The study product UC-II® (Lot 1204004) was derived from chicken sternum. It was manufactured under current good manufacturing practice (cGMP) conditions using a patented process that preserved its native structure (Chick Cart Inc., Fort Smith, AR). Both glucosamine hydrochloride (GH) and chondroitin sulfate (CS) were purchased through Wilke Resources (Lenexa, KS). The Wellable group (Shishi City, Fujian) manufactured GH under cGMP and according to United States Pharmacopeia 26 specifications. Sioux Pharm (Sioux Center, IA) manufactured bovine-derived CS under cGMP. UC-II and GC were encapsulated in opaque, size “00” capsules with sufficient amounts of excipients (microcrystalline cellulose and silicon dioxide) such that they were sensory identical to placebo. InterHealth Nutraceuticals provided all study materials. All American Pharmaceutical (Billings, MT) verified the amount of active ingredients in the study capsules. Study materials were kept in a secure cabinet with access restricted to the site coordinator, the dispensing pharmacist, and the principal investigator.
The objective of this randomized, double-blind, placebo-controlled clinical study was to evaluate the ability of UC-II to improve knee symptoms in OA subjects, as measured by overall WOMAC score, compared to placebo and to GC. The trial was conducted at 13 centers in southern India. Because of a limitation in synovial fluid sampling procedures at multiple clinical sites, the study was conducted under two separate study protocols. Study protocols were approved by each center’s Institutional Ethics Committee (IEC), and listed on the clinical trial registry of India as study protocols 003663 and 003348. Enrollment, randomization, and follow-up visits were identical for both protocols, and were carried out at days 1 (baseline), 7, 30, 60, 90, 120, 150 and 180 (Table 1). All investigators attended the same investigator meetings, used identical intake and data reporting forms, and were trained and monitored by the same group of clinical research associates.
Efficacy measurements were assessed at all visits and included WOMAC, VAS, and LFI indices. The knee flexion range of motion (ROM) test was performed at each visit. Subject diaries and study product were provided at all visits, except day 180 and were collected at all follow-up visits. Subjects were instructed to record daily the consumption of study product, use of rescue medication, as well as concomitant medications in the subject dairy for the entire duration of the study. Blood and urine were collected at screening and day 180. Pregnancy testing was done at screening and follow-up visits. Adverse events (AEs) were recorded using each subject’s diary inputs plus site visit questionnaires administered by intake personnel at all study visits.
The primary endpoint was defined as the change in total WOMAC score from baseline through day 180 for the UC-II group versus placebo and GC. Secondary clinical endpoints for both protocols were similar and included the change from baseline through day 180 versus placebo and GC for all endpoints including the following scores: (1) mean VAS; (2) mean WOMAC subscales; (3) LFI; and (4) knee flexion. Another endpoint included the change from baseline to day 180 for the serum biomarker cartilage oligomeric matrix protein (COMP). In protocol 003348, additional secondary endpoints included the change in serum biomarker, C-reactive protein (CRP) plus synovial fluid biomarkers interleukin (IL)-6, and matrix metalloproteinase (MMP)-3 from baseline to day 180.
A total of 234 subjects were screened and 191 randomized (Fig. 1). Study inclusion criteria were 40–75 years-old male and female subjects, a body-mass index (BMI) of 18–30 kg/m2, moderate-to-severe OA by physical examination (crepitus, bony enlargements, joint swelling, etc.) in one or both knees, knee pain for at least 3 months prior to the start of the study, an LFI score between 6 and 10 and a VAS score of 40–70 mm 7 days after withdrawal from excluded medications, plus a knee radiograph that was graded as Kellgren and Lawrence (K-L) radiograph score of either 2 or 3 . All OA diagnoses were confirmed by each study site investigator and noted in the subject’s case report form (CRF). In the case of bilateral knee involvement, the index knee used for the study was the one that presented with the most severe OA symptoms at baseline. Detailed inclusion–exclusion criteria are summarized in Table 2.
Ethics, consent and permissions
Subjects were recruited after they reviewed, understood the study details, and then signed the IEC-approved consent form. The study conformed to the Declaration of Helsinki (version 1996).
Randomization & blinding
Block randomization, consisting of nine individuals per block, was executed in a 1:1:1 ratio using random numbers generated by an independent statistician (SPSS version 16.0). Knowledge of the randomization code was limited to the statistician plus one QA monitor unrelated with the study. Each investigator was given opaque, sealed envelopes denoting single patient identity numbers, randomization codes, and supplementation regimen to be opened in case of an emergency. The code was broken after the clinical database was locked.
Subjects ingested two blue pills in the morning with breakfast and two white capsules before bedtime. For the UC-II cohort, the two morning capsules were placebo, while the evening capsules contained 20 mg each of UC-II totaling 40 mg, which is identical to previously used clinical dose levels [5, 7]. This dose delivered 1.2 mg of undenatured type II collagen as determined by a newly developed and validated extraction-ELISA protocol (AIBiotech, Richmond, VA & Chondrex, Redmond, WA). For the GC group, the morning and evening doses delivered 750 mg of GH plus 600 mg of CS each totaling a daily dose of 1,500 mg of GH plus 1,200 mg of CS. The placebo group ingested identical numbers of blue and white capsules containing excipients only. Study bottles were labeled according to ICH-GCP and applicable local regulatory guidelines.
Prior and concomitant therapies
Prior medications were documented at the screening visit by the study investigator. At each visit, study personnel reviewed subject diaries and questioned each participant on the use of any concomitant medications including those on the prohibited list. Prohibited medications included ibuprofen, aspirin, other NSAIDS, or any other pain relievers (OTC or prescription), plus any dietary supplements (excluding vitamins) that could support joint health. All concomitant medications used during the study was documented in the subject’s medical record by the study investigator then transcribed into their CRF by study personnel.
Acetaminophen was allowed at a dose of 500 mg twice daily. Participants were instructed to not take this medication within 48 h of an evaluation visit. Usage levels and timing was entered at each visit into the subject’s medical record by the study investigator. Study personnel transcribed this information into the subject’s CRF.
Compliance and safety
Subjects were instructed to bring their bottles to each visit. Remaining capsules were counted and recorded in the subject’s CRF and accountability log. As a secondary measure of compliance, subjects completed a diary indicating daily dosing of the study products. Safety assessments were performed at all visits by the site investigator and staff (see Table 9).
WOMAC scores were determined using the WOMAC VA3.1 questionnaire containing 24 items grouped into three categories: pain, stiffness, and physical function (score range 0–2400). Each respective WOMAC subscale mean scores was determined by dividing the subscale score by the number of questions (5, pain; 2, stiffness; 17, physical function) it contained. The mean VAS score was determined using a VAS questionnaire containing 7 pain-related questions (score range 0–700), and then dividing the overall score by seven. LFI score was determined using an LFI questionnaire that assessed pain, walking distance, and activities of daily living, (score range 0–24). Knee flexion was measured using goniometry with the subject lying in the prone position and the leg to be tested positioned along the edge of the table .
Synovial fluid biomarkers
Synovial fluid (~0.5 mL) was aspirated from the knee joint using an appropriate sized needle (18–24 gauge, depending on joint size). Harvested fluid was stored frozen until tested. IL-6 and MMP-3 levels were determined using the corresponding Duoset ELISA kits (R&D Systems, Minneapolis, MN).
COMP levels (Quantikine ELISA, R&D Systems) were determined in both study protocols. CRP levels (Latex COBAS INTEGRA, Roche Diagnostics GmbH, Mannheim) were assessed in protocol 003348. Serum was stored frozen until analyzed. Interassay and intrassay coefficients of variation for COMP and CRP were <5 %.
We verified, using 2-way analysis of variance (ANOVA), that the results of the two protocols could be combined into a single analysis by demonstrating there was no group by study interaction and that the magnitude of the efficacy observed under the two protocols was similar.
A modified intent-to-treat (mITT) analysis was used to assess the efficacy and safety outcomes that was defined a priori. This included all subjects who were randomized, consumed study product, and had at least one completed post-baseline visit. An analysis of covariance (ANCOVA), that included supplementation as a fixed factor and the corresponding baseline value of the variable being tested as a covariate, was used for assessing the overall statistical significance of the primary and secondary endpoints. Following ANCOVA, the Tukey-Kramer multiple comparison test was used for determining pairwise statistical significance and calculating 95 % confidence intervals. Also, a mixed model repeated measures (MMRM) analysis of the primary endpoint was performed to account for the multiple assessments obtained during this study. In addition, the method of trapezoids was used to calculate incremental area under the curve (iAUC) for all study groups. For iAUC estimation, missing values were imputed using the expectation-maximization algorithm in SAS. Rescue medication usage between groups was compared using logistic regression followed by pairwise comparisons using the Tukey-Kramer test. In addition, a stratified analysis of the primary endpoint was performed according to baseline serum COMP levels above and below the median value for this biomarker. Differences were considered significant if the resultant p-value was ≤0.05. An independent statistician performed the analyses and other calculations using SAS version 9.3 (Cary, NC).