Double blind, placebo controlled trial of the remission inducing and steroid sparing properties of an ICAM-1 antisense oligodeoxynucleotide, alicaforsen (ISIS 2302), in active steroid dependent Crohn's disease.

BACKGROUND AND AIMS: To evaluate the safety and efficacy of the intercellular adhesion molecule 1 (ICAM-1) antisense phosphorothioate oligonucleotide alicaforsen (ISIS 2302) in Crohn's disease. METHODS: Active (Crohn's disease activity index (CDAI) 200-350), steroid dependent (prednisone 10-40 mg) Crohn's patients were randomised into three treatment groups: placebo versus ISIS 2302 (2 mg/kg intravenously three times a week) for two or four weeks. Patients were treated in months 1 and 3, with steroid withdrawal attempted by week 10. The primary end point (steroid free remission) was a CDAI <150 off steroids at the end of week 14. RESULTS: A total of 299 patients were enrolled, with a mean baseline CDAI of 276 and steroid dose of 23 mg/day. Rates of steroid free remission were equivalent for the two and four week ISIS 2302 groups (20.2% and 21.2%) and the placebo group (18.8%). At week 14, steroid withdrawal was successful in more ISIS 2302 patients compared with placebo treated patients (78% v 64%; p=0.032). Steroid free remission was highly correlated with exposure (p=0.0064). Other clinical responses were correlated with exposure, with significant results versus placebo being observed in the highest area under the curve subgroup. CDAI scores decreased by 136 (112) at week 14 versus 52 (107) for placebo (p=0.027) and inflammatory bowel disease score questionnaire improved by 43 (31) versus 15 (36) for placebo (p=0.027). CONCLUSIONS: Although the primary outcomes failed to demonstrate efficacy, pharmacodynamic modelling suggests that alicaforsen (ISIS 2302) may be an effective therapy for steroid dependent Crohn's disease.

Preclinical and clinical pharmacology of antisense oligonucleotides.

Antisense oligonucleotides are short (typically 15-20 bases in length pieces of synthetically manufactured, chemically modified DNA or RNA. They are designed to interact by Watson-Crick base pairing with mRNA transcripts encoding proteins of interest, and by virtue of this interaction inhibit the expression of the protein encoded in the mRNA. Since their first proposed use in 1978, antisense oligonucleotides have become come widely used as tools to modulate gene expression in tissue culture. The great specificity that these compounds exhibited in vitro has also led them to be viewed as potentially therapeutically useful. This interest has resulted in the progression of (to date) a dozen compounds into human clinical trials for a variety of indications ranging from cancer to inflammatory diseases. Here, we will review some of the progress that has been made with antisense pharmacology, both in vitro and in vivo, as well as describe the current status of this class of compound in clinical trials.

Making sense of antisense.

Since the identification of the DNA double-stranded helix, the gene as a target of therapy and, moreover, the use of DNA as a drug have been possibilities. 'Antisense' is used by some living organisms, specifically viruses, to control gene replication. Only recently, the use of antisense DNA as a mechanism to control human gene translation has been appreciated. A recent report on the use of systemically administered oligonucleotides in human Crohn's disease is reviewed. DNA antisense oligonucleotides offer a technology capable of unique use at the laboratory bench as well as for highly specific therapeutic drugs. The conceptualization and possible future directions of these exciting compounds are reviewed.

ISIS 2302, an antisense inhibitor of intercellular adhesion molecule 1.

ISIS 2302 is a 20 base phosphorothioate oligodeoxynucleotide (ODN) that inhibits intercellular adhesion molecule 1 (ICAM-1) expression through an antisense mechanism. Murine and rat analogues have been effective at doses of 0.06 - 10 mg/kg in a spectrum of models of human inflammatory diseases and allograft transplantation, and ISIS 2302 inhibits the upregulation of ICAM-1 expression in a variety of human cells in vitro. In animals, including primates, plasma distribution half-life ranges from 30 - 60 min, but tissue elimination half-lives range from 1 - 5 days, and the compound is metabolised as other nucleic acids. In a Phase I iv. study, the pharmacokinetic behaviour of ISIS 2302 was similar to that in other primates, and single and multiple every other day doses from 0.06 - 2 mg/kg infused over 2 h were well-tolerated. Phase IIa studies have been completed in Crohn's disease, rheumatoid arthritis, and psoriasis, and a combined Phase I/II renal allograft acute rejection prophylaxis study has just completed enrolment. In these studies, ISIS 2302, 0.5 - 2 mg/kg, or placebo was administered iv. every 2 - 3 days over 14 - 26 days (7 - 13 infusions) to 17 - 52 patients, with follow up for 6 months. In the Crohn's study, evidence of highly durable (5+ month) remission-inducing and steroid-sparing properties were demonstrated, without clinically important adverse events. A 300-patient, pivotal quality trial investigating the steroid-sparing and remission-inducing qualities of ISIS 2302 in patients with steroid-dependent Crohn's disease is completely enrolled, with results expected in the first half of 2000. Modest efficacy and excellent tolerability were demonstrated in the psoriasis and rheumatoid arthritis trials. A Phase IIa trial of a topical formulation in patients with psoriasis is expected to commence in late 1999, as is a trial of an enema formulation in distal ulcerative colitis. Administration by nebulisation for asthma is undergoing preclinical evaluation. Execution of future plans in organ transplantation will await the results of the ongoing Phase I/II trial.

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 beta7 and alpha 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.

Phase I safety and pharmacokinetic profile of an intercellular adhesion molecule-1 antisense oligodeoxynucleotide (ISIS 2302).

Healthy male volunteers received single or multiple intravenous infusions of an intercellular adhesion molecule-1 antisense phosphorothioate oligodeoxynucleotide, ISIS 2302, in a rising-dose (0.06-2.00 mg/kg infused over 2 hr), double-blind, placebo-controlled trial. Brief, dose-related increases in activated partial thromboplastin time were seen at the time of peak plasma concentration (C(max)). Clinically insignificant increases in C3a were seen after higher, repeated doses, but C5a, blood pressure and pulse were unaffected. No adverse events or other laboratory abnormalities were related to treatment with the drug. ISIS 2302 C(max) was linearly related to dose and occurred at the end of infusion. Plasma half-life for intact drug (53-54 min) and total oligonucleotide (67-74 min) were similar at the two doses (0.5 and 2.0 mg/kg) at which extensive pharmacokinetic data were collected. Nonlinear changes in area under the plasma concentration/time curve and steady-state volume of distribution with increasing dose suggested a saturable component to disposition. Metabolites co-migrating with n-1, n-2 and n-3 chain-shortened versions of ISIS 2302 appeared very rapidly in plasma, and disposition and metabolism appeared unaltered by repeated dosing. ISIS 2302 was well tolerated and behaved reproducibly with respect to plasma pharmacokinetics and expected side effects.

Reduction of acute-phase proteins with tenidap sodium, a cytokine-modulating anti-rheumatic drug.

Four independent studies have investigated and compared the effects of tenidap sodium, naproxen and placebo on CRP in patients with active RA. One of these studies also investigated the effects of tenidap and naproxen on serum amyloid A (SAA) concentrations and ESR. The duration of the four studies ranged between 2 weeks and 24 weeks, and depending on the study, tenidap sodium was administered orally in doses of 40-120 mg/day and naproxen in doses of 1000 mg/day. In all four studies serum CRP concentrations in tenidap-treated patients had decreased significantly from baseline at the time of final assessment. The decrease in CRP concentration in tenidap-treated patients was observed as early as 1 week after initiation of therapy and was sustained for up to 6 months, the last assessment timepoint. CRP concentrations in naproxen-treated and placebo patients were essentially unchanged. The decreases from baseline observed in tenidap-treated patients were significantly greater than the changes observed in naproxen-treated or placebo patients. After 24 weeks of tenidap treatment the decrease in CRP was paralleled by significant decreases in SAA concentration and ESR. The finding that tenidap sodium rapidly, consistently and significantly lowered CRP serum concentrations differentiates tenidap sodium from the NSAID, naproxen. This could possibly have important therapeutic implications given that other long-term investigations have shown that reducing serum CRP and SAA concentrations correlates with a reduction in radiographically-assessed disease progression.

Expression of IL-1 and tumor necrosis factor by endothelial cells: role in stimulating fibroblast PGE2 synthesis.

We describe the elaboration by endothelial cells of activity that stimulates fibroblast PGE2 production. Culture supernates of human umbilical vein endothelial cells (ECSN) at concentrations of 2.5 to 25% stimulated human foreskin fibroblast PGE2 production 6 to 180-fold. Following molecular sieve chromatography, peak activity eluted with an Mr of 14-18,000. In a standard IL-1 assay, neither ECSN or 14-18,000 Mr fractions possessing PGE2 stimulatory activity were able to stimulate murine thymocyte proliferation in response to PHA. Immunoperoxidase staining of endothelial cells demonstrated intracellular IL-1; after addition of exogenous IL-1 endothelial cells also stained for tumor necrosis factor (TNF). IL-1 and TNF are known to be synergistic in stimulating fibroblast PGE2 synthesis. Thus, elaboration of TNF by endothelial cells may allow detection of IL-1 in fibroblast PGE2 assays when the concentration of IL-1 is inadequate to stimulate thymocyte proliferation. Interactions of cytokines elaborated by cells may play an important role in effects on target cells.

Allogeneic fibroblasts and endothelial cells support mitogen but not antigen responses of tetanus-responsive T-cell lines.

Recent studies suggest a role for endothelial cells (EC) and fibroblasts (FB) in performing certain accessory functions of monocytes in immune responses. We examined the ability of allogeneic adherent cells (AC), umbilical vein EC, and foreskin FB to support antigen and mitogen responses of tetanus toxoid-responsive human T-cell lines (TCTet). Syngeneic AC supported antigen and mitogen (phytohemagglutinin and pokeweed mitogen, PHA and PWM) responses of TCTet. Allogeneic AC, EC, and FB supported mitogen but not antigen responses of TCTet, in a dose-dependent manner. PHA-activated mononuclear cell supernates or EC or FB supernates could not replace accessory cells in mitogen responses. We provide further evidence that EC and FB can function as fully competent accessory cells, a function that may be of significance in in vivo initiation of immune responses.

Endothelial cells enhance the human pokeweed mitogen lymphocyte response.

The effect of endothelial cells (EC) on lymphocyte mitogen responses was examined. Irradiated or mitomycin C treated EC were co-cultured with allogeneic peripheral blood mononuclear cells (PBM), and proliferative responses to pokeweed mitogen (PWM) and phytohemagglutinin (PHA) were assessed by 3H-thymidine incorporation. Compared to lymphocyte responses in the absence of EC, EC co-culture enhanced PWM responses at 72 hours by 55 +/- 28%, 103 +/- 24%, and 96 +/- 9% at EC:PBM ratios of 1:30, 1:10, and 1:3, respectively. The EC co-culture also resulted in significant lymphocyte responses to otherwise submitogenic doses of PWM (10(-4) micrograms/ml) as well as an accelerated kinetics of response. There was no effect of EC on PHA responses. The EC effect appeared not to require cell contact for its expression; however, supernates of EC cultures were not capable of reproducing the effect. On a cell-for-cell basis, EC were more potent in enhancing responses of adherent-cell-depleted lymphocytes than either allogeneic or syngeneic monocytes. Fibroblasts could not substitute for EC in enhancing PWM response, suggesting that the effect was not a nonspecific feeder phenomenon. The EC may play a role in modulating some immune responses in vivo, especially those occurring in areas of inflammation, neovascularization, and endothelial cell proliferation.

Cytotoxic activity of sera from scleroderma and other connective tissue diseases. Lack of cellular and disease specificity.

It has been suggested that serum-mediated endothelial cell injury in scleroderma might contribute to disease pathogenesis. We compared the effect of serum from 28 scleroderma patients on human umbilical cord vein endothelial cell and human foreskin fibroblast proliferation with sera from 28 healthy controls, 13 patients with isolated Raynaud's disease, 22 patients with systemic lupus erythematosus, 19 patients with rheumatoid arthritis, and 15 patients with other connective tissue diseases. Five sera (2 scleroderma, 1 morphea, 12 rheumatoid arthritis, 1 systemic lupus erythematosus) markedly suppressed 3H-thymidine incorporation into both endothelial cells and fibroblasts (to greater than 3 SD below the mean of the control group). These sera were also cytotoxic to endothelial cells and fibroblasts in a 51Cr release assay. Three additional sera (1 Raynaud's, 2 controls) suppressed endothelial cell proliferation moderately (greater than 2 SD but less than 3 SD from control mean) but did not affect fibroblasts. Mean 3H-thymidine incorporation by endothelial cells and fibroblasts in scleroderma serum was comparable to that of the other disease and control groups. In contrast to previous studies, we found serum-mediated endothelial cell cytotoxicity occurred infrequently in scleroderma, occurred also in other connective tissue diseases, and was without target cell specificity. Furthermore, scleroderma serum did not appear to stimulate fibroblast proliferation.

Assessment of longterm benefit of total hip replacement in patients with ankylosing spondylitis.

Clinical and radiographic follow-up examinations were obtained on 12 patients with ankylosing spondylitis (AS) who had undergone total hip replacement (THR). Four of these patients had bilateral THRs performed. The mean follow-up of these 16 THRs was 89 months (range 7-144 months). The THR had achieved excellent pain relief in 15 hips. Postoperative ankylosis did not occur. One arthroplasty has to be removed because of the development of protrusio acetabulum.. Radiographic follow-up was obtained in 14 hips. Six were found to have moderate periarticular ossification which did not significantly affect function. In our experience the risk of postoperative periarticular ossification developing in patients with AS is not a contraindication to hip replacement.