WO2013059642A1

WO2013059642A1 - Methods and compositions for attenuating tnf-a-mediated monocyte-endothelial interaction and improved flow-mediated vasodilation in hyperlipidemia using hops tetrahydro iso-alpha acids and niacin

Info

Publication number: WO2013059642A1
Application number: PCT/US2012/061091
Authority: WO
Inventors: Matthew L. Tripp; Jeffrey S. Bland; Veera R. KONDA; Anuradha Desai; Joseph J. LAMB
Original assignee: Metaproteomics, Llc
Priority date: 2011-10-21
Filing date: 2012-10-19
Publication date: 2013-04-25

Abstract

Disclosed are methods and compositions combining the anti-inflammatory tetrahydro iso-alpha acids (THIAA) from hops with niacin for improving endothelial function. The THIAA/niacin mixture inhibited various TNF-a-induced cytokines in human aortic endothelial cells and in THP-1 monocytic cells, and was significantly more efficacious than niacin alone. The effects of THIAA and niacin on endothelial-regulated flow-mediated vasodilation (FMD) was tested in dyslipidemic volunteers. The 12-week treatment resulted in a clinically relevant FMD increase compared to a trend toward decrease in placebo. THIAA/niacin treatment also improved total cholesterol, low-density lipoprotein cholesterol and uric acid.

Description

METHODS AND COMPOSITIONS FOR ATTENUATING TNF-A-MEDIATED MONOCYTE-ENDOTHELIAL INTERACTION AND IMPROVED FLOW-MEDIATED VASODILATION IN HYPERLIPIDEMIA USING HOPS TETRAHYDRO ISO-ALPHA

ACIDS AND NIACIN

RELATED APPLICATIONS

[001] This application claims priority to U.S. Provisional Patent Application No.

61/549,900 filed October 21 , 201 1 , the entire content of which is hereby incorporated herein by reference as though fully set forth here.

FIELD OF THE INVENTION

[002] The invention relates to methods and compositions using hops tetrahydro iso- alpha acids and niacin for attenuating TNF-a mediated monocyte - endothelial cell interactions in hyperlipidemia. The methods and compositions so disclosed additionally improve flow mediated vasodilation.

BACKGROUND OF THE INVENTION

[003] Atherosclerosis, a chronic inflammatory disease of the arterial wall reflecting dysregulated lipid metabolism, pathological monocyte - endothelial cell interactions, and oxidative stress, is a major contributor to the morbidity and mortality of cardiovascular disease. In the presence of insults, such as oxidized lipoproteins, hyperglycemia, bacterial cell wall components, and pro-inflammatory cytokines, the vascular endothelium mediates early pathophysiological changes resulting in the initiation of atherosclerosis. Shear stress, the drag created by flow of blood across the vascular endothelial, is the primary influence upon production of nitric oxide by a healthy vascular endothelium [Mitchell GF, Parise H. Vita JA, et al., Local shear stress and brachial artery flow-mediated dilation: the Framingham Heart Study. Hypertension, 2004. 44(2): p. 134-139]. In the presence of hyperglycemia and the activation of phosphokinase Cpn, production of nitric oxide (NO) is reduced which results in loss of flow- mediated vasodilation (FMD) [Dimmeler S, Fleming I, Fisslthaler B, et al., Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature, 1999. 399(6736): p. 601 -605.].

[004] Contemporaneously, oxidation of low-density lipoprotein cholesterol (LDL-C) and localized immune/inflammatory signaling alter cell signaling by endothelial cells [Hotamisligil GS, Inflammation and metabolic disorders. Nature, 2006. 444(7121 ): p. 860-867., Tomaselli GF Barth AS, Sudden cardio arrest: oxidative stress irritates the heart. Nat Med, 2010. 16(6): p. 648-649]. Activated endothelial cells express adhesion molecules, resulting in circulating monocyte adhesion to the endothelial cells. Monocyte chemoattractant protein- 1 (MCP-1) secreted by endothelial cells directs migration into subendothelial space, and subsequent monocyte differentiation into macrophages. Rapidly accumulating lipoprotein particles, these lipid-laden macrophages form fatty streaks and secrete matrix metalloproteinases (MMPs), tissue factors, and pro-inflammatory cytokines that further amplify the feed-forward cycle of local inflammatory response [Ait-Oufella H, Taleb S, Mallat Z, et al.. Recent advances on the role of cytokines in atherosclerosis. Arterioscler Thromb Vase Biol, 201 1. 31(5): p. 969- 979],

[005] Nicotinic acid, or niacin, is a water-soluble vitamin (vitamin B ). It has been shown to reduce oxidative stress and vascular inflammation in vivo [Wu BJ, Yan L, Charlton F, et al., Evidence that niacin inhibits acute vascular inflammation and improves endothelial dysfunction independent of changes in plasma lipids. Arterioscler Thromb Vase Biol, 2010. 30(5): p. 968-975], and in clinical studies to favorably modify serum lipids. It also reduces the atherogenic LDL-C and very-low-density lipoprotein cholesterol (VLDL-C), increases the atheroprotective high-density lipoprotein cholesterol (HDL-C) [Hausenloy DJ Yellon DM, Targeting residual cardiovascular risk: raising high-density lipoprotein cholesterol levels. Heart, 2008. 94(6): p. 706-714], and improves atherosclerotic outcomes [Chapman MJ, Redfern JS. McGovern ME. et al., Niacin and fibrates in atherogenic dyslipidemia: pharmacotherapy to reduce cardiovascular risk. Pharmacol Ther, 2010. 126(3): p. 314-345., Lee JM, Robson MD, Yu LM. et al., Effects of high-dose modified-release nicotinic acid on atherosclerosis and vascular function: a randomized, placebo-controlled, magnetic resonance imaging study. J Am Coll Cardiol, 2009. 54(19): p. 1787-1794], A recent study showed that niacin had limited benefit (restricted to in subjects with low HDL-C) for treating endothelial dysfunction [Warnholtz A, Wild P, Ostad MA, et al., Effects of oral niacin on endothelial dysfunction in patients with coronary artery disease: results of the randomized, double-blind, placebo-controlled INEF study. Atherosclerosis, 2009. 204(1): p. 216-221]. Flushing, liver dysfunction and gastrointestinal symptoms associated with higher doses of niacin limit its broad applicability for the treatment of dyslipidemia and cardiovascular disease. Additional clinical concerns include an unfavorable impact on glucose homeostasis and a propensity to elevate uric acid levels [Florentin M, Liberopoulos EN, Kei A, et al., Pleiotropic Effects of Nicotinic Acid: Beyond High Density Lipoprotein Cholesterol Elevation. Curr Vase Pharmacol, 201 1 ].

[006] In addition to niacin associated side effects (e.g., flushing and palpitations) niacin has been shown to have toxicity associated with exacerbation of peptic ulcers, hepatitis, and worsening of glucose tolerance and diabetes control [Crouse, J.. New developments in the use of niacin for treatment of hyperlipidemia: new considerations in the use of an old drug; Coronary Artery Disease, 1996. 7(4) 321-326]. Furthermore, niacin is contraindicated for gout a it has been shown to raise uric acid levels and, at time, precipitating an attack of gout [Kasper, D.. et al. Harrison's Principles of Internal Medicine, 16th edition, McGraw-Hill, New York, USA., 2005., pages 2296 - 2297].

[007] Tetrahydro iso-alpha acids (THIAA), a family of reduced iso-alpha acid compounds derived from Humulus lupulus (hops), have shown potent effects on key cell signaling pathways mediating inflammation [Desai A, Konda VR, Darland G, et al., META060 inhibits multiple kinases in the NF-kappaB pathway and suppresses LPS— mediated inflammation in vitro and ex vivo. Inflamm Res, 2009. 58(5): p. 229-234]. In a mouse model of rheumatoid arthritis (RA), THIAA decreased bone, joint, and cartilage degradation, reduced carrageenan- induced footpad swelling, and reduced plasma levels of IL-6 in a dose-dependent manner [Konda VR, Desai A, Darland G, et al., META060 inhibits osteoclastogenesis and matrix metalloproteinases in vitro and reduces bone and cartilage degradation in a mouse model of rheumatoid arthritis. Arthritis Rheum, 2010. 62(6): p. 1683-1692]. Since systemic chronic inflammation associated with RA in humans has been associated with atherosclerosis and increased cardiovascular events [Villa-Forte A Mandell BF, Cardiovascular Disorders and Rheumatic Disease. Rev Esp Cardiol, 201 1], THIAA's anti-inflammatory properties may be efficacious in ameliorating inflammation-mediated monocyte-endothelial interaction.

[008] Therefore, there still remains a need for improved methods and compositions for promoting healthy uric acid levels and/or attenuating TNF-a-mediated monocyte-endothelial interaction and/or improving flow-mediated vasodilation in a subjects in need thereof.

SUMMARY OF THE INVENTION

[009] A first embodiment describes compositions for promoting healthy uric acid levels in a subject where the compositions comprise therapeutically effective amounts of niacin and tetrahydro iso-alpha acids.

[0010] A second embodiment of the invention describes methods for promoting healthy uric acid levels in a subject in need thereof where the method comprises administering to the subject a composition comprising therapeutically effective amounts of niacin and tetrahydro iso- alpha acids.

[0011] A further embodiment describes compositions for promoting healthy hs-CRP levels in a subject where the compositions comprise therapeutically effective amounts of niacin and tetrahydro iso-alpha acids.

[0012] In yet another embodiment methods for promoting healthy hs-CRP levels in a subject in need thereof are described where the methods comprise administering to the subject a composition comprising therapeutically effective amounts of niacin and tetrahydro iso-alpha acids.

BRIEF DESCRIPTION OF THE DRAWINGS [0013] Figure 1. Effects of test compounds on TNF-a-mediated cytokine release in endothelial cells. HAECs were pre-incubated with various concentrations of THIAA, niacin, and THIAA/niacin (1 - 20 μg/mL) for 1 hour, followed by TNF-a (10 ng/mL) stimulation for 8 hours. The medium was collected for the measurement of MCP-1 , RANTES, IL-6 and IL-8. Results represent mean ± SE and normalized with TNF-a alone stimulation equivalent to 100%. Repeated measures ANOVA was applied to compare the effects of niacin and THIAA/niacin. THIAA-only data (dash line) were included as reference.

[0014] Figure 2. Effects of test compounds on TNF-a-mediated cytokine release in TI IP-

1 monocytic cells. THP-1 cells were pre-incubated with various concentrations of THIAA, niacin, and THIAA/niacin (1 - 20 μg/mL) for 1 hour, followed by TNF-a (10 ng/mL) stimulation for 8 hours. The medium was collected for the measurement of MCP-1, RANTES, IL-Ι β and MMP-9. Results represent mean ± SE and normalized with TNF-a alone stimulation equivalent to 100%. Repeated measures ANOVA was applied to compare the effects of niacin and THIAA/niacin. THIAA-only data (dash line) were included as reference.

[0015] Figure 3. Percent changes in flow-mediated vasodilation (FMD) after a 12-week treatment with THIAA/niacin or placebo. A, data from 11 subjects completed the study (N=7 for THIAA/niacin arm and N=4 for placebo). B, data from the optional follow-up study in which 2 placebo arm participants (after completing the trial) volunteered to receive THIAA/niacin treatment for 10 - 12 weeks and their data were combined with the original THIAA/niacin arm (N=9). Placebo arm data remained unchanged (N=4).

DETAILED DESCRIPTION OF THE INVENTION

[0016] The invention relates to methods and compositions using hops tetrahydro iso- alpha acids and niacin for attenuating TNFa mediated monocyte - endothelial cell interactions in hyperlipidemia. The methods and compositions so disclosed additionally improve flow mediated vasodilation.

[0017] 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.

[0018] 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 ah, 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 the general principles of pharmacology include Goodman and Gilman's The Pharmacological Basis of Therapeutics, 1 1th Ed., McGraw Hill Companies Inc., New York (2006).

[0019] 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." 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%.

[0020] 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 a variable that is inherently discrete, the variable can be equal to any integer value of the numerical range, including the end-points of the range. Similarly, for a variable that is inherently continuous, 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 variables that are inherently discrete, and can be 0.0, 0.1. 0.01 , 0.001 , or any other real value for variables that are inherently continuous.

[0021] 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.

[0022] 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

[0023] A first embodiment describes compositions for promoting healthy uric acid levels in a subject where the compositions comprise therapeutically effective amounts of niacin and tetrahydro iso-alpha acids. [0024] As used herein, "promoting healthy uric acid levels" or variations thereof refers to usages where a reduction in uric acid levels is encouraged. Contemplated usage includes, without limitation, treatment support for individuals with gout.

[0025] As used herein, niacin shall refer to nicotinic acid, or vitamin B₃.

[0026] As used herein, "tetrahydro iso-alpha acid" or "THIAA" refers to any of one or more of tetrahydro-isoadhumulone, tetrahydro-isocohumulone and tetrahvdro-isohumulone. Structurally, THIAA comprises compounds of Genus A:

(Genus A)

wherein tetrahydro-isohumulone (R= -C¾CH(CH₃)2); tetrahydro-isocohumulone (R=

CH(CH₃)₂); and tetrahydro-isoadhumulone (R= -CH(CH₃)(CH₂CH₃)) are shown.

[0027] 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.

[0028] 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, ^ljC, ¹⁴C, N, O, O, etc. Compounds may exist in unsolvated forms as well as solvated forms, including hydrated forms and as 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, unless otherwise indicated, all physical forms are equivalent for the uses contemplated herein and are intended to be within the scope of the present invention.

[0029] Compounds according to the invention may be present as salts. In particular, pharmaceutically acceptable salts of the compounds are contemplated. A "'pharmaceutically acceptable salt" of the invention is a combination of a compound of the invention and either an acid or a base that forms a salt (such as, for example, the magnesium salt, denoted herein as '^"Mg" or ''Mag") with the compound and is tolerated by a subject under therapeutic conditions. In general, a pharmaceutically acceptable salt of a compound of the invention will have a therapeutic index (the ratio of the lowest toxic dose to the lowest therapeutically effective dose) of 1 or greater. The person skilled in the art will recognize that the lowest therapeutically effective dose will vary from subject to subject and from indication to indication, and will thus adjust accordingly. [0030] 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.

[0031] 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, though preferably administration is by inhalation 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.

[0032] Formulations suitable for administration by inhalation include formulations that can be dispensed by inhalation devices known to those in the art. Such formulations may include carriers such as powders and aerosols. The present invention encompasses liquid and powdered compositions suitable for nebulization and intrabronchial use, or aerosol compositions administered via an aerosol unit dispensing metered doses ("MDI").The active ingredient may be formulated in an aqueous pharmaceutically acceptable inhalant vehicle, such as. for example, isotonic saline or bacteriostatic water and other types of vehicles that are well known in the art. The solutions are administered by means of a pump or squeeze-actuated nebulized spray dispenser, or by any other conventional means for causing or enabling the requisite dosage amount of the liquid composition to be inhaled into the patient's lungs. Powder compositions containing the anti-inflammatory compounds of the present invention include, by way of illustration, pharmaceutically acceptable powdered preparations of the active ingredient thoroughly intermixed with lactose or other inert powders acceptable for intrabronchial administration. The powder compositions can be administered via a dispenser, including, but not limited to, an aerosol dispenser or encased in a breakable capsule which may be inserted by the patient into a device that punctures the capsule and blows the powder out in a steady stream. Aerosol formulations for use in the subject method typically include propellants, surfactants, and co-solvents and may be filled into conventional aerosol containers that are closed by a suitable metering valve.

[0033] Formulations of compositions of the present invention suitable for nasal administration, wherein the carrier is a solid, include a coarse powder having a particle size, for example, in the range of 20 to 500 microns which is administered in the manner in which snuff is administered, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations, wherein the carrier is a liquid, for administration, for example via a nasal spray, aerosol, or as nasal drops, include aqueous or oily solutions of the compound of the invention.

[0034] For oral administration, the compositions of the invention may be presented as discrete units such as capsules, caplets, gelcaps, cachets, pills, or tablets each containing a predetermined amount of the active ingredient as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil emulsion and as a bolus, etc. Alternately, administration of a composition of all of the aspects of the present invention may be effected by liquid solutions, suspensions or elixirs, powders, lozenges, micronized particles and osmotic delivery systems.

[0035] Formulations of compositions according to the aspects of the present invention suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, stabilizers, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) conditions requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

[0036] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface active or dispersing agent. Molded tablets may be made by molding, in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may be optionally coated or scored and may be formulated to provide a slow or controlled release of the active ingredient therein.

[0037J Formulations of compositions of the present invention for rectal administration may be prepared as a suppository with a suitable base comprising, such as, for example, cocoa butter.

[0038] Formulations of compositions of the present invention suitable for topical administration in the mouth include lozenges comprising the ingredients in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the ingredient to be administered in a suitable liquid carrier. Formulations of compositions of the present invention suitable for topical administration to the skin may be presented as ointments, creams, gels, lotions and pastes comprising the ingredient to be administered in a pharmaceutical acceptable carrier. A topical delivery system contemplated is a transdermal patch containing the ingredient to be administered.

[0039] Formulations of compositions according to the aspects of the present invention suitable for vaginal administration may be presented as pessaries, suppositories, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the compound of the invention such pharmaceutically acceptable carriers as are known in the art to be appropriate.

[0040] The methods and compositions of the present invention are intended for use with any mammal that may experience the benefits of the methods of the invention. Foremost among such mammals are humans, although the invention is not intended to be so limited, and is applicable to veterinary uses. Thus, in accordance with the invention, "mammals" or "mammal in need" include humans as well as non-human mammals, particularly domesticated animals including, without limitation, cats, dogs, and horses.

[0041] As used herein, by "treating" is meant reducing, preventing, and/or reversing the symptoms in the individual to which a compound of the invention has been administered, as compared to the symptoms of an individual not being treated according to the invention. A practitioner will appreciate that the compounds, compositions, and methods described herein are to be used in concomitance with continuous clinical evaluations by a skilled practitioner (physician or veterinarian) to determine subsequent therapy. Hence, following treatment the practitioners will evaluate any improvement in reducing cardiovascular risk factors or associated dyregularities according to standard methodologies. Such evaluation will aid and inform in evaluating whether to increase, reduce or continue a particular treatment dose, mode of administration, etc.

[0042] 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. Hence, as used herein, by "treating or alleviating the symptoms" is meant reducing, preventing, and/or reversing the symptoms of the individual to which a compound of the invention has been administered, as compared to the symptoms of an individual receiving no such administration. [0043] The term "therapeutically effective amount" is used to denote treatments at dosages effective to achieve the therapeutic result sought. Furthermore, one of 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 concurrent evaluation of beneficial effect.

[0044] It will be appreciated by those of skill in the art that the number of administrations of the compounds according to the invention will vary from patient to patient based on the particular medical status of that patient at any given time including other clinical factors such as age, weight and condition of the mammal and the route of administration chosen.

[0045] A second embodiment of the invention describes methods for promoting healthy uric acid levels in a subject where the method comprises administering to the subject a composition comprising therapeutically effective amounts of niacin and tetrahydro iso-alpha acids.

[0046] A further embodiment describes compositions for promoting healthy hs-CRP levels in a subject where the compositions comprise therapeutically effective amounts of niacin and tetrahydro iso-alpha acids.

[0047] As used herein, "promoting healthy hs-CRP levels" or variations thereof refers to usages where a modulating hs-CRP levels is encouraged. Contemplated usage includes, without limitation, treatment support for individuals with dyslipidemia. As used, hs-CRP refers to high- sensitivity C-reactive protein. [0048] In yet another embodiment methods for promoting healthy hs-CRP levels in a are described where the methods comprise administering to the subject in need a composition comprising therapeutically effective amounts of niacin and tetrahydro iso-alpha acids.

[0049] The following examples are intended to further illustrate certain preferred embodiments of the invention and are not limiting in nature. Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein.

EXAMPLES

[0050] Chemicals and Reagents. The human monocytic cell line THP-1 was purchased from ATCC (Manassas, VA) and maintained in RPMI1640 in the presence of 10% serum according to manufacturer's instructions. Human aortic endothelial cells (HAECs) were purchased from Lonza (Walkersville, MD) and maintained in EBM2 medium in the presence of 10% serum according to manufacturer^'s instructions. TNF-a was purchased from Sigma (St. Louis, MO). THIAA was supplied by Hopsteiner (New York, NY) and the chemical composition has been described [Mitchell GF, Parise H, Vita JA, et al., Local shear stress and brachial artery flow-mediated dilation: the Framingham Heart Study. Hypertension, 2004. 44(2): p. 134-139.]. Niacin was supplied by Glanbia Nutritionals (Carlsbad, CA). The tablet for the clinical trial contained 125 mg THIAA and 500 mg niacin (1 :4 w:w) in an extended release formulation.

Example 1

TNF-a-induced Inflammatory Markers in HAECs

[0051] HAECs were pre-incubated with various concentrations of THIAA, niacin and

THIAA/niacin (1 - 20 μg/mL) for 1 hour, and then stimulated with TNF-a (10 ng/mL) for 8 hours. IL-6, IL-8, MCP- 1 , and RANTES levels in the medium were measured by Milliplex MAP Human Cytokine/Chemokine Panel (Millipore, Billerica, MA) as per manufacturer's instructions. The analytes were quantified using Luminex 100 ^rM IS System and data were analyzed using a 5-parameter logistic method. [0052] Results: TNF-α incubation dramatically increased the expression of MCP-L

RANTES IL-6 and IL-8 in HAECs. Niacin or THIAA/niacin pre-incubation prior to TNF-a stimulation decreased the expression of all 4 markers (Fig. 1). THIAA+niacin was more efficacious (P<0.001) in inhibiting these markers than niacin alone.

Example 2

TNF-a-induced Inflammatory Markers in THP-1 Monocytes

[0053] Cells were pre-incubated with various concentrations of THIAA, niacin and

THIAA/niacin (1 - 20 μg/mL) for 1 hour, and then stimulated with TNF-a (10 l g/mL) for 18 hours. Measurement and quantification of markers in the medium were the same as described in Example 1.

[0054] Results: TNF-a incubation increased the expression of MCP-1 , RANTES IL-Ι β and MMP-9 in THP-1 cells (IL-6 and IL-8 levels were below detectable limits in THP-1 cells). Niacin pre-incubation prior to TNF-a stimulation decreased the expression of MCP-L RANTES, and MMP-9, but not IL-Ι β (Fig. 2). THIAA/niacin pre-incubation. on the other hand, reduced the TNF-a-induced expression of all 4 markers, and was more efficacious in inhibiting these markers than niacin alone (Fig. 2).

Example 3

TNF-a-induced MMP-9 Levels in THP-1 Cells

[0055] THP-1 cells were pre-incubated with various concentrations of THIAA, niacin and THIAA/niacin (1 - 20 μg/mL) for 1 hour, and then stimulated with TNF-a ( 10 ng/mL) for 18 hours. MMP-9 concentration in the medium was determined by Human MMP-9 Immunoassay Kit (GE Healthcare Life Sciences, Piscataway, NJ) as per manufacturer's instructions.

Example 4

Human Clinical Trial [0056] To investigate the effect of THIAA/niacin on FMD in 1 1 volunteers with dyslipidemia. a randomized, placebo-controlled trial was conducted at the Functional Medicine Research Center (Gig Harbor, WA). Inclusion criteria included (1) age between 30 and 60 years old, (2) LDL-C > 130 mg/dL, (3) HDL-C < 50 mg/dL for men or < 60 mg/dL for women, and (4) willingness to maintain current dietary and exercise practice during the study. Key exclusion criteria included (1) use of dietary supplements that contained the active ingredient(s) in this study with the exception of multi-vitamins that contain no more than 25 mg niacin, (2) use of NSAIDs and COX-2 inhibitors in the preceding 2 weeks and oral corticosteroids in the preceding 4 weeks, (3) subjects who were > 300 lbs, (4) subjects with a history of coronary artery disease, arrhythmia, cerebrovascular accident, HIV infection, cancer, or significant liver or kidney disease, and (5) pregnant and nursing women. The study was conducted in accordance with the Declaration of Helsinki, and informed written consent was obtained from each participant prior to enrollment in the trial.

[0057] At the beginning of the trial, eligible participants provided fasting blood samples, and had their brachial FMD measured by the study doctor. Participants were then randomized to receive either the placebo tablet (N=4) or the THIAA/niacin combined tablet (N=7; 1 tablet —125 mg THIAA/500 mg niacin— twice daily with food) for 12 weeks, during which they were instructed to maintain their normal dietary and exercise practice. Participants returned to the clinic every 2 weeks for the evaluation of compliance and potential adverse events. At the end of the 12 weeks, participants provided fasting blood samples and had their FMD measured by the same study doctor.

[0058] Blood samples collected at baseline and at end of trial were sent to Quest

Diagnostics (Seattle, WA) for analyses of lipids, glucose, high-sensitivity C-reactive protein (hs- CRP), and uric acid. The FMD measurements were performed on a MicroMaxx® Ultrasound System (SonoSite, Bothell, WA) using the protocol following the guidelines reported by Corretti et al. [Corretti MC, Anderson TJ, Benjamin EJ, et al., Guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery: a report of the International Brachial Artery Reactivity Task Force. J Am Coll Cardiol, 2002. 39(2): p. 257- 265.]. Participants were instructed to fast for 12 hours and minimize their physical activities, and rest for 15 minutes in supine position prior to the procedure. The participant's blood pressure was monitored every 3-5 minutes for safety purposes utilizing an automated sphygmomanometer with the blood pressure cuff placed around the participant's left arm. An automatic inflation, narrow- width occlusion cuff was place as high as possible on the participant's right arm. The brachial artery of the right arm approximately 5-10 cm above the antecubital fossa was scanned with a 10.5 MHz operating frequency. The cuff was then inflated to 40 mm Hg above the participant's systolic pressure or at least 200 mm Hg for initial occlusion of the brachial artery. After 5 minutes of occlusion, the pressure in the cuff was rapidly released and post-stimulus image acquisition was recorded until 3 minutes after the cuff release. The relative increase in diameter compared to the baseline diameter was calculated (as percentage) using Vascular Research Tools version 5.7.6 (Medical Imaging Associates, LLC. Coralville, IA).

[0059] For in vitro data, repeated measures ANOVA was applied to analyze the effects of

THIAA, niacin and THIAA/niacin. Data were analyzed using GraphPad Prism software (San Diego, CA) and reported as mean ± SD. For the pilot clinical study data, two-sample t-tests were performed to compare biomarkers between THIAA/niacin arm and placebo arm at baseline and at end of trial. T-tests were also utilized for comparing changes from baseline between the two arms. For the within-arm changes from baseline, paired t-tests were conducted to determine the significance. The probability of a type I error was set at the nominal 5 percent level. Data were analyzed using SAS (software version 8.1, SAS Institute) and reported as means ± SE,

[0060] Results: The averaged age in the THIAA/niacin arm, including 3 men and 4 women, was 49.7 ± 8.2 years old (mean ± SD). Their baseline BMI was 33.1 ± 5.4 kg/m². The average age in the placebo arm (1 man and 3 women) was 56.1 ± 1/3 years old and their BMI was 33.0 ± 7.7 kg/m . Baseline serum lipids, glucose, hs-CRP, and uric acid did not differ between study arms. In the THIAA/niacin arm, there was a significant decrease (PO.05) in total cholesterol, LDL-C and uric acid at week 12 compared to baseline (Table 1 ). A trend toward a decrease in apolipoprotein B (apo B) (P=0.07) and a trend toward increase in glucose (P=0.08) was also observed. In contrast, no differences in all measurements were observed in the placebo arm except there was a significant increase in hs-CRP (P=0.03) at week 12. A significant between-arm difference (PO.05) was seen in hs-CRP and glucose.

Table 1.

Values and changes of lipid markers, hs-CRP, glucose, and uric acid by study arms

Variable Visit THIAA/niacin arm Placebo arm P

Value Change Value Change

Cholesterol Baseline 236.1 ± 248.3 ±

(mg/dL) 12 8.0 -17.0 ± 6.5* 18.8 -2.3 ± 9.3 0.22 weeks 219.1 ± 246.0 ±

5.9 23.8

TG Baseline 215.6 ± 185.3 ±

(mg/dL) 12 59.1 -28.7 ± 27.4 31.5 27.0 ± 38.4 0.26 weeks 186.9 ± 212.3 ±

37.0 61.4

LDL Baseline 152.9 ± 160.5 ±

(mg/dL) 12 8.7 -19.1 ± 6.1 * 17.7 -7.3 ± 4.3 0.21 weeks 133.7 ± 153.3 ±

6.6 14.3

HDL Baseline 47.9 ± 50.5 ± 1.6

(mg/dL) 12 2.7 0.3 ± 2.5 50.3 ± 3.4 -0.3 ± 4.4 0.91 weeks 48.1 ±

4.7 Variable Visit THIAA/niacin arm Placebo arm P

Value Change Value Change

Chol/HDL Baseline 5.00 ± 4.98 ±

12 0.23 -0.17 ± 0.34 0.50 0.00 ± 0.35 0.75 weeks 4.83 ± 4.98 ±

0.54 0.64

TG/HDL Baseline 4.95 ± 3.73 ±

12 1.69 -0.36 ± 0.37 0.71 0.56 ± 0.97 0.31 weeks 4.59 ± 4.29 ±

1.45 1.35

Apo Al Baseline 142.3 ± 146.3 ± 3.2

(mg/dL) 12 2.6 -6.7 ± 8.0 143.3 ± 4.5 -3.0 ± 2.6 0.67 weeks 135.6 ±

9.9

Apo B Baseline 1 12.6 ± 131.3 ±

(mg/dL) 12 4.2 -12.3 ± 5.6 12.5 -6.0 ± 4.7 0.47 weeks 1 10.3 ± 125.3 ± 9.5

5.6

ApoB/ApoAl Baseline 0.86 ± 0.89 ±

12 0.02 -0.03 ± 0.06 0.07 -0.02 ± 0.04 0.92 weeks 0.83 ± 0.87 ±

0.06 0.07 Variable Visit THIAA/niacin arm Placebo arm P

Value Change Value Change

Lp(a) Baseline 108.3 ± 95.5 ±

(nmol/L) 12 30.5 -9.3 ± 8.3 72.3 -1.0 ± 1.3 0.36 weeks 99.0 ± 94.5 ±

30.4 72.6 hs-CRP Baseline 3.5 ± 5.0 ± 2.0

(mg/L) 12 1.0 -0.3 ± 0.2 5.7 ± 2.1 0.7 ± 0.2^* 0.03 weeks 3.3 ±

1.0

Glucose Baseline 105.6 ± 100.8 ± 4.7

(mg/dL) 12 12.5 3.3 ± 1.6 96.5 ± 2.4 -4.3 ± 3.5 0.05 weeks 108.9 ±

1 1.9

Uric acid Baseline 6.6 ± 7.6 ± 0.9

(mg/dL) 12 0.7 -0.6 ± 0.2* 7.6 ± 0.9 0.0 ± 0.1 0.1 1 weeks 6.1 ±

0.5

*P<0.05

[0061] Baseline FMD measurements did not differ between arms. By the end of week 12, a trend toward an improvement was observed in the THIAA/niacin arm whereas there was a trend toward a decrease in the placebo arm; the difference between arms was statistically significant (P=0.036, Fig. 3A). In the optional follow-up study in which 2 placebo arm participants (after completing the trial) volunteered to receive THIAA/niacin treatment for 10 - 12 weeks, the combined data (7 data points from the original THIAA/niacin arm plus the 2 new data points) showed a statistically significant improvement in FMD (P=0.049, Fig. 3B) compared to baseline. The between-arm difference was also statistically significant (P=0.031).

[0062] Fourteen adverse events, occurring in 7 subjects, were noted during the stud_}'.

Events were mild to moderate in severity and were deemed to have no or unlikely causality. During study product consumption, 2 respiratory infections, mild headaches, one accidental fall, and back discomfort were noted. THIAA/niacin did not differ from placebo in regards to physical symptoms and reported adverse events.

[0063] While the claimed invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one of ordinary skill in the art that various changes and modifications can be made to the claimed invention without departing from the spirit and scope thereof. Thus, for example, those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention, and are covered by the following claims.

Claims

23 CLAIMS

1. A composition for promoting healthy uric acid levels in a subject in need thereof, said composition comprising therapeutically effective amounts of niacin and tetrahydro iso- alpha acids.

2. A method for promoting healthy uric acid levels in a subject in need thereof, said method comprising administering to the subject in need a composition comprising therapeutically effective amounts of niacin and tetrahydro iso-alpha acids.

3. A composition for promoting healthy hs-CRP levels in a subject in need thereof, said composition comprising therapeutically effective amounts of niacin and tetrahydro iso- alpha acids.

4. A method for promoting healthy hs-CRP levels in a subject in need thereof, said method comprising administering to the subject in need a composition comprising therapeutically effective amounts of niacin and tetrahydro iso-alpha acids.

5. The composition of claims 1 or 3, wherein the ratio of niacin to and tetrahydro iso-alpha acids is 4: 1.

6. The methods of claims 2 or 4, wherein the ratio of niacin to and tetrahydro iso-alpha acids is 4:1.