COMPOSITIONS AND METHODS FOR TREATING JOINT DISORDERS

RELATED APPLICATIONS

This application claims the benefit of priority to US application 60/928,727 filed May 1 1, 2007, which is hereby incorporated by reference in its entirety. FIELD OF THE INVENTION

The present invention relates to methods and compositions for treating joint disorders in vertebrates.

BACKGROUND

Arthritis is a collective term for a number of different conditions that cause pain, swelling and limited movement in joints and connective tissue throughout the body. The condition is usually chronic and progressive. The main symptoms of arthritis are joint pain, joint stiffness or inability to move a joint normally, and sometimes swelling that lasts more than two weeks. Most treatment programs include a combination of medication, exercise, rest, use of heat and cold, joint protection techniques, and sometimes surgery. The two most prevalent forms of arthritis are osteoarthritis (OA), and rheumatoid arthritis (RA). Osteoarthritis (OA, also known as degenerative arthritis or degenerative joint disease, and sometimes referred to as "arthrosis" or "osteoarthrosis") is a degenerative joint disease characterized by a breakdown of the joint's cartilage. Cartilage functions to cushion the ends of the bones at each joint. A breakdown of the cartilage causes the bones to rub against each other, causing pain and loss of movement. Osteoarthritis primarily affects hands and weight-bearing joints, such as the knee, hips, feet and back. Risk factors for osteoarthritis include advanced age, obesity, joint injury, and genetic disposition. Causes for osteoarthritis include an abnormal release of destructive enzymes from the cartilage cells themselves, and inherent defects in the way joints fit together. Conventional treatment of osteoarthritis focuses on decreasing pain and improving joint movement. Medications include aspirin, acetaminophen, ibuprofen and nonsteroidal anti-inflammatory drugs (hereinafter NSAEDs) for pain relief and inflammation reduction. Corticosteriod injection directly into affected joints in acute cases is also employed. Other noninvasive techniques commonly used to control osteoarthritis include heat/cold treatment, exercise, joint protection and weight control.

Rheumatoid arthritis (RA) is an inflammatory disease having many components, including an autoimmune disorder aspect. The autoimmune disorder aspect is generally characterized by inflammation of the membrane lining the joint resulting from an attack upon

the joint by the body's own immune system. The inflammation causes pain, stiffness, warmth, redness and swelling. The inflamed joint lining, called the synovium, can invade and damage surrounding bone and cartilage. The involved joint can lose shape and alignment, resulting in pain, loss of movement and possible destruction of the joint. Early symptoms of the disease include general fatigue, soreness, stiffness and aching. Pain usually occurs in the same joints on both sides of the body and will usually start in the hands or feet. Rheumatoid arthritis can also affect wrists, elbows, shoulders, neck, knees, hips and ankles. Other symptoms include lumps, called rheumatoid nodules, under the skin in areas subjected to pressure, such as the back of elbows. Conventional treatment of rheumatoid arthritis focuses on reducing swelling, relieving pain and stiffness, and maintaining normal joint function. Medications include NSAIDs for controlling inflammation, joint pain, stiffness and swelling. Disease-modifying drugs include low doses of prednisone, methotrexate, hydroxychloroquine, azulfidine, gold salt and cyclosporin, used alone or in combination. Some combination of exercise, rest, medication, joint protection, physical and occupational therapy, and surgery is also used to treat rheumatoid arthritis patients.

Joints Affected by Arthritis:

Synovial joints (or diarthroses, or diarthroidal joints) are the most common and most moveable type of joints in the body and are the joints most afflicted in arthritis sufferers. As with all other joints in the body, synovial joints achieve movement at the point of contact of the articulating bones by encasing the ends of the articulating bones with a cavity and the occupation of a fluid in that cavity which aids movement. The surfaces of the two articulating bones at the joint are covered in cartilage. The thickness of the cartilage varies with each joint, and sometimes may be of uneven thickness. Articular cartilage is multi- layered. A thin superficial layer provides a smooth surface for the two bones to slide against each other. Of all the layers, it has the highest concentration of collagen and the lowest concentration of proteoglycans, making it very resistant to shear stresses. Deeper than that is an intermediate layer, which is mechanically designed to absorb shocks and distribute the load efficiently. The deepest layer is highly calcified, and anchors the articular cartilage to the bone. In joints where the two surfaces do not fit snugly together, a meniscus or multiple folds of fibro-cartilage within the joint correct the fit, ensuring stability and the optimal distribution of load forces. The synovium is a membrane that covers all the non-cartilaginous surfaces within the joint capsule. It secretes synovial fluid into the joint, which nourishes and

lubricates the articular cartilage. The synovium is separated from the capsule by a layer of cellular tissue that contains blood vessels and nerves.

Synovial Fluid

Synovial fluid is a thick, stringy fluid found in the cavities of synovial joints. With its egg-like consistency (synovial comes from Latin for "egg"), synovial fluid reduces friction between the articular cartilage and other tissues in joints to lubricate and cushion them during movement.

The inner membrane of synovial joints is called the synovial membrane, which secretes synovial fluid into the joint cavity. This fluid forms a thin layer (approximately 50 micrometers) at the surface of cartilage, but also seeps into microcavities and irregularities in the articular cartilage surface, filling any empty space. The fluid within articular cartilage effectively serves as a synovial fluid reserve. During normal movements, the synovial fluid held within the cartilage is squeezed out mechanically (so-called weeping lubrication) to maintain a layer of fluid on the cartilage surface. Normal synovial fluid contains 3-4 mg/mL hyaluronan (hyaluronic acid), a polymer of disaccharides composed of D-glucuronic acid and D-N-acetylglucosamine joined by alternating beta- 1,4 and beta- 1,3 glycosidic bonds. Hyaluronan is synthesized by the synovial membrane and secreted into the joint cavity to increase the viscosity and elasticity of articular cartilages and to lubricate the surfaces between synovium and cartilage. Synovial fluid also contains lubricin secreted by synovial cells. It is chiefly responsible for so-called boundary-layer lubrication, which reduces friction between opposing surfaces of cartilage. There is also some evidence that it helps regulate synovial cell growth.

Damages to Joints Due to Arthritis

In disorders such as osteoarthritis, there is destruction of the joints (including synovial joints and synovial fluid) caused by the proteolytic degradation of collagen by collagenase. Collagenase belong to the superfamily of metalloproteinases (MP) or matrix metallproteinases (MMP). The MMP form a group of Zn dependent enzymes which are involved in biological degradation of the extracellular matrix (D. Yip et al., Investigational New Drugs 17 (1999), 387-399 and Michaelides et al., Current Pharmaceutical Design 5 (1999), 787-819). These MMPs are able to degrade fibrillary and non-fibrillary collagen, and proteoglycans, both of which represent important matrix constitutients. In particular, MMP plays an important part in the degradation of the joint matrix in many forms of arthritis.

A selected subgroup within MMP is formed, for example, by collagenases. Only collagenases are able to degrade native collagen, which exercises an important supporting function in the joints. A large number of different inhibitors of MMPs and of collagenases are known (EP 0 606 046; WO94/28889; WO96/27583). Unfortunately, these inhibitors are known for causing substantial side effects, which are manifested as musculoskeletal pain or arthralgias.

It is a longfelt need, therefore, to find effective compositions, particularly natural compositions which can achieve their therapeutic effect with few or no side effects, for the treatment of joint disorders without the side effects of musculoskeletal pain or arthralgias. Disadvantages of some current treatments

Oral glucosamine has been used for the treatment of osteoarthritis. It is thought that since glucosamine is a precursor for glycosaminoglycans, and glycosaminoglycans are a major component of joint cartilage, supplemental glucosamine may help to rebuild cartilage and treat arthritis. Glucosamine is often sold in combination with other supplements such as chondroitin sulfate and methylsulfonylmethane as being beneficial for arthritis sufferers.

The use of glucosamine and chondroitin sulfates has several disadvantages. Glucosamine and N-acetylglucosamine are currently produced by extraction and acid hydrolysis of chitin from shellfish waste. Shellfish derived glucosamine could be limited by the amount of raw material available and the product potentially carries the risk of shellfish protein contamination and potential adverse reaction in people who are allergic to shellfish. Another more significant concern has been that the extra glucosamine could contribute to diabetes by interfering with the normal regulation of the hexosamine biosynthesis pathway (Buse MG. "Hexosamines, insulin resistance, and the complications of diabetes: current status," Am J Physiol Endocrinol Metab, 2006 Jan; 290(1 ):E1-E8). Tests are being conducted to see if glucosamine has an adverse effect in obese patients, since this population may be particularly sensitive to any effects of glucosamine on insulin resistance (National Institutes of Health Clinical Center (CC), ClinicalTrials.gov Identifier: NCT00065377). In addition, one common source of chondroitin sulfates is derived from shark cartilage - a source which is of some controversy and which is harvested from a declining animal population.

The effectiveness of glucosamine and a combination of glucosamine and chondroitin sulfate have been disputed. Clinical studies on the effectiveness of glucosamine and a combination of glucosamine and chondroitin sulfate on arthritis have been inconclusive with some studies showing effectiveness and other studies showing no benefit over the

administration of placebos (Adams ME. "Hype about glucosamine," Lancet, 1999 JuI 31 ; 354(9176):353-4; McAlindon TE et al., "Glucosamine and Chondroitin for Treatment of Osteoarthritis: A Systematic Quality Assessment and Meta-analysis," JAMA, 2000; 283: 1469-1475; Hughes R, Carr A. "A randomized, double-blind, placebo-controlled trial of glucosamine sulphate as an analgesic in osteoarthritis of the knee," Rheumatology (Oxford), 2002 Mar; 41(3):279-84; Cibere J et al., "Randomized, double-blind, placebo-controlled glucosamine discontinuation trial in knee osteoarthritis," Arthritis Rheum. 2004 Oct 15; 51(5):738-45; Clegg DO et al., "Glucosamine, chondroitin sulfate, and the two in combination for painful knee osteoarthritis," New Engl J Med, 2006 Feb 23; 354(8):795- 808.).

In view of the current results with chondroitin sulfate, glucosamine, and a combination of the two, and in view of the potential negative side effect from the use of these supplements, various sources (e.g., Arthritis Foundation) have discouraged their use for children, pregnant women, diabetics, people on blood thinners, and people with shellfish allergies. Hence, there is a longfelt need for the production of a natural product for treatment of arthritis which does not involve chondroitin sulfate or glucosamine.

SUMMARY OF THE INVENTION

One aspect of the present invention provides methods for treating a joint disorder (e.g., joint inflammation or arthritis, including osteoarthritis and rheumatoid arthritis) in a patient, preferably a human patient, comprising administering to the patient a therapeutically effective amount of a composition comprising a proteolytic enzyme inhibitor, preferably proanthocyanidins, lysine, and one or more components selected from proline, hydroxyproline, collagen, collagen derivatives (e.g., collagen hydrolysates) and combinations thereof. In another aspect, the present invention provides compositions for treating or preventing a joint disorder (e.g., joint inflammation or arthritis, including osteoarthritis and rheumatoid arthritis) in a vertebrate, preferably a human, comprising a proteolytic enzyme inhibitor, preferably proanthocyanidins, lysine, and one or more components selected from proline, hydroxyproline, collagen, collagen derivatives (e.g., collagen hydrolysates) and combinations thereof. In some embodiments, compositions of the invention include compositions that are substantially free of glucosamine, as well as compositions that are substantially free of chondroitin sulfate or other products derived from shark cartilage. In some embodiments of the invention, the compositions do not contain any additional amino acids other than proline, hydroxyproline and lysine. In other embodiments of the invention,

the compositions do not contain any additional amino acids other than proline and lysine. Further, some embodiments of the invention are directed to oral and topical compositions which contain only three active ingredients: (1) proanthocyanidins, preferably in the form of Pycnogenol ^® , (2) proline and (3) lysine. It is contemplated that whenever appropriate, any embodiment of the present invention can be combined with one or more other embodiments of the present invention, even though the embodiments are described under different aspects of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses compositions and methods using compositions for the treatment of joint disorders. According to some embodiments, compositions of the present invention are orally administered compositions of protease inhibitors and amino acids which can serve as metabolic precursors for the therapy of joint and connective tissue disorders in vertebrates. The components of these compositions act synergistically to promote the repair and maintenance of connective tissue and to reduce inflammation in these tissues so that the degradation process in the joint tissues, including in the synovial joint and synovial fluid, is halted and repair may be initiated. Further these compositions suppress the autoimmune response which creates the inflammatory response and which further degrades tissue in the affected area.

Some embodiments of the present invention relate to compositions for treating joint disorders in vertebrates. According to some embodiments, such compositions comprise proanthocyanidins, proline/hydroxyproline and lysine - three ingredients that act synergistically to treat joint disorders, prevent degradation of joint tissue and to promote the natural repair of joint tissues.

Proanthocyanidins Proanthocyanidins (also known as oligomeric proanthocyanidin (OPC)) represent a group of plant polyphenols found in roots, barks and fruits with an astringent taste. Proanthocyanidins include the subgroups of procyanidins, prodelphinidins and propelargonidins. Proanthocyanidins are biopolymers composed of flavan subunits. Since the biopolymers are a mixture of different sizes comprising different amounts of subunits, the term is usually used in the plural form (proanthocyanidins) but it is also used in the singular form (proanthocyanidin). Procyanidins are composed of catechin and epicatechin units, also called monomeric procyanidins.

Proanthocyanidins are homogeneous or heterogeneous polymers consisting of the monomer units catechin or epicatechin, which are connected either by 4-8 or 4-6 linkages, to the effect that a great number of proanthocyanidin isomers exist. Typically, the procyanidin oligomers have a chain length of 2-12 monomer units. Proanthocyanidins are extracted from plant material by conventional methods using solvents like water, ethanol or acetone or fluid carbon dioxide. The extracts are purified by solvent/solvent extraction, ultra filtration or chromatographic procedures. The purified extracts are concentrated by solvent evaporation, freeze drying or spray drying.

Proanthocyanidins may be derived from herbal sources or produced by synthesis. Common sources of proanthocyanidins can be found in vegetable extracts, as for example in extracts of the bark of the maritime pine, cones of cypresses, cocoa beans, and grape seeds. It follows that proanthocyanidins-rich extracts may be prepared from these materials. A well- known product containing proanthocyanidins, which is available in trade as a food supplement under the name Pycnogenol ^® (Horphag Research, Switzerland) is an extract of the maritime pine bark (alternatively called Pinus pinaster or Pinus maritima, both terms refer to the same plant.). The Pycnogenol ^® food supplement contains approximately 70-80% (such as, for example, 70% to 75%) of proanthocyanidins and is a complex mixture of phenolic substances which includes other flavanols such as catechin, epicatechin and taxifolin. Pycnogenol ^® is the preferred protease inhibitor of the claimed invention because of the superior results obtained from Pycnogenol ^® when used in our clinical studies. While we do not know the reasons for the superior results when using Pycnogenol ^® , it is possible that the result is attributable to the unique combination (ratios) of proanthocyanidins, catechin and epicatechin in Pycnogenol ^® .

Proanthocyanidins possess a multitude of interesting and useful biochemical and pharmacological qualities. In particular, they are well known for their protective effect against aging associated chronic diseases, such as atherosclerosis and related cardiovascular events such as stroke or heart infarction. In addition, Pycnogenol ^® pine bark extract has been shown to stimulate endothelial nitric oxide synthase and to induce vasodilation (Fitzpatrick, D. F., Bing, B., Rohdewald, P., 1998). Besides proanthocyanidins and its monomeric unit catechin, Pycnogenol ^® food supplement contains taxifolin and a wide range of phenolic acids, e.g., free acids like p-hydroxybenzoic acid, protoacatechic acid, vanillic acid, caffeic acid and ferulic acid as well as glucosides and glucose esters.

Pycnogenol ^® extracts, containing proanthocyanidins, scavenge free radicals in vitro and in vivo. In the context of osteoarthritis treatment, its inhibition of matrix

metal loproteases (MMPs) is of great interest. For example, after intake of a proanthocyanidins extract (Pycnogenol ^® ), release of MMP from macrophage is inhibited, thus blocking the destructive activity of MMPs on cartilage. It is suggested that proanthocyanidins function as a protease inhibitor to stabilize degrading joint tissue, including synovial joint tissue and synovial fluids. Proanthocyanidins achieve this function through a number of mechanisms. Proanthocyanidins were shown to inhibit the body's proteolytic enzymes which break down joint tissue. In this specification, a proteolytic enzyme is an enzyme (e.g., protease) that breaks down non-living tissue. These enzymes include, at least, collagenase, elastase, glycosidases, hyaluronidase, and β-glycuronidase. These enzymes are involved in the destruction of the main structural components of joint tissue such as: collagen, elastin, and hyaluronic acid (Facino, et al, Arzneimittelforschung. 1994 May; 44(5):592-601).

The pine bark extract also inhibits in vitro the activation of NF-κB, the key element of inflammation. Plasma taken from volunteers following intake of Pycnogenol ^® inhibits significantly the activation of NF-κB in inflammatory cells ex vivo, blocking subsequent steps of the inflammatory reactions. Cyclooxygenases initiate the production of pain-producing prostaglandins. Plasma from human volunteers inhibits cyclooxygenases I and II following intake of Pycnogenol ^® . The sum of these antiinflammatory effects suggests that this special pine bark extract could have a positive effect in reducing symptoms of mild to moderate osteoarthritis.

In a range of clinical trials, unwanted effects of the pine bark extract, mainly mild gastrointestinal symptoms or dizziness, were transient in most cases. Pycnogenol ^® is recognized by the United States of America Food and Drug Administration (FDA) as having GRAS status (Generally Recognized As Safe). Proline

L-Proline is an amino acid that is essential for collagen synthesis. Collagen contains about 9% proline (Pro). Multiple prolines and/or hydroxyprolines in a row can create a polyproline helix, the predominant secondary structure in collagen. The hydroxylation of proline (or other additions of electron-withdrawing substituents such as fluorine) increases the conformational stability of collagen significantly. Hence, the hydroxylation of proline is a critical biochemical process for maintaining the connective tissue of higher organisms. Severe diseases such as scurvy can result from defects in this hydroxylation.

While proline is an amino acid in many proteins, the proline of compositions of the present invention should preferably be in amino acid form (i.e., not part of a protein or a

protein hydrolysate). Proline, in amino acid form, provides superior absorption when used topically or orally.

Lysine

Lysine is an essential amino acid, which means that it is essential to human health but cannot be manufactured by the body. For this reason, lysine must be obtained from food.

Lysine appears to help the body absorb and conserve calcium and it also plays an important role in the formation of collagen, a substance important for bones and connective tissues including skin, tendon, and cartilage. Conversely, lysine deficiency negatively affects calcium absorption and cartilage formation, other symptoms of lysine deficiency include kidney stones, fatigue, nausea, dizziness, loss of appetite, agitation, bloodshot eyes, slow growth, anemia, and reproductive disorders.

While lysine makes up only 3 or 4% of the total amino acid in collagen, it nevertheless serves an important function in the constitution of the cross-links between the molecules to build the fibrils and the fibers of collagen. Defects of lysine metabolism are responsible for heritable diseases of the connective tissue as types FV and IX of the Ehlers- Danlos syndrome, lathyrism, the Menkes kinky hair syndrome, Cutis-Laxa or the type II of osteogenesis imperfecta.

While lysine is an amino acid in many proteins, the lysine of compositions of the present invention should preferably be in amino acid form (i.e., not part of a protein or a protein hydrolysate). Lysine, in amino acid form, provides superior absorption when used topically or orally.

Compositions of the present invention

The use of proanthocyanidins, proline/hydroxyproline and lysine is superior to the direct use of chondroitin sulfate or glucosamine for a number of reasons. First, there is a high rate of allergic reactions when chondroitin sulfate or glucosamine is used, which causes undesirable immune reactions in a patients including redness, swelling, and discomfort. Further, it may be desirable to avoid medicinal collagen derived from cows, to avoid the risk of transmitting prion diseases like BSE. Disadvantages of chondroitin sulfate and glucosamine are discussed in the background section of this application. In some embodiments, compositions of the invention present a number of advantages over the prior art including (1) the compositions are substantially free of glucosamine and thus are free from the side effects of glucosamine, (2) the compositions are substantially free of chondroitin sulfate or other products derived from shark cartilage.

In some embodiment of the invention, the compositions do not contain any additional amino acids other than proline/hydroxyproline and lysine. It is our observation that compositions of the present invention are suitable for oral and/or topical administration when only two amino acids (lysine and proline — in either or both the hydroxylated or unhydroxylated forms) are added to proanthocyanidins. Thus, some embodiments of the invention are directed to oral and topical compositions which contain only three active ingredients: (1) proanthocyanidins, preferably in the form of Pycnogenol ^® , (2) proline and/or hydroxyproline and (3) lysine. Some embodiments of the invention are directed to oral and topical compositions which contain only three active ingredients: (1) Pycnogenol ^® , (2) proline and (3) lysine.

We surprisingly find that a combination of proanthocyanidins, proline/hydroxyproline and lysine to have synergistic effect that is superior to the effect of any one or any combination of two of the three ingredients.

The dosages listed in this specification are for the average person of about 80 kg, such as between 50 to 100 kilograms. For patients weighing more or less than the foregoing, it may be necessary to adjust the dosage accordingly, as is well known to one of skill in the art.

By an "effective" amount or a "therapeutically effective amount" of a drug or pharmacologically active agent is meant a nontoxic but sufficient amount of the drug or agent to provide the desired effect, e.g., reduction of a symptom associated with a joint disorder. An appropriate "effective" amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

The term "extract", as used herein includes any preparation obtained from plants, fruits or vegetables using an extraction method.

One aspect of the present invention is a method for treating a joint disorder in a patient, preferably a human patient, comprising administering to the patient a therapeutically effective amount of a composition comprising a proteolytic enzyme inhibitor, lysine and one or more components selected from proline, hydroxyproline, collagen, collagen derivatives, such as collagen hydrolysates, or combinations thereof. According to some embodiments of this method, examples of the joint disorder include, but is not limited to, osteoarthritis, rheumatoid arthritis, psoriatic arthritis, inflammatory bowel disease, ankylosing spondylitis, Sjogren's syndrome and osteoporosis. According to some embodiments of this method, the joint disorder is joint inflammation. According to some embodiments of this method, the joint disorder is arthritis, such as osteoarthritis or rheumatoid arthritis. According to embodiments of this method, the degradation of synovial joint proteins is diminished.

According to some embodiments of this method, the therapeutically effective amount of the composition is administered orally.

According to some embodiments of this method, the proteolytic enzyme inhibitor inhibits the action of a proteolytic enzyme selected from the group consisting of collagenase, elastase, glycosidases, hyaluronidase, β-glucuronidase and combinations thereof. According to some embodiments of this method, the one or more components selected from proline, hydroxyproline, collagen, collagen derivatives is selected from proline, hydroxyproline and combinations thereof.

According to preferred embodiments of this method, the proteolytic enzyme inhibitor comprises proanthocyanidins. According to some embodiments of this method, the proanthocyanidins are an extract from a plant material or are synthesized. In some preferred embodiments, the plant material is selected from the group consisting of grape seeds, grape skin, cinnamon bark, cypress cones, pine barks, ginkgo leaves, cocoa beans, tamarind, tomato, peanut, almond, apple, cranberry, blueberry and tea leaves. In more preferred embodiments, the proanthocyanidins are from a pine bark extract, e.g., a pine bark extract from a pine of the P inus pinaster species.

According to some embodiments of this method, about 20 mg to about 10 grams per day of proanthocyanidins are administered. In some preferred embodiments, about 50 mg to about 500 milligrams per day of proanthocyanidins are administered. According to some embodiments of this method, about 12 mg/kg/day to about 9 g/kg/day of lysine is administered. According to some embodiments of this method, about 12 mg/kg/day to about 9 g/kg/day of proline and/or hydroxyproline is administered.

According to some embodiments of this method, the therapeutically effective amount of the composition is substantially free of glycosaminoglycans. According to some embodiments of this method, the therapeutically effective amount of the composition is substantially free of chondroitin sulfate and/or glucosamine.

According to some embodiments of this method, the therapeutically effective amount of the composition consists essentially of a proteolytic enzyme inhibitor, lysine and either or both of proline and hydroxyproline. According to some embodiments of this method, the therapeutically effective amount of the composition consists essentially of proanthocyanidins, proline/hydroxyproline and lysine. According to some embodiments of this method, the therapeutically effective amount of the composition is administered in the absence of glycosaminoglycans. According to some embodiments of this method, the therapeutically

effective amount of the composition is administered in the absence of chondroitin sulfate and/or glucosamine.

Another aspect of the present invention is a composition for treating or preventing a joint disorder in a vertebrate, preferably a human, comprising a proteolytic enzyme inhibitor, lysine and one or more components selected from proline, hydroxyproline, collagen, collagen derivatives, such as collagen hydrolysates, or combinations thereof. According to some embodiments, this composition is for oral administration. According to some embodiments, this composition diminishes the degradation of synovial joint proteins.

According to some embodiments, the composition is for a joint disorder that includes, but is not limited to, osteoarthritis, rheumatoid arthritis, psoriatic arthritis, inflammatory bowel disease, ankylosing spondylitis, Sjogren's syndrome and osteoporosis. According to some embodiments, the joint disorder is joint inflammation. According to some embodiments, the joint disorder is arthritis, such as osteoarthritis or rheumatoid arthritis.

According to some embodiments, the proteolytic enzyme inhibitor of this composition inhibits the action of a proteolytic enzyme selected from the group consisting of collagenase, elastase, glycosidases, hyaluronidase, β-glucuronidase and combinations thereof. According to some embodiments of this composition, the one or more components selected from proline, hydroxyproline, collagen, collagen derivatives is selected from proline, hydroxyproline and combinations thereof. According to preferred embodiments, the proteolytic enzyme inhibitor of this composition comprises proanthocyanidins. According to some embodiments, the proanthocyanidins are an extract from a plant material or are synthesized. In some preferred embodiments, the plant material is selected from the group consisting of grape seeds, grape skin, cinnamon bark, cypress cones, pine barks, ginkgo leaves, cocoa beans, tamarind, tomato, peanut, almond, apple, cranberry, blueberry and tea leaves. In more preferred embodiments, the proanthocyanidins are from a pine bark extract, e.g., a pine bark extract from a pine of the P inus pinaster species.

According to some embodiments, a unit dosage of this composition contains about 20 mg to about 10 grams of proanthocyanidins. In some preferred embodiments, a unit dosage of this composition contains about 50 mg to about 500 milligrams of proanthocyanidins. According to some embodiments, a unit dosage of this composition contains about 12 milligrams to about 9 grams of lysine. According to some embodiments, a unit dosage of this composition contains about 12 milligrams to about 9 grams of proline and/or hydroxyproline.

According to some embodiments, this composition is a unit dosage for consumption for a person of between 40 to 100 kg weight.

According to some embodiments, this composition contains between 1% proanthocyanidins to 80% proanthocyanidins by weight. According to some embodiments, this composition contains between 1% to 50% proline and/or hydroxyproline by weight. According to some embodiments, this composition contains between 1% lysine to 50% lysine by weight. In some preferred embodiments, this composition contains between 50% to 80% proanthocyanidins by weight, between 1% proline and/or hydroxyproline to 25% proline and/or hydroxyproline by weight, and between 1% lysine to 25% lysine by weight. According to some embodiments, the composition is substantially free of glycosaminoglycans. According to some embodiments, the composition is substantially free of chondroitin sulfate and/or glucosamine. According to some embodiments, the composition consists essentially of proanthocyanidins, lysine and either or both of proline and hydroxyproline. According to some embodiments, the composition consists essentially of proanthocyanidins, proline and lysine.

It will be appreciated by those skilled in the art that various omissions, additions and modifications may be made to the invention described above without departing from the scope of the invention, and all such modifications and changes are intended to fall within the scope of the invention, as defined by the appended claims. All references, patents, patent applications or other documents cited are herein incorporated by reference in their entireties.

EXAMPLE l Treatment of Osteoarthritis with Proanthocyanidins, Lysine and Proline.

Study Design Patients and Methods A total of 40 knee osteoarthritic patients (37 female and 3 male) were enrolled in this prospective, randomized, parallel group double blind study. Of these 5 were dismissed due to non-compliance and 35 were enrolled in the study. Forty patients were enrolled in the study. Twenty patients (2 male and 18 female) aged between 36 and 61 years (mean ± SD: 47.5 ± 7.4 years), received Pycnogenol ^® + Lysine + Proline ("Pycnogenol+Lys/Pro"), while the other 20 patients (1 male and 19 female) aged between 29 and 63 years (48.9 ± 9.6 years), received the placebo. All patients fulfilled the American College of Rheumatology radiological and clinical criteria for knee osteoarthritis (Altman, R. et al. "Development of criteria for the classification of osteoarthritis of the knee" Arthritis Rheum, 1986, 29: 1039-

49.)- Inclusion criteria were as follows: Age between 25-65 years; primary osteoarthritis of the knee (grade 1 or 2); pain in the target knee for minimum of 3 months; the use of NSAIDs or COX-2 inhibitors; and, informed consent form signed by the subject. Exclusion criteria: secondary osteoarthritis (owing to previous trauma); arthroscopy of the target knee performed less than 6 months prior to enrollment or during the trial period; or, other chronic inflammatory process.

Patients received a daily dose of Pycnogenol ^® + Lysine + Proline ("Pycnogenol+Lys/Pro") (two (2) tablets each with 60 mg Pycnogenol ^® and 250 mg of proline and 250 mg of lysine) or two (2) placebo tablets. Patients also received 4 copies of the Western Ontario McMaster Universities Osteoarthritis Index (WOMAC) form, containing twenty-four 10 mm visual analogue scales to assess pain, stiffness, and physical function, and a composite. High scores indicate greater disease severity. Each subject was contacted by telephone once every 7 days to verbally complete the WOMAC form. Each patient visited the trial center every 30 days (maximum, 33 days), with the final visit to be accomplished after 12 weeks of using the Pycnogenol+Lys/Pro or placebo. Medications were counted and the WOMAC forms evaluated exactly every 30 days. Each patient's intake of NSAIDs, COX-2 inhibitors, or other drugs, was catalogued in each patient's diary, and drug use was counted at each visit to the trial center. Routine blood examinations were conducted on day 0 and day 90, to include red blood cell count, hemoglobin, white blood cell count and platelets. Biochemical parameters included alanine aminotransferase, AST, UREA, creatinine and fasting plasma glucose were done by the clinical laboratory of Mashhad Medical School.

Data analysis was performed with SPSS, version 1 1.5. The results were expressed as a mean ± standard deviation. Parametric data were compared using students' t-test. Differences between groups were determined using X square tests. A P-value below 0.050 was considered significant.

Results

No side effects were reported during the study by patients in either group. There were no statistically significant differences between the groups in hematology and blood chemistry at the beginning or end of the study. The WOMAC osteoarthritis Index composite score measured no statistically significant difference between the placebo and Pycnogenol+Lys/Pro supplemented groups prior to and after 1 month of treatment based upon self-reported pain, stiffness, and physical function (Table 1). Significant reductions in self-reported pain, physical function and

WOMAC composite scores occurred in the Pycnogenol+Lys/Pro treated group after 2 months of supplementation compared to patients treated with placebo (Table 1).

TABLE l Pain, stiffness and physical function scores

Table IA PAIN SCORE

Treatment Pycnogenol+Lys/Pro Placebo p value

Mean ± SD Mean ± SD

O days 292.30 ± 101.16 301.00 ± 1 19.62 <0.805

30 days 225.50 ± 1 16.45 251.41 ± 132.86 <0.543

60 days 165.00 ± 82.51 264.82 ± 129.18 O.010

90 days 164.66 ± 72.46 306.00 ± 103.53 O.001

Table IB STIFFNESS SCORE

Treatment Pycnogenol+Lys/Pro Placebo p value Mean ± SD Mean ± SD

0 days 1 10 .30 ± 66 .10 120 .15 ± 63 .44 <0.633

30 days 86. 22 ± 56. 83 82. 64 ± 62. 66 <0.861

60 days 75. 38 ± 57. 98 92. 52 ± 61. 34 <0.402

90 days 75. 50 ± 54. 58 108 .82 ± 56 .85 <0.086

Table 1C PHYSICAL FUNCTION SCORE

Treatment Pycnogenol+Lys/Pro Placebo p value Mean ± SD Mean ± SD

O days 997.90 ± 352.58 1042.00 ± 420.08 O.721

30 days 707.33 ± 331.99 912.94 ± 481.88 <0.149

60 days 512.00 ± 272.87 909.52 ± 458.23 <0.004

90 days 485.50 ± 346.22 1014.00 ± 385.16 O.001

Table ID WOMAC COMPOSITE SCORE

Treatment Pycnogenol+Lys/Pro Placebo p value

Mean ± SD Mean ± SD

O days 1400.50 482.06 1463.15 552.31 <0.704

30 days 1019.05 463.01 1247.00 461.32 <0.235

60 days 752.38 347.44 1266.88 620.60 <0.004

90 days 725.50 346.22 1455.82 509.13 <0.001

At the end of month 2 significant differences between the Pycnogenol+Lys/Pro and placebo treated groups were observed for self-reported pain (p<0.010), physical function (p<0.004), and composite WOMAC (p<0.004) scores (Table 1).

After 3 months of supplementation with Pycnogenol+Lys/Pro that group showed reductions of 43, 35, 52, and 49%, respectively, as to self-reported pain, stiffness, physical dysfunction, and composite WOMAC scores. The placebo group demonstrated only reductions of 4, 15, 5, and 6%, respectively, as to self-reported pain, stiffness, physical dysfunction and composite WOMAC scores by month 3 of supplementation (Table 2).

TABLE 2 Change in WOMAC scores after 60 and 90 days of Pycnogenol ^® +Lys/Pro Therapy

After 1 month of supplementation, the number of NSAIDS and COX-2 inhibitor drugs used by the patients in both groups was unchanged. However after 2 and 3 months of Pycnogenol+Lys/Pro consumption, drug use was significantly reduced (p<0.001) (Table 3). On the other hand the placebo group significantly increased its use of NSAIDS and COX-2 inhibitors (p<0.037) by month 3 of intake. While no change was observed after 1 month of supplementation, in the number of days that patients used NSAIDS and COX-2 inhibitors in either group; by the completion of 2 months, the number of days of NSAIDS pill use was

significantly reduced (p<0.001) in the Pycnogenol+Lys/Pro group (Table 4). The number of days the placebo group used NSAIDS and COX-2 inhibitors increased significantly (p<0.001).

TABLE 3

Change in the number of pills of NSAEDS or COX-2 inhibitory drugs used per patient/month

TABLE 4

Change in the number of days using NSAIDS or COX-2 inhibitory drugs/month

This study clearly showed that a concentrate of proanthocyanidins combined with Lysine and Proline lowered clinical symptoms of osteoarthritis. In particular, the regimen of NSAIDS and COX-2 inhibitors was decreased, demonstrating the clinical benefits from the dietary supplement. Self-reported pain, stiffness, physical function and overall WOMAC scores all gradually improved and reached statistical significance from 60 days onwards of supplementation with proanthocyanidins and lysine and proline compared to placebo.

This result is surprising in that proanthocyanidins and lysine and proline, administered as the only active ingredients, are able to induce a beneficial effect on patients suffering from osteoarthritis.

In vitro, proanthocyanidins had significant activity in lowering nitric oxide production by activated macrophages (Virgili F et al.; 1998. In: Flavonoids in health and disease. Rice- Evans CA, Packer LM, eds. Marcel Dekker: New York. 421-36.), resulting from inhibition of NF-κB controlled inducible nitric oxide synthetase (iNOS) expression and demonstrated to lower generation of peroxides from activated macrophages ("oxidative burst"). A significantly lowered generation of reactive oxygen species by peripheral neutrophils was found in Lupus erythematosus patients in response to Pycnogenol ^® administration (Stefanescu M et al., Phytother Res 2001; 15: 698-704).

A lowering of inducible nitric oxide by proanthocyanidins (Rohdewald P., Int J Clin Pharmacol Therap, 2002, 40 (4):158-168; Blazso G et al., Pharm Pharmacol, 1994, 3:217-20; Packer L et al., 1999 Free Radic Biol Med, 704-24; Virgili F 1998 In: Flavonoids in health and disease. Rice-Evans CA, Packer LM, eds. Marcel Dekker: New York. 421-36; Grimm T, et al., Free Radic Biol, 2004, 36: 811-22) helps to explain symptom improvement in osteoarthritis. Nitric oxide is a major catabolic factor produced by chondrocytes in response to proinflammatory cytokines (Pelletier J.P., Arthritis Rheum, 2001, 44: 1237-47). Overproduction of nitric oxide by chondrocytes plays a major role in the perpetuation of cartilage destruction in osteoarthritis (Pelletier J.P. et al., Osteoarthritis Cartilage, 1996, 4: 77-84; Grabowski P. S., et al., Br. J. Rheumatol, 1997, 36: 651-55). Increased concentrations of nitrites have been demonstrated in synovial fluids of patients with osteoarthritis, and iNOS has been demonstrated in osteoarthritis synoviocytes and chondrocytes (Hayashi T. et al., Arthritis Rheum, 1997, 40: 261-69; Loeser R.F. et al., Arthritis Rheum, 46: 2349-57). Nitric oxide and reactive oxygen species, combined to nitro peroxides, exert multiple effects on chondrocytes that promote the degradation of articular cartilage including activation of MMPs and apoptosis (Abramson S. B. et al., Curr Rheumatol Rep, 2001, 3: 535-41 ; Hirai. et al., Life Sci, 2001, 68: 913-20). Chondrocyte apoptosis is a particular feature of osteoarthritis and studies implicated that nitric oxide and oxidative stress are important mediators in this process (Lotz, M. "The role of nitric oxide in articular cartilage damage. " Rheum Dis Clin Norh Am, 1999, 25: 269-82). Importantly nitric oxide levels are unchanged in patients without degenerative alterations. In contrast, nitric oxide levels in fluid taken from painful joints are higher than in joints without pain. Thus reduction of nitric oxide and reactive oxygen species by Pycnogenol ^® should reduce tissue damage and this helps to explain the lowered clinical symptoms of osteoarthritis.

Osteoarthritic chondrocytes release matrix metalloproteinases (MMP) which promote cleavage of articular collagen and gelatin. Excessive MMP expression exacerbates articular

connective tissue and cartilage degradation and plays a critical role in the development of inflammatory joint disease. A broad spectrum of MMP species are expressed in osteoarthritic cartilage and these contribute significantly to tissue damage, pain and stiffness (Sholpov B. V. et al., Arthritis Rheum, 40: 2065-74; Imai K. et al., Am J Pathol, 1997, 151 : 245-56; Mohtai M. et al., Lab Invest, 1992, 66: 680-90; Ohta S. et al., Lab Invest, 1998, 78 :79-87; Mitchell P.G. et al., J Clin Invest, 1996, 97: 761-68).

Human metabolites of proanthocyanidins, δ-(3,4-dihydroxy-phenyl)-γ-valerolactone (Ml) and δ-(3-methoxy-4-hydroxyphenyl)-γ-valerolactone (M2), were shown to be more potent than the parent molecules for inhibition of MMP-I, MMP-2 and MMP-9 (Grimm T et al., Free Radic Biol, 2004, 36: 81 1-22). The inhibitory activity of proanthocyanidins metabolites Ml and M2 against MMP-2 and MMP-9 was found to be 100-fold more potent than that of captopril, a well documented inhibitor of these MMPs. Moreover, proanthocyanidin metabolites were as potent as hydrocortisone for inhibition of MMP-9 released from activated macrophages. The pronounced MMP inhibitory activity of proanthocyanidin and its metabolites should provide significant help for allowing recovery of articular tissue. From our experiments it was striking that all clinical symptoms improved gradually during the three months treatment course. It may be speculated that anti-inflammatory and MMP-inhibitory activity of proanthocyanidins allowed for progressive articular tissue recovery which resulted in significant symptom improvement only after 2 of months treatment.