Composition comprising hydroxytyrosol and masticinic acid

Abstract

Compositions comprising hydroxytyrosol and masticinic acid are provided. Compositions comprising a synergistic combination of hydroxytyrosol and 3-O-acetyl-11-one-beta-masticinic acid are described. The hydroxytyrosol can be derived from an olive extract, and the 3-O-acetyl-11-keto-beta-boswellic acid can be derived from a Boswellia serrata extract, and the 3-O-acetyl-11-keto-beta-boswellic acid can be derived from a Boswellia serrata extract. The composition may be formulated for oral administration to a mammalian or avian subject. Methods are provided for treating, repairing, or reducing connective tissue damage caused by one or more inflammatory mediators, and reducing the level of one or more inflammatory mediators in connective tissue, comprising orally administering the composition to a subject in need thereof.

Description

[0001] This application is a divisional application of the application with the filing date of April 21, 2017, application number 201780061735.7, and invention title "Composition Comprising Hydroxytyrosol and Boswellic Acid".

[0002] This application claims the priority of U.S. Provisional Patent Application Serial No. 62 / 403,807, filed on October 4, 2016, the entire content of which is incorporated herein by reference. TECHNICAL FIELD

[0003] The present invention provides a method comprising administering to a mammalian or avian subject: (i) 3-O-acetyl-11-keto-β-boswellic acid (AKBA) and (ii) hydroxytyrosol. The present invention also provides an orally administrable composition comprising AKBA and hydroxytyrosol. BACKGROUND OF THE INVENTION

[0004] Connective tissue is the structural framework tissue that connects cartilage, bone, synovium, ligaments, menisci, and tendons in joints. The components of connective tissue are produced by resident cells and then secreted to form the extracellular matrix (ECM) characteristics of the tissue. In addition to serving as a structural framework tissue, the ECM also plays a key role in cell communication and function. In articular cartilage, chondrocytes are arranged in a unique pattern within a type II collagen ECM framework tissue. Osteoblasts and osteocytes that form bone, as well as osteoclasts that resorb bone, are organized in a mineralized type I collagen ECM. A few fibroblast-like and macrophage-like cells in the synovium are also held in place by the ECM. Similarly, tenocytes and ligament cells are assembled together within the ECM. The synthesis and breakdown of connective tissue ECM are controlled by a network of regulatory molecules, which are also produced by resident tissue cells. This network includes growth factors and various molecules known as pro-inflammatory mediators.

[0005] They include cytokines, chemokines, prostaglandins, and nitric oxide. These molecules exhibit many biological activities. They can induce cell proliferation or cell death. These substances can also induce anabolic pathways for the production of ECM or induce catabolic enzymes that can break down ECM. Under physiological conditions of cell survival or death, the production or breakdown of connective tissue ECM is strictly controlled to maintain a balanced homeostasis. The production and function of regulatory molecules are regulated by many factors, including mechanical forces, physiological factors such as temperature and pH, chemicals, microorganisms, and their products. Under certain conditions, these factors can lead to the production of excessive and untimely regulatory molecules, resulting in irreparable tissue damage, loss of function, and death.

[0006] Tissues respond to mechanical, physical, chemical insults, and infection through an inflammatory response. The inflammatory process is known to result in recovery, healing, defense against infection, and generally the maintenance of life. The inflammatory response in humans and animals consists of two phases. The initial phase is characterized by the local synthesis of pro-inflammatory mediators such as prostaglandins and leukotrienes. They are derived from arachidonic acid through the action of cyclooxygenase and lipoxygenase. These pro-inflammatory mediators increase local blood flow and enhance the permeability of endothelial cells to allow 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 nitric oxide. During the second phase of resolution, the prostaglandins generated during the initial phase activate an enzymatic pathway along which arachidonic acid is converted into chemical mediators with anti-inflammatory properties. It has been reported that prostaglandin E2 (PGE2) activates the expression of 15-lipoxygenase, which generates anti-inflammatory lipoxins from arachidonic acid. Thus, the resolution of inflammation is driven by the pro-inflammatory response. These studies suggest that the initiation, progression, and termination of the inflammatory process are tightly controlled. Prolonged and exaggerated inflammation is associated with many disorders, including osteoarthritis (OA), rheumatoid arthritis (RA), Alzheimer's disease, and cardiovascular diseases.

[0007] In joint tissues, chondrocytes, synoviocytes, osteoblasts, osteoclasts, ligament cells, and tendon cells produce large amounts of pro-inflammatory mediators. One of these is PGE2, which is known to play a regulatory role by inducing the production of other mediators, including cytokines, nitric oxide, and matrix metalloproteinases (MMPs) that degrade connective tissue. Due to its ability to induce MMPs, PGE2 contributes to the breakdown of cartilage ECM. In addition, PGE2 promotes bone resorption and osteophyte formation. PGE2 sensitizes nociceptors on peripheral nerve endings and thus contributes to the development of inflammatory pain. The level of PGE2 is locally regulated by cyclooxygenase-2 (COX-2). Under pathological conditions such as osteoarthritis, the expression of COX-2 is upregulated, accompanied by an increase in PGE2 production.

[0008] TNF-α is a major mediator of inflammation and plays an important role in tissue regeneration / dilation and destruction during inflammation. Under normal conditions, inflammation is well regulated by these factors. That is, after these factors cause inflammation and induce an immune response, their levels decrease to normal. However, dysregulated TNF-α production causes chronic inflammation, which is directly associated with various diseases such as arthritis.

[0009] Although inflammation is a key immune process necessary to resolve tissue injury or infection, the long-term release of pro-inflammatory mediators such as IL-1β and TNF-α can continue to induce the production of additional inflammatory mediators. If levels do not return to normal, dysregulation of TNF-α production can potentially lead to harmful pathophysiological processes, including osteoarthritis (OA).

[0010] TNF-α plays a key role in the initiation of the inflammatory process. TNF-α is produced by multiple cell types in the joint (i.e., chondrocytes, osteoblasts, cells in the synovium, and resident immune cells in the joint) or those cells that infiltrate the joint during an inflammatory response. Elevated levels of TNF-α have been detected in the synovial fluid, synovium, cartilage, and subchondral bone of patients with osteoarthritis.

[0011] TNF-α, together with IL-1β, is able to induce nuclear factor-κB (NF-κB), which is the master regulator of the inflammatory response. TNF-α induces the production of PGE2 by increasing the production of key enzymes involved in its synthesis, including COX-2, microsomal PGE synthase (mPGES-1), and soluble phospholipase A2 (sPLA2). Additionally, TNF-α induces the production of inducible nitric oxide synthase (iNOS), leading to elevated levels of nitric oxide (NO). The production of other cytokines (including IL-6, IL-17, and IL-18) and the chemokine IL-8 are positively regulated by TNF-α. Collectively, the production of these pro-inflammatory mediators (prostaglandins, NO, cytokines, and chemokines) ultimately leads to cartilage degradation associated with osteoarthritis.

[0012] TNF-α is able to inhibit the production of two key components of the extracellular matrix, aggrecan and type II collagen. In addition, TNF-α induces the expression of the aggrecanases ADAMTS4 and ADAMTS-5, enzymes that degrade aggrecan. The combination of these two effects disrupts the normal biochemical balance between cartilage matrix synthesis and degradation in the joint, ultimately leading to cartilage degradation. It has also been shown that TNF-α plays a role in mitochondrial dysfunction, reduced ATP production, and apoptosis, further promoting cartilage destruction. Although TNF-α plays a central role in initiating the basic immune response to injury and infection, its harmful effects when dysregulated make TNF-α a target for the development of inflammation management products.

[0013] The role of other tissues during the inflammatory process has also been well established. It is now recognized that synovial inflammation plays a key role in cartilage degradation in osteoarthritis, particularly in the early stages of the disease. Synovitis is characterized by the activation of resident macrophage-like cells and fibroblast-like cells in the synovium, leading to the production of an excess of pro-inflammatory mediators (including TNF-α, IL-1β, and PGE2). Recent evidence suggests that synovial macrophages are the main source of cytokines in the earliest stages of osteoarthritis and that they are important contributors to cartilage damage throughout the disease process. Cytokines also induce the production of PGE2 and active matrix metalloproteinases (MMPs). It is now generally accepted that these mediators control the balance between ECM destruction and repair, making these molecules preferred targets for therapeutic intervention. Other tissues in the joint, such as subchondral bone, also produce pro-inflammatory mediators that regulate joint health.

[0014] In addition to pro-inflammatory mediators such as cytokines and prostaglandins, reactive oxygen species (ROS) are also involved in joint degradation observed in osteoarthritis. It has been shown that oxidative stress induced by ROS (such as nitric oxide and hydrogen peroxide) causes chondrocyte apoptosis and degradation of the cartilage ECM. Moreover, it has been reported that ROS activate signal transduction pathways, leading to an increase in the production of pro-inflammatory mediators (including cytokines and prostaglandins). In vitro studies have confirmed the association between the pathways involved in the production of ROS and pro-inflammatory mediators. These studies support the idea that substances that can simultaneously inhibit oxidative stress and inflammatory pathways will be particularly useful for the regulation of inflammation.

[0015] The widespread use of selective COX-2 inhibitors and various non-selective non-steroidal anti-inflammatory drugs (NSAIDs) in the treatment of osteoarthritis reflects the central role of COX-2 and PGE2 in the pathophysiology of osteoarthritis. However, long-term administration of these drugs has adverse side effects, including gastrointestinal lesions and disruption of cartilage proteoglycan metabolism. Studies in humans and animal models have confirmed that the use of COX inhibitors impairs bone healing and repair. Therefore, alternative therapies for managing inflammation are needed that do not focus on using NSAIDs to inhibit the production of PGE2 and other pro-inflammatory mediators.

[0016] To date, drugs developed for 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 linked to the Fc portion of human IgG), golimumab, CDP571, and thalidomide. However, in addition to inhibiting the positive functions of TNF-α, these drugs may also trigger unwanted outcomes, including the development of lymphoma and infections. Therefore, a therapeutic agent is needed that regulates the production of excess reactive oxygen species and cell death induced by TNF-α without blocking the positive physiological functions of TNF-α. SUMMARY OF THE INVENTION

[0017] In one aspect of the present invention, there is provided a composition comprising a synergistic combination of hydroxytyrosol and 3-O-acetyl-11-keto-β-boswellic acid, for the purposes and benefits described herein. In an embodiment, the hydroxytyrosol is derived from an olive extract, and the 3-O-acetyl-11-keto-β-boswellic acid is derived from an extract of Boswellia serrata. The composition can be formulated for oral administration to a mammalian subject, which can be selected from a human, dog, cat, horse, camel, or cow. In other embodiments, the composition can be formulated for oral administration to an avian subject.

[0018] In an embodiment, the composition formulated for oral administration to a human subject can comprise from about 0.67 to about 2.70 mg / kg body weight of 3-O-acetyl-11-keto-β-boswellic acid and from about 0.15 to about 2.50 mg / kg body weight of hydroxytyrosol. In an embodiment, the composition formulated for oral administration to a dog subject can comprise from about 1.24 to about 4.98 mg / kg body weight of 3-O-acetyl-11-keto-β-boswellic acid and from about 0.28 to about 4.60 mg / kg body weight of hydroxytyrosol.

[0019] In another aspect, the present invention provides a method for treating, repairing, or reducing connective tissue damage caused by one or more inflammatory mediators, which comprises administering to a subject in need thereof an orally administrable composition comprising a synergistic combination of hydroxytyrosol and 3-O-acetyl-11-keto-β-boswellic acid as described above. In an embodiment, the subject is a mammalian subject. In an embodiment, the subject is an avian subject. In an embodiment, the method comprises obtaining hydroxytyrosol from an olive extract. In an embodiment, the method comprises obtaining 3-O-acetyl-11-keto-β-boswellic acid from an extract of Boswellia serrata. In an embodiment, the method comprises providing an orally administrable composition formulated for a human subject, the composition comprising from about 0.67 to about 2.70 mg / kg body weight of 3-O-acetyl-11-keto-β-boswellic acid. In an embodiment, the method comprises providing an orally administrable composition formulated for a dog subject, the composition comprising from about 1.24 to about 4.98 mg / kg body weight of 3-O-acetyl-11-keto-β-boswellic acid. In an embodiment, the method comprises providing an orally administrable composition formulated for a human subject, the composition comprising from about 0.15 to about 2.50 mg / kg body weight of hydroxytyrosol. In an embodiment, the method comprises providing an orally administrable composition formulated for a dog subject, the composition comprising from about 0.28 to about 4.60 mg / kg body weight of hydroxytyrosol.

[0020] In yet another aspect, the present invention provides a method for reducing the level of one or more inflammatory mediators in connective tissue, which comprises administering to a subject in need thereof an orally administrable composition comprising a synergistic combination of hydroxytyrosol and 3-O-acetyl-11-keto-β-boswellic acid as described above. In an embodiment, the subject is a mammalian subject. In an embodiment, the subject is an avian subject. In an embodiment, the method comprises selecting one or more inflammatory mediators from tumor necrosis factor α, prostaglandin E2, cyclooxygenase-2, and nitric oxide. In an embodiment, the method comprises providing an orally administrable composition formulated for a human subject, the composition comprising from about 0.67 to about 2.70 mg / kg body weight of 3-O-acetyl-11-keto-β-boswellic acid. In an embodiment, the method comprises providing an orally administrable composition formulated for a dog subject, the composition comprising from about 1.24 to about 4.98 mg / kg body weight of 3-O-acetyl-11-keto-β-boswellic acid. In an embodiment, the method comprises providing an orally administrable composition formulated for a human subject, the composition comprising from about 0.15 to about 2.50 mg / kg body weight of hydroxytyrosol. In an embodiment, the method comprises providing an orally administrable composition formulated for a dog subject, the composition comprising from about 0.28 to about 4.60 mg / kg body weight of hydroxytyrosol.

[0021] In the following description, embodiments of the disclosed compositions and methods are shown and described. It should be recognized that the described compositions and methods can have other different embodiments, and that several details thereof can be modified in various, obvious aspects, all of which do not depart from the subject matter set forth and described in the claims below. Accordingly, the drawings and the specification are to be regarded as illustrative in nature and not restrictive. Brief Description of the Drawings

[0023] The drawings incorporated herein and forming a part of the specification illustrate several aspects of the disclosed compositions and, together with the description, serve to explain certain principles thereof. In the drawings:

[0024] Figure 1 illustrates the effect of hydroxytyrosol and AKBA at certain concentrations on TNF-α production in lipopolysaccharide-stimulated RAW 264.7 murine macrophages;

[0025] Figure 2 illustrates the effect of hydroxytyrosol and AKBA at certain concentrations on TNF-α production in lipopolysaccharide-stimulated RAW 264.7 murine macrophages; and

[0026] Figure 3Illustrates the effects of hydroxytyrosol and AKBA on TNF-α production in lipopolysaccharide-stimulated RAW 264.7 murine macrophages at certain concentrations.

[0027] Reference will now be made in detail to embodiments of the disclosed compositions and related methods, examples of which are illustrated in the accompanying drawings. DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention provides a method comprising administering to a mammalian or avian subject (i) 3-O-acetyl-11-keto-β-boswellic acid (AKBA) and (ii) hydroxytyrosol. AKBA and hydroxytyrosol can be administered together in a composition or dosage form, or they can be administered separately. In certain embodiments, AKBA and hydroxytyrosol are administered together or separately in a composition or dosage form during a period in which their therapeutic efficacies overlap. In an embodiment, the composition is administered separately within 1 hour. In other embodiments, the composition is administered separately within 30 minutes. In other embodiments, the composition is administered separately within 5 minutes.

[0030] The term "mammalian subject" is any mammal, including but not limited to humans, dogs, cats, horses, cows, and camels. The term "avian subject" refers to a bird.

[0031] Hydroxytyrosol is a type of phenolic phytochemical found in parts of the olive tree. The IUPAC name of hydroxytyrosol is 4-(2-hydroxyethyl)-1,2-benzenediol, and it refers to a compound having the following structure:

[0032]

[0033] As used herein, hydroxytyrosol can be of synthetic origin or can be obtained by extraction and / or purification from natural sources (such as from products and by-products derived from the olive tree). Additionally, hydroxytyrosol can be administered in the form of an extract that can contain hydroxytyrosol, which is obtained from products or by-products of the olive tree. Products and by-products of the olive tree include olives, olive leaves, olive pulp, olive oil, olive-derived plant water, and olive pomace, but are not limited thereto. Based on the extraction procedure, those skilled in the art can easily adjust the amount and respective ratios of hydroxytyrosol. In an embodiment, hydroxytyrosol is derived from olives, which can be obtained from conventional and commercially available sources such as growers.

[0034] The hydroxytyrosol used herein can be prepared by many methods known in the art. The olives can be processed by any suitable means to obtain the composition. For example, the olives and / or olive leaves can be pressed to obtain a mixture comprising olive oil, plant water, and solid by-products. Hydroxytyrosol can be obtained directly from this mixture, or the mixture can be separated and / or purified to obtain hydroxytyrosol. The composition can be separated and / or purified by many methods known to those skilled in the art. Examples of separation methods include partitioning with an organic solvent, chromatography, high performance liquid chromatography (HPLC), or using supercritical fluids.

[0035] Examples of references related to the extraction of hydroxytyrosol from olive leaves are WO02 / 18310A1, US2002 / 0198415A1, WO2004 / 005228A1, U.S. Patent No. 6,416,808, and US 2002 / 0058078A1, which disclose a method of acid-hydrolyzing olive plant water for 2 to 12 months until at least 90% of the oleuropein present is converted. Methods for extracting hydroxytyrosol from olives, olive pulp, olive oil, and olive mill wastewater are described in U.S. Patent No. 6,361,803, WO01 / 45514A1, and US2002 / 0004077 A1. EP 1582512 A1 describes the extraction of hydroxytyrosol from olive leaves. A method for obtaining hydroxytyrosol from the plant water of pitted olives is disclosed in paragraphs

[0080] -

[0091] of US 2004 / 0039066A1. Similarly, commercially available olive extracts containing hydroxytyrosol are suitable for the present invention.

[0036] The oral bioavailability of a single 2.5 mg / kg dose of hydroxytyrosol in human subjects has been reported in the literature, with an observed peak plasma concentration of 1.11 ± 0.20 μmol / L. González-Santiago et al., Pharmacological research, 61.4 (2010): 364-370. By evaluating body weight, surface area, and species differences, those skilled in the art can determine dose calculations. Similarly, those skilled in the art can calculate cross-species extrapolated doses using conventional dose conversion methods.

[0037] Typical dose ratios of hydroxytyrosol are from about 0.001 mg / kg to about 2.0 mg / kg. In some embodiments, for human and non-human subjects, a typical daily dose is at least 0.1 mg and at most 300 mg. The daily dose refers to the total dose administered over a 24-hour period.

[0038] According to some exemplary embodiments, hydroxytyrosol can be administered at a dose of 0.15 to 2.50 mg / kg of human subject body weight (i.e., 9-250 mg for a 60 kg human subject).

[0039] According to some exemplary embodiments, hydroxytyrosol can be administered at a dose of 0.28 to 4.60 mg / kg of the body weight of a canine subject (i.e., 2.8 - 46 mg for a 10 kg canine subject).

[0040] Hydroxytyrosol can be administered at a frequency of once a week to five times a day. In an embodiment, hydroxytyrosol is administered once every two days to three times a day. In an alternative embodiment, hydroxytyrosol is administered one to two times a day. In other embodiments, hydroxytyrosol is administered once a day. Hydroxytyrosol can be taken with or without food.

[0041] It has been reported that the phytochemical constituents extracted from Boswellia serrata are active in treating many ailments and diseases. The gum resin of Boswellia serrata has long been used by practitioners of Ayurvedic medicine, an Indian system of medicine, to treat rheumatoid arthritis and gout. Various extracts of the gum resin have shown effective anti-inflammatory and anti-atherosclerotic activities in laboratory animals. The biological activity of the extracts is related to the components of the boswellic acid moiety. 3-O-acetyl-11-keto-β-boswellic acid (AKBA) has been identified as the most active compound in the Boswellia serrata extract. It has been reported that the Boswellia serrata extract containing AKBA inhibits 5-lipoxygenase and matrix metalloproteinase-3 (MMP-3) in vitro, as described in WO2010 / 029578A2. WO2010 / 029578A2 similarly reported the anti-inflammatory potency in vivo of a composition comprising a Boswellia serrata extract selectively enriched in AKBA up to 30%, including a significant reduction in the serum biomarkers TNF-α and IL-1β.

[0042] The bioavailability of a Boswellia serrata extract standardized to 30% AKBA at a single dose of 100 mg / kg has been reported in the literature in rat serum, with a peak serum concentration of 2.0 μg / mL observed. Sengupta et al., Molecular and cellular biochemistry, 354.1 - 2 (2011): 189 - 197. Dose calculations can be determined by those skilled in the art by evaluating body weight, surface area, and species differences. Similarly, those skilled in the art can calculate cross-species extrapolated doses using conventional dose conversion methods.

[0043] A typical dose rate of AKBA is from about 0.01 mg / kg to about 10.0 mg / kg. In some embodiments, for human and non-human subjects, a typical daily dose is at least 1 mg and at most about 1 g. The daily dose refers to the total dose administered within 24 hours.

[0044] According to some exemplary embodiments, AKBA can be administered at a dose of 0.67 to 2.70 mg / kg of human subject body weight (i.e., 40 - 162 mg for a 60 kg human subject).

[0045] According to some exemplary embodiments, AKBA can be administered at a dose of 1.24 to 4.98 mg / kg of dog subject body weight (i.e., 12.4 - 49.8 mg for a 10 kg dog subject).

[0046] AKBA can be administered at a frequency of once a week to five times a day. In certain embodiments, AKBA is administered at a frequency of once every two days to three times a day. In alternative embodiments, AKBA is administered one to two times a day. In other embodiments, AKBA is administered once a day. AKBA can be taken with or without food.

[0047] In some embodiments, the combination of (i) hydroxytyrosol and (ii) AKBA shows a synergistic effect. Synergistic effect means that the combination of two or more components provides a result whose benefit is greater than the sum of the effects produced when each substance is used alone. In certain embodiments, the result is statistically significant and greater than the additive effect. In some embodiments, the combination of hydroxytyrosol and AKBA has a statistically significant and greater effect than each component used alone. In certain embodiments, the combination of hydroxytyrosol and AKBA shows a synergistic effect in one or more of the following aspects: preventing, treating, repairing or reducing connective tissue damage; alleviating symptoms associated with connective tissue damage in avian or mammalian subjects; and reducing the levels of one or more inflammatory mediators in connective tissue.

[0048] The present invention provides a method for preventing, treating, repairing, reducing connective tissue damage or controlling connective tissue inflammation, protecting cartilage or alleviating symptoms associated with connective tissue damage in avian or mammalian subjects, the method comprising administering to the subject: (i) hydroxytyrosol and (ii) AKBA. The term "connective tissue" includes but is not limited to cartilage, bone, synovium, ligament, meniscus and tendon. In some embodiments, the administration of (i) hydroxytyrosol and (ii) AKBA can prevent, treat, repair or reduce connective tissue damage. Connective tissue damage can be the result of physical injury or can represent "wear and tear" caused by continuous use, body weight and age (such as by osteoarthritis). Connective tissue damage can also be caused by diseases such as rheumatoid arthritis, synovial disorders, rheumatic diseases related to infection and inflammatory connective tissue disorders. In some embodiments, the administration of (i) hydroxytyrosol and (ii) AKBA can alleviate symptoms associated with connective tissue damage in avian or mammalian subjects. Symptoms associated with connective tissue damage include but are not limited to: pain, discomfort, pressure, inflammation, stiffness and / or swelling.

[0049] The present invention also provides a method for reducing the level of one or more inflammatory mediators in connective tissue, comprising administering to an avian or mammalian subject: (i) hydroxytyrosol and (ii) AKBA. 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) and chemokines, leukotrienes, nitric oxide, and reactive oxygen species.

[0050] The administration of hydroxytyrosol and AKBA can also be used to treat, prevent, and reduce injury associated with conditions affecting the cardiovascular system, nervous system, musculoskeletal system, and gastrointestinal system or to alleviate symptoms associated with conditions affecting the cardiovascular system, nervous system, musculoskeletal system, and gastrointestinal system. In one aspect, the present invention provides compositions and methods for preventing and / or reducing an inflammatory response and / or inflammation in a subject. In one aspect, the present invention provides compositions and methods for managing an inflammatory disorder or generally reducing the inflammatory burden in a human or non-human animal. Thus, in one embodiment, the present invention provides a method for preventing and / or reducing an inflammatory response and / or inflammation in one or more tissues, the method comprising delivering a composition of the present invention to the one or more tissues.

[0051] The present invention also provides an orally administrable composition comprising: (i) hydroxytyrosol and (ii) AKBA. The orally administrable composition is any orally administrable dosage form known in the art, such as, but not limited to: capsules; tablets; powders dispersible in beverages; pastes; pellet forms; liquids such as solutions, suspensions, or emulsions; soft gels / chewable capsules; chew sticks; or other convenient dosage forms, such as oral liquids in capsules.

[0052] The orally administrable composition may contain one or more inactive pharmaceutical ingredients (also commonly referred to herein as "excipients"). Inactive ingredients are used, for example, to dissolve, suspend, thicken, dilute, emulsify, stabilize, preserve, protect, color, flavor, and shape the active ingredient into a suitable and effective formulation that is safe, convenient, and otherwise acceptable upon use. Excipients may be pharmaceutically acceptable excipients. Examples of the types of pharmaceutically acceptable excipients include lubricants, buffers, stabilizers, foaming agents, pigments, colorants, flavoring agents, fillers, bulking agents, fragrances, release regulators, adjuvants, plasticizers, flow promoters, demolding agents, polyols, granulating agents, diluents, binders, buffers, adsorbents, glidants, adhesives, anti-adhesives, acidifying agents, softeners, resins, emollients, solvents, surfactants, emulsifiers, elastomers, and mixtures thereof.

[0053] An orally administrable composition may further comprise one or more active ingredients. For example, the composition may further comprise one or more pharmaceuticals or nutritional supplements. In some embodiments, the composition may further comprise compounds beneficial to connective tissue. Examples include, but are not limited to, glycosaminoglycans such as chondroitin; amino sugars such as glucosamine, methylsulfonylmethane (MSM), collagen (including type II collagen), green tea extract, Scutellaria extract, Robinia extract, turmeric extract, curcumin, cetyl myristoleate complex (CMO), and eggshell membrane.

[0054] All references cited herein are incorporated by reference in their entirety. Examples

[0055] Example 1: Effects of hydroxytyrosol and AKBA on TNF-α production in lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophages.

[0056] RAW 264.7 murine macrophages were pretreated for 24 hours with 60 nM, 160 nM, or 1 μM hydroxytyrosol (HT) (98% purity, Sigma-Aldrich, St. Louis, MO) alone; 0.28 μg / mL, 0.56 μg / mL, or 1.124 μg / mL AKBA (administered normalized to 30% AKBA, PLT Health Solutions, Inc.) alone for 24 hours; or each of the three concentrations of HT in combination with each of the three concentrations of AKBA for 24 hours. The cells were then stimulated with 1 μg / mL lipopolysaccharide (LPS) for an additional 24 hours. LPS is an endotoxin in the bacterial cell wall that can induce an inflammatory response, including increased production of TNF-α. The TNF-α production in the cell supernatants was analyzed. Statistical comparisons were made using one-way analysis of variance (ANOVA), and Tukey post hoc analysis was performed, where differences with P < 0.05 were considered significant. Data are represented as mean + / - 1 SD.

[0057] When each of the three concentrations of HT was combined with AKBA, a statistically significantly greater reduction in TNF-α levels was observed compared to the reduction with either substance alone. The combinations of 60 nM HT with 0.28 μg / mL, 0.56 μg / mL, or 1.124 μg / mL AKBA all resulted in a greater reduction in TNF-α production than HT alone (P < 0.001) or AKBA alone (P < 0.001) ( Figure 1)。Compared with HT alone (P < 0.001) or AKBA alone (P = 0.001), the reduction of TNF-α in cells treated with a combination of 160 nM HT and 0.56 μg / mL AKBA reached statistical significance ( Figure 2 )。Treatment of cells with 1 μM HT and 0.28 μg / mL, 0.56 μg / mL, or 1.124 μg / mL AKBA also resulted in statistically significant reductions compared with HT alone (P < 0.001, P = 0.002, and P < 0.001, respectively) and AKBA alone (P < 0.001, P = 0.02, and P = 0.004, respectively). Figure 3 )。

Claims

1. A composition for reducing the level of tumor necrosis factor α in the connective tissue of a subject in need thereof, comprising hydroxytyrosol in a daily dose of at least 0.1 mg and at most 300 mg and 3-O-acetyl-11-keto-β-boswellic acid in a daily dose of at least 1 mg and at most 1 g.

2. The composition according to claim 1, wherein the hydroxytyrosol is derived from olive extract.

3. The composition according to claim 1, wherein the 3-O-acetyl-11-keto-β-boswellic acid is derived from Boswellia serrata extract.

4. The composition according to claim 1, which is formulated for oral administration to a mammalian subject.

5. The composition according to claim 4, wherein the mammalian subject is selected from the group consisting of humans, dogs, cats, horses, camels or cows.

6. The composition according to claim 1, which is formulated for oral administration to an avian subject.

7. Use of an orally administrable composition in the preparation of a medicament for reducing the level of tumor necrosis factor α in the connective tissue of a subject in need thereof, the orally administrable composition comprising hydroxytyrosol in a daily dose of at least 0.1 mg and at most 300 mg and 3-O-acetyl-11-keto-β-boswellic acid in a daily dose of at least 1 mg and at most 1 g.

8. The use according to claim 7, wherein the subject is a mammalian subject or an avian subject.

9. The use according to claim 7, wherein the hydroxytyrosol is derived from olive extract.

10. The use according to claim 7, wherein the 3-O-acetyl-11-keto-β-boswellic acid is derived from Boswellia serrata extract.

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