Figure 7 From Icam-1 (cd54): A Counter-receptor For Mac

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. Intercellular adhesion molecule 1 (ICAM-1), a member of the immunoglobulin superfamily, is an inducible transmembrane glycoprotein that is constitutively expressed at low levels on vascular endothelial cells and a subset of leukocytes.

1. Figure 7 From Icam-1 (cd54) A Counter-receptor For Mac Os X

The common form of ICAM-1 contains five extracellular Ig-like domains, a hydrophobic domain, and a short cytoplasmic domain 5.The first and third Ig-like domains are responsible for binding to LFA-1 and Mac-1, respectively, both members of the β2 family of leukocyte integrins 6, 7.On the cell surface, ICAM-1 has been shown to exist as a non-covalently-linked dimer and larger multimer 8, 9.

X 1 Dustin, ML, Rothlein, R, Bhan, AK, Dinarello, CA, and Springer, TA. Induction by IL 1 and interferon gamma: tissue distribution, biochemistry, and function of a natural adherence molecule (ICAM-1). 1986; 137: 245–254 , x 2 Rothlein, RM, Dustin, L, Marlin, SD, and Springer, TA.

A human intercellular adhesion molecule (ICAM-1) distinct from LFA-1. 1986; 137: 1270–1274 In response to proinflammatory mediators, many cell types up-regulate expression of ICAM-1 on their surface. The primary counterligands for ICAM-1 are the β 2-integrins, leukocyte function–associated antigen 1 (LFA-1), and Mac-1, expressed on leukocytes. X 3 Marlin, SD and Springer, TA. Purified intercellular adhesion molecule–1 (ICAM-1) is a ligand for lymphocyte function associated antigen–1 (LFA-1). 1987; 51: 813–819 , x 4 Diamond, MS, Staunton, DE, deFougerolles, AR, Stacker, SA, Garcia-Aguilar, J, Hibbs, ML, and Springer, TA.

ICAM-1 (CD54): a counter-receptor for Mac-1 (CD11b/CD18). 1990; 111: 3129–3139 ICAM-1 serves multiple functions in the propagation of inflammatory processes, including facilitatory roles in leukocyte activation and in leukocyte emigration from the intravascular space in response to inflammatory stimuli. X 5 Butcher, EC. Leukocyte-endothelial cell recognition: three (or more) steps to specificity and diversity.

1991; 67: 1033–1036 , x 6 Furie, MB, Tancinco, MCA, and Smith, CW. Monoclonal antibodies to leukocyte integrins CD11a/CD18 and CD11b/CD18 or intercellular adhesion molecule–1 inhibit chemoattractant-stimulated neutrophil transendothelial migration in vitro. 1991; 78: 2089–2097 , x 7 Oppenheimer-Marks, N, Davis, LS, Bogue, DT, Ramberg, J, and Lipsky, PE. Differential utilization of ICAM-1 and VCAM-1 during the adhesion and transendothelial migration of human T lymphocytes. 1991; 147: 2913–2921 , x 8 Altmann, DM, Hogg, N, Trowsdale, J, and Wilkinson, D. Cotransfection of ICAM-1 and HLA-DR reconstitutes human antigen-presenting cell function in mouse L cells. 1989; 338: 512–514 , x 9 Van Seventer, GA, Shimizu, Y, Horgan, KJ, and Shaw, S.

The LFA-1 ligand ICAM-1 provides an important costimulatory signal for T cell receptor–mediated activation of resting T cells. 1990; 144: 4579–14458 , x 10 Entman, ML, Youker, K, Shoji, T, Kukielka, G, Shappell, SB, Taylor, AA, and Smith, CW. Neutrophil induced oxidative injury of cardiac myocytes. J Clin Invest.

1992; 90: 1335–1345 , x 11 Damle, NK, Klussman, K, Linsley, PS, and Aruffo, A. Differential costimulatory effects of adhesion molecules B7, ICAM-1, LFA-3, and VCAM-1 on resting and antigen-primed CD4 + T lymphocytes. 1992; 148: 1985–1992 , x 12 Poudier, J and Owens, T. CD54/Intercellular adhesion molecule 1 and major histocompatibility complex II signalling induces B cells to express interleukin 2 receptors and complements help provided through CD40 ligation. 1994; 179: 1417–1427 Current knowledge of the immunology of inflammatory bowel disease recognizes the recruitment and activation of inflammatory cells as a result of proinflammatory cytokine production, and the subsequent up-regulation of a spectrum of cell adhesion molecules, including ICAM-1, vascular cell adhesion molecule 1 (VCAM-1), selectins, and integrins, on mucosal endothelial and lamina propria mononuclear cells.

X 13 Montefort, S and Holgate, ST. Adhesion molecules and their role in inflammation. 1991; 85: 91–99 , x 14 Berlin, C, Berg, EL, Briskin, MJ, Andrew, DP, Kilshaw, PJ, Holzman, B, Weismann, IL, Hamann, A, and Butcher, EC. Alpha 4 beta 7 integrin mediates lymphocyte binding to the mucosal vascular addressin MAdCAM-1. 1993; 74: 185–195 , x 15 Malizia, G, Calabrese, A, Cottone, M, Raimondo, M, Trejdosiewicz, LK, Smart, CJ, Oliva, L, and Pagliaro, L. Expression of leukocyte adhesion molecules by mucosal mononuclear phagocytes in inflammatory bowel disease.

1991; 100: 150–159 , x 16 Ohtani, H, Nakamura, S, Watanabe, Y, Fukushima, K, Mizoi, T, Kimura, M, Hiwatashi, N, and Nagura, H. Light and electron microscopic immunolocalization of endothelial leukocyte adhesion molecule–1 in inflammatory bowel disease.

Virchows Arch A Pathol Anat Histopathol. 1992; 420: 403–409 , x 17 Nakamura, S, Ohtani, H, Wanatabe, Y, Fukushima, K, Matsumoto, T, Kitano, A, Kobayashi, K, and Nagura, H. In situ expression of the cell adhesion molecules in inflammatory bowel disease: evidence of immunologic activation of vascular endothelial cells. 1993; 69: 77–85 , x 18 Yacyshyn, BR, Lazarovits, A, Tsai, V, and Matejko, K. Crohn's disease, ulcerative colitis, and normal intestinal lymphocytes express integrins in dissimilar patterns. 1994; 107: 1364–1371 , x 19 Bernstein, CN, Sargent, M, Gallatin, WM, and Wilkins, J.

Β2 integrin/intercellular adhesion molecule (ICAM) expression in the normal intestine. Clin Exp Immunol. 1996; 106: 160–169 , x 20 Hokari, R, Miura, S, Fujimoro, H, Nagata, H, Sekizuka, E, Tsuzuki, Y, Kurose, I, Serizawa, H, Suematsu, M, Yagita, H, Granger, DN, and Ishii, H. Lymphocyte outflux to intestinal microlymphatics is an intercellular adhesion molecule–1 dependent process in Peyer's patches (abstr).

1997; 112: A369 Expression of the integrins α 2–α 6 CD49b–CD49f, α dβ 2, and α 4β 7 have been reported to be altered on lymphocytes isolated from patients with Crohn's disease and/or ulcerative colitis. X 18 Yacyshyn, BR, Lazarovits, A, Tsai, V, and Matejko, K. Crohn's disease, ulcerative colitis, and normal intestinal lymphocytes express integrins in dissimilar patterns. 1994; 107: 1364–1371 , x 19 Bernstein, CN, Sargent, M, Gallatin, WM, and Wilkins, J.

Β2 integrin/intercellular adhesion molecule (ICAM) expression in the normal intestine. Clin Exp Immunol. 1996; 106: 160–169 , x 21 Meenan, J, Spaans, J, Grool, TA, Pals, ST, Tytgat, GNJ, and van Deventer, SJH. Altered expression of alpha 4 beta 7, a gut homing integrin, by circulating and mucosal T cells in colonic mucosal inflammation. 1997; 40: 241–246 ISIS 2302 is a 20-base phosphorothioate oligodeoxynucleotide designed to specifically hybridize to a sequence in the 3' untranslated region of the human ICAM-1 message.

X 22 Bennett, CF, Condon, T, Grimm, S, Chan, H, and Chiang, MY. Inhibition of endothelial cell–leukocyte adhesion molecule expression with antisense oligonucleotides.

1994; 152: 3530–3540 , x 23 Nestle, F, Mitra, RS, Bennett, CF, and Nickoloff, BJ. Cationic lipid is not required for uptake and inhibitory activity of ICAM-1 phosphorothioate antisense oligonucleotide in keratinocytes. J Invest Dermatol. 1994; 103: 569–575 The oligodeoxynucleotide-RNA heterodimer so formed serves as a substrate for the ubiquitious nuclease RNase-H, with subsequent cleavage and reduction in cellular specific message content and consequent reduction in ICAM-1 expression. X 24 Chiang, MY, Chan, H, Zounes, MA, Freier, SM, Lima, WF, and Bennett, CF. Antisense oligonucleotides inhibit intercellular adhesion molecule 1 expression by two distinct mechanisms. 1991; 266: 1 , x 25 Bennett, CF and Crooke, ST.

Regulation of endothelial cell adhesion molecule expression with antisense oligonucleotides. Adv Pharmacol. 1994; 28: 1–43 Phosphorothioate oligodeoxynucleotides achieve relative nuclease resistance over DNA by the substitution of a sulfur molecule for one of the nonbridging oxygen molecules in each phosphodiester linkage. X 26 Wickstrom, E. Oligonucleotide stability in subcellular extracts and culture media. J Biochem Biophys Methods. 1986; 13: 97–102 , x 27 Hoke, GD, Draper, K, Freier, S, Gonzales, C, Driver, VB, Zounes, MC, and Ecker, DJ.

Effects of phosphorothioate cappping on antisense oligonucleotide stability, hybridization and antiviral efficacy versus herpes simplex virus infection. Nucleic Acid Res. 1991; 19: 5743–5748 ISIS 2302 selectively inhibits cytokine-induced ICAM-1 expression in a variety of human cells in vitro and in vivo. X 22 Bennett, CF, Condon, T, Grimm, S, Chan, H, and Chiang, MY.

Inhibition of endothelial cell–leukocyte adhesion molecule expression with antisense oligonucleotides. 1994; 152: 3530–3540 , x 23 Nestle, F, Mitra, RS, Bennett, CF, and Nickoloff, BJ. Cationic lipid is not required for uptake and inhibitory activity of ICAM-1 phosphorothioate antisense oligonucleotide in keratinocytes. J Invest Dermatol. 1994; 103: 569–575 , x 28 Mielo, ME, Bennett, CF, Miller, BE, and Welch, DR. Enhanced metastatic ability of TNF-α treated malignant melanoma cells is reduced by intercellular adhesion molecule–1 (CD54) antisense olgonucleotides.

Exp Cell Res. 1994; 214: 231–241 , x 29 Christofidou-Solomidou, M, Albelda, SM, Bennett, CF, and Murphy, GF. Experimental production and modulation of human cytotoxic dermatitis in human-murine chimeras.

1997; 150: 631–639 A murine analogue, ISIS 3082, has been shown to be active in multiple models of inflammation, including dextran sulfate–induced colitis. X 30 Stepkowski, SM, Tu, Y, Condon, TP, and Bennett, CF.

Blocking of heart allograft rejection by ICAM-1 antisense oligonucleotides alone or in combination with other immunosuppresive modalities. 1994; 153: 5336–5346 , x 31 Bennett, CF, Kornbrust, D, Henry, S, Stecker, K, Howard, R, Cooper, S, Dutson, S, Hall, W, and Jacoby, HI. An ICAM-1 antisense oligonucleotide prevents and reverses dextran sulfate sodium–induced colitis in mice. J Pharmacol Exp Ther. 1997; 280: 988–1000 , x 32 Kumasaka, T, Quinlan, WM, Doyle, NA, Condon, TP, Sligh, J, Takei, F, Beaudet, AL, Bennett, CF, and Doerschuk, CM.

The role of ICAM-1 in endotoxin-induced pneumonia evaluated using ICAM-1 antisense oligonucleotides, anti–ICAM-1 monoclonal antibodies, and ICAM-1 mutant mice. J Clin Invest. 1996; 97: 2362–2369 Control oligonucleotides failed to show pharmacological activity, and there was down-regulation of either ICAM-1 message or protein in involved tissue in the endotoxin pneumonitis and the dextran-induced colitis models. In primates, phosphorothioate oligodeoxynucleotides as a chemical class have shown a similar, sequence-independent pharmacokinetic behavior, with dose-dependent plasma clearance half-lives of 30–80 minutes and metabolic “half-lives” ranging from 1 to 5 days in different tissues. Clearance from plasma is primarily through distribution to the extravascular space, with subsequent uptake of oligonucleotide by tissues and then slow metabolism via nucleases and normal nucleotide degradation pathways.

X 33 Geary, RS, Leeds, JM, Henry, SP, Monteith, DK, and Levin, AA. Antisense oligonucleotide inhibitors for the treatment of cancer: 11. Pharmacokinetic properties of phosphorothioate oligodeoxynucleotides.

Anticancer Drug Des. 1997; 12: 383–393 , x 34 Geary, RS, Leeds, JM, Fitchett, J, Burckin, T, Truong, L, Spainhour, C, Creek, M, and Levin, AA. Pharmacokinetics and metabolism of a phosphorothioate oligonucletide antisense inhibitor of c-RAF-1 kinase expression. Drug Metab Dispos. 1997; 25: 1272–1281 In a phase 1 study, a single intravenous dose of ISIS 2302 from 0.06 to 2 mg/kg and four doses every other day ranging from 0.5 to 2 mg/kg was administered over 2 hours, were well tolerated in normal volunteers, and had a pharmokinetic behavior in plasma similar to that in primates. X 35 Glover, JM, Leeds, JM, Mant, TGK, Amin, D, Kisner, DL, Zuckerman, JE, Geary, RS, Levin, AA, and Shanahan, WR.

Phase 1 safety and pharmacokinetic profile of an intercellular adhesion molecule–1 antisense oligonucleotide (ISIS 2302). J Pharmacol Exp Ther. 1997; 282: 1173–1180 The only drug-related adverse events observed were dose related, modest 1.5-fold at the 2-mg/kg dose, and transient (2–4 hours after dosing) increases in the activated partial thromboplastin time (aPTT) and threshold effects on complement C3 but not C5 conversion at the highest doses.

Both of these dose-related effects (prolongation of the intrinsic coagulation pathway and alternative pathway complement activation) have been observed in monkeys given ISIS 2302 and a spectrum of other phosphorothioate oligodeoxynucleotides. X 36 Henry, SP, Monteith, D, and Levin, AA. Antisense oligonucleotide inhibitors for the treatment of cancer: 2. Toxicological properties of phosphorothioate oligodeoxynucleotides. Anticancer Drug Des.

1997; 12: 395–408 , x 37 Henry, SP, Bolte, H, Auletta, C, and Kornbrust, DJ. Evaluation of the toxicity of ISIS 2302, a phosphorothioate oligonucleotide, in a 4-week study in cynomolgus monkeys. 1997; 120: 145–155 Alternative pathway complement activation seems to be mediated by an interaction with the inhibitory protein factor H. X 38 Henry, SP, Giclas, PC, Leeds, J, Pangburn, M, Auletta, C, Levin, AA, and Kornbrust, DJ. Activation of the alternative pathway of complement by a phosphorothioate oligonucleotide: potential mechanism of action.

J Pharmacol Exp Ther. 1997; 281: 810–816 The prolongation of aPTT appears to be effected through inhibition of intrinsic tenase complex (factors IXa and VIIIa, phospholipid, and calcium) activity. X 39 Sheehan JP, Lan HC. Phosphorothioate oligonucleotides inhibit the intrinsic tenase complex. Blood (in press).

In aggregate, tissue culture, animal, and phase 1 studies support the safety and efficacy of anti–ICAM-1 antisense phosphorothioate oligodeoxynucleotide therapy. X 22 Bennett, CF, Condon, T, Grimm, S, Chan, H, and Chiang, MY. Inhibition of endothelial cell–leukocyte adhesion molecule expression with antisense oligonucleotides. 1994; 152: 3530–3540 , x 23 Nestle, F, Mitra, RS, Bennett, CF, and Nickoloff, BJ. Cationic lipid is not required for uptake and inhibitory activity of ICAM-1 phosphorothioate antisense oligonucleotide in keratinocytes. J Invest Dermatol.

1994; 103: 569–575 , x 29 Christofidou-Solomidou, M, Albelda, SM, Bennett, CF, and Murphy, GF. Experimental production and modulation of human cytotoxic dermatitis in human-murine chimeras. 1997; 150: 631–639 , x 30 Stepkowski, SM, Tu, Y, Condon, TP, and Bennett, CF. Blocking of heart allograft rejection by ICAM-1 antisense oligonucleotides alone or in combination with other immunosuppresive modalities.

1994; 153: 5336–5346 , x 31 Bennett, CF, Kornbrust, D, Henry, S, Stecker, K, Howard, R, Cooper, S, Dutson, S, Hall, W, and Jacoby, HI. An ICAM-1 antisense oligonucleotide prevents and reverses dextran sulfate sodium–induced colitis in mice. J Pharmacol Exp Ther. 1997; 280: 988–1000 , x 35 Glover, JM, Leeds, JM, Mant, TGK, Amin, D, Kisner, DL, Zuckerman, JE, Geary, RS, Levin, AA, and Shanahan, WR. Phase 1 safety and pharmacokinetic profile of an intercellular adhesion molecule–1 antisense oligonucleotide (ISIS 2302). J Pharmacol Exp Ther. 1997; 282: 1173–1180 , x 37 Henry, SP, Bolte, H, Auletta, C, and Kornbrust, DJ.

Evaluation of the toxicity of ISIS 2302, a phosphorothioate oligonucleotide, in a 4-week study in cynomolgus monkeys. 1997; 120: 145–155 , x 38 Henry, SP, Giclas, PC, Leeds, J, Pangburn, M, Auletta, C, Levin, AA, and Kornbrust, DJ. Activation of the alternative pathway of complement by a phosphorothioate oligonucleotide: potential mechanism of action. J Pharmacol Exp Ther. 1997; 281: 810–816 , x 40 Henry, SP, Larkin, R, Novotny, WF, and Kornbrust, DJ. Effects of ISIS 2302, a phosphorothioate oligonucleotide, on in vitro and in vivo coagulation parameters.

1994; 11: S-353, x 41 Henry, SP, Taylor, J, Midgley, L, Levin, AA, and Kornbrust, DJ. Evaluation of the toxicity of ISIS 2302, a phosphorothioate oligonucleotide, in a 4-week study in CD-1 mice. Antisense Nucleic Acid Drug Dev. 1997; 7: 473–481 The present study was undertaken to obtain preliminary evidence of the safety, efficacy, and pharmacology of ISIS 2302 in patients with steroid-dependent, active Crohn's disease. In addition to clinical measures, serum soluble ICAM-1 (sICAM-1), peripheral blood lymphocyte expression of α 1-α 6 (CD49a-CD49f), α d, and β 7 and intestinal mucosal expression of ICAM-1 (CD54) were measured serially, and the pharmacokinetic behavior of ISIS 2302 was assessed. The study was a single-center, double-blinded, placebo-controlled, randomized (3:1; ISIS 2302 to placebo), fixed-dose within patient, and dose-escalation trial, approved by the Ethics Committee of the University of Alberta. We enrolled 20 patients between the ages of 18 and 80 who had moderately active Crohn's disease (Crohn's Disease Activity Index CDAI, 200–350) despite stable background therapy of moderate-dose corticosteroids (prednisone or equivalent, ≤40 mg/day) and mesalamine drugs for at least 1 month before enrollment.

Patients were treated with ISIS 2302 or placebo for 26 days and then followed up for an additional 5 months. Patients received either ISIS 2302 or saline placebo in 13 doses over 26 days by 2-hour intravenous infusions.

Four patients were assigned to the 0.5- and 1-mg/kg dose groups and the remaining 12 patients to the 2-mg/kg dose group. Introduction of new anti-inflammatory or immunosuppressive therapy was not permitted at any time during the study. Corticosteroid doses were kept stable during the treatment period but were adjusted thereafter by the investigator according to blinded clinical judgment. Because this was the first study of ISIS 2302 in Crohn's disease and it was not known whether evidence of efficacy would be found, a mandated schedule of corticosteroid dosage tapering was not considered to be appropriate in this study. All but 4 patients (all ISIS 2302–treated) took aminosalicylates (15 mesalamine, 1 sulfasalazine) throughout the study. Dosages of aminosalicylates (4 g/day except in 1 patient taking 3 g/day) remained unchanged throughout the 6-month study in all but 4 patients, 3 of whom were in the ISIS 2302 group. Three of these patients transiently (1–2 months) decreased dosage on their own after the treatment period (to 3 g/day in 1 patient, to 0 mg/day in 1, and to suppositories and then back to tablets in 1), and 1 patient increased dosage from 3 to 4 g/day during the third month.

ISIS 2302 was manufactured by Isis Pharmaceuticals, Inc. (Carlsbad, CA) and supplied as a sterile 10-mg/mL saline solution in 2- and 5-mL vials. The appropriate amount of ISIS 2302 or saline placebo (matching vials) was diluted in 100 mL of normal saline for infusion by a university hospital pharmacist; the investigator was not involved in ISIS 2302 preparation or administration.

The investigator remained blinded as to treatment until the study was completed and all patient data were collected. In addition, the investigator was shielded from results of aPTT testing because knowledge of these results could have compromised the blinding. Outcome was assessed by changes from baseline in the CDAI, x 42 Best, WR, Bectel, JM, Singleton, JW, and Kern, F. Development of a Crohn's Disease Activity Index: National Cooperative Crohn's Disease Study. 1976; 70: 439–444 , x 43 Best, WR, Bectel, JM, and Singleton, JW.

Rederived values of the eight coefficients of the Crohn's Disease Activity Index. 1979; 77: 483–486 corticosteroid usage, the Endoscopic Index of Severity (EIS), x 44 Ma, X, Lefer, DJ, Lefer, AM, and Rothlein, R.

Coronary endothelial and cardiac protective effects of a monoclonal antibody to intercellular adhesion molecule–1 in myocardial ischemia and reperfusion. 1992; 86: 937–946 , x 45 Mary, JY and Modigliani, R.

Development and validation of a Crohn's disease endoscopic index: a prospective multicentric study. 1989; 30: 983–989 and the Inflammatory Bowel Disease Questionnaire (IBDQ). X 46 Guyatt, G, Mitchell, A, Irvine, EJ, Singer, J, Williams, N, Goodacre, R, and Tompkins, C.

A new measure of health status for clinical trials in inflammatory bowel disease. 1989; 96: 804–810 , x 47 Irvine, EJ, Feagan, B, Rochon, J, Archambault, A, Fedorak, RN, Groll, A, Kinnear, D, Siabil, F, and McDonald, JW. Quality of life: a valid and reliable measure of therapeutic efficacy in the treatment of inflammatory bowel disease.

1994; 106: 287–296 A CDAI of. Peripheral blood samples (15 mL) for determination of cell adhesion molecule expression were obtained from all patients enrolled before treatment with ISIS 2302 (day 1) and again at days 13, 26, 56, and 116 at approximately the same time each morning; sampling on days 13 and 26 was done before ISIS 2302 infusion. Peripheral blood mononuclear cells (PBMCs) were immediately purified by density centrifugation over Ficoll-Hypaque (Pharmacia, Dorval, Quebec) followed by two washes. Serum for sICAM-1 expression was obtained on days 1, 3, 7, 13, 19, 26, 33, 40, 60 and monthly thereafter.

Pinch mucosal biopsy specimens were obtained from the same area of the intestine (distance from anal verge) at baseline and day 26. Ten representative specimens were each taken from diseased and nondiseased colon and/or small bowel at the index region. Specimens were kept on ice and then immediately snap-frozen in OCT compound for storage at −80°C until use. CD18, CD49c, CD3PE, and Leukogate were purchased from Becton Dickinson (Mountain View, CA); CD49a, CD49f, and CD20 fluorescein isothiocyanate from Immunotech/Coulter (Westbrook, ME/Miami, FL); and CD54, CD49b, CD49d, and CD49e from Pharmingen (San Diego, CA).

Β 7 was a gift from Dr. Lazarovits (University of Western Ontario). 169a (α d) was a gift from Dr. Gallatin (ICOS Corp., Bothell, WA). X 19 Bernstein, CN, Sargent, M, Gallatin, WM, and Wilkins, J. Β2 integrin/intercellular adhesion molecule (ICAM) expression in the normal intestine.

Clin Exp Immunol. 1996; 106: 160–169 The following antibodies were purchased from Southern Biotech (Birmingham, AL): mouse immunoglobulin (Ig) G2a fluorescein isothiocyanate, mouse IgG1 phycoerythrin, mouse IgG1 unlabeled, mouse IgG2 unlabeled, goat anti-mouse IgG1 biotin labeled, goat anti-mouse IgG2a and goat anti-mouse IgG2b biotin labeled, rat IgG, goat anti-rat IgG biotin labeled, and the third color reagent, streptavidin spectral red. Cell surface antigens present on the isolated mononuclear cells were evaluated by three-color immunofluorescence using a four-stage combined direct and indirect staining procedure.

X 18 Yacyshyn, BR, Lazarovits, A, Tsai, V, and Matejko, K. Crohn's disease, ulcerative colitis, and normal intestinal lymphocytes express integrins in dissimilar patterns. 1994; 107: 1364–1371 The cells were centrifuged and analyzed by using a fluorescence-activated cell sorter (FACScan; Becton Dickinson). Dead cells were excluded by gating on forward and side light scatter. All samples included staining with isotype-matched control antibodies.

Files of 20,000 cells were collected for each sample, and measurements of all three fluorochromes as well as forward and side scatter were recorded. The mucosal biopsy specimens (1 × 1 mm) were placed in OCT and snap-frozen. Representative frozen sections (4–6 μm thick) were air-dried, fixed in acetone at 4°C for 10 minutes, and immunohistochemically stained for CD54 using the 3,3'-diaminobenzidine tetrahydrochloride procedure.

Slides were interpreted in blinded fashion by a gastrointestinal pathologist (L.J.). Day 26 slides were compared with day 0 slides and scored as improved (1+), without change (0), or worse (−1) for anti-CD54 staining. Noninflamed tissue was compared with noninflamed tissue and inflamed with inflamed tissue where possible. Twenty patients were enrolled in this study: 15 patients were randomized to treatment with one of three doses of ISIS 2302 and 5 patients to treatment with placebo. Analysis was by intention-to-treat, and efficacy variables were censored at the time of study discontinuation.

All patients were categorized at each time point at which CDAI data were collected as having met remission criteria (CDAI. Mean values, with the range for each group in parentheses.Patients had a spectrum of disease locations: ileitis only, colitis only, and ileocolitis. Two patients had gastroduodenal as well as intestinal involvement, and 2 patients had a single, open enterocutaneous fistula at baseline. All but 3 patients completed the full 6 months of the study. One placebo-treated patient, although having active disease at screening, was in remission at baseline (CDAI, 129) and remained in remission with very low CDAI scores (4–53) until he was discontinued from the study before month 4 visit because of a mechanical bowel obstruction (stricture plus dietary indiscretion) that resulted in a partial bowel resection. In addition to this patient, 2 ISIS 2302–treated patients (dose for both, 2 mg/kg) were prematurely discontinued from the study, after visits at month 2 (patient moved) and 4 (disease progression), respectively.

At the end of the treatment period (day 33), 7 of 15 ISIS 2302–treated patients (2 of 3 receiving 0.5 mg/kg, 1 of 3 receiving 1 mg/kg, and 4 of 9 receiving 2 mg/kg) and 1 placebo-treated patient (the patient in remission at baseline) were in remission by CDAI criteria (47% vs. At the end of month 6, 5 of these ISIS 2302–treated remitters were still in remission, and a 6th patient had a CDAI score of 156.

Remission in the 7th patient lasted 41 days. There was no apparent dose response in either time to response or duration of response (data not shown). After the treatment period, in the 6 ISIS 2302–treated patients with prolonged remissions, steroids were slowly tapered in 5 patients (to 0 mg/day in 3 patients) and maintained (at 10 mg/day) in 1 patient. Two additional ISIS 2302–treated patients and 3 additional placebo-treated patients met remission criteria by CDAI at some later time during the trial, but these late remissions were short lived. Only one of these five late remissions, in a placebo patient, was associated with an increase in corticosteroid dosage. The 8th ISIS 2302–treated remitter went into remission on day 60 and the 9th at day 150, and the durations were both of 30 days. For the 3 additional placebo-treated patients experiencing remission, remissions occurred on days 60, 60, and 150 and were of relatively short duration (30, 60, and 30 days, respectively) compared with most ISIS 2302–associated remissions.

Mean CDAIs over time for the ISIS 2302 and placebo groups are shown in Figure 1. After the treatment period, mean corticosteroid dose steadily decreased from baseline (15.5 mg/day) in the ISIS 2302 group to a nadir of 8.75 mg/day on day 90, and then slowly increased to a level of 13.35 mg/day at day 180. In the placebo group (baseline, 13.6 mg/day), mean corticosteroid dose increased after the treatment period to a peak level of 18.43 mg/day on day 40, then rapidly decreased to 11.80 mg/day on day 60 and a nadir of 11.43 mg/day on day 90, and gradually climbed thereafter to a peak level of 18.47 mg/day at day 180. At the end of the trial (month 6), 33% (5 of 15) ISIS 2302 patients and no placebo patients were completely tapered off of all corticosteroids. However, prednisone was successfully discontinued in the the placebo-treated patient in remission at baseline on day 41, and the patient remained off corticosteroids through his discontinuation from the study after month 3 visit.

Maximal concentration of drug in plasma (C max) occurred at or near the end of infusion. Mean C max on different days ranged from 1.43 to 2.07 μg/mL for the 0.5-mg/kg group, from 3.13 to 5.90 μg/mL for the 1-mg/kg group, and from 9.28 to 18.08 μg/mL for the 2-mg/kg group, without evidence of drug accumulation.

Figure 7 From Icam-1 (cd54) A Counter-receptor For Mac Os X

Plasma half-life (0.56–0.94 hours), area under the curve, volume of distribution, and plasma clearance were dose dependent, suggesting a saturable component to distribution. The full-length parent compound (ISIS 2302, 20-mer) was the predominant oligonucleotide present at all time points. Percent change in absolute lymphocyte counts did not differ significantly between the ISIS 2302 and placebo treatment groups. From baselines of 1.51 ± 0.25 (SD) and 1.85 ± 0.51 × 10 3/μL, the changes were +41.8% and +31.0% on day 26, +19.6% and +0.2% on day 56, and −12.9% and +27.5% on day 112. The proportion of PBMCs that were CD3 + at baseline were 76.0% ± 2.0% (SD) and 84.2% ± 3.4% for the ISIS 2302 and placebo groups, respectively; relative percent changes in CD3 + lymphocytes were significant on day 56 (+2.0% and −9.4%; P = 0.034) but not on days 13 (+6.9% and −7.9%; P = 0.150), 26 (+7.0% and −2.9%; P = 0.138), and 112 (+1.8% and −3.0%). The percentage of lymphocytes expressing CD20 (baseline, 10.4% ± 1.6% SD and 5.6% ± 1.0% for ISIS 2302 and placebo, respectively) fluctuated similarly in each treatment group.

From baseline (71.6% ± 2.8% SD for ISIS 2302; 80.5% ± 4.1% for placebo) to day 26, the percentage of CD3 + lymphocytes expressing β 7 increased by 13.9% ± 5.3% (SD) and decreased by 5.5% ± 4.6% in the ISIS 2302–treated and placebo-treated patients, respectively ( P = 0.007). On day 56, differences in the relative changes from baseline approached significance ( P = 0.053; Figure 5). P = 0.033.Reductions in CD54 expression on day 26 compared with baseline were observed more frequently in intestinal frozen sections of ISIS 2302–treated compared with placebo-treated patients ( P = 0.033). In this variable there was evidence of a dose response: ICAM-1 expression from baseline was reduced in only 1 of 6 patients treated with 0.5 or 1 mg/kg of ISIS 2302, but in 7 of 9 patients treated with 2 mg/kg. Interestingly, increased ICAM-1 expression was only observed in placebo-treated patients (2 of 5 patients) and reduction was only noted in 1 placebo patient. Representative day 1 and day 26 mucosal biopsy specimens stained immunohistochemically for CD54 are shown in Figure 7.

ISIS 2302 was well tolerated. There was no evidence of a drug effect on routine safety laboratory (complete blood count, sequential multiple analyzer, urinalysis), electrocardiography, or vital signs, and no increases in complement C3a or C5a were seen. The only apparent drug-related adverse events were transient, dose-related elevations in aPTT and transient facial flushing at the end of several infusions of ISIS 2302 in a single patient.

As predicted from animal and normal volunteer data, mean increases in aPTT of approximately 3, 5, and 10 seconds were produced by 2-hour infusions of 0.5, 1, and 2 mg/kg. Episodes of nausea and vomiting occurring in 5 ISIS 2302–treated patients were mild, single events occurring twice in 1 patient and once in the others (during treatment).

Because these events were not reported in any placebo patient, a relationship to ISIS 2302 cannot be ruled out, but nausea and vomiting did not occur in the normal volunteer study. X 35 Glover, JM, Leeds, JM, Mant, TGK, Amin, D, Kisner, DL, Zuckerman, JE, Geary, RS, Levin, AA, and Shanahan, WR. Phase 1 safety and pharmacokinetic profile of an intercellular adhesion molecule–1 antisense oligonucleotide (ISIS 2302). J Pharmacol Exp Ther. 1997; 282: 1173–1180. This study provided a novel opportunity to use systemically administered antisense in a human disease process, offering us a means to study a very specific manipulation in the treatment of Crohn's disease: the inhibition of ICAM-1 through an antisense mechanism. In addition to following clinical measures, the study was designed to allow an examination of the effects of ISIS 2302 on peripheral blood lymphocyte and intestinal mucosal ICAM-1 expression and the effects of manipulating ICAM-1 on additional variables, specifically the expression of other adhesion molecules in peripheral blood.

In this pilot study, we report on the clinical effects in 15 ISIS 2302–treated patients. In addition to evidence of clinical efficacy that has allowed us to expand to an ongoing larger study of 300 patients, this study has also provided us with data pertaining to safety and immune mechanisms. ISIS 2302 appears to be a very safe modality; in treating 15 patients, there was no evidence of a drug effect on routine safety laboratory indices.

As expected, because of a class effect of systemically administered phosphorothioate oligodeoxynucleotide, transient increases in aPTT of approximately 3, 5, and 10 seconds were observed at doses of 0.5, 1, and 2 mg/kg, respectively. Mild, single episodes of nausea and vomiting were observed in 5 ISIS 2302–treated patients, occurring twice in 1 patient and once in the others during treatment. Such episodes did not occur in the normal volunteer phase 1 study.

The plasma pharmacokinetics of this compound in patients with Crohn's disease are very similar to those observed in normal volunteers, with a C max occurring at the end of infusion and ranging from 9.3 to 18.1 μg/mL at the 2-mg/kg dose, a plasma elimination half-life of 1 hour, and evidence of a saturable component to disposition. Changes in clinical measures were observed with ISIS 2302 treatment, including statistically significant reductions in steroid usage. Change in steroid dose was at the discretion of a blinded investigator (B.R.Y.), who adjusted the dosage according to clinical activity after the drug infusion period. Improvements in the CDAI, EIS, and the quality of life assessments (IBDQ) were also noted, with a significantly greater improvement from baseline in IBDQ for ISIS 2302–treated patients on day 40. Seven of 15 ISIS 2302–treated patients were in remission at the end of the 1-month drug infusion period; surprisingly, 5 of these 7 remitters were still in remission at the end of the study (end of month 6), and a 6th patient had a CDAI score of 156. Mean CDAI scores only showed transient and nonsignificant differences between the treatment groups, and the greatest difference, on day 60, actually favored the placebo group.

There are at least two explanations for this: (1) that ISIS 2302 is no more effective than placebo in reducing the CDAI; (2) that ISIS 2302 is effective, but differences in treatment were masked by the small size of the placebo group and/or the increase in corticosteroid dosage after the treatment period in this group (mean increase from baseline of almost 5 mg/day 37% on day 40 coupled with a reduction of almost 7 mg/day from baseline 44% by day 90 in the ISIS 2302–treated group). A small placebo group was included in this study, not so much to provide an adequate control group but to provide blinded assessment of responses to therapy. Mean CDAI score in the placebo group was substantially influenced by the patient in remission at baseline, and the behavior of the placebo group in this study may not be reflective of how a larger placebo-treated group might respond. Nevertheless, the durability of remissions, significant differences in steroid doses, trends in EIS and IBDQ (significant on day 40), and significant differences in the expression of mucosal ICAM-1 and two lymphocyte cell adhesion molecules all suggest a different response in the ISIS 2302–treated patients; an answer to the question of whether ISIS 2302 is effective in Crohn's disease will require a much larger study. Changes during the drug treatment period in peripheral blood lymphocyte and intestinal mucosal expression of adhesion molecules were significantly different between drug- and placebo-treated patients with Crohn's disease. During this period, the percentage of CD3 + peripheral blood lymphocytes expressing β 7 and the proportion expressing α d increased in the ISIS 2302–treated patients and decreased in the placebo-treated patients. In addition, the absolute lymphocyte count and the proportion of CD3-bearing lymphocytes tended to increase in the ISIS 2302–treated patients, but the changes relative to the placebo group were not generally significant in this small study.

Decreases in qualitative ICAM-1 expression in intestinal mucosa, judged by a blinded pathologist (L.J.), were also seen in significantly more ISIS 2302 patients than in placebo patients during drug treatment; in this measure, there was evidence of a dose response. The mechanism(s) behind these changes may be multifactorial, including suppressed inflammation due to loss of inflammatory homing sites in the gut with intestinal release of β 7 and α d bearing lymphocytes, or a direct pharmacological effect in the case of CD54 expression in the intestine. The lack of an effect on CD54 expression in peripheral blood lymphocytes may represent relatively poor uptake of drug by this cell population or a balance between direct pharmacological effect and intestinal release. These findings are consistent with the recent findings of Hokari et al., x 20 Hokari, R, Miura, S, Fujimoro, H, Nagata, H, Sekizuka, E, Tsuzuki, Y, Kurose, I, Serizawa, H, Suematsu, M, Yagita, H, Granger, DN, and Ishii, H. Lymphocyte outflux to intestinal microlymphatics is an intercellular adhesion molecule–1 dependent process in Peyer's patches (abstr). 1997; 112: A369 who speculated that ICAM-1 is needed for retention of gut lymphocytes in the intestinal compartment. They found that in the rat, antibody to ICAM-1, but not to α 4, CD18, or L-selectin, led to release of lymphocytes from Peyer's patches.

In the present study, observed differences in lymphocyte adhesion molecule expression support the suggestion of a therapeutic effect with ISIS 2302, and our findings provide the first evidence of an interplay between β 7, α d, and CD54 in human therapeutics. Across a spectrum of clinical and pharmacological measures, evidence of a dose response was found only for inhibition of intestinal mucosal ICAM-1 expression, and this evidence was qualitative only. Potential reasons for the general lack of a dose response include the possibility that all doses in this study exceed the plateau threshold on the dose-response curve, the small size of the lower dose cohorts, and the fact that only a fourfold range in dose was explored in this study. It is also not uncommon for even larger studies of biological agents to fail to show a dose response. The durability of remission associated with ISIS 2302 treatment was unexpected, and the reasons for this durability can only be speculated on presently.

Persistence of drug is an unlikely explanation: tissue half-lives of ISIS 2302 and other phosphorothioate oligodeoxynucleotides range from 1 to 5 days, so that within 15–30 days after end of therapy, ISIS 2302 should be essentially cleared from all tissues. The observed changes in circulating T cell, α d, and β 7 expressing populations, and mucosal ICAM-1 expression, are supportive of a hypothesis that, by suppression of mucosal ICAM-1 on endothelium and perhaps other cells, the pattern of T-cell trafficking and retention critical to perpetuation of the disease process is interrupted, allowing restoration of normal mucosal integrity and immune function. It is conceivable that this restoration leads to relatively prolonged suppression of disease. In addition to its role in leukocyte trafficking and retention, ICAM-1 appears to provide an important secondary signal during antigen presentation x 8 Altmann, DM, Hogg, N, Trowsdale, J, and Wilkinson, D. Cotransfection of ICAM-1 and HLA-DR reconstitutes human antigen-presenting cell function in mouse L cells. 1989; 338: 512–514 , x 9 Van Seventer, GA, Shimizu, Y, Horgan, KJ, and Shaw, S. The LFA-1 ligand ICAM-1 provides an important costimulatory signal for T cell receptor–mediated activation of resting T cells.

1990; 144: 4579–14458 , x 49 Kuhlman, P, Moy, VT, Lollo, BA, and Brian, AA. The accessory function of murine intercellular adhesion molecule–1 in T lymphocyte activation. 1991; 146: 1773–1782 and to play an important facilitatory role in cytoxic T cell–, x 50 Makgoba, MW, Sanders, ME, Luce, GEG, Gugel, EA, Dustin, ML, Springer, TA, and Shaw, S. Functional evidence that intercellular adhesion molecule–1 (ICAM-1) is a ligand for LFA-1–dependent adhesion in T cell–mediated cytotoxicity. Eur J Immunol.

1988; 18: 637–640 NK cell–, x 51 Allavena, P, Paganin, C, Martin-Padura, I, Peri, G, Gaboli, M, Dejana, E, Marchisio, PC, and Mantovani, A. Molecules and structures involved in the adhesion of natural killer cells to vascular endothelium. 1991; 173: 439–448 and neutrophil-mediated x 10 Entman, ML, Youker, K, Shoji, T, Kukielka, G, Shappell, SB, Taylor, AA, and Smith, CW. Neutrophil induced oxidative injury of cardiac myocytes.

J Clin Invest. 1992; 90: 1335–1345 damage to target cells. Inhibition of ICAM-1 function during antigen presentation results in impaired T-cell responses, and inhibition of both ICAM-1 and LFA-1 function by antibodies or a combination of antibody and antisense produces apparent specific tolerance in murine cardiac allograft models. X 30 Stepkowski, SM, Tu, Y, Condon, TP, and Bennett, CF.

Blocking of heart allograft rejection by ICAM-1 antisense oligonucleotides alone or in combination with other immunosuppresive modalities. 1994; 153: 5336–5346 , x 52 Isobe, M, Yaghita, H, Okumura, K, and Ihara, A.

Specific acceptance of cardiac allograft after treatment with antibodies to ICAM-1 and LFA-1. 1992; 255: 1125–1127 It is therefore possible that treatment with ISIS 2302 produced a partial and temporary tolerance to antigens involved in the disease process. Interestingly, in an open-label trial of a murine anti–ICAM-1 antibody in patients with rheumatoid arthritis, prolonged clinical responses seemed to correlate with prolonged T-cell hyporesponsiveness. X 53 Davis, LS, Kavanaugh, AF, Nichols, LA, and Lipsky, PE. Induction of persistent T cell hyporesponsiveness in vivo by monoclonal antibody to ICAM-1 in patients with rheumatoid arthritis. 1995; 154: 3525–3537 This study represents the first report of clinical, peripheral blood, and intestinal immune activity for a systemically administered antisense compound.

ISIS 2302 appears to be a promising and well-tolerated new therapeutic approach to Crohn's disease, producing a significant steroid-sparing effect while inducing highly durable remissions in 6 of 15 treated patients in this small study. A large, controlled trial of patients with Crohn's disease is underway to definitively assess the efficacy and tolerability of this novel drug. References.

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