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Clinical and epidemiological research
Extended report
A 2-year randomised, double-blind, placebo-controlled, multicentre study of oral selective iNOS inhibitor, cindunistat (SD-6010), in patients with symptomatic osteoarthritis of the knee
  1. Marie-Pierre Hellio le Graverand1,
  2. Ray S Clemmer1,
  3. Patricia Redifer1,
  4. Robert M Brunell1,
  5. Curtis W Hayes2,
  6. Kenneth D Brandt3,
  7. Steven B Abramson4,
  8. Pamela T Manning5,
  9. Colin G Miller6,
  10. Eric Vignon7
  1. 1Primary Care Medicines Development Group, Pfizer Inc, Groton, Connecticut, USA
  2. 2Department of Radiology, Virginia Commonwealth University Health System, Richmond, Virginia, USA
  3. 3Department of Rheumatology, Kansas University Medical Center, Kansas City, Kansas, USA
  4. 4Division of Rheumatology, New York School of Medicine, New York, New York, USA
  5. 5Vasculox, Inc, St Louis, Missouri, USA
  6. 6Department of Medical Affairs, BioClinica, Inc., Newtown, Pennsylvania, USA
  7. 7Claude Bernard University, Lyon, France
  1. Correspondence to Dr Marie-Pierre Hellio Le Graverand, Medicines Development Group, Primary Care Business Unit, Pfizer Inc., 445 Eastern Point Road, Groton, CT 06340, USA; helliomp{at}pfizer.com

Abstract

Objective To determine if inhibition of inducible nitric oxide synthase (iNOS) with cindunistat hydrochloride maleate slows progression of osteoarthritis (OA)

Methods This 2-year, multinational, double-blind, placebo-controlled trial enrolled patients with symptomatic knee OA (Kellgren and Lawrence Grade (KLG) 2 or 3). Standard OA therapies were permitted throughout. Patients were randomly assigned to cindunistat (50 or 200 mg/day) or placebo. Randomisation was stratified by KLG. Radiographs to assess joint space narrowing (JSN) were acquired using the modified Lyon-schuss protocol at baseline, week 48 and 96.

Results Of 1457 patients (50 mg/day, n=485; 200 mg/day, n=486; placebo, n=486), 1048 (71.9%) completed the study. Patients were predominantly women; 56% had KLG3. The primary analysis did not demonstrate superiority of cindunistat versus placebo for rate of change in JSN. In KLG2 patients, JSN after 48 weeks was lower with cindunistat 50 mg/day versus placebo (p=0.032). Least-squares mean±SE JSN with cindunistat 50 mg/day ( −0.048±0.028 mm) and 200 mg/day (−0.062±0.028 mm) were 59.9% (95% CI 6.8% to 106.9%) and 48.7% (95% CI -8.4% to 93.9%) of placebo, improvement was not maintained at 96 weeks. No improvement was observed for KLG3 patients at either time-point. Cindunistat did not improve joint pain or function, but was generally well tolerated.

Conclusions Cindunistat (50 or 200 mg/day) did not slow the rate of JSN versus placebo. After 48-weeks, KLG2 patients showed less JSN; however, the improvement was not sustained at 96-weeks. iNOS inhibition did not slow OA progression in KLG3 patients.

Clinical trial listing NCT00565812

  • Knee Osteoarthritis
  • Inflammation
  • Treatment

https://doi.org/10.1136/annrheumdis-2012-202239

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    Introduction

    Osteoarthritis (OA) is a progressive disease considered as a failure of the entire synovial joint1 that may be initiated by abnormality in any tissue of the joint.1 Both mechanical and biochemical factors contribute to the pathology and progressive degradation of the joint in OA. Accumulating evidence has linked an over-production of nitric oxide (NO), an inflammatory mediator involved in OA, to the progressive degradative changes in OA joints.2–5 NO is synthesised enzymatically by three distinct isoforms of NO synthase (NOS), two of which (endothelial NOS [eNOS]; neuronal NOS [nNOS]) are constitutively expressed and produce low, transient concentrations of NO.6 ,7 The inducible form (iNOS) is responsible for sustained and excessive NO production when induced by endotoxin, cytokines and/or other pathological stresses. iNOS has been localised to the menisci and cartilage in non-clinical models of OA3 ,8–10 and in synoviocytes, chondrocytes and chondro-osteophytes in OA patients.3 ,11–14 When stimulated, human chondrocytes induce iNOS expression, leading to production of NO and NO metabolites, a reduction in proteoglycan synthesis, an increased activity of the matrix degrading metalloproteinases (MMPs) and chondrocyte apoptosis.5 ,15–17 This ability of NO to stimulate cartilage matrix degradation and chondrocyte cell death suggests that continued over-production of NO may lead to a net loss of cartilage matrix and alterations in normal physiological function of the joint.3 Selective inhibition of iNOS may reduce cytotoxicity and tissue damage in OA17–20 without affecting the physiological activities mediated by eNOS and nNOS.6 ,7 The clinical utility of selective iNOS inhibitors as disease-modifying OA drugs (DMOADs) therefore warranted investigation.

    Cindunistat hydrochloride maleate (SD-6010) is an orally-administered, selective, time-dependent and irreversible inhibitor of human iNOS (hiNOS).21 ,22 In vivo studies using the canine anterior cruciate ligament-transection model have demonstrated that cindunistat improves synovial fluid nitrite and nitrotyrosine biomarkers, osteophyte formation and cartilage lesions (Pfizer Inc, data on file). Cindunistat has also been shown to improve pain behaviour in rodent models of inflammatory and neuropathic pain.21 Based on its potential to inhibit NO production and an early clinical development programme, the disease-modifying efficacy and safety profile of cindunistat was studied in a 2-year proof-of-concept clinical trial of patients with knee OA.

    Patients and methods

    Patient population

    In total 5077 patients were screened from 131 investigational sites in 14 countries (USA, Canada, Australia, Belgium, Czech Republic, Germany, Hungary, Italy, Poland, Russian Federation, Slovakia, Spain, Argentina and Peru). Eligible patients were: greater than or equal to 40 years old; had body mass index (BMI) greater than or equal to 25 to less than or equal to 40 kg/m2; had symptomatic and radiographic knee OA per American College of Rheumatology (ACR) criteria; and in the past year had pain, aching or stiffness on most days, and/or use of medication (other than potent opioids) for treatment of knee pain. Radiographic eligibility criteria for selecting the study knee were: (1) Kellgren and Lawrence Grade 2 (KLG2) and KLG323 with a medial tibiofemoral joint space width (JSW) ≥2 mm and more narrowing than the lateral compartment JSW; (2) disease severity in the contralateral knee that, in the opinion of the investigator, would not lead to arthroplasty or otherwise incapacitate the patient within the duration of the trial; (3) an anatomic axis angle (AAA) of 174–184°; and (4) no radiographic findings in the study knee such as, but not limited to, osteonecrosis, fracture, infection, gout, Paget's disease, osteopetrosis, osteochondritis or other pathology. If knees were identical with respect to radiographic severity, the patient's dominant kicking leg was selected as the study knee.

    Exclusion criteria included: systolic blood pressure (SBP)/diastolic blood pressure (DBP) greater than 150/90 mm  Hg; a diagnosis of any other rheumatic disease (eg, spondyloarthropathies, rheumatoid arthritis, systemic lupus erythematosus); a systemic metabolic condition or endocrinopathy affecting bone or cartilage; a history of severe, uncontrolled renal, hepatic, haematological, gastrointestinal, metabolic, endocrine, pulmonary, cardiac or neurological disease, or another condition that would make the patient unsuitable for the study; use of any investigational drug within 1-month of screening; and positive tuberculosis screening or current treatment for tuberculosis; subjects considered at risk of becoming incapacitated in the near future due to KLG 4 changes such that activities of daily living would be severely curtailed.

    Standard medical therapies (pharmacological and non-pharmacological) for OA were permitted for all patients, if they had been stable for 4 weeks or more before the first dose of study medication. The use of chronic potent opioids, oral, intramuscular or intra-articular corticosteroids, and intra-articular hyaluronic acid were discouraged. If these medications were employed, their use was documented. Rescue medications (eg, acetaminophen, non-steroidal anti-inflammatory drugs (NSAIDs), or tramadol) were allowed for patient-reported breakthrough pain.

    Study design

    This was a 2-year, phase IIb/III, proof-of-concept, multinational, multicentre, randomised, double-blind, placebo-controlled, parallel group, clinical trial (see online supplementary figure S1). Eligible patients were assigned (1 : 1 : 1) to once-daily cindunistat (50 mg/day or 200 mg/day) or placebo, according to a computer-generated randomisation schedule. Patients were stratified according to KLG (2 or 3) in the study knee at randomisation. KLG2 was defined as minimal OA, and categorised according to a definite osteophyte and mild diminution of the joint space. KLG3 was defined as moderate OA, and categorised according to a definite diminution of the joint space with at least a minimal osteophyte. Regularly scheduled clinic visits occurred every 2 weeks in the first 4 weeks of treatment, and then at 12-week intervals to the end of treatment, and at 4 weeks post-treatment. When study medication was prematurely discontinued, subjects returned for an early withdrawal visit for safety and final x-rays.

    The study was conducted in compliance with the Declaration of Helsinki and Good Clinical Practice guidelines. The final protocol and informed consent documentation were reviewed and approved by the Institutional Review Board(s) and/or an Independent Ethics Committee(s) at each study centre. Written informed consent was received from all eligible patients before trial procedures were initiated. The trial was registered and the full trial protocol can be accessed at ClinicalTrials.gov #NCT00565812.

    Modified Lyon-schuss x-ray methodology

    For assessment of joint space narrowing (JSN), radiographs were acquired using the modified Lyon-schuss (ml/S) protocol24 ,25 at baseline, weeks 48 and 96. The first image was acquired with 10° caudal beam angulation, and superimposition of the anterior and posterior margins of the medial tibial plateau (MTP) was assessed. Three radiographs with adjustments of knee positioning and x-ray tube angulation were acquired to achieve an image that met the criteria for knee centring, flexion, rotation and an intermargin distance (IMD) ≤1.5 mm. A central imaging laboratory (BioClinica, Inc., Newtown, Pennsylvania, USA) was responsible for the implementation, standardisation, quality control and analysis of the images. Longitudinal measurements of IMD and JSW were performed by an experienced reader (EV) blinded to sequence and treatment assignment, using digitised image analysis software (Holy's Software, UCLB, Lyon, France).26 Intra-observer and inter-observer intra-class correlation coefficients for reproducibility of minimal JSW measurements have been previously reported as 0.99.24 Repeat images were requested when required, if the MTP was not aligned (≤1.5 mm) or for other quality issues related to centring of the knee on the film, knee rotation or knee flexion.

    Radiographic end point

    The primary objective was to determine the efficacy of cindunistat (50 mg/day or 200 mg/day) for reducing the rate of radiographic JSN in the medial tibiofemoral compartment of the study knee of OA patients.

    Assessment of clinical benefit

    Clinical assessment questionnaires, including Western Ontario McMaster Osteoarthritis Index (WOMAC) subscales (pain, physical function) and Patient Global Assessment of Arthritic Condition, were collected at week 12 and 24, and every 24 weeks thereafter.

    Western Ontario McMaster Osteoarthritis Index (WOMAC)

    The WOMAC V.LK 3.1 was administered.27 The pain subscale ranged from 0–20 and the physical function subscale from 0–68, where higher scores represent worse pain and physical function, respectively.

    Patient Global Assessment of Arthritic Condition

    The Patient Global Assessment of Arthritic Condition was administered as a Likert scale, where patients rated their arthritis as: Very Good; Good; Fair; Poor; Very Poor (range 1–5; higher scores represent poorer condition).

    Safety and tolerability

    For all patients receiving 1 or more dose of study medication, safety and tolerability assessments were conducted through spontaneous reporting of adverse events (AEs), physical examinations (blood pressure (BP), vital signs), ECG, and clinical laboratory results. BP was measured in triplicate, with the mean of the second and third BP readings used. AEs were coded according to the MedDRA (V.14.1) dictionary. Blinded safety data were reviewed by internal sponsor clinicians on a daily basis, and monthly by an internal Risk Management Committee. An independent, external Data Monitoring Committee (DMC) also conducted quarterly, unblinded safety reviews during the course of the trial.

    Statistical methods

    This study had ∼85% power to detect a difference in JSN progression of 0.08 mm/year. The assumed rate of JSN in the placebo group was 0.2 mm/year; 0.08 mm/year represented a 40% improvement over placebo. The common SD of 0.60 mm was based on data from previous radiographic studies.24 ,25 ,28 The experiment-wise type I error rate was set at α=0.05, using the Hochberg procedure to adjust for multiple comparisons to placebo.29 The sample size was estimated as 467 per group using a simulation which accounted for 30% dropout and three pre-planned interim analyses, all which were conducted by the external independent DMC.

    The primary analysis of the rate of JSN included all randomised patients and employed a mixed model for repeated measures (MMRM) on JSW with the intercept and slope as random effects and fixed effects for treatment group, time, a time×treatment group interaction term, KLG, a time×KLG interaction term, geographic region, age, gender and baseline BMI. JSW was modelled as a linear function of time and the slope over the entire 96-week period was used to assess the rate of change in JSW. As JSW, not JSN, was the dependent variable, all subjects with at least one measurement were included in the primary analysis. After spending α=0.0001 for the planned interim analyses, none of which could have resulted in termination of the study for early efficacy, the Hochberg procedure was used with α=0.0499. Sensitivity analyses for the primary endpoint examined the assumptions of linearity, uninformative missingness and model adequacy.

    JSN progressors were identified according to the Osteoarthritis Research Society International-Outcome Measures in Rheumatology (OARSI-OMERACT) definition.30 Test-retest radiographs were obtained in 22 patients at four centres to determine the smallest detectable difference (SDD). Patients with a decrease from baseline in JSW at the last post-baseline observation greater than SDD were considered to be JSN progressors.30 The SDD was defined as: 1.96×√2 × the within-patient SD determined from the test-retest JSW data.31 JSN progression was analysed using a logistic regression model similar to the linear time MMRM model, except all terms involving time were deleted and baseline JSW was added as a fixed effect.

    Changes from baseline in continuous secondary endpoints were analysed using a discrete time MMRM model similar to the linear time model, except that time was considered discrete and the baseline value was included as an additional fixed effect. Patients without both a baseline and post-baseline observation dropped out of all change from baseline analyses. The MMRM analyses for the primary and continuous secondary endpoints do not require the assumption that data are missing completely at random, only that data are missing at random.

    Analyses presented in this paper were pre-specified. Statistical significance was set at 5% (2-sided) for the secondary, sensitivity and supplemental analyses; no adjustments for multiplicity were made. Data were analysed using SAS V.9.2. Data are given as mean±SD, unless indicated otherwise.

    Results

    Patient disposition

    Among the 5077 patients screened, 1457 were assigned to treatment, of which 1452 (n=482, 50 mg/day; n=485, 200 mg/day; n=485, placebo) were treated (figure 1). The majority of screen failures (68%) were related to radiographic criteria; more specifically, related to KLG scores (47%), AAA (13%), minimum JSW <2 mm (7%), or lateral compartment OA (2%). Altogether, 1048 patients (71.9%) completed the trial. The number of patients who discontinued was similar in each treatment arm (50 mg/day, n=144; 200 mg/day, n=135; placebo, n=130) (figure 1).

    Figure 1
    Figure 1

    Flow of patients through the study. AE, adverse event.

    Baseline characteristics

    Patients (61.0 years; BMI 31.8 kg/m2) were predominantly women and 56% had KLG3. Baseline characteristics were similar across treatment arms (table 1). Minimum JSW at baseline was 3.2±0.7 mm and was consistent across treatment groups. Most patients (1100/1457 (75.5%)) were taking analgesics for knee OA. Demographics and other baseline characteristics for completers and dropouts were similar.

    View this table:
    Table 1

    Baseline demographics and clinical characteristics for all randomised patients

    Efficacy analyses

    Quantitative measurement of JSN

    The primary analysis did not support the superiority of cindunistat over placebo for either treatment group (table 2). Improvement in the rate of JSN with cindunistat 50 mg/day versus placebo was 0.012 mm/year (95% CI –0.023 to 0.046; p=0.509), and for cindunistat 200 mg/day was 0.005 mm/year (95% CI −0.029 to 0.040; p=0.754). In an exploratory discrete time analysis of change from baseline in JSW, the loss of JSW after 48 weeks in KLG2 patients was smaller with cindunistat 50 mg/day than with placebo (least squares (LS)-mean vs placebo (95% CI): 0.072 (0.006 to 0.138); p=0.032), but did not differ for cindunistat 200 mg/day versus placebo (0.058 (–0.007 to 0.124); p=0.081) (figure 2A). LS-mean (±SE) losses in JSW with cindunistat 50 mg/day (–0.048±0.028 mm) and 200 mg/day (–0.062±0.028 mm) were 59.9% (95% CI 6.8% to 106.9%) and 48.7% (95% CI –8.4% to 93.9%) of placebo (–0.120±0.028 mm). After 96 weeks, no significant difference in JSN relative to that in the placebo group was observed with either cindunistat 50 mg/day or 200 mg/day. The mean losses in JSW of –0.132±0.036 mm and –0.156±0.037 mm, respectively, were 20.9% (95% CI –50.5% to 64.0%) and 6.8% (95% CI –72.7% to 51.7%) of the mean JSW loss in the placebo group (–0.167±0.036 mm). In a similar analysis of KLG3 patients, no improvement in JSN was observed with cindunistat (figure 2B).

    View this table:
    Table 2

    Linear time MMRM analysis of JSW for all randomised patients and analysis of JSN progressors† for all randomised patients, and KLG2 and KLG3 patients

    Figure 2
    Figure 2

    Discrete time MMRM LS-Mean (95% CI) change from baseline in JSW for (A) KLG2, and (B) KLG3 patients. BL, baseline; JSW, joint space width; KLG, Kellgren and Lawrence Grade; LS, least squares; MMRM, mixed model for repeated measures.

    In an analysis of JSN progressors, an improvement with cindunistat treatment in all randomised patients was observed (table 2). Using an SDD of 0.199 mm, the model-adjusted probability of progression in placebo-treated patients was 40.6% (95% CI 35.1% to 46.3%), compared with 33.7% (95% CI 28.5% to 39.5%) with cindunistat 50 mg/day and 37.1% (95% CI 31.8% to 42.8%) with cindunistat 200 mg/day. The adjusted OR versus placebo for the cindunistat 50 mg/day group indicated significant improvement (OR: 0.745 (95% CI 0.557 to 0.997); p=0.047), but the OR for cindunistat 200 mg/day versus placebo did not (OR: 0.864 (95% CI 0.650 to 1.150)). The analyses of JSN progressors by KLG indicated that the improvement was attributable to KLG2 patients, with an adjusted OR versus placebo of 0.553 (95% CI 0.341 to 0.896; p=0.016) with cindunistat 50 mg/day, but 0.708 (95% CI 0.441 to 1.135); p=0.152) with cindunistat 200 mg/day for KLG2 patients. For patients with KLG3, no significant reduction in the adjusted OR versus placebo was observed for either group. Subjects without a baseline or post-baseline observation dropped out of each of these analyses (table 2).

    To understand the inconsistency between the continuous time MMRM and JSN progressor analyses, the distributions of change in JSW from baseline for cindunistat (50 mg/day) and placebo treatment were assessed. No difference in location was found; however, the distributions had different shapes. For cindunistat 50 mg/day versus placebo, fewer patients had changes of –0.6 mm to –0.2 mm and more had changes from 0.0 mm to 0.3 mm (see online supplementary figure S2).

    Clinical benefit endpoints

    Although significant improvements over baseline in WOMAC pain, WOMAC physical function and Patient Global Assessment of Arthritic Condition were observed as early as 3 months post-baseline and throughout the study, no difference between cindunistat and placebo was established for these endpoints (figure 3).

    Figure 3
    Figure 3

    Discrete time MMRM LS-Mean (95% CI) change from baseline in WOMAC measures of (A) pain and (B) physical function, and (C) Patient Global Assessment of Arthritic Condition, for all randomised patients. BL, baseline; LS, least squares; MMRM, mixed model for repeated measures; WOMAC, Western Ontario McMaster Osteoarthritis Index.

    Safety and tolerability

    The incidences of AEs, withdrawals due to AEs and serious AEs were similar between cindunistat 50 mg/day and placebo treatment although the incidence of these was slightly higher with cindunistat 200 mg/day (table 3). The most common treatment-emergent AEs leading to discontinuation from the study overall were arthralgia and hypertension (1.0% each for subjects in the cindunistat 50 mg once daily (QD) treatment group, 1.0% and 0.6%, respectively, for subjects in the cindunistat 200 mg QD treatment group and 0.6% and 0.4%, respectively, for subjects in the placebo group). The majority of AEs leading to discontinuation from the study were mild or moderate in severity. Changes from baseline in SBP and DBP were observed and differed across treatment groups (see online supplementary figure S3). The cindunistat 200 mg/day group showed small, but statistically significant, increases in SBP and DBP at multiple time points, in comparison with placebo treatment, primarily in the first 6 months of the study. This effect was driven by patients who were normotensive at baseline and who were not receiving antihypertensive medication. The cindunistat 50 mg/day group was similar to the placebo group for changes in SBP/DBP.

    View this table:
    Table 3

    AEs reported by ≥5% of patients in any treatment group

    Discussion

    To date, regulatory authorities have not approved any therapy as a DMOAD and major efforts to develop such treatments have been hampered by technical challenges. Of the biochemical factors that contribute to the progressive degradation of the entire joint in OA1 iNOS was considered an attractive molecular target with the potential to affect mechanisms involved in both structural deterioration and inflammation.2–5 17–20 In this 2-year study, cindunistat (50 or 200 mg/day) did not reduce OA progression, as assessed by a reduction in the rate of radiographic JSN. During the first 48-weeks of treatment, however, an exploratory analysis determined that patients with KLG2 treated with cindunistat 50 mg/day showed less JSN than placebo-treated patients. This improvement was not sustained at 96 weeks, and iNOS inhibition did not slow OA progression in patients with KLG3, at either 48- or 96 weeks. These observations may indicate that at some point in the progression of OA, joint tissue deterioration may not be reversible through inhibition of iNOS and its downstream products, which might explain the lack of effect on JSN in patients with KLG3. It should be noted that we attempted to select patients with less aberrant biomechanics (no valgus and only mild to moderate varus), although patients were overweight and obese. In addition, significantly fewer JSN progressors were observed with cindunistat 50 mg/day compared with placebo, driven by a greater response in KLG2 patients. However, the progressor analyses should be interpreted with caution. JSN is dependent on time, and when adjusted for time, the difference in JSN progression was no longer significant. Also, the OARSI-OMERACT definition of JSN progression uses the sample within-patient SD,30 which is known to be biased (too small on average). Using an unbiased estimator would result in a patient's change from baseline being compared to a larger SDD value for determining JSN progression. When the JSN progressor analysis was repeated with any larger value, it was again no longer significant.

    Certain pathophysiological pathways, such as the iNOS-NO pathway, may be more relevant targets for pharmacological intervention early in disease, an idea supported by some other DMOAD studies, where efficacy for reducing disease progression was suggested after 1-year but lost after 2,32 ,33 or improvement was suggested in less severe OA.28 ,34 Although the quality of the x-ray acquisition techniques in these studies has been questioned,35 earlier DMOAD studies support the view that disease modification may be possible if mild OA is treated early on before integral pathophysiological and/or biomechanical processes overwhelm any drug effect. Interestingly, a 30-month trial of doxycycline reported a 33% reduction in JSN in the index knee of older, obese women, but no reduction in the contralateral knee, in which OA was not apparent radiographically in the anteroposterior view but mild disease was seen in lateral and/or patellofemoral views.28 These studies targeted different pathological pathways and used different methodologies, but collectively can be used to illustrate the complexities involved in the study of disease progression, and the need to consider the influence of other mediators, imaging protocols and/or early versus late disease.

    Our study represents the first large-scale field test of the utility of the non-fluoroscopic ml/S protocol for imaging OA of the knee. Historically, the reproducibility of x-rays has been a limiting factor in trials using JSN as a clinical end point,24 ,25 as variability in the assessment of JSW limits conclusion regarding efficacy. As a result of tightly controlled methodology, we demonstrated progression of JSN in our placebo population. Besides limiting variability in radiographic technique, the power of our study was maintained by aggressively engaging in subject retention, and recruiting a homogeneous population resulting from strict entry criteria. The observed mean loss of JSW in both KLG2 and KLG3 patients was consistent with the population enrolled, which excluded patients with strong risk factors for OA progression, including genu varum and valgum, and severe JSN (<2 mm) at baseline. While the observed JSN slowed for both groups from week 48 to week 96, JSN in KLG3 patients was twice as much as that observed in KLG2 patients. Our study suggests that wider implementation of the m/LS protocol in clinical trials is justified, particularly to strengthen the method for differentiating clearly KLG in the target knee.

    Our focus on structural modification based on JSN measured using the ml/S method has its limitations. We did not measure changes in other joint tissues, such as synovium or bone, and the possibility that iNOS inhibition was beneficial to other tissues in the OA joint that were not monitored in our protocol remains unexplored. JSN is the recommended outcome measure in clinical trials for registration purposes. As suggested by Felson and Kim,36 the current method of staging the disease for clinical trials using JSN may be insufficient to adequately identify suitable candidates for DMOAD therapies. Research efforts must therefore continue to validate biomarkers and MRI parameters to improve patient selection and the detection of structure modification, particularly for patients with early OA where the potential for slowing progression is greater.

    Demonstration of structure modification in OA must be related to clinical benefit,37 ,38 even though the relationship between OA severity and pain and/or function is not well understood.39–42 Our study detected a 25–30% improvement in WOMAC pain and physical function in the placebo group, but no additional improvement with cindunistat over placebo treatment was noted. As discussed above, by the time an OA joint becomes symptomatic, damage may already have progressed beyond the capability of pharmacological modification, making DMOAD trials in patients presenting with OA pain inappropriate.40 An important limitation of our study was that in order to study structure modification over 2 years, treatment with cindunistat or placebo was given in conjunction with standard-of-care analgesics, which may have introduced noise and/or bias in the evaluation of pain relief and physical function.43

    Cindunistat was generally safe and well-tolerated, but a small, albeit significant, increase in BP was noted with 200 mg/day, suggesting loss of iNOS selectivity at the higher dose. eNOS and nNOS both play major roles in the physiological regulation of vascular tone and BP regulation in humans.44 ,45 Endothelial and neuronal NOS knock-out mice are hypertensive and lack a vasorelaxatory response to acetylcholine.44 ,46 Transgenic mice with over-expression of eNOS are hypotensive.47 Potential secondary pharmacodynamic effects related to loss of iNOS selectivity and inhibition of eNOS and nNOS were studied in non-clinical safety pharmacology studies with cindunistat. Elevations in BP with a corresponding decrease in heart rate likely related to vasoconstriction were observed at high concentrations in various species. BP was monitored as a biomarker in the clinical cindunistat programme. Dose selection for the present study took into consideration BP effects observed in OA subjects with higher doses of cindunistat. The observed numerical differences in JSN between 50 and 200 mg/day may therefore be related to loss of selectivity, suggesting that eNOS/nNOS might play an essential role in joint homeostasis. Taking all points into consideration, our results provide important insight into inhibition of the iNOS pathophysiological pathway within the OA joint.

    The current failure of long-term DMOAD studies suggests that the altered mechanics in even radiographic ‘mild-to-moderate’ OA are of such overwhelming significance that the outcome may not be effectively altered with a pharmacological or biological structure-modifying agent.36 Our data do not exclude the possibility that iNOS was too limited a target to retard progression of OA in established disease, and that a broad-based target would merit consideration as a DMOAD. Evidence from surgical approaches to OA, such as distraction procedures48 and hip, ankle, and knee osteotomy, indicate that modification of the structural damage in OA is possible, with reversal of cartilage loss and improvement in symptoms in the affected joint if intra-articular mechanical stresses are normalised.49 This suggests that under the correct circumstances, joints can heal.49 ,50 Therefore, the debate continues as to whether structure-modifying treatments alone can have a long-term effect on disease progression, or only in conjunction with interventions that normalise the altered mechanics of the OA joint. Further studies are thus warranted to investigate this hypothesis, particularly in patients with advanced disease.

    In conclusion, irreversible iNOS inhibitor cindunistat (50 or 200 mg/day) did not reduce the rate of JSN in patients with knee OA in comparison with placebo. During the first 48-weeks of treatment, patients with KLG2 treated with cindunistat 50 mg/day showed less JSN; however, this improvement was not sustained at 96 weeks and iNOS inhibition did not slow OA progression in patients with KLG3. The early effect on JSN in KLG2 patients supports a role of iNOS in the pathogenesis of joint damage in OA. However, the loss of efficacy over time and lack of effect in KLG3 patients suggest that alternative biochemical catabolic pathways overcame the effects of NO inhibition and/or that the consequences of the increased intra-articular stress may not have been amenable to iNOS inhibition alone.

    Acknowledgments

    This study was sponsored by Pfizer Inc. Medical writing support was provided by Karen Burrows, MPhil, of UBC Scientific Solutions and was funded by Pfizer Inc. The authors thank the study investigators for their participation. We also express our gratitude to the following x-ray technologists whose skills and dedication were essential in assuring the successful conduct of the study: Elaine Dawson, Sonia Morges, Debbie Peterson, Rebecca McCarrie, Mohsen Haddad-Kaveh, and Gene Dunkle. We are also grateful to the dedicated Pfizer Study Team for their successful conduct of this study: Claire Johnston, Charles Packard, Nicola Schreiber, Evelyn Moy, Kris Kokomoor, and Bradley Wyman.

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    Supplementary materials

    • Supplementary Data

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    Footnotes

    • Contributors M-PHLG, RSC, CWH, CGM and EV were involved in the conception and execution of the study. RMB was involved in the analysis of the study. All authors had access to the data. All authors were involved in the data interpretation, writing of the manuscript and critically revising during development. All authors approved the final draft of the manuscript for submission.

    • Funding The study was sponsored by Pfizer Inc.

    • Competing interests M-PHLG, RSC, PR and RMB are full-time employees of Pfizer Inc. CWH acted as a paid consultant to BioClinica, Inc., in relation to the blinded analysis of radiographs. KB and SBM declare no competing interests. PTM was an employee of Pfizer Inc at the time of study design and initiation. CGM is an employee and shareholder of BioClinica, Inc., and was a consultant to Pfizer Inc in relation to the blinded analysis of radiographs. EV acted as a paid consultant to Pfizer Inc in relation to the blinded analysis of radiographs.

    • Ethics approval The study was conducted in compliance with the Declaration of Helsinki and Good Clinical Practice guidelines. The final protocol and informed consent documentation were reviewed and approved by Institutional Review Board(s) and/or an Independent Ethics Committee(s) at each study centre. Written informed consent was received from all eligible patients before trial procedures were initiated.

    • Provenance and peer review Not commissioned; externally peer reviewed.

    • Data sharing statement All authors had access to the data.

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