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Title:
A-TYPE PROCYANIDINS AND INFLAMMATION
Document Type and Number:
WIPO Patent Application WO/2007/053641
Kind Code:
A3
Abstract:
This invention relates to compositions, and methods of use thereof, containing polyphenols such as A-type procyanidins and derivatives thereof, for treating inflammation and/or inflammation-related or associated disease or condition, and/or for the relief of pain, in a subject sensitive to a selective cyclooxygenase-2 (COX-2) inhibitor and/or a subject sensitive to a COX-nonselective nonsteroidal anti-inflammatory drug (NSAID).

Inventors:
SCHMITZ HAROLD H (US)
KWIK-URIBE CATHERINE L (US)
Application Number:
PCT/US2006/042546
Publication Date:
November 29, 2007
Filing Date:
November 01, 2006
Export Citation:
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Assignee:
MARS INC (US)
SCHMITZ HAROLD H (US)
KWIK-URIBE CATHERINE L (US)
International Classes:
A61K31/35
Foreign References:
US6696485B12004-02-24
US20040204471A12004-10-14
Attorney, Agent or Firm:
JAIN, Nada (P.C.560 White Plains Road, Suite 46, Tarrytown NY, US)
Download PDF:
Claims:

What is claimed is:

1. A method of treating inflammation and/or inflammation-related or associated disease or condition, which comprises administering to a subject in need thereof, an effective amount of a compound which is an oligomer composed of n monomeric, flavan-3-ol units, which has the following formula:

wherein

(i) the monomeric units are connected via interflavan linkages 4~→6 and/or 4→8;

(ii) at least two of the monomeric units are additionally linked by an A- type interflavan linkage (4→8; 2→O→7) or (4→6; 2→O→7); and (iii) n is 2 to 12; or a pharmaceutically acceptable salt or derivative thereof; wherein the subject is a human or a veterinary animal and is sensitive to a selective COX-2 inhibitor and/or COX-nonselective NSAID.

2. The method of claim 1 , wherein the subject is a human.

3. The method of claim 2, wherein the composition is a pharmaceutical composition.

4. The method of claim 3 , wherein n = 2-4.

5. The method of claim 3 , wherein n = 2-5. 6. The method of claim 3, wherein n = 2.

7. The method of claim 6, wherein the compound is dimer Al .

8. The method of claim 6, wherein the compound is dimer A2.

9. The method of claim 3, wherein the subject suffers from arthritis.

10. The method of claim 3, wherein the subjects suffers from a gastrointestinal disease or condition; a respiratory/pulmonary disease or condition; a musculoskeletal disease or condition; a dermal disease or condition; an allergic disease or condition; pain associated with dysmenorrhea, menstrual cramps, headache, toothache, low back and neck pain; an ocular disease or condition; a diabetes-associated condition; fever; systemic lupus erythematos s; an inflammation-associated with burns; and/or an inflammatory disease or condition affecting multiple organs.

11. The method of claim 3 , wherein the subject suffers from osteoporosis.

12. The method of claim 3, wherein the subject suffers from diabetes-associated inflammatory conditions.

13. The method of claim 9 , wherein the compound is dimer A 1.

14. The method of claim 10, wherein the compound is dimer Al .

15. The method of claim 11 , wherein the compound is dimer Al .

16. The method of claim 12, wherein the compound is dimer Al .

17. The method of claim 9, wherein the compound is dimer A2.

18. The method of claim 10, wherein the compound is dimer A2.

19. The method of claim 11, wherein the compound is dimer A2.

20. The method of claim 12, wherein the compound is dimer A2.

Description:

A-TYPE PROCYANIDINS AND INFLAMMATION

FIELD QF THE INVENTION

This invention relates to compositions, and methods of use thereof, containing certain polyphenols such as A-type procyanidins and derivatives thereof for treating inflammation and/or inflammation-related or associated disease or condition, and/or methods for the relief of pain, in a subject sensitive to a selective cyclooxygenase-2 (COX-2) inhibitor and/or a subject sensitive to a COX-nonselective nonsteroidal antiinflammatory drug (NSAID).

BACKGROUND OF THE INVENTION The A-type procyanidins have attracted a great deal of attention in the fields of medicine and nutrition due to the wide range of their biological activities (e.g. U. S. Pat. App. Pub. No. 20050164956). Applicants have now discovered specific antiinflammatory properties of A-type procyanidins and derivatives thereof and their effect on cyclooxygenase-2 (COX-2) gene transcription, a key regulating enzyme in the biosynthesis of prostaglandins in humans and other mammals (Simmons et al., ' Pharmacol. Rev., 2004, 56:387-437).

At least two distinct isoforms of cyclooxygenase are known: COX-I and COX- 2. COX-I is constitutively expressed in many tissues, where it regulates physiological functions. In contrast, COX-2 is not normally expressed by most tissues, but is induced rapidly and transiently by proinflammatory mediators and mitogenic stimuli including cytokines, growth factors and tumor promoters. Up-regulation of COX-2 expression is observed at inflammatory sites, where it mediates the generation of prostaglandins responsible for pain and inflammation. The role of excessive inflammation as a critical factor in a wide range of human diseases is well established and therefore one of ordinary skill in the art will appreciate that the compounds of invention have utility in treating a diverse array of diseases, pathologies and conditions.

Most of the previously-known COX-2 inhibitors work primarily by blocking COX-2 enzyme activity either directly or indirectly. A disadvantage of inhibition at the enzyme level is that the loss of COX-2 enzyme activity is compensated for (by the body's natural response) by a bio-feedback loop which leads to an increased production of enzyme. The present inventive compounds offer a clear advantage as COX-2 synthesis is inhibited at the level of gene transcription thereby circumventing the formation of additional, undesirable COX-2 via the biofeedback loop mechanism. While the existing selective COX-2 inhibitors were found to be efficacious in blocking COX-2 activity and in reducing severe gastrointestinal events associated with use of nonselective NSAIDs, their safety following clinical administration has been questioned. The use of several commercially available selective COX-2 inhibitors has been shown to be associated with serious side effects, most notably those within the cardiovascular system such as myocardial infarction, strokes, and elevations in blood pressure.

SUMMARY OF THE INVENTION

The invention relates to compositions, and methods of use thereof, containing certain polyphenols such as A-type procyanidins and derivatives thereof for treating inflammation and/or inflammation-related or associated disease or condition, and/or methods for the relief of pain, in a subject sensitive to a selective cyclooxygenase-2 (COX-2) inhibitor and/or a subject sensitive to a COX-nonselective nonsteroidal anti- irtflamrnatory drug (NSAID).

In one aspect, the invention relates to a composition, such as a pharmaceutical, a food, a food additive, or a dietary supplement comprising the compound of the invention such as an A-type procyanidin or a derivative thereof. The composition may optionally contain an additional COX-2 selective inhibitor and/or an additional COX- nonselective NSAID, or may be administered in combination with an additional COX-2 selective inhibitor and/or an additional COX-nonselective NSAID. Packaged products containing the above-mentioned composition and a label and/or instructions for use as described herein, e.g. to treat inflammation and/or inflammation-related or associated disease or condition, and/or for the relief of pain, in a subject sensitive to a selective COX-2 inhibitor and/or a subject sensitive to a COX-nonselective NSAID are also within the scope of the invention.

In another aspect, the invention relates to a method for treating inflammation and/or inflammation-related or associated disease or condition, and/or a method for the relief of pain, in a subject sensitive to a selective COX-2 inhibitor and/or a subject sensitive to a COX-nonselective NSAID, which comprises administering to a mammal, such as a human or a veterinary animal, an effective amount of a compound of the invention such as an A-type procyanidin or a derivative thereof.

In a further aspect, the invention relates to a method comprising (i) profiling or diagnosing a subject for sensitivity to a selective COX-2 inhibitor and/or to a COX- nonselective NSAID, and (ii) treating the sensitive subject by administering an effective amount of the compound of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1 A-C show the effects of Al dimer in comparison with Bl dimer, B2 dimer, (-)-catechin and (+)-epicatechin (each lOμM) on the mRNA expression of COX- 2 in LPS and fiVILP-pretreated macrophages, and their potency comparison. n=3-4, Mean ± SD. **P<0.01 versus control, ##P<0.01 versus LPS, $$P<0.01 versus LPS + fMLP. The normal macrophages without LPS or fMLP treatment were considered as controls.

DETAILED DESCRIPTION

AU patents, patent applications and references cited in this application are hereby incorporated herein by reference. In case of any inconsistency, the present disclosure governs.

The invention relates to compositions comprising an effective amount of an A- type procyanidin, or a pharmaceutically acceptable salt or derivative thereof.

The present invention relates to a compound and, a composition comprising an effective amount of the compound which is an oligomer composed of n monomeric, flavan-3τθl units, which has the following formula:

wherein

(i) the monomeric units are connected via interflavan linkages 4— »6 and/or 4-→8; (ii) at least two of the monomeric units are additionally linked by an A-type interflavan linkage (4→8; 2-→O→7) or (4→6; 2→O→7); and (iii) n is 2 to 12.

It will be understood by a person of skill in the art that one of the two flavanol units linked by the A-type interflavanoid linkage must comprise two bonds at the 2- and 4-positions. Both of these have either α or β stereochemistry, i.e., the bonds are either 2α, 4α or 2β, 4β. These bonds connect to the 6- and 7-0-positions, or the 8- and 7-0- positions of the second flavanol unit linked by the A-type interflavan linkage. In constituent flavanol units of the oligomer which do not comprise A-type interflavan linkages at positions C-2 and C-4, the linkage at position C-4 can have either alpha or beta stereochemistry. The OH group at position C-3 of flavanol units has either alpha or beta stereochemistry. Flavan-3-ol (monomeric) units may be (+)-catechin or (-)- epicatechin.

An A-type procyanidin as defined above may be derivatized, for instance esterified, at one or more of the OH groups on one or more of the constituent flavan-3- ol units. A given flavan-3-ol unit may thus comprise one or more ester groups, preferably gallate ester groups, at one or more of the 3-, 5-, 7-, 3'- and 4'- ring positions. It may in particular be a mono-, di-, tri-, tetra- or penta-gallated unit.

Examples of the compounds useful for products, and in the methods of the present invention, include the compounds wherein the integer n is 3 to 12; 4 to 12; 5 to 12; 4 to 10; or 5 to 10. In some embodiments, n is 2 to 4, or 2 to 5, for example n is 2 or 3.

In one embodiment, the invention relates to epicatechin-(4β—»8; 2β→O-→7)- catechin (i.e., Al dimer), or a pharmaceutically acceptable salt or derivative thereof, which has the

In another embodiment, the invention relates to epicatechin-(4β-→8; 2β— »0— >7)-epicatechin (i.e., A2 dimer), or a pharmaceutically acceptable salt or derivative thereof, which has the following formula:

In yet another embodiment, the invention relates to A-type trimer, or a pharmaceutically acceptable salt or derivative thereof, which has the following formula:

Methods of Use

The invention relates to methods of treating inflammation and/or mflammation- related or associated disease or condition, and/or to methods for the relief of pain, in a subject sensitive to a selective cyclooxygenase-2 (COX-2) inhibitor and/or a subject sensitive to a COX-nonselective nonsteroidal anti-inflammatory drug (NSAID). Any compound described in the application may be used to practice the methods described herein.

As used herein, "treatment" or "treating" means improving an existing medical condition, for example an inflammatory disease or condition, for example by slowing down the disease progression, prolonging survival, reducing the risk of death, and/or inducing a measurable reduction in inflammation.

As used herein, treatment of "inflammation and/or inflammation-related or associated disease or condition" refers to treatment of inflammation other than the inflammation associated with diseases or conditions of the vascular system (inclusive of the heart, the brain and the renal system). Examples of such diseases or conditions are: gastrointestinal diseases or conditions other than GI complications of NSAIDs (e.g. inflammatory bowel diseases, Crohn's disease, regional enteritis, ulcerative colitis, diverticulitis, pancreatitis); respiratory/pulmonary diseases or conditions (e.g. emphysema, acute respiratory distress syndrome, asthma, bronchitis, chronic obstructive pulmonary disease); musculoskeletal diseases or condition (e.g. arthritis including rheumatoid arthritis, osteoarthritis, gouty arthritis, juvenile arthritis, degenerative joint diseases, and spondyloarthropathies, muscle or joint strains or sprains, osteoporosis, loosening of artificial joint implants, myositis, polymyositis, bursitis, synovitis, ankylosing spondylitis, tendonitis); dermal diseases or conditions (e.g. psoriasis, eczema, scleroderma, dermatitis, epidermolysis bullosa); allergic diseases or conditions (e.g. allergic reactions, allergic contact hypersensitivity); pain associated with dysmenorrhea, menstrual cramps, headache (including migraine), toothache, low back and neck pain; ocular diseases or conditions (e.g. macular degeneration, conjunctivitis, corneal scarring, scleritis, ocular angiogenesis); diabetes- associated conditions (e.g. diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, inflammatory conditions associated with type I and type II diabetes); fever (e.g. fever associated with influenza and other viral infections, rheumatic fever, common cold); systemic lupus erythematosis; inflammation-associated with burns;

inflammatory diseases or conditions affecting multiple organs (e.g. Sarcoidosis, Behcet's syndrome).

As used herein, a "subject sensitive to selective COX-2 inhibitor" is a subject in whom an existing health condition is exacerbated by, or an adverse health condition/reaction results from, the use of selective COX-2 inhibitor, or a subject for whom use of selective COX-2 inhibitor is contraindicated due to existence of previous health history of known risk factors which may increase the likelihood of adverse side effects associated with use of selective COX-2 inhibitors. Examples of these subjects are: elderly (e.g. age >65 for example >75 ); subjects with history of ischemic heart disease, hypertension or heart failure, subjects showing symptoms of an unstable coronary heart disease, subjects who have recently undergone heart surgery, subjects showing symptoms of an imminent cerebral ischemia, subjects with decompensated heart failure, subjects with uncontrolled arterial hypertension, subjects with previous history of NSAID-induced urticaria or angioedema, subjects with history of sulfonamide hypersensitivity (note: in these subjects only use of selective COX-2 inhibitors which have a sulfonamide group is contraindicated), subjects with previous history of NSAID-related nephrotoxicity, salt-depleted healthy subjects, elderly subjects (e.g. age >65 for example >75) with compromised renal function, pregnant women in third trimester of pregnancy, subjects with liver problems/hepatic dysfunction.

As used herein, a "subject sensitive to COX-nonselective NSAIDS" is a subject in whom an existing health condition is exacerbated or an adverse health condition/reaction results from the use of a COX-nohselective NSAID, or for whom use of COX-nonselective NSAID is contraindicated due to existence of previous health history of known risk factors which may increase the likelihood of adverse side effects associated with use of a COX non-selective NSAID. Examples of these subjects are: subjects with history of gastrointestinal complications (e.g. gastroduodenal perforations, ulcers and bleeding), elderly subjects (e.g. age >65 for example >75), subjects with history of NSAID-sensitive asthma or respiratory disease, subjects with history of NSAID-induced cutaneous reactions (e.g. urticaria, angeoedema, non- urticarial rash), subjects/children with chicken pox or influenza, subjects with NSAID- induced nephrotoxicity, subjects with liver failure, subjects with sulfonamide hypersensitivity (note: only use of NSAIDS with sulfonamide group is

contraindicated), subjects undergoing treatment with anticoagulants, subjects undergoing treatment with corticosteroids, subjects with concurrent illnesses (e.g. rheumatoid arthritis, heart disease).

In certain embodiments, the invention provides a method of treating inflammation and/or inflammation-related or associated disease or condition, and/or a method for the relief of pain, which comprises administering to a human or a veterinary animal in need thereof an effective amount of a compound, or a composition comprising an effective amount of the compound, which is an oligomer composed of n monomeric, fiavan-3-ol units, which has the following formula:

wherein

(i) the monomeric units are connected via interflavan linkages 4→6 and/or 4→8;

(ii) at least two of the monomeric units are additionally linked by an A-type interflavan linkage (4→8; 2→O→7) or (4→6; 2→O→7); and

(iii) n is 2 to 12, or a pharmaceutically acceptable salt or derivative thereof, wherein the subject is sensitive to a selective COX-2 inhibitor and /or a COX- nonselective NSAID.

It will be understood by a person of skill in the art that all compounds described in this application can be used for treatment of inflammation and/or inflammation- related or associated disease or condition, and/or for the relief of pain,

Examples of the compounds useful for products, and in the methods of the present invention, include the compounds wherein the integer n is 3 to 12; 4 to 12; 5 to 12; 4 to 10; or 5 to 10. In some embodiments, n is 2 to 4, or 2 to 5, for example n is 2 or 3.

In one embodiment, a method of treating inflammation and/or inflammation- related or associated disease or condition, and/or a method for the relief of pain, comprises administering to a human or a veterinary animal in need thereof an effective amount of an A-type procyanidin which is epicatechin-(4β→8; 2β→O— »7)-catechin (i.e., Al dimer), or a pharmaceutically acceptable salt or derivative thereof, which Al dimer has the following formula:

wherein the subject is sensitive to a selective COX-2 inhibitor and /or a COX- nonselective NSAID.

In another embodiment, a method of treating inflammation and/or inflammation-related or associated disease or condition, and/or a method for the relief of pain, comprises administering to a human or a veterinary animal in need thereof an effective amount of an the A-type procyanidin which is epicatechin-(4β→8;

2β→O→7)-epicatechin (i.e., A2 dimer), or a pharmaceutically acceptable salt or derivative thereof, which A2 dimer has the following formula:

wherein the subject is sensitive to a selective COX-2 inhibitor and /or a COX- nonselective NSAID.

In yet another embodiment, a method of treating inflammation and/or inflammation-related or associated disease or condition, and/or a method for the relief of pain, comprises administering to a human or a veterinary animal in need thereof an effective amount of an A-type procyanidin which is an A-type trimer, or a pharmaceutically acceptable salt or derivative thereof, which A-type trimer may have the following formula:

wherein the subject is sensitive to a selective COX-2 inhibitor and /or a COX- nonselective NSAID. Examples of the compounds for use in the methods described herein are dimers

Al and A2.

The present compounds may be administered alone, or as a combination therapy with other COX-2 selective inhibitor(s), most of which primarily target the COX-2 enzyme activity. Examples of COX-2 inhibitors include: meloxicam, etodolac, nimesulide, flosulide, lumiracoxib, celecoxib, valdecoxib, rofecoxib, deracoxib, parecoxib, etoricoxib, darbufelone, and meclofenamate esters and amides.

The present compounds may also be administered in combination with COX- nonselective NSAIDs. Examples of COX-nonselective NSAIDs include: nabumetone, meclofenamic acid, mefenamic acid, ibuprofen, flurbiprofen, suprofen, ketoprofen, naproxen, piroxicam, tenoxicam, phenylbutazone, diclofenac, ketorolac, tolmetin, indomethacin, sulindac, and acetamiophen.

When used for the above-mentioned combination therapies, COX-2 selective inhibitor and/or COX-nonselective NSAID may be administered in reduced amounts,

i.e., amounts lower than when they are administered alone thereby reducing the side effects of these compounds.

The present compounds may be administered, in some embodiments, in combination with and/or to enhance responsiveness of immunomodulatory agents (other than NSAIDs and selective COX-2 inhibitors). Examples of such agents include: biological therapeutic agents (e.g. tumor necrosis factor (TNF)-α inhibitors such as anti-TNF monoclonal antibodies and TNF receptors (examples of TNF-blockers include entanercept, infliximab and adalimumas), matrix metalloproteinase inhibitors, aggrecanase inhibitors, tumor necrosis alpha converting enzyme (TACE) inhibitors, leukotriene receptor antagonists, interleukin-1 (IL-I) processing and release inhibitors, prostaglandin inhibitors such as PGD-, PGF-, PGI 2 and PGE-receptor antagonists); anti-osteoporosis agents (e.g. raloxifene, lasofoxifene, droloxifene, zoledronate, alendronate, risedronate, ibandronate, etidomate, teriparatide, miacalcin); anti-gout agents (e.g. colchicines, xanthine oxidase inhibitors, uricosuric agents such as probenecid, sufinpyrazole and benbromane); anti-arthritis drugs (e.g. leflunomide, oral gold, sulfasalazine, mycophenolate, injectable gold, cyclosporine, cyclophospahmide, azathioprine, chlorambucil, methotrexate, minocycline, cuprimine, hydroxychloroquine); anti-inflammatory glucocorticoids (e.g. betamethasone, cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, prednisone); antacids; histamine (H2) receptor blockers (e.g. Axid®, Pepcid®, Zantac®); proton pump inhibitors ( e.g. rabeprazole, esomeprazole, lansoprazole, omeprazole, pantoprazole). For that purpose, dosage forms other than pharmaceuticals, e.g. dietary supplements and foods, may also be used (e.g. chondroprotective nutraceuticals such as polysulfated glycosaminoglycan, glucosamine, chondroitin sulfate, hyaluronic acid, and pentosan polysulfate).

The methods described herein may be used in a human or a veterinary animal, such as a dog, a cat, and a horse.

Thus, the following uses are within the scope of the invention. Use of compounds of the invention or a pharmaceutically acceptable salt or derivative thereof, as defined above, in the manufacture of a medicament, food, nutraceutical or dietary supplement for inhibiting COX-2 expression in a subject sensitive to a selective COX-2 inhibitor and/or a COX-nonselective NSAID. Use of compounds of the invention or a pharmaceutically acceptable salt or derivative thereof, as defined herein, in the

manufacture of a medicament, food, nutraceutical or dietary supplement for use in treating inflammation and/or inflammation-related or associated disease or condition in a subject sensitive to a selective COX-2 inhibitor and/or a COX-nonselective NSAID.

The above described methods may further comprise determining the effectiveness of the treatment, for example, by measuring the level of COX-2 expression in a tissue sample using techniques known in the art.

The advantage of the present invention is that it can offer a personalized medicine approach to the treatment of inflammation and/or inflammation-related or associated disease or condition. Each patient/subject can be profiled or diagnosed for his/her sensitivity to COX-2 selective inhibitors and/or COX-nonselective NSAIDs and treating according to the methods described herein. It will be understood by a person of skill in the art that the sensitive subjects can be identified as described herein and as is known in the art.

The effective amount may be determined by a person of skill in the art using the guidance provided herein and general knowledge in the art. For example, the effective amount may be such as to achieve a physiologically relevant concentration in the body of a mammal. Such a physiologically relevant concentration may be at least 20 nanomolar (nM), preferably at least about 100 nM, and more preferably at least about 500 nM. In one embodiment, at least about one micromole in the blood of the mammal, such as a human, is achieved. The compounds of the invention, as defined herein, may be administered at from about 50 mg/day to about 1000 mg/day, preferably from about 100-150 mg/day to about 900 mg/day, and most preferably from about 300 mg/day to about 500 mg/day. However, amounts higher than stated above may be used. The amounts may be measured as described in Adamson, G.E. et al., J. Ag. Food Chem.; 1999; 47 (10) 4184-4188.

The treatment/administration may be continued as a regimen, i.e., for an effective period of time, e.g., daily, monthly, bimonthly, biannually, annually, or in some other regimen, as determined by the skilled medical practitioner for such time as is necessary. The administration may be continued for at least a period of time required to reduce inflammation to therapeutically relevant levels. Preferably, the composition is administered daily, most preferably two or three times a day, for example, morning and evening to maintain the levels of the effective compounds in the body of the mammal. To obtain the most beneficial results, the composition may be administered

for at least about 30, or at least about 60 days. These regimens may be repeated periodically.

Compositions and Formulations The compounds of the invention may be administered as a pharmaceutical, a food, a food additive, or a dietary supplement.

As used herein, a "pharmaceutical" is a medicinal drug. See Merriam- Webster's Collegiate Dictionary, 10th Edition, 1993. A pharmaceutical may also be referred to as a medicament. A "food" is a material containing protein, carbohydrate and/or fat, which is used in the body of an organism to sustain growth, repair and vital processes and to furnish energy. Foods may also contain supplementary substances, for example, minerals, vitamins and condiments. See Merriam- Webster's Collegiate Dictionary, 1 Oth Edition, 1993. The term food includes a beverage adapted for human or animal consumption. As used herein a "food additive" is as defined by the FDA in 21 C.F.R. 170.3(e)(l) and includes direct and indirect additives. As used herein, a "dietary supplement" is a product (other than tobacco) that is intended to supplement the diet that bears or contains the one or more of the following dietary ingredients: a vitamin, a mineral, an herb or other botanical, an amino acid, a dietary substance for use by man to supplement the diet by increasing the total daily intake, or a concentrate, metabolite, constituent, extract or combination of these ingredients. The above compositions may be prepared as is known in the art.

The compositions may contain a carrier, a diluent, or an excipient. Depending on the intended use, the carrier, diluent, or excipient may be chosen to be suitable for human or veterinary use, food, additive, dietary supplement or pharmaceutical use. The composition may optionally contain an additional anti-inflammatory agent. Also depending on use, a person of skill in the art may select the degree of purity of the compound of the invention. For example, when used to prepare pharmaceutical dosage forms, the compound should be as pure as commercially possible, while when preparing food, additive, or supplement, less pure or mixtures of compounds (e.g. plant extracts) may be used.

The compound of the invention may be "isolated and purified," i.e., it may be separated from compounds with which it naturally occurs (e.g. when the compound is of natural origin), or it may be synthetically prepared, in either case such that the level

of contaminating compounds and/or impurities does not significantly contribute to, or detract from, the effectiveness of the compound. For example, an "isolated and purified Aldimer" is separated from A2 dimer, with which it may occur in nature (e.g. peanut skins, cranberries) to the extent achievable by the available commercially viable purification and separation techniques. Such compounds are particularly suitable for pharmaceutical applications.

The compound may also be less pure, i.e., "substantially pure," i.e., it may possess the highest degree of homogeneity achievable by available purification, separation and/or synthesis technology but need not be separated from the like compounds. As used herein, "the like compounds" are the compounds having the same degree of polymerization. For example, a "substantially pure dimer" refers to a mixture of dimers (e.g. Al and A2, as it would occur in a "plant extract fraction"). While less suitable for pharmaceutical applications, such "substantially pure" compounds may be utilized for food, food additive and dietary supplement applications. In some embodiments, the compound of the invention is at least 80% pure, at least 85% pure, at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure. Such compounds are particularly suitable for pharmaceutical applications.

Pharmaceuticals containing the inventive compounds, optionally in combination with another anti-inflammatory agent, may be administered in a variety of ways such as orally, sublingually, bucally, nasally, rectally, intravenously, parenterally and topically. A person of skill in the art will be able to determine a suitable mode of administration to maximize the delivery of the compounds of the invention, and optionally another anti-inflammatory agent, to the site of the inflammation. Thus, dosage forms adapted for each type of administration are within the scope of the invention and include solid, liquid and semi-solid dosage forms, such as tablets, capsules, gelatin capsules (gelcaps), bulk or unit dose powders or granules, emulsions, suspensions, pastes, creams, gels, foams or jellies. Sustained-release dosage forms are also within the scope of the invention. Suitable pharmaceutically acceptable carriers, diluents, or excipients are generally known in the art and can be determined readily by a person skilled in the art. The tablet, for example, may comprise an effective amount of the compound of the invention and optionally a carrier, such as sorbitol, lactose, cellulose, or dicalcium phosphate.

The foods comprising the compounds described herein and optionally another anti-inflammatory agent may be adapted for human or veterinary use, and include pet foods. The food may be other than a confectionery, for example, a beverage (e.g. cocoa flavored beverage). A confectionery such as a standard of identity (SOI) and non-SOI chocolate, such as milk, sweet and semi-sweet chocolate including dark chocolate, low fat chocolate and a candy which may be a chocolate covered candy are also within the scope of the invention. Other examples include a baked product (e.g. brownie, baked snack, cookie, biscuit) a condiment, a granola bar, a toffee chew, a meal replacement bar, a spread, a syrup, a powder beverage mix, a cocoa or a chocolate flavored beverage, a pudding, a rice cake, a rice mix, a savory sauce and the like. If desired, the foods may be chocolate or cocoa flavored. Food products may be chocolates and candy bars, such as granola bars, containing nuts, for example, peanuts, walnuts, almonds, and hazelnuts.

A-type procyanidins may be of natural origin or synthetically prepared. For example, A-type procyanidins may be isolated from peanut skins as described in Lou et al, Phytochemistry, 51: 297-308 (1999), or Karchesy and Hemingway, J. Agric. Food Chem., 34:966-970 (1986), the relevant portions of each being hereby incorporated herein by reference. Mature red peanut skin contain about 17% by weight procyanidins, and among the dimeric procyanidins epicatechin-(4β→8; 2β→O—»7)- catechin dominates, with smaller proportion of epicatechin-(4β— »8; 2β→O-→7)- epicatechin being present. However, in addition to procyanidins having (4— »8; 2— >O— >7) double linkages, procyanidins having (4→6; 2—»O→7) double linkages are also found in peanut skins.

Other sources of the above compounds are cranberries as described, for example in Foo et al, J, Nat. Prod, 63: 1225-1228, and in Prior et al, J. Agricultural Food Chem., 49(3): 1270-76 (2001), the relevant portions of each being hereby incorporated herein by reference. Other sources include Ecdysanthera utϊlis (Lie-Chwen et al., J. Nat. Prod., 65:505-8 (2002)) and Aesculus hippocastanum (U.S. Pat. No. 4,863,956), the relevant portions of each being hereby incorporated herein by reference. A-type compounds may also be obtained from B-type procyanidins via oxidation using l,l-diphenyl-2-pycrylhydrazyl (DPPH) radicals under neutral conditions as described in Kondo et al, Tetrahedron Lett., 41 : 485 (2000), the relevant portions of which are hereby incorporated herein by reference. Methods of obtaining

natural and synthetic B-type procyanidins are well known in the art and are described, for example, in U.S. Pat. Nos. 6,670,390 to Romanczyk et al.\ 6,207,842 to Romanczyk et ah; 6,420,572 to Romanczyk et al. ; and 6,156,912 to Romanczyk et ah

The A-type procyanidins may be used in the compositions described herein and administered in the form of an extract (e.g. peanut skins extract) comprising A-type procyanidins as the main component.

A daily effective amount of the compound of the invention may be provided in a single serving in case of a food or a single dosage in case of a pharmaceutical or a dietary supplement. For example, a confectionery (e.g. chocolate) may contain at least about 100 mg/serving (e.g. 150-200, 200-400 mg/serving).

The dietary supplement containing the compounds of the invention, and optionally another anti-inflammatory agent, may be prepared using methods known in the art and may comprise, for example, nutrients such as dicalcium phosphate, magnesium stearate, calcium nitrate, vitamins, and minerals. Further within the scope of the invention is an article of manufacture such as a packaged product comprising the composition of the invention (e.g. a food, a dietary supplement, a pharmaceutical) and a label indicating the presence of, or an enhanced content of the inventive compounds or directing use of the composition to treat inflammation and/or inflammation-related or associated disease or condition, and/or a for the relief of pain. The packaged product may contain the composition and the instructions for use to treat inflammation and/or inflammation-related or associated disease or condition, and/or for the relief of pain. The label and/or instructions for use may refer to any of the methods of use described in this application. In certain embodiments, the label and/or the instructions for use direct use of the compounds of the invention for treating inflammation and/or inflammation-related or associated disease or condition, and/or for the relief of pain.

The invention also relates a method of manufacturing an article of manufacture comprising any of the compositions described herein, packaging the composition to obtain an article of manufacture and instructing, directing or promoting the use of the composition/article of manufacture for any of the uses described herein. Such instructing, directing or promoting includes advertising.

The invention is further described in the following non-limiting examples.

EXAMPLES Example 1: Effect of Procyanidins Al and A2 on Cox-2 expression

Materials Procyanidin dimers Al and A2 are obtained. Phorbol 12-myristate 13-acetate

(PMA) 5 Lipopolysaccharides (LPS, from Escherichia coli serotype 0111: B4) and NS398 are purchased from Sigma (St. Louis, MO). RPMI 1640, L-glutamine, HEPES, 2-mercaptoethanol, fetal bovine serum, and penicillin/streptomycin are purchased from Gibco BRL (Grand Island, NY). Anti-COX-2 is purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA). SuperSignal West Pico chemiluminescent substrate and PGE 2 -specific RIA kit are purchased from Beijing East Asia Institute of Immunology (Beijing CN). All other chemicals used are in the purest form available commercially.

Cell culture

Human monocytic THP-I cells (American Type Culture Collection, Manassas, VA) in RPMI 1640 medium (Life Technologies, Rockville, MD), with 4.5 g/L glucose, 10 mM HEPES, 1 mM sodium pyruvate, and 50 μM 2-ME supplemented with 10% FBS, are cultured under a humidified 5% CO 2 atmosphere at 37 °C. For differentiation, THP-I cells are plated at IxIO 6 cells/ml in the medium containing 100 nM PMA and

> allowed to adhere for 48 h, after which they are fed with PMA-free medium and cultured for 24 h prior to use. LPS is used at concentration of 1 μg/ml in the medium. sf9 cells are cultured in monolayer at 28 °C in Grace's supplemented medium with 10 % heat- inactivated fetal bovine serum.

Determination of COX-2 Enzyme Activity

The effect of procyanidin dimers Al and A2, and NS398 on the activity of COX-2 is measured using baculovirus-expressed recombinant human COX-2 enzyme as previously described (Zhang et al, Acta. Pharmacol. Sin. 25(8): 1000-1006, 2004). Briefly, 24 h after infecting sf9 cells with hCOX-2 recombinant baculovirus, the cells are collected and washed in HHBS. The assays are performed as follows. One milliliter of Hank's solution containing IxIO 5 COX-2 expressing cells plus 9xlO 5 uninfected sf9 cells is dispensed per well of 24-well polypropylene plates. Inhibitor or DMSO vehicle

(10 μL) is added to the appropriate well containing the cell suspension. Following a 15- min drug or DMSO preincubation at 37 °C, the cells are challenged with 10 μmol/L arachidonic acid (Sigma) in ethanol and incubated for 10 min. Reactions are terminated by the addition of 100 μL of 1 mol/L HCl, neutralized with 100 μL of 1 mol/L NaOH. The cells are pelleted for 10 min at 300xg and the level of PGE 2 in the supernatant is determined by a PGE 2 -specific RIA (Beijing East Asia Institute of Immunology). The concentration OfPGE 2 is then determined by interpolation from a standard curve and inhibition is calculated by comparison of the PGE 2 production by drug-treated cells with that of DMSO-treated cells.

Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to assess COX-2 mRNA expression

Total RNA is extracted from macrophages with TRIzol reagent (Invitrogen Corporation, Carlsbad, CA). Real-time quantitative RT-PCR is performed using the Opticon 2 (MJ Research Inc., Waltham, MA). Sequence specific PCR primers for COX-2 [accession no. NM_000963; forward primer: 5'- GGGCAAAGACTGCGAAGAAG-3' [SEQ ID NO:1]; reverse primer: 5'- CCCATGTGACGAAATGACTG-3' [SEQ ID NO:2] and GAPDH [accession no. NM_002046; forward primer: S'-ACGGATTTGGTCGTATTGGG-S' [SEQ ID N0:3]; reverse primer: 5'-CGCTCCTGGAAGATGGTGAT-S' [SEQ ID NO:4]] are designed using the Primer Premier software version 5.00. Standard curves are run on the same plate and the relative standard curve method is used to calculate the relative gene expression.

Western Blot analysis

5x10 6 cells are resuspended in modified RIPA lysis buffer (Tris-HCl 50 mM, pH 7.4 NaCl 150 mM, EDTA 1 mM, Na-deoxycholate 0.25%, NP-40 1%, PMSF 1 mM, Na 3 VO4 1 mM, NaF 1 mM, Aprotinin 10 μg/ml, leupeptin 5 μg/ml, pepstatin 5 μg/ml), and lysed cells on ice for 45 min. The lysate is centrifuged at 14,000xg for 15 min to sediment the particulate materials. The protein concentration of the supernatant is measured by the method of Lowry (Lowry et al., J. Biol. Chem. 193:265-267, 1951). Samples are electrophoresed in SDS/PAGE gels and separated proteins are transferred

onto a PVDF membrane. The blots are blocked with 5% non-fat dry milk in Tris- buffered saline (TBS) for 1 h at room temperature and subsequently incubated overnight at 4 °C with primary antibodies diluted (1 :1000) in TBST [TBS, 1% (v/v) Tween 20 and 5% (w/v) BSA]. Following three washes of 10 min each with TBST, the blots are incubated with horseradish peroxidase-conjugated secondary antibodies in blocking buffer for 1 h at room temperature. After three washes with TBST, the blots are developed with chemiluminescence reagent and exposed to X-ray film (Kodak XAR5, Eastman Kodak, Rochester, NY, U.S.A.).

Example 2: Effect of procyanidin dimer Al in comparison with Bl dimer, B2 dimer and flavanols (Y-)- catechin and (+Vepicatechin) on COX-2 expression

Materials

Phorbol 12-myristate 13-acetate (PMA), lipopolysaccharide (LPS), and N- formyl-methionyl-leucyl-phenylalanine (fMLP), were obtained from Sigma (St. Louis, MO). Chemicals employed for gel electrophoresis were purchased from Bio-Rad (Hercules, CA). Trypsin sequencing grade was obtained from Promega (Southampton, United Kingdom). EDTA 5 EGTA, and PMSF were purchased from Amresco (Solon, OH). Al dimer was prepared from peanut skins as described in the U.S. Application Serial No. 11/045,648 (published as US 2005/0164956), which is hereby incorporated herein by reference. Flavanols (+)-catechin and (-)-eρicatechin were purchased from Sigma, and (-) catechin and (+)-epicatechin were prepared by thermally-treating (+)- catechin and (-)-epicatechin, respectively, in an aqueous solution as is known in the art. Procyanidin dimer B2 was prepared from cocoa by solvent extraction, using gel permeation chromatography, followed by further purification/isolation of a dimer enriched fraction using Normal-Phase HPLC (described in detail in Adamson et al., J. Ag. Food Chem., 1999, 47 (10):4184-4188), see also US Pat No. 5,554,645, both of which are hereby incorporated herein by reference. This material was then passed over a Cl 8 column to further enrich B2 dimer (98.3%) in the fraction which was used in the experiments described below. Bl dimer was prepared synthetically as described in US Pat No. 6,420,572, hereby incorporated herein by reference.

Cell Culture, RNA Extraction and Polymerase Chain Reaction (RT-PCR)

The human monocyte line U937 was obtained from the cell bank in Shanghai Institute of Biological Sciences, Chinese Academy of Sciences. Monocytic cells of 3-4 passages were grown to confluence in RPMI 1640 (GIBCO-BRL, Glasgow, Scotland) containing 10 % fetal bovine serum at 37 0 C in a 5 % CO 2 humidified incubator during all experimental procedures. Macrophages were pretreated with procyanidins Al dimer, Bl dimer, B2 dimer, and flavanols (-)-catechin and (+)-epicatechin (lOμM) for 2 h before LPS (lμg.ml "1 ) priming or fMLP (lμM) stimulation for 2 h. The normal macrophages without LPS or fMLP treatment were considered as controls. They were then differentiated to macrophages in the medium containing 100 nM PMA and allowed to adhere for 48 h, after which they were fed with PMA-free medium and cultured for 24 h prior to use. Cells from different groups were collected and total RNA was prepared with TRI-REAGENT-LS extraction kit. The expression of RNAs was determined by RT-PCR. Complementary DNA was created from RNA using TrueScript MMLV reverse transcriptase and oligo d (T) 18 primers. 5 μg RNA was included in each reaction. The primers of COX-2 and GAPDH are shown in the following table.

Products

Gene Name Primer sequnce

(bp)

COX-2 sense: 5'-TATACTAGAGCCCTTCCTCCTGTGCC-S' [SEQ 503

ID NO: 5] antisense: 5'-ACATCGCATACTCTGTTGTGTTCCC-S' [SEQ BD NO: 6] GAPDH sense: 5'-AAGAAGGTGGTGAAGCAGGC-S' [SEQ ID NO: 200

7] antisense: 5'-CCACCACCCTGTTGCTGTAG-S' [SEQ ID

NO: 8]

Statistical analysis of data. The data are presented as means ± SD and compared with ANOVA and least significant difference test using SPSS statistical program. The level of the statistical significance was set at P < 0.05.

Results

The anti-inflammatory effects of Al dimer in comparison with Bl dimer, B2 dimer, (-)-catechin and (+)-epicatechin were investigated. As shown in Fig. 1 A-C, (- )-catechin, (+)-epicatechin, Al, Bl and B2 showed significant inhibition of the mRNA expression of COX-2. Further, the order of suppression potency of the COX-2 mRNA expression induced by LPS and fMLP was: (-)-catechin, (+)-epicatechin > Bl, B2 >A1 (Fig. 4C).