DESC:FIELD OF THE INVENTION
[0001] The present invention provides adipocyte modifying, anti-inflammatory botanical products, compounds, compositions, kits, and related methods for the treatment of arthritis. These compositions would be useful in the prevention, treatment, or co-treatment of all major forms of arthritis includingosteoarthritis, rheumatoid arthritis,fibromyalgia, gout, and juvenile rheumatoid arthritis.
BACKGROUND OF THE INVENTION
Description of the Related Art
[0002] Arthritis means inflammation or swelling of one or more joints. It describes over 100 conditions that affect the joints, tissues around the joint, and other connective tissues. Of the 100 forms of arthritis, five are most prevalent. These are osteoarthritis, rheumatoid arthritis,fibromyalgia, gout, and juvenile rheumatoid arthritis.
[0003] Osteoarthritis (OA) is the most common form of arthritis. Some people call it a degenerative joint disease or "wear and tear" arthritis. It occurs most frequently in the hands, hips, and knees. With OA, the cartilage within a joint begins to break down and the underlying bone begins to change. These changes usually develop slowly and get worse over time. OA can cause pain, stiffness, and swelling. In some cases, it also causes reduced function and disability; some people are no longer able to do daily tasks or work. OA affects over 32.5 million US adults. There is no cure for OA, so doctors usually treat OA symptoms with a combination of therapies, which may include the following: (1) Increasing physical activity, (2) Physical therapy with muscle strengthening exercises, (3) Weight loss, (4) Medications, including over-the-counter pain relievers and prescription drugs, (5) Supportive devices such as crutches or canes, and (6) Surgery (if other treatment options have not been effective)
[0004] Rheumatoid arthritis, or RA, is an autoimmune and inflammatory disease, which means that your immune system attacks healthy cells in your body by mistake, causing inflammation (painful swelling) in the affected parts of the body.
[0005] Fibromyalgia is a condition that causes pain all over the body (also referred to as widespread pain), sleep problems, fatigue, and often emotional and mental distress. People with fibromyalgia may be more sensitive to pain than people without fibromyalgia. This is called abnormal pain perception processing. Fibromyalgia affects about 4 million US adults, about 2% of the adult population. The cause of fibromyalgia is not known, but it can be effectively treated and managed.
[0006] Gout is a common form of inflammatory arthritis that is very painful. It usually affects one joint at a time (often the big toe joint). There are times when symptoms get worse, known as flares, and times when there are no symptoms, known as remission. Repeated bouts of gout can lead to gouty arthritis, a worsening form of arthritis. There is no cure for gout, but it can be effectively treated and managed with medication and self-management strategies.
[0007] Arthritis in children is called childhood arthritis or juvenile arthritis. The most common type of childhood arthritis is juvenile idiopathic arthritis (JIA), also known as juvenile rheumatoid arthritis. JIA can cause permanent physical damage to joints. This damage can make it hard for the child to do everyday things like walking or dressing and can result in disability. Although there is no cure, some children with arthritis achieve permanent remission, which means the disease is no longer active. Any physical damage to the joint will remain.
[0008] Pro-inflammatory cytokine mediatorsare common in all forms of arthritis. For example, two key pro-inflammatory cytokines in OA and RA are tumor necrosis factor-alpha (TNFa) and Interleukin-6 (IL-6). Each of these cytokines, in turn, also figure to some extent in all other arthritic conditions. Regulating their cellular autocrine or paracrine receptor interactions in arthritis (Figure1) has become a focus of therapeutic management.
[0009] Until recently it was believed that adipose tissue functioned mainly for excess energy storage. This original view of adipose tissue changed with the identification of leptin and adiponectin as specific adipocyte-derived hormones and numerous publications confirming that the adipocyte produces and secretes dozens of factors to regulate food intake, energy expenditure, and a series of metabolic processes. More than 100 secretory factors have been described as being produced and released by adipocytes. Through paracrine and autocrine functioning, adipocytes are capable of influencing multiple physiological and pathophysiological processes. Additionally, compartmentalization of lipids into adipocytes functions to protect nonadipose tissue from lipotoxicity or pro-inflammatory cytokines. Moreover, dysregulation of adipokine production with alteration of adipocyte mass has recently been implicated in metabolic complications of arthritis[Eymard F, Pigenet A, Rose C, et al.: Contribution of adipocyte precursors in the phenotypic specificity of intra-articular adipose tissues in knee osteoarthritis patients. Arthritis Res Ther. 2019; 21(1):252]. We hypothesize that differences in reactivity of intraarticular and subcutaneous adipose tissues to pro-inflammatory stimulus reflect dissimilarity in the intensity of disease-specific inflammation and thus support the contribution of intraarticular adipose tissues to arthritic pathological processes (Figure 1).
[00010] Obesity is a disease resulting from a prolonged positive imbalance between energy intake and energy expenditure. Excess body weight is one of the most important risk factors for all-cause morbidity and mortality. Further, obesity is a primary risk factor for OA, involving complex interactions among the metabolic, biomechanical, and inflammatory factors caused by increased adiposity. Circulating adipokines, including leptin, adiponectin, and visfatin, as well as TNFa-stimulatedincreases in IL-6and free fatty acid (FFA) release, and adiponectin decreases have been observed in arthritis (Figure 1). These, in turn, activate joint-associated neutrophils to release elastase, TGFß1, VEGF, MMP-3, TIMP-1, sICAM-1, sVCAM-1, and MCP-1leading to joint erosion and pain. Under the hypothesis that adipocyte-derived, inflammatory mediators are responsible for arthritic pain and tissue destruction, we conducted a screening program to assess the inhibitory effects ofbotanical products against TNF??stimulated adipose-free fatty acid and IL-6 and adiponectin release.
Summary of Botanical Products Tested
[00011] To test the relationship between adipocyte stimulation of inflammatory mediators of arthritis, our objective was to screen commercial botanical samples for their capacity to modify adipocyte functioning through differentiation and as mature adipocytes in the presence of TNFa-. A total of 207 commercial products representing 94 unique botanicals were tested for their ability to induce preadipocyte differentiation thereby increasing triglyceride incorporation in 3T3-L1 preadipocytes (lipogenesis). Each of the commercial botanical samples had published reports supporting disparate anti-inflammatory properties in various model systems in modern and Ayurvedic medicine. Sixty-one of the 94 unique botanicals (64.9%) exhibited a statistically significant capacity to enhance preadipocyte differentiation (lipogenesis) over the solvent control. Due to cost and complexity, a random subset of 20(33% of the positives) of these lipogenic botanical products were further tested for their effects on TNFa-stimulated FFA, IL-6, and adiponectin secretion in mature 3T3-L1 adipocytes.Only 15 of these 20 (75%) were positive modifiers of adipocyte inflammation.
[00012] It is now generally accepted that adipose tissue acts as an endocrine organ producing many biologically active peptides with an important role in the regulation of food intake, energy expenditure, and a series of metabolic processes. Adipose tissue secretes several bioactive peptides collectively termed adipokines. Through their secretory function, adipocytes lie at the heart of a complex network capable of influencing several physiological processes. Dysregulation of adipokine production with alteration of adipocyte mass has been implicated in metabolic and cardiovascular complications of obesity.
[00013] Excessive production of adipocyte TNFa, IL-6, or FFA deteriorates synovial joints through stimulation of neutrophils. Adiponectin blocks neutrophil activation and exerts an anti-arthritic effect. Further, adiponectin is the only known adipokine whose circulating levels are decreased in the obese state, characterized by excessive pro-inflammatory excretion of adipocytes. The thiazolidinedione anti-diabetic drugs increase plasma adiponectin, supporting the idea that adipokine-targeted pharmacology represents a promising therapeutic approach to control arthritis. Treatment with a PPAR? agonist appears to be a promising therapeutic principle in psoriatic arthritis, but the use of PPAR? ligands might be limited by side effects such as an increase in weight and fluid retention [T. Bongartz, B. Coras, T. Vogt, J. Schölmerich, U. Müller-Ladner, Treatment of active psoriatic arthritis with the PPAR? ligand pioglitazone: an open-label pilot study, Rheumatology, Volume 44, Issue 1, January 2005, Pages 126–129]but has no substantive clinical support.
[00014] To date, no method consisting of the administration of a preadipocyte differentiating composition comprising botanical extracts individually capable of inhibiting free fatty acid and IL-6 secretion and increasing adiponectin secretion from inflammation-stimulated adipocytes has been described for the treatment of arthritis.
[00015] The procedures and compositions described herein can be used for the treatment of all forms of arthritis including osteoarthritis, rheumatoid arthritis, fibromyalgia, gout, and juvenile rheumatoid arthritis.
SUMMARY OF THE INVENTION
[00016] Before invention embodiments are disclosed and described, it is to be understood that no limitation to the particular structures, process steps, or materials disclosed herein is intended, but also includes equivalents thereof as would be recognized by those ordinarily skilled in the relevant arts. It should also be understood that terminology employed herein is used to describe particular examples only and is not intended to be limiting. The same reference numerals in different drawings represent the same element. Numbers provided in flow charts and processes are provided for clarity in illustrating steps and operations and do not necessarily indicate a particular order or sequence. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the specification and the appended claims, the singular forms include plural referents unless the context clearly dictates otherwise.
OBJECT OF THE INVENTION
[00017] The present invention relates to the unexpected discovery that the administration of a preadipocyte differentiating composition comprising botanical extracts individually capable of inhibiting free fatty acid and IL-6 secretion and increasing adiponectin secretion from inflammation-stimulated adipocytes can effectively treat multiple forms of arthritis including osteoarthritis, rheumatoid arthritis, fibromyalgia, gout, and juvenile rheumatoid arthritis.
[00018] The invention provides methods for modifying adipocyte physiology in a subject, comprising administering to the subject a pharmaceutical composition comprising botanical extracts individually capable of inhibiting free fatty acid and IL-6 secretion, and increasing adiponectin secretion from inflammation-stimulated adipocytes.
[00019] The present invention further provides novel compositionscomprising five botanical extracts individually capable of inhibiting free fatty acid and IL-6 secretion, and increasing adiponectin secretion from inflammation-stimulated adipocytes to reduce signs or symptoms of arthritis in a subject in need thereof.
[00020] The present invention also provides a composition for treating diseases or pathologies related to inflammation, oxidative stress, and arthritisin an animal comprising administering to an animal exhibiting signs, signalments, or symptoms of the pathology or disease an effective amount of a formulation comprising five botanical extracts individually capable of inhibiting free fatty acid and IL-6 secretion, and increasing adiponectin secretion from inflammation-stimulated adipocytes and continuing the administration of the composition until the signs, signalments or symptoms are reduced.
[00021] The present invention further provides a method of treating diseases or pathologies related to inflammation, arthritis, and oxidative stress, in an animal comprising administering to an animal exhibiting signs, signalments, or symptoms of the pathology or disease an effective amount of a formulation comprising five botanical extracts individually capable of inhibiting free fatty acid and IL-6 secretion, and increasing adiponectin secretion from inflammation-stimulated adipocytes and continuing the administration of the composition until the signs, signalments, or symptoms are reduced.
[00022] Additionally, the present invention relates to the unexpected discovery that formulations comprising botanical extracts individually capable of inhibiting free fatty acid and IL-6 secretion, and increasing adiponectin secretion from inflammation-stimulated adipocytes wherein, when administered to a subject in need, the formulation has an additive effect of treating arthritis.
[00023] Moreover, the present invention relates to the unexpected discovery that novel, combinations of a preadipocyte-differentiating composition comprising five botanical extracts individually capable of inhibiting free fatty acid and IL-6 secretion and increasing adiponectin secretion from inflammation-stimulated adipocytes has an additive effect of treating arthritis wherein, when administered to a subject in need within 3months and continue these effects through 6 months.
BRIEF DESCRIPTION OF THE DRAWINGS
[00024] FIG. 1 represents [A] the relationship of fat and inflammatory arthritis (IA), and [B] reduction of inflammatory mediators through the inhibition of adipocyte FFA, IL-6, and increased adiponectin secretion.
[00025] FIG. 2 depicts a schematic of a single botanical with one target versus multiple botanicals hitting multiple targets in the mechanisms underlying additive or agonistic effects and supra-additive or synergistic effects, respectively.
[00026] FIG.3illustrates the screening program used to identify botanicals capable of enhancing preadipocyte differentiation, as well as inhibiting free fatty acid and IL-6 secretion, and increasing adiponectin secretion from inflammation-stimulated adipocytes.
[00027] FIG. 4 is a schematic representation of subject disposition in the arthritis clinical trial.
DETAILED DESCRIPTION OF THE INVENTION
[00028] The compositions of the invention include unique preadipocyte-differentiating formulations comprising five botanical extracts individually capable of inhibiting free fatty acid and IL-6 secretion and increasing adiponectin secretion from inflammation-stimulated adipocytes. These preadipocyte-differentiating compositions may be used for treating osteoarthritis, rheumatoid arthritis, fibromyalgia, gout, and juvenile rheumatoid arthritis as well as for their antioxidant or anti-inflammatory properties. The resulting compositions can be consumed as a capsule, medical food, or dietary supplement to address osteoarthritis, rheumatoid arthritis, fibromyalgia, gout, and juvenile rheumatoid arthritis.
[00029] The patents, published applications, and scientific literature referred to herein establish the knowledge of those with skill in the art and are hereby incorporated by reference in their entirety to the same extent as if each was specifically and individually indicated to be incorporated by reference. Any conflict between any reference cited herein and the specific teachings of this specification shall be resolved in favor of the latter. Likewise, any conflict between an art-understood definition of a word or phrase and a definition of the word or phrase as specifically taught in this specification shall be resolved in favor of the latter.
[00030] Technical and scientific terms used herein have the meaning commonly understood by one of skill in the art to which the present invention pertains unless otherwise defined. Reference is made herein to various methodologies and materials known to those of skill in the art. Standard reference works setting forth the general principles of recombinant DNA technology include Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, New York (1989); Kaufman et al., Eds., Handbook of Molecular and Cellular Methods in Biology in Medicine, CRC Press, Boca Raton (1995); McPherson, Ed., Directed Mutagenesis: A Practical Approach, IRL Press, Oxford (1991). Standard reference works setting forth general definitions of medical terms and the general principles of pharmacology, respectively, including Stedman's Medical Dictionary [26th edition] and Goodman and Gilman's The Pharmacological Basis of Therapeutics, 11th Ed., McGraw Hill Companies Inc., New York (2006).
[00031] As used herein, the following abbreviations are defined as BMI: Body Mass Index; DBP: Diastolic blood pressure; eAG: estimated average glucose; FBG: Fasting blood glucose; HCA: hydroxycitric acid; HDL: High Density Lipoprotein; ICO: Index of central obesity; LDL: Low Density Lipoprotein; MAP: Mean arterial pressure; MCP: metacarpophalangeal joints; MMP-3: matrix metalloproteinase-3; ND: Not determined; PP: Pulse pressure; PPG: Postprandial glucose; SBP: Systolic blood pressure; sICAM-1: Soluble intercellular adhesion molecule-1; sVCAM-1:Soluble vascular cell adhesion molecule-1; TC: Total cholesterol; TGFß1: Transforming growth factor-beta 1; TIMP-1: Tissue inhibitors of metalloproteinases; VEGF: Vascular endothelial growth factor;
[00032] In the specification and the appended claims, the singular forms include plural referents unless the context clearly dictates otherwise. As used in this specification, the singular forms "a," "an" and "the" specifically also encompass the plural forms of the terms to which they refer, unless the content clearly dictates otherwise. Additionally, as used herein, unless specifically indicated otherwise, the word "or" is used in the "inclusive" sense of "and/or" and not the "exclusive" sense of "either/or.”
[00033] The term "about" is used herein to mean approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 20%.
[00034] As used herein, the recitation of a numerical range for a variable is intended to convey that the invention may be practiced with the variable equal to any of the values within that range. Thus, for an inherently discrete variable, the variable can be equal to any integer value of the numerical range, including the end-points of the range. Similarly, for an inherently continuous variable, the variable can be equal to any real value of the numerical range, including the end-points of the range. As an example, a variable that is described as having values between 0 and 2 can be 0, 1, or 2 for inherently discrete variables, and can be 0.0, 0.1, 0.01, 0.001, or any other real value for inherently continuous variables.
[00035] As used in this specification, whether in a transitional phrase or the body of the claim, the terms "comprise(s)" and "comprising" are to be interpreted as having an open-ended meaning. That is, the terms are to be interpreted synonymously with the phrases "having at least" or "including at least". When used in the context of a process, the term "comprising" means that the process includes at least the recited steps, but may include additional steps. When used in the context of a compound or composition, the term "comprising" means that the compound or composition includes at least the recited features or compounds, but may also include additional features or compounds.
[00036] Reference is made hereinafter in detail to specific embodiments of the invention. While the invention will be described in conjunction with these specific embodiments, it will be understood that it is not intended to limit the invention to such specific embodiments. On the contrary, it is intended to cover alternatives, modifications, and equivalents as may be included within the spirit and scope of the invention as defined by the appended claims. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. The present invention may be practiced without some or all of these specific details. In other instances, well-known process operations have not been described in detail, in order not to unnecessarily obscure the present invention.
[00037] Any suitable materials and/or methods known to those of skill can be utilized in carrying out the present invention. However, preferred materials and methods are described. Materials, reagents, and the like to which reference are made in the following description and examples are obtainable from commercial sources, unless otherwise noted.
[00038] The term “phytoceutical” or its equivalent “nutraceutical” as well as verbal variants refer to any plant, combinations of plants, or microbiological material that is consumed for beneficial effects on metabolic functioning in a subject.
[00039] As used herein, "synergistic" means more than the additive effect of the individual components against a mechanism of action. For example, if F1 produces response X, F2 produces response Y, then the combination of F1+F2>X+Y. In some situations, F2 produces no response, and the value for Y is equal to zero.
[00040] As used herein, “arthritis” refers to any of the more than 100 variants of the disease including, but not limited to, osteoarthritis, rheumatoid arthritis, fibromyalgia, gout, and juvenile rheumatoid arthritis.
[00041] The term "treat" and its verbal variants refer to palliation or amelioration of an undesirable physiological state. Thus, for example, where the physiological state is poor glucose tolerance, "treatment" refers to improving the glucose tolerance of a treated subject. As another example, where the physiological state is obesity, the term "treatment" refers to reducing the body fat mass, improving the body mass, or improving the body fat ratio of a subject. Treatment of diabetes means improvement of blood glucose control. Treatment of inflammatory diseases means reducing the inflammatory response either systemically or locally within the body. Treatment of osteoporosis means an increase in the density of bone mineralization or a favorable change in metabolic or systemic markers of bone mineralization. The person skilled in the art will recognize that treatment may, but need not always, include remission or cure.
[00042] The term "prevent" and its variants refer to prophylaxis against a particular undesirable physiological condition. The prophylaxis may be partial or complete. Partial prophylaxis may result in the delayed onset of a physiological condition. The person skilled in the art will recognize the desirability of delaying the onset of a physiological condition and will know to administer the compositions of the invention to subjects who are at risk for certain physiological conditions in order to delay the onset of those conditions. For example, the person skilled in the art will recognize that obese subjects are at elevated risk for arthritic diseases. Thus, the person skilled in the art will administer compositions of the invention in order to inhibit adipocyte inflammation in an obese or overweight subject, whereby the onset of an arthritic disease may be prevented entirely, treated, or delayed.
[00043] As used herein the term “oxidative stress” is used to describe the effect of oxidation in which an abnormal level of reactive oxygen species (ROS), such as the free radicals (e.g. hydroxyl, nitric acid, superoxide) or the non-radicals (e.g. hydrogen peroxide, lipid peroxide) lead to damage (called oxidative damage) to specific molecules with consequential injury to cells or tissue. Increased production of ROS occurs as a result of fungal or viral infection, inflammation, aging, UV radiation, pollution, excessive alcohol consumption, cigarette smoking, etc. Removal or neutralization of ROS is achieved with antioxidants, endogenous (e.g. catalase, glutathione, superoxide dismutase), or exogenous (e.g. vitamins A, C, E, bioflavonoids, carotenoids). Oxidative damage to the eye, particularly the retina and the lens, is a contributing factor to age-related macular degeneration and cataract.
[00044] All forms of life maintain a reducing environment within their cells. This reducing environment is preserved by enzymes that maintain the reduced state through constant input of metabolic energy. Disturbances in this normal redox state can cause toxic effects through the production of peroxides and free radicals that damage all components of the cell, including proteins, lipids, and DNA.
[00045] In humans, oxidative stress is involved in the etiology of many diseases, such as obesity, arthritis, atherosclerosis, metabolic syndrome, type 1 and type 2 diabetes, hepatic steatosis, Parkinson's disease, cardiac arrest, myocardial infarction, Alzheimer's disease, Fragile X syndrome, and chronic fatigue syndrome. Short-term oxidative stress, however, may also be important in the prevention of aging by induction of a process named mitohormesis. ROS can be beneficial, as they are used by the immune system as a way to attack and kill invading pathogens. ROS are also used in cell signaling. This is dubbed redox signaling and is a critical component of such pathognomic conditions as obesity, arthritis, metabolic syndrome, colitis, irritable bowel syndrome, and adaptive thermogenesis.
[00046] The methods of the present invention are intended for use with any subject that may experience the benefits of the methods of the invention. Thus, in accordance with the invention, "subjects" include humans as well as non-human subjects, particularly domesticated animals. It will be understood that the subject to which a compound of the invention is administered need not suffer from a specific traumatic state. Indeed, the compounds of the invention may be administered prophylactically, prior to any development of symptoms. The term "therapeutic," "therapeutically," and permutations of these terms are used to encompass therapeutic, palliative as well as prophylactic uses.
[00047] As used herein, the term "solvent" refers to a liquid or gaseous, aqueous, or organic nature possessing the necessary characteristics to extract solid material from the hop plant product. Examples of solvents would include but are not limited to, water, steam, superheated water, methanol, ethanol, hexane, chloroform, liquid CO2, supercritical CO2, liquid N2, propane, or any combinations of such materials.
[00048] As used herein, “decreased secretion or biosynthesis,” means to decrease by at least 3%, the rate of secretion or amount of biosynthesis of the referent compound. The invention further provides a method of decreasing adipocyte or myocyte concentrations of inflammatory mediators in a subject, comprising administering to the subject an amount of the composition sufficient to decrease free fatty acid (FFA)and IL-6 secretion from adipocytes in the subject. In general, a decrease in adipocyte FFA or IL-6 secretion or biosynthesis will result in improvements in such conditions as osteoarthritis, rheumatoid arthritis, fibromyalgia, gout, and juvenile rheumatoid arthritis.
[00049] As used herein, “linear inhibitory effect” refers to a linear decrease in secretion or biosynthesis resulting from all concentrations of the inhibiting material over a dose-response curve. For example, inhibition at low concentrations followed by a failure of inhibition or increased secretion at higher concentrations represents a lack of a linear inhibitory effect.
[00050] In some aspects, the compositions further comprise a pharmaceutically acceptable excipient where the pharmaceutically acceptable excipient is selected from the group consisting of coatings, isotonic and absorption delaying agents, binders, adhesives, lubricants, disintegrants, coloring agents, flavoring agents, sweetening agents, absorbents, detergents, and emulsifying agents. In yet further aspects, the composition additionally comprises one or more members selected from the group consisting of antioxidants, vitamins, minerals, proteins, fats, and carbohydrates.
[00051] The term "therapeutically effective amount" is used to denote treatments at dosages effective to achieve the therapeutic result sought. Furthermore, one of the skill will appreciate that the therapeutically effective amount of the compound of the invention may be lowered or increased by fine-tuning and/or by administering more than one compound of the invention, or by administering a compound of the invention with another compound. See, for example, Meiner, C.L., "Clinical Trials: Design, Conduct, and Analysis," Monographs in Epidemiology and Biostatistics, Vol. 8 Oxford University Press, USA (1986). The invention, therefore, provides a method to tailor the administration/treatment to the particular exigencies specific to a given mammal. As illustrated in the following examples, therapeutically effective amounts may be easily determined, for example, empirically by starting at relatively low amounts and by step-wise increments with the concurrent evaluation of beneficial effect.
[00052] As used herein, “more effectively” is used to describe relative biological responses of compounds or formulations wherein the response elicited by one formulation is greater per unit dose than the other.
[00053] The term "pharmaceutically acceptable" is used in the sense of being compatible with the other ingredients of the compositions and not deleterious to the recipient thereof.
[00054] As used herein, “compounds” may be identified either by their chemical structure, chemical name, or common name. When the chemical structure and chemical or common name conflict, the chemical structure is determinative of the identity of the compound. The compounds described herein may contain one or more chiral centers and/or double bonds and therefore, may exist as stereoisomers, such as double-bond isomers (i.e., geometric isomers), enantiomers, or diastereomers. Accordingly, the chemical structures depicted herein encompass all possible enantiomers and stereoisomers of the illustrated or identified compounds including the stereoisomerically pure form (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) and enantiomeric and stereoisomeric mixtures. Enantiomeric and stereoisomeric mixtures can be resolved into their component enantiomers or stereoisomers using separation techniques or chiral synthesis techniques well known to the skilled artisan. The compounds may also exist in several tautomeric forms including the enol form, the keto form, and mixtures thereof. Accordingly, the chemical structures depicted herein encompass all possible tautomeric forms of the illustrated or identified compounds. The compounds described also encompass isotopically labeled compounds where one or more atoms have an atomic mass different from the atomic mass conventionally found in nature. Examples of isotopes that may be incorporated into the compounds of the invention include, but are not limited to, 2H, 3H, 13C, 14C, 15N, 18O, 17O, etc. Compounds may exist in unsolvated forms as well as solvated forms, including hydrated forms and N-oxides. In general, compounds may be hydrated, solvated, or N-oxides. Certain compounds may exist in multiple crystalline or amorphous forms. Also contemplated within the scope of the invention are congeners, analogs, hydrolysis products, metabolites, and precursor or prodrugs of the compound. In general, all physical forms are equivalent for the uses contemplated herein and are intended to be within the scope of the present invention.
[00055] The compounds according to the invention are optionally formulated in a pharmaceutically acceptable vehicle with any of the well-known pharmaceutically acceptable carriers, including diluents and excipients (see Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, Mack Publishing Co., Easton, PA 1990 and Remington: The Science and Practice of Pharmacy, Lippincott, Williams & Wilkins, 1995). While the type of pharmaceutically acceptable carrier/vehicle employed in generating the compositions of the invention will vary depending upon the mode of administration of the composition to a mammal, generally pharmaceutically acceptable carriers are physiologically inert and non-toxic. Formulations of compositions according to the invention may contain more than one type of compound of the invention), as well as any other pharmacologically active ingredient useful for the treatment of the symptom/condition being treated.
[00056] The compounds of the present invention may be provided in a pharmaceutically acceptable vehicle using formulation methods known to those of ordinary skill in the art. The compositions of the invention can be administered by standard routes. The compositions of the invention include those suitable for oral, inhalation, rectal, ophthalmic (including intravitreal or intracameral), nasal, topical (including buccal and sublingual), vaginal, or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, and intratracheal). In addition, polymers may be added according to standard methodologies in the art for sustained release of a given compound.
[00057] It is contemplated within the scope of the invention that compositions used to treat a disease or condition will use a pharmaceutical-grade compound and that the composition will further comprise a pharmaceutically acceptable carrier. It is further contemplated that these compositions of the invention may be prepared in unit dosage forms appropriate to both the route of administration and the disease and patient to be treated. The compositions may conveniently be presented in dosage unit form be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active ingredient into association with the vehicle that constitutes one or more auxiliary constituents. In general, the compositions are prepared by uniformly and intimately bringing the active ingredient into association with a liquid vehicle or a finely divided solid vehicle or both, and then, if necessary, shaping the product into the desired composition.
[00058] The term "dosage unit" is understood to mean a unitary, i.e. a single dose which is capable of being administered to a patient, and which may be readily handled and packed, remaining as a physically and chemically stable unit dose comprising either the active ingredient as such or a mixture of it with solid or liquid pharmaceutical vehicle materials.
[00059] Compositions suitable for oral administration may be in the form of discrete units as capsules, sachets, tablets, soft gels, or lozenges, each containing a predetermined amount of the active ingredient; in the form of a powder or granules; in the form of a solution or a suspension in an aqueous liquid or non-aqueous liquid, such as ethanol or glycerol; or in the form of an oil-in-water emulsion or a water-in-oil emulsion. Such oils may be edible oils, such as e.g. cottonseed oil, sesame oil, coconut oil, or peanut oil. Suitable dispersing or suspending agents for aqueous suspensions include synthetic or natural gums such as tragacanth, alginate, gum arabic, dextran, sodium carboxymethylcellulose, gelatin, methylcellulose, and polyvinylpyrrolidone. The active ingredient may also be administered in the form of a bolus, electuary, or paste.
[00060] In addition to the compositions described above, the compositions of the invention may also be formulated as a depot preparation. Such long-acting compositions may be administered by implantation (e.g. subcutaneously, intraabdominally, or intramuscularly) or by intramuscular injection. Thus, for example, the active ingredient may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in a pharmaceutically acceptable oil), or an ion exchange resin.
[00061] The compounds of this invention either alone or in combination with each other or other compounds generally will be administered in a convenient composition. The following representative composition examples are illustrative only and are not intended to limit the scope of the present invention. In the compositions that follow, "active ingredient" means a compound of this invention.
[00062] As used herein, "therapeutically effective time window" means the time interval wherein administration of the compounds of the invention to the subject in need thereof reduces or eliminates the deleterious effects or symptoms. In a preferred embodiment, the compound of the invention is administered proximate to the deleterious effects or symptoms.
[00063] A total of 207 commercial products representing 94 unique botanicals (Figure 3/201) were tested for their ability to induce preadipocyte differentiationthereby increasing triglyceride incorporation in 3T3-L1 preadipocytes (Figure 3/203). Each of the commercial botanical samples had published reports supporting disparate, anti-inflammatory properties in various model systems in modern and Ayurvedic medicine. Although all of the candidate botanicals were selected based on potential anti-inflammatory activity, only slightly more than half of the 94 unique botanicals (64.9%) exhibited a statistically significant capacity to enhance preadipocyte differentiation (Figure 3/205) over the solvent control. Due to cost and complexity, asubset of 20 (33% of the positives) of these lipogenic botanical products was further tested for their effects on TNFa-stimulatedFFA (Figure 3/207a), IL-6 (Figure 3/207b), and adiponectin secretion (Figure 3/207c) in mature 3T3-L1 adipocytes. Moreover, only 15 of these 20 (75%) were positive modifiers of adipocyte inflammation. Five of those 15 were selected to create a formulation for clinical evaluation in OA and RA subjects. These five were selected based on optimizing the preadipocyte differentiating composition and individually capable of inhibiting free fatty acid and IL-6 secretion and increasing adiponectin secretion from inflammation-stimulated adipocytes (Figure 3/207a,b,c).
[00064] Boswellia Extract 65% (Shallaki) - Resins from various Boswellia species have a long track record in different cultures as a treatment for inflammatory diseases. The resin of Boswellia species has been used as incense in religious and cultural ceremonies and medicines since time immemorial. Boswellia serrata (Salai/Salai guggul), is a moderate to large-sized branching tree of the family Burseraceae (Genus Boswellia), which grows in dry mountainous regions of India, Northern Africa, and the Middle East. In India, the States of Andhra Pradesh, Gujarat, Madhya Pradesh, Jharkhand, and Chhattisgarh are the main source of Boswellia serrata. Regionally, it is also known by different names. The oleo gum-resins contain 30-60% resin, 5-10% essential oils, which are soluble in the organic solvents, and the rest is made up of polysaccharides. Gum-resin extracts of Boswellia serrata have been traditionally used in folk medicine for centuries to treat various chronic inflammatory diseases. The resinous part of Boswellia serrata possesses monoterpenes, diterpenes, triterpenes, tetracyclic triterpenic acids, and four major pentacyclic triterpenic acids i.e. beta-boswellic acid, acetyl-beta-boswellic acid, 11-keto-beta-boswellic acid, and acetyl-11-keto-beta-boswellic acid, responsible for inhibition of pro-inflammatory enzymes. Out of these four boswellic acids, acetyl-11-keto-beta-boswellic acid is the most potent inhibitor of 5-lipoxygenase, an enzyme responsible for inflammation [Siddiqui MZ. Boswellia serrata, a potential antiinflammatory agent: an overview. Indian J Pharm Sci. 2011 May;73(3):255-61. doi: 10.4103/0250-474X.93507. PMID: 22457547; PMCID: PMC3309643].
[00065] Cissus quadrangularis Extract (20:1) - Cissus quadrangularis L. is a succulent plant of family Vitaceae usually found in tropical and subtropical xeric wood. It is a beefy desert plant like liana generally utilized as typical nourishment in India, where it finds numerous applications in medicine. Experts have made efforts to test the plant's suitability using rational analysis. Some of the pharmacological uses of the plant are linked to cell reinforcement, free radical search, hostile to microbials, bone regeneration, ulceration, pain relief, mitigation, and diuretics [Sundaran J, Begum R, Vasanthi M, Kamalapathy M, Bupesh G, Sahoo U. A short review on pharmacological activity of Cissus quadrangularis. Bioinformation. 2020 Aug 31;16(8):579-585. doi: 10.6026/97320630016579. PMID: 33214745; PMCID: PMC7649020]. Recent studies report Cissus quadrangularis inhibits preadipocyte differentiation.
[00066] Curcumin Extract 95% (Turmeric)- Curcumin is a natural polyphenol and the main compound from the rhizome of Turmeric (Curcuma longa) and other Curcuma species. It has been widely used for different medical purposes, such as improvement of pain and inflammatory conditions in various diseases. Most studies have shown the curative effects of curcumin on clinical and inflammatory parameters of rheumatoid arthritis and reported different mechanisms; inhibition of mitogen-activated protein kinase family (MAPK), extracellular signal-regulated protein kinase (ERK1/2), activator protein-1 (AP-1), and nuclear factor kappa B (NF-kB) in macrophage and neutrophils cell models are the main mechanisms associated with the anti-inflammatory function of curcumin in rheumatoid arthritis [Pourhabibi-Zarandi F, Shojaei-Zarghani S, Rafraf M: Curcumin and rheumatoid arthritis: A systematic review of the literature. Int J Clin Pract. 2021:e14280]. To date, no studies have been reported on the effects of curcumin on adipocytes and its association with rheumatoid arthritis or any other arthritic conditions.
[00067] Ginger Extract 5% - Ginger, the rhizome of Zingiber officinalis, one of the most widely used species of the ginger family, is a common condiment for various foods and beverages with a long history of medicinal use dating back 2500 years. Clinical applications of ginger with an expectation of clinical benefits are receiving significant attention. Of the 109 randomized controlled trials on the effects of ginger that examined the improvement of nausea and vomiting in pregnancy, inflammation, metabolic syndromes, digestive function, and colorectal cancer's markers,positive results were consistently supported; but less than half of the reported studies met the criterion of having a 'high quality of evidence [Anh NH, Kim SJ, Long NP, et al.: Ginger on Human Health: A Comprehensive Systematic Review of 109 Randomized Controlled Trials. Nutrients. 2020;12(1)]. A wide variety of antioxidant properties are also attributed to ginger and several randomized controlled trials have investigated the effect of ginger intake on major oxidative stress parameters. Ginger intake was shown to significantly increase glutathione peroxidase activity, total antioxidant capacity, and significantly decrease malondialdehyde levels compared to control groups [Morvaridzadeh M, Sadeghi E, Agah S, et al.: Effect of ginger (Zingiber officinale) supplementation on oxidative stress parameters: A systematic review and meta-analysis. J Food Biochem. 2021;45(2):e13612]. Further studies with adequate designs are warranted to validate the reported clinical functions of ginger.
[00068] Withania somnifera Extract 4% (Ashwagandha) -Ashwagandha is a small evergreen shrub. It grows in India, the Middle East, and parts of Africa. The root and berry are used to make medicine. W. somnifera is famous for the anti-cancerous activity, low back pain treatment, and muscle strengthening, which may be attributed to the withanolide alkaloids. Ashwagandh is also rich in numerous valued secondary metabolites such as steroids, alkaloids, flavonoids, phenolics, saponins, and glycosides. A wide range of preclinical trials such as cardioprotective, anticancer, antioxidant, antibacterial, antifungal, anti-inflammatory, hepatoprotective, anti-depressant, and hypoglycemic have been attributed to various parts of the plant. Different parts of the plant have also been evaluated for the clinical trials such as male infertility, obsessive-compulsive disorder, antianxiety, bone and muscle strengthening potential, hypolipidemic, and antidiabetic [Saleem S, Muhammad G, Hussain MA, Altaf M, Bukhari SNA: Withania somnifera L.: Insights into the phytochemical profile, therapeutic potential, clinical trials, and future prospective. Iranian journal of basic medical sciences. 2020;23(12):1501-1526]. The most common use of Ashwagandha is for stress. It is also used as an "adaptogen" for many other conditions [Lopresti AL, Smith SJ, Malvi H, Kodgule R: An investigation into the stress-relieving and pharmacological actions of an ashwagandha (Withania somnifera) extract: A randomized, double-blind, placebo-controlled study. Medicine (Baltimore). 2019;98(37):e17186]. Moreover, the mechanisms of action for these properties are not fully understood. Ashwagandha commercial products have demonstrated an unfavorable safety factor and combinations with other phytochemicals present an unmet need to reduce the adverse side effects and increase efficacy.
[00069] The present compositions can be provided in any convenient form. It can be provided as a dietary supplement in capsule or tablet form. It can be formulated into a food or drink, and provided, for example, as a snack bar, a cereal, a drink, a gum, or in any other easily ingested form. It can also be provided as a cream or lotion for topical application. One trained in the art can readily formulate the present composition into any of these convenient forms for oral or topical administration.
[00070] The amounts of other additives per unit serving are a matter of design and will depend upon the total number of unit servings of the nutritional supplement daily administered to the patient. The total amount of other ingredients will also depend, in part, upon the condition of the patient. Preferably, the number of other ingredients will be a fraction or multiplier of the RDA or DRI amounts. For example, the nutritional supplement will comprise 50% RDI (Reference Daily Intake) of vitamins and minerals per unit dosage, and the patient will consume two units per day.
[00071] Flavors, coloring agents, spices, nuts, and the like can be incorporated into the product. Flavorings can be in the form of flavored extracts, volatile oils, chocolate flavorings (e.g., non-caffeinated cocoa or chocolate, chocolate substitutes such as carob), peanut butter flavoring, cookie crumbs, crisp rice, vanilla, or any commercially available flavoring. Flavorings can be protected with mixed tocopherols. Examples of useful flavorings include but are not limited to pure anise extract, imitation banana extract, imitation cherry extract, chocolate extract, pure lemon extract, pure orange extract, pure peppermint extract, imitation pineapple extract, imitation rum extract, imitation strawberry extract, or pure vanilla extract; or volatile oils, such as balm oil, bay oil, bergamot oil, cedarwood oil, cherry oil, walnut oil, cinnamon oil, clove oil, or peppermint oil; peanut butter, chocolate flavoring, vanilla cookie crumb, butterscotch or toffee. In a preferred embodiment, the nutritional supplement contains berry or other fruit flavors. The food compositions may further be coated, for example with a yogurt coating if it is as a bar.
[00072] Emulsifiers may be added for the stability of the final product. Examples of suitable emulsifiers include, but are not limited to, lecithin (e.g., from egg or soy), or mono- and di-glycerides. Other emulsifiers are readily apparent to the skilled artisan and the selection of suitable emulsifier(s) will depend, in part, upon the formulation and final product.
[00073] Preservatives may also be added to the nutritional supplement to extend product shelf life. Preferably, preservatives such as potassium sorbate, sodium sorbate, potassium benzoate, sodium benzoate, or calcium disodium EDTA are used.
[00074] In addition to the carbohydrates described above, the nutritional supplement can contain natural or artificial sweeteners, e.g., glucose, sucrose, fructose, saccharides, cyclamates, aspartame, sucralose, aspartame, acesulfame K, or sorbitol.
Manufacture of the Preferred Embodiments
[00075] The medical foods or nutritional supplements of the present invention may be formulated using any pharmaceutically acceptable forms of the vitamins, minerals, and other nutrients discussed above, including their salts. They may be formulated into capsules, tablets, powders, suspensions, gels, or liquids optionally comprising a physiologically acceptable carrier, such as but not limited to water, milk, juice, soda, starch, vegetable oils, salt solutions, hydroxymethyl cellulose, carbohydrate. In a preferred embodiment, the nutritional supplements may be formulated as powders, for example, for mixing with consumable liquids, such as milk, juice, sodas, water, or consumable gels or syrups for mixing into other nutritional liquids or foods. The nutritional supplements of this invention may be formulated with other foods or liquids to provide pre-measured supplemental foods, such as single-serving chapatis, beverages, or bars, for example.
[00076] In a particularly preferred embodiment, the medical food or nutritional supplement will be formulated into a capsule, a form that has consumer appeal, is easy to administer, and incorporate into one's daily regimen, thus increasing the chances of patient compliance. To manufacture a beverage, the ingredients are dried and made readily soluble in water. For the manufacture of other foods or beverages, the ingredients comprising the nutritional supplement of this invention can be added to traditional formulations or they can be used to replace traditional ingredients. Those skilled in food formulating will be able to design appropriate foods or beverages with the objective of this invention in mind.
[00077] The medical food can be made in a variety of forms, such as flatbreads, puddings, confections, (i.e. candy), nutritional beverages, ice cream, frozen confections and novelties, or non-baked, extruded food products such as bars. The preferred form is a mixture to add to a beverage or a non-baked extruded nutritional bar. In another embodiment, the ingredients can be separately assembled. For example, certain of the ingredients (e.g., N. sativa EO and HCA) can be assembled into a tablet or capsule using known techniques for their manufacture. The remaining ingredients can be assembled into a powder or nutritional bar. For the manufacture of a food product, the dry ingredients are added with the liquid ingredients in a mixer and mixed until the dough phase is reached; the dough is cooked, and the product is cooled and consumed. The two assembled forms comprise the nutritional supplement and can be packaged together or separately, such as in the form of a kit, as described below. Further, they can be administered together or separately, as desired.
Use of Preferred Embodiments
[00078] The preferred embodiments contemplate the treatment of disorders related to arthritis, inflammation, and oxidative stress. A pharmaceutically acceptable carrier may also be used in the present compositions and formulations. The recommended daily amounts of each ingredient, as described herein, serve as a guideline for formulating the medical foods and nutritional supplements of this invention. The actual amount of each ingredient per unit dosage will depend upon the number of units administered daily to the individual in need thereof. This is a matter of product design and is well within the skill of the nutritional supplement formulator.
[00079] The ingredients can be administered in a single formulation or they can be separately administered. For example, it may be desirable to administer the compounds in a form that masks their taste (e.g., capsule or pill form) rather than incorporating them into the nutritional composition itself (e.g. chapatis). Thus, the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the nutritional compositions of the invention (e.g., nutritional supplement in the form of a powder and capsules containing a preadipocyte differentiating composition comprising five botanical extracts individually capable of inhibiting free fatty acid and IL-6 secretion and increasing adiponectin secretion from inflammation-stimulated adipocytes). Optionally associated with such container(s) can be a notice in the form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceutical products, which notice reflects approval by the agency of manufacture, use, or sale for human administration. The pack or kit can be labeled with information regarding mode of administration, sequence of administration (e.g., separately, sequentially, or concurrently), or the like. The pack or kit may also include means for reminding the patient to take the therapy. The pack or kit can be a single unit dosage of the combination therapy or it can be a plurality of unit dosages. In particular, the agents can be separated, mixed in any combination, present in a formulation or tablet.
[00080] The preferred embodiments provide compositions and methods to promote normal, adipocytefunctioning relating to subjects exhibiting arthritis or its co-morbidities. All preferred embodiments provide varying amounts of a preadipocyte differentiating composition comprising five botanical extracts individually capable of inhibiting free fatty acid and IL-6 secretion and increasing adiponectin secretion from inflammation-stimulated adipocytes.
[00081] Generally, the formulations comprise a preadipocyte differentiating composition comprising five botanical extracts individually capable of inhibiting free fatty acid and IL-6 secretion and increasing adiponectin secretion from inflammation-stimulated adipocytes.
EXAMPLES
Example 1
[00082] Increased Preadipocyte Differentiationin 3T3-L1 Adipocytes Elicited by Commercial Botanical Products
[00083] Objective – The objective of this Example was to screen commercial botanical samples for their capacity to modify adipocyte functioning through differentiation and as mature adipocytes.Specifically, we compared 207 botanical products and the thiazolidinedione troglitazone with respect to triglyceride incorporation during differentiation in the presence of each botanical product.Enhanced FFA incorporation through differentiation of adipocytes is an indication of anti-inflammatory activity, as this process protects other cells from the proinflammatory activity of FFA.
[00084] Test materials and chemicals – Commercially available botanical products including plants, spices, herbs, and phytochemicals were used as test materials. Samples were analyzed before their expiration dates as stated on their packages and kept at storage conditions as specified on their labels before analysis. All test products that were in dry form were ground to a fine powder before dissolving in dimethyl sulfoxide (DMSO), whereas oils were simply dissolved in DMSO. By using commercially available material, it was assured that the test materials were identical to those purchased by consumers. A total of 207 products representing 94 unique botanicals were tested for their ability to increase triglyceride incorporation in 3T3-L1 adipocytes through preadipocyte differentiation. A selectedsubset of thepositive botanical products was further tested for their effects on TNFa-stimulated FFA, IL-6, adiponectin secretion in mature 3T3-L1 adipocytes (Figures 2 and 3).
[00085] Fetal bovine serum (FBS) and Dulbecco’s modification of Eagle’s medium (DMEM) were purchased from Mediatech (Herndon, VA, USA). Troglitazone and 15-Deoxy-?12,14-prostaglandin J2. were from Cayman Chemical (Ann Arbor, MI, USA). Murine TNFa and all standard chemicals, unless noted, were obtained from Sigma (St. Louis, MO, USA) and were of the highest purity commercially available.
[00086] Cell culture and Treatment - The murine fibroblast cell line 3T3-L1 was purchased from the American Type Culture Collection (Manassas, VA) and sub-cultured according to instructions from the supplier. Before experiments, cells were cultured in DMEM containing 10% FBS-HI added 50 units penicillin/ml and 50 µg streptomycin/ml, and maintained in log phase before experimental setup. Cells were grown in a 5% CO2 humidified incubator at 37°C. Components of the pre-confluent medium included: (1) 10% FBS/DMEM (Fetal Bovine Serum/Dulbecco's Modified Eagle's Medium) containing 4.5 g glucose/L; (2) 50 U/ml penicillin; and (3) 50 µg/ml streptomycin. Growth medium was made by adding 50 ml of heat-inactivated FBS and 5 ml of penicillin/streptomycin to 500 ml DMEM. This medium was stored at 4°C. Before use, the medium was warmed to 37°C in a water bath.
[00087] 3T3-T1 cells were seeded at an initial density of 6x104 cells/cm2 in 96-well plates. For two days, the cells were allowed to grow to reach confluence. Following confluence, the cells were forced to differentiate into adipocytes by the addition of differentiation medium; this medium consisted of (1) 10% FBS/DMEM (high glucose); (2) 0.5 mM methyl isobutyl xanthine; (3) 0.5 µM dexamethasone and (4) 10 µg/ml insulin (MDI medium). After three days, the medium was changed to a post-differentiation medium consisting of 10 µg/ml insulin in 10% FBS/DMEM.
[00088] Preadipocyte differentiation activity of test materials (lipogenesis) - A cell-based assay for screening of the commercial botanical products for lipogenic (anti-inflammatory) activity was performed using a preadipocyte differentiation assay as described by Xu et al. [Xu ME, Xiao SZ, Sun YH, et al.: A preadipocyte differentiation assay as a method for screening potential anti-type II diabetes drugs from herbal extracts. Planta Med 2006;72:14–19] with the modifications that 3T3-L1 preadipocytes were used in place of freshly isolated rat preadipocytes and that intracellular lipid was quantified using Oil Red O and BODIPY (Invitrogen, Carlsbad, CA, USA). Additionally, troglitazone was used as the positive control instead of rosiglitazone. In brief, murine 3T3-T1 preadipocytes were seeded at an initial density of approximately 3 x 104 cells/cm2 in 24-well plates. For 3 days, the cells were allowed to grow to reach confluence. Following confluence, the cells were forced to differentiate into adipocytes by addition of differentiation medium consisting of (1) 10% FBS/DMEM (high glucose), (2) 0.5 mM methyl isobutyl xanthine, (3) 0.5 µM dexamethasone, or (D) 10 µg/mL insulin. After 3 days, the medium was changed to a post-differentiation medium consisting of 10 µg/mL insulin in 10% FBS/DMEM.
[00089] Test materials or troglitazone were added in DMSO at Day 0 of differentiation and every 2 days throughout the maturation phase (Day 6/Day 7). Whenever fresh medium was added, the fresh test material was also added. In addition to the solvent control, 10 µM troglitazone was used as a positive control in every assay. Microscopic evaluation was performed at Day 0 and every 2 days throughout testing to assess preadipocyte differentiation to adipocyte morphology. Intracellular lipid was quantified with Oil Red O staining.
[00090] Intracellular lipid staining - Differentiated 3T3-L1 cells were stained with Oil Red O according to the method of Kasturi and Joshi [Kasturi R, Joshi VC: Hormonal regulation of stearoyl Coenzyme A desaturase activity and lipogenesis during adipose conversion of 3T3-l1 cells. J Biol Chem 1982;257: 12224–12230]. Stained oil droplets were extracted from the cells with isopropanol and quantified by spectrophotometric analysis at 540 nm (EL312e Bio-Kinetics Reader, Bio-Tek Instruments, Winooski, VT, USA). Results were represented relative to stained cells in the solvent controls within each experiment as the lipogenic index (LI).
[00091] Statistical analyses - The commercial botanical products were each assayed in duplicate a minimum of three independent times. The LI was computed for each sample by normalizing Oil Red O values to the solvent control within each experiment. For determining positive samples in this screening program, the sample test results were compared to the upper or lower 95% confidence interval (CI) of the log-normal distribution of all experimental solvent controls. This resulted in a least significant difference of 17%. Thus, an LI greater than 1.17 or less than 0.83 was considered significantly different (p80% 3.0 1.00
Hops hexahydrocolupulone 4.0 0.57#
Ilex paraguariensis Green mate leaf powder extract 50 1.40*
Ipomoea batatas Caiapo AF 50 1.45*
Juniperus communis Juniper berry powder AN1286 25 0.64#
Lagerstoemia speciosa Banaba leaf extract 50 2.07*
Larix occidentali Fiber Aid 50 1.06
Lavandula angustifolia Lavender flower PE 2%
essential oil 50 1.00
Lycium barbarum Lycium berry 4:1 50 1.11
Lycopersicon esculentum Lycopene paste 50 1.74*
Tomato extract 50 1.36*
Medicago sativa Alfalfa herb PE 4:1 50 1.85*
Momordica charantia Bitter melon 50 1.73*
Bitter melon 5% 50 1.59*
M. charantia extract 50 1.17*
Karela (bitter melon) 25 1.14
M. charantia extract 50 1.05
Morinda citrifolia Morinda extract powder 50 1.09
Mucuna pruriens M. puriens dry extract 50 1.36*
M. pruriens 50 1.10
Murraya koenigii Curry leaf oil 50 1.12
Curry leaves 50 1.03
Ocimum sanctum Tulsi extract 50 1.59*
Oenothera biennis Evening primrose extract 50 1.08
Olea europaea ssp.Europaea Olive leaf extract 18% 50 1.56*
Olivo (Olea Eur.) 50 0.95
Opuntia ficus-indica Cactus extract Pharmline 50 1.08
Origanum vulgare Oregano essential oil 50 1.03
Origanum vulgare/Salvia officinalis Origanox WS 50 1.42*
Oryza sativa Oryzbran 50 1.24*
Rice extract 50 1.15
O. sativa/Monascus purpureus Red yeast rice 50 1.18*
Panax ginseng Ginseng, Korean root 50 1.12
Panax quinquefolius American ginseng PE 6% ginsenosides 50 1.57*
P. ginseng extract 50 1.33*
Ginseng root PE 16% ginsenosides 50 1.21*
Petroselinum crispum Parsley leaf PE 4:1 50 1.33*
Phaseolus vulgaris Red kidney bean extract 50 2.37*
Northern kidney bean extract 4:1 50 1.48*
Kidney bean 10:1 50 1.39*
White kidney bean phase 2 50 1.14
White kidney bean extract 10:1 50 1.09
Phyllanthus emblica Amla dry extract 50 1.74*
Amla fruit extract 50 1.17*
Emblica extract 50 1.51*
Phyllanthus amarus P. amarus extract 50 2.10*
P. amarus extract 50 1.60*
P. amarus extract 50 1.52*
Picrorhiza kurroa Picroliv 50 1.49*
Picrorhira root 50 1.46*
Pinus radiate Enzogenol 25 1.27*
Pisum sativum Pea powder 50 1.08
Polygonum cuspidatum Protykin 50 0.78#
Punica granatum Pomegranate powder 5% drum dried 50 1.40*
Pomegranate extract 70% ellagic 50 1.28*
Pomegranate extract 40% 25 1.19*
Pomegranate seed grade A sweet 50 1.06
Pomegranate seed grade B sour 50 0.98
Rosmarinus officinalis Rosemary extract 50 1.32*
Herblox seasoning 50 1.29*
Rosemary leaf PE 2% essential 50 1.24*
Oleoresin rosemary 50 1.08
Rubus occidentalis Evergreen (blackberry seed oil) 50 1.15
Black raspberry seed oil 50 1.09
Blackberry fruit PE 12:1 50 1.09
Rubus idaeus Red raspberry seed oil 50 1.62*
Ellagic acid extract from red raspberry seed 50 0.97
Salacia oblonga S. oblonga extract 50 1.33*
S. oblonga extract 50 1.21*
Salix alba Willow bark powder extract 50 0.99
Taraxacum officinale Dandelion root PE 4:1 50 1.06
Dandelion herb extract 50 1.01
Terminalia arjuna T. arjuna extract 50 1.47*
Tinospora cordifolia Tinofolin 50 1.41*
Tinospora 50 1.24*
T. cardifolia extract 50 0.98
T. cordifolia 50 0.81#
Trachyspermum ammi Ajwain seed 25 0.99
Tribulus terrestris T. terrestris dry extract 50 1.33*
Trigonella foenum graecum Fenugreek seed PE 4:1 50 1.08
Fenugreek seed 50 1.01
Fenugreek extract 4946a 50 0.85
Fenugreek extract 4945 50 0.85
Oleoresin fenugreek 25 0.88
Vaccinium macrocarpon Cranberry seed oil 50 1.22*
Cranberry protein 50 1.11
Cranberry seed extract 50 0.98
Vaccinium myrtillus Bilberry fruit PE 25% anthocyanidins 50 0.99
Vitis vinifera Grape seed extract phytosome 25 1.89*
Grape seed extract 50 1.58*
Grape pomace extract 25 1.42*
Grape juice extract 50 1.29*
Grape seed extract, powder 50 1.12
W. japonica Wasabi powder 50 1.74*
Withania somnifera Ashwagandha root 50 1.53*
Ashwagandha root 50 1.33*
Withania somnifera Extract 4% 50 1.31*
Ashwagandra extract powder 50 1.14
Ashwagandha extract powder 50 1.13
W. somnifera extract 50 0.97
Zingiber officinale Ginger Extract 5% 50 1.51*
Zea mays Cornsilk PE 4:1 50 0.84
[00092] The test material was added in DMSO at Day 0 of differentiation and every 2 days throughout the maturation phase (Day 6/Day 7). Whenever fresh medium was added, fresh test material was also added. PE, Powdered Extract; XN, xanthohumol.
aLI = (optical density)Test/(optical density)DMSO control.
LI values *>1.17 or # <0.83 are different from than the solvent control with P< .05.
[00093] Preadipocyte-differentiating results - As assessed microscopically, none of the products inhibited differentiation of the preadipocytes into adipocytes at the apparent concentrations reported. All testing resulted in greater than 95% of the cells exhibiting adipocyte morphology before Oil Red O or BIODIPY staining. Lipogenic results are presented alphabetically in Table 1 along with the apparent concentration tested and the LI. Sixty-one of the 94 unique botanicals (64.9%) exhibited an LI greater than the upper 95% CI of the mean of all solvent controls (>1.17). The mean LI of the troglitazone positive control was 1.43 (a 43% increase in intracellular lipid relative to the solvent controls), whereas the top 20% of lipogenic test materials included 23 botanical products and phytochemicals with an LI ranging from 1.59 to 2.45. The endogenous peroxisome proliferator-activated receptor ? agonist PGJ2 at 3.2 µg/mL produced an LI of 1.88 and fell within the range of the top 20% of botanicals.
Example 2
Selected, Positive Lipogenic Botanical Extracts Exhibit Anti-inflammatory Activity in 3T3-L1 Adipocytes
[00094] Rationale - Elevated plasma FFA and IL-6 levels induce neutrophilic inflammation at jointsand are highly associated with the development and severity of arthritis. TNF? can impact the endocrine functions of adipose tissue through stimulation of lipolysis, IL-6 secretion, and inhibition of adiponectin secretion (Figure 1). Targeting inhibitors of TNF?-stimulated fatty acid and IL-6 release from adipose tissue constitutes critical aspects of a multifunctional therapeutic strategy.
[00095] The model- The 3T3-L1 murine fibroblast model as used in Example 1 was employed in this example.
[00096] Chemicals - All chemicals used in this example were purchased from Sigma (St. Louis, MO) or otherwise noted and were of the highest purity commercially available. Test materials used in this example are listed in Table 3.
[00097] Anti-inflammatory activity of test materials - Post-differentiation, D6/D7 adipocytes were treated with test material 4 hours before the addition of TNF? at a final concentration of 10 ng/mL. Following overnight incubation of approximately 18 hours, the supernatant medium was removed and assayed for glycerol as a biomarker for FFA release, IL-6, and adiponectin.
[00098] Glycerol assay - Free fatty acid release (lipolysis) from 3T3-L1 adipocytes was quantified by measuring glycerol secretion into the medium. Glycerol was measured spectrophotometrically using the Free Glycerol Determination Kit (F6428, Sigma) and an EL 312e Microplate BIO-KINETICS spectrophotometer (BioTek, Winooski, VT). Glycerol release from adipocytes and was expressed as relative glycerol content (glycerol index) of the DMSO controls of eight observations.
[00099] Cytokine assays- IL-6 and adiponectin secretion into the medium in response to TNF? stimulation were quantified, respectively, using the Quantikine® mouse IL-6 immunoassay kit or the mouse adiponectin Quantikine immunoassay kit with no modifications (R&D Systems, Minneapolis, MN, USA).
[000100] Statistical analyses - Experiments assessing the anti-inflammatory activity of the random subset of test samples were repeated three times. For statistical analysis, the effects of test materials on glycerol, IL-6, or adiponectin secretion were normalized to the TNF? control to generate glycerol, IL-6, and adiponectin indexes. Differences among the treatments were determined by analysis of variance and Student’s least significant difference (LSD) test without correction for multiple comparisons; the nominal 5% probability of a type I error was used. The 95% CI values were computed using the analysis of variance error mean square estimate of variance (Excel®, Microsoft, Redmond, WA, USA).
Table 2
Relative Glycerol Secretion in TNF?-stimulated 3T3-L1 Adipocytes Treated with Selected Positive Lipogenic Botanical Products
Test Material
TNF? Common name Relative Glycerol
Dimethyl Sulfoxide 0 DMSO Control 0.54*
Dimethyl Sulfoxide + DMSO+ TNF? 1.0
Troglitazone + Positive Control 0.60*
Acacia nilotica + A. nilotica heartwood extract 0.69*
Aloe barbadensis + Aloe vera PE 40:1 0.80*
Areca catechu + Bettle nut 0.63*
Azadirachta indica + Neem leaves 0.69*
Boswellia serrata + Boswellia extract 65% 0.70*
Camellia sinensis + Tea polyphenol 0.52*
Centella asiatica + Centellin 0.71*
Cissus quadrangularis + Cissus extract (20:1) 0.76*
Curcuma longa + Turmeric extract 95% 0.70*
Foeniculum vulgare + Oleoresin fennel 0.76*
Humulus lupulus + Hops isoalpha acids 0.76*
Humulus lupulus + Hops rho-isoalpha acids 0.82*
Lagerstoemia speciosa + Banaba leaf extract 0.74*
Lycopersicon esculentum + Lycopene paste 0.69*
Medicago sativa + Alfalfa herb PE 4:1 0.85*
Phaseolus vulgaris + Red kidney bean extract 0.77*
Phyllanthus emblica + Amla dry extract 0.68*
Vitis vinifera + Grape seed extract phytosome 0.80*
Withania somnifera + Withania extract 4% 0.76*
Zingiber officinale + Ginger extract 4% 0.57*
[000101] Relative glycerol secretion in TNF?-stimulated 3T3-L1 adipocytes treated with selected botanical products. Day 6/Day 7 adipocytes were treated with test material 4 hours before the addition of TNF? at a final concentration of 10 ng/mL. Troglitazone, rho-isoalpha acids, and isoalpha acids were assayed at 5.0 µg/mL; all other samples were tested at 50 µg/mL. Following overnight incubation of approximately 18 hours, the supernatant medium was removed and assayed for glycerol. Relative glycerol secretion was calculatedas GlycerolTest+TNF?]/[GlycerolTNF?? Values are means of eight observations blocked over two independent experiments. (*) indicates statically different (p<0.05) from the TNF?-stimulated control.
[000102] Results – TNF? treatment of D6/D7 3T3-L1 adipocytes increased glycerol secretion 1.9-fold (Table 2) relative to the DMSO controls. Troglitazone as well as all 20 commercial botanicals decreased glycerol secretion relative to the TNF? treatment alone (P < 0.05).
Table 3
Relative IL-6 Secretion in TNF?-stimulated 3T3-L1 Adipocytes Treated with Selected Positive Lipogenic Botanical Products
Test Material
No.
TNF? Common name Relative IL-6
Dimethyl Sulfoxide 0 DMSO Control 0.15*
Dimethyl Sulfoxide + DMSO+ TNF? 1.0
Troglitazone + Positive Control 0.71*
Acacia nilotica 1 + A. nilotica heartwood extract 0.50*
Aloe barbadensis 2 + Aloe vera PE 40:1 0.69*
Areca catechu 3 + Bettle nut 0.87*
Azadirachta indica 4 + Neem leaves 0.58*
Boswellia serrata 5 + Boswellia extract 65% 0.57*
Camellia sinensis 6 + Tea polyphenol 0.91
Centella asiatica 7 + Centellin 0.74*
Cissus quadrangularis 8 + Cissus extract (20:1) 0.61*
Curcuma longa 9 + Turmeric extract 95% 0.70*
Foeniculum vulgare 10 + Oleoresin fennel 0.89
Humulus lupulus 11 + Hops isoalpha acids 0.60*
Humulus lupulus 12 + Hops rho-isoalpha acids 0.54*
Lagerstoemia speciosa 13 + Banaba leaf extract 0.73*
Lycopersicon esculentum 14 + Lycopene paste 2.09*
Medicago sativa 15 + Alfalfa herb PE 4:1 0.77*
Phaseolus vulgaris 16 + Red kidney bean extract 0.97
Phyllanthus emblica 17 + Amla dry extract 1.16*
Vitis vinifera 18 + Grape seed extract phytosome 0.69*
Withania somnifera 19 + Withania extract 4% 0.73*
Zingiber officinale 20 + Ginger extract 4% 0.57*
[000103] Relative IL-6 secretion in TNF?-stimulated 3T3-L1 adipocytes treated with randomly selected botanical products. Day 6/Day 7 adipocytes were treated with test material 4 hours before the addition of TNF? at a final concentration of 10 ng/mL. Troglitazone, rho-isoalpha acids, and isoalpha acids were assayed at 5.0 µg/mL, whereas. all other samples were tested at 50 µg/mL. Following overnight incubation of approximately 18 hours, the supernatant medium was removed and assayed for IL-6. Relative IL-6 secretion was calculated as (IL-6Test+TNF?]/[IL-6TNF?); values are means of eight observations blocked over two independent experiments.(*) indicates statically different (p<0.05) from the TNF?-stimulated control.
[000104] Results – TNF? increased IL-6 secretion from D6/D7 3T3-L1 adipocytes approximately 6.6-fold relative to DMSO controls (Table 3). Fifteen botanical products all inhibited IL-6 secretion (P < 0.05), whereas amla dry extract and lycopene paste increased IL-6 secretion, respectively, 20% and 109% relative to the TNF? control.Tea polyphenol, oleoresin fennel, and red kidney bean extract had no effect (P> 0.05).
Table 4
Relative Adiponectin Secretion in TNF?-stimulated 3T3-L1 Adipocytes Treated with Selected Positive Lipogenic Botanical Products
Test Material
No.
TNF? Common name Relative Adiponectin
Dimethyl Sulfoxide 0 DMSO Control 1.73
Dimethyl Sulfoxide + DMSO+ TNF? 1.00*
Troglitazone + Positive Control 1.94*
Acacia nilotica 1 + A. nilotica heartwood extract 1.31*
Aloe barbadensis 2 + Aloe vera PE 40:1 1.65*
Areca catechu 3 + Bettle nut 1.45*
Azadirachta indica 4 + Neem leaves 1.72*
Boswellia serrata 5 + Boswellia extract 65% 1.65*
Camellia sinensis 6 + Tea polyphenol 1.38*
Centella asiatica 7 + Centellin 1.47*
Cissus quadrangularis 8 + Cissus extract 20:1 1.49*
Curcuma longa 9 + Turmeric extract 95% 1.41*
Foeniculum vulgare 10 + Oleoresin fennel 1.54*
Humulus lupulus 11 + Hops isoalpha acids 1.39
Humulus lupulus 12 + Hops rho-isoalpha acids 1.45*
Lagerstoemia speciosa 13 + Banaba leaf extract 1.74*
Lycopersicon esculentum 14 + Lycopene paste 1.35*
Medicago sativa 15 + Alfalfa herb PE 4:1 1.80*
Phaseolus vulgaris 16 + Red kidney bean extract 1.87*
Phyllanthus emblica 17 + Amla dry extract 1.28*
Vitis vinifera 18 + Grape seed extract phytosome 1.56*
Withania somnifera 19 + Withania extract 4% 1.42*
Zingiber officinale 20 + Ginger extract 5% 1.58*
[000105] Relative adiponectin secretion in TNF?-stimulated 3T3-L1 adipocytes treated with selected botanical products. Day 6/Day 7 adipocytes were treated with test material 2 hours before the addition of TNF?at a final concentration of 10 ng/mL. Troglitazone, rho-isoalpha acids, and isoalpha acids were assayed at 5.0 µg/mL, whereas all other samples were tested at 50 µg/mL. Following overnight incubation of approximately 18 hours, the supernatant medium was removed and assayed for adiponectin. Relative adiponectin secretion was calculated as (AdiponectinTest+TNF?]/[AdiponectinTNF?); values are means of eight observations blocked over two independent experiments.(*) indicates statically different (p<0.05) from the TNF?-stimulated control.
[000106] Results – TNF? treatment of D6/D7 3T3-L1 adipocytes decreased adiponectin secretion 58% (Table 4) relative to the DMSO controls. Troglitazone as well as all 20 commercial botanicals increased adiponectin secretion relative to the TNF? treatment alone (P < 0.05).
Example 3
A Preadipocyte Differentiating Composition Clinically Improved Osteoarthritis, and Rheumatoid Arthritis
[000107] Objective– The primary endpoint of this study was to evaluate the effects of administration of capsules containinga preadipocyte differentiating compositioncomprising five botanical extracts individually capable of inhibiting free fatty acid and IL-6 secretion, and increasing adiponectin secretion from inflammation-stimulated adipocytes on subjects exhibiting signs and symptoms of osteoarthritis, and rheumatoid arthritis.
[000108] Test material – The test material consisted of five preadipocyte differentiating botanical extracts individually capable of inhibiting free fatty acid and IL-6 secretion and increasing adiponectin secretion from inflammation-stimulated adipocytes and excipients as described in Table 5. Clinical testing of our adipocyte-modifying hypothesis role in arthritis of the 15 botanical extracts meeting all criteria posed an undue burden, as a calculation of the possible number of combination of 15 botanical products taken 5 at a time would necessitate the undue burden of clinical testing 3,003 formulations. The selection of five of the 15 botanical products and dosage was based on an algorithm that incorporated the responses in the adipocyte screening assays, relative safety, and commercial availability.
[000109] Study design- The study was a single-arm, open-label, six-month safety and efficacy study of capsules t.i.d. as detailed in Table 5.
Table 5
Composition of Anti-arthritic Formulation
Ingredient % Per capsule
[mg]
Withania somnifera Extract 4% (Ashwagandha) 20.9 90
Boswellia Extract 65% (Shallaki) 20.9 90
Curcumin Extract 95% (Turmeric) 5.8 25
Ginger Extract 5% 2.3 10
Cissus quadrangularis Extract (20:1) 29.1 125
Citric acid 0.50 2.0
Di-calcium Phosphate 10.1 43.6
Starch 3.5 15
Sodium benzoate 0.50 2.0
Aerosil (Degussa) 4.65 20
Butylhydroxytoluene 0.05 0.20
Polyvinylpyrrolidone 1.4 6.0
Magnesium stearate 0.30 1.2
Total 100 430
[000110] Study plan –This was an open-label, single-arm, clinical study therefore investigator as well as subjects were aware of the investigational product allocated to individual subjects.The composition capsule (Table 5) was taken orally three times a day for a period of six months. Health evaluations were made at the beginning of the study(screening Days -7 to -2), at the first month, third month, and at the end of the study i.e., sixth month (Table 6). At each visit, subjects were evaluated for efficacy and any adverse effects, vital examination, and laboratory investigation.
Table 6
Subject Variables and Scheduling

Variable Day
(-7 to -2) Day
(0) Month
(1) Month
(3) Month
(6)
General Physical Examination
Blood Pressure (mm/Hg) Y Y Y Y Y
Pulse rate (beats/min) Y Y Y Y Y
Temperature (°F) Y Y Y Y Y
Respiratory rate Y Y Y Y Y
Pallor Y Y Y Y Y
Cyanosis Y Y Y Y Y
Icterus Y Y Y Y Y
Clubbing Y Y Y Y Y
Systemic Examination
Cardiovascular system Y Y Y Y Y
Respiratory system Y Y Y Y Y
Pre-abdominal Y Y Y Y Y
Arthritis Specific Assessment
Swelling Y Y Y Y Y
Joint pain Y Y Y Y Y
Restricted movement Y Y Y Y Y
Morning stiffness Y Y Y Y Y
Migratory joint pain Y Y Y Y Y
Signs of inflammation Y Y Y Y Y
Fever Y Y Y Y Y
Deformity Y Y Y Y Y
Other symptoms Y Y Y Y Y
Clinical assessment will be Nil-0, moderate -2 and severe-3
The overall response: Symptoms became worse-1: No change-2: Slight improvement-3 Moderate improvement - 4, Marked improvement-5, Cured – 6
Laboratory Investigations
Rheumatoid factor (RF) Y Y Y Y
C-reactive protein Y Y Y Y
Erythrocyte sedimentation rate (ESR) Y Y Y Y
IL-2 Y Y Y Y
IL-6 Y Y Y Y
Complete blood count Y Y
Compete metabolic profile Y Y
Urine analysis- routine and microscopy Y Y
Serum Beta- HCG (only female patients) Y
Overall Impression by the Subject
Symptoms became worse Y Y Y
No change Y Y Y
Slight improvement Y Y Y
Moderate improvement Y Y Y
Marked improvement Y Y Y
Cured Y Y Y
Overall Impression by the Physician
Symptoms became worse Y Y Y
No change Y Y Y
Slight improvement Y Y Y
Moderate improvement Y Y Y
Marked improvement Y Y Y
Cured Y Y Y
Subjects –After screening (Figure 4 -301) the total number of the patients taking part in the study was 34 (Figure 4 – 303), with 6 lost to follow-upand 28 patients (82.3%) completed the treatment (Figure 4 – 305).
[000111] Physical examination - A physical examination was performed at each visit as follows: general appearance, respiratory, skin, eyes ears, nose, throat, heart, abdomen, reflexes, lymph nodes and extremities, body temperature, heart rate, respiratory rate, and blood pressure.
[000112] Laboratory examination - The safety variables included hematology, biochemistry, urinalysis, complete blood count, kidney function, aminotransferases, and alkaline phosphatase were performed by a central laboratory at the time of screening and after the study.
Table 7
Description of Subjects
Subject Number Gender Age
[yr]
Diagnosis
1 F 45 OA
2 M 52 OA
3 F 58 OA
4 M 42 OA
5 F 57 OA
6 M 58 OA
7 M 39 OA
8 M 64 OA
9 F 65 RA
10 F 75 OA
11 F 65 RA
12 F 40 RA
13 M 57 OA
14 F 60 RA
15 M 57 OA
16 F 54 OA
17 M 54 OA
18 F 43 OA
19 F 41 OA
20 M 64 OA
21 F 60 OA
22 F 44 OA
23 M 35 OA
24 F 55 OA
25 F 66 RA
26 F 62 RA
27 F 47 RA
28 M 47 OA
[000113] Early discontinuation of individual patients - Patients were informed that they have the right to withdraw from the study at any time without giving a reason. In addition, the investigator had the right to remove a patient from the study if, in the investigator's opinion, it was not in the best medical interest of the patients to continue in the study.
[000114] Treatment period – Eligible patients at Day -7 to -2were asked to return to the clinic for follow up on Day - 0 (Visit 1), month 1 (Visit 2), month 3 (Visit 3), and month 6 (Visit 4) for clinical assessment (Table 6). Eligible patients were given test material at Baseline Visit 1. At all these visits, subjects were assessed for treatment efficacy and adverse events.
[000115] Patients were considered forremoval from the study for the following reasons: (1) Protocol violation by the patient, which may result in improper conclusion; (2) At the discretion of the investigator; (3) Lack of efficacy; and (4) Adverse Events.
[000116] For discontinuation of a patient, the visit V8 assessments were performed as far as possible, at the earliest opportunity. Alternative therapy was to be provided to the subject upon discontinuation from the study. The date of discontinuation from the study and the reason for discontinuation were recorded.
[000117] Criteria for removal from the study: Following were the criteria that were utilized by the subject, attending physician, or Principal Investigator to remove a subject from the study: (1) Disease progression/recurrence; (2) Intercurrent illness that prevents further administration of treatment; (3) Unacceptable adverse event(s); (4) Patient's decision to withdraw from the study; (5) Change in the patient's condition rendering the patient unacceptable for further treatment in the judgment of the investigator.
[000118] Protocol amendments - (1) Any amendment had to be agreed upon between the investigator and sponsor in the form of a written amendment. Any changes were signed and dated by the Principal Investigator. and the sponsor and attached to the original protocol; and (2) All amendmentswere notified to the local ethical committee.
[000119] Ethics - The clinical protocol was approved by the Ethical Committee of the JSS Ayurveda Medical College & Hospital, Mysur, Mysore, Karnataka India. This study was conducted based on good clinical practice International Conference on Harmonisation guidance and the ethical principles of the Declaration of Helsinki. Before enrollment, every participant received complete instructions concerning the protocol and the objectives of the study in nontechnical terms and they then executed a written, informed consent document. A personal copy of the executed, informed consent document was provided to each subject.
[000120] Results – The formulation was well tolerated and compliance was excellent. Out of 34 patients, participated in the study, 24 patients completed the treatment (71.6%) and 10 were lost to the study for reasons unrelated to the test formulation. The results were based on clinical improvement and biochemical values assessed. According to clinical assessment, 12 patients out of 24, showed good improvement, 4 patients got complete relief and the remaining 8 patients had moderate improvement. The assessment was based on different signs and symptoms like pain, swelling, range of movements, deformity, and other inflammatory signs. The biochemical assessment was done mainly on the values of ESR, CRP, RF value, Serum calcium, BUN, IL2 & IL6.
[000121] According to the clinical assessment 8 patients got complete relief from the signs and symptoms of arthritis, 5 patients showed marked improvement, 8 patients showed moderate improvement while 3 patients had slight improvement. The assessment was based on different signs and symptoms like pain, swelling, range of movements, deformities, crepitations in the joints, and other inflammatory signs.
[000122] The biochemical assessment was done based mainly on the values of ESR, CRP, Serum Calcium, Serum Urea, BUN, IL-1 (interleukin 1), and IL6 -(interleukin 6). Ten patients (29.4%) showed a marked reduction in ESR at the end of the treatment showing anti-inflammatory action of the drug.In 5 cases the positive CRP initially became negative at the end of the study (100%) showing the reduction in the inflammation.IL2 and IL6. Out of 24 patients, 17 showed a gradual reduction in the values of IL2 and IL6, which are inflammatory mediators. This supports the decreases in adipocyte secretion of IL-6 seen in our in vitro studies in Example 2.
[000123] Serum calcium was seen to improve in 7 cases.BUN values remained within normal limits at the end of the 6th month indicating the safety of the drug on kidneys.
[000124] Overall, the results were encouraging with marked improvement in a majority of patients and 8 patients with initial stages of arthritis got complete relief. Further, these clinical findings strongly support our hypothesis that the adipocyte is a primary target for alleviating the signs and symptoms of arthritis.
Example 4
Proposed Formulations of a Preadipocyte Differentiating Composition Capable of Clinically Improving Arthritis
[000125] While Example 3 only presents the combination of five of the 15 preadipocyte differentiating botanicals individually capable of inhibiting free fatty acid and IL-6 secretion, and increasing adiponectin secretion from inflammation-stimulated adipocytes, it would be obvious for one skilled in the art to expect the same results with other combinations of botanicals with similar properties. Examples of botanicals and phytochemicals useful for clinically effective, anti-arthritic formulations of five components resulting from examples presented herein are presented in Table 8.
Table 8
Botanical Extracts and phytochemicals with Anti-inflammatory Activity in TNF?Stimulated
for Improving Joint Function in Arthritic Subjects

TNF?-Stimulated Adipocytes Positive
Ingredients† FFA IL-6 Adiponectin
Acacia nilotica Y Y Y
Aloe barbadensis Y Y Y
Areca catechu Y Y Y
Azadirachta indica Y Y Y
Boswellia serrata Y Y Y
Centella asiatica Y Y Y
Cissus quadrangularis Y Y Y
Curcuma longa Y Y Y
Hops isoalpha acids Y Y Y
Hops rho-isoalpha acids Y Y Y
Lagerstoemia speciose Y Y Y
Medicago sativa Y Y Y
Vitis vinifera Y Y Y
Withania somnifera Y Y Y
Zingiber officinale Y Y Y
Inactive Ingredients††
Colloidal silicon dioxide (Aerosil/Cabot)
Sodium lauryl sulfate
Talcum
Magnesium stearate
Polyvinylpyrrolidone
Isopropyl alcohol
†Five active ingredients and doses can be selected based upon
††Amounts in similar, final proportions to Table 7
[000126] Potential formulations for improving joint function in arthritic patients are presented in Table 8. Five active ingredients of the 15 presented can be formulated in combinations based on an algorithm that incorporates the responses in the adipocyte screening assays, relative safety, and commercial availability. These selected active ingredients can be added with inactive ingredients added in amounts known to one skilled in the art as necessary to allow for formulating capsules, bars, medical foods, etc.
[000127] Thus, there has been disclosednovel combinations of a preadipocyte differentiating composition comprising five botanical extracts individually capable of inhibiting free fatty acid and IL-6 secretion, and increasing adiponectin secretion from inflammation-stimulated adipocytes. Methods for the production of these formulations and uses have been described. It will be readily apparent to those skilled in the art, however, that various changes and modifications of an obvious nature may be made without departing from the spirit of the invention, and all such changes and modifications are considered to fall within the scope of the invention as defined by the appended claims. Such changes and modifications would include, but not be limited to, the incipient ingredients added to affect the food, capsule, tablet, powder, lotion, or bar manufacturing process as well as additional herbs, phytoceuticals, vitamins, flavorings, and carriers. Other such changes or modifications would include the use of herbs or other botanical products containing the combinations of the preferred embodiments disclosed above. Many additional modifications and variations of the embodiments described herein may be made without departing from the scope, as is apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only.

,CLAIMS:We Claim:
1 A therapeutically effective preadipocyte differentiating composition comprising about 20.9% Boswellia extract (65% - Shallaki), about 29.1% Cissus quadrangularis Extract (20:1), about 5.8%Turmeric extract (95% - Turmeric), about 20.9% Withania somnifera extract (4% - Ashwagandha), and about 2.3%Ginger extract powder (5%), wherein each component is individually capable of inhibiting free fatty acid and IL-6 secretion and increasing adiponectin secretion from inflammation-stimulated adipocytes and when administered to a subject in need, the formulation has an additive effect of treating aninflammation-mediated metabolic disorder.
2 The composition of claim 1, wherein the Boswellia extract (65% - Shallaki) comprises from about 0.01 wt % to about 80 wt %, the Cissus quadrangularis Extract (20:1) comprises from about 0.01 wt % to about 80 wt %,), the Turmeric extract (95% - Turmeric) ) comprises from about 0.01 wt % to about 80 wt %,), the Withania somnifera extract (4% - Ashwagandha) comprises from about 0.01 wt % to about 80 wt %,), and the Ginger extract powder (5%), comprises from about 0.01 wt % to about 80 wt % of the composition.
3 The composition of claim 1, wherein the five botanical extracts comprises from about 1 wt % to about 80 wt % of the composition.
4 The composition according to claim 1, wherein the five botanical extracts are chosen from the group comprising Acacia nilotica heartwood extract, Aloe vera PE 40:1, Areca catechu, Neem leaves, Boswellia extract 65%, Centellin, Cissus extract (20:1), Turmeric extract 95%, Hops isoalpha acids, Hops rho-isoalpha acids, Banaba leaf extract, Alfalfa herb PE 4:1, Grape seed extract phytosome, Withania somnifera extract 4%, Ginger extract powder 5%, wherein each component is individually capable of inhibiting free fatty acid and IL-6 secretion and increasing adiponectin secretion from inflammation-stimulated adipocytes.
5 The composition according to claim 1, wherein the five botanical extracts are chosen from the group comprising Acacia nilotica, Aloe barbadensis, Areca catechu, Azadirachta indica, Boswellia serrata, Centella asiatica, Cissus quadrangularis, Curcuma longa, Hops isoalpha acids, Hops rho-isoalpha acids, Lagerstoemia speciose, Medicago sativa. Vitis vinifera, Withania somnifera, and Zingiber officinale, wherein each component is individually capable of inhibiting free fatty acid and IL-6 secretion and increasing adiponectin secretion from inflammation-stimulated adipocytes.
6 The composition according to claim 1, wherein the inflammation-mediated, metabolic disorder comprises at least one member of the group comprising osteoarthritis, rheumatoid arthritis, fibromyalgia, gout, and juvenile rheumatoid arthritis.
7 The composition according to claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier.
8 The composition of claim 1, wherein the composition is an oral or topical dosage formulation.
9 The composition of claim 1, wherein the oral dosage form comprises a capsule, a tablet, a powder, a beverage, a syrup, a suspension, or a food.
10 The composition of claim 1, wherein the topical dosage form comprises an aerosol, gel, lotion, liquid, cream, foam, tape, or suspension.
11 The composition according to claim 1, further comprising administering to the subject at least one secondary therapeutic agent.
12 The composition according to claim 1, wherein the secondary therapeutic agent is selected from the group comprising aspirin, ibuprofen, naproxen sodium, acetaminophen, celecoxib, diclofenac, fenoprofen, indomethacin, ketorolac tromethamine, meclofenamate sodium, diflunisal, tolmetin, ketoprofen, flurbiprofen, hydrocortisone, betamethasone, prednisolone, dexamethasone, hydrocortisone, and methylprednisolone.
13 The composition of claim 1, wherein the composition comprises greater than 1.5 times the ability to inhibit free fatty acid and IL-6 secretion and increase adiponectin secretion from inflammation-stimulated adipocytes than an equivalent amount of any one extract or concentrate or a sum of the extracts and concentrate.
14 A method of treating an inflammation-mediated, metabolic disorder in a subject in need thereof by administering a therapeutically effective preadipocyte differentiating composition comprising about 20.9% Boswellia extract (65% - Shallaki), about 29.1% Cissus quadrangularis Extract (20:1), about 5.8%Turmeric extract (95% - Turmeric), about 20.9% Withania somnifera extract (4% - Ashwagandha), and about 2.3% Ginger extract powder (5%), wherein each component is individually capable of inhibiting free fatty acid and IL-6 secretion and increasing adiponectin secretion from inflammation-stimulated adipocytes.
15 The method of claim 14, wherein the Boswellia extract (65% - Shallaki) comprises from about 0.01 wt % to about 80 wt %, the Cissus quadrangularis Extract (20:1) comprises from about 0.01 wt % to about 80 wt %,), the Turmeric extract (95% - Turmeric) ) comprises from about 0.01 wt % to about 80 wt %,), the Withania somnifera extract (4% - Ashwagandha) comprises from about 0.01 wt % to about 80 wt %,), and the Ginger extract powder (5%), comprises from about 0.01 wt % to about 80 wt % of the composition.
16 The method of claim 14, wherein the five botanical extracts comprises from about 1 wt % to about 80 wt % of the composition.
17 The method according to claim 14, wherein the five botanical extracts are chosen from the group comprising Acacia nilotica heartwood extract, Aloe vera PE 40:1, Areca catechu, Neem leaves, Boswellia extract 65%, Centellin, Cissus extract (20:1), Turmeric extract 95%, Hops isoalpha acids, Hops rho-isoalpha acids, Banaba leaf extract, Alfalfa herb PE 4:1, Grape seed extract phytosome, Withania somnifera extract 4%, Ginger extract powder 5%, wherein each component is individually capable of inhibiting free fatty acid and IL-6 secretion and increasing adiponectin secretion from inflammation-stimulated adipocytes.
18 The method according to claim 14, wherein the five botanical extracts are chosen from the group comprising Acacia nilotica, Aloe barbadensis, Areca catechu, Azadirachta indica, Boswellia serrata, Centella asiatica, Cissus quadrangularis, Curcuma longa, Hops isoalpha acids, Hops rho-isoalpha acids, Lagerstoemia speciose, Medicago sativa. Vitis vinifera, Withania somnifera, and Zingiber officinale, wherein each component is individually capable of inhibiting free fatty acid and IL-6 secretion and increasing adiponectin secretion from inflammation-stimulated adipocytes.
19 The method according to claim 14, wherein the inflammation-mediated, metabolic disorder comprises at least one member of the group comprising osteoarthritis, rheumatoid arthritis, fibromyalgia, gout, and juvenile rheumatoid arthritis.
20 The method according to claim 14, wherein the composition further comprises a pharmaceutically acceptable carrier.
21 The method according to claim 14, wherein the composition is an oral or topical dosage formulation.
22 The method according to claim 14, wherein the oral dosage form comprises a capsule, a tablet, a powder, a beverage, a syrup, a suspension, or a food.
23 The method according to claim 14, wherein the topical dosage form comprises an aerosol, gel, lotion, liquid, cream, foam, tape, or suspension.
24 The method according to claim 14, further comprising administering to the subject at least one secondary therapeutic agent.
25 The method according to claim 14, wherein the secondary therapeutic agent is selected from the group comprising aspirin, ibuprofen, naproxen sodium, acetaminophen, celecoxib, diclofenac, fenoprofen, indomethacin, ketorolac tromethamine, meclofenamate sodium, diflunisal, tolmetin, ketoprofen, flurbiprofen, hydrocortisone, betamethasone, prednisolone, dexamethasone, hydrocortisone, and methylprednisolone.
26 The method according to claim 14, wherein the composition comprises greater than 1.5 times the ability to inhibit free fatty acid and IL-6 secretion and increase adiponectin secretion from inflammation-stimulated adipocytes than an equivalent amount of any one extract or concentrate or a sum of the extracts and concentrate.