Results of a proof of concept, double-blind, randomized trial of a second generation antisense oligonucleotide targeting high-sensitivity C-reactive protein (hs-CRP) in rheumatoid arthritis

Introduction This randomized, double-blind, phase II study evaluated the pharmacodynamics, safety and tolerability of ISIS 329993 (ISIS-CRPRx), an antisense oligonucleotide, in patients with active rheumatoid arthritis (RA). Methods Patients with active RA of at least six months duration were randomized into three cohorts to receive ISIS-CRPRx (100 mg, 200 mg or 400 mg) or placebo (3 active:1 placebo within each cohort) via subcutaneous (SC) injection on Days 1, 3, 5 and 8 and then once weekly for the next 11 weeks. The effects of study treatment on high-sensitivity C-reactive protein (hs-CRP) level were evaluated. An exploratory analysis on disease activity was assessed via the American College of Rheumatology 20% improvement criteria (ACR20). Safety was evaluated via adverse events and laboratory measures. Results Fifty-one patients received one of the following treatments: ISIS-CRPRx 100 mg, n = 12; 200 mg, n = 13, 400 mg, n = 14; placebo n = 12. In the ISIS-CRPRx treatment groups there were dose-dependent reductions in hs-CRP. At Day 36 the mean percent change from baseline was: placebo: −14.4%; ISIS-CRPRx 100 mg: −19.5%; 200 mg: −56.6% and 400 mg: −76.7%, (P = 0.0015 placebo compared to 400 mg). There were no differences between treatment groups and placebo in the ACR20 at Day 36 or Day 92. There were no serious infections and no elevations in liver function tests, lipids, creatinine or other lab abnormalities related to ISIS-CRPRx. Conclusions In this study, ISIS-CRPRx selectively reduced hs-CRP in a dose-dependent manner, and was well-tolerated in patients with RA. Its utility as a therapy in RA remains unclear. Trial registration Clinicaltrials.gov NCT01414101. Registered 21 July 2011. Electronic supplementary material The online version of this article (doi:10.1186/s13075-015-0578-5) contains supplementary material, which is available to authorized users.


Introduction
Studies of patients with rheumatoid arthritis (RA) document a correlation between C-reactive protein (CRP) blood concentration and worsening of RA symptoms. Inflammation in RA is closely related to the production of CRP and proinflammatory cytokines [1]. Levels of CRP correlate closely with changes in inflammation/disease activity, radiological damage and progression, and functional disability [1]. Although CRP is considered to be a marker of inflammation in RA, it may also function in the promotion of inflammation through complement activation [2]. CRP, and in particular the high-sensitivity CRP (hs-CRP) assay, has been shown to be more closely associated with disease activity variables than erythrocyte sedimentation rate (ESR) [3].
ISIS 329993 (ISIS-CRP Rx ) is an antisense drug targeted to human CRP. ISIS-CRP Rx has been tested in a rodent model of RA (that is, CRP transgenic mice with collageninduced arthritis) and was shown to improve the clinical signs of arthritis [4]. Further, in a previously conducted clinical study in healthy human volunteers (N = 8), whose blood hs-CRP levels ranged from ≥2 to 10 mg/L on two qualifying examinations within a two-week period, treatment with ISIS-CRP Rx achieved significant hs-CRP lowering [4]. The next step in the clinical development process was to determine if ISIS-CRP Rx would be effective in reducing hs-CRP in patients with a chronic inflammatory disease, RA.
The objectives of this clinical study were to evaluate the pharmacodynamics, safety and tolerability of ISIS-CRP Rx in patients with active RA.

Methods
This Phase II, international, multi-center, double-blind, placebo-controlled, parallel group study in patients with RA was conducted at three sites in Canada and ten sites in Russia with enrollment beginning on 3 October 2011 and ending on 18 December 2012. The Institutional Review Board at each site approved the study protocol and the informed consent (see Additional file 1 for details of the ethical bodies). This study was performed in accordance with globally accepted standards of Good Clinical Practice (as defined in the International Conference on Harmonisation E6 Guidelines for Good Clinical Practice, 1 May 1996), and in agreement with the Declaration of Helsinki and in keeping with local regulations. Prior to screening, all subjects provided written informed consent. The trial was registered with clinicaltrials.gov (www.clinicaltrials.gov) [Identification number: NCT01414101].

Patients
Patients (18 to 75 years of age) with active RA, as defined by the American Rheumatism Association 1987 revised criteria [5], for at least six months, and a function Class I-III classified according to the American College of Rheumatology 1991 criteria were enrolled [6]. Patients were required to have hs-CRP of ≥5 mg/L at screening (upper limit of normal >3 mg/L) with at least six swollen joints and at least six tender joints, based on a 28-joint count. All patients had received at least three months of methotrexate therapy at a stable dose of ≥10 mg unless they could not tolerate that dose. Methotrexate, at stable dose, was continued throughout the study. Other concomitant disease-modifying antirheumatic drugs (DMARDs), non-steroidal anti-inflammatory drugs (NSAIDs) and prednisone at 10 mg daily or less were stable prior to and during the study.

Interventions
Eligible patients were randomized in an equal ratio to one of the three dose cohorts (100 mg ISIS-CRP Rx or placebo; 200 mg ISIS-CRP Rx or placebo; or 400 mg ISIS-CRP Rx or placebo). Within each dose cohort, patients were randomized to receive ISIS-CRP Rx or placebo in a 3:1 ratio. Enrolled patients received subcutaneous (SC) injections of ISIS-CRP Rx or placebo on Days 1, 3, 5 and 8 and then once weekly for the next 11 weeks.

Outcomes Pharmacodynamic
The effects of treatment with ISIS-CRP Rx versus placebo on hs-CRP and other markers of inflammation (for example, ESR, serum amyloid A (SAA), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), tumor necrosis factor receptor 1 (TNF-RI), tumor necrosis factor receptor 2 (TNF-RII), fibrinogen and complement) were evaluated throughout the study. During screening, patients had to have at least two hs-CRP measurements taken at least one week apart and no more than one week prior to initiation of dosing and the average of these values was used as the subjects' baseline hs-CRP level. Patient hs-CRP levels and other markers of inflammation were assessed during the treatment and posttreatment evaluation period at regular intervals.

Exploratory activity in RA
The American College of Rheumatology 20% improvement criteria (ACR20) is a widely accepted index of improvement in RA [7] that refers to an improvement of at least 20% in a composite measure of the disease. ACR20 response criteria were assessed at baseline on Day 1 prior to dosing and on Day 36 and Day 92. Disease activity was also assessed using the Disease Activity Score Activity Score Calculator for Rheumatoid Arthritis using the ESR (DAS28-ESR) [8]. DAS28-ESR criteria were assessed at baseline on Day 1 prior to dosing and on Day 36 and Day 92. Patients were considered to be in remission if they had a score of less than 2.6 on the DAS28-ESR.

Safety
Safety was assessed via adverse events (defined as an adverse event that occurred on or after the first dose of treatment) and clinical laboratory parameters throughout the study.

Sample size
It was estimated that the standard deviation of the percent change of hs-CRP was approximately 40% based on the results of a Phase I study [9]. With 12 subjects in each treatment group, the study was designed to have approximately 74% power to detect a 45% difference in hs-CRP between the treated group and the placebo group, assuming the treated group would have a 50% reduction in hs-CRP while the control group would have a 5% reduction. An alpha level of 0.05 was utilized.

Randomization
Eligible subjects were randomized equally to one of three dose cohorts (ratio = 1:1:1). Each dose cohort was to consist of 16 subjects. Within each dose cohort, subjects were randomized to receive ISIS-CRP Rx or placebo in a 3:1 ratio, respectively. Subjects were centrally randomized prior to study drug administration (ISIS-CRP Rx or placebo) using an automated system.

Blinding
The study was blinded with respect to the treatment allocation for subjects, investigators, coordinators and sponsor. The dose cohort was not blinded.

Statistical methods
The safety population consisted of all patients who were randomized and received at least one dose of study drug. The per protocol population consisted of all subjects who were randomized and received at least 60% of doses (≥9 doses), completed the Day 92 or early termination evaluations and did not have any clinically significant protocol deviations.
Demographic and baseline characteristic information was summarized descriptively for each treatment group. Pharmacodynamic endpoints were summarized using the per protocol population. The effects of treatment (ISIS-CRP Rx versus placebo) on hs-CRP and other markers of inflammation were evaluated at multiple time points after dosing. Baseline hs-CRP was determined as the average of all of the values during the screening period, prior to dosing on Day 1. In addition, the effects of treatment (ISIS-CRP Rx versus placebo) were evaluated on the proportion of subjects achieving clinical improvement in RA disease activity at Day 36 and Day 92 compared to baseline using the ACR20 response criteria in the per protocol population.

Medication exposure
The mean number of doses that patients received was similar across treatment groups in the per protocol population (14.

Pharmacodynamics
Robust, durable, selective and dose dependent reductions of hs-CRP were observed by Day 36, with the effects preserved at Day 92 (Table 2 and Additional file 3). There was a large placebo response (45% reduction at Day 92) observed in hs-CRP. There were no meaningful differences or trends between placebo and the ISIS-CRP Rx treatment in the other markers of inflammation (that is, fibrinogen, SAA, TNF-α, cytokine IL-6, TNF-RI and TNF-RII) at either Day 36 or Day 92. Mean baseline, Day 36 and 92 results are presented in Table 2 for TNF-α, IL-6 and SAA.

Exploratory disease activity
As compared with placebo, there were no statistically significant effects seen with ISIS-CRP Rx on disease symptoms as assessed via ACR20, ACR50 or DAS28-ESR ( Table 2) at Day 36 or Day 92.

Safety
ISIS-CRP Rx was generally safe and well tolerated with no serious infections due to treatment. There were similar numbers of patients who reported treatment emergent adverse events (TEAEs) across the treatment groups (8/ 12 placebo, 7/12 ISIS-CRP Rx 100 mg, 11/13 ISIS-CRP Rx 200 mg and 12/14 ISIS-CRP Rx 400 mg) ( Table 3). There were a similar number of infections reported in each group (Table 3). Infections were reported as mostly mild in severity with only a few reported as moderate (Table 3).
There were seven patients who discontinued treatment either due to TEAEs or meeting the requirements for one of the stopping rules. One patient on ISIS-CRP Rx 200 mg discontinued treatment due to fatal pulmonary edema which also met the criteria for a serious adverse event (SAE). This patient had known atherosclerotic vascular disease. The investigator rated this as possibly related to study drug. Two patients discontinued treatment due to worsening of RA symptoms. One was on ISIS-CRP Rx 200 mg and the other was on ISIS-CRP Rx 400 mg. Three patients (one placebo, one ISIS-CRP Rx 200 mg and one ISIS-CRP Rx 400 mg) met the protocol pre-specified stopping criteria for renal function (that is, serum creatinine (Cr) increase >0.2 mg/dL). However, the absolute serum Cr levels remained within the normal limits for all of these patients. One patient on ISIS-CRP Rx 200 mg discontinued treatment due to an exacerbation of chronic calculous cholecystitis although this was considered unlikely related to study drug by the investigator.

Discussion
ISIS 329993 (ISIS-CRP Rx ) is a novel approach to reducing the inflammatory marker CRP and to potentially down regulate inflammation. As an antisense drug targeting human CRP it was hypothesized that ISIS-CRP Rx should be non-immunosuppressant and it, therefore, might be a valuable adjunct to standard DMARDs, or even biologic DMARDs. Previous studies of ISIS-CRP Rx in rodent models of RA (that is, CRP transgenic mice with collagen-induced arthritis) demonstrated improvement in the clinical signs of arthritis [4]. Subsequently, initial clinical studies in healthy human volunteers demonstrated that treatment with ISIS-CRP Rx achieved significant hs-CRP lowering [4]. Before embarking on a large RA study to definitively address the question of clinical improvement in symptoms and signs, it was decided to first explore whether this approach would reproducibly decrease hs-CRP in RA patients and to assess its potential impact on other markers of inflammation. Given the results of this study, ISIS-CRP Rx has demonstrated that the antisense oligonucleotide platform can selectively reduce circulating levels of hs-CRP. The results at 36 days demonstrate separation between dosing cohorts with respect to percent change in hs-CRP and achieved statistical significance at the highest dose, with a 76.7% reduction in hs-CRP in the ISIS CRP Rx 400 mg arm compared with a 14.4% reduction in the placebo arm at Day 36 (P = 0.0015). However, by 92 days this response does not appear as statistically robust given the significant decreases in hs-CRP seen in the placebo group. It is unclear what drove the decreases in hs-CRP in the placebo group but it could be speculated that it was in part regression to the mean, and possibly related to increased compliance with background regimens that can also be seen in clinical trials as a consequence of closer follow-up.
It remains unclear whether ISIS-CRP Rx is truly antiinflammatory in an active human disease state. Despite the encouraging data in the animal models, there was little change in the other inflammatory markers studied in this trial including ESR, SAA, IL-6, TNF-α, fibrinogen and complement. This invites the question as to whether inflammation was down regulated or whether the drug was simply effective at blocking a surrogate marker of inflammation. Additionally, the exploratory endpoints of ACR20/50 and DAS28-ESR were frankly difficult to interpret in the setting of such small numbers and such high placebo response rates. That said, the reductions     DAS28-ESR, disease activity score 28-erythrocyte sedimentation rate (DAS28-ESR = 0.56*square root oftender joint count + 0.28* square root of swollen joint count + 0.014*subject assessment of pain + 0.70*ln(ESR)); ESR, erythrocyte sedimentation rate; hs-CRP, high sensitivity C-reactive protein; SD, standard deviation. Swelling, number (%) 0 0 0 1 ( 7 )