WO2020227424A1

WO2020227424A1 - Omega-3 and eggshell membrane compositions, dosage forms, and methods of use

Info

Publication number: WO2020227424A1
Application number: PCT/US2020/031699
Authority: WO
Inventors: David Barnes
Original assignee: Marine Ingredients LLC
Current assignee: Marine Ingredients LLC
Priority date: 2019-05-06
Filing date: 2020-05-06
Publication date: 2020-11-12
Anticipated expiration: 2021-11-06

Abstract

Compositions and dosage forms that include eggshell membrane and omega fatty acids, as well as methods of using them in addressing musculoskeletal effects of exercise are described herein.

Description

OMEGA-3 AND EGGSHELL MEMBRANE COMPOSITIONS, DOSAGE FORMS, AND

METHODS OF USE

Related Applications

[0001] This application claims priority to United States Provisional Application No. 62/843,957 filed on May 6, 2019 and titled OMEGA-3 AND EGGSHELL MEMBRANE COMPOSITIONS, DOSAGE FORMS, AND METHODS OF USE” which is hereby incorporated herein by reference in its entirety.

Field of the Invention

[0002] The present disclosure relates generally to nutritional and medicinal compositions, more particularly to compositions of omega fatty acids and eggshell membrane, and their use in treating musculoskeletal conditions induced by physical activity.

Background

[0003] Musculoskeletal conditions are common throughout life, although their prevalence increases with age. They affect muscles, bones, joints, and associated tissues such as tendons, and ligaments. Musculoskeletal conditions are typically painful and frequently limit mobility, dexterity, and functional ability. The most disabling musculoskeletal conditions are associated with osteoarthritis (OA), rheumatoid arthritis (RA), back and neck pain, and bone fractures.

[0004] Although numerous studies have demonstrated the beneficial effects of exercise in relieving symptoms of OA and improving balance and joint flexibility, injuries from sports and exercise can induce sprains and strains and other injuries, especially when exercise is done too intensely, with too great a load, or too long a period. The discomfort often manifests as either pain or stiffness in joints. For both the novice and experienced athlete, delayed onset muscle soreness (DOMS) also may occur following unaccustomed physical activity or when individuals return to exercise after a period of reduced activity. Strenuous exercise can result in degradation of articular cartilage which, together with the resulting inflammatory response, contribute to joint discomfort. Due to the importance of exercise for overall health and disease prevention, there is a need to evaluate new treatment alternatives that can not only protect joints from potential detrimental effects of exercise but also reduce the resulting pain and stiffness.

Brief Description of the Drawings

[0005] FIG. 1 shows a protocol for selecting subjects in a study of an investigational formulation (ES-OM3) in accordance with an embodiment.

[0006] FIG. 2 shows graphs of the percent change in C-terminal crosslinked telopeptide of type-ll collagen in the urine (uCTX-ll) from resting in ES-OM3 and placebo subject groups after 1 week and 2 weeks of exercise. (*P < 0.05 versus placebo) [0007] FIG. 3 shows graphs of subject joint pain scores immediately after exercise and joint pain scores 12-hours post exercise in ES-OM3 and placebo subject groups during 2 weeks of exercise. (P < 0.05; # P < 0.10)

[0008] FIG. 4 shows graphs of subject joint stiffness scores immediately after exercise and joint stiffness scores 12-hours post exercise in ES-OM3 and placebo subject groups during 2 weeks of exercise. (* P < 0.05; # P < 0.10)

Detailed Description

[0009] The following detailed description of various embodiments is not intended to limit the scope of the present disclosure, but is merely representative of various embodiments. The present disclosure relates to medicinal compositions, and more particularly, to omega fatty acids and eggshell membrane compositions, dosage forms, and methods of use.

[0010] Recent studies have shown that eggshell membrane supplementation can reduce exercise-induced joint pain and stiffness and lower the urinary excretion of C-terminal cross- linked telopeptide of type-ll collagen (CTX-II), a marker of cartilage degradation. Eggshell membrane lies between the albumin and the calcified shell of the egg. It is characterized by its bi-layered, mesh-like structure formed by fibrous proteins such as collagen Type I. Eggshell membrane contains chondroitin sulfate, dermatan sulfate, hyaluronic acid, and other glycosaminoglycans (GAGs), and has been shown to reduce the expression of pro- inflammatory cytokines such as interleukin- 1 beta (I L-1 b) and tumor necrosis factor-alpha (TNF-a). It has been observed that eggshell membrane supplementation can reduce joint pain and stiffness in humans with OA, and reduce CTX-II levels in rat models of OA and RA, and in dogs with naturally-occurring joint disease.

[0011] The omega-3 (n-3) and omega-6 (n-6) long-chain polyunsaturated fatty acids (PUFAs) have been shown to play an important role in the hormonal regulation of bone formation. The omega-3 PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are found mainly in marine foods, particularly in cold water fish like salmon, sardines, and herring. The modern diet in many parts of the world, particularly the western hemisphere, is typically low in omega-3 PUFAs and high in omega-6 PUFAs. Diets having a high n-6 content (n-6 to n-3 ratio >15: 1) have been associated with numerous inflammatory disorders such as heart disease, diabetes, colitis, and RA.

[0012] PUFAs are essential fatty acids and precursors to eicosanoids such as prostaglandins, thromboxanes, leukotrienes, and resolvins. Omega-6 and omega-3 PUFAs each produce a different series of eicosanoids. The eicosanoids produced by omega-6 PUFAs tend to be more pro-inflammatory than those generated by omega-3 PUFAs. A product of omega-6 PUFAs, prostaglandin PGE2_, is a potent mediator and regulator of cartilage formation and resorption. At high levels, PGE2 increases the degradation of cartilage. At low levels, PGE2 stimulates bone formation and cartilage production by increasing insulin-like growth factor. Omega-3 PUFAs (EPA and DHA) mediate the degradation effects of high levels of PGE2. An omega-3 PUFA diet has been observed to reduce disease in OA-prone animals by increasing GAGs content. It has also been observed in animals fed omega-3 PUFAs that most cartilage and subchondral bone parameters were changed toward those seen in a non-pathological strain of animals.

[0013] DOMS frequently occurs following physical activity. The intensity of joint discomfort (e.g., stiffness) usually develops in a delayed fashion, increasing within the first 24 hours after exercise and peaks between 24 and 72 hours following exercise. Improving stiffness soon after exercise can be clinically meaningful, e.g., as stiffness is commonly used as an indirect measure of joint inflammation. There is strong evidence that eggshell membrane and particularly omega-3 PUFAs mediate the highly pro-inflammatory effects of certain cytokines, eicosanoids, and reactive oxygen species associated with inflammation. Thus, treatments and compositions utilizing a combination of these materials can play an important role in relieving localized joint stiffness and/or inflammation resulting from exercise.

[0014] The term“about,” when used with reference to ratios, amounts, or percentages of one or more elements of a composition, dosage form, or portion thereof, encompasses both the actual ratios, amounts, and percentages of the elements (as measured) and the ratios, amounts, and percentages after correction using standard correction methods.

[0015] The term "exercise" refers to physical activity, particularly activity that involves musculoskeletal movement including joint flexion and extension. Exercise includes physical activity specifically directed to obtaining various health benefits arising from said physical activity and engaged in for at least that purpose. Exercise also includes physical activity that may be attendant to other activities in which a subject is engaged, including but not limited to labor, personal transportation, and recreation.

[0016] Methods of realizing the potential benefits of omega PUFAs and eggshell membrane in addressing negative musculoskeletal effects of exercise are provided in the present disclosure. Also provided are compositions and dosage forms that can be employed to these ends.

[0017] In some embodiments, a composition comprises an eggshell membrane product and a marine oil comprising omega-3 PUFAs and omega-6 PUFAs. The eggshell membrane product can include naturally occurring eggshell membrane material obtained e.g., by separating the membrane from the shell and other components of an egg. The eggshell membrane product can include materials obtained after mechanical or chemical processing of harvested eggshell membrane. Methods of processing harvested eggshell membrane material are known in the art, and can involve mechanical processing steps including drying, grinding, and milling, and/or chemical processing steps including chemical hydrolysis and enzymatic hydrolysis. Examples of such eggshell membrane product include eggshell membrane mechanically reduced to a powder or to flakes, eggshell membrane isolates, and eggshell membrane hydrolyzates.

[0018] The composition can comprise an amount of the eggshell membrane product selected in view of the properties or benefits sought and based on the form of the eggshell membrane product. In some embodiments, the composition comprises eggshell membrane product in an amount from about 15 wt% to about 35 wt%. In particular embodiments, the amount is from about 20 wt% to about 30 wt%, or from about 15 wt% to about 25 wt%, or from about 25 wt% to about 35 wt%. In an embodiment, the amount of eggshell membrane product is about 25 wt% of the composition.

[0019] As indicated above, the composition further comprises a marine oil, which can be derived from one or more marine organisms having a PUFA content suitable for the applications described herein, including, but not limited to, fish and cephalopods. In some embodiments, the marine oil comprises a blend of oils from two or more marine organisms. In certain embodiments, the marine oil includes fish oil.

[0020] In some embodiments, the marine oil includes omega PUFAs, particularly omega-3 PUFAs and omega-6 PUFAs. In an aspect, the marine oil can be selected to provide particular absolute or relative concentrations of omega-3 and omega-6 PUFAs to the composition. In a particular aspect, the marine oil can be selected to provide a omega-3 PUFAs in a higher concentration relative to omega-6 PUFAs. In some embodiments, the weight ratio of omega- 3 PUFAs to omega-6 PUFAs in the composition is from about 3: 1 to about 5: 1. In more particular embodiments, the weight ratio of omega-3 PUFAs to omega-6 PUFAs in the composition is from about 3.5: 1 to about 4.5: 1 , or more particularly about 4: 1.

[0021] In some embodiments, omega-3 PUFAs can include EPA and DHA. In an aspect, the marine oil can be selected to provide particular absolute or relative concentrations of EPA and DHA to the composition. In some embodiments, the marine oil is relatively rich in EPA as compared to DHA. In other embodiments, the marine oil is relatively rich in DHA. In some embodiments, a ratio of EPA to DHA in the composition is from about 5:1 to about 1 :5. In certain embodiments, the ratio of EPA to DHA is from about 5:1 to about 1.5: 1 , or from about 3.5: 1 to about 2: 1 , or from about 5: 1 to about 3: 1 , or from about 4:1 to about 1.5: 1. In other embodiments, the ratio of EPA to DHA is from about 1 : 1.5 to about 1 :5, or from about 1 :2 to about 1 :3.5, or from about 1 :3 to about 1 :5, or from about 1 :1.5 to about 1 :4. In an embodiment, the ratio of EPA to DHA is about 4: 1.

[0022] The present disclosure also encompasses dosage forms based on the components described above and incorporating various aspects thereof. In some embodiments, a dosage form comprises an eggshell membrane product and a marine oil comprising omega-3 PUFAs and omega-6 PUFAs, wherein the eggshell membrane product is present in a selected amount. In some embodiments, the dosage form includes eggshell membrane product in an amount from about 150 mg to about 350 mg. In particular embodiments, the amount of eggshell membrane product is from about 200 mg to about 300 mg, or from about 150 mg to about 250 mg, or from about 250 mg to about 350 mg. In an embodiment, the dosage form comprises about 250 mg.

[0023] The dosage forms of the present disclosure further comprise a marine oil comprising omega-3 PUFAs and omega-6 PUFAs. In some embodiments, the dosage form comprises an amount of omega-3 PUFAs from about 500 mg to about 700 mg. In particular embodiments the amount of omega-3 PUFAs is from about 550 mg to about 650 mg, or from about 500 mg to about 600 mg, or from about 600 mg to about 700 mg. In an embodiment, the amount of omega-3 PUFAs is about 600 mg.

[0024] In some embodiments, the omega-3 PUFAs include EPA and DHA. In an aspect, the marine oil can be selected to provide particular absolute or relative amounts of EPA and DHA. In some embodiments, the marine oil is relatively more abundant in EPA as compared to DHA. In some embodiments, the dosage form comprises EPA in an amount from about 250 mg to about 450 mg. In particular embodiments, the amount of EPA is from about 300 mg to about 400 mg, or from about 250 mg to about 350 mg, or from about 350 mg to about 450 mg. In an embodiment, the amount of EPA present in the dosage form is about 360 mg. In some embodiments, the dosage form comprises DHA in an amount from about 150 mg to about 300 mg. In particular embodiments, the amount of DHA is from about 150 mg to about 250 mg, or from about 200 mg to about 300 mg, or from about 200 mg to about 250 mg. In an embodiment, the amount of DHA present in the dosage form is about 240 mg. In other embodiments, the marine oil is relatively abundant in DHA.

[0025] Dosage forms encompassed by the present disclosure include various forms suitable for oral administration. In some embodiments, the dosage form is a liquid formulation. In some embodiments, the dosage form is a discrete dosage form composed to facilitate delivery of a unit dose of one or more of the agents described herein. In certain embodiments, the dosage form is a capsule. In particular embodiments the dosage form is a liquid-filled capsule. In various embodiments, the dosage form is selected to provide a particular amount of the active agents in a single administration. Depending on the dosing regime, the dosage form can be selected to provide a desired daily dose in a single administration, or alternatively facilitate provision of a desired daily dose by plural administration.

[0026] In various embodiments, the compositions or dosage forms described herein can comprise one or more additional agents suitable for inclusion in a nutritional preparation including, but not limited to, vitamins and minerals. In various embodiments, the compositions or dosage forms described herein can comprise one or more excipients including, but not limited to, diluents, disintegrants, lubricants, glidants, odorants, flavors, and colorants. [0027] The present disclosure encompasses methods of mitigating potential negative effects of exercise on a subject's health or comfort. In some embodiments, a method of enhancing a subject's post-exercise recovery comprises administering to the subject a dose of an eggshell membrane product and a dose of a marine oil comprising omega-3 PUFAs and omega-6 PUFAs. In some embodiments, a method of reducing delayed onset muscle soreness in a subject comprises administering to the subject a dose of an eggshell membrane product and a dose of a marine oil comprising omega-3 PUFAs and omega-6 PUFAs.

[0028] The present disclosure encompasses methods of reducing post-exercise pain and stiffness. In some embodiments, methods of reducing post-exercise joint pain and methods of reducing post-exercise joint stiffness comprise administering a dose of an eggshell membrane product and a dose of a marine oil comprising omega-3 PUFAs and omega-6 PUFAs. It is common for exercise-induced joint pain and/or stiffness to exhibit a time course in which the onset of pain and/or stiffness may occur some time after the commencement--or even completion--of the exercise, and the pain and/or stiffness may continue for a period of hours or days. During this period the pain and/or stiffness may increase for some time before beginning to diminish. It has been found that administration of agents, compositions and dosage forms described herein can reduce the level of joint pain and/or joint stiffness a subject experiences shortly after exercise, and further can reduce the level of joint pain and or joint stiffness a subject experiences a number of hours after exercise. Accordingly in some embodiments, a method comprising administration of eggshell membrane product and marine oil comprising omega-3 PUFAs and omega-6 PUFAs to a subject is used to reduce the level of joint stiffness and/or joint pain the subject experiences shortly after exercise, such as less than about one hour after exercise. In some embodiments, the method is used to reduce the level of joint stiffness and/or joint pain the subject experiences later post-exercise, such as from about 8 hours to about 24 hours after exercise.

[0029] The present disclosure encompasses methods of reducing the effects of exercise- induced wear on cartilage. In some embodiments, a method of reducing cartilage turnover in a subject comprises administering to the subject a dose of an eggshell membrane product and a dose of a marine oil comprising omega-3 PUFAs and omega-6 PUFAs. As discussed above, cartilage degradation can be indicated by the presence of certain biomarkers in a subject's tissues or fluids. One such biomarker of cartilage degradation is CTX-I I , which can be detected in the serum or urine of a subject and the level of which is correlative of cartilage degradation. In some embodiments, a method of reducing production of CTX-II in a subject comprises administering to the subject a dose of an eggshell membrane product and a dose of a marine oil comprising omega-3 PUFAs and omega-6 PUFAs.

[0030] As discussed above, omega PUFAs are involved in the natural cycle of immune response to exercise. While not wishing to be bound by a particular theory, it is believed that this immune response is implicated in post-exercise discomfort. In some embodiments, a method of modulating an inflammatory response to exercise in a subject comprises administering to the subject a dose of an eggshell membrane product and a dose of a marine oil comprising omega-3 PUFAs and omega-6 PUFAs.

[0031] The methods described herein encompass various modes of administration of the active agents to a subject, which include the use of various regimens involving formulations and forms of the agents and dosing schemes selected to bring about a contemplated therapeutic effect. In some embodiments, the dose of eggshell membrane product and dose of marine oil are each administered to the subject via separate formulations or dosage forms. In other embodiments, the eggshell membrane product and marine oil are formulated into a single composition or dosage form such as are described above. In some embodiments, the dose of eggshell membrane product administered to the subject per day is from about 400 mg to about 600 mg. In a particular embodiment, the dose of eggshell membrane product is about 500 mg/day. In some embodiments, the dose of marine oil administered to the subject per day is from about 1200 mg to about 1700 mg. In a particular embodiment, the dose of marine oil is about 1500 mg/day. In various embodiments, the agents are administered to the subject from one to four times per day. It will be understood that an effective dose of the agents may depend upon other factors including, but not limited to, body mass, activity level, exercise intensity, and baseline musculoskeletal health, and the present disclosure encompasses dosing regimens and forms to achieve desired outcomes in view of such factors.

Examples

Example 1.

[0032] A randomized, double-blind, placebo-controlled study was conducted to evaluate the ability of a formulation comprising a combination of eggshell membrane product (NEM^®, ESM Technologies, LLC, Carthage, Missouri, USA) and fish oil concentrate (EPA and DHA [kd- pur^®], KD Pharma, Germany; called“ES-OM3” herein) versus placebo to reduce cartilage turnover (as determined by CTX-II) and alleviate joint pain or stiffness immediately following exercise and 12 hours post exercise in healthy adults.

Study design and population

[0033] This was a single-center, randomized, double-blind, placebo-controlled, parallel-group trial (NCT 04215198) conducted in accordance with the United States Food and Drug Administration’s principles of Good Clinical Practice (Title 21 , U.S. Code of Federal Regulations, Parts 50 and 56 and ICH E6) and the Helsinki Declaration of 1975 as revised in 1983. Clinical procedures were conducted in a single clinical site located in Springfield, Missouri. The study was approved by the QPS Bio-Kinetic Clinical Applications Investigational Review Board (Springfield, Missouri, USA). Written informed consent was obtained from each subject. [0034] One-hundred-forty-three (143) male and female subjects from Missouri were screened to participate in the study. Inclusion criteria were: aged between 40-75 years without a diagnosis of joint or connective tissue disorder of the lower extremities (i.e. , ankles, knees, or hips); no persistent lower-extremity joint pain at rest, but could have experienced mild periodic lower-extremity joint pain rated £ 2 on an 1 1-point ordinal pain scale (0 = no pain, 10 = worst pain imaginable); sufficiently healthy to perform moderate exercise (e.g., using an aerobics step platform) as judged by a medical examination including vital signs (i.e. resting heart rate, blood pressure) and an electrocardiogram (ECG). Subjects who were currently taking analgesic or related medications were eligible to participate following a 14-day washout period for non-steroidal anti-inflammatory drugs (NSAIDs), a 7-day washout period for opioids, and a 90-day washout period for injected steroids or anti-depressants. Subjects currently taking dietary supplements for joints such as glucosamine, chondroitin sulfate, curcumin, Boswellia, etc. were eligible after a 3-month washout period. Exclusion criteria were: any continued use of prescriptions or over-the-counter (OTC) pain relief medications including NSAIDs, analgesics, opioids, and anti-depressants prescribed for painful conditions such as fibromyalgia; taking remission-inducing drugs such as methotrexate or immunosuppressive medications or had received them within the past 3 months. Subjects were also excluded if they were currently receiving or planning to receive blood-thinning drugs during the study period; had a confounding inflammatory disease or condition (gout, pseudo gout, lupus, Paget’s disease, chronic pain syndrome, etc.) that would interfere with assessment of lower- extremity joint pain; participated in activities involving intensive use of the lower extremities (i.e. running, jogging, sports, bicycling, dancing, etc.) two or more days per week or participated in activities that involved moderate use of the lower extremities (i.e. walking, golfing, yoga, etc.) three or more days per week. Other exclusionary criteria were a body weight ³ 275 pounds (125 kg) and a known allergy to eggs/egg products or fish/fish products, or being pregnant or breastfeeding. Subjects previously enrolled in a research study involving an investigational product (drug, device, or biologic) or a new application of an approved product within 30 days of screening were also not eligible to participate.

[0035] At screening, using a 9-inch tall aerobics step platform, subjects performed 40 steps per leg, increasing by ten at a time per leg up to 100 steps per leg, until they experienced at least a one-unit change in discomfort (either pain or stiffness) from their resting rating. This number of steps was then assigned to that subject for the remainder of the study.

[0036] After satisfying all inclusion/exclusion criteria at screening, eligible subjects were then randomized to receive either ES-OM3 or placebo using a permuted block randomization table consisting of four subjects per block. Eighty-five (85) subjects between the ages of 40 and 70 years were eligible to participate (n = 85) in the study and were randomly assigned to either the ES-OM3 treatment group (n = 43) or placebo (n = 42). All but one subject (assigned to the ES-OM3 group) completed the study as per the protocol. The subject who dropped out developed choledocholithiasis just prior to the final exercise visit. The protocol flow diagram is shown in FIG. 1.

[0037] Treatment consisted of two daily oral capsules containing a total of either 2,000 mg of ES-OM3 (500 mg of eggshell membrane + 1 ,500 mg fish oil concentrate containing 1 ,200 mg EPA and DHA) or 2,000 mg placebo. Treatment compliance was checked during clinic visits by an interview conducted by study personnel and by counting the number of unused study capsules. Acetaminophen was allowed for pain relief rescue, if necessary. Subjects recorded the time and amount of acetaminophen taken in subject diaries. All participants, clinical staff, and study management staff remained blinded to treatment assignment throughout the study. Clinical procedures

[0038] The aim of the study was to evaluate whether ES-OM3 would reduce cartilage turnover (via the CTX-II biomarker) or alleviate joint pain or stiffness, either directly following exercise or 12 hours post exercise, versus placebo. An 1 1 -point ordinal scale (0 = no pain or stiffness, 10 = worst pain or stiffness imaginable) was used to assess the amount of pain/stiffness experienced at baseline and at rest before beginning the exercise regimen and after each exercise event.

[0039] Subjects followed their assigned exercise regimen at the clinical site on alternating days for 2 consecutive weeks (i.e. Sunday, Tuesday, Thursday, Saturday, Monday, Wednesday, Friday). Resting (before exercise) pain and stiffness scores were recorded in a questionnaire. The questionnaire consisted of recording a resting score, an assessment of pain/stiffness immediately after exercise (just after and up to 1 hour post exercise), and an approximately 12-hour post exercise assessment. Subjects provided blood and urine samples (from the 2^nd void in the morning) at the clinical site about 24 hours after each weekly exercise period, i.e., on Friday of Week 1 and on Saturday of Week 2.

[0040] The primary endpoint was any statistically significant change from baseline in the urinary CTX-II biomarker (uCTX-ll) (corrected for creatinine) between treatment groups after 1 week and 2 weeks of exercise. Secondary endpoints were any statistically significant changes from baseline in either exercise-induced joint pain or stiffness (immediately after exercise and 12 hours post exercise) between treatment groups as recorded in the subject questionnaire.

[0041] Safety assessments included physical examinations, adverse event (AE) monitoring, assessment of tolerability of investigational products, and changes in vital signs. Subjects underwent a thorough medical examination at screening and upon study completion by a licensed physician. A complete medical history, vital signs (resting heart rate, blood pressure, respirations, and temperature), and ECG were recorded. Subject diaries were reviewed during each clinic visit. Any discomfort beyond what would normally be expected with exercise, tolerability problems, or any other AEs were recorded and reported in accordance with applicable regulations.

Formulation and administration

[0042] ES-OM3 consisted of NEM^® (provided by ESM Technologies, LLC; Carthage, MO) USA), which is derived from eggshell membrane, and kd-pur^®fish oil concentrate (provided by KD Pharma, Germany, Lot #1709055), that contained ³480 mg of EPA and 320 mg DHA per gram in the form of triglycerides to form ES-OM3. Two #18 oblong softgel capsules, brown in color, contained a total 500 mg of NEM^® plus 1 ,500 mg of kd-pur^® (720 mg of EPA and 480 mg DHA). The placebo consisted of two #18 oblong softgel capsules, brown in color, that contained 2,000 mg of corn oil. Treatment and placebo softgel capsules were identical in appearance, odor, and taste and were stored in closed containers at ambient temperature. Subjects were instructed to take two softgels daily with water, approximately the same time each morning prior to eating breakfast.

Assessment of urinary CTX-II (uCTX-ll)

[0043] Analysis of uCTX-ll was performed using a known methodology and is briefly summarized here. Concentrations of uCTX-ll were measured via enzyme-linked immunosorbent assay (ELISA) using a commercial immunoassay (Urine CartiLaps® EIA; Immunodiagnostic Systems, Inc.; Gaithersburg, MD). Urinary creatinine (Cr) was measured via a colorimetric assay (Creatinine Urinary Colorimetric Assay Kit; Cayman Chemical Company, Inc.; Ann Arbor, Ml) using a SpectraMax Plus 384 microplate reader (Molecular Devices, LLC; Sunnyvale, CA). uCTX-ll concentrations were subsequently normalized by dividing uCTX-ll expressed in micrograms per liter ^g/L) by Cr expressed in millimoles per liter (mmol/L), the results of which were reported as nanograms of uCTX-ll per millimole of Cr (ng/mmol Cr). Samples were assayed in duplicate and all assays were repeated (n = 4). Duplicate assay values that were within 90% agreement were subsequently averaged. Repeated assay means that were within 90% agreement were further averaged. If repeat assay means differed by more than 10%, an additional assay was performed in duplicate with the remaining frozen aliquot of urine. The additional repeat assay mean was either substituted for the original outlier, or if sufficient agreement wasn’t reached with either original repeat assay mean, then all three were averaged. About 33% of the uCTX-ll assays were repeated three times, primarily due to inter-assay variability. Overall, intra-assay coefficients of variation were 5.32 and 1.90 for uCTX-ll and Cr, respectively.

Sample size calculation and statistical analysis

[0044] The hypothesis for this study was that the ES-OM3 treatment group would be superior to that of the placebo group in limiting the increase in uCTX-ll levels resulting from moderate exercise. A 17% absolute change in the mean treatment response (i.e., uCTX-ll would increase by an average of at least 17% more in the placebo group than in the ES-OM3 treatment group) was expected based upon results from the previously published study using NEM^® alone [3] A sample size of 84 subjects was needed to achieve a statistical power of 80% to detect a clinically meaningful difference between treatment groups, assuming a rate of response of -8% for the ES-OM3 treatment group and a rate of response of +9% for the placebo group, with no dropouts.

[0045] Descriptive statistics were calculated for mean age, height, weight, body mass index (BMI), and number of steps per leg. Kruskal-Wallis test for multiple independent samples was used to compare treatment group baseline data and to validate randomization. Following evaluation for normality (D’Agostino-Pearson), to determine the overall trend, post-baseline between-group statistical analyses were completed using either an independent group t-test (for uCTX-ll) or repeated measures univariate analysis of variance (rm-ANOVA) for levels of pain and stiffness. Items found to have statistical significance with rm-ANOVA were then compared using a Kruskal-Wallis test for multiple independent samples. Post-baseline within- group statistical analyses were completed using either a paired sample t-test (level of stiffness immediately after exercise) or a paired-sample Wilcoxon test (levels of pain and stiffness immediately after exercise and levels of pain and stiffness 12 hours post exercise). Absolute Treatment Effects (TEabs) were calculated for each outcome value as the net difference between ES-OM3 versus placebo for the mean change (as a percent) from baseline (or resting) to end of Week 1 and Week 2. Negative values for percent differences within groups and between groups and TE_abs values represent superior results for the treatment group. Selected statistically significant TE_abs values are presented in the results section. All statistical tests used a significance level of p < 0.05. Analysis of the primary and all secondary endpoints was conducted on the per protocol population. MedCalc^® Software (version 18.1 1.3) was used for the statistical analyses.

Results

[0046] Demographic data for the treatment groups, including mean pain scores at baseline/resting, are provided in Table 1. The compliance rate, determined from capsule count, was 99.8% (601/602) in the ES-OM3 group and 99.8% (587/588) in the placebo group. Two subjects in the placebo group were unable to provide urine samples. One subject in the placebo group underwent a colonoscopy near the end of Week 1 and was given fentanyl and benzodiazepine (Versed^®); therefore, the subject’s data collected just prior to the colonoscopy were carried forward for the remainder of the study. There were no statistically significant differences between treatment groups in any demographic or baseline parameter (see Table 1)· ES-OM3 Placebo

(n = 43) (n = 42)

Age (years) 55.3 ± 7.6 54.5 ± 8.3

Sex

Female (%) 31 (72) 31 (74)

Male (%) 12 (28) 11 (26)

Height (cm) 167 ± 13 168 ± 7

Weight (kg) 84.7 ± 21.1 84.0 ± 18.6

BMI (kg/m²) 29.9 ± 6.6 29.7 ± 6.5 uCTX-ll, ng/mmol Cr 315 ± 185 273 ± 212

Resting Pain Score 0.6 ± 0.8 0.9 ± 0.8

Resting Stiffness Score 1.0 ± 1.1 1.3 ± 1.2

Number of Steps per Leg 47.9 ± 12.8 45.0 ± 10.9 Baseline Pain Score 2.2 ± 1.3 2.4 ± 1.3

Baseline Stiffness Score 2.1 ± 1.5 2.5 ± 1.7

Table 1. Participant baseline demographic data. Except where indicated otherwise, values are reported as mean ± standard deviation. BMI: body mass index, uCTX-ll·. urinary c-terminal crosslinked telopeptide of type-ll collagen.

[0047] Table 2 shows the mean scores and within-group differences for each of the outcome measures (uCTX-ll, pain, and stiffness) at baseline/resting and after 1 and 2 weeks of exercise. Within-group statistically significant changes (improvements) from baseline in the ES-OM3 treatment group were observed for uCTX-ll at 2 weeks (-12.4%, p = 0.009). No significant within-group differences were observed for uCTX-ll in the placebo group. For the ES-OM3 group, within-group statistically significant changes (improvements) were also observed for pain experienced immediately after exercise at 1 week (-31.8%, p = 0.012) and at 2 weeks (-40.9%, p < 0.001), and for stiffness experienced immediately after exercise at 2 weeks (-32.0%, p = 0.016) (Table 2). For the placebo group, within-group statistically significant changes (improvements) from baseline were observed for pain experienced immediately after exercise at 2 weeks (-25.0%, p= 0.029) and for stiffness experienced immediately after exercise at 2 weeks (-28.6%, p = 0.014).

[0048] Large between-group (placebo versus ES-OM3) statistically significant changes (improvements) were observed for pain immediately after exercise (-28.6%, p = 0.049) at 1 week, pain 12 hours after exercise (-46.7%, p = 0.035) at 1 week, and stiffness 12 hours after exercise at 1 week (-40%, p = 0.042). [0049] As measured by TE_abs, for the primary outcome measure (uCTX-ll), supplementation with ES-OM3 produced a significant absolute treatment effect versus placebo after 1 (TE_abs - 12.9%, p = 0.035) and 2 weeks of exercise (TE_abs -17.7%, p = 0.019) (see FIG. 2).

Weeks Treatment

Post-treatment ES-OM3 (%) Placebo (%)

uCTX-ll Resting (n = 43, 40) 315 ± 185 273 ± 212

1 (n = 43, 40) 296 ± 193 (-6.0) 292 ± 228 (+7.0)

2 (n = 43, 40) 276 ± 175 (-12.4)^* 288 ± 269 (+5.5)

Immediate Pain Baseline (n = 43, 42) 2.2 ± 1 .3 2.4 ± 1 .3

1 (n = 43, 42) 1 .5 ± 1 .5 (-31 .8)^* 2.1 ± 1 .5 (-12.5)

2 (n = 43, 42) 1 .3 ± 1 .4 (-40.9)^** 1 .8 ± 1 .5 (-25.0)^*

12-hour Pain Resting (n = 43, 42) 0.6 ± 0.8 0.9 ± 0.8

1 (n = 43, 42) 0.8 ± 1 .2 (+33.3) 1 .5 ± 1 .7 (+66.7)

2 (n = 43, 42) 0.6 ± 0.9 (0.0) 1 .0 ± 1 .5 (+1 1 .1)

Immediate Baseline (n = 43, 42) 2.1 ± 1 .5 2.5 ± 1 .7

stiffness

1 (n = 43, 42) 1 .7 ± 1 .4 (-19.0) 2.0 ± 1 .5 (-20.0)

2 (n = 43, 42) 1 .5 ± 1 .4 (-28.6)^* 1 .7 ± 1 .7 (-32.0)^*

12-hour Stiffness Resting (n = 43, 42) 1 .0 ± 1 .1 1 .3 ± 1 .2

1 (n = 43, 42) 0.9 ± 1 .1 (-10.0) 1 .5 ± 1 .6 (+15.4)

2 (n = 43, 42) 0.7 ± 0.9 (-30.0) 1 .1 ± 1 .4 (-15.4)

Table 2 Mean uCTX-ll, pain and stiffness scores immediately after exercise and 12-hours post exercise for the ES-OM3 treatment and placebo groups at baseline (or resting) and after 1 and 2 weeks of exercise. Except where indicated otherwise, values are reported as mean ± standard deviation. ^*P < 0.05, **P <0.01 , within-group from resting or baseline, percent difference in parentheses.

[0050] Trend data indicated that significant differences between treatment groups for level of pain immediately after exercise occurred on Days 3, 5 and 7 (FIG. 3A). However, the overall trend for pain immediately after exercise in the ES-OM3 group versus placebo fell just shy of statistical significance (p = 0.065) (FIG. 3A). The overall trend for pain 12 hours post exercise (recovery) in the ES-OM3 group was significantly different from placebo (p = 0.019); significant differences in levels of pain 12 hours post exercise occurred on Days 2 and 8 (FIG. 3B). [0051] The overall trend for stiffness immediately after exercise in the ES-OM3 group was not significantly different from placebo (p = 0.442) (Fig. 4A), but a significant difference in the level of stiffness immediately after exercise occurred on Day 3 (p < 0.05). The overall trend for stiffness 12 hours post exercise in the ES-OM3 group was significantly different from placebo (p = 0.031); significant differences in levels of stiffness 12 hours post exercise occurred on Days 2, 4, 6 and 8 (FIG. 4B). Both treatment groups experienced less stiffness 12 hours post exercise (recovery) as the study progressed. However, the ES-OM3 treatment group showed a greater benefit (Day 14, TE_abs -11.8%).

[0052] The investigational formulations were well tolerated by all study participants. There were fifteen (15) non-exercise-related AEs reported in the ES-OM3 treatment group, the most common of which were 6 instances of headache and 2 instances each of cold/flu/sinus congestion, and nausea. None of the ES-OM3 treatment group AEs were judged by the principal investigator to be associated with the investigational formulation. There were eighteen (18) non-exercise-related AEs reported in the placebo group, the most common of which were 7 instances of headache and 2 instances of cold/flu/sinus congestion. None of the placebo group AEs were judged to be associated with the investigational formulation. There was one serious adverse event (SAE) reported during the study in the ES-OM3 treatment group. A subject developed acute choledocholithiasis that required surgical intervention. This was deemed unrelated to the treatment by the principal investigator due to the patient’s medical history. There were 7 instances of acetaminophen use in the placebo group (avg. 10.4 mg/subject/day) and no instances of acetaminophen use in the ES-OM3 treatment group.

[0053] The use of a different population and study design may explain why the administration of ES-OM3 did not achieve statistical significance versus placebo for the overall trend for stiffness immediately after exercise. In this study, both men and women were considered. Men who, on average, are taller than women have a smaller knee flexion angle when performing the step exercise. The smaller knee flexion angle likely results in less knee strain. Post menopausal women may have cartilage that is more sensitive to strain than their pre menopausal counterparts. This is supported by the fact that the uCTX-ll has been reported to be about 2-fold higher in post-menopausal women compared with age-matched pre menopausal women. The present study included women irrespective of menopausal status. Another possible reason for the difference in results is that there were several non-responders: 5 subjects (3F, 2M) in the placebo group who experienced no stiffness immediately after performing the exercise regimen. This reduced the sample size of the placebo group by 12%. As a result, a statistically significant treatment effect was not achieved.

[0054] It will be apparent to those having skill in the art that many changes may be made to the details of the above-described embodiments without departing from the underlying principles of the invention. The scope of the present invention should, therefore, be determined only by the following claims.

Claims

1. A composition comprising:

an eggshell membrane product; and

a marine oil comprising omega-3 polyunsaturated fatty acids (PUFAs) and omega-6

PUFAs.

2. The composition of claim 1 , wherein the omega-3 PUFAs include eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).

3. The composition of claim 1 or claim 2, comprising the eggshell membrane product in an amount from about 15 wt% to about 35 wt%.

4. The composition of any one of claims 1-3, comprising the marine oil in an amount from about 50 wt% to about 85 wt%.

5. The composition of claim 4, comprising a total amount of EPA and DHA from about 30 wt% to about 75 wt%.

6. The composition of claim 5, wherein the total amount of EPA and DHA is from about 55 wt% to about 65 wt%.

7. The composition of any one of claims 1-6, wherein the eggshell membrane product comprises one or more of naturally occurring eggshell membrane material, an eggshell membrane isolate, and an eggshell membrane hydrolyzate.

8. The composition of any one of claims 1-7, wherein a weight ratio of omega-3 PUFAs to omega-6 PUFAs is from about 3:1 to about 5:1.

9. A dosage form comprising the composition of any one of claims 1-8.

10. A dosage form comprising:

an eggshell membrane product; and

a marine oil comprising omega-3 polyunsaturated fatty acids (PUFAs) and omega-6

PUFAs,

wherein the eggshell membrane product is present in an amount from about 150 mg to about 350 mg.

11. The dosage form of claim 10, wherein omega-3 PUFAs are present in an amount from about 500 mg to about 700 mg.

12. The dosage form of claim 10 or claim 11 , wherein the omega-3 PUFAs include eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).

13. The dosage form of claim 12, comprising EPA in an amount from about 250 mg to about 450 mg.

14. The dosage form of claim 12 or claim 13, comprising DHA in an amount from about 200 mg to about 300 mg.

15. The dosage form of any one of claims 10-14, wherein omega-6 PUFAs are present in an amount from about 50 mg to about 200 mg.

16. The dosage form of any one of claims 10-15, wherein the amount of eggshell membrane product is from about 200 mg to about 300 mg

17. The dosage form of any one of claims 9-16, further comprising one or more additional agents selected from vitamins, minerals, and combinations thereof.

18. The dosage form of any one of claims 9-17, wherein the dosage form is a liquid.

19. The dosage form of any one of claims 9-18, wherein the dosage form is a capsule.

20. The dosage form of any one of claims 9-19, wherein the dosage form is a liquid-filled capsule.

21. A method of enhancing post-exercise recovery in a subject, comprising administering to the subject a dose of an eggshell membrane product and a dose of a marine oil comprising omega-3 polyunsaturated fatty acids (PUFAs) and omega-6 PUFAs.

22. A method of reducing delayed onset muscle soreness in a subject, comprising administering to the subject a dose of an eggshell membrane product and a dose of a marine oil comprising omega-3 polyunsaturated fatty acids (PUFAs) and omega-6 PUFAs.

23. A method of reducing a level of post-exercise joint pain in a subject, comprising administering to the subject a dose of an eggshell membrane product and a dose of a marine oil comprising omega-3 polyunsaturated fatty acids (PUFAs) and omega-6 PUFAs.

24. The method of claim 23, wherein the level of post-exercise joint pain is present less than about one hour after exercise.

25. The method of claim 23, wherein the level of post-exercise joint pain is present from about 8 hours to about 24 hours after exercise.

26. A method of reducing a level of post-exercise joint stiffness in a subject, comprising administering to the subject a dose of an eggshell membrane product and a dose of a marine oil comprising omega-3 polyunsaturated fatty acids (PUFAs) and omega-6 PUFAs.

27. The method of claim 26, wherein the level of post-exercise joint stiffness is present less than about one hour after exercise.

28. The method of claim 26, wherein the level of post-exercise joint pain is present from about 8 hours to about 24 hours after exercise.

29. A method of reducing cartilage turnover in a subject, comprising administering to the subject a dose of an eggshell membrane product and a dose of a marine oil comprising omega-3 polyunsaturated fatty acids (PUFAs) and omega-6 PUFAs.

30. A method of modulating an inflammatory response to exercise in a subject, comprising administering to the subject a dose of an eggshell membrane product and a dose of a marine oil comprising omega-3 polyunsaturated fatty acids (PUFAs) and omega-6 PUFAs.

31. A method of reducing production of C-terminal cross-linked telopeptide of type II collagen (CTX-II) in a subject, comprising administering to the subject a dose of an eggshell membrane product and a dose of a marine oil comprising omega-3 polyunsaturated fatty acids (PUFAs) and omega-6 PUFAs.

32. The method of any one of claims 21-31 , wherein the dose of eggshell membrane product is from 400 mg/day to about 600 mg/day.

33. The method of any one of claims 21-31 , wherein the dose of marine oil is from about 1200 mg/day to about 1700 mg/day.

34. The method of any one of claims 21-31 , comprising administering the composition of any one of claims 1-8 or the dosage form of any one of claims 9-20.

35. Use of the composition of any one of claims 1-8 or the dosage form of any one of claims 9-20 for the manufacture of a medicament for enhancing post-exercise recovery in a subject, reducing delayed onset muscle soreness in a subject, reducing a level of post exercise joint pain in a subject, reducing a level of post-exercise joint stiffness in a subject, reducing cartilage turnover in a subject, modulating an inflammatory response to exercise in a subject, or reducing production of C-terminal cross-linked telopeptide of type II collagen (CTX-II) in a subject.

36. A composition of any one of claims 1-8 or dosage form of any one of claims 9-20 for use in enhancing post-exercise recovery in a subject, reducing delayed onset muscle soreness in a subject, reducing a level of post-exercise joint pain in a subject, reducing a level of post-exercise joint stiffness in a subject, reducing cartilage turnover in a subject, modulating an inflammatory response to exercise in a subject, or reducing production of C-terminal cross-linked telopeptide of type II collagen (CTX-II) in a subject.