Effect Size Statistics to Inform an Exploratory Analysis of a Double-Blinded, Randomised, Placebo

Background: Potential effects of different doses of specific fish collagen peptides (Naticol®) on the signs of skin ageing were firstly assessed in double-blind, randomised and placebo controlled clinical studies. The studies showed benefits of fish collagen peptides (Naticol®) on skin firmness, skin hydration and wrinkle appearance. In addition to these benefits, some animal experiments have suggested that ingestion of specific fish collagen peptides (Naticol®) might also have beneficial effects on joint health such as osteoarthritis.


Introduction
Osteoarthritis (OA) results from an imbalance of anabolism and catabolism in the joint, which may be influenced by the biological and mechanical environment. Excessive forces in specific regions of the joint surfaces and alterations in tissue responses, leading to the development of osteoarthritis are also exacerbated with abnormal loading of the knee joint due to overweight or a joint disorder [1][2][3].
Today, the total cost of osteoarthritis has been estimated to be between 1% and 2.5% of the total gross domestic product in western countries [4]. In Europe, over 100 million people have arthritis, and in the United States, the direct costs of arthritis were $51.1 billion in 2004 [5]. As the number of elderly people increases, osteoarthritis represents a major economic burden on the health care systems and is a serious threat to the quality of life within the populations. For instance, in the UK in 2010, there were 4.7 million doctor appointments due to knee osteoarthritis for patients over 45 years. It has been predicted that this number will increase to 6.7 million by 2051 [6].
This pilot study was designed to assess the tolerability and efficacy of oral doses of fish collagen peptides (Naticol ® , 10g/day) in volunteers with knee osteoarthritis. The collagen hydrolysates or peptides (Naticol ® ) which were the object of this efficacy study were products derived from fish hydrolysed collagen (gelatin) suitable for use in human food. Previous clinical studies aimed at assessing the effects of oral intakes of fish collagen peptides, Naticol ® , on skin firmness and hydration, and the decrease of wrinkles were conducted using different dosages (10g/day; 5g/day; 2,5g/day) [7,8]. The results from Duteil et al. [7] and Sibilla et al. [8] confirmed preclinical and clinical human trials investigating the effects of oral supplementation with collagen peptides that have indicated the possibility that dietary compounds can modulate skin function. For example, Matsuda et al. [9] reported that in pigs a 9-week oral ingestion of collagen hydrolysates (CH) induced increased fibroblast density and enhanced formation of collagen fibrils in the dermis in a proteinspecific manner. Literature also indicates benefits of collagen hydrolysates in joints as they are absorbed, distributed to joint tissues and have analgesic and anti-inflammatory properties [10][11][12][13][14].
The aim of the study was to determine the effect of 8 weeks' supplementation of Naticol ® , Fish collagen peptides on change in WOMAC scores; Quality of Life, using the Participant Global Assessment (SF-36v2 questionnaire) and Physical performance using the Short Physical Performance Battery (SPPB), along with supporting the safety of the product by recording any reported adverse events.

Participants
Subjects underwent an initial phone screen and were asked questions regarding their age, general health and osteoarthritis diagnosis. Forty eligible subjects were scheduled for an initial visit. The pilot study involved 2 visits over an 8-week period. At the initial visit, subjects were included if they were: Aged 30 to 75 years (inclusive); Had documented diagnosis of primary osteoarthritis (OA) of the target knee made at least 12 months prior to the visit; Had radiographic evidence of OA in the tibiofemoral compartment of the target knee with at least 1 definite osteophyte and a measurable joint space, as diagnosed by standard X-rays; Had a Kellgree-Lawrence grade 2-4. Potential female subjects were excluded if they were pregnant, lactating or wished to become pregnant during the study. Subjects were also excluded if they: Had viscosupplementation in any joint including the target knee or other joint within 6 months prior to the initial visit; Had concomitant inflammatory disease or other condition that affects the joints; Had taken any calcium supplements within the previous 4-weeks; Had a disease/illness that would preclude supplement ingestion and/or assessment of safety and the study objectives and/or had a known allergy to components of the test product; Had lost > 5% of their total body weight in the last 3 months.
The site, investigator, protocol, informed consent form and other pertinent documents for this study were approved by the Cork Research Ethics Committee of the Cork Teaching Hospitals, Lancaster Hall, 6 Little Hanover Street, Cork. All subjects gave their written consent according to the Helsinki Declaration. The pilot study was a double-blind, randomised, placebocontrolled clinical study in which adults, male or female, between 30 and 75 years of age, with a diagnosis of primary osteoarthritis of the target knee were included. The study occurred between May 9, 2017 (first subject, first visit) and December 12, 2017 (last subject, last visit). Based on findings from Kumar et al. [15], 30 subjects were randomised into one of two groups. 10 subjects were randomly assigned to the placebo group, with 20 subjects to the active group -i.e., receiving the investigational product (Naticol ® , fish collagen peptides). The investigational product (NATICOL ® , fish collagen peptides) was manufactured and released under the responsibility of Weishardt and was provided to the Coordinator/Investigator in boxes containing single dose sachets. A 10g sachet of the investigational products and the comparator product (maltodextrin) were to be consumed one daily intake in 20cl of cold liquid (fruit juice, milk or cold tea) 15 minutes before breakfast, for 8 weeks. The Naticol® reveals the presence of numerous amino acids including proline, hydroxyproline, glycine and arginine, see Table 1. Nutritional value is also summarized in Table 1.

Design and conduct of study
At baseline, subjects were provided with a box containing 8-weeks supply of the study product, along with an additional 4-day supply in case of loss. Subjects were instructed to one sachet each morning, with their breakfast, for the duration of the study. Subjects returned after 8 weeks. At each visit the vitals, anthropometric measurements, concomitant medication and any adverse events were recorded. Subjects completed the following questionnaires: Western Ontario and McMaster Universities Osteoarthritis Index Likert Scale (WOMAC) and participant global assessment short form 36 (SF-36v2). Subjects returned any unused study product and compliance was assessed and new study product administered. Subjects also underwent the Short Physical Performance Battery (SPPB) test at each visit. Subjects were instructed to follow their standard diet and exercise routine and not consume medications that could interfere with the assessment of the study product for the duration of the study. Any symptoms and changes in health status or medications were recorded.

Statistical analysis
Subjects were randomized by a computer-generated schedule to either the active or placebo groups (2:1). All statistical analyses were carried out using SPSS Version 24 for Windows. All tests were twosided and performed at the 5% level of significance. Cohen's d was used to determine the strength (standardised mean difference) of statistical results: No effect: 0 ≤ d <0.2; Small effect: 0.2 ≤ d < 0.5; Medium effect 0.5 ≤ d < 0.8; Large effect d ≥ 0.8 [16]. Categorical parameters were summarized by means of absolute numbers. Numerical data were summarized by means of standard statistics (i.e., number of available data, mean, standard deviation, minimum, maximum). The change in subject's measurements (WOMAC scores; SF-36v2; SPPB) between treatment groups from baseline to the end of treatment were analyzed using a repeated measures ANOVA (with the appropriate test of normality, homogeneity of variance and sphericity carried out initially). The overall incidence of treatment-related adverse events was compared across groups by Fisher's exact test and the chi squared test.

Measurements
Analysis of WOMAC scores, SF-36v2 and SPPB were based on [17][18][19], respectively. The higher scores on the WOMAC indicate worse pain, stiffness, and functional limitations [20]. The SF6 36 consists of eight scaled scores, which are the weighted sums of the questions in their section. The lower the score the more disability [18]. In relation to the SPPB test, higher scores indicate better lower body function [21]. The clinical team for the study assessed each subject's-reported adverse event (AE) and categorized it according to Intensity; Relationship to study; Action taken; Outcome; Serious Adverse Event (SAE).

Results
Exploratory analysis on the effect of 8 weeks' supplementation of specific fish collagen peptides (Naticol ® ), on osteoarthritis measurements, for subjects that completed the study With the exception of gender, there was no difference in subjects across treatment groups at baseline, see Table 2. One subject from each treatment group had treatment discontinued due to nausea (active group) and an allergic reaction to the product (placebo group), see Table 3. Hence, results on measurements are reported on subjects that completed the study -i.e., 9:19 Placebo: Active.  Upon investigation it was apparent that subjects in the active group experienced more of an improvement across the majority of measurements than subjects in the placebo group over the 8 weeks of the study, see Table 4, Table 5 & Figure 1. The magnitude of the differences observed were compared using effect sizei.e., the standardized mean difference in measurements from baseline to 8 weeks, with a larger difference, in magnitude of the effect size statistics, found in the active group than the placebo group across the majority of measurements. Due to the exploratory nature of the study and the small sample size, comparing effect sizes are a reliable approach to inform the statistical power of future studies, [22].    Figure 1(a), with the WOMAC Pain sub-score, both groups experienced a large improvement, d = 0.81, 0.88, respectively [16]. For WOMAC Physical Function the placebo group experienced a small improvement (d = 0.68) against the active group's large improvement (d = 1.08). Finally, for the WOMAC Composite score the placebo group experienced a medium improvement (d = 0.82) against the active groups even larger improvement (d = 1.07). The WOMAC stiffness sub score is not presented Table 5 and Figure 1(a).

SF-36v2
Noting the lower the score the more disability [18]. Hence, a negative effect size (Baseline -Week 8) represents less disability (an improvement) over the 8 weeks. In terms of Role Limitations due to Physical Problems score, Role Limitations due to Emotional Problems score active group experienced medium improvements, see Table 5. Physical functioning, energy and vitality scores are not presented in Table 5 and Figure 1

SPPB
Noting higher scores indicate better lower body function [21]. Hence, a negative effect size (Baseline -Week 8) represents an improvement in lower body function over the 8 weeks. Both groups experienced an improvement in the Chair test and Total score. While the placebo group experienced a small and negligible improvements (d = -0.25 and -0.10, respectively), while the active group experienced a small improvement in both tests (d = -0.47 and d = -0.33, respectively). Gait score results are not presented in Table 5 and Figure 1(c).

Adverse events
Fisher's exact test found there was a statistically insignificant association between the number of adverse events reported per visit and treatment group (p = 1.000), see Table 3. Furthermore, there was a statistically insignificant association between treatment group and the: Intensity, Relationship to study; Action taken and Outcome of an adverse event (p = 1.000, 0.894, 0.638 and 0.051, respectively).

Discussion
This double-blinded, placebo-controlled pilot clinical study demonstrates the potential for once daily oral doses of Naticol®, specific fish collagen peptides, Naticol®, to alleviate symptoms of osteoarthritis in the knee. The study design was exploratory in nature, and effect size calculations show that subjects in the active group experienced more of an improvement across the majority of measurements than subjects in the placebo group over the 8 weeks of the study. These results corroborate those of previous preclinical and clinical studies to assess the potential effects of collagen hydrolysates on joint pain and physical mobility as assessed by the Western Ontario McMaster Universities (WOMAC) scoring system.
Clinical studies suggest that the ingestion of 10g collagen hydrolysates daily reduces pain in patients with osteoarthritis of the knee or hip while blood concentration of hydroxyproline is increased. Clinical use was associated with minimal adverse effects, mainly gastrointestinal, characterized by fullness or unpleasant taste [23].
In a prospective, randomized, double-blind, placebocontrolled study in elderly women with mild-to moderate knee osteoarthritis, oral intake of collagen peptides for a duration of 6 months showed to significantly reduce joint pain and improve physical mobility as assessed by the Western Ontario McMaster Universities (WOMAC) scoring system. This clinical study confirmed that collagen peptides are a highly efficient nutraceutical to improve joint health in patients with osteoarthritis which can help to maintain an active lifestyle throughout ageing [24].
We can also indicate a double-blind, placebo-controlled, randomized trial with collagen hydrolysates isolated from pork skin (PCP) and bovine bone (BCP) sources carried out to study the effectiveness of orally supplemented collagen peptide to control the progression of osteoarthritis in patients diagnosed with knee osteoarthritis. Improvement in treatment with reduction in WOMAC and quality of life (QOL) scores from baseline to 13 weeks were recorded. Indeed, all the score levels of WOMAC and QOL decreased significantly (p<0.01) in the study group compared to placebo group after week 13. Safety and tolerability were also good [15].
Additionally, research in mice has demonstrated that after oral administration of radiolabeled gelatin hydrolysate, the radioactivity was specifically found in cartilage indicating an accumulation of these peptides within the connective tissue [25]. The study demonstrated the intestinal absorption and cartilage accumulation of collagen peptide. Thus, the potential role of collagen hydrolysates in repair of damaged cartilage could be associated with the accumulation of orally administered collagen hydrolysates. A recent animal study has also suggested that oral ingestion of collagen hydrolysates 9 might have beneficial effects on joint health such as osteoarthritis (OA) as collagen hydrolysates might reduce the morphological changes associated with osteoarthritic cartilage destruction in knee joints, [26].
This mechanism about absorption, distribution and collagen peptide accumulation in cartilage may explain the benefits we clinically recorded on joint pain and physical mobility. However, anti-inflammatory properties may also be an additional way for benefits. Indeed, the literature reports that intra-articular administration of collagen in the knee of subjects with osteoarthritis demonstrated a significant local inflammation decrease (increase in T regs, IL-10 and decrease in IL-1β) and a mitigation in the symptoms inherent to the pathology [13]. Similarly, oral administration of collagen hydrolysates in mice with post-traumatic osteoarthritis inhibited synovial inflammation and induced cartilage regeneration [12].
Moreover, in this pilot clinical trial study, we may indicate that from the 28 (9:19 Placebo: Active) subjects that completed the study, 5 (55.6%) of the placebo group and 14 (73.7%) of the active group had a body weight greater than 70kg at baseline. From Table 2, the maximum weight of subjects (that completed the study) at baseline was 85.5kg and 100.0kg for the placebo and active groups, respectively. In Shigemura et al. [27], the daily ingested dose of collagen hydrolysates is linked to the body weight. These authors examined the relationship between ingested dose/kg body weight and food-derived hydroxyproline levels in human plasma to estimate the effective beneficial dose of the collagen peptides. Hence, healthy volunteers ingested 30.8, 153.8 and 384.6mg per kg body weight of collagen hydrolysates. The average plasma concentration of hydroxyproline containing peptides was dose-dependent, reaching maximum levels of 6.43, 20.17 and 32.84nmol/ml following ingestion of 30.8, 153.8 and 384.6mg doses of collagen hydrolysate, respectively. Ingesting over 153.8mg of collagen hydrolysate significantly increased the average concentrations of the free and peptide forms of hydroxyproline in plasma. The hydroxyproline absorption limit was not reached with ingestion of as much as 384.6mg of collagen hydrolysate per kg body weight. These finding suggest that ingestion of less than 30.8mg of collagen hydrolysate per body weight is not effective for health benefits. They also show that the dose is calculated taking account of body weight.
Hence, doses may vary from one subject to another due to the variations of body weight. In this way, in order to maintain a similar dose per kg body weight between all subjects, the 100kgsubjects who received a daily dose of 10g of specific fish collagen peptides (Naticol ® ) should have received more than 10g/day compared to the ones who received 10g/day for a body weight equivalent to 70kg. Here, the dose of collagen hydrolysates per body weight was not equivalent between all subjects due to the huge body weight variations. However, all subjects received the minimum dose of collagen hydrolysates reported by Shigemura et al. [27] for benefits. This dose of collagen hydrolysates ingested by the subjects with a very high body weight was nevertheless enough to demonstrate significant benefits and larger benefits in the active group.

Conclusion
This pilot clinical trial study, which consisted in a once daily treatment for 8 consecutive weeks, indicates that oral supplement specific fish collagen peptides have some interesting effects on knee osteoarthritis symptoms. The results demonstrated that daily oral doses of specific fish collagen peptides (Naticol ® ) in healthy subjects with knee osteoarthritis has the potential to reduce pain, improve quality of life and lower body function. Further studies with an extension of duration to 12weeks or more and a higher dosage of product for subjects with a body weight greater than 70kg should be performed to confirm these preliminary results.