Inclusion criteria were as follows: i) Japanese male or female aged 35–75 years; ii) radiographic severity of knee joints grade 0 or 1, based on Kellgren and Lawrence grades (32) (grade 0, no radiographic features of osteoarthritis; grade 1, doubtful joint space narrowing and possible osteophytic lipping) without the diagnosis of osteoarthritis by orthopedists; iii) one knee joint with visual analog scale (VAS) of ≥20 mm (33), based on at least one of four VAS subscales, including subscale I (degree of knee pain evaluated by VAS) of the Japan Knee Osteoarthritis Measure (JKOM) (33) and three VAS subscales (pain at rest, pain at walking and pain at going up and down stairs). Each VAS subscale was scored from 0 to 100 mm, where 0 mm indicates ‘no pain at all’ and 100 mm indicates ‘the most severe pain ever experienced’.

Exclusion criteria were as follows: i) Total VAS subscale of ankle, elbow, shoulder and hip joints being greater than the subscale of knee joint (the subscale I of JKOM); ii) a score of ≥30 points on subscale I (pain on walking) of the Japanese Orthopedic Association criteria (34), which indicates that patients can walk ≥1 km without apparent pain; iii) a diagnosis of gout/hyperuricemia or rheumatoid arthritis; iv) surgical treatment of joint(s) performed or required; v) clinical history of bone or cartilage disorders including fracture and distortion within one year prior to enrollment; vi) routine use of dietary supplements containing proteoglycans, hyaluronic acid, N-acetyl glucosamine, glucosamine, chondroitin sulfate, collagen peptides or any other constituents of the test supplement within three months prior to enrollment; vii) hypersensitivity or allergy to the test component; viii) diagnosis or current medication of disorders including malignancies, hypertension (atherosclerosis), cardiac, renal, thyroid, lung and hepatic disorders, or cerebral infarction; ix) routine use of external medicine including poultices and taking prescribed medicine (>3 days/week); x) intra-articular injections of either corticosteroids or hyaluronic acid within one year prior to enrollment; xi) excessive exercise, which places loads on the joints; xii) daily drinking of >60 g alcohol/day; xiii) pregnant women, nursing mothers or women intending to have children in the future; xiv) participation in any other clinical studies within one month prior to enrollment; and xv) the presence of any clinical conditions judged by the medical investigator to preclude the participation of subjects in the study.

Following assessment of 180 subjects for eligibility, 115 subjects were excluded based on the exclusion criteria and 5 subjects declined to participate of their own volition. Finally, 60 Japanese adults (26 males and 34 females; aged 36–71 years; mean age, 52.4±1.1 years) with knee discomfort but without a diagnosis of knee osteoarthritis (Kellgren and Lawrence grades 0–1) (32) were enrolled as eligible subjects. The research co-coordinators created an allocation table and randomly assigned the eligible subjects to receive a salmon nasal proteoglycan-containing capsule (n=30, proteoglycan group) or a placebo capsule (n=30, placebo group; Fig. 1). The allocation table was sealed, and all research staff and participants were blinded to the allocation during the test period. Following completion of the study, the allocation table was made available for analysis of the data. During the intervention, two subjects in the placebo group discontinued the study of their own volition (one for a long-term business trip, and the other for the treatment of right knee joint pain). A further three subjects (two subjects in the placebo group and one subject in the proteoglycan group) were excluded by the medical investigator, due to the onset of renal stone and joint disease, taking prescribed medicine including inhaled steroids and the use of external medicine (non-steroidal anti-inflammatory drug-containing poultice) for lower back pain during the intervention, which may have affected the efficacy of test supplements. Thus, 55 subjects (mean age, 52.6±1.1 years; n=26 in the placebo group, n=29 in the proteoglycan group) were judged to be eligible for assessment of the efficacy of the test supplement (Fig. 1 and Table I).

A prospective randomized double-blind placebo-controlled, parallel-group comparative study was designed to compare the actions of salmon nasal cartilage proteoglycan and a placebo on cartilage metabolism. Markers for type II collagen synthesis (PIICP) and degradation (C1,2C), aggrecan (CS846) and COMP in individuals with knee joint discomfort were measured. In addition, the safety of the test was evaluated throughout. Subjects were enrolled and screened from February 2015 to April 2015, and the test supplements were administered from May 2015 to August 2015 in the Nihonbashi Sakura Clinic (Tokyo, Japan). The study protocol was registered at the UMIN Clinical Trials Registry (trial no. UMIN000016470), and involved the Nihonbashi Sakura Clinic. The study protocol was approved by the Aisei Hospital Ueno Clinic Research Ethics Committee (Tokyo, Japan) and was conducted in accordance with the principles of the amended Declaration of Helsinki and Ethical Guidelines for Epidemiological Research (established by the Japanese Government in 2008). Written informed consent was obtained from all participants prior to their enrollment in the study. The study consisted of a 4-week run-in (screening) period and a 16-week intervention period. Subjects were screened at a baseline visit by physical examination, knee radiograph according to a standardized method, a symptom questionnaire and routine laboratory tests. In addition, medical examinations were performed at weeks 4, 8, 12 and 16 during the intervention, and laboratory tests were performed at weeks 12 and 16 during the intervention for the enrolled subjects.

The test supplement was manufactured in the form of a hard capsule (220 mg in a capsule) by Ichimaru Pharcos, Co., Ltd. (Gifu, Japan) and consisted of 12.5 mg salmon nasal cartilage extract (containing 10 mg proteoglycan) and 207.5 mg dextrin (as a vehicle). The placebo capsule contained only dextrin powder (Ichimaru Pharcos, Co., Ltd.). Salmon proteoglycan was extracted from salmon (Oncorhynchus keta) nasal cartilage, as previously reported (35). Briefly, frozen (−20°C) salmon nasal cartilage was dissolved in a solution of 4–5% acetic acid and salmon proteoglycan was extracted. High-performance liquid chromatography analysis was performed and detected a salmon proteoglycan peak as 450,000 molecular size, as previously reported (36,37).

Subjects were randomly assigned to receive a 10-mg salmon nasal cartilage proteoglycan-containing capsule (proteoglycan group) or a placebo capsule containing only vehicle (placebo group). All subjects were instructed to take the test supplement or placebo with water once a day between breakfast and lunch for 16 weeks. The daily dose of salmon nasal cartilage proteoglycan (10 mg/day) was determined based on the results of a previous study (29). Adherence to the intervention was evaluated on the basis of a consumption record in the study diary and <80% adherence was considered a protocol violation.

Serum and second void of morning urine were collected from the subjects in a fasting state at baseline, and at weeks 12 and 16 during the intervention. Blood samples were collected by venipuncture, and incubated for 20–30 min at 25°C, followed by centrifugation at 1,000 × g for 10 min at 4°C to isolate sera. Serum and urine samples were immediately used for routine laboratory tests; sera were also aliquoted and stored at −80°C until the assays for C1,2C, PIICP, CS846 and COMP were conducted.

To evaluate the effect of the test supplement on cartilage metabolism, the levels of type II collagen degradation (C1,2C) and synthesis (PIICP) markers, CS846 and COMP were evaluated in serum samples. Serum C1,2C and PIICP were measured using a Collagen Type I and II Cleavage ELISA kit (60-1002-001; IBEX Technologies Inc., Montreal, QC, Canada) and ELISA kit for Procollagen II C-Terminal Propeptide (SEA964Hu; Wuhan USCN Business Co., Ltd., Wuhan, China), respectively. The C1,2C/PIICP ratio was then calculated. Serum CS846 and COMP were measured using an Aggrecan Chondroitin Sulfate 846 Epitope ELISA kit (60–1004; IBEX Technologies, Inc.) and Human COMP Quantikine ELISA kit (DCMP0; R&D Systems, Inc., Minneapolis, MN, USA), respectively.

The efficacy of the test supplement was also evaluated based on the changes in subscale scores of JKOM (33). This was measured at baseline, and at weeks 4, 8, 12 and 16 during the intervention. JKOM is a self-answered evaluation questionnaire that includes five subcategories: i) Degree of knee pain as determined by VAS; ii) Pain and stiffness in knees (8 questions); iii) Condition in daily life (10 questions); iv) General activities (5 questions); and v) Health conditions (2 questions). The responses to each question (ii-v subcategories) are scored from 1 to 5 points, with 1 point indicating the best functional status and 5 points indicating the worst functional status. The JKOM score is higher in subjects with increased pain and physical dysfunction, and is reliable and valid for studying the clinical outcomes of knee osteoarthritis (38). The outcome of JKOM is closely correlated with that of other arthritis-related scales, including the Western Ontario and McMaster Universities Arthritis Index (38).

Safety and tolerability were assessed throughout the study. The incidence and severity of intervention-related adverse events (side effects) were recorded, as well as abnormal changes in physical parameters, such as blood pressure and pulse rate. The results of laboratory tests, including hematology, biochemical profile and urinalysis, were also recorded. Any changes in physical conditions and the use of pharmaceutical products were recorded in a diary by the enrolled subjects.

Data are expressed as the mean ± standard error of the mean, unless otherwise noted. Regarding the baseline characteristics of subjects, the distributions of males and females, and Kellgren and Lawrence grades were analyzed using Pearson's χ2 test, and other parameters were analyzed using the Student's t-test. Subcategory I of JKOM and VAS subscales were compared using the Student's t-test. Subcategories II–V of JKOM were compared between different time points using the Wilcoxon rank sum test, and between different groups using the Mann Whitney-U test. Safety data, C1,2C, PIICP, CS846 and COMP levels, and C1,2C/PIICP ratios were compared using the Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

Table I presents the baseline characteristics of the 55 subjects (n=26 in the placebo group; n=29 in the proteoglycan group) who completed the study and fulfilled the eligibility criteria. The baseline characteristics included demographic characteristics (age, and distribution of male and female subjects), physiological measurement parameters (body height, body weight and body mass index), physiological examinations (systolic blood pressure, diastolic blood pressure and pulse rate), distribution of Kellgren and Lawrence grades, JKOM (total score), VAS subscales (pain at rest, pain at walking, and pain at going up and down stairs), and levels of biomarkers for type II collagen metabolism (C1,2C, PIICP and C1,2C/PIICP ratio), CS846 and COMP. There were no significant differences in these parameters between the placebo and proteoglycan groups at the baseline. Adherence to the allotted supplement was 96–100% among the 58 subjects who completed the study.

In addition to the markers for type II collagen degradation (C1,2C) and synthesis (PIICP), aggrecan (CS846) and COMP, the ratio of type II collagen degradation to synthesis may be used to predict the progression of joint damage in patients with knee osteoarthritis (39,40). The effect of salmon cartilage proteoglycan on cartilage metabolism was evaluated by measuring the C1,2C/PIICP ratio as well as levels of C1,2C, PIICP, CS846 and COMP using sera collected at baseline, and at weeks 12 and 16 of the intervention. The levels of C1,2C and PIICP did not significantly change in the placebo and proteoglycan groups after 16 weeks intervention. There were also no significant differences in C1,2C and PIICP levels between the placebo and proteoglycan groups. However, C1,2C levels slightly decreased from baseline levels of 0.77±0.04 and 0.79±0.03 µg/ml to 0.76±0.03 and 0.73±0.03 µg/ml after the intervention for 16 weeks in the placebo and proteoglycan groups, respectively. Thus, C1,2C levels decreased slightly more in the proteoglycan group (−0.06±0.04 µg/ml) compared with the placebo group (−0.01±0.03 µg/ml) after the intervention for 16 weeks (Fig. 2A). By contrast, PIICP levels were marginally increased from the baseline levels of 47.20±2.00 and 47.66±2.15 ng/ml to 49.31±2.57 and 50.14±2.58 ng/ml after the intervention for 16 weeks in the placebo and proteoglycan groups, respectively. PIICP levels increased slightly more in the proteoglycan group (2.49±1.80 ng/ml) compared with the placebo group (2.11±2.52 ng/ml) after 16 weeks intervention, although this difference was not significant (Fig. 2B). Furthermore, the C1,2C/PIICP ratios decreased from the baseline after 16 weeks intervention in both groups. The C1,2C/PIICP ratios decreased slightly more in the proteoglycan group (−0.002±0.001) compared with the placebo group (−0.001±0.001) after the intervention for 16 weeks (Fig. 2C), although there was no significant difference between the two groups.

Levels of CS846 were not markedly altered during the intervention period in the placebo and proteoglycan groups (Fig. 2D), while levels of COMP decreased somewhat more in the proteoglycan group than in the placebo group (Fig. 2E). However, there were no significant differences in COMP levels between the placebo and proteoglycan groups at weeks 12 and 16.

In order to improve clarity regarding the effect of the test supplement, further assessments focused on subjects with a high level of pain and physical dysfunction, as determined by their JKOM score (33). Subjects with a total JKOM score of <41 (25% of the 55 subjects) were excluded. Thus, 41 subjects (mean age, 52.3±1.2 years; n=19 in placebo group, n=22 in proteoglycan group) with a total score of JKOM ≥41 (increased pain and physical dysfunction) were evaluated. Table II presents the baseline characteristics of these subjects, including demographic characteristics, physiological characteristics, distribution of Kellgren and Lawrence grades, JKOM (total score), VAS subscales and levels of biomarkers. There were no significant differences between these parameters in the placebo and proteoglycan groups.

In the proteoglycan group, C1,2C levels decreased from the baseline levels of 0.80±0.04 to 0.71±0.03 µg/ml after 16 weeks intervention. In the placebo group, C1,2C levels increased from the baseline levels of 0.74±0.04 to 0.77±0.04 µg/ml after 16 weeks intervention in the placebo group. Thus, the C1,2C levels were significantly decreased in the proteoglycan group (−0.09±0.04 µg/ml) compared with the placebo group (0.03±0.02 µg/ml) after 16 weeks intervention (P<0.05; Fig. 3A). By contrast, PIICP levels were slightly increased from the baseline levels of 47.34±2.30 and 48.03±2.55 ng/ml to 48.96±3.14 and 50.86±3.12 ng/ml in the placebo and proteoglycan groups, respectively following 16 weeks intervention. PIICP levels increased slightly more in the proteoglycan group (2.83±2.20 ng/ml) compared with the placebo group (1.63±3.21 ng/ml) after 16 weeks intervention (Fig. 3B). Furthermore, C1,2C/PIICP ratios were markedly decreased in the proteoglycan group (−0.003±0.001) compared with the placebo group (0.001±0.001) after 16 weeks intervention, though this difference was not significant (Fig. 3C).

Levels of CS846 were markedly decreased during the intervention period in the placebo group relative to the proteoglycan group (Fig. 3D), while levels of COMP were reduced to a greater extent in the proteoglycan group than in the placebo group (Fig. 3E). However, levels of CS846 and COMP did not differ significantly between the placebo and proteoglycan groups at weeks 12 and 16.

The effect of the test supplement was also evaluated in subjects with constant knee joint discomfort, based on the subscale of VAS (pain at rest). Thus, 25 subjects without pain at rest (VAS subscale of 0) were excluded. A total of 30 subjects (mean age, 50.1±1.4 years n=12 in the placebo group and n=18 in the proteoglycan group) with a subscale of VAS (pain at rest) ≥1 mm were evaluated. Table III presents the baseline characteristics of these subjects. No significant differences in these parameters were found between the placebo and proteoglycan groups.

In the proteoglycan group, C1,2C levels decreased from the baseline level of 0.80±0.04 to 0.70±0.03 µg/ml after 16 weeks intervention. In the placebo group, C1,2C levels increased from the baseline levels of 0.79±0.04 to 0.83±0.05 µg/ml after 16 weeks intervention. Thus, C1,2C levels were significantly decreased in the proteoglycan group (−0.10±0.05 µg/ml) compared with the placebo group (0.03±0.02 µg/ml) after 16 weeks intervention (P<0.05; Fig. 4A). By contrast, PIICP levels were slightly increased from the baseline levels of 48.37±2.77 and 47.46±2.59 ng/ml to 49.70±3.89 and 52.42±3.69 ng/ml in the placebo and proteoglycan groups respectively, after 16 weeks intervention. Thus, PIICP levels from the baseline were slightly increased in the proteoglycan group (4.96±2.64 ng/ml) compared with the placebo group (1.33±3.90 ng/ml) after 16 weeks intervention (Fig. 4B). Furthermore, the C1,2C/PIICP ratio decreased in the proteoglycan group (−0.003±0.001) whereas it increased in the placebo group (0.001±0.002) after 16 weeks intervention, although this difference was not significant (Fig. 4C).

By contrast, levels of CS846 were markedly decreased during the intervention period in the placebo group relative to the proteoglycan group (Fig. 4D), while levels of COMP were reduced to a greater extent in the proteoglycan group than in the placebo group (Fig. 4E). However, levels of CS846 and COMP did not differ significantly between the placebo and proteoglycan groups at weeks 12 and 16 (Fig. 4D and E).

The test supplement was evaluated based on changes in the subscale scores of JKOM (33) and VAS. However, there were no significant differences between the placebo and proteoglycan groups during the intervention at weeks 4, 8, 12 or 16 in the subscale scores of JKOM and VAS subscales, among the subjects of the initial analysis (Table I), the subjects with a total score of JKOM ≥41 (Table II) and the subjects with pain at rest (Table III) (data not shown).

Together these observations suggest that oral administration of test supplement containing salmon nasal cartilage proteoglycan may have a protective effect on cartilage metabolism in subjects with severe and constant knee pain, by improving the C1,2C/PIICP ratio (lowering type II collagen degradation and increasing type II collagen synthesis).

Out of all 60 enrolled subjects, 6 (26.7%) in the placebo group and 11 (36.7%) in the proteoglycan group experienced one or more adverse events during the intervention period. The total number of adverse events reported was 11 and 21 in the placebo and proteoglycan groups, respectively, and there was no significant difference in the frequency of adverse events occurring between the two groups (P=0.580). Major adverse events reported from the subjects of the placebo and proteoglycan groups were symptoms of a common cold (sore throat, cough and/or bronchitis), headache, back pain and neck/shoulder pain. All adverse events were of mild or moderate intensity and judged by the medical investigator to be unrelated to the intervention.

Furthermore, changes in physical measurement parameters (body weight and body mass index), physiological examinations (systolic and diastolic blood pressures, and pulse rate) and laboratory tests (urinalysis, hematology and blood chemistry) were minimal and within the reference values during the intervention in both groups.