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WO2007139887A2 - Methods for treating bone or joint inflammation - Google Patents

Methods for treating bone or joint inflammation Download PDF

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Publication number
WO2007139887A2
WO2007139887A2 PCT/US2007/012388 US2007012388W WO2007139887A2 WO 2007139887 A2 WO2007139887 A2 WO 2007139887A2 US 2007012388 W US2007012388 W US 2007012388W WO 2007139887 A2 WO2007139887 A2 WO 2007139887A2
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WO
WIPO (PCT)
Prior art keywords
methyl
enyl
hydroxy
acacia
chromen
Prior art date
Application number
PCT/US2007/012388
Other languages
French (fr)
Other versions
WO2007139887A3 (en
Inventor
Sekhar Boddupalli
Khalid Mahmood
David N. Liebowitz
Original Assignee
Cargill, Incorporated
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cargill, Incorporated filed Critical Cargill, Incorporated
Publication of WO2007139887A2 publication Critical patent/WO2007139887A2/en
Publication of WO2007139887A3 publication Critical patent/WO2007139887A3/en

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • RA rheumatoid arthritis
  • JRA juvenile onset rheumatoid arthritis
  • the joint inflammation aspects of RA are present as persistent inflammatory synovitis, usually involving peripheral joints in a symmetric distribution.
  • SLE systemic lupus erythematosis
  • Joint inflammation exerts both local and systemic effects on skeletal tissues.
  • osteoarthritis which is a degenerative joint disorder primarily affecting cartilage that covers and cushions the ends of the bones causing it to fray, wear, ulcerate, and in extreme cases, to disappear entirely leaving a bone on bone joint.
  • Osteoarthritis mostly affects the weight-bearing joints such as spine, knees and hips, but thumb and finger joints may also be affected.
  • the disorder can result in severe disability particularly in the weight bearing joints such as the knees, hips, and spine.
  • Repetitive mechanical injury of the cartilage eventually results in loss of cartilage and damage to joint surfaces and adjacent bone.
  • As a result of the tissue destruction inflammatory cells invade the joint and the synovial membrane which is manifested by pain, swelling and stiffness of the affected joints.
  • the occurrence of osteoarthritis frequently increases with advancing years. When all ages are considered, men are as frequently affected as women. But in people under age 45, more than twice as many men as women have it and between 55 and 65 more women than men have it.
  • osteoarthritis In the above 65 group, there is hardly any difference in the incidence of occurrence. [004] The prevalence of osteoarthritis will inexorably rise due to the estimated increase of life expectancy. In the developed countries, osteoarthritis is the major cause for hip and knee replacement and, as a cause of invalidism, is surpassed only by the coronary diseases. Osteoarthritis is a major issue for national health
  • U.S. Patents have been issued for herbal medicinals used for the treatment of various disorders and other health-related problems afflicting humans and animals.
  • U.S. Pat. No. 5,595,743 discloses various herbal compositions which include licorice extract (Glycyrrhiza) and Siegesbeckia, Sophora, Stemona and Tetrandra herbs, used for the treatment of various mammalian diseases, including inflammation and rheumatoid arthritis.
  • 5,683,697 discloses a pharmaceutical composition having anti-inflammatory, anti-fever, expectorant or anti-tussive action, wherein the composition includes plant parts from the species Melia, Angepica, Dendrobium, Impatiens, Citrus Medica, Loranth ⁇ s, Celosia, Cynanchum and Glehnia.
  • An herbal formulation comprising extracts of the roots, rhizomes, and/or vegetation of Alphinia, Smilax, Tinospora, Tribulus, Withania and Zingiber has been found to reduce or alleviate the symptoms associated with rheumatoid arthritis, osteoarthritis, reactive arthritis and for reducing the production of proinflammatory cytokines (U.S. Pat. No.
  • U.S. Patent Publication 2004/0185122 discloses selective COX-2 inhibition from certain edible plants and U.S. Pat. No. 6,194,469 discloses a method for inhibiting cyclooxygenase and inflammation using cherry bioflavonoids.
  • Pharmaceutical compositions containing Parthenium integrifolium or extracts thereof, are disclosed for. the suppression of COX-2 and the treatment of inflammatory diseases.
  • U.S. Pat No. 5,650,433 discloses flavonoids as chondroprotective agents, but this application neither teaches nor discloses the compositions of this invention.
  • Cyclooxygenase inhibitors have been used in therapy of joint and bone inflammation for a long time, especially those referred to as non-steroidal anti-inflammatory agents (NSAIDS), such as aspirin or ibuprofen. It is known today that these products, by inhibiting the cyclooxygenase enzyme, prevent or make difficult the conversion of arachidonic acid into prostaglandins and thromboxanes.
  • NSAIDS non-steroidal anti-inflammatory agents
  • lipoxygenases are essential enzymes for the conversion of arachidonic acid into leukotrienes, and the later compounds and their metabolites play an important role in the genesis and development of numerous inflammatory disorders.
  • the present invention relates to a method for the treatment or regulation of bone and joint inflammation, thus promoting bone and joint health, with compositions comprising compounds described in Table I.
  • the invention relates to a method of treating, preventing, or inhibiting bone or joint inflammation in a subject, which comprises administering to the subject a therapeutically effective amount of at least one plant extract comprising at least one compound selected from
  • the plant extract is selected from at least one plant genus selected from: Acacia; Anaphalis; Artocarpus; Berberis; Bro ⁇ ssonetia; Cajanus; Cudrania; Dal ⁇ a; Desmodium; Erythrina; Fagopyrum; Flemingia; Helichrysum; Hum ⁇ lus; Hypericum; Lonchocarpus; Lotusi; L ⁇ pinus; Madura; Melicope; Melilotus; Mundul ⁇ a; Platanus ; Psolarea; Scoparia; Bitumina ⁇ a; Dalbergia; Dolichos; Genista; Hardenbergia; Mucuna; Neonotonia;; Pueraria; Artemisia; Myrsine; Rapanea; Astragalus; Baphia; Centrosema; Cicer, Eclipta; Eclipta; Hedysarium; Lens; Myroxylon; Ononis; Pericopsis; Pisum; Pterocar
  • the method of treating, preventing, or inhibiting bone or joint inflammation in a subject comprises administering to the subject a therapeutically effective amount of a nutraceutical composition comprising at least one compound of the compounds in Table I, and in other embodiments the nutraceutical composition comprises at least one plant extract selected from Acacia; Anaphalis; Artocarpus; .Berberis; Broussonetia; Cajanus; Cudrania; Dalea; D ⁇ smodium; Erythrina; Fagopyrum; Flemingia; Helichrysum; Humulus; Hypericum; Lonchocarpus; Lotusi; Lupinus; Madura; Melicope; Melilotus; Mundulea; Platanus ; Psolarea; Scoparia; Bituminaria; Dalbergia; Dolichos; Genista; Hardenbergia; Mucuna; Neonotonia; Pueraria; Artemisia; Myrsine; Rapanea;
  • the nutraceutical compositions comprise plant extracts that are enriched with at least one compound selected from:
  • compositions are useful for the treatment of arthritis, such as gout, childhood arthritis, lupus arthritis, rheumatoid arthritis, or osteoarthritis. In other embodiments the compositions are useful for the treatment of joint inflammation, joint swelling or joint pain.
  • the route of administration is oral, and in other embodiments the route of administration is a medical food, a functional food, a special nutrition food, or a dietary supplement which may additionally include one or more ingredients comprising vitamins, minerals, herbs, botanicals, amino acids, and/or dietary substances intended to supplement the diet by increasing total dietary intake.
  • the administration is a medical food, a functional food, a special nutrition food, or a dietary supplement which may additionally include one or more ingredients comprising glucosamine, glucosamine hydrochloride, glucosamine sulfate, chondroitin, chondroitin sulfate, calcitonin, collagen, collagen proteins, or hydrolyzed collagen.
  • the administration is a medical food, a functional food, a special nutrition food, or a dietary supplement which may additionally include an analgesic or an NSAID.
  • the composition is administered as a regular or controlled release formulation. In other embodiments, the composition is administered topically on the epidermis covering the joint in need of such treatments.
  • the subject is a human; in other embodiments the subject is an animal, such as, but not limited to farm animals such as cattle, horses, sheep, goats and swine; or domestic animals such as rabbits, dogs, cats, rats, mice and guinea pigs.
  • the plant extract is selected from the group including, but not limited to, stems, stem barks, trunks, trunk barks, twigs, tubers, roots, root barks, young shoots, seeds, grains, nuts, nut skins, rhizomes, tubers, fruits, fruit skins, flowers and other reproductive organs, leaves and other aerial parts, callus cultures, and cell cultures.
  • analgesic refers to but is not limited to, acetaminophen, ibuprofen, aspirin, salicyclamide, trolamine salicylate, methyl salicylate, salicyalte salts, N,N-dimethyl aspartic acid, N.N-dimethyl glutamic acid, and antipyrine
  • Amelioration refers to prevention, reduction or palliation of a state, or improvement of the state of a subject in need of improvement or the reduction of the symptoms associated with irritation. Amelioration includes, but does not require complete recovery or complete prevention.
  • arthritis refers to the inflammation of a joint or joints which results in pain and swelling.
  • the two most common forms of arthritis are osteoarthritis and rheumatoid arthritis.
  • Osteoarthritis is characterized by chronic degeneration of the cartilage of the joints, mainly in older persons.
  • Rheumatoid arthritis sometimes called arthritis deformans, is a chronic and progressive systemic disorder, especially common in women, characterized by stiffness. swelling and inflammation of the joints and sometimes leading to deformity and permanent disability.
  • formulation refers to a combination of active components or ingredients that are administered together or separately under a coordinated dosing regimen.
  • a formulation need not consist of admixed components. Rather, it may include components that are given separately in different oral forms or even via different modes of administration, for example as a combination of oral and parenteral treatments.
  • a formulation may also comprise a "kit” whereby components are bundled together in a combination packaging format.
  • joint disorders include, but are not limited to: joint inflammation, joint swelling, joint pain, and arthritis, which consists of more than 100 different joint-related disorders including, rheumatoid arthritis, juvenile rheumatoid arthritis, childhood arthritis, psoriatic arthritis, Reiter's syndrome (also referred to as reactive arthritis), sarcoidosis; osteoporosis; osteoarthritis, gout, pseudogout, lupus arthritis, calcification in joints, synovial osteochondromatosis (SOC), chronic back injury, diffuse idiopathic skeletal hyperostosis, ankylosing spondylitis, synovitis, including, but not limited to, rheumatic synovitis, pigmented villonodular synovitis, and chronic synovitis, and temporomand
  • joint disorders through association, such as, but not limited to: congenital and acquired torticollis, osteochondroma, osteoporosis, Sjogren's syndrome, avascular necrosis (also referred to as osteonecrosis), osteomyelitis, carpal tunnel syndrome, repetitive stress injury, dysplasia of joints, fibromyalgia, shin splints, broken bones, stress fractures, scleroderma, scoliosis, bursitis, lyme disease, and Paget's disease.
  • congenital and acquired torticollis such as, but not limited to: congenital and acquired torticollis, osteochondroma, osteoporosis, Sjogren's syndrome, avascular necrosis (also referred to as osteonecrosis), osteomyelitis, carpal tunnel syndrome, repetitive stress injury, dysplasia of joints, fibromyalgia, shin splints, broken bones, stress fractures, s
  • NSAID refers to any compound acting as a nonsteroidal anti-inflammatory agent identifiable as such by one of ordinary skill in the art. NSAIDs are categorized by virtue of their ability to inhibit cyclooxygenase.
  • the term NSAID includes, but is not limited to, salicylates, indomethaci ⁇ , flurbiprofen, diclofenac, ketorolac, naproxen, piroxicam, tebufelone, ibuprofen, etodolac, nabumetone, tenidap, alcofenac, antipyrine, aminopyrine, dipyrone. aminopyrone, phenylbutazone, clofezone.
  • the salicylates may include acetylsalicylic acid, sodium acetylsalicylic acid, calcium acetylsalicylic acid, salicylic acid, and sodium salicylate.
  • NSAID also includes known spices and herbs with anti-inflammatory activity, such as Boswellia serrata, ginger, turmeric, holy basil, green tea extract, resveratrol, white willow bark, devil's claw, capsicum, stinging nettle, wintergreen, and pineapple (bromelain).
  • nutraceutical refers to any compounds or chemicals from natural sources that can provide dietary or health benefits when consumed by humans or animals. Examples of nutraceuticals include, but are not limited to plant extracts.
  • rheumatism refers to any of several pathological conditions of the muscles, tendons, joints, bones, or nerves, characterized by discomfort and disability. The term includes but is not limited to arthritis.
  • rheumatism refers to arthritis, articular or acute rheumatism, Besnier's rheumatism, Heberden's rheumatism, nodose rheumatism, osseous rheumatism, palindromic rheumatism, and Poncet's rheumatism.
  • subject includes, but is not limited to, humans and animals, such as farm animals (cattle, horses, sheep, goats, and swine) and domestic animals (rabbits, dogs, cats, rats, mice and guinea pigs).
  • treatment relates to any treatment of a disorder, in a mammal, including: preventing or protecting against the disorder, that is, causing the clinical symptoms of the disorder not to develop; inhibiting the disorder, that is, arresting or suppressing the development of clinical symptoms; and/or relieving the disorder, that is, causing the regression of clinical symptoms It will be understood by those skilled in the art that it is not always possible to distinguish between “preventing” and “suppressing” since the ultimate inductive event or events may be unknown, latent, or the patient is not ascertained until well after the occurrence of the event or events.
  • prophylaxis is intended as an element of “treatment” to encompass both “preventing” and “suppressing” as defined herein.
  • protection as used herein, is meant to include “prophylaxis.” [031] As used herein, the term “comprising” and its cognates are used in their inclusive sense; that is, equivalent to the term “including” and its corresponding cognates.
  • compositions and methods of the present invention are useful for promoting joint and bone health by, for example, preventing and/or treating joint and bone inflammation in a subject in need of such treatment.
  • the compounds of the present invention may target certain enzymes such as cyclooxygenases or lipoxygenases that function across a variety of physiological processes.
  • compositions of the present invention may be useful for the treatment of disorders involving inflammation of the joints, such as, but not limited to, rheumatism, joint inflammation, joint swelling, joint pain, rheumatoid arthritis, juvenile rheumatoid arthritis, childhood arthritis, psoriatic arthritis, lupus arthritis, Reiter's syndrome (also referred to as reactive arthritis), sarcoidosis, osteoporosis, osteoarthritis, gout; pseudogout, ankylosing spondylitis, calcification in joints, synovial osteochondromatosis (SOC), chronic back injury, diffuse idiopathic skeletal hyperostosis, ankylosing spondylitis, rheumatic synovitis, pigmented villonodular synovitis, chronic synovitis, and temporomandibular joint disorder (TMJ).
  • rheumatism joint inflammation
  • joint swelling joint pain
  • rheumatoid arthritis juvenile rhe
  • compositions may also be useful in the treatment of disorders that may develop joint disorders through association, such as, but not limited to: congenital and acquired torticollis, osteochondroma, osteoporosis, Sjogren's syndrome, avascular necrosis (also referred to as osteonecrosis), osteomyelitis, carpal tunnel syndrome, repetitive stress injury, dysplasia of joints, fibromyalgia, shin splints, broken bones, stress fractures, scleroderma, scoliosis, bursitis, lyme disease, and Paget's disease.
  • disorders may develop joint disorders through association, such as, but not limited to: congenital and acquired torticollis, osteochondroma, osteoporosis, Sjogren's syndrome, avascular necrosis (also referred to as osteonecrosis), osteomyelitis, carpal tunnel syndrome, repetitive stress injury, dysplasia of joints, fibromyalgia, shin splin
  • compositions can be prepared as foods for humans or animals, including medical foods, functional food, special nutrition foods, and dietary supplements.
  • a "medical food” is a product prescribed by a physician that is intended for the specific dietary management of a disorder or health condition for which distinctive nutritional requirements exist, and may include formulations fed through a feeding tube (referred to as enteral administration or gavage administration).
  • a "dietary supplement” shall mean a product that is intended to supplement the human diet and may be provided in the form of a pill, capsule, tablet, or like formulation.
  • a dietary supplement may include one or more of the following dietary ingredients: vitamins, minerals, herbs, botanicals, amino acids, and dietary substances intended to supplement the diet by increasing total dietary intake, or a concentrate, metabolite, constituent, extract, or combinations of these ingredients, not intended as a conventional food or as the sole item of a meal or diet.
  • Dietary supplements may also be incorporated into food stuffs, such as functional foods designed to promote joint and bone health or to prevent inflammation.
  • a "functional food” is an ordinary food that has one or more components or ingredients incorporated into it to give a specific medical of physiological benefit, other than a purely nutritional effect.
  • Specific nutrition food means ingredients designed for particular diet related to conditions or to support treatment of nutritional deficiencies.
  • Formulations of the invention may be administered in nutritionally accepted vehicles for oral ingestion, such as, capsules, tablets, or pills, soft gel caps, powders, solutions, dispersions, or liquids.
  • nutritionally accepted vehicles for oral ingestion such as, capsules, tablets, or pills, soft gel caps, powders, solutions, dispersions, or liquids.
  • any of the usual media may be employed.
  • media containing, for example, water, oils, alcohols, flavoring agents, preservatives, coloring agents and the like may be used.
  • Carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like may be used to prepare oral solids (e.g., powders, capsules, pills, tablets, and lozenges).
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free- flowing form such as a powder or granules, optionally mixed with a binder (e.g. povidone, gelatin, hydroxy propylmethylcellulose), lubricant, inert diluent, preservative, disintegrant (e.g.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide controlled release of the active ingredients therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide the desired release profile.
  • Soft gel caps may contain lipophilic substances, such as tocopherols and polyunsaturated fatty acids. Methods for preparing gel caps are well known in the art.
  • the subject formulations may be compounded with other physiologically acceptable materials which can be ingested including foods, for example, food bars, beverages, powders, cereals, cooked foods, food additives and candies.
  • the food can be a dietary supplement (such as a snack or wellness dietary supplement) or, especially for animals, comprise the nutritional bulk (e.g., when incorporated into a primary animal feed, a grain ration, or incorporated into a salt block).
  • the dietary supplements and medical foods of the present invention can be used in powder form which can be dissolved in a liquid suitable for human consumption, such as water or a fruit juice.
  • the compositions of the present invention may be formulated with a liquid foodstuff, for example, milk, juices, liquid vitamin supplements, and oral rehydration(electrolyte) solutions.
  • the dietary supplements and medical foods of the present invention are preferably administered two times per day, preferably once in the morning and once in the afternoon.
  • a typical treatment regime for the dietary supplements or medical foods will continue for four to eight weeks. Depending on such factors as the medical condition being treated and the response of the patient, the treatment regime may be extended.
  • a medical food of the present invention will typically be consumed in two servings per day as either a meal replacement or as a snack between meals.
  • a serving size for a medical food of the present invention will preferably be in the range of from about 45 grams to about 60 grams and will provide from about 180 calories to about 220 calories to the consumer.
  • a person in need of treatment is provided with two servings of a medical food of the present invention per day.
  • composition of the present invention may further comprise vitamins, minerals, coenzymes, organic or inorganic antioxidants or precursors thereof.
  • the coenzyme may be coenzyme Q10
  • the organic antioxidant may be selected from the group comprising lipoic acid, resveratrol and glutathione
  • the inorganic antioxidant may be selenium.
  • the vitamins may be selected from the group vitamin A (palmitate), vitamin B-1, vitamin B-2, vitamin B-6, vitamin B-12, vitamin C (ascorbic acid), vitamin D-3, vitamin E (alpha, beta, gamma, delta tocopherol or mixtures thereof), vitamin K-1, niacin, folic acid, biotin, choline, inositol, and pantothenic acid; and the amino acids may be selected from the group L-alanine, L-arginine, L-aspartic acid, L-cystine, L-glutamic acid, L- glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-proline.
  • the minerals may be selected from the group calcium, potassium, magnesium, phosphorous, zinc, manganese, selenium, chromium, and vanadium.
  • flavoring agents may be added to the compositions of the invention for tasting purposes.
  • flavors are present in an amount from about 0.05 to about 0.5, preferably from about 0.1 to about 0.3 percent by weight, based on the total weight of the compositions.
  • the amount of the composition ingested, consumed or otherwise administered will depend on the desired final concentration. Typically, the amount of a single administration of the formulation of the invention can be about 0.1 to about 10,000 mg per day. Any of these doses can be further subdivided into separate administrations, and multiple dosages can be given to any individual patient.
  • compositions are administered in one dosing of a single formulation and in other embodiments, compositions are administered in multiple dosing of a single formulation.
  • the components are administered to a subject within a time period between about 3 hours to about 6 hours. In other embodiments, the time period is between about 6 hours and 12 hours. In additional embodiments, the time period is between about 12 hours and 24 hours. In yet further embodiments, the time period is between about 24 hours and 48 hours.
  • the administration of separate formulations can be simultaneous or staged throughout a specified time period.
  • the composition and may comprise 0.2-2% of the active agent in solution.
  • a method for alleviating joint and bone inflammation encompasses co-administering a dietary supplement of the invention with a separately formulated therapeutically active agent or compound (e.g., a drug such as an analgesic or an NSAID 1 or a bio-active molecule such as such as glucosamine, glucosamine hydrochloride, glucosamine sulfate, chondroitin, chondroitin sulfate, calcitonin, collagen, collagen proteins, or hydrolyzed collagen) to obtain an enhanced and/or more rapid relief of the condition sought to be alleviated.
  • a therapeutically active agent or compound e.g., a drug such as an analgesic or an NSAID 1 or a bio-active molecule such as such as glucosamine, glucosamine hydrochloride, glucosamine sulfate, chondroitin, chondroitin sulfate, calcitonin, collagen, collagen proteins, or hydrolyze
  • compositions may be administered topically.
  • a topical administration refers to application of the present compositions by spreading, dabbing, dusting, swabbing, sponging, dispensing, covering and coating the affected area.
  • the compositions of the present invention may be formulated for topical administration to the epidermis covering the joint in need of such treatment as ointments, creams, gels, lotions or transdermal patches.
  • Topical formulations of the present invention typically comprise the composition of the invention and optionally, a polar solvent.
  • Solvents suitable for use in the formulations of the present invention include any polar solvent capable of dissolving the compositions of the invention.
  • Suitable polar solvents include: water; alcohols (such as ethanol, propyl alcohol, isopropyl alcohol, hexanol, and benzyl alcohol); polyols (such as propylene glycol, polypropylene glycol, butylene glycol, hexylene glycol, sorbitol, and glycerin); and panthenol dissolved in glycerin, flavor oils and mixtures thereof. Mixtures of these solvents can also be used.
  • An emollient may also be added to the topical compositions of the present invention.
  • the emollient component can comprise fats, oils, fatty alcohols, fatty acids and esters which aid application and adhesion, yield gloss, and most importantly, provide occlusive moisturization.
  • Suitable emollients for use are isostearic acid derivatives, isopropyl palmitate, lanolin oil, diisopropyl dimerate, maleated soybean oil, octyl palmitate, isopropyl isostearate, cetyl lactate, cetyl ricinoleate, tocopheryl acetate, acetylated lanolin alcohol, cetyl acetate, phenyl trimethicone, glyceryl oleate, tocopheryl linoleate, wheat germ glycerides, arachidyl propionate, myristyl lactate, decyl oleate, propylene glycol ricinoleate, isopropyl lanolate, pentaerythrityl tetrastearate, neopentylglycol dicaprylate/dicaprate, hydrogenated coco- glycerides, isononyl isononanoate, is
  • solidify refers to the physical and/or chemical alteration of the liquid base material so as to form a solid or semi-solid at ambient conditions, i.e., to form a final composition that has a stable physical structure and can be deposited on the skin under normal use conditions.
  • the selection of the particular solidifying agent will depend upon the particular type of composition desired, i.e., gel or wax-based, the desired rheology, the liquid base material used and the other materials to be used in the composition.
  • compositions and methods of administration are meant to describe but not limit the methods and compositions of the present invention.
  • the methods of producing various compositions and devices are within the ability of one skilled in the art and are not described in detail here.
  • Anti-inflammatory activity of a composition of the present invention, or activity of components administered in methods of the present invention, can be experimentally tested in vitro or in vivo. Inflammation may, for example, be assessed in an assay which measures the ability of the composition to inhibit the cyclooxygenase or lipoxygenase enzymes.
  • the 5-lipoxygenase pathway is a major synthetic pathway relevant to human inflammatory disease. 5-lipoxygenase catalyses the two first steps in the oxygenation of arachidonic acid (a polyunsaturated 20-carbon fatty acid) to leukotrienes. Leukotrienes are known to be important mediators of inflammatory and allergic reactions.
  • the first step in the synthesis of leukotrienes which is catalyzed by 5-lipoxygenase, is the formation of 5-HPETE.
  • the rearrangement of 5-HPETE to form the unstable LTA 4 is also catalyzed by 5-lipoxygenase.
  • LTA 4 is then converted to either LTB 4 or LTC 4 .
  • LTC 4 is rapidly metabolized to LTD 4 and then to LTE 4 .
  • LTC 4 , LTD 4 and LTE 4 are collectively referred to as the cysteinyl (Cys) leukotrienes.
  • LTB 4 Biosynthesis of LTB 4 , C 4 , D 4 and E 4 occurs predominantly in leukocytes, in response to a variety of immunological stimuli.
  • the primary target of LTB 4 is the leukocyte where it elicits enzyme release, chemotaxis, adherence, and aggregation in nM concentrations.
  • LTB 4 modulates immune responses and participates in the host-defense against infections.
  • LTB 4 is an important chemical mediator in the development and maintenance of inflammatory reactions and disease states.
  • a useful cell screening assay exemplified herein in Examples is the E-selectin
  • ELAM ELAM production assay, which measures activity of test compounds in reducing expression of E-selectin in activated endothelial cells.
  • endothelial cells are activated by adding known activators such as lipopolysaccharide, TNF, or IL-1 ⁇ , alone or in some combination.
  • Activated cells produce E-selectin, which can be measured using, for example, an E-selectin monoclonal antibody-based ELISA assay.
  • ELAM production was decreased by formulations containing enriched forms of gamma-tocopherol, beta-tocopherol, and delta-tocopherol but not by formulations enriched in alpha-tocopherol.
  • compositions and methods of administration are meant to describe but not limit the methods and compositions of the present invention.
  • the methods of producing various compositions and devices are within the ability of one skilled in the art and are not described in detail here.
  • FOX reagent 25mM sulphuric acid, 100 ⁇ M xylenol orange, 100 ⁇ M iron (II) sulphate, methanolrwater 9:1).
  • the yellow color of acidified xylenol orange was converted to a blue color by the lipid hydroperoxide-mediated oxidation of Fe 2+ ions and the interaction of the resulting
  • Fe 3* ions with the dye.
  • the complex was allowed to form during a one hour incubation at room temperature with shaking. Absorbance of the Fe 3+ complex was then measured at 62OnM using a spectrophotometer.
  • Negative controls contained enzyme during the incubation step but substrate was not added until after the FOX reagent.
  • Porcine Leukocyte 12/15-lipoxygenase (Cayman Cat # 60300) was used in this assay. Test compound and/or vehicle was added to 1.3U 12/15-lipoxygenase in 5OmM Tris-HCI buffer, pH 7.4. The reaction was initiated by addition of 70 ⁇ M arachidonic acid in Tris-HCI buffer, pH 7.4, and terminated after a 10 minute incubation at room temperature by addition of FOX reagent (25mM sulphuric acid, 100 ⁇ M xylenol orange, 100 ⁇ M iron (II) sulphate, methanohwater 9:1).
  • FOX reagent 25mM sulphuric acid, 100 ⁇ M xylenol orange, 100 ⁇ M iron (II) sulphate, methanohwater 9:1).
  • the yellow color of acidified xylenol orange was converted to a blue color by the lipid hydroperoxide-mediated oxidation of Fe 2 " ions and the interaction of the resulting Fe 3* ions with the dye.
  • the complex was allowed to form during a one hour incubation at room temperature with shaking. Absorbance of the Fe 3+ complex was then measured at 62OnM using a spectrophotometer. Negative controls contained enzyme during the incubation step but substrate was not added until after the FOX reagent. [066] Compounds were screened at 5 concentrations in triplicate starting at 10 ⁇ M.
  • This example provides exemplary assays for measuring inflammatory reaction in a cell line. Specifically, this assay provides a predictive measure of anti-inflammatory activity of compositions of the present invention.
  • the Hep3B cell line was derived from liver tissue of an 8-year-old Black male.
  • the cells are epithelial in morphology and produce tumors in nude mice.
  • the cells produce ⁇ -fetoprotein, hepatitis B surface antigen, albumin, ⁇ -2-macroglobulin, ⁇ -1 -antitrypsin, transferrin, plasminogen, complement C3 and ⁇ -lipoprotein (Knowles BB, et al., Science, , 209:497-499, 1980).
  • HEP3B cells are grown in Minimum Essential Medium (MEM; GlBCO) supplemented with 10% Fetal Bovine Serum (FBS; Hyclone). 1x Penicillin/Streptomycin (GIBCO, Cat #. 15140-122) and 0.1mM non-essential amino acids (GIBCO, Catalog No. 11140-050). Cells are thawed and transferred to warm medium according to standard methods known in the art. [069] Cells are incubated in flasks at 37°C with 5% CO 2 in an air atmosphere incubator.
  • HEP3B growth media is changed every 2 days until the cells reach 70-80% confluence (approx. 3-4 days).
  • the cells are transferred to 96-well plates, seeded at 5000 cells per well in culture media, and left to grow for 7 days in a 37°C incubator (air supplemented with 5% CO 2 ). Media is replaced daily until assay.
  • Test compounds are diluted into "Stimulus Buffer” (MEM medium containing 0.1 mM non-essential amino acids, 1X penicillin/streptomycin, 10% FBS with 10 ng/ml IL-1 ⁇ , 20 ng/ml lL-6 and 1 ⁇ M dexamethasone. Media is removed from the cells and is replaced with 200 ⁇ l of test dilution. Cells are returned to the incubator for three days at 37 * C. CRP ELISA is then performed on supernatant from the cells, as described below.
  • Stimulus Buffer MEM medium containing 0.1 mM non-essential amino acids, 1X penicillin/streptomycin, 10% FBS with 10 ng/ml IL-1 ⁇ , 20 ng/ml lL-6 and 1 ⁇ M dexamethasone. Media is removed from the cells and is replaced with 200 ⁇ l of test dilution. Cells are returned to the incubator for three days at 37 * C. CRP ELISA is then performed
  • CRP measured as above is normalized to cell count per well, using a cell viability assay, such as the Cell Tracker Green assay. To do this, the remainder of the medium is from the cell test plates, cells are washed with 200. ⁇ L of pre-warmed 1x Hanks Basic Salt Solution (HBSS; GIBCO) 1 and 100 ⁇ L of 5 ⁇ M Cell Tracker Green (Molecular Probes, Eugene, OR) is added to each well. Plates are then incubated at 37°C for 30 minutes. Cells are then washed twice with prewarmed 1x HBSS. Plates are immediately read using a Fluoroskan® fluorometer with a 485 excitation/538 emission filter pair.
  • HBSS Hanks Basic Salt Solution
  • Endothelial-Leukocyte Adhesion Molecule (ELAM) 1 also known as E-selectin, is expressed on the surface of endothelial cells.
  • LPS lipopolysaccharide
  • IL-1 ⁇ IL-1 ⁇
  • test agents were tested for their abilities to reduce this expression, in accordance with studies showing that reduction of leukocyte adhesion to endothelial cell surface is associated with decreased cellular damage (e.g., Takada, M., Et al., Transplantation 64: 1520-25, 1997; Steinberg, J.B., et al., J. Heart Lung Trans. 13:306-313. 1994).
  • Endothelial cells may be selected from any of a number of sources and cultured according to methods known in the art; including, for example, coronary artery endothelial cells, human brain microvascular endothelial cells (HBMEC; Hess, D.C., et al., Neurosci. Lett. 213(1): 37-40, 1996), or lung endothelial cells.
  • HBMEC human brain microvascular endothelial cells
  • HBMEC human brain microvascular endothelial cells
  • et al. Neurosci. Lett. 213(1): 37-40, 1996)
  • lung endothelial cells were conveniently cultured in 96-well plates. Cells were stimulated by adding a solution to each well containing 10 ⁇ g/ml LPS and 100 pg/ml IL-1 ⁇ for 6 hours in the presence of test agent (specific concentrations and time may be adjusted depending on the cell type).
  • Treatment buffer was removed and replaced with pre-warmed Fixing Solution® (100 ⁇ l/well) for 25 minutes at room temperature. Cells were then washed 3X, then incubated with Blocking Buffer (PBS + 2% FBS) for 25. minutes at room temperature. Blocking Buffer containing Monoclonal E-Selectin Antibody (1 :750, Sigma Catalog #S-9555) was added to each well. Plates were sealed and stored at 4 0 C overnight. Plates were washed 4X with 160 ⁇ L Blocking Buffer per well. Second Antibody-HRP diluted 1 :5000 in Blocking Buffer was then added (100 ⁇ LVwell), and plates were incubated at room temperature (protected from light) for two hours.
  • Blocking Buffer PBS + 2% FBS
  • This assay measures the ability of test compounds to prevent or reduce inflammation secondary to oxazolone or arachidonic acid.
  • CD-1 mice are induced by applying 3% oxazolone (Sigma) (30 mg/ml prepared in corn oikaceto ⁇ e) to the shaved abdomen. Five days later, the mice are challenged with 2% oxazolone (20 mg/ml) in acetone on the left ear (right ear was untreated control). One hour after challenge, test compounds are applied to the left ear. in 70% ethanol/30% propylene glycol. Animals are sacrificed 24 hours later and 7 mm ear punches are removed. The ear punches are placed on a balance scale, and the difference between the untreated and treated ears is determined. Percent inhibition is calculated by comparing the means of each group to the vehicle group. (Hydrocortisone serves as a positive control in this test.)
  • Cyclooxygenase-2 (human recombinant, expressed in Sf9 cell, Cayman 60122) is used. Test compound and/or vehicle is pre-incubated with 0.11 U cyclooxygenase-2, 1 mM reduced GSH, 500 ⁇ M phenol and 1 ⁇ M hematin for 15 minutes at 37 0 C. The reaction is initiated by addition of 0.3 ⁇ M arachidonic acid as substrate in Tris-HCI pH 7.7 and terminated after 5 minutes incubation at 37 0 C by addition of 1N HCI. Following centrifugation, substrate conversion to PGE 2 is measured by an Amersham EIA kit. Compounds are screened at 10 ⁇ M.
  • PBMC peripheral blood mononuclear cells
  • Cells were activated with phorbol ester to express cyclooxygenase-2 and produce prostaglandins.
  • Prostaglandin production was quantified by enzyme linked immunosorbent assay (ELISA).
  • 2-enyl)-chroman-4-one was isolated by methanol extraction of the suspension cell culture of L ⁇ pinus micranthus, followed by ethyl acetate/water partitioning.
  • the crude ethyl acetate extract was then passed through an HP-20 resin column using an aqueous acetonitrile gradient, and was purified over silica gel using mixtures of dichloromethane and methanol, and the purified mixture was then subjected to preparative HPLC/MS on C18 to isolate the compound with the right.

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Abstract

The present invention provides methods for promoting bone and joint health with certain compositions described herein or with optionally enriched extracts of plant material comprising such compositions. The present invention provides methods for the treatment or regulation of, bone and joint conditions such as joint inflammation, joint swelling, joint pain, gout, childhood arthritis, lupus arthritis, rheumatoid arthritis, and osteoarthritis.

Description

METHODS FOR PROMOTING JOINT AND BONE HEALTH
BACKGROUND
[001] The prevention or inhibition of bone and joint inflammation is of significant concern because these disorders can cause severe pain, stiffness, fatigue and sometimes, a loss of motion to its sufferers. Joint inflammation is associated with a group of disorders that are referred to as arthritis, and exerts both local and systemic effects on skeletal tissues. Disorders commonly associated with joint inflammation are rheumatoid arthritis (RA) and juvenile onset rheumatoid arthritis (JRA), both chronic multisystem disorders of unknown cause. The joint inflammation aspects of RA are present as persistent inflammatory synovitis, usually involving peripheral joints in a symmetric distribution. Many patients with systemic lupus erythematosis (SLE) also develop joint inflammation referred to as lupus arthritis. [002] Joint inflammation exerts both local and systemic effects on skeletal tissues.
Three forms of bone disorder have been described in rheumatoid arthritis, namely: focal bone toss affecting the immediate subchondral bone and bone at the joint margins; periarticular osteopenia adjacent to inflamed joints; and generalized osteoporosis involving the axial and appendicular skeleton (Goldring, S. R. and Gravallese, E. M. Mechanisms of bone loss in inflammatory arthritis: diagnosis and therapeutic implications. Arthritis Res. 2000; 2(1):33-7). [003] Another common form of arthritis is osteoarthritis which is a degenerative joint disorder primarily affecting cartilage that covers and cushions the ends of the bones causing it to fray, wear, ulcerate, and in extreme cases, to disappear entirely leaving a bone on bone joint. Osteoarthritis mostly affects the weight-bearing joints such as spine, knees and hips, but thumb and finger joints may also be affected. The disorder can result in severe disability particularly in the weight bearing joints such as the knees, hips, and spine. Repetitive mechanical injury of the cartilage eventually results in loss of cartilage and damage to joint surfaces and adjacent bone. As a result of the tissue destruction, inflammatory cells invade the joint and the synovial membrane which is manifested by pain, swelling and stiffness of the affected joints. The occurrence of osteoarthritis frequently increases with advancing years. When all ages are considered, men are as frequently affected as women. But in people under age 45, more than twice as many men as women have it and between 55 and 65 more women than men have it. In the above 65 group, there is hardly any difference in the incidence of occurrence. [004] The prevalence of osteoarthritis will inexorably rise due to the estimated increase of life expectancy. In the developed countries, osteoarthritis is the major cause for hip and knee replacement and, as a cause of invalidism, is surpassed only by the coronary diseases. Osteoarthritis is a major issue for national health
[005] U.S. Patents have been issued for herbal medicinals used for the treatment of various disorders and other health-related problems afflicting humans and animals. For example, U.S. Pat. No. 5,595,743 discloses various herbal compositions which include licorice extract (Glycyrrhiza) and Siegesbeckia, Sophora, Stemona and Tetrandra herbs, used for the treatment of various mammalian diseases, including inflammation and rheumatoid arthritis. U.S. Pat. No. 5,683,697 discloses a pharmaceutical composition having anti-inflammatory, anti-fever, expectorant or anti-tussive action, wherein the composition includes plant parts from the species Melia, Angepica, Dendrobium, Impatiens, Citrus Medica, Loranthυs, Celosia, Cynanchum and Glehnia. An herbal formulation comprising extracts of the roots, rhizomes, and/or vegetation of Alphinia, Smilax, Tinospora, Tribulus, Withania and Zingiber has been found to reduce or alleviate the symptoms associated with rheumatoid arthritis, osteoarthritis, reactive arthritis and for reducing the production of proinflammatory cytokines (U.S. Pat. No. 5,683,698). U.S. Patent Publication 2004/0185122 discloses selective COX-2 inhibition from certain edible plants and U.S. Pat. No. 6,194,469 discloses a method for inhibiting cyclooxygenase and inflammation using cherry bioflavonoids. Pharmaceutical compositions containing Parthenium integrifolium or extracts thereof, are disclosed for. the suppression of COX-2 and the treatment of inflammatory diseases. U.S. Pat No. 5,650,433 discloses flavonoids as chondroprotective agents, but this application neither teaches nor discloses the compositions of this invention. [006] Cyclooxygenase inhibitors have been used in therapy of joint and bone inflammation for a long time, especially those referred to as non-steroidal anti-inflammatory agents (NSAIDS), such as aspirin or ibuprofen. It is known today that these products, by inhibiting the cyclooxygenase enzyme, prevent or make difficult the conversion of arachidonic acid into prostaglandins and thromboxanes. [007] It has also been found that lipoxygenases are essential enzymes for the conversion of arachidonic acid into leukotrienes, and the later compounds and their metabolites play an important role in the genesis and development of numerous inflammatory disorders. [008] Unfortunately, many known anti-inflammatory agents have more or less severe adverse side effects, such as effects of gastrointestinal type and in some cases of cardiovascular type. Therefore, the search for new products with anti-inflammatory activity for the treatment of bone and joint inflammation is still very important, and there is a need for novel safe compositions for the treatment of inflammation associated with rheumatoid arthritis and osteoarthritis. There is also a need for a dietary supplement and/or medical food that ameliorates at least one of the symptoms, preferably all of the symptoms of joint inflammation- related disorders such as arthritis. The novel compositions of the present invention fulfill those requirements.
DISCLOSURE OF THE INVENTION
[009] The present invention relates to a method for the treatment or regulation of bone and joint inflammation, thus promoting bone and joint health, with compositions comprising compounds described in Table I.
Figure imgf000004_0001
and single stereoisomers, mixtures of stereoisomers, or pharmaceutically acceptable salts thereof.
[010] In another embodiment, the invention relates to a method of treating, preventing, or inhibiting bone or joint inflammation in a subject, which comprises administering to the subject a therapeutically effective amount of at least one plant extract comprising at least one compound selected from
3-[2,4-dihydroxy-3-(3-methyl-but-2-enyl)-phenylj-5,7-dihydroxy-chromen-4-one;
• 5,7-dihydroxy-3-[4-hydroxy-3-(3-methyl-but-2-enyl)-pheny!]-6-(3-methyl-but-2-enyl)- chromen-4-one; 3,5,7-trihydroxy*2-[4-hydroxy-3-(3-methyl-but-2-enyl)-phenyl]-chromen-4-one;
• i-methoxy^-CS-methyi-but^-enylJ-eH-benzotA.SJfuroIS^-clchromene-S.Θ-diol; 2-[3-hydroxy-5-methoxy-4-(3-methyl-but-2-enyl)-phenyl]-benzofuran-3,6-diol;
• 4-[7-hydroxy-5-methoxy-6-(3-methyl-but-2-enyl)-2H-chromen-3-yl]-benzene-1 ,3-diol; and single stereoisomers, mixtures of stereoisomers, or pharmaceutically acceptable salts thereof.
[011] In some embodiments, the plant extract is selected from at least one plant genus selected from: Acacia; Anaphalis; Artocarpus; Berberis; Broυssonetia; Cajanus; Cudrania; Dalβa; Desmodium; Erythrina; Fagopyrum; Flemingia; Helichrysum; Humυlus; Hypericum; Lonchocarpus; Lotusi; Lυpinus; Madura; Melicope; Melilotus; Mundulβa; Platanus ; Psolarea; Scoparia; Bituminaήa; Dalbergia; Dolichos; Genista; Hardenbergia; Mucuna; Neonotonia;; Pueraria; Artemisia; Myrsine; Rapanea; Astragalus; Baphia; Centrosema; Cicer, Eclipta; Eclipta; Hedysarium; Lens; Myroxylon; Ononis; Pericopsis; Pisum; Pterocarpus; trifolium; and Tήgonell; and in other embodiments the plant extract is selected from Acacia anβυra; Acacia aulacocarpa; Acacia concurrens; Acacia cyanophylla; Acacia Cyclops; Acacia deanei; Acacia holosericea; Acacia karroo; Acacia mellifera; Acacia mucronata; Anaphalis cinnamomea; Artocarpus integer; Berberis chinensis; Berbeήs lyceum; Broussonetia papyrifβra; Cajanus cajan; Citrus aurantium; Citrus sinensis; Citrus x paradise; Cudrania tricuspidata; Dalea purpurea; Desmodium uncinatum; Erythrina abyssinica; Erythrina berteroana; Erythrina fυsca; Erythrina indica; Erythrina poeppigiana; Erythrina variegate; Fagopyrum esculentum; Flemingia macrophylla; Helichrysum italicum; Humulus lupulus; Hypericum ascyron; Hypericum perforatum; Lonchocarpus cappassa; Lotus uliginosus; Lotus wrightii; Lupinus albicaulis; Lυpinus lυteus; Lupinus perennis; Lupinus polyphyllus; Madura pomifera; Melicope ternate; Melilotus alba; Melilotus officinalis; Moots nigra; Mundulea sericea; Platanus occidentalis; Psolarea esculenta; Scoparia dulcis; Bituminaria bituminosa; Dalbergia boehmii; Dalbergia latifolia; Dalbergia melanoxylon; Dolichos biflonis; Dolichos lablab; Genista linifolia; Genista tinctoήa; Hardenbergia violacβa; Mucuna deeringiana; Mucuna gigantean; Mucuna pruriens; Neonotonia wightii; Phaseolus aureus; Phaseolus coccineus; Pυeraria lobata; Pueraria phaseoloides; Vigna aconitifolia; Vigna luteola; Vigna mungo; Vigna oblongifolia; Vigna radiate; Artemisia scopaήa; Myrsine Africana; Rapanea mβlanophloeos; Albizzia procera; Astragalus sinicυs; Baphia nitida; Centrosema pubescβns; Cicer arietinum; Eclipta alba; Eciipta prostrate; Glycine tomentella; Hedysarium coronarium; Lens culinaris; Myroxyion balsamum; Ononis spinosa; Peπcopsis angolensis; Pisum sativum; Pterocarpus soyauxii; Trifoliυm cherieri; Tn folium hybridum; Trifoliυm pretense; Trifoliυm repens; and Trigoπella comicυlata.. [012] In some embodiments, the plant extract is enriched with at least one compound selected from
• 3-[2,4-dihydroxy-3-(3-methyl-but-2-enyl)-phenyl]-5,7-dihydroxy-chromen-4-one;
5,7-dihydroxy-3-[4-hydroxy-3-(3-methyl-but-2-enyl)-phenyl]-6-(3-methyl-but-2-enyl)- chromen-4-one;
3,5,7-trihydroxy-2-[4-hydroxy-3-(3-methyl-but-2-enyl)-phenyl]-chromen-4-one; i-methoxy^-CS-methyl-but^-enyO-δH-benzoH.SJfuroβ^-clchromene-S.Θ-diol; ■ 2-[3-hydroxy-5-methoxy-4-(3-methyl-but-2-enyl)-phenyl]-benzofuran-3,6-diol;
4-[7-hydroxy-5-methoxy-6-(3-methyl-but-2-enyl)-2H-chromen-3-yl]-benzene-1,3-diol; and single stereoisomers, mixtures of stereoisomers, or pharmaceutically acceptable salts thereof.
[013] In other embodiments, the method of treating, preventing, or inhibiting bone or joint inflammation in a subject, comprises administering to the subject a therapeutically effective amount of a nutraceutical composition comprising at least one compound of the compounds in Table I, and in other embodiments the nutraceutical composition comprises at least one plant extract selected from Acacia; Anaphalis; Artocarpus; .Berberis; Broussonetia; Cajanus; Cudrania; Dalea; Dβsmodium; Erythrina; Fagopyrum; Flemingia; Helichrysum; Humulus; Hypericum; Lonchocarpus; Lotusi; Lupinus; Madura; Melicope; Melilotus; Mundulea; Platanus ; Psolarea; Scoparia; Bituminaria; Dalbergia; Dolichos; Genista; Hardenbergia; Mucuna; Neonotonia; Pueraria; Artemisia; Myrsine; Rapanea; Astragalus; Baphia; Centrosema; Cicer, Eclipta; Eclipta; Hedysarium; Lens; Myroxyion; Ononis; Pericopsis; Pisum; Pterocarpus; and Trifolium; for example the plant extracts are selected from Acacia aneura; Acacia aulacocarpa; Acacia concuπrens; Acacia cyanophylla; Acacia Cyclops; Acacia deanei; Acacia holosericea; Acacia karroo; Acacia mellifera; Acacia mucronata; Anaphalis cinnamomea; Artocarpus integer, Berberis chinensis; Berberis lyceυm; Broussonetia papyrifera; Cajanus cajan; Citrus aυrantium; Citrus sinensis; Citrus x paradise; Cudrania thcuspidata; Dalea purpurea; Desmodium uncinatum; Erythrina abyssinica; Erythrina berteroana; Erythrina fusca; Erythrina indica; Erythήna poeppigiana; Erythrina variegate; Fagopyrum esculentum; Flemingia macrophylla; Helichrysum italicum; Humulus lupulυs; Hypericum ascyron; Hypericum perforatum; Lonchocarpus cappassa; Lotus uliginosus; Lotus wrightii; Lupinus albicaulis; Lupinus luteus; Lupin us perennis; Lupinus polyphyllus; Madura pomifera; Meiicope temate; Melilotus alba; Melilotus officinalis; Moms nigra; Mundulea sericea; Platanus occidentalis; Psolarea esculenta; Scoparia dυlcis; Bituminaria bituminosa; Dalbergia boehmii; Dalbergia latifolia; Dalbergia melanoxylon; Dolichos biflorus; Dolichos lablab; Genista linifolia; Genista tinctoria; Hardenbergia violacea; Mucuna deeringiana; Mucuna gigantean; Mucuna pruriens; Neonotonia wightii; Phasβolus aureus; Phaseolus coccineus; Pueraria lobata; Pueraria phaseoloides; Vigna aconitifolia; Vigna luteola; Vigna mungo; Vigna oblongifolia; Vigna radiate; Artemisia scoparia; Myrsinβ Africana; Rapanea mβlanophloeos; Albizzia procera; Astragalus sinicus; Baphia nitida; Cβntrosema pubescens; Cher arietinum; Eclipta alba; Eclipta prostrate; Glycine tomentella; Hedysarium coronarium; Lens culinaris; Myroxylon balsamum; Ononis spinosa; Pericopsis angolensis; Pisum sativum; Pterόcarpus soyauxii; Trifolium cherleri; Trifolium hybridυm; Trifolium pretense; Trifolium repens; and Trigonella corniculata.
[014] In some embodiments, the nutraceutical compositions comprise plant extracts that are enriched with at least one compound selected from:
3-[2,4-dihydroxy-3-(3-methyl-but-2-enyl)-phenyl]-5,7-dihydroxy-chromen-4-one; • 5,7-dihydroxy-3-[4-hydroxy-3-(3-methyl-but-2-enyl)-phenyl]-6-(3-methyl-but-2-enyl)- chromen-4-one;
3,5,7-trihydroxy-2-[4-hydroxy-3-(3-methyl-but-2-enyl)-phenyl]-chromen-4-one;
1-methoxy-2-(3-methyl-but-2-enyl)-6H-benzo[4,5]furo[3,2-c]chromene-3,9-diol;
2-[3-hydroxy-5-methoxy-4-(3-methyl-but-2-enyl)-phenyl]-benzofuran-3,6-diol;
4-[7-hydroxy-5-methoxy-6-(3-methyl-but-2-enyl)-2/-/-chromen-3-yl]-benzene-1,3-diol; and single stereoisomers, mixtures of stereoisomers, or pharmaceutically acceptable salts thereof.
[015] In some embodiments, the compositions are useful for the treatment of arthritis, such as gout, childhood arthritis, lupus arthritis, rheumatoid arthritis, or osteoarthritis. In other embodiments the compositions are useful for the treatment of joint inflammation, joint swelling or joint pain.
[016] In some embodiments, the route of administration is oral, and in other embodiments the route of administration is a medical food, a functional food, a special nutrition food, or a dietary supplement which may additionally include one or more ingredients comprising vitamins, minerals, herbs, botanicals, amino acids, and/or dietary substances intended to supplement the diet by increasing total dietary intake. In other embodiments the administration is a medical food, a functional food, a special nutrition food, or a dietary supplement which may additionally include one or more ingredients comprising glucosamine, glucosamine hydrochloride, glucosamine sulfate, chondroitin, chondroitin sulfate, calcitonin, collagen, collagen proteins, or hydrolyzed collagen. In other embodiments the administration is a medical food, a functional food, a special nutrition food, or a dietary supplement which may additionally include an analgesic or an NSAID. In some embodiments the composition is administered as a regular or controlled release formulation. In other embodiments, the composition is administered topically on the epidermis covering the joint in need of such treatments.
[017] In some embodiments, the subject is a human; in other embodiments the subject is an animal, such as, but not limited to farm animals such as cattle, horses, sheep, goats and swine; or domestic animals such as rabbits, dogs, cats, rats, mice and guinea pigs. [018] In some embodiments, the plant extract is selected from the group including, but not limited to, stems, stem barks, trunks, trunk barks, twigs, tubers, roots, root barks, young shoots, seeds, grains, nuts, nut skins, rhizomes, tubers, fruits, fruit skins, flowers and other reproductive organs, leaves and other aerial parts, callus cultures, and cell cultures.
MODE FOR CARRYING OUT THE INVENTION Definitions
[019] The term "analgesic" refers to but is not limited to, acetaminophen, ibuprofen, aspirin, salicyclamide, trolamine salicylate, methyl salicylate, salicyalte salts, N,N-dimethyl aspartic acid, N.N-dimethyl glutamic acid, and antipyrine
[020] The term "amelioration" refers to prevention, reduction or palliation of a state, or improvement of the state of a subject in need of improvement or the reduction of the symptoms associated with irritation. Amelioration includes, but does not require complete recovery or complete prevention.
[021] The term "arthritis" refers to the inflammation of a joint or joints which results in pain and swelling. The two most common forms of arthritis are osteoarthritis and rheumatoid arthritis. Osteoarthritis is characterized by chronic degeneration of the cartilage of the joints, mainly in older persons. Rheumatoid arthritis, sometimes called arthritis deformans, is a chronic and progressive systemic disorder, especially common in women, characterized by stiffness. swelling and inflammation of the joints and sometimes leading to deformity and permanent disability.
[022] The term "formulation" refers to a combination of active components or ingredients that are administered together or separately under a coordinated dosing regimen. For purposes of the present invention, a formulation need not consist of admixed components. Rather, it may include components that are given separately in different oral forms or even via different modes of administration, for example as a combination of oral and parenteral treatments. A formulation may also comprise a "kit" whereby components are bundled together in a combination packaging format.
[023] The term "effective amount" refers to an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations [024] The term "joint disorders" include, but are not limited to: joint inflammation, joint swelling, joint pain, and arthritis, which consists of more than 100 different joint-related disorders including, rheumatoid arthritis, juvenile rheumatoid arthritis, childhood arthritis, psoriatic arthritis, Reiter's syndrome (also referred to as reactive arthritis), sarcoidosis; osteoporosis; osteoarthritis, gout, pseudogout, lupus arthritis, calcification in joints, synovial osteochondromatosis (SOC), chronic back injury, diffuse idiopathic skeletal hyperostosis, ankylosing spondylitis, synovitis, including, but not limited to, rheumatic synovitis, pigmented villonodular synovitis, and chronic synovitis, and temporomandibular joint disorder (TMJ). [025] Many patients that suffer from various other disorders may develop joint disorders through association, such as, but not limited to: congenital and acquired torticollis, osteochondroma, osteoporosis, Sjogren's syndrome, avascular necrosis (also referred to as osteonecrosis), osteomyelitis, carpal tunnel syndrome, repetitive stress injury, dysplasia of joints, fibromyalgia, shin splints, broken bones, stress fractures, scleroderma, scoliosis, bursitis, lyme disease, and Paget's disease.
[026] The term "NSAID," as used herein, refers to any compound acting as a nonsteroidal anti-inflammatory agent identifiable as such by one of ordinary skill in the art. NSAIDs are categorized by virtue of their ability to inhibit cyclooxygenase. The term NSAID includes, but is not limited to, salicylates, indomethaciπ, flurbiprofen, diclofenac, ketorolac, naproxen, piroxicam, tebufelone, ibuprofen, etodolac, nabumetone, tenidap, alcofenac, antipyrine, aminopyrine, dipyrone. aminopyrone, phenylbutazone, clofezone. oxyphenbutazone, prexazone, apazone, benzydamine. bucolome, cinchopen, clonixin, ditrazol, epirizole, fenoprofen, floctafeninl, flufenamic acid, glaphenine, indoprofen, ketoprofen, meclofenamic acid, mefenamic acid, niflumic acid, phenacetin, salidifamides, sulindac, suprofen and tolmetin. The salicylates may include acetylsalicylic acid, sodium acetylsalicylic acid, calcium acetylsalicylic acid, salicylic acid, and sodium salicylate. The term NSAID also includes known spices and herbs with anti-inflammatory activity, such as Boswellia serrata, ginger, turmeric, holy basil, green tea extract, resveratrol, white willow bark, devil's claw, capsicum, stinging nettle, wintergreen, and pineapple (bromelain).
[027] The term "nutraceutical" refers to any compounds or chemicals from natural sources that can provide dietary or health benefits when consumed by humans or animals. Examples of nutraceuticals include, but are not limited to plant extracts. [028] The term "rheumatism" refers to any of several pathological conditions of the muscles, tendons, joints, bones, or nerves, characterized by discomfort and disability. The term includes but is not limited to arthritis. By way of examples not of limitation, the term rheumatism refers to arthritis, articular or acute rheumatism, Besnier's rheumatism, Heberden's rheumatism, nodose rheumatism, osseous rheumatism, palindromic rheumatism, and Poncet's rheumatism. [029] The term "subject" includes, but is not limited to, humans and animals, such as farm animals (cattle, horses, sheep, goats, and swine) and domestic animals (rabbits, dogs, cats, rats, mice and guinea pigs). The term "subject "does not denote a particular age or sex. [030] The term "treatment" or "treating" relates to any treatment of a disorder, in a mammal, including: preventing or protecting against the disorder, that is, causing the clinical symptoms of the disorder not to develop; inhibiting the disorder, that is, arresting or suppressing the development of clinical symptoms; and/or relieving the disorder, that is, causing the regression of clinical symptoms It will be understood by those skilled in the art that it is not always possible to distinguish between "preventing" and "suppressing" since the ultimate inductive event or events may be unknown, latent, or the patient is not ascertained until well after the occurrence of the event or events. Therefore, as used herein the term "prophylaxis" is intended as an element of "treatment" to encompass both "preventing" and "suppressing" as defined herein. The term "protection," as used herein, is meant to include "prophylaxis." [031] As used herein, the term "comprising" and its cognates are used in their inclusive sense; that is, equivalent to the term "including" and its corresponding cognates. General Utility
[032] The compositions and methods of the present invention are useful for promoting joint and bone health by, for example, preventing and/or treating joint and bone inflammation in a subject in need of such treatment. Without subscribing to a particular theory of mechanism of action, the compounds of the present invention may target certain enzymes such as cyclooxygenases or lipoxygenases that function across a variety of physiological processes. [033] Compositions of the present invention may be useful for the treatment of disorders involving inflammation of the joints, such as, but not limited to, rheumatism, joint inflammation, joint swelling, joint pain, rheumatoid arthritis, juvenile rheumatoid arthritis, childhood arthritis, psoriatic arthritis, lupus arthritis, Reiter's syndrome (also referred to as reactive arthritis), sarcoidosis, osteoporosis, osteoarthritis, gout; pseudogout, ankylosing spondylitis, calcification in joints, synovial osteochondromatosis (SOC), chronic back injury, diffuse idiopathic skeletal hyperostosis, ankylosing spondylitis, rheumatic synovitis, pigmented villonodular synovitis, chronic synovitis, and temporomandibular joint disorder (TMJ). [034] The compositions may also be useful in the treatment of disorders that may develop joint disorders through association, such as, but not limited to: congenital and acquired torticollis, osteochondroma, osteoporosis, Sjogren's syndrome, avascular necrosis (also referred to as osteonecrosis), osteomyelitis, carpal tunnel syndrome, repetitive stress injury, dysplasia of joints, fibromyalgia, shin splints, broken bones, stress fractures, scleroderma, scoliosis, bursitis, lyme disease, and Paget's disease. Administration
[035] The compositions, as described above, can be prepared as foods for humans or animals, including medical foods, functional food, special nutrition foods, and dietary supplements. A "medical food" is a product prescribed by a physician that is intended for the specific dietary management of a disorder or health condition for which distinctive nutritional requirements exist, and may include formulations fed through a feeding tube (referred to as enteral administration or gavage administration). A "dietary supplement" shall mean a product that is intended to supplement the human diet and may be provided in the form of a pill, capsule, tablet, or like formulation. By way of example, but not limitation, a dietary supplement may include one or more of the following dietary ingredients: vitamins, minerals, herbs, botanicals, amino acids, and dietary substances intended to supplement the diet by increasing total dietary intake, or a concentrate, metabolite, constituent, extract, or combinations of these ingredients, not intended as a conventional food or as the sole item of a meal or diet. Dietary supplements may also be incorporated into food stuffs, such as functional foods designed to promote joint and bone health or to prevent inflammation. A "functional food" is an ordinary food that has one or more components or ingredients incorporated into it to give a specific medical of physiological benefit, other than a purely nutritional effect. "Special nutrition food" means ingredients designed for particular diet related to conditions or to support treatment of nutritional deficiencies. [036] Formulations of the invention may be administered in nutritionally accepted vehicles for oral ingestion, such as, capsules, tablets, or pills, soft gel caps, powders, solutions, dispersions, or liquids. In preparing the compositions in oral dosage form, any of the usual media may be employed. For oral liquid preparations (e.g., suspensions, elixirs, and solutions), media containing, for example, water, oils, alcohols, flavoring agents, preservatives, coloring agents and the like may be used. Carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like may be used to prepare oral solids (e.g., powders, capsules, pills, tablets, and lozenges). A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free- flowing form such as a powder or granules, optionally mixed with a binder (e.g. povidone, gelatin, hydroxy propylmethylcellulose), lubricant, inert diluent, preservative, disintegrant (e.g. sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxy-methylcellulose) surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide controlled release of the active ingredients therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide the desired release profile. Soft gel caps may contain lipophilic substances, such as tocopherols and polyunsaturated fatty acids. Methods for preparing gel caps are well known in the art.
[037] The subject formulations may be compounded with other physiologically acceptable materials which can be ingested including foods, for example, food bars, beverages, powders, cereals, cooked foods, food additives and candies. The food can be a dietary supplement (such as a snack or wellness dietary supplement) or, especially for animals, comprise the nutritional bulk (e.g., when incorporated into a primary animal feed, a grain ration, or incorporated into a salt block). The dietary supplements and medical foods of the present invention can be used in powder form which can be dissolved in a liquid suitable for human consumption, such as water or a fruit juice. The compositions of the present invention may be formulated with a liquid foodstuff, for example, milk, juices, liquid vitamin supplements, and oral rehydration(electrolyte) solutions.
[038] Typically, the dietary supplements and medical foods of the present invention are preferably administered two times per day, preferably once in the morning and once in the afternoon. A typical treatment regime for the dietary supplements or medical foods will continue for four to eight weeks. Depending on such factors as the medical condition being treated and the response of the patient, the treatment regime may be extended. A medical food of the present invention will typically be consumed in two servings per day as either a meal replacement or as a snack between meals. A serving size for a medical food of the present invention will preferably be in the range of from about 45 grams to about 60 grams and will provide from about 180 calories to about 220 calories to the consumer. In an exemplary treatment regime a person in need of treatment is provided with two servings of a medical food of the present invention per day.
[039] The composition of the present invention may further comprise vitamins, minerals, coenzymes, organic or inorganic antioxidants or precursors thereof. As way of example not of limitation, the coenzyme may be coenzyme Q10, the organic antioxidant may be selected from the group comprising lipoic acid, resveratrol and glutathione, and the inorganic antioxidant may be selenium. The vitamins may be selected from the group vitamin A (palmitate), vitamin B-1, vitamin B-2, vitamin B-6, vitamin B-12, vitamin C (ascorbic acid), vitamin D-3, vitamin E (alpha, beta, gamma, delta tocopherol or mixtures thereof), vitamin K-1, niacin, folic acid, biotin, choline, inositol, and pantothenic acid; and the amino acids may be selected from the group L-alanine, L-arginine, L-aspartic acid, L-cystine, L-glutamic acid, L- glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-proline. L- serine, L-threonine, L-tryptophan, L-tyrosine and L-valine. The minerals may be selected from the group calcium, potassium, magnesium, phosphorous, zinc, manganese, selenium, chromium, and vanadium.
[040] Any flavoring agents may be added to the compositions of the invention for tasting purposes. When utilized, flavors are present in an amount from about 0.05 to about 0.5, preferably from about 0.1 to about 0.3 percent by weight, based on the total weight of the compositions.
[041] The amount of the composition ingested, consumed or otherwise administered will depend on the desired final concentration. Typically, the amount of a single administration of the formulation of the invention can be about 0.1 to about 10,000 mg per day. Any of these doses can be further subdivided into separate administrations, and multiple dosages can be given to any individual patient.
[042] In some embodiments, compositions are administered in one dosing of a single formulation and in other embodiments, compositions are administered in multiple dosing of a single formulation. In some embodiments, the components are administered to a subject within a time period between about 3 hours to about 6 hours. In other embodiments, the time period is between about 6 hours and 12 hours. In additional embodiments, the time period is between about 12 hours and 24 hours. In yet further embodiments, the time period is between about 24 hours and 48 hours. The administration of separate formulations can be simultaneous or staged throughout a specified time period. The composition and may comprise 0.2-2% of the active agent in solution.
[043] In some embodiments of the invention, a method for alleviating joint and bone inflammation encompasses co-administering a dietary supplement of the invention with a separately formulated therapeutically active agent or compound (e.g., a drug such as an analgesic or an NSAID1 or a bio-active molecule such as such as glucosamine, glucosamine hydrochloride, glucosamine sulfate, chondroitin, chondroitin sulfate, calcitonin, collagen, collagen proteins, or hydrolyzed collagen) to obtain an enhanced and/or more rapid relief of the condition sought to be alleviated. "Co-administration" within the context of this embodiment should be understood as meaning simultaneous or sequential administering of the dietary supplement and the therapeutic agent or drug, either by the same or different route of administration.
[044] In some embodiments of the invention, the compositions may be administered topically. A topical administration refers to application of the present compositions by spreading, dabbing, dusting, swabbing, sponging, dispensing, covering and coating the affected area. The compositions of the present invention may be formulated for topical administration to the epidermis covering the joint in need of such treatment as ointments, creams, gels, lotions or transdermal patches.
[045] Topical formulations of the present invention typically comprise the composition of the invention and optionally, a polar solvent. Solvents suitable for use in the formulations of the present invention include any polar solvent capable of dissolving the compositions of the invention. Suitable polar solvents include: water; alcohols (such as ethanol, propyl alcohol, isopropyl alcohol, hexanol, and benzyl alcohol); polyols (such as propylene glycol, polypropylene glycol, butylene glycol, hexylene glycol, sorbitol, and glycerin); and panthenol dissolved in glycerin, flavor oils and mixtures thereof. Mixtures of these solvents can also be used.
[046] An emollient may also be added to the topical compositions of the present invention. The emollient component can comprise fats, oils, fatty alcohols, fatty acids and esters which aid application and adhesion, yield gloss, and most importantly, provide occlusive moisturization. Suitable emollients for use are isostearic acid derivatives, isopropyl palmitate, lanolin oil, diisopropyl dimerate, maleated soybean oil, octyl palmitate, isopropyl isostearate, cetyl lactate, cetyl ricinoleate, tocopheryl acetate, acetylated lanolin alcohol, cetyl acetate, phenyl trimethicone, glyceryl oleate, tocopheryl linoleate, wheat germ glycerides, arachidyl propionate, myristyl lactate, decyl oleate, propylene glycol ricinoleate, isopropyl lanolate, pentaerythrityl tetrastearate, neopentylglycol dicaprylate/dicaprate, hydrogenated coco- glycerides, isononyl isononanoate, isotridecyl isonόnanoate, myristal myristate, triisocetyl citrate, cetyl alcohol, octyl dodecanol, oleyl alcohol, paπthenol, lanolin alcohol, linoleic acid, linolenic acid, sucrose esters of fatty acids, octyl hydroxystearate and mixtures thereof: [047] The compositions of this invention may contain one or more materials, herein singly or collectively referred to as a "solidifying agent", that is effective to solidify the particular liquid base materials to be used. As used herein, the term "solidify" refers to the physical and/or chemical alteration of the liquid base material so as to form a solid or semi-solid at ambient conditions, i.e., to form a final composition that has a stable physical structure and can be deposited on the skin under normal use conditions. As is appreciated by those skilled in the art, the selection of the particular solidifying agent will depend upon the particular type of composition desired, i.e., gel or wax-based, the desired rheology, the liquid base material used and the other materials to be used in the composition.
[048] The above-mentioned compositions and methods of administration are meant to describe but not limit the methods and compositions of the present invention. The methods of producing various compositions and devices are within the ability of one skilled in the art and are not described in detail here.
Testing
[049] Anti-inflammatory activity of a composition of the present invention, or activity of components administered in methods of the present invention, can be experimentally tested in vitro or in vivo. Inflammation may, for example, be assessed in an assay which measures the ability of the composition to inhibit the cyclooxygenase or lipoxygenase enzymes. [050] The 5-lipoxygenase pathway is a major synthetic pathway relevant to human inflammatory disease. 5-lipoxygenase catalyses the two first steps in the oxygenation of arachidonic acid (a polyunsaturated 20-carbon fatty acid) to leukotrienes. Leukotrienes are known to be important mediators of inflammatory and allergic reactions. The first step in the synthesis of leukotrienes, which is catalyzed by 5-lipoxygenase, is the formation of 5-HPETE. The rearrangement of 5-HPETE to form the unstable LTA4, the rate-limiting step in the synthesis of the leukotrienes, is also catalyzed by 5-lipoxygenase. LTA4 is then converted to either LTB4 or LTC4. LTC4 is rapidly metabolized to LTD4 and then to LTE4. LTC4, LTD4 and LTE4 are collectively referred to as the cysteinyl (Cys) leukotrienes. [051] Biosynthesis of LTB4, C4, D4 and E4 occurs predominantly in leukocytes, in response to a variety of immunological stimuli. The primary target of LTB4 is the leukocyte where it elicits enzyme release, chemotaxis, adherence, and aggregation in nM concentrations. LTB4 modulates immune responses and participates in the host-defense against infections. Hence, LTB4 is an important chemical mediator in the development and maintenance of inflammatory reactions and disease states.
[052] In vitro evaluation of the ability of a composition to inhibit the enzymes 5- lipoxygenase, 15-lipoxygeπase, or 12/15 lipoxygenase as described in Walidge, N. B. et al Anal. Biochem., 231: 354-358 ,1995) using a high throughput colorimetric method; as well as in vitro evaluation of LTB4 inhibition, is described in Examples.
[053] A useful cell screening assay, exemplified herein in Examples is the E-selectin
(ELAM) production assay, which measures activity of test compounds in reducing expression of E-selectin in activated endothelial cells. Briefly, endothelial cells are activated by adding known activators such as lipopolysaccharide, TNF, or IL-1β, alone or in some combination. Activated cells produce E-selectin, which can be measured using, for example, an E-selectin monoclonal antibody-based ELISA assay. In studies carried out in support of the present invention, ELAM production was decreased by formulations containing enriched forms of gamma-tocopherol, beta-tocopherol, and delta-tocopherol but not by formulations enriched in alpha-tocopherol. [054] In vitro evaluation of the ability of a composition to inhibit COX-2 can be determined using standard COX- 1 and COX-2 assays performed by employing ELISA procedures generally known to those skilled in the art. In such procedures, COX- 1 and COX-2 activities are assayed as PGE2 formed/μg protein/time using ELISA to detect the amount of PGE2 synthesized from arachidonic acid. PGE2 formation may be measured using PGE2 specific antibody. Indomethacin, a non selective COX-2/COX-1 inhibitor may be employed as a positive control. An exemplary assay to evaluate COX inhibition is described in Examples. [055] In vivo evaluation of anti-inflammatory activity can be determined by well characterized assays such as reduction of carrageenan-induced paw edema in rats (Gabor, M., Mouse Ear Inflammation Models and their Pharmacological Applications, 2000). Carrageenan-induced paw edema is a model of inflammation, which causes time-dependent edema formation following carrageenan administration into the intraplantar surface of a rat paw. [056] Test formulations can also be tested for anti-inflammatory properties in models of inflammation including the carrageenan paw edema model (Winter et al Proc. Soc. Exp. Biol. Med. 111:544, 1962); R. A. Scherrer and M.W. Whitehouse, Eds., 1974; Antiinflammatory Agents, Chemistry and Pharmacology, Vol. 13; 11:33, 1977, Academic, New York) and collagen induced arthritis (Trentham et al J. Exp. Med. 146:857; 1980).
[057] Data in support of the present invention, showing that the compounds are active in various inflammatory assays is included in Table II.
Figure imgf000017_0001
[058] The above-described compositions and methods of administration are meant to describe but not limit the methods and compositions of the present invention. The methods of producing various compositions and devices are within the ability of one skilled in the art and are not described in detail here.
EXAMPLES
[059] The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. They should not be considered as limiting the scope of the invention, but merely as being illustrative and representative thereof.
Example 1
5-Lipoxygenase Enzyme Assay
[060] This procedure was used for measuring the enzymatic activity of human recombinant 5-lipoxygenase using a colorimetric method based on the ferric oxidation of xylenol orange. Materials
- 96 well flat bottom microfilter plates (VWR, Catalog # 62402-933 9295) Lipoxygenase screening assay buffer (Cayman, Catalog # 760710) Human recombinant 5-lipoxygenase (Cayman, Catalog # 60402) Arachidonic Acid (Sigma, Catalog # A3555)
Xylenol orange tetrasodium salt (Aldrich, Catalog # 227854)
- Iron (II) sulfate heptahydrate (Sigma, Catalog # F7002)
- Sulfuric acid (95-98%) [18M] Methanol
Procedure
[061] Human recombinant 5-lipoxygenase (Cayman Cat # 60402) was used in this assay. The test compound and/or vehicle was added to 0.5U 5-lipoxygenase in 5OmM Tris-HCI buffer, pH 7.4. The reaction was initiated by addition of 70μM arachidonic acid in Tris-HCI buffer, pH 7.4, and terminated after a 10 minute incubation at room temperature by addition of
FOX reagent (25mM sulphuric acid, 100μM xylenol orange, 100μM iron (II) sulphate, methanolrwater 9:1). The yellow color of acidified xylenol orange was converted to a blue color by the lipid hydroperoxide-mediated oxidation of Fe2+ ions and the interaction of the resulting
Fe3* ions with the dye. The complex was allowed to form during a one hour incubation at room temperature with shaking. Absorbance of the Fe3+ complex was then measured at 62OnM using a spectrophotometer.
[062] Negative controls contained enzyme during the incubation step but substrate was not added until after the FOX reagent.
[063] Compounds were screened at 5 concentrations in triplicate starting at 10 μM.
Example 2
12/15-Lipoxygenase Enzyme Assay
[064] This procedure was used for measuring the enzymatic activity of porcine leukocyte 12/15-lipoxygenase using a colorimetric method based on the ferric oxidation of xylenol orange.
Materials
96 well flat bottom microfilter plates (VWR, Catalog # 62402-933 9295) Lipoxygenase screening assay buffer (Cayman, Catalog # 760710) - Porcine leukocyte 12/15-lipoxygenase (Cayman, Catalog # 60300)
- Arachidonic Acid (Sigma, Catalog # A3555)
- Xylenol orange tetrasodium salt (Aldrich, Catalog # 227854)
- Iron (II) sulfate heptahydrate (Sigma, Catalog # F7002)
- Sulfuric acid (95-98%) (18M]
- Methanol Procedure
[065] Porcine Leukocyte 12/15-lipoxygenase (Cayman Cat # 60300) was used in this assay. Test compound and/or vehicle was added to 1.3U 12/15-lipoxygenase in 5OmM Tris-HCI buffer, pH 7.4. The reaction was initiated by addition of 70μM arachidonic acid in Tris-HCI buffer, pH 7.4, and terminated after a 10 minute incubation at room temperature by addition of FOX reagent (25mM sulphuric acid, 100μM xylenol orange, 100μM iron (II) sulphate, methanohwater 9:1). The yellow color of acidified xylenol orange was converted to a blue color by the lipid hydroperoxide-mediated oxidation of Fe2" ions and the interaction of the resulting Fe3* ions with the dye. The complex was allowed to form during a one hour incubation at room temperature with shaking. Absorbance of the Fe3+ complex was then measured at 62OnM using a spectrophotometer. Negative controls contained enzyme during the incubation step but substrate was not added until after the FOX reagent. [066] Compounds were screened at 5 concentrations in triplicate starting at 10 μM.
Example 3
Cellular Inflammation Assays
[067] This example provides exemplary assays for measuring inflammatory reaction in a cell line. Specifically, this assay provides a predictive measure of anti-inflammatory activity of compositions of the present invention.
A. Human Hep3B Cells - CRP Assay
[068] Hep3B Cell Line is obtained from the American Type Culture Collection (ATCC
Catalog No. HB-8064). The Hep3B cell line was derived from liver tissue of an 8-year-old Black male. The cells are epithelial in morphology and produce tumors in nude mice. The cells produce α-fetoprotein, hepatitis B surface antigen, albumin, α-2-macroglobulin, α-1 -antitrypsin, transferrin, plasminogen, complement C3 and β-lipoprotein (Knowles BB, et al., Science, , 209:497-499, 1980). This cell line has been widely used to study hepatocyte cytokine and acute phase protein release (e.g., Damtew B, et al., J Immunol 150:4001-4007, 1993,). HEP3B cells are grown in Minimum Essential Medium (MEM; GlBCO) supplemented with 10% Fetal Bovine Serum (FBS; Hyclone). 1x Penicillin/Streptomycin (GIBCO, Cat #. 15140-122) and 0.1mM non-essential amino acids (GIBCO, Catalog No. 11140-050). Cells are thawed and transferred to warm medium according to standard methods known in the art. [069] Cells are incubated in flasks at 37°C with 5% CO2 in an air atmosphere incubator. HEP3B growth media is changed every 2 days until the cells reach 70-80% confluence (approx. 3-4 days). For assay, the cells are transferred to 96-well plates, seeded at 5000 cells per well in culture media, and left to grow for 7 days in a 37°C incubator (air supplemented with 5% CO2). Media is replaced daily until assay.
[070] Test compounds are diluted into "Stimulus Buffer" (MEM medium containing 0.1 mM non-essential amino acids, 1X penicillin/streptomycin, 10% FBS with 10 ng/ml IL-1β, 20 ng/ml lL-6 and 1 μM dexamethasone. Media is removed from the cells and is replaced with 200 μl of test dilution. Cells are returned to the incubator for three days at 37*C. CRP ELISA is then performed on supernatant from the cells, as described below.
[071] Costar EIA/RIA plates are coated with rabbit anti-human CRP (DAKO) diluted
1 :4000 in carbonate buffer (100 μl/well) for 45 minutes at 37°C. Plates are then washed 5x with CRP washing buffer (50 mM Tris-HCI, 0.3M NaCI, 0.5 Ml Tween-20, pH 8.0) using an automatic plate washer. Plates may be dried, covered and refrigerated until use. Supernatant (100 μl) is removed from each well of the test plates and added to the corresponding well of a pre-coated ELISA plate.
[072] 100 μl HRP-conjugated rabbit anti-human CRP (DAKO) diluted 1 :500 (in CRP wash buffer) is added to each well, followed by incubation for 30 minutes at 37°C. Plates are washed 5x with CRP washing buffer using the automatic plate washer. 200 μl of 3,3',5,5'- Tetramethyl Benzidine (TMB) liquid Substrate System (Sigma, St. Louis, MO) is added to each well, followed by incubation in the dark for 15 minutes at room temperature. Finally, 50 μl of 1M H2SO4 is added to each well and absorbance at 450 nm is immediately measured in a microtiter spectrophotometer.
[073] CRP measured as above is normalized to cell count per well, using a cell viability assay, such as the Cell Tracker Green assay. To do this, the remainder of the medium is from the cell test plates, cells are washed with 200. μL of pre-warmed 1x Hanks Basic Salt Solution (HBSS; GIBCO)1 and 100 μL of 5μM Cell Tracker Green (Molecular Probes, Eugene, OR) is added to each well. Plates are then incubated at 37°C for 30 minutes. Cells are then washed twice with prewarmed 1x HBSS. Plates are immediately read using a Fluoroskan® fluorometer with a 485 excitation/538 emission filter pair. B. CeII-ELAM Assay
[074] Endothelial-Leukocyte Adhesion Molecule (ELAM)1 also known as E-selectin, is expressed on the surface of endothelial cells. In this assay, lipopolysaccharide (LPS) and IL-1β were used to stimulate the expression of ELAM; test agents were tested for their abilities to reduce this expression, in accordance with studies showing that reduction of leukocyte adhesion to endothelial cell surface is associated with decreased cellular damage (e.g., Takada, M., Et al., Transplantation 64: 1520-25, 1997; Steinberg, J.B., et al., J. Heart Lung Trans. 13:306-313. 1994).
[075] Endothelial cells may be selected from any of a number of sources and cultured according to methods known in the art; including, for example, coronary artery endothelial cells, human brain microvascular endothelial cells (HBMEC; Hess, D.C., et al., Neurosci. Lett. 213(1): 37-40, 1996), or lung endothelial cells. Cells were conveniently cultured in 96-well plates. Cells were stimulated by adding a solution to each well containing 10 μg/ml LPS and 100 pg/ml IL-1β for 6 hours in the presence of test agent (specific concentrations and time may be adjusted depending on the cell type). Treatment buffer was removed and replaced with pre-warmed Fixing Solution® (100 μl/well) for 25 minutes at room temperature. Cells were then washed 3X, then incubated with Blocking Buffer (PBS + 2% FBS) for 25. minutes at room temperature. Blocking Buffer containing Monoclonal E-Selectin Antibody (1 :750, Sigma Catalog #S-9555) was added to each well. Plates were sealed and stored at 40C overnight. Plates were washed 4X with 160 μL Blocking Buffer per well. Second Antibody-HRP diluted 1 :5000 in Blocking Buffer was then added (100 μLVwell), and plates were incubated at room temperature (protected from light) for two hours. Plates were then washed 4X with Blocking Buffer before addition of 100 μL of ABTS Substrate solution at room temperature (Zymed, Catalog #00-2024). Wells were allowed to develop for 35 minutes, before measurement at 402 nm in a Fluoroskan® Reader with shake program for 10 seconds. Positive results were recorded as a decrease in ELAM concentration in tested wells, as compared to control wells.
Example 4
In vivo Model of Cellular Inflammation
[076] This assay measures the ability of test compounds to prevent or reduce inflammation secondary to oxazolone or arachidonic acid.
A. Arachidonic Acid
[077] Albino male CD-1 mice, 7-9 weeks old are used in this test. A 20% (w/v) arachidonic acid solution in acetone is prepared. Twenty microliters of the arachidonic acid solution is applied to the dorsal left ear of the mouse. Immediately thereafter, test compounds (20 μL in 70% ethanol/30% propylene glycol) are applied to the left ear. The untreated right ears serve as control. Mice are sacrificed by CO2 inhalation, one hour, after treatment. The left and right ears are removed and 7 mm punch biopsies taken from each. The punch biopsies are weighed, and the differences calculated.
B. Oxazolone
[078] CD-1 mice are induced by applying 3% oxazolone (Sigma) (30 mg/ml prepared in corn oikacetoπe) to the shaved abdomen. Five days later, the mice are challenged with 2% oxazolone (20 mg/ml) in acetone on the left ear (right ear was untreated control). One hour after challenge, test compounds are applied to the left ear. in 70% ethanol/30% propylene glycol. Animals are sacrificed 24 hours later and 7 mm ear punches are removed. The ear punches are placed on a balance scale, and the difference between the untreated and treated ears is determined. Percent inhibition is calculated by comparing the means of each group to the vehicle group. (Hydrocortisone serves as a positive control in this test.)
Example S
Cyclooxygeπase-2 (COX-2) assay
[079] Cyclooxygenase-2 (human recombinant, expressed in Sf9 cell, Cayman 60122) is used. Test compound and/or vehicle is pre-incubated with 0.11 U cyclooxygenase-2, 1 mM reduced GSH, 500 μM phenol and 1 μM hematin for 15 minutes at 370C. The reaction is initiated by addition of 0.3 μM arachidonic acid as substrate in Tris-HCI pH 7.7 and terminated after 5 minutes incubation at 370C by addition of 1N HCI. Following centrifugation, substrate conversion to PGE2 is measured by an Amersham EIA kit. Compounds are screened at 10 μM.
Example 6 PGE2 Assay
[080] Rat Peripheral blood mononuclear cells
Whole blood was collected from aspirin treated rats.
[081] Protocol
Functional COX-2 inhibitory activity of test compounds was assessed by using whole blood cells with activated COX-2.
Anti-coagulant treated whole blood was collected form aspirin treated animals. Peripheral blood mononuclear cells (PBMC) were separated from other blood cells by centrifugation of fresh whole blood in the presence of a density gradient.
Cells were activated with phorbol ester to express cyclooxygenase-2 and produce prostaglandins.
Prostaglandin production was quantified by enzyme linked immunosorbent assay (ELISA).
Cell viability was assayed by counting cells stained with a vital dye to assess cytotoxicity, of test articles.
[082] Assay details
Compounds were screened at 5 concentrations in quadruplicate.
Reference compound (Nimuselide) run for QC.
Example 7
Isolation of
3-[2,4-Dlhydroxy-3-(3-methyI-but-2-enyl)-phenyl]-5,7-dihydroxy-6-{3-methyl-but-2-enyl)- chroman-4-oπe
[083] 3-[2,4-Dihydroxy-3-(3-methyl-but-2-enyl)-phenyl]-5,7-dihydroxy-6-(3-methyl-but-
2-enyl)-chroman-4-one was isolated by methanol extraction of the suspension cell culture of Lυpinus micranthus, followed by ethyl acetate/water partitioning. The crude ethyl acetate extract was then passed through an HP-20 resin column using an aqueous acetonitrile gradient, and was purified over silica gel using mixtures of dichloromethane and methanol, and the purified mixture was then subjected to preparative HPLC/MS on C18 to isolate the compound with the right.
[084] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto. All publications cited above are hereby incorporated by reference.

Claims

We claim:
1. A method of treating, preventing, or inhibiting bone or joint inflammation in a subject comprising administering to the subject a therapeutically effective amount of a composition comprising at least one of the following compounds:
S-^^-dihydroxy-S-CS-rπethyl-but^-enyO-phenyll-SJ-dihydroxy-chromeπ^-oπe;
5,7-dihydroxy-3-[4-hydroxy-3-(3-methyl-but-2-enyl)-phenyl]-6-(3-methyl-but-2-enyl)- chromen-4-one;
3,5,7-trihydroxy-2-[4-hydroxy-3-(3-methyl-but-2-enyl)-phenyl]-chromen-4-one; • 1τmethoxy-2-(3-methyl-bυt-2-enyl)-6H-benzo[4,5]furo[3,2-c]chromene-3,9-diol;
2-[3-hydroxy-5-methoxy-4-(3-methyl-but-2-enyl)-phenyl]-benzofuran-3,6-diol;
4-[7-hydroxy-5-methoxy-6-(3-methyl-but-2-enyl)-2/-/-chromen-3-yl]-benzene-1,3-diol; and single stereoisomers, mixtures of stereoisomers, or pharmaceutically acceptable salts thereof.
2. A method of treating, preventing, or inhibiting bone or joint inflammation in a subject which comprises administering to the subject a therapeutically effective amount of at least one plant extract comprising at least one compound selected from
3-[2,4-dihydroxy-3-(3-methyl-but-2-enyl)-phenyl]-5,7-dihydroxy-chromen-4-oήe;
5,7-dihydroxy-3-[4-hydroxy-3-(3-methyl-but-2-enyl)-phenyl]-6-(3-methyl-but-2-enyl)- chromen-4-one;
3,5,7t-2-[4-hydroxy-3-(3-methyl-but-2-enyl)-phenyl]-chromen-4-one; i-methoxy^-CS-methyl-buW-enyO-βH-benzoK.SlfuroIS^-clchromene-S.Θ-diol;
2-[3-hydroxy-5-methoxy-4-(3-rπethyl-but-2-enyl)-phenyl]-benzofuran-3,6-diol;
4-[7-hydroxy-5-methoxy-6-(3-methyl-but-2-enyl)-2H-chromen-3-yl]-benzene-1,3-diol; and single stereoisomers, mixtures of stereoisomers, or pharmaceutically acceptable salts thereof.
3. The method of Claim 2, wherein the plant extract is selected from the plant genus Acacia; AnaphaHs; Artocarpus; Berberis; Broυssonetia; Cajanυs; Cudrania; Dalea; Desmodiυm; Erythrina; Fagopyrυm; Flemingia; Helichrysum; Humulυs; Hypericum; Lonchocarpus; Lotusi; Lυpinus; Madura; Melicopβ; Melilotus; Mundulea; Platanus ; Psolarea; Scopaήa; Bituminaria; Dalbergia; Dolichos; Genista; Hardenbergia; Mucuna; Neonotonia; Pueraria; Artemisia; Myrsine; Rapanea; Astragalus; Baphia; Ceπtrosema; Cicer, Eclipta; Eclipta; Hedysaήum; Lens; Myroxylon; Ononis; Pericopsis; Pisum; Pterocarpus; and Trifolium.
4. The method of Claim 3, wherein the plant extract is selected from the plants Acacia anβura; Acacia aυlacocarpa; Acacia concurrens; Acacia cyanophylla; Acacia Cyclops; Acacia dβanei; Acacia holosericea; Acacia karroo; Acacia mellifera; Acacia mucronata; Anaphalis cinnamomβa; Artocarpus integer, Berberis chinensis; Bβrbeήs lyceυm; Broussonetia papyrifera; Cajanus cajan; Citrus aurantium; Citrus sinensis; Citrus x paradise; Cudrania thcuspidata; Dalea purpurea; Desmodium uncinatum; Erythrina abyssinica; Erythrina berteroana; Erythrina fυsca; Erythrina indica; Erythrina poeppigiana; Erythrina variegate; Fagopyrum esculentum; Flemingia macrophylla; Helichrysum italicum; Humulus lupulus; Hypericum ascyron; Hypericum perforatum; Lonchocarpus cappassa; Lotus uliginosus; Lotus wrightii; Lupinus albicaυlis; Lupinus luteus; Lupinus perennis; Lupinus polyphyllus; Madura pomifera; Melicope ternate; Melilotus alba; Melilotus officinalis; Morus nigra; Mundulea sericea; Platanus occidentalis; Psolarea esculenta; Scoparia dυlcis; Bituminaria bituminαsa; Dalbergia boehmii;. Dalbergia latifolia; Dalbergia melanoxylon; Dolichos biflorus; Dolichos lablab; Genista linifolia; Genista tinctoria; Hardenbergia violacea; Mucuna deeringiana; Mucuna gigantean; Mucuna pruriens; Neonotonia wightii; Phaseolus aureus; Phaseolus coccineus; Pυeraria lobata; Pueraria phaseoloides; Vigna aconitifolia; Vigna lυteola; Vigna mungo; Vigna oblongifolia; Vigna radiate; Artemisia scoparia; Myrsine Africana; Rapanea melanophloeos; Albizzia procera; Astragalus sinicυs; Baphia nitida; Cβntrosema pubescens; Cicer arietinum; Eclipta alba; Eclipta prostrate; Glycine tomentella; Hedysarium coronarium; Lens culinaris; Myroxylon balsamum; Ononis spinosa; Pericopsis angolensis; Pisum sativum; Pterocarpus soyauxii; Trifolium cherieri; Trifolium hybridum; Trifolium pretense; Trifolium repens; and Trigonella corniculata.
5. The method of Claim 3, wherein the plant extract is enriched with at least one compound selected from
3-[2,4-dihydroxy-3-(3-methyl-but-2-enyl)-phenyl]-5,7-dihydroxy-chromen-4-one; 5,7-dihydroxy-3-[4-hydroxy-3-(3-methyl-but-2-enyl)-phenyl]-6-(3-methyl-but-2-enyl)- chromen-4-one;
S.SJ-trihydroxy-Σ-^-hydroxy-S-tS-methyl-but-Σ-enyty-phenylj-chromen^-one; , • 1-methoxy-2-(3-methyl-but-2-enyl)-6H-benzo[4,5]furo[3,2-c]chromene-3,9-diol; 2-[3-hydroxy-5-methoxy-4-(3-methyl-but-2-enyl)-phenyl]-benzofuran-3,6-diol; 4-[7-hydroxy-5-methoxy-6-(3-methyl-but-2-enyl)-2W-chromen-3-yl]-benzene-1,3-diol; and single stereoisomers, mixtures of stereoisomers, or pharmaceutically acceptable salts thereof.
6. The method of Claim 4, wherein the plant extract is enriched with at least one compound selected from
• 3-[2,4-dihydroxy-3-(3-methyl-but-2-enyl)-phenyl]-5,7-dihydroxy-chromen-4-one;
• 5,7-dihydroxy-3-{4-hydroxy-3-(3-methyl-but-2-enyl)-phenyl]-6-(3-methyl-but-2-enyl)- chromen-4-one;
• 3,5,7-trihydroxy-2-[4-hydroxy-3-(3-methyl-but-2-enyl)-phenyl]-chromen-4-one;
• i-methoxy-2-(3-methyl-but-2-enyl)-6H-benzo[4,5]furo[3,2-c]chromene-3.9-diol;
• 2-[3-hydroxy-5-methoxy-4-(3-methyl-but-2-enyl)-phenyl]-benzofuran-3,6-diol; 4-[7-hydroxy-5-methoxy-6-(3-methyl-but-2-eπyl)-2/-/-chromen-3-yl]-benzene-1,3-diol; and single stereoisomers, mixtures of stereoisomers, or pharmaceutically acceptable salts thereof.
7. The method of Claim 1, Claim 2, Claim 3, Claim 4, Claim 5, or Claim 6, wherein the route of administration is oral.
8. The method of Claim 1, Claim 2, Claim 3, Claim 4, Claim 5, or Claim 6, wherein the route of administration is topical.
9. The method of Claim 7, wherein the route of administration is a medical food, a functional food, a special nutrition food, or a dietary supplement which may additionally include one or more ingredients comprising vitamins, minerals, herbs, botanicals, amino acids, or/and dietary substances intended to supplement the diet by increasing total dietary intake.
10. The method of Claim 7, wherein the administration is a medical food, a functional food, a special nutrition food, or a dietary supplement which may additionally include one or more ingredients comprising glucosamine, glucosamine hydrochloride, glucosamine sulfate, chondroitin, chondroitin sulfate, calcitonin, collagen, collagen proteins, or hydrolyzed collagen.
11. The method of Claim 7, wherein the administration is a medical food, a functional food, a special nutrition food, or a dietary supplement which may additionally include an analgesic or a non-steroidal anti-inflammatory agent (NSAID).
12. A method of treating, preventing, or inhibiting bone or joint inflammation in a subject which comprises administering to the subject a therapeutically effective amount of a nutraceutical composition comprising at least one compound selected from
S-l∑^-dihydroxy-S-CS-methyl-but^-enylJ-phenyll-S.Z-dihyd.roxy-chromen^-one;
• 5,7^dihydroxy-3-t4-hydroxy-3-(3-methyl-but-2-enyl)-phenyl]-6-(3-methyl-but-2-enyl)- chromen-4-one;
• 3,5,7-trihydroxy-2-[4-hydroxy-3-(3-methyl-but-2-enyl)-phenyl]-chromen-4-one; i-methoxy^-φ-methyl-but^-enylJ-βH-benzoμ.Slfuroβ^-cJchromene-S.S-diol; 2-[3-hydroxy-5-methoxy-4-(3-methyl-but-2-enyl)-phenyl]-benzofuran-3,6-diol; 4-[7-hydroxy-5-methoxy-6-(3-methyl-but-2-enyl)-2H-chrornen-3-yl]-benzene-1,3-diol; and single stereoisomers, mixtures of stereoisomers, or pharmaceutically acceptable salts thereof
13. A method of treating, preventing, or inhibiting bone or joint inflammation in a subject which comprises administering to the subject a therapeutically effective amount of a nutraceutical composition comprising at least one plant extract selected from the genus Acacia; Anaphalis; Artocarpus; Berberis; Broussonetia; Cajanυs; Cυdrania; Dalea; Dθsmodiυm; Erythrina; Fagopyrum; Flemiπgia; Helichrysum; Humulus; Hypericum; Lonchocarpus; Lotusi; Lupinus; Madura; Melicopβ; Melilotυs; Mundulea; Platanus ; Psolarβa; Scoparia; Bituminaria; Dalbergia; Dolichos; Genista; Hardenbergia; Mucυna; Neonotonia; Pυeraria; Artemisia; Myrsine; Rapanea; Astragalus; Baphia; Centrosema; Cicer, Eclipta; Eclipta; Hedysarium; Lens; Myroxylon; Ononis; Pericopsis; Pisυm; Pterocarpus; and Trifoliυm.
14. The method of Claim 13, comprising administering to the subject a therapeutically effective amount of a nutraceutical composition with at least one plant extract selected from the plants Acacia aneυra; Acacia aυlacocarpa; Acacia concurrens; Acacia cyanophylla; Acacia Cyclops; Acacia deanei; Acacia holosericea; Acacia karroo; Acacia mellifera; Acacia mucronata; Anaphalis cinnamomea; Artocarpus integer, Berberis chinensis; Berberis lyceυm; Broussonetia papyrifera; Cajanυs cajan; Citrus aurantiυm; Citrus sinensis; Citrus x paradise; Cυdrania tricuspidata; Dalea purpurea; Desmodium uncinatυm; Erythrina βbyssinica; Erythrina berteroana; Erythrina fusca; Erythrina indica; Erythrina poβppigiana; Erythrina variegate; Fagopyrυm escυlentum; Flemingia macrophylla; Helichrysum italicum; Humulus lupulus; Hypericum ascyron; Hypericum perforatum; Lonchocarpus cappassa; Lotus uligino' sus; Lotus wrightii; Lυpinυs albicaulis; Lupinus luteus; Lupin us perennis; Lupinus polyphyllus; Madura pomifera; Mέlicope temate; Melilotus alba; Melilotus officinalis; Morus nigra; Mυndulea sericea; Platanus occidentalis; Psolarea esculenta; Scopaha dulcis; Bituminaπa bituminosa; Dalbergia boehmii; Dalbergia latifolia; Dalbergia melanoxylon; Dolichos biflorus; Dolichos lablab; Genista linifolia; Genista tinctoria; Hardenbergia violacea; Mucuna deeringiana; Mucuna gigantean; Mucuna pruriens; Neonotonia wightii; Phasβolus aureus; Phaseolus coccineus; Pueraria lobata; Pueraria phaseoloides; Vigna aconitifolia; Vigna luteola; Vigna mungo; Vigna oblongifolia; Vigna radiate; Artemisia scoparia; Myrsine Africana; Rapanea melanophloeos; Albizzia procera; Astragalus sinicus; Baphia nitida; Centrosema pubescens; Cicer arietinum; Eclipta alba; Eclipta prostrate; Glycine tomentella; Hedysarium coronaήum; Lens culinaris; Myroxylon balsamum; Ononis spinosa; Pericopsis angolensis; Pisum sativum; Pterocarpus soyauxii; Trifolium cherieri; Trifolium hybridum; Trifolium pretense; Trifolium repens; and Trigonella corniculata.
15. The method of Claim 13 or Claim 14, comprising administering to the subject a therapeutically effective amount of a nutraceutical composition comprising at least one plant extract enriched with at least one compound selected from
3-[2,4-dihydroxy-3-(3-methyl-but-2-enyl)-phenyl]-5,7-dihydroxy-chromen-4-one;
• 5,7-dihydroxy-3-[4-hydroxy-3-(3-methyl-but-2-enyl)-phenyl]-6-(3-methyl-but-2-enyl)- chromen-4-one;
S.S^-trihydroxy^-^-hydroxy-S-CS-methyl-but^-enylJ-phenyll-chromerM-one; 1-methoxy-2-(3-methyl-but-2-enyl)-6H-benzo[4,5]furo[3,2-c]chromene-3,9-diol; 2-[3-hydroxy-5-methoxy-4-(3-methyl-but-2-enyl)-phenyl]-benzofuran-316-diol;
• 4-[7-hydroxy-5-methoxy-6-(3-methyl-but-2-enyl)-2H-chromen-3-yl]-benzene-1,3-diol; and single stereoisomers, mixtures of stereoisomers, or pharmaceutically acceptable salts thereof.
16. The method of Claim 1, Claim 2, Claim 12, or Claim 13, wherein the disorder is arthritis.
17. The method of Claim 16, wherein the arthritis comprises gout, childhood arthritis, lupus arthritis, rheumatoid arthritis, or osteoarthritis.
18. The method of Claim 1 , Claim 2, Claim 12, or Claim 13, wherein the joint disorder comprises joint inflammation, joint swelling, or joint pain.
19. The method of Claim 2, wherein said plant extract is selected from the group consisting of stems, stem barks, trunks, trunk barks, twigs, tubers, roots, root barks, young shoots, seeds, grains, nuts, nut skins, rhizomes, tubers, fruits, fruit skins, flowers and other reproductive organs, leaves and other aerial parts, callus cultures, and cell cultures.
20. The method of Claim 13, wherein said plant extract is selected from the group consisting of stems, stem barks, trunks, trunk barks, twigs, tubers, roots, root barks, young shoots, seeds, grains, nuts, nut skins, rhizomes, tubers, fruits, fruit skins, flowers and other reproductive organs, leaves and other aerial parts, callus cultures, and cell cultures.
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