Collagen hydrolysate composition for reducing joint pain after exercise

Abstract

The present invention relates to a composition comprising chondroitin sulfate and collagen hydrolysate, for use in ameliorating pain, preferably for use in ameliorating joint pain after exercise. The present invention furthermore relates to a method of preparing said composition by hydrolysis of a collagen-containing material comprising different types of collagen. The composition is preferably administered as a food supplement.

Claims

1. A method for ameliorating joint pain in a subject after exercise, comprising: administering a composition to the subject, wherein the composition comprises 10-40 wt. % chondroitin sulfate and 60-90 wt. % collagen hydrolysate, calculated on the total weight of the chondroitin sulfate and the collagen hydrolysate, and wherein the collagen hydrolysate is derived from the hydrolysis of a mixture of collagen comprising type I, type II, and type Ill collagen such that the collagen hydrolysate comprises hydroxyproline and hydroxylysine in a molar ratio (hydroxyproline:hydroxylysine) of more than 8 and less than 15.

2. The method according to claim 1, wherein the subject is a healthy individual.

3. The method according to claim 1, wherein the collagen hydrolysate comprises 2-20 wt. % hydroxyproline, wherein the wt. % is calculated on the total weight of the amino acids in the collagen hydrolysate.

4. The method according to claim 1, wherein the collagen hydrolysate comprises 0.1-5 wt. % hydroxylysine, wherein the wt. % is calculated on the total weight of the amino acids in the collagen hydrolysate.

5. The method according to claim 1, wherein the composition comprises 15-40 wt. %, or 20-40 wt. %, or 30-40 wt. % chondroitin sulfate, calculated on the total weight of the chondroitin sulfate and the collagen hydrolysate.

6. The method according to claim 1, wherein the collagen hydrolysate has an average molecular weight of more than 1000 Da and less than 10000 Da.

7. The method according to claim 1, wherein the composition is administered orally.

8. The method according to claim 1, wherein the composition is provided as a food supplement.

9. The method according to claim 1, wherein the composition is administered at a daily dose of 0.2-2 g, wherein the daily dose is the total dry weight amount of the composition.

Description

BRIEF DESCRIPTION OF FIGURES

(1) FIG. 1. Effect of composition based on collagen hydrolysate (HC) versus placebo on joint pain feel over a period of 16 weeks. Intake of the supplements was stopped after 12 weeks (represented by the vertical dashed line). The numbers in the plot corresponds with the number of subjects.

(2) FIG. 2. Effect of composition based on collagen hydrolysate (HC) versus placebo on joint pain by gender. The study period was 16 weeks. Intake of the supplements was stopped after 12 weeks (represented by the vertical dashed line). The number of participants throughout the study is depicted in the figure.

(3) FIG. 3. Effect of composition based on collagen hydrolysate (HC) versus placebo on joint pain by age category. The study period was 16 weeks. Intake of the supplements was stopped after 12 weeks (represented by the vertical dashed line). The number of participants throughout the study is depicted in the figure.

(4) FIG. 4. Effect of composition based on collagen hydrolysate (HC) versus placebo on joint pain according to physical activity impact. The study period was 16 weeks. Intake of the supplements was stopped after 12 weeks (represented by the vertical dashed line). The number of participants throughout the study is depicted in the figure.

EXAMPLES

Example 1

(5) The following protocol is a general protocol used to prepare a composition comprising collagen hydrolysate and chondroitin sulfate, starting with a mixture comprising porcine trachea and sternum tissue.

(6) The amount of type I, II, and Ill collagen in the tissues was established by ELISA. The relative amounts of trachea and sternum tissue were combined as to achieve a weight ratio of Type I, Type II, Type III collagen of 5:5:1 in the starting mixture.

(7) The general protocol involves the steps: a) Grinding of the collagen-containing material; b) Enzymatic hydrolysis (pH=6-7, T=60-65 C., t=5-6 h); c) First separation step and heat treatment (T=70-80 C.); d) Enzyme activation and heat treatment (T>90 C., t>15 min); e) Second separation step using one or more filtration sheets with a water permeability of 20-500 (l/min*m.sup.2 at 1 bar), a degree of separation of 0.2-10 (mK), and a weight per unit area of 1000-2000 (g/m.sup.2), and heat treatment (T=85-99 C.); f) Concentration under vacuum; g) Drying.

(8) Above protocol yields a composition comprising collagen hydrolysate and chondroitin sulfate with the general characteristics according to Table 1. The product is referred to as HC in Examples 2-4.

(9) Variations in the starting material and protocol can be applied to adjust the composition and bioactivity (as shown in Examples 3 and 4).

(10) TABLE-US-00001 TABLE 1 Product characteristics and nutritional information of the composition based on collagen hydrolysate and chondroitin sulfate (also referred to as HC in the Examples). AA = amino acids. Typical Product Characteristics Standard parameters Specifications Total collagen 55-70% Average Molecular Weight protein rich fraction 1200-3000 Da Total chondroitin sulfate 12-25% Average Molecular Weight chondroitin sulfate 17-27 kDa Viscosity (20%, 25 C.) 8-15 mPa .Math. s Loss on drying 8-10% Residue on ignition 8-10% Hydroxyproline (per protein content) 6-12 g/100 g protein Hydroxyproline (per amino acid content) 50-110 AA/1000 AA Hydroxylysine (per protein content) 0.5-2 g/100 g protein Hydroxylysine (per amino acid content) 2-10 AA/1000 AA Hydroxyproline/Hydroxyline molar ratio ~8.5-15 Typical nutritional information Standard parameters For 100 g Protein 65 g Fat 1 g Carbohydrates 18 g Fiber 0 g Sodium 1560 mg Converted to salt 3.9 g Vitamins 0 g Minerals Potassium 560 mg Calcium 130 mg Magnesium 40 mg Energy 1431 kJ

Example 2

(11) The aim of this study is to understand how the general consumer experiences a supplement based on collagen hydrolysate in terms of ameliorating joint pain. The supplement is prepared from porcine trachea and sternum according to Example 1. The supplement, herein denoted as HC, is provided by Rousselot BV (Ghent, Belgium).

Materials and Methods

(12) Study Population and Design

(13) This consumer lifestyle study is a placebo-controlled (ratio 5:1, 177 participants with HC and 36 with placebo), participant-blinded randomized parallel trial to evaluate the effect of collagen hydrolysate versus placebo on joint pain.

(14) In order to represent the general consumer who may seek a nutritional supplement to reduce joint pain, the research aimed to gather data on a wide number of people with diverse demographics. Participants were recruited via various sports and health clubs across the United Kingdom. In order to have significant relevant data, the researchers aimed to gather data from a minimum of 200 participants.

(15) Through a placebo-controlled design, the experiment was set-up for the participants to take either 20.5 g of HC (experiment group) or 20.5 g of maltodextrin (placebo group) on a daily basis, during a trial period of 12 weeks and 4 weeks without run-out period. Participants had to fill in the self-assessment tool for all 16 weeks. The HC and the placebo matched taste, color, odor characteristics. Similar packaging was used for both.

(16) All communication between researchers and participants was done via an online app in which they had to fill in their self-assessment and other observations.

(17) Inclusion criteria for the participants to be included in the study were 18 years of age, no medical diagnosis, not taking any medication, frequent physical activity and not pregnant or currently breast feeding. For physical activity, it was meant all levels of activity from gardening, brisk walking to marathon training.

(18) The exclusion criteria were defined as aged under 18 years, currently under a medical diagnosis, currently taking/prescribed medication, advised not to or does not frequently participate in physical activity and currently pregnant or breast feeding.

(19) If participants were already using any type of supplementation with Vitamin D, Curcumin/turmeric, other collagen, Green Mussel Extract, Boswellia, Glucosamine, Chondroitin Sulfate, Hyaluronic Acid or Folic Acid, they were excluded from the experiment as well.

(20) Participants that were already supplementing Multivitamin, Iron, Omega 3, Vitamin B complex, Vitamin C, Calcium, Rosehip extract, Amino acid complex or creatine were not excluded as these supplements are not specifically targeting joint health and the results could potentially show any relationship with HC.

(21) Materials

(22) The product used in this study is derived from hydrolyzed porcine trachea and sternum. The starting material was hydrolyzed (according to Example 1) to yield a composition with collagen hydrolysate and chondroitin sulfate (onwards referred to as HC). The typical product characteristics of the product are according to Example 1. The HC dosage form was in 0.5 g hydroxypropyl methylcellulose (HPMC) capsules, whilst 0.5 g of maltodextrin in HPMC capsules was used as placebo. Maltodextrin is a food grade material commonly used as a placebo in clinical trials. This product is a tasteless and odourless white powder that holds no micronutrient value and therefore could be used as placebo in this experiment.

(23) Questionnaire Design

(24) In order to have continuous follow-up with the participants, a smartphone app was developed for the participants to hand in their feedback on a weekly basis.

(25) This weekly reporting was composed of several questions. First, they were asked which physical activity they recently had completed and the duration of the activity. A pre-set of activities was provided with e.g. gardening, Brisk Walking, Running, Cycling, Tennis, Swimming, Strengthening sports, Weightlifting (average weight squat 70 kg), Flexibility/balance, Yoga, Pilates or an option box to fill out any unlisted activities. On duration, participants were asked to look at the total on weekly basis and then indicate the amount of time spent (0-5h, 6-10h, 11-15h, 16-20h or 20h or more).

(26) Next, they received a silhouette of a body diagram. Here they could indicate the area of experienced joint pain. Then participants were asked to give their feedback on joint pain using the validated pain measuring instrument Visual Analogue Scale (VAS) (Breivik et al., Br. J. Anaest. 2008 July; 101(1):17-24). The validated measure VAS, combined with a numeric rating system, enables the research to convert data into a pain scoring chart.

(27) The weekly reporting section is designed to make it very simple for participants to give in scores. The scoring tool that was used was chosen based on the accuracy demonstrated in previous similar studies to track subjective pain measures of participants (e.g. Hawker et al. Arthritis Care Res. (Hoboken). 2011 November; 63 Suppl 11:S240-52, Kumar et al. J. Sci. Food Agric. 2015 Mar. 15; 95(4):702-7). At the beginning of the study, each participant had to define their baseline scoring on joint pain. Each week, participants were asked to indicate their pain scores starting at week 1, throughout the trial until week 16.

(28) Participants also had to fill in a short demographic questionnaire asking for gender, age (in ranges), height, weight and waist measurement.

(29) The participants individual pain score portfolio was anonymous in line with the General Data Protection Regulation (EU GDPR).

(30) Weighting Matrix to Calculate Average Activity Impact

(31) To assess the average impact of physical activity, participants were categorized according to the intensity (low (L), moderate (M) and high (H)) and number of activities (1, 2, 3) using a weighting matrix. Walking, swimming, flexibility/balance, gardening, cycling, dancing and horse riding were considered activities of low joint impact. Weightlifting, gym cardio and boxing were considered activities of moderate joint impact. Running, ball sports, skipping and heavy weightlifting were considered activities of high joint impact. Activities' classification was based on the UK National Health Services (NHS) guidelines (Physical activity guidelines for older adults. Available online: https://www.nhs.uk/live-well/exercise/physical-activity-guidelines-older-adults/#moderate (accessed on 3 March)). Regarding the number of activities, LLL means that the participant practiced three different types of low intensity activities, LLM means that the participant practiced two different types of low intensity activities and one type of moderate intensity activity, and LLH means that the participant practiced two different types of low intensity activities and one type of high intensity activity. The other subgroups follow the same rationale. Given that only one participant fitted into category MHH (1 Moderate and 2 High) and would not have statistical relevance alone, this participant was considered in category 5 (LHH or MMH or MHH)).

(32) Statistical Analyses

(33) The Full Analysis population contained all participants eligible for the study, who had taken supplement at least once and who reported at least one weekly VAS measure post exercise.

(34) Continuous data were summarized by their mean, standard deviation (SD), and range. Categorical data were summarized by frequencies and percentages.

(35) The effect of treatment (HC vs. placebo) on changes in mean joint pain score during follow-up in comparison with baseline was studied in detail using generalized linear mixed-effects model (LME) with random intercept (bi) for each subject (each subject has its own intercept).

(36) Follow-up time was treated as a factor variable and since the intake of supplements was stopped at week 12, the analysis was based on the period from baseline until week 12.

(37) Full Model with Interaction:

(38) $Y_{\mathrm{ij}}=\left({}_0+b_i\right)+{}_1{\mathrm{Time}}_{\mathrm{ij}}+{}_2{\mathrm{Treatment}}_{\mathrm{ij}}+{}_3{\mathrm{Time}}_{\mathrm{ij}}{\mathrm{Treatment}}_{\mathrm{ij}}+{}_{\mathrm{ij}}$

(39) The interaction term represents the difference between the changes in the expected joint pain, comparing a subject from the HC group to a subject from the Placebo group for a given follow-up time. In case of a non-significant interaction term, it can be concluded that there was not enough evidence of differences between the treatment groups. A second analysis was then performed that includes only the main effects.

(40) Systematic departures from the normality assumptions were verified using QQ-plots and scatterplots of the residuals versus the predictions were used to check for systematic departures from the mean model.

(41) The LME analysis were performed using the Imne package (Pinheiro et al. 2021. nlme: Linear and Nonlinear Mixed Effects Models. R package version 3.1-153) in the R environment (R Core Team, 2019). All analyses were performed in R version 3.6.0. (R Core Team, 2019). A value of p<0.05 was considered statistically significant. No imputation of missing data is performed. The amount of missing data is presented in the tables wherever appropriate.

(42) Results

(43) Subjects Included in the Study

(44) In total, 213 subjects were enrolled in the study (HC/Placebo: 177/36). The drop-out rate was 19% (7/36) in the placebo group and 3% (5/177) in the HC group. Thus, the application tool that was used for follow-up was engaging for participants.

(45) Subject Characteristics

(46) Table 2 summarizes subject demographic and baseline characteristics overall and by treatment group. Subjects ranged in age from 18 to 72 years (mean age: 41 years), with 52.6% of the subjects being females.

(47) TABLE-US-00002 TABLE 2 Demographic characteristics of subjects included in the study Variable Total (N = 213) HC (N = 177) Placebo (N = 36) Gender Female 112 (52.6%) 94 (53.1%) 18 (50.0%) Male 101 (47.4%) 83 (46.9%) 18 (50.0%) Age (years) Mean (SD) 40.7 (11.0) 40.2 (11.0) 42.1 (11.0) Range 18-72 18-67 23 72 Age category <30 39 (18.3%) 35 (19.8%) 4 (11.1%) 30-39 59 (27.7%) 48 (27.1%) 11 (30.6%) 40-49 69 (32.4%) 57 (32.2%) 12 (33.3%) 50-55 27 (12.7%) 22 (12.4%) 5 (13.9%) 55+ 19 (8.9%) 15 (8.5%) 4 (11.1%)
Joint Pain Alleviation by HC Vs Placebo

(48) FIG. 1 shows the changes in the participants' joint pain over a period of 16 weeks. A linear mixed effects model revealed a significant interaction between treatment and time on treatment (p<0.001). The results suggest that there are differences between the placebo and HC group in terms of subject-specific changes in expected joint pain feel. Subjects that took HC showed a significant (p<0.001) reduction in mean joint pain feel starting from week 3 when compared to the subjects from the placebo group. Note also that the difference between the HC and placebo group increased with time on treatment.

(49) After 12 weeks, the treatment was stopped and the average joint pain score starts to increase again in the HC group (FIG. 1). At week 16, the average joint pain score for the HC group was still significantly lower compared to baseline (paired sample t-test, p<0.001) and there was also a significant difference between HC and the placebo group (independent sample t-test, p<0.001). A decrease in joint pain was sustained 6-8 weeks after stopping the treatment.

(50) FIG. 2 shows the effect of HC versus placebo on joint pain by gender. There was no evidence of a significant three-way interaction, and no interaction between gender and treatment and between gender and time (p>0.05). Furthermore, the LME model revealed that joint pain scores were statistically similar between genders (p=0.469).

(51) FIG. 3 shows the effect of HC versus placebo on joint pain by age category. The LME model revealed a significant interaction between age and time on treatment (p=0.005), highlighting that different age categories differ in their joint pain levels for HC treatment over time. Therefore, the pattern of joint pain alleviation among age categories for HC treatment was compared using as reference the participants with age <30 years, who reported the lowest joint pain scores among age categories. After 10 weeks on treatment, subjects between 40-49 years had on average higher joint pain scores compared to subjects <30 years (mean difference 0.71, 95% CI 0.25-1.18, p<0.01). This difference was recorded late in the study. Study participants below age 30 reported the lowest joint pain scores among age categories. Overall, all subgroups had joint pain levels significantly below the levels reported at the baseline of the study in all age categories.

(52) FIG. 4 shows the effect of HC versus placebo on joint pain according to physical activity level. The statistical analysis revealed that joint pain scores were consistent among the different subgroups of physical activity (p=0.091). The range of treatment differences suggests that the effect of HC treatment is consistent across different physical activity subgroups.

(53) Overall, a reduction in joint pain was seen following intake of HC, regardless of gender, age group and activity intensity. This indicates that HC intake has a positive effect on the general population, including active adults of different age groups and lifestyles. The general trend was that the reduction in joint pain is observed starting 3 weeks after the begin of HC intake, and is sustained for 6-8 weeks after stopping HC intake.

(54) The reduction in joint pain appears largest in subjects in the age category <30 years; these may be subjects which are most active and healthy.

(55) Table 3 shows the reduction in pain by HC in subjects experiencing different types of joint pain. The reduction in joint pain is seen regardless of the nature of the joint pain, but the effect is most pronounced in (healthy) subjects post-exercise.

(56) TABLE-US-00003 TABLE 3 Change in VAS score in subjects experiencing different types of joint pain, after daily oral administration with HC for 12 weeks Type of joint pain Reduction VAS score Joint pain in septic arthritis Lowest Joint pain in osteoarthritis Average/High Joint pain after exercise Highest

Example 3

(57) The method of Example 1 was used, wherein the comparison was made between porcine bone, porcine hide, porcine trachea, or porcine sternum as starting material for hydrolysis.

(58) Table 4 shows that daily treatment with a composition derived from porcine bone or porcine hide does not lead to a large reduction in joint pain after 12 weeks. In comparison, daily treatment with a collagen hydrolysate composition derived from porcine trachea or porcine sternum leads to more than 50% reduction in the VAS score after 12 weeks. Similar results were obtained with bovine-derived tissues. Chicken sternum was found to be less effective in reducing joint pain as compared to porcine or bovine sternum, indicating that there may be species-dependency in the pain-reducing effect.

(59) TABLE-US-00004 TABLE 4 VAS score after 12 weeks daily intake of HC derived from different collagen-containing materials. VAS score Placebo 4.5 HC derived from porcine bone 4.0 HC derived from porcine hide 4.0 HC derived from porcine trachea 2.0 HC derived from porcine sternum 2.0

Example 4

(60) The method of Example 1 was used, but using different combinations of porcine bone, hide, trachea, and sternum to obtain a starting material for hydrolysis with different relative amounts of collagen type I, II and III.

(61) Table 5 shows that a composition prepared from a starting material comprising predominantly type I or type II collagen has a pain-reducing effect, albeit limited. In comparison, a composition prepared from a starting material comprising type I, II, and Ill collagen in a weight ratio of 5:5:1 has a far greater pain-reducing effect. The collagen hydrolysate obtained from this starting material has a Hyp/Hyl molar ratio of more than 8 and less than 15.

(62) The pain-reducing effect is highest for a relatively high amount of chondroitin sulfate in the composition. In the current example, the highest reduction in pain is found for a composition comprising 70% collagen hydrolysate and 30% chondroitin sulfate.

(63) TABLE-US-00005 TABLE 5 Starting material, obtained composition and the effect on lowering pain. Type I:II:III Hyp/Hyl HC/CS (%) Reduction Group col starting in in VAS # material composition composition score 1. 1:5:1 4.0-8.5 82.5/17.5% Very low 2. 5:1:1 15-25 82.5/17.5% Very low 3. 5:5:1 8.5-15 82.5/17.5% High 4. 1:5:1 4.0-8.0 70/30% Low 5. 5:1:1 15-25 70/30% Low 6. 5:5:1 8.5-15 70/30% Highest HC: collagen hydrolysate, CS: chondroitin sulfate, Hyp/Hyl: molar ratio of hydroxyproline and hydroxylysine.

(64) Table 6 shows that type III collagen contributes in reducing pain. A ten-fold reduction type III collagen in the starting material leads to a lower pain-reducing effect.

(65) TABLE-US-00006 TABLE 6 Starting material, obtained composition and the effect on lowering pain. Type I:II:III col Hyp/Hyl HC/CS (%) Reduction Group # starting material output material in composition VAS score 1. 5:5:1 8.5-15 82.5/17.5% High 2. 5:5:0.1 7.0-14 82.5/17.5% Average 3. 5:5:1 8.5-15 70/30% Highest 4. 5:5:0.1 7.0-14 70/30% Average HC: collagen hydrolysate, CS: chondroitin sulfate, Hyp/Hyl: molar ratio of hydroxyproline and hydroxylysine.