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An Overview of a Novel, Water-Soluble Undenatured Type II Collagen (NEXT-II) a

a

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Orie Yoshinari PhD, FACN , Hiroyoshi Moriyama PhD, FACN & Yoshiaki Shiojima MS a

Development Division, Ryusendo Co. Ltd., Tokyo, JAPAN Published online: 09 Mar 2015.

Click for updates To cite this article: Orie Yoshinari PhD, FACN, Hiroyoshi Moriyama PhD, FACN & Yoshiaki Shiojima MS (2015) An Overview of a Novel, Water-Soluble Undenatured Type II Collagen (NEXT-II), Journal of the American College of Nutrition, 34:3, 255-262, DOI: 10.1080/07315724.2014.919541 To link to this article: http://dx.doi.org/10.1080/07315724.2014.919541

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An Overview of a Novel, Water-Soluble Undenatured Type II Collagen (NEXT-II) Orie Yoshinari, PhD, FACN, Hiroyoshi Moriyama, PhD, FACN, Yoshiaki Shiojima, MS Development Division, Ryusendo Co. Ltd., Tokyo, JAPAN

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Key words: Type II collagen, osteoarthritis, safety Background: Osteoarthritis, the most common form of arthritis, is a crippling, chronic debilitating bone disease that commonly affects humans, dogs, and horses. Inflammation and inflammatory responses are key factors for causing swelling, redness, pain, and loss of movement in arthritic animals and humans. Methods and Results: We developed a novel, water-soluble, undenatured type II collagen (NEXT-II) for osteoarthritis. NEXT-II demonstrated broad-spectrum safety and nonmutagenicity. NEXT-II exhibited significant efficacy in ameliorating pain and inflammation in collagen-induced arthritis in mice. NEXT-II enhanced the proportion of CD4+CD25+T cells, and gene expressions of stimulated dendritic cells induced markers for regulatory T cell such as forkhead box p3, transforming growth factor-b1, and CD25. Furthermore, NEXT-II was assessed in moderately arthritic dogs receiving either placebo or 10 mg NEXT-II over a period of 150 days. NEXT-II exhibited a significant reduction in overall pain, pain after limb manipulation, and pain after physical exertion compared to the control dogs. Physical health and serum chemistry (alanine aminotransferase, blood urea nitrogen, and creatine kinase) were not altered when these arthritic dogs were treated over a period of 150 days. Conclusions: These results demonstrate the broad-spectrum safety and efficacy of NEXT-II in ameliorating the symptoms of arthritis. Key Teaching Points:  A novel, water-soluble, undenatured type II collagen (NEXT-II) was developed for osteoarthritis.  The safety studies including acute oral and dermal toxicity, primary dermal and primary eye irritation, Ames’ bacterial reverse mutation assay, mouse lymphoma assay, and 150-day long-term safety studies were conducted.  NEXT-II exhibited significant efficacy in ameliorating pain and inflammation in collagen-induced arthritis in mice.  NEXT-II exhibited a significant reduction in overall pain in moderately arthritic dogs without changing physical parameters.

INTRODUCTION

pain and joint inflammation. Cartilage breakdown occurs because of a chronic condition in which the bones rub against each other, leading to a gradual and progressive impairment of joint structures and collagen degradation, causing joint stiffness, pain, degeneration of the bone, a dramatic change in the texture of synovial fluid, and swelling and ultimately leads to loss of mobility [2,3]. In OA patients, the synovial fluid breaks down and the joint space becomes depolymerized and fragmented. The synovial fluid

Osteoarthritis (OA) is an inflammatory disorder that is due to advancing age and wear and tear on a joint. OA may result from obesity, advancing age, and sedentary lifestyle or autoimmune inflammation. OA causes limited movement and a decrease in the quality of life in humans and animals and leads to disability and long-term absence from work [1]. OA causes breakdown of the cartilage and buildup of synovial fluid, which results in

Address correspondence to: Orie Yoshinari, PhD, FACN, Ryusendo Co. Ltd., 1-5-3 Nishi-ikebukuro, Toshima-ku, Tokyo 171-0021, JAPAN. E-mail: [email protected] Abbreviations: ALP D alkaline phosphatase, ALT D alanine aminotransferase, BUN D blood urea nitrogen, CIA D collagen-induced arthritis, CK D creatine kinase, Foxp3 D forkhead box p3, TGF-b1 D transforming growth factor-b1, C II D type II collagen.

Journal of the American College of Nutrition, Vol. 34, No. 3, 255–262 (2015) Ó American College of Nutrition Published by Taylor & Francis Group, LLC 255

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Arthritis and Type II Collagen (NEXT-II) in OA sufferers becomes less viscous, and the shockabsorbing and filtering abilities are reduced [4]. As a result, the coat over the joint surface breaks down, leaving the cartilage exposed to mechanical and inflammatory injury. Thus, the cartilage in the joint gradually wears away and is ultimately destroyed. The synovial membrane becomes inflamed and causes more pain and mobility is hindered [5]. The American College of Rheumatology describes OA as a heterogeneous group of conditions characterized by degeneration of articular cartilage and changes in the underlying bone at the joints. Approximately 46 million people suffer from arthritis resulting in disability in the United States alone and it costs the health care industry a huge amount [6]. This number is expected to rise to 67 million by 2030 [7], which is a 46% increase due to increase in obesity, sedentary lifestyle, lack of exercise, and increase longer life span [8]. Though osteoarthritis is the most common form of arthritis, there are several other types of arthritis, including juvenile arthritis, rheumatoid arthritis, gout, viral arthritis, psoriatic arthritis, etc. It is well known that dogs often develop arthritis, especially OA, due to advancing age, lack of walking and exercise, overweight leading to obesity [9], compromised immune disorders, and accidental injury. Hielm-Bj€orkman et al. demonstrated that certain breeds of dogs are genetically predisposed to developing arthritis [10]. Considerable medical advances in recent years have been made, but there is limited treatment for OA. Nonsteroidal antiinflammatory drugs target the amelioration of pain and inflammation, but unfortunately many of these exert some adverse effects due to toxicity with little amelioration of the pain and inflammation. Furthermore, pharmaceutical therapies are very expensive [11]. Several adverse effects have been reported, including renal failure, nephrotoxicity, gastrointestinal bleeding, congestive heart failure, and elevated blood pressure [12,13]. Certain COX-2 inhibitors and nonsteroidal antiinflammatory drugs have been withdrawn from the market due to their serious adverse effects. Thus, novel complementary therapies are warranted [14]. Alternative and complementary therapies have become essential and hence the development of a safe and efficacious water-soluble collagen was required. NEXT-II is a unique, water-soluble, undenatured type-II collagen. It was developed in our laboratory to ameliorate arthritis therapy and used in all of the studies reported here. We conducted extensive shortand long-term safety (150 days) studies including mutagenicity assessment, mechanistic assessments, and in vivo rat and dog studies to demonstrate the safety and efficacy of NEXT-II [15]. The efficacy of NEXT-II was assessed in collagen-induced arthritis (CIA) in DBA/1J mice. The rat study demonstrated that orally administered NEXT-II reduced the antibody response to type II collagen (CII), which clearly showed that orally administered NEXT-II is effective in CIA mice. NEXT-

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II also resulted in the reduction of arthritis in mice [16]. NEXT-II treatment was carried out over a period of 150 days in moderately arthritic dogs and significant benefits were obtained in ameliorating overall pain, pain after limb manipulation, and pain after physical exertion.

METHOD Preparation of NEXT-II This unique, water-soluble, undenatured CII (creme nonfibrous powder; NEXT-II) was manufactured by Ryusendo Co. Ltd. (Tokyo, Japan) and used in all evaluations [15]. Basically, sternum cartilages of commercially available chickens (Thailand, age 4–6 weeks) were sterilized in 5% sodium hypochlorite and pulverized using a food grinder. The ground cartilage was extracted with 0.05 M acetic acid and reaction initiated by adding 1.0% pepsin at 4 C for 60 hours under stirring conditions and filtered. The solution was evaporated under vacuum and diluted with deionized water. The presence of epitope in NEXT-II was evaluated by using a commercial kit (Native Type II Collagen Capture Kit, Astarte Biologics, LLC, WA) [15,16].

Safety and Toxicological Evaluation A battery of safety studies including acute oral and dermal toxicity, primary dermal and primary eye irritation, Ames’ bacterial reverse mutation assay, mouse lymphoma assay, and 150-day long-term safety studies were conducted at Eurofins/ Product Safety Laboratories (Dayton, NJ), INTOX Pvt Ltd. (Pune, India), and Bioservice Scientific Laboratories (Planegg, Germany) in compliance with the Good Laboratory Practices as defined in 21CFR58 by the U.S. Food and Drug Administration [17] and in accordance with the Organization for Economic Cooperation and Development guidelines for testing of chemicals [18]. Animals were cared for in accordance with the most recent Guide for the Care and Use of Laboratory Animals DHEW (National Institutes of Health). The safety of NEXT-II was extensively evaluated by conducting acute oral toxicity, acute dermal toxicity, primary skin irritation, primary eye irritation, and Ames’ bacterial reverse mutation assay, which demonstrated its broad-spectrum safety [15]. Long-term safety studies on dogs were carried out in 20 large breed dogs (age between 7 and 12 years) over a period of 150 days. Dogs were randomly assigned to placebo and NEXTII-treated groups (dose 40 mg/dog) and efficacy was evaluated at different time points over a period of 150 days. Furthermore, extensive blood chemistry was assessed, including blood urea nitrogen (BUN, a marker of nephrotoxicity), alanine aminotransferase (ALT, a marker of hepatotoxicity) and creatine kinase (CK, a marker of cardiotoxicity) at several time points including 150 days. No toxicity or adverse events were observed over a period of 150 days [15,16].

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Arthritis and Type II Collagen (NEXT-II)

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Efficacy of NEXT-II in Collagen-Induced Arthritis Mice The efficacy of NEXT-II was assessed in CIA mice [16]. All animal experiments were performed according to the Guidelines on Animal Experimentation of Showa Pharmaceutical University (Japan). DBA1/J mice (8 weeks old, Sankyo Labo Service, Tokyo, Japan) were divided into 2 groups, a normal group and collagen-induced group. Bovine C II was dissolved in 0.05 M acetic acid at 4 mg/ml and emulsified in an equal volume of Freund’s complete adjuvant and 100 ml of this emulsion was injected intradermally into the dorsal root of the tail of the collagen-induced group. This was regarded as day 0 of treatment. On day 21, the same booster injection was administered to the collagen-induced group. On day 39, the collagen-induced group was divided into two groups (CIA group: n D 8; NEXTII group: n D 9) and administered with saline only (for the CIA group or NEXT-II, 1 mg/kg, as undenatured CII level) up to day 48. Oral dose of NEXT-II was decided by measuring undenatured CII. The control group was treated with saline only as the CIA group from day 39 to day 48 [16]. Histopathology of the hind limbs was conducted to demonstrate the extent of damage and repair following NEXT-II treatment in the joint tissues using hematoxylin and eosin [19]. Anti-CII immunoglobulin G (IgG), interleukin (IL)-2, IL-6, and tumor necrosis factor-a (TNF-a) levels were measured in the serum of normal, CIA, and NEXT-II groups of mice using a commercially enzyme-linked immunosorbent assay kit [16]. Immunofluorescence flow cytometry was performed on spleen tissues for IL-10 detection [16]. Flow cytometric analysis was conducted on lymphocytes and stained for the presence of CD4+IL-10+T and CD4+CD25+T cells [16].

Efficacy of NEXT-II in Moderately Arthritic Dogs This study was done to determine whether NEXT-II can ameliorate arthritic symptoms in dogs, including inflammation, flexibility, and improvement of daily lifestyle following supplementation of NEXT-II (10 mg/day, as active undenatured C II level) over a period of 150 days. A total of 20 adult clientowned large breed dogs (body weight range: 18 to 34 kg, age between 7 and 12 years) was selected for this efficacy study based on signed consent from the owners. All necessary regulatory approval procedures regarding animal care and handling were strictly followed in this study [20]. Dogs were chosen for this long-term safety and efficacy study because dogs are monogastric and have a single chamber stomach similar to humans. Dogs were randomly assigned to the placebo and NEXT-II groups. Body weight, body temperature, heart rate, and respiration rate were assessed in both the control and NEXT-II-treated groups on 0, 30, 60, 90, 120 and 150 days of treatment. Body temperature was measured using a digital thermometer. Heart rate (beats/min) and respiration rate (breaths/min) were obtained using a stethoscope and a wrist watch. All methods of

JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION

evaluation of pain were strictly followed and approved by veterinarians. Blood samples were collected from both untreated controls and NEXT-II-treated groups on days 0, 30, 60, 90, 120, and 150 of treatment by jugular venipuncture using needles (22 gauge) and 12-ml syringes. Serum samples from the dogs were prepared and frozen at ¡80 C for analysis. Serum samples were analyzed for hepatic (alkaline phosphatase (ALP), ALT, and bilirubin), renal (creatinine and BUN), and cardiovascular (CK) functions. Overall pain was evaluated in each dog every 30 days, including day 0. Overall pain, pain from limb manipulation, and pain after physical exertion were assessed on days 0, 30, 60, 90, 120, and 150 of treatment. Overall pain was based on a scale from 1 to 10, with 1 being very slight pain, 5 being a moderate level of pain, and 10 being constant and severe pain (unable to stand or walk). Overall pain was evaluated by observing the following: (a) overall quality of gait, (b) effort exerted during movement, (c) rising from a sitting or lying position, (d) posture while standing, and (e) overall temperament and body language. Overall pain was also graded from vocalization. Pain from limb manipulation was assessed by flexing or extending the limbs, while evaluating for stiffness and popping within the limbs. Each dog involved in the study was evaluated for pain from limb manipulation each month. Pain was evaluated on a scale from 0 to 4, with 0 indicating no pain, 1 indicating mild pain, 2 indicating moderate pain, 3 indicating severe pain, and 4 indicating severe and constant pain. Each canine’s pain level was determined after flexing and extending each limb, examining joints for grinding/ popping, observing breathing, grunting, and vocalization. Pain from physical exertion was evaluated each month after jogging dogs for 3 minutes. Pain was evaluated on a scale from 0 to 4, with 0 indicating no pain, 1 indicating mild pain, 2 indicating moderate pain, 3 indicating severe pain, and 4 indicating severe and constant pain. Each dog was evaluated throughout exercise for exercise tolerance, lameness, and vocalization. Each dog was examined during and immediately after exertion by observing the dog’s willingness to jog, limping, and whimpering.

Statistical Analysis Data within groups were compared using a one-way analysis of variance (ANOVA), followed by comparison of the treated groups to control by Dunnett’s multiple comparisons test. Data were evaluated for homogeneity of variances and normality by the Bartlett test. Data that were considered significant by Bartlett’s test were further evaluated with a nonparametric method (Kruskal-Wallis or Dunn’s test). The nonparametric Kruskal-Wallis test was used to determine statistical significance of the differences in the arthritis index between groups. Other data were statistically analyzed

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Arthritis and Type II Collagen (NEXT-II) by one-way ANOVA. A post hoc analysis of significance was made by using Tukey’s test. p < 0.05 was considered significant for all data comparisons.

RESULTS Safety and Toxicological Evaluation

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Acute Oral and Dermal Toxicity A single oral dose of 5000 mg/kg body weight (b.w.) to female Sprague-Dawley rats did not cause any mortality and did not demonstrate any signs of gross toxicity or mortality. The acute oral LD50 of NEXT-II was found to be greater than 5000 mg/kg b.w. in rats. Acute dermal toxicity of NEXT-II was tested in male and female Sprague-Dawley rats following a single topical application. The acute dermal LD50 of NEXTII was found to be greater than 2000 mg/kg b.w. in both male and female rats. No adverse pharmacological effects or abnormal behavior were observed following dosing and during the observation period of 14 days posttreatment. All animals gained normal body weight and appeared active and healthy during the study. No gross abnormalities or pathological alterations were noted for any of the rats when necropsied at the conclusion of the 14-day observation period [15].

Primary Skin Irritation and Primary Eye Irritation Primary skin irritation was investigated in female New Zealand albino rabbits to evaluate irritation after a single topical application. The overall incidence and severity of irritation decreased with time. The mean primary dermal irritation index for NEXT-II was calculated to be 1.8 using Draize primary dermal irritation scoring criteria, thus classifying NEXT-II to be slightly irritating to the skin. All animals were free from dermal irritation within 24 hours and appeared active and healthy, and there were no signs of gross toxicity, adverse pharmacological effects, or abnormal behavior [15]. Primary eye irritation was tested in New Zealand albino rabbits from a single instillation via the ocular route in accordance with the Draize scale for scoring eye lesions and the Kay and Calandra scheme for classifying eye irritants [21,22]. All animals were free of ocular irritation within 48 hours. The maximum mean total score of NEXT-II was determined to be 7.3, classifying NEXT-II to be minimally irritating to the eye. All animals used in the study were active and healthy, and no signs of gross toxicity, adverse pharmacological effects, or any abnormal behaviors were noted [15].

Mutagenicity Test Ames’ Bacterial Reverse Mutation Assay The Salmonella typhimurium reverse mutation test (Ames test) [23] was carried out in compliance with Organization for

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Economic Cooperation and Development guidelines for testing of chemicals (No. 471, Section 4: Health effects) on conduct of bacterial reverse mutation assay, adopted July 21, 1997, and International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) draft consensus guideline S2 (R1) on genotoxicity testing. NEXT-II was evaluated in the 5 tester strains of Salmonella typhimurium, such as TA 1535, TA 97a, TA 98, TA 100, and TA 102 in the presence and absence of a metabolic activation system (S9) [24] and NEXT-II was used in triplicate cultures at doses of 5000, 1500, 500, 150, and 50 mg/plate. No significant increases in 5 of the colony tested strains following NEXT-II treatment. NEXT-II also did not cause gene mutation by base pair changes or frame shifts in the genome. The results demonstrate that NEXT-II is nonmutagenic and noncytotoxic [15].

Mutagenicity Test: Mouse Lymphoma Assay NEXT-II was assessed by in vitro mammalian cell gene mutation assay for its potential to induce mutation at the mouse lymphoma thymidine kinase locus (Thymidine Kinase Locus/ TK+/¡) using the mouse (Mus musculus) lymphoma cell line L5178Y. In experiment I with metabolic activation, the relative total growth (RTG) was 92.50% for the highest concentration (5000 mg/ml) evaluated. The highest concentration evaluated without metabolic activation was 5000 mg/ml with an RTG of 86.13%. In experiment II with metabolic activation, the RTG was 96.97% for the highest concentration (5000 mg/ml) evaluated. The highest concentration evaluated without metabolic activation was 5000 mg/ml with an RTG of 115.88%. No biologically relevant increases of mutants were found after treatment with NEXT-II (with or without metabolic activation) in both experiments I and II. No dose–response relationship was observed. Additionally, in experiments I and II colony size showed no clastogenic effects induced by NEXT-II. Thus, NEXT-II is considered to be nonmutagenic in the mouse lymphoma thymidine kinase locus using the cell line L5178Y [15].

Long-term Safety Studies in Dogs Compared to the control group, all safety parameters were evaluated from blood samples collected on days 0, 30, 60, 90, 120, and 150 to evaluate ALT, BUN, and CK. No significant differences were observed. Furthermore, body weight, heart rate, and respiration rate were evaluated in both groups on dats 0, 30, 60, 90, 120 and 150. There were no significant differences between the control and treated groups or between the treated groups at each time point over a period of 150 days. Thus, NEXT-II was well tolerated and no adverse events were reported [15].

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Arthritis and Type II Collagen (NEXT-II) Table 1. Effect of NEXT-II on Overall Pain in Moderately Arthritic Dogsa Days of Treatment Group

Day 0

Day 30

Day 60

Day 90

Day 120

Day 150

Control Treated

5.70 § 0.30 5.90 § 0.35

5.85 § 0.22 5.30 § 0.37

5.95 § 0.21 4.60 § 0.38*

5.93 § 0.20 3.60 § 0.43*

5.98 § 0.23 3.00 § 0.41*

6.25 § 0.20 2.70 § 0.37*

Each value represents mean § SEM (n D 10). Statistical significance was determined by analyzing the analysis of variance in conjunction with Tukey-Kramer test. *Significantly different compared to control (p < 0.05).

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Efficacy of NEXT-II in Collagen-Induced Arthritis Mice

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Histological Findings Mice in the CIA group demonstrated inflammatory cell infiltration similar to rheumatoid arthritis pathogenesis with pannus formation, cartilage degradation, and irregular joint space compared to the untreated control group. The NEXT-II group demonstrated limited synovial infiltration, well-defined articular cartilage, and improved joints. Furthermore, the histopathological findings demonstrated the anti-arthritic efficacy of NEXT-II as exhibited by less swelling and inflammation and improved joint integrity [16].

Changes in the Level of Serum Anti-CII IgG, IL-2, IL-6, and TNF-a Anti-CII IgG, IL-2, IL-6, and TNF-a levels were measured in the serum of normal, CIA, and NEXT-II groups of mice. Anti-CII IgG production in the NEXT-II group significantly decreased compared to that of the CIA group [16]. IL-2 levels in the NEXT-II group significantly increased compared to the CIA group [16], and IL-6 and TNF-a levels of the NEXT-II group were remarkably inhibited compared to the increased level in the CIA group.

Analysis of T Cells and Expression Levels of Transforming Growth Factor-b1, Forkhead box p3, and CD25 Immunofluorescence flow cytometry was performed on spleen tissues for IL-10 detection. Flow cytometric analysis was conducted on lymphocytes and stained for the presence of CD4+IL-10+T and CD4+CD25+T cells. The proportion of

CD4+IL-10+T cells in the CIA group was found to be lower compared to the normal control group, but these parameters in the NEXT-II group were increased compared to the CIA group. On the contrary, flow cytometric analysis demonstrated a significant decrease in CD4+CD25+T cells in CD4+T cells in the CIA mice compared to the normal control mice. However, administration of NEXT-II significantly increased the proportion of CD4+CD25+T cells in CD4+T cells. The expression levels of transforming growth factor (TGF)bforkhead box p3 (Foxp3), and CD25 in splenocytes were measured using primers specific to each gene and reverse transcription polymerase chain reaction. Administration of NEXTII significantly elevated the expression levels of these mRNAs in spleen lymphocytes compared to the CIA group, whereas the expression levels of TGF-b [16].

Efficacy of NEXT-II in Moderately Arthritic Dogs Overall Pain, Pain from Limb Manipulation, and Pain after Physical Exertion In overall pain assessment, dogs receiving placebo exhibited no significant change in arthritic conditions at any time point over the period of 150 days. In the NEXT-II group, overall pain reduced by approximately 10%, 22%, 39%, 49.2%, and 54.3% on days 30, 60, 90, 120, and 150 days of treatment, respectively (Table 1). Pain upon limb manipulation decreased by approximately 11%, 22%, 41.3%, 59.6%, and 65.2% on days 30, 60, 90, 120, and 150 of treatment, respectively, following treatment with NEXT-II (Table 2), whereas under these same conditions pain after physical exertion was reduced by approximately 8.3%, 19.6%, 42.5%, 61.3%, and 62.5% on days 30, 60, 90, 120, and

Table 2. Effect of NEXT-II on Pain after Limb Manipulation in Moderately Arthritic Dogsa Days of Treatment Group

Day 0

Day 30

Day 60

Day 90

Day 120

Day 150

Control Treated

2.45 § 0.14 2.30 § 0.13

2.46 § 0.12 2.05 § 0.11*

2.47 § 0.11 1.80 § 0.13*

2.45 § 0.11 1.35 § 0.18*

2.55 § 0.10 0.93 § 0.16*

2.61 § 0.10 0.80 § 0.17*

Each value represents mean § SEM (n D 10). Statistical significance was determined by analyzing the analysis of variance in conjunction with Tukey-Kramer test. *Significantly different compared to control (p < 0.05).

a

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Arthritis and Type II Collagen (NEXT-II) Table 3. Effect of NEXT-II on Pain after Physical Exertion in Moderately Arthritic Dogsa Days of Treatment Group

Day 0

Day 30

Day 60

Day 90

Day 120

Day 150

Control Treated

2.43 § 0.14 2.40 § 0.15

2.47 § 0.13 2.20 § 0.15

2.50 § 0.11 1.93 § 0.16*

2.57 § 0.11 1.38 § 0.18*

2.60 § 0.10 0.93 § 0.23*

2.74 § 0.09 0.90 § 0.21*

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a Each value represents mean § SEM (n D 10). Each value represents mean § SEM (n D 10). Statistical significance was determined by analyzing the analysis of variance in conjunction with Tukey-Kramer test. *Significantly different compared to control (p < 0.05).

150 of treatment, respectively (Table 3). Maximum reduction in overall pain, pain upon limb manipulation, and pain after physical exertion was observed at the end of 120 days of treatment (Tables 1–3). Dogs consuming NEXT-II (10 mg active/day) exhibited no signs of adverse effects. NEXT-II did not cause any significant change in body weight, body temperature ( C), heart rate (beats/min), or respiration rate (breaths/min) as well as overall health (Table 4). Furthermore, NEXT-II did not cause any significant changes in total blood chemistry or biomarkers of liver function (ALP, ALT, and bilirubin), renal (creatinine and BUN), and cardiovascular (CK) functions [15].

DISCUSSION In recent years, many nutraceuticals have been used to treat arthritis in humans and animals because they have little or no adverse effects. These popular nutraceuticals include avacado saponins, curcumin, bromelain, Boswellia serrata extract, fish oil enriched with omega-3, glucosamine hydrochloride, chondroitin sulfate, denatured and undenatured collagen, shark cartilage, red ginger extract, green lipid mussel, nobiletin, citrus fruit, pomegranate, genistein, soy protein, pycnogenol, Methyl Sulfonyl Metane (MSM), and many others [5,8].

A multicenter, randomized, double-blind placebo-controlled clinical trial was funded by National Institutes of Health to confirm the efficacy of CII. This research was also conducted to test the safety of CII with different dosage for 24 weeks [24]. Two hundred and seventy-four patients suffering from rheumatoid arthritis (RA) were enrolled in 6 different sites and randomized to receive placebo or CII in 4 different doses (0, 20, 100, 500, or 2500 mg/d). Clinical evaluation and assessment for efficacy were carried out at 0 (baseline), 2, 4, 8, 12, 16, 20, and 24 weeks. Safety parameters were evaluated by questionnaire and by observing adverse event. Blood was collected and safety parameters for kidney (BUN), CK (heart), and liver (ALT, AST). There were no significant changes in blood parameters, which demonstrated the safety of CII. Paulus and American College of Rheumatology (ACR) criteria for improvement in rheumatoid arthritis were used to determine the efficacy of CII treatment. Eighty-three percent of patients completed 24 weeks of treatment, which indicated that CII was well tolerated. A statistically significant change (30% reduction, p < 0.05) was noted with 20 mg/d treatment for swollen and tender joints compared to placebo. The safety profile of CII was excellent compared to placebo [25]. In another study, 503 RA patients were chosen to study the efficacy of chicken CII [26]. Patients received either 0.1 mg daily of chicken CII (n D 326) or 10 mg once a week of

Table 4. Effect of NEXT-II on Physical Parameters in Moderately Arthritic Dogsa Days of Treatment Group Body weight (kg) Control Treated Body temperature ( C) Control Treated Heart rate (beats/min) Control Treated Respiration rate (breaths/min) Control Treated a

Day 0

Day 30

Day 60

Day 90

Day 120

Day 150

25.5 § 0.7 26.6 § 1.4

25.3 § 0.7 26.7 § 1.4

25.6 § 0.7 27.0 § 1.6

26.1 § 0.6 26.8 § 1.5

25.8 § 0.7 26.9 § 1.5

26.0 § 0.7 27.0 § 1.3

38.6 § 0.4 38.5 § 0.5

38.6 § 0.4 38.5 § 0.5

38.7 § 0.1 38.5 § 0.4

101.5 § 0.2 38.3 § 0.4

101.7 § 0.1 38.4 § 0.6

101.6 § 0.1 38.5 § 0.5

114.0 § 4.8 114.4 § 5.2

118.9 § 2.3 124.8 § 7.3

113.4 § 3.5 127.8 § 7.9

113.9 § 4.2 128.6 § 10.1

116.2 § 1.7 139.4 § 7.5

115.0 § 2.2 124.4 § 8.0

57.5 § 5.1 89.0 § 18.2

55.5 § 4.9 65.6 § 17.8

54.5 § 7.5 105.2 § 24.2

71.0 § 8.3 92.6 § 22.1

65.5 § 7.0 106.4 § 24.2

60.7 § 6.8 91.8 § 21.6

Each value represents mean § SEM (n D 10).

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Arthritis and Type II Collagen (NEXT-II) methotrexate (n D 177) for 24 weeks. Each patient was evaluated for pain, morning stiffness, tender and swollen joint count, health assessment questionnaire, assessments by investigator and patient, erythrocyte sedimentation rate, and C-reactive protein at baseline (week 0) and at weeks 12 and 24. Rheumatoid factor was evaluated at weeks 0 and 24. A total of 454 subjects completed the study. There were decreases in pain, morning stiffness, and inflammation and the differences were statistically significant at 24 weeks of treatment. Significant improvements were observed with minor side effects. Thus, the authors indicated that chicken CII is safe and efficacious in RA [26]. In 2 other studies conducted on water-insoluble undenatured type II collagen, researchers demonstrated significant beneficial effects in osteoarthritic dogs and humans [27,28]. These studies also demonstrated the safety and efficacy of a combination of chondroitin and glucosamine in dogs and humans. The above studies encouraged us to develop this unique undenatured water-soluble CII with active epitope (NEXT-II), which demonstrated significant efficacy in CIA mice and moderately arthritic dogs. Broad-spectrum safety and toxicity studies of NEXT-II were conducted in several in vitro and in vivo models for short- and long-term periods of treatment. Acute oral LD50 of NEXT-II was found to be >5000 mg/kg b.w. in female rats, and acute dermal LD50 was found to be >2000 mg/kg b.w. in male and female rats. In the primary dermal irritation assay, NEXT-II was classified as slightly irritating to the skin, and NEXT-II was classified as minimally irritating to the eye in primary eye irritation study. There were no other signs of gross toxicity, adverse pharmacological effects, or abnormal behavior in any of these tests [16]. Ames’ bacterial reverse mutation assay using 7 strains of Salmonella typhimurium (TA 1535, TA 97a, TA 98, TA 100, and TA 102) was evaluated in the presence and absence of metabolic activation. NEXT-II did not induce mutagenic effects either with or without metabolic activation. Cell gene mutation assay in mouse lymphoma cells was conducted to test the mutagenic potential of NEXT-II in the L5178Y mouse lymphoma cell line. NEXT-II did not induce mutagenic effects either with or without metabolic activation. Thus, NEXT-II was classified as nonmutagenic [16]. Furthermore, long-term safety studies over a period of 150 days in dogs demonstrated no adverse events and no significant changes in the blood chemistries, body weight, heart rate, or respiration rate. Overall, these results confirmed the broad-spectrum safety of NEXT-II. In another study, a low dose of NEXT-II caused significant improvement in pain amelioration in CIA mice. In the histopathological study, the NEXT-II-treated group demonstrated well-kept articular cartilage, improved degenerative joints, and limited synovial infiltration. Administration of NEXT-II resulted in a reduction of RA symptoms in CIA mice in conjunction with decreased production of anti-IgG and IL-6, which

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resulted in enhanced secretion of IL-2. Furthermore, gene expression of TGF-b1, Foxp3, and CD25 in the NEXT-IItreated group significantly increased. From this study, it was concluded that oral administration of NEXT-II may induce CD4+CD25+T cells in ameliorating the signs of arthritis [16]. It has been well demonstrated that Treg cell (TGF-b, Foxp3, and CD-25) is related to autoimmune diseases, rheumatoid arthritis, and different types of arthritis. There are several publications demonstrating the association of arthritis with Treg cells with arthritis [29]. This study encouraged us to conduct a study in arthritic dogs, because dogs suffer from moderate to severe arthritis due to advancing age, obesity, lack of physical exercise, and joint degeneration, which is very similar to human arthritis. NEXTII treatment was carried out over a period of 150 days in dogs. NEXT-II exhibited significant efficacy in ameliorating overall pain, pain after limb manipulation, and pain after physical exertion. Significant improvement in arthritic conditions was noted after 90 day of treatment with NEXT-II (10 mg/day, as active undenatured C II level). The maximum efficacy was observed on 120 days of treatment. NEXT-II did not cause any adverse events as demonstrated by extensive blood chemistry analysis and physical assessments. In addition, NEXT-II has the effect with small quantity in comparison with other supplements; for example, glucosamine.

CONCLUSIONS These studies clearly emphasize that orally administered NEXT-II has broad-spectrum safety and is efficacious in ameliorating the symptoms of joint pain and arthritis. Furthermore, human clinical studies are warranted to demonstrate the safety and efficacy of NEXT-II in humans.

ACKNOWLEDGMENTS The safety studies were conducted in Eurofins/Product Safety Laboratories (Dayton, NJ), INTOX Pvt Ltd. (Pune, India), and Bioservice Scientific Laboratories (Planegg, Germany). Molecular and mechanistic studies were conducted in Showa Pharmaceutical University (Tokyo, Japan). Dog studies were conducted in the Veterinary Division of Murray State University (Murray, Kentucky).

FUNDING These studies were supported by a research grant from Ryusendo Co. Ltd., Japan.

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Received February 24, 2014; accepted April 25, 2014.

VOL. 34, NO. 3

An overview of a novel, water-soluble undenatured type II collagen (NEXT-II).

Osteoarthritis, the most common form of arthritis, is a crippling, chronic debilitating bone disease that commonly affects humans, dogs, and horses. I...
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