Compositions and methods for reducing the levels of inflammatory mediators

Abstract

To provide a composition and a method configured to alleviate pain, discomfort, pressure, inflammation, tension, and / or a feeling of swelling associated with connective tissue damage caused by inflammatory mediators.SOLUTION: Hydroxytyrosol and 3-O-acetyl-11-keto-β-boswellic acid are used in combination. The hydroxytyrosol may be sourced from olive extract and the 3-O-acetyl-11-keto-β-boswellic acid may be sourced from Boswellia serrata extract.SELECTED DRAWING: Figure 1

Description

Related Applications

[0001] This application claims the priority of U.S. Provisional Patent Application No. 62 / 403,807 filed on October 4, 2016, and incorporates the entire disclosure of this U.S. Provisional Patent Application by reference.

Technical Field

[0002] The present invention provides a method of administering (i) 3-O-acetyl-11-keto-β-boswellic acid (AKBA) and (ii) hydroxytyrosol to mammals and birds. The present invention also provides an orally administrable composition comprising AKBA and hydroxytyrosol.

Background Art

[0003] Connective tissue is the structural framework of cartilage, bone, synovium, ligaments, menisci and tendons in joints. The components of connective tissue are produced by resident cells and then secreted to exert the properties of the extracellular matrix (ECM) of the tissue. In addition to acting as a structural framework, the ECM plays a critical role in cell signaling and function. In articular cartilage, chondrocytes are arranged in a distinct pattern within the type II collagen ECM structure. Osteogenic osteoblasts and osteocytes as well as bone-resorbing osteoclasts are organized in the mineralized type I collagen ECM. Some fibroblast-like cells and macrophage-like cells in the synovium are also retained in place by the ECM. Similarly, tendon cells and ligament cells are arranged within the ECM. The synthesis and degradation of connective tissue ECM are controlled by a network of regulatory molecules produced by resident tissue cells. This network has numerous molecules known as growth factors and inflammatory mediators.

[0004] These mediators include cytokines, chemokines, prostaglandins, and nitric oxide. These molecules exhibit numerous biological activities and induce cell proliferation and cell death. Furthermore, these substances can induce anabolic pathways that produce the extracellular matrix (ECM) and can induce catabolic enzymes that can degrade the ECM. Under physiological conditions, cell survival or death, and the production or degradation of the connective tissue ECM, are strictly controlled to maintain a balanced homeostasis. The production and function of regulatory molecules are modulated by numerous factors, including mechanical forces, physical factors such as temperature and pH, chemical substances, microorganisms, and their products. Under certain conditions, these factors can lead to excessive and premature production of regulatory molecules, resulting in irreparable tissue damage, loss of function, and cell death.

[0005] Tissues respond to mechanical, physical, and chemical factors, as well as infection, through inflammatory responses. Inflammatory processes are known to contribute to recovery, healing, defense against infection, and preservation of life. Inflammatory responses in humans and animals consist of two phases. Phase 1 is characterized by the local synthesis of pro-inflammatory mediators such as prostaglandins and leukotrienes. These are induced from arachidonic acid through the action of cyclooxygenase and lipoxygenase. These pro-inflammatory mediators increase local blood flow, enhance the permeability of endothelial cells, and lead to leukocyte recruitment and accumulation. Other pro-inflammatory mediators that are subsequently produced include cytokines (interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α)), chemokines (IL-8), and (1) nitric oxide. In phase 2, or the split phase, prostaglandins produced in phase 1 activate an enzymatic pathway in which arachidonic acid is converted into anti-inflammatory chemical mediators. It has been reported that prostaglandin E2 (PGE2) activates the expression of 15-lipoxygenase, producing anti-inflammatory lipoxins from arachidonic acid. Thus, the splitting of inflammation is promoted by the pro-inflammatory response. These studies indicate that the onset, progression, and termination of the inflammatory response are strictly controlled. If inflammation persists and worsens, numerous disorders can develop, such as osteoarthritis (OA), rheumatoid arthritis (RA), Alzheimer's disease, and cardiovascular disease.

[0006] In joint tissues, chondrocytes, synovial cells, osteoblasts, osteoclasts, ligament cells, and tendinocytes produce numerous pro-inflammatory mediators. Among these is PGE2, which is known to play a regulatory role by inducing the production of cytokines, (i) nitric oxide, and other mediators, including connective tissue degrading metalloproteinase (MMP) enzymes. Because PGE2 can induce metalloproteinases (MMPs), it contributes to the degradation of the cartilage extracellular matrix (ECM). Furthermore, PGE2 promotes bone resorption and osteophyte formation. PGE2 also sensitizes nociceptors in peripheral nerve endings, exacerbating inflammatory pain. PGE2 levels can be locally regulated by the cyclooxygenase-2 (COX-2) enzyme. In pathological conditions such as osteoarthritis, COX-2 expression is upregulated, and consequently, PGE2 production increases.

[0007] TNF-α is a major mediator of inflammation, playing a crucial role in tissue regeneration / expansion and degradation during inflammation. Under normal conditions, inflammation is well regulated by these factors. That is, inflammation develops due to these factors, and after the accompanying immune response is induced, the levels of these factors decrease to normal levels. However, if unregulated TNF-α is produced, inflammation becomes chronic, and various diseases such as arthritis directly develop.

[0008] Inflammation is a necessary and important immunological process for resolving tissue damage and infection, but chronic release of pro-inflammatory mediators such as IL-1β and TNF-α leads to the continued production of additional inflammatory mediators. If levels do not return to normal, dysregulation of TNF-α production occurs, potentially leading to adverse pathophysiological processes such as osteoarthritis (OA).

[0009] TNF-α plays a key role in initiating inflammatory processes. TNF-α stimulates the production of various cells present in the joint, namely chondrocytes, osteoblasts, synovial cells, intrinsic immune cells, or cells that infiltrate the joint during inflammatory responses. When TNF-α levels are elevated, it can be detected in the synovial fluid, synovial membrane, ligaments, and subchondral bone of patients with osteoarthritis.

[0010] TNF-α, along with IL-1β, can induce nuclear factor-kappa B (NF-κB), a major regulator of the inflammatory response. TNF-α induces PGE2 production by increasing the production of key enzymes involved in PGE2 synthesis. These key enzymes include COX-2, microsomal PGE synthase (mPGES-1), and soluble phospholipase A2 (sPLA2). Furthermore, TNF-α induces the production of inducible nitric oxide synthase (iNOS), resulting in increased nitric oxide (NO) levels. The production of other cytokines and chemokines, including IL-6, IL-17, and IL-18, as well as IL-8, is also reliably modulated by TNF-α. When these pro-inflammatory mediators—prostaglandins, NO, cytokines, and chemokines—combine and are produced, it ultimately leads to fractures in cartilage affected by osteoarthritis.

[0011] TNF-α can suppress the production of two key components of the extracellular matrix: aggrecan and type II collagen. Furthermore, TNF-α induces the expression of aggrecanases ADAMTS4 and ADAMTS-5, which degrade aggrecan. The combined action of these two disrupts the normal biochemical balance between the synthesis and degradation of cartilage matrix in the joints, ultimately resulting in cartilage degradation. TNF-α is also involved in mitochondrial dysfunction, reduced ATP production, and apoptosis, and further plays a role in causing cartilage degradation. While TNF-α plays a central role in initiating essential immune responses to injury and infection, it is a target for the development of anti-inflammatory drugs because dysregulation can trigger adverse effects.

[0012] The roles of other tissues in the inflammatory process are also well established. Synovial inflammation is now recognized as a key symptom in osteoarthritis, particularly in the early stages of osteoarthritis, leading to cartilage degradation. A characteristic of synovitis is the activation of endogenous macrophage-like and fibroblast-like cells within the synovium, resulting in the overproduction of pro-inflammatory mediators, including TNF-α, IL-1β, and PGE2. Recent evidence suggests that synovial macrophages are the primary source of cytokines in the very early stages of osteoarthritis, and that these macrophages are important contributors to cartilage damage throughout the progression of osteoarthritis. Cytokines also induce the production of PGE2 and active metalloproteinases (MMPs). Currently, there is a growing recognition that these mediators regulate the balance between ECM degradation and repair, and through this recognition, these molecules have become preferred targets for therapeutic intervention. Other intra-articular tissues, such as subchondral bone, also produce pro-inflammatory mediators that modulate joint health.

[0013] In addition to pro-inflammatory mediators such as cytokines and prostaglandins, reactive oxygen species (ROS) are also involved in joint degradation observed in osteoarthritis. Oxidative stress induced by ROS, such as nitric oxide and hydrogen peroxide, is known to cause chondrocyte apoptosis and chondrogenic extracellular matrix (ECM) degradation. Furthermore, reports indicate that ROS activate signaling pathways that lead to increased production of pro-inflammatory mediators, including cytokines and prostaglandins. In vitro studies have demonstrated a link between the pathways involved in the production of ROS and pro-inflammatory mediators. These studies support the idea that drugs capable of suppressing both oxidative stress and inflammatory pathways would be particularly useful in regulating inflammation.

[0014] The central roles of COX-2 and PGE2 in the pathophysiology of osteoarthritis are reflected in the widespread use of selective COX-2 inhibitors and various non-selective nonsteroidal anti-inflammatory drugs (NSAIDs) for treating the injury. However, long-term use of these drugs can lead to side effects, including gastrointestinal pathology and disruption of cartilage proteoglycan metabolism. Studies using human and animal models have demonstrated that COX inhibitors promote healing and repair of damaged bone. Thus, there is a need for alternative treatment methods for inflammation management that suppress the production of other inflammatory mediators, such as PGE2 and TNF-α, without relying primarily on NSAIDs.

[0015] Drugs that have been conventionally developed targeting TNF-α include Infliximab (a chimeric monoclonal antibody against human TNF), Adalimumab (a fully human monoclonal antibody), Etanercept (a dimeric TNFRII(p75) fusion protein bound to the Fc portion of human IgG), Golimumab, CDP571, and Thalidomide. However, these drugs not only suppress the positive functions of TNF-α but can also lead to undesirable consequences, including the development of lymphoma and infection. Thus, there is a need for therapeutic agents that can regulate the excessive generation of reactive oxygen species and cell death induced by TNF-α without inhibiting its positive physiological functions. [Prior art documents] [Patent Documents]

[0016] [Patent Document 1] WO2001 / 045514 A1 [Patent Document 2] WO2002 / 018310 A1 [Patent Document 3] WO2004 / 005228 A1 [Patent Document 4] WO2010 / 029578 A2 [Patent Document 5] US2002 / 0004077 A1 [Patent Document 6] US2002 / 0058078 A1 [Patent Document 7] US2002 / 0198415 A1 [Patent Document 8] US2004 / 0039066 A1 [Patent Document 9] USP6,361,803 [Patent Document 10] USP6,416,808 [Patent Document 11] EP1582512 A1 [Non-Patent Document]

[0017] [Non-Patent Document 1] Gonzalez-Santiago,et al., Pharmacological research, 61.4(2010):364-370 [Non-Patent Document 2] Sengupta,et al. Molecular and cellular biochemistry, 354.1-2(2011):189-197 [Summary of the Invention]

[0018] According to the object and the effect of the present invention, the first aspect of the present invention provides a synergistic composition using hydroxytyrosol and 3-O-acetyl-11-keto-β-boswellic acid in combination. In an embodiment, the hydroxytyrosol source is olive extract, and the 3-O-acetyl-11-keto-β-boswellic acid source is Boswellia serrata extract. This composition can be formulated for oral administration to mammals. Regarding mammals, it can be selected from the group consisting of humans, dogs, cats, horses, camels or cows. In other embodiments, the composition of the present invention can be formulated for oral administration to birds.

[0019] In an embodiment, a composition formulated for oral administration to humans may have 3-O-acetyl-11-keto-β-boswellic acid in an amount of about 0.67 mg to about 2.70 mg per kg of body weight and hydroxytyrosol in an amount of about 0.15 mg to about 2.50 mg per kg of body weight. In an embodiment, a composition formulated for oral administration to dogs may have 3-O-acetyl-11-keto-β-boswellic acid in an amount of about 1.24 mg to about 4.98 mg per kg of body weight and hydroxytyrosol in an amount of about 0.28 mg to about 4.60 mg per kg of body weight.

[0020] Another aspect of the present invention provides a method of treating, repairing or alleviating damage to connective tissue caused by one or more inflammatory mediators, comprising administering to a mammal or bird in need thereof an orally administrable synergistic composition comprising hydroxytyrosol and 3-O-acetyl-11-keto-β-boswellic acid as described above in combination.

[0021] Yet another aspect of the present invention provides a method of reducing the level of one or more inflammatory mediators in connective tissue, comprising administering to a mammal or bird in need thereof an orally administrable composition comprising hydroxytyrosol and 3-O-acetyl-11-keto-β-boswellic acid in a synergistic form as described above in combination.

[0022] Embodiments of the composition and method of the present invention are shown below. It should be understood that the composition and method of the present invention can also be implemented in different alternative embodiments, and details can be changed in various obvious ways without departing from the gist described in the claims. That is, the accompanying drawings and description are merely illustrative and not intended to be limiting. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings incorporated herein and constituting part thereof illustrate several aspects of the disclosed compositions and are useful in illustrating certain principles of the compositions of the present invention together with this disclosure.

[0024] [Figure 1] This study demonstrates the effects of certain concentrations of hydroxytyrosol and AKBA on TNF-α production in lipopolysaccharide-stimulated RAW264.7 mouse macrophage cells. [Figure 2] This study demonstrates the effects of certain concentrations of hydroxytyrosol and AKBA on TNF-α production in lipopolysaccharide-stimulated RAW264.7 mouse macrophage cells. [Figure 3] This study demonstrates the effects of certain concentrations of hydroxytyrosol and AKBA on TNF-α production in lipopolysaccharide-stimulated RAW264.7 mouse macrophage cells.

[0025] The embodiments of the compositions and corresponding methods of the present invention will be described in detail below, and examples of these embodiments are shown in the accompanying drawings. [Modes for carrying out the invention]

[0026] The present invention provides a method for administering (i) 3-O-acetyl-11-keto-β-boswellic acid (AKBA) and (ii) hydroxytyrosol to mammals and birds. AKBA and hydroxytyrosol may be administered simultaneously in a single composition or in a single dosage form, or individually. In some embodiments, AKBA and hydroxytyrosol are administered simultaneously or individually in a single composition or in a single dosage form within a period of overlapping therapeutic properties. In some embodiments, the compositions are administered individually within one hour. In other embodiments, the compositions are administered individually within 30 minutes. In yet another embodiment, the compositions are administered individually within 5 minutes.

[0027] As used herein, the term “mammal” refers to any mammal, and is not intended to be limited to them, but includes humans, dogs, cats, horses, cattle, and camels, and the term “bird” refers to birds.

[0028] Hydroxytyrosol is a type of phenolic phytochemical found in various parts of the olive tree. Its IUPAC name is 4-(2-hydroxyethyl)-1,2-benzenediol, and it is represented by the following structural formula. TIFF0007876938000001.tif52126

[0029] In this invention, hydroxytyrosol may be synthesized or obtained from natural sources such as products or by-products derived from extraction and / or purification of olive trees. Furthermore, hydroxytyrosol can be administered in the form of a hydroxytyrosol-containing extract obtainable from products or by-products derived from olive trees. Products and by-products obtained from olive trees include, but are not limited to, olives (olive fruits), olive leaves, olive pulp, olive oil, olive-derived plant water, and olive oil lees. Those skilled in the art should be able to easily adjust the amount and proportions of hydroxytyrosol based on the extraction method. In embodiments, hydroxytyrosol is derived from olives that can be obtained from conventionally commercially available sources such as producers.

[0030] The hydroxytyrosol used in this invention can be produced by many conventionally known methods. The composition used in this invention can be obtained by processing olives by any suitable means. For example, pressing olives and / or olive leaves can yield a mixture containing olive oil, plant water, and solid by-products. Hydroxytyrosol may be obtained directly from this mixture, or it may be obtained by fractional distillation and / or purification of the mixture. Fractional distillation and / or purification of the composition can be carried out by numerous methods known to those skilled in the art. Examples of fractional distillation methods include partition treatment using organic solvents, chromatography, high-pressure liquid chromatography (HPLC), and the use of supercritical fluids.

[0031] Reference examples for the extraction of hydroxytyrosol from olive leaves are WO02 / 18310 A1, US2002 / 0198415 A1, WO2004 / 005228 A1, USP6,416,808, and US2002 / 0058078 A1, all of which disclose a method of acid hydrolyzing olive plant water for 2 to 12 months until at least 90% of oleuropein is converted. Methods for extracting hydroxytyrosol from olives, olive pulp, olive oil, and wastewater from essential oil plants are disclosed in USP6,361,803, WO01 / 45514 A1, and US2002 / 0004077 A1. EP1582512 A1 also discloses the extraction of hydroxytyrosol from olive leaves. Paragraphs

[0080] to

[0091] of US2004 / 0039066 A1 disclose a method for obtaining hydroxytyrosol from olive plant water in which the nucleus has not been removed.

[0032] Similarly, commercially available hydroxytyrosol-containing olive extracts are also suitable for use in the present invention.

[0033] The literature reports that oral bioavailability was observed when hydroxytyrosol was administered to humans at a single dose of 2.5 mg / kg, with an observed peak plasma concentration of 1.11 ± 0.20 μM mol / L. Gonzalez-Santiago, et al., Pharmacological Research, 61.4 (2010), pp. 364-370, states that the dosage can be determined by a person skilled in the art by evaluating body weight, surface area, and species differences. Similarly, the dosage for interspecies extrapolation can be calculated by a person skilled in the art using the usual dosage conversion method.

[0034] Typical doses of hydroxytyrosol range from approximately 0.001 mg / kg to approximately 2.0 mg / kg. In some embodiments, when used in humans or non-human animals, typical daily doses are at least 0.1 mg to 300 mg. Here, the daily dose refers to the total dose administered over a 24-hour period.

[0035] In some exemplary embodiments, when administered to humans, the dose of hydroxytyrosol can be set to 0.15 to 2.50 mg per kg of body weight (i.e., 9 to 250 mg for a person weighing 60 kg).

[0036] In some exemplary embodiments, when applied to dogs, the dose of hydroxytyrosol can be set to 0.28 kg to 4.60 mg per kg of body weight (i.e., 2.8 to 46 mg for a dog weighing 10 kg).

[0037] The frequency of hydroxytyrosol administration can be set from once a week to five times a day. In one embodiment, the frequency of hydroxytyrosol administration is set from once every two days to three times a day. In yet another embodiment, the frequency of hydroxytyrosol administration is set from once to twice a day. In yet another embodiment, the frequency of hydroxytyrosol administration is set to once a day. When administering hydroxytyrosol, feeding may or may not be performed.

[0038] Phytochemicals extracted from Boswellia serrata have been reported to be active in treating numerous diseases and ailments. The gum resin of Boswellia serrata has been used for many years in the Indian pharmaceutical system by Ayurvedic physicians to treat rheumatoid arthritis and gout. Various extracts of the gum resin have shown potential anti-inflammatory and anti-atherosclerotic activity in experimental animals. The biological activity of this extract is related to the components of the boswellic acid fraction. 3-O-acetyl-11-keto-β-boswellic acid (AKBA) is recognized as the most potent compound in Boswellia serrata extracts. Boswellia serrata extracts containing AKBA have been reported to inhibit 5-lipoxygenase and matrix metalloproteinase-3 (MMP-3) in vitro, as described in WO2010 / 029578 A2. Similarly, WO2010 / 029578 A2 reports on the anti-inflammatory effects of a composition containing Boswellia serrata extract selectively concentrated to 30% in vivo with AKBA, including a significant reduction in serum biomarkers TNF-α and IL-1β.

[0039] Furthermore, there is a report in the literature regarding the bioavailability of a single dose of 100 mg / kg of Boswellia serrata extract standardized to 30% AKBA in rat serum. In that report, a peak serum concentration of 2.0 μg / mL was observed. (Sengupta, et al. Molecular and Cellular Biochemistry, 354.1-2(2011):189-197). The dosage can be determined by those skilled in the art by evaluating body weight, surface area, and species differences. Similarly, the dosage for interspecies extrapolation can be calculated by those skilled in the art using standard dosage conversion methods.

[0040] Typical doses of AKBA range from approximately 0.01 mg / kg to approximately 10.0 mg / kg. In some embodiments, when used in humans or non-human animals, the typical daily dose is at least 1 mg to approximately 1 g. Here, the daily dose refers to the total dose administered over a 24-hour period.

[0041] In some exemplary embodiments, when administered to humans, the dose of AKBA can be set to 0.67 to 2.70 mg per kg of body weight (i.e., 40 to 162 mg for a person weighing 60 kg).

[0042] In some exemplary embodiments, when administered to dogs, the dosage of AKBA can be set to 1.24 to 4.98 mg per kg of body weight (i.e., 12.4 to 49.8 mg for a dog weighing 10 kg).

[0043] The frequency of AKBA administration can be set from once a week to five times a day. In some embodiments, the frequency of AKBA administration can be set from once every two days to three times a day. In yet another embodiment, the frequency of AKBA administration can be set from once to twice a day. In yet another embodiment, the frequency of AKBA administration can be set to once a day. When administering AKBA, feeding may or may not be performed.

[0044] Some embodiments demonstrate that a synergistic effect is achieved when (i) hydroxytyrosol and (ii) AKBA are used in combination. Here, synergy refers to the effect where the combined use of two or more components yields a result greater than the sum of the effects of the individual components when used individually. In some embodiments, the results are statistically significant and greater than the additive effect. In some embodiments, the combined use of hydroxytyrosol and AKBA yields a statistically significant effect greater than the combined use of each component individually. In some embodiments, the combined use of hydroxytyrosol and AKBA can achieve one or more of the following synergistic effects: prevention, treatment, repair, or mitigation of damage to connective tissue; mitigation of symptoms associated with connective tissue damage in birds and mammals; and a reduction in the level of one or more inflammatory mediators in connective tissue.

[0045] The present invention provides a method for preventing, treating, repairing, and mitigating damage, or a method for suppressing inflammation of connective tissue, protecting cartilage, or mitigating symptoms associated with connective tissue damage in birds and mammals, wherein the method involves administering (i) hydroxytyrosol and (ii) AKBA to the target animal. Herein, the term “connective tissue” includes, but is not limited to, cartilage, bone, synovial membrane, ligaments, meniscus, and tendons. In some embodiments, the administration of (i) hydroxytyrosol and (ii) AKBA can prevent, treat, repair, or mitigate connective tissue damage. Connective tissue damage may result from physical injury or manifest as “wear and tear” caused by continuous use, weight, and age. Examples include osteoarthritis. Connective tissue damage can also cause diseases such as rheumatoid arthritis, synovial disorders, infections associated with rheumatic diseases, and inflammatory connective tissue disorders. In some embodiments, the administration of (i) hydroxytyrosol and (ii) AKBA can mitigate symptoms associated with connective tissue damage in birds and mammals. Symptoms associated with connective tissue damage include, but are not limited to, pain, discomfort, pressure, inflammation, tightness, and / or swelling.

[0046] Furthermore, the present invention provides a method for reducing the level of one or more inflammatory mediators in connective tissue, comprising administering (i) hydroxytyrosol and (ii) AKBA to birds or mammals. Inflammatory mediators include, but are not limited to, tumor necrosis factor-α (TNF-α), prostaglandins such as prostaglandin E2 (PGE2), cytokines such as interleukin-1β (IL-1β), chemokines, leukotrienes, (i) nitric oxide, and reactive oxygen species.

[0047] Administration of hydroxytyrosol and AKBA is useful for treating, preventing, and alleviating damage, as well as for alleviating symptoms associated with conditions that adversely affect the cardiovascular, nervous, musculoskeletal, and gastrointestinal systems. In one embodiment, the present invention provides compositions and methods for preventing and / or alleviating inflammatory responses and / or inflammation in an administered subject. In one embodiment, the present invention provides compositions and methods for managing inflammatory disorders, or for suppressing the overall inflammatory burden in humans or non-human animals. Accordingly, in one embodiment, the present invention provides a method for preventing and / or alleviating inflammatory responses and / or inflammation in one or more tissues, and a method for delivering the compositions of the present invention to these one or more tissues.

[0048] The present invention also provides an orally administrative composition having (i) hydroxytyrosol and (ii) AKBA. The orally administrative composition may take any orally administrative form, but is not intended to be limited, including but exemplify capsules, tablets, powders that can be dispersed in beverages, pastes in pellet form, liquids such as solutions, suspensions and emulsions, soft gels / chewing capsules, chewable bars and conventionally known encapsulated oral liquids, and other convenient forms of administration.

[0049] Orally administered compositions may contain one or more inactive pharmaceutical ingredients (commonly known as “excipients”). Non-active ingredients can, for example, solubilize, suspend, concentrate, dilute, emulsify, stabilize, preserve, protect, color, and flavor the active ingredients, and finally formulate them into a safe, convenient, and otherwise acceptable doseable and effective preparation. Excipients may be any pharmaceutically acceptable excipients. Examples of pharmaceutically acceptable excipients include lubricants, buffers, stabilizers, foaming agents, pigments, colorants, flavoring agents, fillers, bulking agents, fragrances, release modifiers, auxiliaries, plasticizers, flow enhancers, release agents, polyols, granulators, diluents, binders, buffers, absorbents, lubricants, adhesives, anti-adhesion agents, acidulants, softeners, resins, analgesics, solvents, surfactants, emulsifiers, elastomers, and mixtures thereof.

[0050] Orally administered compositions may further contain one or more active ingredients. For example, a composition may further contain one or more drugs or nutritional supplements. In some embodiments, a composition may further contain compounds beneficial to connective tissue. Examples, though not intended to be limiting, include glycosaminoglycans such as chondroitin, amino sugars such as glucosamine, methylsulfonylmethane (MSM), collagen (including collagen type II), green tea extract, Scutellaria extract, acacia extract, turmeric extract, curcumin, cetylmyristrein complex (CMO), and eggshell membrane.

[0051] All references cited herein should be taken at face value. [Examples]

[0052] Example 1: Effects of hydroxytyrosol and AKBA on TNF-α production in lipopolysaccharide (LPS)-stimulated RAW264.7 mouse macrophage cells

[0053] RAW264.7 mouse macrophage cells were pre-treated for 24 hours with 60 nM, 160 nM, or 1 μM hydroxytyrosol (HT) (98% purity, manufactured by Sigma-Aldrich, St. Louis, Missouri) alone, or with 0.28 μg / mL, 0.56 μg / mL, or 1.124 μg / mL AKBA (administered as 5-LOXIN (trademark) standardized to 30% AKBA, manufactured by PLT Health Solutions) alone, or in combination with each of the three concentrations of HT and each of the three concentrations of AKBA. The cells were then stimulated for another 24 hours with 1 μg / mL lipopolysaccharide (LPS). LPS is an endotoxin present in bacterial cell walls and can induce an inflammatory response that increases TNF-α production. TNF-α production was analyzed in the cell supernatant. Statistical comparisons were performed using one-way analysis of variance (ANOVA), followed by Tukey post-hoc analysis, with p<0.05 considered statistically significant. Data were scaled to mean + / - 1SD.

[0054] When each of the three concentrations of HT was used in combination with AKBA, a statistically significant reduction in TNF-α levels was observed compared to when each was used alone. When 60 nM HT was used in combination with AKBA at 0.28 μg / mL, 0.56 μg / mL, or 1.124 μg / mL, TNF-α production was significantly reduced compared to when HT (P<0.001) or AKBA (P<0.001) was used alone (Figure 1). The reduction in TNF-α in cells treated with 160 nM HT in combination with 0.56 μg / mL AKBA was statistically significant compared to when HT (P<0.001) or AKBA (P=0.001) was used alone (Figure 2). Cells treated with 1 μM HT in combination with 0.28 μg / mL, 0.56 μg / mL, or 1.124 μg / mL AKBA showed a statistically significant reduction in TNF-α compared to the use of HT alone (P<0.001, P=0.002, and P<0.001, respectively) and AKBA alone (P<0.001, P=0.002, and P=0.004, respectively) (Figure 3).

Claims

1. A composition configured to alleviate pain, discomfort, pressure, inflammation, tightness, and / or bloating associated with connective tissue damage caused by one or more inflammatory mediators, characterized in that hydroxytyrosol and 3-O-acetyl-11-keto-β-boswellic acid act synergistically in amounts that prevent or reduce the inflammatory response in the connective tissue of an animal in need thereof.

2. The hydroxytyrosol source is at least one of olive extract, a product obtained from the olive tree, or a by-product obtained from the olive tree. The composition according to claim 1, wherein the source of 3-O-acetyl-11-keto-β-boswellic acid is an extract of Boswellia serrata.

3. The composition according to claim 1, formulated for oral administration to mammals or birds.

4. The composition according to claim 3, wherein the mammal is selected from the group consisting of humans, dogs, cats, horses, camels, or cattle.

5. The composition according to claim 4, wherein the composition formulated for oral administration to humans contains 3-O-acetyl-11-keto-β-boswellic acid in an amount of about 0.67 mg to about 2.70 mg per kg of body weight, and the composition formulated for oral administration to dogs contains 3-O-acetyl-11-keto-β-boswellic acid in an amount of about 1.24 mg to about 4.98 mg per kg of body weight.

6. The composition according to claim 4, wherein the composition formulated for oral administration to humans contains hydroxytyrosol in an amount of about 0.15 mg to about 2.50 mg per kg of body weight, and the composition formulated for oral administration to dogs contains hydroxytyrosol in an amount of about 0.28 mg to about 4.60 mg per kg of body weight.

7. The composition according to claim 1, wherein the hydroxytyrosol and the 3-O-acetyl-11-keto-β-boswellic acid are administered to the target animal together as separate compositions.

8. The composition according to claim 1, wherein the hydroxytyrosol and the 3-O-acetyl-11-keto-β-boswellic acid are administered individually to the target animal within one hour.

9. The composition according to claim 1, wherein the hydroxytyrosol and the 3-O-acetyl-11-keto-β-boswellic acid are administered individually to the target animal within 30 minutes.

10. The composition according to claim 1, wherein the hydroxytyrosol and the 3-O-acetyl-11-keto-β-boswellic acid are administered individually to the target animal within 5 minutes.

11. A method for alleviating pain, discomfort, pressure, inflammation, tightness, and / or bloating associated with connective tissue damage caused by one or more inflammatory mediators, characterized by administering to a non-human target animal in a combination of hydroxytyrosol and 3-O-acetyl-11-keto-β-boswellic acid in an amount that reduces the level of one or more inflammatory mediators in the connective tissue of the non-human target animal in need thereof.

12. The hydroxytyrosol source is at least one of olive extract, a product obtained from the olive tree, or a by-product obtained from the olive tree. The method according to claim 11, wherein the source of 3-O-acetyl-11-keto-β-boswellic acid is an extract of Boswellia serrata.

13. The method according to claim 11, wherein a composition formulated for oral administration is administered to mammals other than humans or birds.

14. The method according to claim 13, wherein the mammal other than humans is selected from a group consisting of dogs, cats, horses, camels, or cattle.

15. The method according to claim 13, wherein the hydroxytyrosol and the 3-O-acetyl-11-keto-β-boswellic acid are administered together as separate compositions.

16. The method according to claim 13, wherein the hydroxytyrosol and the 3-O-acetyl-11-keto-β-boswellic acid are administered individually within one hour.

17. The method according to claim 13, wherein the hydroxytyrosol and the 3-O-acetyl-11-keto-β-boswellic acid are administered individually within 30 minutes.

18. The method according to claim 13, wherein the hydroxytyrosol and the 3-O-acetyl-11-keto-β-boswellic acid are administered individually within 5 minutes.

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