Journal of Clinical Trials
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UC-II^® Undenatured Type II Collagen Reduces Knee Joint Discomfort and Improves Mobility in Healthy Subjects: A Randomized, Double-Blind, Placebo-Controlled Clinical Study

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Introduction

Osteoarthritis (OA) is a major public health risk and challenge. According to the Centers for Disease Control and Prevention (CDC), one in four U.S. adults have some form of arthritis, a figure projected to reach 78 million by the year 2040 [1]. Knee OA contributes to more than $27 billion in health care costs each year [1,2]. Overall the burden of OA and complications are increasing in most countries [3].

Over the past few years, the health and wellness industry has witnessed large number of millennials participating in exercise on a regular basis as they pursue more active lifestyles including use of dietary supplements to improve their activities [4-6].

UC-II® undenatured type II collagen (Undenatured Collagen) is a joint health ingredient derived from chicken sternum cartilage, and has been shown in clinical and preclinical studies to support knee joint comfort and reduce overall joint pain by lowering markers of inflammation [7-10]. In addition, laboratory animal study results demonstrate that a clinically relevant daily dose of Undenatured Collagen can improve the mechanical function of the injured knee and prevent excessive deterioration of articular cartilage and reduces inflammation [11-13].

Undenatured (native) Collagen is believed to exert its effects via a unique mechanism in the gut known as oral tolerance, which involves induction of T regulatory cells thereby allowing the knee joint to repair and rebuild the cartilage. The presence of active epitopes on the Undenatured Collagen are required to interact with Peyer’s patches in the gut and induce oral tolerance [14-15].

Some research groups examined people with activity-related joint discomfort and showed improvement of joint pain in response to stresses from the daily life activity [16-21]. Such an approach has limitations because daily life activity is characterized by variable intensities and therefore by the variable load on the joints resulting in different levels of stress on joint-related structures. To overcome these shortcomings, stress models (joint or muscle-related) have been used to induce a standardized load on physical structures to observe changes in perceived stress as accurately as possible [19-21].

We recently reported the effect of Undenatured Collagen on improving knee Range of Motion (ROM) flexion and extension in a 24-week study in healthy subjects who experienced activity-related join discomfort [22]. The current paper reports the data from the same study specifically focused on the ability of Undenatured Collagen supplementation to improve knee joint discomfort and mobility in healthy subject with Activity-related Joint discomfort (ArJD). In addition, we also present the sub-analysis of the joint outcome measures based on age and gender.

Methodology

Study design

This study is a randomized, parallel design and double-blind twoarm study. Subjects signed the informed consent forms prior to participating in the study. Table 1 provides the summary of the study design.

Table 1: Study Design; measures and outcomes based on visits during the period of the study.

We recently validated a Single-Leg-Step-Down (SLSD) test as a reliable model to induce joint discomfort [21]. Subjects with previous history of joint discomfort during sports activities, and who reported joint pain while performing the SLSD test were enrolled in the study. Joint discomfort during daily life and physical activity was evaluated using the subjective Knee Injury and Osteoarthritis Outcome Score (KOOS) questionnaire [23]. Joint mobility was measured using the daily number of steps taken as recorded by a step counter.

Subjects were asked to maintain their normal activities, nutrition and lifestyle habits plus avoid taking any medications or dietary supplements for pain relief within 36 hours prior to the study visits. Additionally, on the day of study visits, subjects were instructed to consume their last meal at least one hour before the performance of the step test. During the study, subjects documented the type of frequency of their physical activity, as well as the pain level.

This study was conducted following the guidelines for Good Clinical Practice (GCP) set forth by the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use International Council for Harmonisation (ICH E6 (R2), Nov 2016) and following the Declaration of Helsinki (E8) regarding the treatment of human subjects in a study. This study was approved by the local ethics committee (Institutional Review Board of the Landesärztekammer Baden-Württemberg, file number F-2019-072) and the clinical trial was registered at Deutsches Register Klinischer Studien (DRKS)-German Clinical Trials Register DRKS (ID: DRKS00018792).

Investigational products

Undenatured Collagen sourced from chicken sternum (40 mg UCII ® collagen per day, providing ≥ 3% undenatured type II collagen) was provided by Lonza Consumer Health Inc. Placebo capsule was sensory identical and contained only the excipient, microcrystalline cellulose. Subjects were asked to take one capsule of study product daily with water before bedtime. Subjects documented their intake time in a diary.

Study product compliance

Compliance was calculated based on dispensed and returned study products. In the case of missing products or no return, compliance was estimated from subject diaries.

Study population

In the current study, 178 men and women were screened for eligibility using the inclusion-exclusion criteria described in detail in Table 2. Inclusion criteria was based on subjects reporting joint discomfort of at least 5 on an 11-point Likert scale while performing between 30 and 150 repetitions during the SLSD and performing joint bearing sports (at least 2 times per week for at least 20 min). Ninety-six (96) subjects who met the eligibility were randomized to receive either Undenatured Collagen or PLA. The study was conducted at BioTeSys GmbH, Esslingen, and at the Institute of Sport and Movement Science of the University of Stuttgart from September 2019 to January 2021.

Table 2: Inclusion and exclusion criteria of the subjects.

Subjects' medical history, concomitant medications use, anthropometric measurements and vital signs (blood pressure and heart rate) were recorded during the course of the study. General joint health conditions were assessed based on small orthopedic tests (knock knees, bandy legs, McMurray Test, Steinman I and Steinman II, Drawers Test, Zohlen sign). Additionally, routine blood parameters such as liver enzymes, kidney function, hematocrit, hs-CRP, HbA1c and lipid profiles were assessed for safety at baseline and the end of the study. Adverse Events (AEs) were recorded during the study period.

Joint discomfort

Steps to reach pain 5 and 2 during SLSD test: For the SLSD test Figure 1, subjects were asked to performed as many repetitions (step number) as possible until they reported the knee joint pain level of 2 (first onset) as well as of level 5 on an 11-point Likert scale, where 0 meant “no pain” and 10 meant “worst pain possible”. The number of total repetitions was documented. The leg with the highest pain level after regular physical activity was defined as the target knee. At the initial screening, the SLSD test was used to assess suitability for study inclusion. At visit one (baseline), only subjects who met the inclusion criteria concerning the SLSD test were enrolled. SLSD test was performed at each visit and the number of repetitions (step number) to reach a pain level of 2 and 5 on Likert scale were recorded (Figure 2).

Figure 1: Single-leg-step-down (SLSD) test.

Figure 2: Likert rating scale to rate knee pain while doing the singleleg- step-down test.

Duration after sports activity

Starting with the initial screening, subjects documented the type and frequency of their activity and reported duration of joint discomfort after sports activity.

KOOS questionnaire

The KOOS questionnaire is a validated instrument used to assess subject's opinion about their knee and associated problems [23]. It consists of five subscales: (1) Pain, (2) Other symptoms, (3) Activities of Daily Living (ADL), (4) Function in sport and recreation (Sport/ Rec), and (5) Knee-related Quality Of Life (QOL). Subjects are asked to consider their previous week when answering the questions. For the retrospective measurement of joint discomfort during daily life and physical activity, the KOOS was used and filled in by subjects at initial screening, visit one, visit four, and visit seven. Subscale Sport/Rec was filled in at each study visit.

Daily step count

A step counter was used between visits (OMRON Walking style IV) during the study to measure the daily steps count. The daily step count was documented in the subjects’ diaries.

Statistical analysis

Sample size calculations were based on the results of a previously performed pilot study [7,21]. Assuming an effect size of 0.636, a sample size of 40 subjects per group would provide approximately 80% power at a significance level of 5%. Considering a drop-out rate of 15%, the study was performed with 48 subjects per group.

Analysis of Covariance (ANCOVA) with baseline value as covariate was used for between group comparisons. Changes over time within study groups were evaluated with repeated measures ANOVA or Friedman test as appropriate. Post hoc analysis for comparison between baseline and each study visit was performed applying Dunnett's Multiple Comparison Test or Dunn's Multiple Comparison Test as appropriate. Additionally, analysis between and within groups was assessed applying a linear mixed model with repeated measures considering baseline and gender as covariates. All analysis was evaluated in the Intention-To-Treat population (ITT). The missing data were imputed by the Last Observation Carried Forward (LOCF) method. All statistical tests were performed two-sided with p value ≤ 0.05 was considered significant. The analysis was performed with IBM SPSS statistics 24 statistical software (Armonk, NY, USA) as well as with SAS software Version 9.4 (Cary, NC, USA).

Results

Baseline characteristics

There were no significant difference between the groups at baseline with respect to the age (Undenatured Collagen: 34 ± 1.5 y; PLA: 37.8 ± 1.6 y) and BMI (Undenatured Collagen: 23.91 ± 0.43 kg/m2; PLA: 24.9 ± 0.38 kg/m2) (p>0.05).

Daily step count

Joint mobility was measured based on the daily number of steps taken by subjects using a step counter. It is a direct determinant of posture and movements that influences daily activities.

The Undenatured Collagen supplemented group reported taking a higher number of daily steps at week 24 compared to the baseline value (+217 steps), whereas the PLA groups reported reduction in number of steps versus the baseline value (-530 steps). However, this change failed to achieve statistical significance between the groups (p=0.2113). A sub-analysis based on gender showed a significantly higher number of daily steps in males from the Undenatured Collagen group versus the PLA group over the study period (+669 steps vs. -526 steps, p=0.0374, Table 3).

Table 3: Daily step count between the study groups delta change from baseline to 24 weeks of intervention.

Joint discomfort

Steps to reach pain 5 during SLSD test: After 24 weeks of supplementation, the Undenatured Collagen group demonstrated an average increase in the number of steps (2.85 steps) prior to reporting pain 5, whereas the PLA group showed a slight decrease in number of steps (-0.65 steps). However, these changes were not significantly different between the groups (p>0.05). In a subgroup analysis based on age, subjects aged 20 and 35 years old in the Undenatured Collagen group showed a significant increase in number of steps change overtime (p=0.0409) and no changes were observed in PLA group during the study period (p>0.05).

Steps to reach pain 2 during SLSD test: The Undenatured Collagen group showed a non-significant increase in the number of steps to reach pain 2 (first onset of pain) than the PLA group after 24 weeks of supplementation (p>0.05). Undenatured Collagen group showed a significant increase of step number to first onset of pain over the baseline (p<0.05). Both study groups further showed a significant change overtime (p<0.05, Table 4). A significant product effect (p=0.0427) as well as a significant time effect (p=0.0013) was further observed. Table 4 summarizes the results of number of repetitions for both study groups.

Table 4: Steps to reach pain 5 and 2 after SLSD test (delta changes from baseline to 24 weeks of intervention).

Knee Injury Osteoarthritis and Outcome Score (KOOS)

The analysis of KOOS subscale data demonstrated a significant improvement of subscale function in sport and recreation over time in the Undenatured Collagen group (p=0.0009, Figure 3), while no change was observed in PLA group (p>0.05). The Undenatured Collagen group also showed improved quality of life over the study period versus the baseline (p<0.05) and no significant change observed in PLA group. Significant differences for such selected KOOS individual items as knee pain (p=0.0482), knee twisting/pivoting (p=0.0346), walking while descending stairs (p=0.0215) and walking on a flat surface (p=0.0241) were observed at visit 7 after 24 weeks of supplementation of Undenatured Collagen compared with the PLA group.

Figure 3: Distribution of KOOS Sport/Rec score between baseline and week 24 in the Undenatured Collagen and Placebo group.

Duration after sporting activity

Subjects were instructed to document joint discomfort and duration of joint discomfort after their regular sporting activity in a daily diary. From this diary, the mean joint discomfort over 14 days before each study visit was calculated. No significance was observed between the study groups at baseline for the pain duration (p>0.05). A significant difference in the duration of pain after sporting activity over baseline was observed in Undenatured Collagen and PLA groups (p<0.05).

When performing the subgroup analysis, a significant decrease of pain after sporting activity over baseline in subgroup of subjects aged 20-35y old in Undenatured Collagen group (p<0.05, Table 5) was observed, while no such significance was seen in PLA group (p>0.05).

Table 5: Duration of pain after sporting activity (min) in the Undenatured Collagen and placebo group.

Safety

Analysis of the vital signs and routine blood parameters showed no clinically abnormal findings or product related changes. None of the reported AEs were related to the study product consumption. The tolerability of the Undenatured Collagen and PLA study products were rated as “well” by almost all subjects. The results of this study further support the tolerability of Undenatured Collagen supplementation.

Discussion

Joint and bone health supplements are increasingly finding their place in the functional foods and nutraceuticals markets. Joint discomfort and joint overload are common problems not only for athletes but also in the daily lives of healthy, active people. Knee joint stress exerted by overload leads to micro-damage of the cartilage those results in localized joint pain. Stress-related joint pain can be caused by anatomical weaknesses (e.g., unequal leg length, bandy legs, small patella, etc.), mechanical overload, or unstable joints resulting from previous ligament injuries. It has also been suggested that the load on knee joints during physical exercise may result in immunological responses that mimic those seen in arthritic disease [24]. Earlier studies have shown Undenatured Collagen supplementation can moderate joint pain and stiffness in osteoarthritic subjects [8-15]. Likewise, Undenatured Collagen has been shown to improve joint function and mobility in healthy subjects who experience activity-related joint pain [7].

There is a need to develop standardized stress tests that would allow evaluation of joint health efficacy of an ingredient in healthy subjects who report ArJD. Our group recently validated a SLSD test as a reliable model to induce joint discomfort and select for participants who are healthy and have ArJD [21]. One parameter to assess joint discomfort with SLSD test is to measure the change in number of repetitions (steps) required to reach a pain of five on an 11-point Likert scale. This test also provides a good measure to study the impact of intervention on improving knee joint mobility and discomfort. Hence, an intervention that increase the number of repetitions until one reaches a defined pain level, it means the intervention is successful in reducing joint discomfort thereby it may allow subjects to move more, and move farther.

In the current study, the SLSD test was used to investigate the effect of Undenatured Collagen group supplementation on joint health in healthy subjects. After 24 weeks of supplementation, subgroup of men in Undenatured collagen group took higher number of steps on SLSD test before experiencing the pain level of 5. Regarding the subgroup analysis by age, an increase in step count to reach pain 5 was seen in subjects of age 20-35 years of the Undenatured Collagen group, whereas, in the PLA group, the increase was only minor. In subjects over 35 years old, a decrease in step count was seen at the end of the study in both the Undenatured Collagen and the placebo group. The decrease in step count observed in both groups might be attributed to the imbalance in the gender distribution with the subgroup comprising of two-third proportion of females in each group. Another possible explanation could be that younger subjects were more responsive to the Undenatured Collagen supplementation.

Interestingly, when comparing the number of repetitions performing the SLSD test to the first onset of pain (pain 2 on the Likert scale), a significant effect of Undenatured Collagen supplementation was observed (p=0.0427). This indicates that Undenatured Collagen supplementation may increase time to the first onset of pain. This observation is in agreement with the previous study where 40 mg Undenatured collagen supplementation for 120 days was shown to extend the pain free exertion in healthy subjects with ArJD [7], where subjects performed a standardized step mill test until reaching a discomfort level of five on an 11-point Likert scale. When evaluating the results at the end of the study in Undenatured Collagen, a significant increase in time to the first onset of pain was observed over baseline [7]. Extrapolating the results of the current study to daily activity, one could say that reduction in joint discomfort translates into increased mobility or movement. This is also further supported by the results of the daily step counting, which showed that Undenatured Collagen group took more daily steps at the end of the study compared to the baseline. Undenatured Collagen group took over 700 steps more than the placebo group, which in practical terms equates to about 1/4th of a mile per day.

Additionally, the KOOS questionnaire was used to assess knee joint discomfort. When comparing the changes between the intervention groups, no differences were seen between the study groups after 24 weeks of intervention in all subscales. Nevertheless, the evaluation of the KOOS questionnaire indicated, that even if the knee pain only occurred during heavy load or sporting activities, this already had some impact on knee related quality of life plus sport and recreation. This is consistent with the observations made during the pilot study [21]. In this study, the KOOS questionnaire was identified as a good tool to differentiate between healthy people, subjects with OA, and subjects with ArJD. Significant differences could be seen between healthy and ArJD subjects in the subscales of Pain, Sport/Rec, and QOL [16]. Nevertheless, some differences were seen when evaluating individual KOOS items in the current study. Regarding individual items of KOOS activities of daily living as descending stairs or walking on a flat surface, Undenatured Collagen group showed lower joint discomfort over PLA group at 24 weeks after supplementation.

Overall, the findings from the current study confirms the previous observation [7,8] that Undenatured Collagen supplementation helps to alleviate the onset of joint discomfort. Previous studies have shown that intense physical activity or exercise may activate processes similar to that seen in arthritis such as increase in production of proinflammatory mediators [11-13]. This is an interesting observation considering the fact that Undenatured Collagen supplementation has been shown in previous studies to reduce inflammation markers and prevent degradation of articular cartilage [11]. In terms of mechanism, Undenatured Collagen is proposed to work via induction of oral tolerance because of the presence of active epitopes on the native type II collagen molecule. When consumed orally, Undenatured collagen passes through the gut and is believed to be taken up by the Peyer’s patches in the small intestine, where it activates immune cells leading to generation of type II collagen specific T regulatory (Treg) cells. Treg cells then migrate through the circulation (blood stream), and when they arrive at an inflamed knee joint, they secrete anti-inflammatory cytokines, TGF- β and IL-10 that helps reduce joint inflammation and promotes cartilage repair [14-16,25-29].

A recent study reported that walking 6000 or more steps per day reduces risk of developing knee OA related mobility issues, such as difficulty getting up from a chair and climbing stairs [30]. The results further suggest that walking an additional 1000 steps per day was associated with 16% to 18% reduction in incidence of functional limitation two years later [30]. In the current study, we observed increase in daily step count with Undenatured Collagen supplementation suggesting that it may enhance joint mobility typically by increasing ambulatory activity.

The current study has some limitations, during the study there was a global outbreak of COVID-19 pandemic. Most of the sports training centers were all closed as part of a lockdown to control the spread of the virus. Hence, subjects performed alternative sport activities at home or outdoors where possible to maintain consistency during the period of the study. These changes were considered as minor deviations. Another limitation of the study is, we observed prominent changes in few parameters in subgroup analysis for the Undenatured Collagen group, however, it was still realized that the overall time and product effect favored the Undenatured Collagen compared to the PLA group.

Conclusion

In short, future larger size studies are warranted to further strengthen the clinically significant outcomes seen in subgroup analysis based on age and gender. In conclusion, the results of this study suggest that a daily dose of 40 mg of UC-II® Undenatured Collagen supplementation may reduce joint discomfort during daily physical activities and supports joint mobility. The Undenatured Collagen supplementation improved daily step count which may help to enhance the daily activities and health related quality of life. However, a larger sample size and longer evaluation period is necessary for future studies.

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