-1) in ulcerative colitis: Presence, visualization, and significance

The intensive research of recent years has suggested that the cause of ulcerative colitis (UC) involves a genetic predisposition to an uncontrolled or unbalanced immune response to luminal or epithelial antigens or against other external factors. Intercellular adhesion molecule-1 (ICAM-1) is pivotal for the influx of neutrophil granulocytes into colonic mucosa, and gene analyses have found polymorphisms in the gene encoding ICAM-1, indicating that changes in ICAM-1 function may be involved in the pathogenesis of UC. Clinical trials of the ICAM-1 antisense oligonucleotide Alicaforsen, which inhibits the synthesis of ICAM-1, have shown positive results in the treatment of patients with left-sided (distal) UC. In addition to emphasizing the central role of ICAM-1 in active stages of UC, the results provide hope for the development and introduction of a more specific and efficient treatment for UC than those currently available. This review discusses the results from studies on the expression of ICAM-1 in colonic tissue from patients with UC.

Role of ICAM-1 in chronic hepatic allograft rejection in the rat.

The pathogenesis of hepatic allograft rejection remains unclear. We aimed to clarify the early role of intercellular adhesion molecule-1 (ICAM-1)-mediated cell recruitment in chronic hepatic rejection. Liver transplantation was performed from Lewis to Lewis rats (isograft controls) and from Lewis to Brown Norway rats (allograft rejection group). The allografted rats were treated with either ICAM-1 antisense oligonucleotides (10 mg. kg(-1). day(-1) x 6 days ip) or a control preparation (either ICAM-1 missense oligonucleotide or normal saline). Hepatic leukocyte recruitment in vivo was studied on day 6 by using intravital microscopy. Liver histology, biochemistry, and survival rates were also examined. Leukocyte adhesion in terminal hepatic venules was significantly increased in the rejection group compared with isograft controls. Antisense ICAM-1 in the allografted group effectively reduced leukocyte adhesion. Histology and liver chemistry were less deranged in the antisense-treated groups compared with control-treated allografted rats. In the allograft groups, survival was significantly prolonged in the antisense-treated rats (42.3 +/- 1.2 days) compared with the controls (25.2 +/- 2.7 days). These results showed that early leukocyte recruitment in the hepatic microvasculature of rejecting allografts is ICAM-1 dependent and suggest that impacting on early cell recruitment can significantly ameliorate chronic rejection.

Effects of ICAM-1 antisense oligonucleotide on the tubulointerstitium in mice with unilateral ureteral obstruction

Effects of ICAM-1 antisense oligonucleotide on the renal tubulointerstitium in mice with unilateral ureteral obstruction. To extend our previous study of the therapy of the renal lesions of unilateral ureteral obstruction (UUO) in mice by an inhibitor of intercellular adhesion molecule-1 (ICAM-1), we investigated the blocking effects of ICAM-1 antisense oligonucleotides (ASONs) on the ICAM-1 expression in mouse kidney. First, ICAM-1 ASON was transducted into mouse renal tubular epithelial cells to investigate the effects of ICAM-1 ASON in vitro. Second, fluorescein isothiocyanate (FITC)-labeled ICAM-1 ASON was injected intravenously to determine the distribution of the ASON in vivo. Third, the expression of ICAM-1 in kidney and the changes of renal morphology were observed to investigate the therapeutic effects of ICAM-1 ASON on the UUO mice in vivo. The expressions of ICAM-1 in the epithelial cells induced by interleukin-1beta were inhibited by ICAM-1 ASON at the dosages of 100 and 200 nmol/L. Twenty-four hours after an introvenous injection with FITC-labeled ICAM-1 ASON, the highest level of fluorescein was detected within the proximal tubules in mouse kidney. Results of immunohistology and Northern blot showed that the ICAM-1 expression was markedly reduced in the obstructed kidney after treatment with ICAM-1 ASON. The ASON also alleviated the infiltration of inflammatory cells and accumulation of the extracellular matrix in the tubulointerstitium of UUO mice without apparent side effects. Our data demonstrate that ICAM-1 ASON is taken up primarily by the proximal tubular cells of mouse kidney. ICAM-1 ASON can selectively inhibit the ICAM-1 expression of the renal tubular cells both in vitro and in vivo.

A placebo-controlled trial of ICAM-1 antisense oligonucleotide in the treatment of Crohn's disease

Background & Aims: Intercellular adhesion molecule 1 (ICAM-1) plays an important role in the trafficking and activation of leukocytes and is up-regulated in inflamed mucosa in Crohn's disease. ISIS 2302 is an antisense phosphorothioate oligodeoxynucleotide that inhibits ICAM-1 expression. The aim of this study was to obtain preliminary assessment of tolerability, pharmacology, and efficacy of ISIS 2302 in Crohn's disease. Methods: Twenty patients with active, steroid-treated Crohn's disease were randomized (3:1, ISIS 2302 to placebo) to receive over 26 days 13 intravenous infusions of ISIS 2302 (0.5, 1, or 2 mg/kg) or saline placebo in a double-blinded study. The patients were followed up for 6 months. Results: At the end of treatment, 47% (7 of 15) of ISIS 2302–treated and 20% (1 of 5) of the placebo-treated patients were in remission (Crohn's Disease Activity Index [CDAI] < 150). At the end of month 6, 5 of these 7 ISIS 2302–treated remitters were still in remission, and a 6th patient had a CDAI of 156. Corticosteroid usage was significantly lower ( P = 0.0001) in the ISIS 2302–treated patients. These findings were corroborated by significant increases in β 7 and α d bearing CD3 + peripheral blood lymphocytes and by decreases in intestinal mucosal ICAM-1 expression during the treatment period. Conclusions: ISIS 2302 seems to be a well-tolerated and promising therapy for steroid-treated Crohn's disease. GASTROENTEROLOGY 1998;114:1133-1142

Structural Requirements for Cationic Lipid Mediated Phosphorothioate Oligonucleotides Delivery to Cells in Culture

A series of 2,3-dialkyloxypropyl quaternary ammonium lipids containing hydroxyalkyl chains on the quaternary amine were synthesized, formulated with dioleoylphosphatidylethanolamine (DOPE) and assayed for their ability to enhance the activity of an intercellular adhesion molecule 1 (ICAM-1) antisense oligonucleotide, ISIS 1570. Cationic liposomes prepared with hydroxyethyl, hydroxypropyl and hydroxybutyl substituted cationic lipid all enhanced the activity of the ICAM-1 antisense oligonucleotide. Cationic lipids containing hydroxypentyl quaternary amines only marginally enhanced the activity of ISIS 1570. Hydroxyethyl cationic lipids synthesized with dimyristyl (C14:0) and dioleyl (C18:1) alkyl chains were equally effective. Activity of cationic lipids containing saturated alkyl groups decreased as the chain length increased, i.e. the dimyristyl (C14:0) was more effective than dipalmityl (C16:0) lipid, which was more effective than distearyl (C18:0). The phase transition temperature of cationic lipids containing saturated aliphatic chains was 56°C for the distearyl lipid, 42°C for the dipalmityl lipid and 24°C for the dimyristyl lipid. Cationic lipids with dioleyl alkyl chains required DOPE for activity, with optimal activity occurring at 50 mole%. In contrast, a dimyristyl containing cationic lipid did not require DOPE to enhance the activity of ISIS 1570. Formulation with different phosphatidylethanolamine derivatives, revealed that optimal activity was obtained with DOPE. These studies demonstrate that several cationic lipid species enhance the activity of phosphorothioate antisense oligonucleotides and provide further information on the mechanism by which cationic lipids enhance the activity of phosphorothioate oligodeoxynucleotides.

Role of the intercellular adhesion molecule-1(ICAM-1) in endotoxin-induced pneumonia evaluated using ICAM-1 antisense oligonucleotides, anti-ICAM-1 monoclonal antibodies, and ICAM-1 mutant mice.

This study examined the effectiveness of antisense oligonucleotides targeted to intercellular adhesion molecule-1 (ICAM-1) to inhibit endotoxin-induced upregulation of ICAM-1 and neutrophil emigration and compared the apparent role of ICAM-1 when examined using antisense oligonucleotides, anti-ICAM-1 antibodies, and ICAM-1 mutant mice. Antisense oligonucleotides inhibited upregulation of ICAM-1 mRNA at 4 and 24 h after instillation of endotoxin in a dose-dependent manner. Neutrophil emigration into the alveolar spaces at 24 h was inhibited by 59%, similar to inhibition using the anti-ICAM-1 antibodies 3E2 (58%) and YN1/1 (75%). No inhibition was observed in the ICAM-1 mutant compared to wild-type mice. These data show that antisense oligonucleotides targeted to ICAM-1 inhibit the endotoxin-induced upregulation of ICAM-1 in the lung and are as effective as anti-ICAM-1 antibodies in preventing neutrophil emigration. The incomplete inhibition by either antisense oligonucleotides or antibodies suggests that alternative adhesion pathways that do not require ICAM-1 are important in neutrophil emigration in the lungs. The disparity in the role of ICAM-1 when evaluated using antisense or antibodies compared to mutant mice suggests that either these inhibitors are exerting additional effects on endothelial cells other than blockade of ICAM-1 or mutant mice have upregulated the ICAM-1-independent pathways to compensate for the long-term loss of ICAM-1.

Role of the intercellular adhesion molecule-1 (ICAM-1) in endotoxin-induced pneumonia evaluated using ICAM-1 antisense oligonucleotides, anti-ICAM-1 monoclonal antibodies, and ICAM-1 mutant mice

Role of the intercellular adhesion molecule-1 (ICAM-1) in endotoxin-induced pneumonia evaluated using ICAM-1 antisense oligonucleotides, anti-ICAM-1 monoclonal antibodies, and ICAM-1 mutant mice

Blocking of heart allograft rejection by intercellular adhesion molecule-1 antisense oligonucleotides alone or in combination with other immunosuppressive modalities

Blocking of heart allograft rejection by intercellular adhesion molecule-1 antisense oligonucleotides alone or in combination with other immunosuppressive modalities

Blocking of heart allograft rejection by intercellular adhesion molecule-1 antisense oligonucleotides alone or in combination with other immunosuppressive modalities.

Intercellular adhesion molecule-1 (ICAM-1) binds circulating leukocytes through interactions with beta 2 integrins, LFA-1, and macrophage Ag-1. The phosphorothioate antisense oligodeoxynucleotide, IP-3082, specific for ICAM-1 mRNA inhibited ICAM-1, but not vascular cell adhesion molecule-1, mRNA induction and expression of ICAM-1 molecules by mouse endothelioma cells. Scrambled control oligonucleotides were ineffective. Untreated C3H (H-2k) mice rejected C57BL/10 (H-2b) heart allografts with a mean survival time of 7.7 +/- 1.4 days. Administration i.v. of IP-3082 by a 7-day osmotic pump prolonged the survival of heart allografts in a dose-dependent fashion: 1.25 mg/kg, to 11 +/- 0 days; 2.5 mg/kg, to 12 +/- 2.7 days; 5 mg/kg, to 14.1 +/- 2.7 days; and 10 mg/kg, to 15.3 +/- 5.8 days (all p < 0.01). Control IP-1082 (10 mg/kg) was ineffective (7 +/- 0.8 days). Although 7-day anti-LFA-1 mAb (50 micrograms/day; i.p.) prolonged allograft survival to 14.1 +/- 2.7 days, the addition of IP-3082 (5.0 mg/kg x 7 days) induced donor-specific transplantation tolerance (> 150 days). Furthermore, IP-3082 (5 mg/kg x 7 days) acted synergistically with antilymphocyte serum, rapamycin, and brequinar, but not cyclosporin A: a single antilymphocyte serum (0.2 ml) i.p. injection alone prolonged graft survival to 10 +/- 0.5 days (p < 0.01) and in combination with IP-3082 (5 mg/kg), to 32.2 +/- 8.3 days (p < 0.001); rapamycin (0.1 mg/kg x 7 days; i.v.) alone prolonged survival to 13 +/- 7.5 days (p < 0.01), and with IP-3082, to 32.4 +/- 8.9 days (p < 0.03); brequinar (0.5 mg/kg x 7 days; oral gavage) alone to 12 +/- 2.4 days (p < 0.05), and with IP-3082 (5 mg/kg), to 38.8 +/- 30.2 days (p < 0.01); in contrast, cyclosporin A (5 mg/kg x 7 days; i.v.) alone produced graft survival of 9.8 +/- 1.3 days (p < 0.1 and in combination with IP-3082 (5 mg/kg), produced survival of 7.8 +/- 3.5 days (NS). Thus, antisense oligonucleotides may proffer a selective gene-targeted immunosuppressive therapy for organ transplantation.

Cationic Lipid Is Not Required for Uptake and Selective Inhibitory Activity of ICAM-1 Phosphorothioate Antisense Oligonucleotides in Keratinocytes

Keratinocyte intercellular adhesion molecule-1 (ICAM-1) is important in mediating retention of T cells within the epidermal compartment. To determine if antisense oligonucleotides designed to hybridize to various ICAM-1 mRNA regions could selectively influence cultured keratinocyte ICAM-1 expression following gamma interferon (IFN-gamma), cells were exposed to several antisense compounds, in the absence and presence of cationic lipid (lipofectin). Keratinocytes rapidly internalized sense and antisense compounds (within 30-60 min), even in the absence of lipofectin with approximately 30% of the cell possessing positive nuclei. Such nuclear accumulation was not observed in the absence of lipofectin in cultured fibroblasts, smooth muscle cells, or endothelial cells, even though total cellular uptake within the cytoplasm was significantly increased in all these cell types. Using flow cytometry, IFN-gamma-inducible ICAM-1 expression was reduced 50% by antisense compounds with lipofectin, and by 30% without lipofectin. This inhibition was specific as no change was observed for HLA-DR or tumor necrosis factor-alpha receptor expression. Northern blot hybridization studies confirmed that ICAM-1 antisense oligonucleotides selectively and significantly inhibited ICAM-1 expression. These results suggest that such antisense compounds interact with keratinocytes differently than other cell types, and provide the in vitro basis for clinical trials in which reduction (or elimination) of ICAM-1 expression by epidermal keratinocytes could be selectively accomplished without necessarily influencing dermal cell types such as fibroblasts, endothelial cells, or smooth muscle cells.

Inhibition of endothelial cell adhesion molecule expression with antisense oligonucleotides.

In response to inflammatory stimuli, expression of a group of proteins that bind circulating leukocytes (endothelial-leukocyte adhesion molecules) are induced on the luminal surface of vascular endothelium. A series of phosphorothioate oligonucleotides 18 to 21 bases in length were designed and synthesized to hybridize selectively to the mRNA, which encodes three such endothelial-leukocyte adhesion molecules; human intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. Antisense oligonucleotides were identified that selectively inhibited ICAM-1, VCAM-1, and E-selectin expression in HUVEC. Oligonucleotides that hybridized to the 3'-untranslated region of either ICAM-1, VCAM-1, or E-selectin mRNAs promoted a selective reduction in the respective mRNA levels. In contrast, oligonucleotides that hybridized to 5'-untranslated sequences did not significantly reduce target mRNA levels, although they did promote a reduction in protein expression. With the use of flow cytometry to measure cell surface expression, ICAM-1 and E-selectin were selectively inhibited by their respective antisense oligonucleotide. At low concentrations of oligonucleotides, only VCAM-1 antisense oligonucleotides inhibited VCAM-1 expression. However, at an oligonucleotide concentration of 50 nM or greater, phosphorothioate oligonucleotides not predicted to hybridize to VCAM-1 mRNA also reduced VCAM-1 expression. The sequence-independent inhibition of VCAM-1 expression by phosphorothioate oligonucleotides could be the result of a perturbation in the transcriptional regulation of the VCAM-1 gene. ICAM-1, VCAM-1, and E-selectin antisense oligonucleotides reduced adhesion of HL-60 cells to TNF-activated HUVEC. These data demonstrate that phosphorothioate oligonucleotides are capable of selectively inhibiting the expression of ICAM-1, VCAM-1, and E-selectin in HUVEC.