Compositions for the effective management of fibroblast-like synoviocyte-mediated rheumatoid arthritis - Patent Application 20070122999

Abstract

The disclosed invention relates to methods and compositions for use in inhibiting the proliferation and migration of fibroblast-like synoviocytes (FLS) in a subject. The invention also relates to T-helper 17 (T h 17) also discloses a composition and method for regulating imbalances in T cells. The disclosed composition contains 20-50% BDMC, 10-25% DMC by mass, and 30-50% curcumin by mass, and further contains boswellic acid and polysaccharides in the ranges of 35-50% by mass and 35-45% by mass, respectively.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a PCT application claiming priority to U.S. Provisional Patent Application No. 63,126,920, filed December 17, 2020, the contents of which are incorporated herein by reference. The present invention generally relates to methods and compositions for use in inhibiting the proliferation and migration of fibroblast-like synoviocytes (FLS) in a subject. The present invention also relates to T-helper 17 (T h 17) Also disclosed are compositions and methods for modulating imbalances in T cells. Even more particularly, the present invention discloses a composition comprising 20-50% BDMC, 10-25% DMC by weight, and 30-50% curcumin by weight, further comprising boswellic acid and polysaccharides from Boswellia serrata. [Background technology]

[0002] Rheumatoid arthritis (RA), an autoimmune synovial disease caused by a complex interplay between genetic and environmental factors, is characterized by synovial inflammation and joint damage. Fibroblast-like synoviocytes (FLS) are mesenchymal cells located within the synovial membrane, the soft tissue lining the diarthrodial joint space, tendon sheaths, and bursae (Nygaard, G., Firestein, G.S. Restoring synovial homeostasis in rheumatoid arthritis by targeting fibroblast-like synoviocytes. Nat Rev Rheumatol 16, 316-333 (2020)). FLS in the synovial lining play a key role in the production of cytokines that perpetuate inflammation and proteases that contribute to cartilage destruction. The lining is composed of two cell types: type A or macrophage-like synoviocytes and type B or FLS. The role of FLS present in the synovial lining is to regulate the composition of the extracellular matrix (ECM) and synovial fluid to lubricate and nourish the cartilage surface. Various studies have shown that in RA, type A cells predominate and produce proinflammatory cytokines, chemokines, and growth factors, which in turn activate local FLS and induce the production of IL-6, prostanoids, and matrix metalloproteinases (MMPs). This process leads to the destruction of the extracellular matrix, driven by an autocrine / paracrine network, perpetuating synovitis. It has also been documented that RA promotes the survival of FLS and prevents their loss through apoptosis (Bartok B, Firestein GS: Fibroblast-like synoviocytes: key effector cells in rheumatoid arthritis. Immunol Rev. 2010; 233:233-255). Regulatory T cells (Treg) / T-helper 17 (T h17) A balance between these cells has been implicated in the progression of RA. Th17 cells mediate pro-inflammatory responses by secreting IL-17A and TNF-α, which contribute to tissue destruction and articular cartilage damage, while Tregs mediate anti-inflammatory responses and maintain a state of self-tolerance. This imbalance is thought to be the underlying cause of several autoimmune diseases, including multiple sclerosis, atherosclerosis, and SLE, to name a few (Li, C. et al. (Arsenic trioxide improves Treg and Th17 balance by modulating STAT3 in treatment-naive rheumatoid arthritis patients. Int. Immunopharmacol. 73, 539-551, 2019)). Furthermore, current treatments for RA using synthetic disease-modifying antirheumatic drugs (DMARDs) and biologic DMARDs, either individually or in combination, offer a therapeutic strategy but exhibit significant side effects, including hypersensitivity, particularly immune-related. Considerable research efforts are being made in this area to discover effective, non-immunomodulatory therapeutic targets (Kohler et al. Current Therapeutic Options in the Treatment of Rheumatoid Arthritis, J. Clin. Med. 8, 1-15 (2019)).

[0003] Rizaldy et al. have shown that extracts of Curcuma longa and Boswellia serrata have therapeutic benefits in osteoarthritis (Rizaldy et al. A randomized controlled trial of Curcuma Longa and Boswellia Serrata Extract in Osteoarthritis. Global Journal of Medical Research: H Orthopedic and Musculoskeletal System 19(3)), but this paper does not clarify the pathways and factors responsible for this activity. Furthermore, targeting an isolated joint in osteoarthritis is quite different from targeting several joints in RA. Svensson et al. reaffirmed that combination therapy, using a non-immunomodulatory anti-FLS approach including TNF inhibitors and Fc-Ig1 and Ig2, was effective against arthritis with better efficacy and fewer side effects (Svensson et al. Synoviocyte-targeted therapy synergizes with TNF inhibition in arthritis reversal. Sci. Adv. 6, eaba4353, Pgs. 1-17 (2020)).

[0004] Shaikh et al. demonstrated that curcumin had a stronger anti-inflammatory effect than other curcuminoids in the treatment of rheumatoid arthritis. Furthermore, they showed that DMC and BDMC blocked the effects of curcumin in turmeric (Shaikh et al. Does Curcumin Analogues, Demethoxycurcumin and Bisdemethoxycurcumin (BDMC), Enhance the Therapeutic Efficacy of Curcumin in the Treatment of Rheumatoid Arthritis (RA), Nat. Prod. Chem. 8(6), Pgs. 1-9, 2020). Makuch et al. provided a comprehensive review of curcumin in RA, but revealed that its mechanism of action and effects on cell populations require further study (The Immunomodulatory and Anti-Inflammatory Effect of Curcumin on Immune Cell Populations, Cytokines, and In Vivo Models of Rheumatoid Arthritis, Pharmaceuticals, 14, pp. 1-18, 2021). Kloesch et al. showed that treatment of FLS with high concentrations of curcumin resulted in a dramatic decrease in cell viability and induced apoptosis. However, the molecular mechanism of curcumin remained unclear (Kloesch et al. Anti-Inflammatory and Apoptotic Effects of the Polyphenol Curcumin on Human Fibroblast-like Synoviocytes, 15, pp. 400-405 (2013)).

[0005] WO 2021 / 090154 includes a combination treatment for osteoarthritis that includes withanolide-enriched ashwagandha, BDMC-enriched curcumin, and AKBA-enriched Boswellia, but does not include the FLS pathway of non-immunomodulatory effects. Similarly, AU2010 / 345338 B2 covers synergistic compositions comprising Boswellia extract and Curcuma species for use in inflammatory conditions including arthritis, but does not cover any specific route. Essentially, there is an unmet need for safe, less toxic, and effective treatments for RA that work with current approaches and can improve disease control without suppressing the immune system. Furthermore, if the treatments were based on natural herbs, many of the side effects associated with the immunomodulatory effects of RA treatments could be overcome. Summary of the Invention

[0006] Object of the invention The primary object of the present invention is to disclose a method and composition for use in inhibiting the proliferation and migration of fibroblast-like synoviocytes (FLS) in a subject, comprising individual concentrations of BDMC, DMC, curcumin, curcuminoid, boswellic acid (B), or polysaccharide (PS). Another object is to disclose a method and composition for use in inhibiting the proliferation and migration of fibroblast-like synoviocytes in a subject, comprising a composition of concentrated BDMC curcuminoid and BPS. Yet another primary object of the present invention is to provide a method for the treatment of T-helper 17 (T 17) in the spleen of a mammal, comprising administering a composition comprising individual concentrations of BDMC, DMC, curcumin, curcuminoids, boswellic acid, or polysaccharides. h Another object of the present invention is to disclose methods and compositions for use in modulating the imbalance between T-helper 17 (T 17) and T cells (Treg) in mammals using a composition comprising a combination of concentrated BDMC curcuminoids and BPS. h 17) discloses methods and compositions for use in alleviating imbalances in T cells (Tregs). Another primary object of the present invention is to disclose methods and compositions for use in the therapeutic management of rheumatoid arthritis in mammals, said methods comprising administering a composition comprising individual concentrations of BDMC, DMC, curcumin, curcuminoids, boswellic acid, or polysaccharides. Another object is to disclose methods and compositions for use in the therapeutic management of rheumatoid arthritis in mammals, said methods comprising administering a composition comprising a combination of concentrated BDMC curcuminoids and BPS.

[0007] overview The present invention relates to the use of BDMC, DMC, curcumin, curcuminoids, boswellic acids, or polysaccharides, either individually or in combination with a concentrated BDMC curcuminoid composition and boswellic acid polysaccharides, to inhibit the proliferation and migration of FLS in a subject and to inhibit the proliferation and migration of T-helper 17 (T) in the spleen of a mammal. h 17) broadly solves the aforementioned problems mentioned in the background by encompassing methods and compositions for use in regulating imbalances in T cells (Tregs). A first aspect of the present invention relates to a composition for use in inhibiting the proliferation and migration of fibroblast-like synoviocytes (FLS) in a subject, comprising individual concentrations of BDMC, DMC, curcumin, curcuminoid, boswellic acid, or polysaccharides. Also encompassed is a composition comprising a combination of concentrated BDMC curcuminoid and BPS.

[0008] Yet another aspect of the present invention is a method for enhancing T-helper 17 (T ) expression in the spleen of a mammal, comprising administering to the mammal a therapeutically effective amount of BDMC, DMC, curcumin, curcuminoids, boswellic acid, or a polysaccharide at individual concentrations. h 17) and T cells (Tregs). Also included are compositions for use in modulating an imbalance in T cells (Tregs). Also included are administering a combination of concentrated BDMC curcuminoid and BPS. Another aspect of the present invention includes a composition for use in the therapeutic management of rheumatoid arthritis in a mammal, comprising individual concentrations of BDMC, DMC, curcumin, curcuminoid, boswellic acid, or polysaccharides, including administering a combination of concentrated BDMC curcuminoid and BPS. Another aspect of the present invention includes a method for inhibiting the proliferation and migration of fibroblast-like synoviocytes (FLS) in a mammal, comprising contacting the mammalian FLS with a composition comprising individual concentrations of BDMC, DMC, curcumin, curcuminoids, boswellic acid, or a polysaccharide. Also included is a composition comprising a combination of concentrated BDMC curcuminoids and BPS. Yet another aspect of the present invention is directed to a) T-helper 17 (T h a) identifying a mammal having a Treg imbalance; and b) administering a composition comprising individual concentrations of BDMC, DMC, curcumin, curcuminoids, boswellic acid, and polysaccharides to the mammal to detect T-helper 17 (T 17) in the spleen of the mammal. h 17), which includes a method for modulating T cell (Treg) imbalance. Also included is a composition comprising a combination of concentrated BDMC curcuminoid and BPS.

[0009] Yet another aspect of the present invention encompasses a method for the therapeutic management of rheumatoid arthritis in a subject, comprising: a) identifying a subject with rheumatoid arthritis; and b) administering a composition comprising individual concentrations of BDMC, DMC, curcumin, curcuminoid, boswellic acid, or a polysaccharide. Also encompassed are compositions comprising a combination of concentrated BDMC curcuminoid and BPS. The broader applicability of the present invention will become apparent from the following detailed description. However, while the following detailed description and specific examples illustrate preferred embodiments of the present invention, they should not be construed as limitations on the present invention, and it should be understood that various changes and modifications, such as changing the concentration range of the sample used, the curcuminoid derivative / analog, BPS, experimental conditions, and the selection of mammals, are within the skill of those skilled in the art and are well within the spirit and scope of the present invention from this detailed description.

[0010] The patent or patent application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows immunostaining of vimentin in FLS. [Figure 2] FIG. 1 shows immunostaining of vimentin in FLS. [Figure 3] Figure 1 shows concentration-dependent inhibition of FLS proliferation using 10, 20, and 40 μg / mL AC3. P<0.01 at 20 and 40 μg / ml compared to FLS control. [Figure 4] Figure 1 shows the percentage of inhibition of AC3 in a dose-dependent manner at 10, 20, and 40 μg / mL. P<0.01 compared to FLS control at 20 and 40 μg / ml. [Figure 5] Figure 1 shows the comparative effect of inhibiting FLS proliferation by any of curcuminoids, AC3, C3, and BPS, P<0.01 compared to FLS control except for BPS. [Figure 6] Figure 1 shows the effect of AC3 and BPS in inhibiting FLS proliferation. P<0.01 compared to FLS control for AC3 and BPS in combination. [Figure 7] FIG. 1 shows cell cycle inhibition of curcuminoids, AC3, C3, and BPS. [Figure 8] FIG. 1 shows the percentage of cells in S phase when treated with either curcuminoids, AC3, C3, or BPS. [Figure 9] Figure 1 shows the percentage of cells treated with AC3, BPS individually and in combination. [Figure 10] Figure 17 shows cell cycle inhibition using FACS for untreated (Figure 10), curcumin (Figure 11), BDMC (Figure 12), DMC (Figure 13), AC3 (Figure 14), C3 (Figure 15), BPS (Figure 16), 40 and 80 μg / mL, respectively (Figure 17), and the combination of AC3 and BPS at 10 and 40 μg / mL, respectively (Figure 18). [Figure 11] Same as above [Figure 12] Same as above [Figure 13] Same as above [Figure 14] Same as above [Figure 15] Same as above [Figure 16] Same as above [Figure 17] Same as above [Figure 18] Same as above [Figure 19] Figure 1 shows the Bax / Bcl2 ratio, suggesting the promotion of apoptosis using either curcuminoids, AC3, C3, BPS, and the combination of AC3+BPS. P<0.01 for AC3, Cur, and BDMC compared to FLS control. P<0.05 for DMC and AC3+BPS. [Figure 20]Figures 30 and 31 show the migration and invasion of FLS in a migration assay when treated with control (Figure 20), BDMC (Figure 21), curcumin (Figure 22), DMC (Figure 23), AC3 (Figure 24), C3 (Figure 25) at 15 μg / mL, BPS (Figure 26), AC3 (10 μg / mL, Figure 27), BPS (40 μg / mL, Figure 28), and a combination of AC3 and BPS (10 μg / mL + 40 μg / mL, Figure 29). Figures 30 and 31 show the percentage inhibition of migration. [Figure 21] Same as above [Figure 22] Same as above [Figure 23] Same as above [Figure 24] Same as above [Figure 25] Same as above [Figure 26] Same as above [Figure 27] Same as above [Figure 28] Same as above [Figure 29] Same as above [Figure 30] Same as above [Figure 31] Same as above [Figure 32] Figure 1 shows arthritis scores for treatment with control, RA control, celecoxib control, curcuminoid, AC3, C3, BPS, and the combination of AC3+BPS. p<0.05 and **P<0.01. [Figure 33] Figure 33 shows the regulatory effects on Th17 (Figure 33), Treg (Figure 34), and the ratio of Th17 / Treg (Figure 35) of treatment with control, RA, celecoxib, AC3, BPS, and a combination of AC3+BPS. FACS studies of normal (Figure 36), AC3 (50 mg / kg, Figure 37), AC3 (100 mg / kg, Figure 38), BPS (40 mg / kg, Figure 39), AC3+BPS (50+40 mg / kg, Figure 40), AC3+BPS (100+40 mg / kg, Figure 41), and celecoxib (Figure 42). [Figure 34] Same as above [Figure 35] Same as above [Figure 36] Same as above [Figure 37] Same as above [Figure 38] Same as above [Figure 39] Same as above [Figure 40] Same as above [Figure 41] Same as above [Figure 42] Same as above DETAILED DESCRIPTION OF THE INVENTION

[0012] Selected Definitions All terms used in this application have their ordinary meanings as known in the art unless otherwise specified. Some other specific definitions used in the present invention are set forth below and apply throughout the specification. The claims provide broader definitions unless otherwise specified.

[0013] In this application, any reference to a sample refers to one or a combination of the following agents that provide a therapeutic effect. The agents include curcuminoids, generally referred to as curcumin, bisdemethoxycurcumin, and demethoxycurcumin, or combinations thereof whenever appropriately referred to. A concentrated BDMC composition refers to a curcuminoid composition containing at least 20% BDMC by weight. More specifically, AC3 refers to a composition that is 20-50% bisdemethoxycurcumin, 10-25% demethoxycurcumin, and 30-50% curcumin by weight. The C3 complex is enriched in curcumin and is 75-81% curcumin, 15-19% demethoxycurcumin, and 2.2-6.5% bisdemethoxycurcumin. Boswellic acids are natural extracts isolated from Boswellia serrata as disclosed in US Pat. No. 60 / 268,713, and PS is a polysaccharide from the gum resin of Boswellia serrata. Specifically, whenever boswellic acids (B) are mentioned, this refers to a total boswellic acid content of 35-50% by weight, standardized to contain 20-30% β-boswellic acids and 12% 3-acetyl-11-keto-β-boswellic acid (AKBA). Whenever polysaccharides (PS) are mentioned, this refers to 35-45% polysaccharides (PS) by weight, containing neutral sugars consisting of galactose, arabinose, and D-glucuronic acid. (Boswellic acid compositions are commercially available as Boswellin® PS from Sami-Sabinsa Group Limited, Bangalore, India.)

[0014] Therapeutic management or management refers to a condition that effectively alleviates the conditions disclosed in this invention. Any reference to a control herein refers to either an untreated, RA control, or celecoxib control depending on the experiment and example included, and wherever appropriate, details of the control are provided. The present invention generally relates to a method for inhibiting the proliferation, migration, and activation of fibroblast-like synoviocytes, including the use of individual concentrations of BDMC, DMC, curcumin, curcuminoids, boswellic acid (B), or polysaccharides (PS), and the use of T-helper 17 (T hThe present invention encompasses methods and compositions for use in modulating the imbalance in rheumatoid arthritis (RA) cells (BPS) and T cells (Tregs). Also encompassed are compositions comprising a combination of concentrated BDMC curcuminoids and BPS. The present invention also encompasses methods for the therapeutic management of rheumatoid arthritis in a subject using the aforementioned compositions. In a most preferred embodiment, the present invention discloses a composition for use in inhibiting the proliferation and migration of fibroblast-like synoviocytes (FLS) in a subject, comprising concentrated bisdemethoxycurcumin (BDMC) present at 20% by weight or greater.

[0015] In another preferred embodiment of the present invention, the present invention provides a method for the treatment of T-helper 17 (T 17) in the spleen of a mammal, comprising administering 20% ​​by weight or more of bisdemethoxycurcumin (BDMC). h 17) discloses a composition for use in modulating an imbalance in T cells (Tregs). In a related aspect of this embodiment, the imbalance is measured by measuring the number of T cells in a patient with a RA of 3.5 compared to a control. h The ratio of 17 / Treg is preferably 1, or more preferably T h The ratio of 17 / Treg was set to 0 to 1 (Figure 35). h This was regulated by lowering 17 cells and increasing T cells (Treg) (Figures 33-35, Example 9), and was almost as good as the celecoxib control at 0.5 (Figure 35). In yet another preferred embodiment, the present invention discloses a composition for use in the therapeutic management of rheumatoid arthritis in a mammal, comprising concentrated bisdemethoxycurcumin (BDMC) present at 20% by weight or greater. In a related aspect of this embodiment, the management of rheumatoid arthritis in a mammal is achieved by inhibiting the proliferation and migration of fibroblast-like synoviocytes, increasing T-helper 17 (T ) expression in the spleen of the mammal, and inhibiting the proliferation and migration of fibroblast-like synoviocytes in the mammal. h This is achieved by regulating the imbalance between T-helper 17 (T 17) and T cells (Treg), reducing the levels of inflammatory markers, and promoting apoptosis. A related aspect of this embodiment is the improvement of arthritis scores. h17), the scope and details of regulating imbalance in T cells (Treg), reducing levels of inflammatory markers, promoting apoptosis, and improving arthritis scores are described in the relevant embodiments.

[0016] In another most preferred embodiment, the present invention discloses a method for inhibiting proliferation and migration of fibroblast-like synoviocytes (FLS) in a mammal, comprising contacting the mammalian FLS with a composition comprising concentrated bisdemethoxycurcumin (BDMC) present at 20% by weight or greater. In another most preferred embodiment of the present invention, the present invention provides a method for the treatment of a) T-helper 17 (T h a) identifying a mammal having an imbalance between T-helper 17 (T 17) and Tregs in the spleen of the mammal; and b) administering a composition comprising concentrated bisdemethoxycurcumin (BDMC) present at 20% by weight or greater. h 17) discloses a method for modulating an imbalance in T cells (Tregs). In a related aspect of this embodiment, the imbalance is measured by measuring the number of T cells in a patient with RA of 3.5 compared to a control. h The ratio of 17 / Treg is preferably 1, or more preferably T h The ratio of 17 / Treg was set to 0 to 1 (Figure 35). h It is regulated by decreasing IgE-17 cells and increasing T cells (Treg) (Figures 33 to 35, Example 9).

[0017] In yet another most preferred embodiment of the present invention, a method for the therapeutic management of rheumatoid arthritis in a subject is disclosed, comprising the steps of: a) identifying a subject having rheumatoid arthritis; and b) administering a composition comprising concentrated bisdemethoxycurcumin (BDMC) present at 20% by weight or greater. In related aspects of this embodiment, the management of rheumatoid arthritis in a mammal is achieved by inhibiting the proliferation and migration of fibroblast-like synoviocytes, increasing T-helper 17 (T ) expression in the spleen of the mammal, and / or inhibiting the proliferation and migration of fibroblast-like synoviocytes. hThis is achieved by regulating the imbalance between T-helper 17 (T 17) and T cells (Treg), reducing the levels of inflammatory markers, and promoting apoptosis. A related aspect of this embodiment is the improvement of arthritis scores. h 17), the scope and details of regulating imbalance in T cells (Treg), reducing levels of inflammatory markers, promoting apoptosis, and improving arthritis scores are described in the relevant embodiments.

[0018] In an embodiment related to the proliferation and inhibition of FLS, the composition contains 20-50% by weight BDMC, 10-25% by weight demethoxycurcumin (DMC), and 30-50% by weight curcumin, and the total curcuminoids in the composition range from 20-95% by weight. In a related aspect of this embodiment of the invention, the composition further contains boswellic acid (B) and polysaccharide (PS) from Boswellia serrata, wherein the boswellic acid and polysaccharide (BPS) are present in the ranges of 35-50% by weight and 35-45% by weight, respectively. In another related aspect of this and other embodiments of the invention, when used individually, the concentrations of BDMC, DMC, curcumin, curcuminoids, boswellic acid, and polysaccharide in the composition are selected from the range of 10-100 μg / mL. In this and other related embodiments of the present invention, the composition comprises a composition enriched with BDMC curcuminoids, and BPS are present in a 1:1 ratio, or preferably a 1:2 ratio, or preferably a 1:3 ratio, or preferably a 1:4 ratio, or preferably a 4:1 ratio, or preferably a 3:1 ratio, or preferably a 2:1 ratio. In a related aspect of this embodiment of the present invention, C3 and BPS are used in combination as specified in the above-mentioned ranges. Finding combinations within the specified ranges, or finding suitable ranges, is common knowledge and known to those skilled in the art. Furthermore, in this and other embodiments of the present invention, AC3 is more preferably used alone or in combination with BPS. In a further aspect of this and other embodiments of the present invention, when used individually, the concentrations of BDMC, curcumin, DMC, BPS, AC3, and C3 are in the range of 10-100 μg / mL, or preferably in the range of 20-80 μg / mL, or preferably in the range of 40-60 μg / mL.

[0019] In related aspects of this embodiment and other previously described embodiments of the present invention, inhibition of fibroblast-like synoviocytes (FLS) results from cell cycle inhibition, reduced levels of inflammatory markers, enhanced apoptosis, and improved arthritis scores. In related aspects of this embodiment, after treatment with the sample, inhibition of FLS proliferation is in the range of 10-90%, or more preferably in the range of 50-90%, or most preferably in the range of 60-90%, compared to untreated controls (Figures 3-6, Example 2). In related aspects of this embodiment of the present invention, after treatment with the sample, cell cycle inhibition is in the G0 / G1 phase, and the percentage of cells in S phase is in the range of 1-20%, or more preferably in the range of 1-10%, or most preferably in the range of 1-5% (Figures 7-9, Example 3). In related aspects of this embodiment of the present invention, the inflammatory marker is selected from the group consisting of TNF-α, IFN-γ, CCL-5, MMP-3, and cathepsin. In a further aspect of this embodiment, after treatment with each sample, the expression level of the inflammatory marker is reduced by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60% compared to the untreated control (Table 3, Example 4). In a further aspect of this embodiment, ELISA studies of the inflammatory marker IL6 showed that the combination of AC3 and BPS showed better inhibition compared to AC3 or BPS used individually (Example 4). In a further aspect of this embodiment, after treatment with the sample, apoptosis is promoted with an increase in the Bax / Bcl2 ratio in the range of 1 to 1.5, or preferably an increase in the ratio in the range of 1.25 to 1.5, compared to the untreated control of 1 (Figure 19, Example 5). In a further aspect of this embodiment, FLS migration was inhibited after treatment with the samples (Figures 20-31, Example 6), with migration inhibition in the range of 40-80%, or preferably in the range of 50-80%, or more preferably in the range of 50-70%, compared to untreated controls (Figure 30), and the combination of AC3+BPS was more effective than the individual treatments (Figure 31).In another aspect of this embodiment, after treatment with the sample, the arthritis score is in the range of 1 to 3, more preferably in the range of 1 to 2, compared to the untreated control (Figure 32, Example 8, RA control is 3.5 and positive control celecoxib is 1).

[0020] T-helper 17 (T) in mammalian spleens h In embodiments relating to the modulation of T cell (Treg) imbalances and the therapeutic management of RA in mammals, the compositions, when used individually, preferably have a concentration of BDMC, DMC, curcumin, curcuminoid, boswellic acid, or polysaccharide selected from the range of 40 mg / kg to 100 mg / kg of mammalian body weight, or preferably 50 to 100 mg / kg of mammalian body weight. In related embodiments, the concentrated BDMC-curcuminoid composition and BPS are present in a 1:1 ratio, or preferably a 1:2 ratio, or preferably a 1:3 ratio, or preferably a 1:4 ratio, or preferably a 4:1 ratio, or preferably a 3:1 ratio, or preferably a 2:1 ratio. In another related embodiment of the present invention, the composition further comprises stabilizers, bioavailability enhancers and antioxidants, pharmaceutically or nutraceutical or cosmeceutical acceptable excipients and enhancers, and is suitably formulated for oral administration in the form of a tablet, capsule, syrup, gummy, powder, suspension, emulsion, chewable, candy, or foodstuff (Example 10). Finding the appropriate formulation for administration is well within the skill of one of ordinary skill in the art. [Example]

[0021] Example 1 Isolation and culture of fibroblast-like synoviocytes (FLS): Synovial tissue was obtained under aseptic conditions from rats with collagen-induced arthritis according to the following method (Jinjun Zhao, Qingqing Ouyang, Ziyou Hu, Qin Huang, Jing Wu, Ran Wang and Min Yang. A protocol for the culture and isolation of murine synovial fibroblasts. Biomedical Reports 5: 171-175, 2016). FLS were isolated from synovial tissue by enzymatic digestion. Briefly, the tissue was cut into 1 mm pieces with microsurgical scissors. 3 After dividing into blocks, the tissue was then incubated for enzymatic digestion with 0.1% collagenase type II enzyme in DMEM supplemented with 10% FBS at 37°C for 45 minutes in an orbital agitation incubator (200 rpm).

[0022] After the incubation period, the tubes were vigorously vortexed for 1–2 min to release the cells, which were then filtered through a 100 μm mesh strainer. The filtrate was centrifuged at 1200 rpm for 5 min, and the cells were resuspended in DMEM supplemented with 10% FBS, 100 μg / ml streptomycin, and 100 U / ml penicillin and cultured at 37°C in a humidified incubator under 5% CO2. After FLS identification based on cell morphology and immunocytochemical staining using an anti-vimentin antibody (PA5-27231, Thermo Fisher Scientific) (Figures 1 and 2), cells from passages 4–7 were used for further studies.

[0023] Example 2 WST-1 proliferation assay: The WST-1 assay was used to measure the effect of various samples on FLS proliferation. FLS were cultured at 5 x 10 3Cells were seeded into 96-well plates at a density of 10 cells / well and incubated overnight at 37°C under 5% CO2. The cells were then treated for 72 hours with various concentrations of samples (AC3, C3, Cur, BDMC, DMC, and BPS) as shown in Figures 3–6. After the treatment period, 100 μl of WST-1 reagent (product number 5015944001, Sigma, USA) diluted 1:10 in serum-free DMEM culture medium was added to each well and incubated for 2 hours at 37°C under 5% CO2. Absorbance was measured at 450 nm with a reference wavelength of 610 nm. AC3 showed better inhibition of FLS proliferation at 20 μg / mL and 40 μg / mL in a dose-dependent manner (Figures 3 and 4). Comparative studies of curcuminoid analogs in inhibiting FLS proliferation showed a trend of BDMC > curcumin > DMC at 20 μg / mL (Figure 5), while BPS showed only 10% inhibition, and the combination was much more effective than Boswellin PS (BPS) or AC3 used individually (Figure 6).

[0024] Example 3 Cell cycle analysis: FLS cells (8 × 10 4 Cells (cells / well) were seeded into 24-well plates and incubated overnight at 37°C under 5% CO2. 。After 24 hours of synchronization in serum-free medium, cells were treated for 24 hours in DMEM 10% FBS with or without the test compound. After the treatment period, cells were collected and suspended in ice-cold PBS. After centrifugation, cells were fixed by adding 70% ice-cold ethanol dropwise to the pellet while vortexing and stored at 4°C for 30 minutes. Cells were stained with propidium iodide (PI) solution (50 μg / ml) for 30 minutes at 37°C in the dark. DNA content was analyzed by flow cytometry (BD FACS Celesta flow cytometer). Among curcuminoids, AC3 and BDMC showed the greatest inhibition of FLS replication (Figures 7, 8, 12, and 14), arresting FLS in the G0 / G1 phase. Boswellin PS (BPS) showed inhibition only at 80 μg / ml (Figures 8 and 16). As illustrated in Figure 9, the effect of the combination (AC3+BPS) on cell cycle inhibition was better than the individual samples used (AC3 and BPS).

[0025] Example 4 Measurement of inflammatory markers: FLS cells (8 × 10 4 Cells (cells / well) were seeded into 24-well plates and incubated overnight at 37°C under 5% CO2. 。 The cells were induced with 10 ng / ml recombinant rat TNF-α (Cat. No. 400-14, Peprotech, NJ, USA) in DMEM 10% FBS for 6 and 24 hours in the presence or absence of various concentrations of the samples to evaluate the anti-inflammatory effects of the samples. Curcumin showed better anti-inflammatory activity by suppressing inflammatory markers (IFNγ, CCL-5, MMP-3, and cathepsins, Table 2), while BDMC was most effective in suppressing TNF-α, and most importantly, the combination of AC3 and BPS was the most effective of all (Table 2). ELISA: After a 24-hour incubation period, culture supernatants were collected and evaluated for the inflammatory marker IL6 by ELISA. At concentrations of 10 μg / mL (AC3) and 40 μg / mL (BPS), inhibition was 20% when used in combination, compared to 3 and 1% when AC3 and BPS were used individually.

[0026] The kit used was Rat IL-6 DuoSet ELISA, R&D Systems, DY506-05. RT-PCR Expression Studies: After a 6-hour incubation, total cellular RNA from untreated and treated cells was isolated using Trizol Reagent® (Ambion, Life Technologies) according to the manufacturer's instructions, followed by RNase-free DNase I treatment (ThermoFisher Scientific) to remove any genomic DNA. Messenger RNA quality and concentration were analyzed spectrophotometrically (NanoDrop Lite, ThermoFisher Scientific). One microgram of total RNA was reverse transcribed into cDNA using the Revert-aid First Strand cDNA Synthesis Kit (ThermoFisher Scientific) according to the manufacturer's instructions and stored at -80°C until use.

[0027] 20 μl of the reaction mixture was then subjected to PCR using SYBR green qPCR master mix for amplification of the cDNA using specifically designed primers obtained from Eurofins India. The housekeeping gene β-actin was co-amplified with each reaction as an internal control (Table 2). PCR was performed in a Light cycler 96 (Roche Life Sciences). The PCR conditions for the gene were an initial denaturation at 95°C for 10 minutes, followed by 35 cycles consisting of denaturation at 95°C for 30 seconds, primer annealing at 60°C for 30 seconds, extension at 72°C for 30 seconds, and a final cooling at 72°C for 30 seconds (Table 1).

[0028] [Table 1]

[0029] [Table 2]

[0030] Example 5 Apoptosis analysis—FLS apoptosis was assessed using RT-PCR expression studies. Briefly, FLS cells (8 × 10 4 Cells (cells / well) were seeded into 24-well plates and incubated overnight at 37°C under 5% CO2. 。 After 24 hours of synchronization in serum-free medium, cells were treated for 24 hours in DMEM 10% FBS with or without the presence of test compounds. After the treatment period, cells were collected, and total cellular RNA was isolated and processed for RT-PCR analysis as previously described (Table 3). The Bcl-2 and Bax protein families play a central role in regulating apoptosis. The Bax / Bcl-2 ratio can act as a rheostat that determines the susceptibility of cells to apoptosis, with cells undergoing apoptosis having a higher Bax / Bcl-2 ratio (Figure 19). Compared to the control, the order of curcuminoids was BDMC > DMC > curcumin, and the AC3 complex was more favorable than the C3 complex (Figure 19).

[0031] [Table 3]

[0032] Example 6 Migration assay—FLS migration and infiltration play an important role in synovitis and bone destruction. FLS migrate locally and may infiltrate distal areas and joints via the bloodstream. RA-FLS secrete MMPs, which further exacerbate cartilage matrix degradation and ultimately lead to bone erosion. The migratory potential of FLS can be studied by a simple scratch assay and assessing wound closure by FLS migration. Untreated FLS migrate and close the wound, whereas treated FLS delays closure (J Immunol March 1, 2014, 192 (5) 2063-2070). FLS cells (5 × 10 4Cells (number of cells / well) were seeded into 24-well plates and incubated at 37°C under 5% CO2 to reach approximately 80-90% confluency. A wound was then created by scratching with a 200 μl pipette tip. To ensure complete removal of debris and floating cells, the cells were washed with PBS. The cells were then incubated with various concentrations of the sample in DMEM medium containing 2% FBS. Control samples contained cells and culture medium without any sample. Cell migration was assessed by evaluating the closure of the monolayer gap using Magvision software. The percentage of inhibition of wound closure was calculated compared to control wells. Compared to the control (Figure 20), among the curcuminoids at 15 μg / mL each, BDMC (Figure 21) was better than DMC (Figure 23) and curcumin (Figure 22), while AC3 complex (Figure 24) was better than C3 complex (Figure 25), and AC3 + Boswellin PS (BPS) (Figure 29) was better than AC3 (Figure 27) and BPS (Figure 28).

[0033] Example 7 Arthritis studies in rats Mouse model (Table 4). Collagen-induced arthritis (CIA) is a widely studied autoimmune model of rheumatoid arthritis. In this model, autoimmune arthritis is induced by immunization with type II collagen (CII) emulsified in complete Freund's adjuvant. These animals develop an autoimmune-mediated polyarthritis that shares several clinical, histological, and immunological features with the human autoimmune disease rheumatoid arthritis. The immune response to CII is characterized by both the stimulation of collagen-specific T cells and the production of high-titer antibodies specific for both the immunogen (heterologous CII) and the autoantigen (mouse or rat CII).

[0034] [Table 4]

[0035] Arthritis was induced by administering type 2 collagen (chicken sternal cartilage, Sigma catalog number: C9301) emulsified at a 1:1 ratio with incomplete Freund's adjuvant (FIA). On day 0, rats were injected intradermally at the base of the tail with 200 μL (200 μg / animal) of collagen-FIA emulsion (primary immunization). For booster injections, emulsions were prepared as above and injected with 100 μL (100 μg / animal) of collagen-FIA emulsion given on day 7. Test samples were administered from day 0 to day 20. Example 8 Arthritis Score Standard methods for arthritis scoring were used to assess the degree of swelling and erythema in all four paws. 0 - no signs, 1 - redness and no edema, 2 - redness and mild edema, 3 - redness and severe edema, 4 - redness, severe edema, and stiff movements. The combination of AC3 and BPS (Figure 32) was effective at concentrations of 100 and 40 mg / kg, respectively. Celecoxib was used as a positive control along with a rheumatoid arthritis control.

[0036] Example 9 Analysis of Treg and Th17 in spleen isolation of splenocytes: For lymphocyte isolation, spleens were obtained under aseptic conditions from rats with collagen-induced arthritis. Spleens were washed twice with PBS containing 100 μg / ml streptomycin and 100 U / ml penicillin, cut into small pieces with microsurgical scissors, homogenized using the plunger end of a syringe, and filtered through a 100 μm mesh strainer. The filtrate was centrifuged at 1200 rpm for 5 minutes, washed once or twice with PBS, and then collected in 1×10 spleens. 6 Cells / ml were suspended in RPMI medium supplemented with 10% FBS. Imbalance was observed at T compared to the RA control of 3.5. h The ratio of 17 / Treg is preferably 1, or more preferably T h The ratio of 17 / Treg was set to 0 to 1 (Figure 35). h This is achieved by reducing inflammatory cytokines (e.g., inflammatory bowel disease, inflammatory bowel disease, and inflammatory bowel disease) and increasing T cells (Tregs) (Figures 33-35). Flow cytometry (FCM) analysis: To assess Th17 and Treg cells, isolated lymphocytes were stimulated for 4 hours with 20 ng / mL phorbol myristate acetate (P8139, Sigma) and 500 ng / mL ionomycin, and BD GolgiStop (554724, BD), a protein transport inhibitor containing monensin, at concentrations suggested by the manufacturer.

[0037] After stimulation, cells were collected and 2 x 10 cells were cultured to assess Th17 and Treg populations. 5 Cells were divided into different tubes. Antibody dilutions used for analysis were as recommended by the respective manufacturers. To assess the Th17 population, cells were first stained with FITC mouse anti-rat CD3 (559975, BD pharmingen) and APC mouse anti-rat CD4 (550057, BD pharmingen) antibodies. To assess the Treg population, cells were first stained with APC mouse anti-rat CD4 (550057, BD pharmingen) and BV421 mouse anti-rat CD25 (565608, BD pharmingen) antibodies. Following the addition of the respective surface staining antibodies, the tubes were incubated for 30 minutes at 4°C in the dark. After surface staining, cells were fixed and permeabilized using fixation and permeabilization solution (BD Cytofix / Cytoperm, 554722) according to the manufacturer's instructions and resuspended in saponin-containing buffer (BD Perm / Wash, 554723). Following fixation and permeabilization, Th17 and Treg cells were incubated with PE-conjugated anti-mouse / rat IL-17A (12-7177-81, eBioscience) and PE-conjugated anti-FOXP3 (12-5773-80, eBioscience) antibodies, respectively, for 30 minutes at 4°C in the dark. Cells were then washed twice with saponin-containing buffer, resuspended in staining buffer, and subjected to flow cytometry analysis. Flow cytometry was performed on a BD FACSCelesta system (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed using FlowJo software.

[0038] Example 10 Formulations: The composition is formulated with pharmaceutically / nutraceutical acceptable excipients, adjuvants, diluents, stabilizers, dispersible gums, bioavailability enhancers or carriers and administered orally in the form of a tablet, capsule, syrup, gummy, powder, suspension, emulsion, chewable tablet, candy, or foodstuff.

[0039] In a related aspect, the bioavailability enhancer is selected from the group consisting of piperine (BioPerine®), quercetin, garlic extract, ginger extract, and naringin. In another related aspect, the stabilizer is selected from the group consisting of rosmarinic acid, butylated hydroxyanisole, butylated hydroxytoluene, sodium metabisulfite, propyl gallate, cysteine, ascorbic acid, and tocopherol. In yet another related aspect, the dispersible gum is selected from the group consisting of agar, alginate, carrageenan, gum arabic, guar gum, locust bean gum, konjac gum, xanthan gum, and pectin. Tables 5-9 provide illustrative examples of dietary supplement formulations containing bisdemethoxycurcumin.

[0040] [Table 5]

[0041] [Table 6]

[0042] [Table 7]

[0043] [Table 8]

[0044] [Table 9]

[0045] The above formulations are merely illustrative examples, and any formulation intended for the above purposes and containing the above active ingredients is considered equivalent. Other modifications and variations of the present invention will be apparent to those skilled in the art from the foregoing disclosure and teachings. Thus, while only certain embodiments of the present invention have been specifically described herein, it will be apparent that numerous modifications may be made thereto without departing from the spirit and scope of the invention, which should be read only in conjunction with the appended claims.

Claims

1. A composition for use in inhibiting fibroblast-like synoviocyte (FLS) proliferation and migration in a mammal, comprising concentrated bisdemethoxycurcumin (BDMC) present at 20% by weight or greater.

2. 2. The composition for use according to claim 1, wherein the concentration of BDMC, DMC, curcumin, or curcuminoid in the composition when used individually is selected from the range consisting of 10 to 100 μg / mL.

3. 3. The composition for use according to claim 2, wherein the composition further comprises boswellic acids (B) and polysaccharides (PS) from Boswellia serrata, the boswellic acids comprising in the range of 35-50% by weight boswellic acids, 20-30% by weight β-boswellic acids, 12% by weight 3-acetyl-11-keto-β-boswellic acid (AKBA), and 35-45% by weight polysaccharides.

4. The composition for use according to claim 3, wherein the concentration of the boswellic acid, polysaccharide, when used individually, is selected from the range consisting of 10 to 100 μg / mL.

5. The composition for use according to claim 1, wherein the inhibition of fibroblast-like synoviocytes (FLS) is brought about by cell cycle inhibition, reduction in the levels of inflammatory markers, and promotion of apoptosis.

6. The composition for use according to claim 5, wherein the cell cycle inhibition is in the G0 / G1 phase.

7. The composition for use according to claim 5, wherein apoptosis is measured as the ratio of Bax to Bcl-2.

8. The composition for use according to claim 5, wherein the inflammatory marker is selected from the group consisting of TNF-α, IFN-γ, CCL-5, MMP-3, and cathepsin.

9. The composition for use according to claim 1, wherein inhibition of fibroblast-like synoviocytes results in an improvement in arthritis score.

10. A method for detecting T-helper 17 (T17) in a mammalian spleen, comprising: h 17) and a composition for use in regulating imbalances in T cells (Treg).

11. Imbalance, T h 17 cells and increase T cells (Treg), thereby modulating T h The composition for use according to claim 10, which reduces the .17 / Treg ratio.

12. 1. A composition for use in the therapeutic management of rheumatoid arthritis in a mammal comprising concentrated bisdemethoxycurcumin (BDMC) present at 20% by weight or greater.

13. 13. The composition for use according to any one of claims 1, 10 or 12, wherein the composition comprises 20-50% by weight of bisdemethoxycurcumin, 10-25% by weight of demethoxycurcumin, and 30-50% by weight of curcumin, and the total curcuminoids in the composition are in the range of 20-95% by weight.

14. 13. The composition for use according to claim 10 or 12, wherein the concentration of BDMC, DMC, curcumin or curcuminoid in the composition when used individually is selected from 40 mg / kg (body weight of the mammal) to 100 mg / kg (body weight of the mammal).

15. 13. The composition for use according to claim 10 or 12, wherein the composition further comprises boswellic acids (B) and polysaccharides (PS) from Boswellia serrata, the boswellic acids and polysaccharides (BPS) being present in the range of 35-50% by weight and 35-45% by weight, respectively.

16. The composition for use according to claim 15, wherein the concentration of boswellic acid, polysaccharide is selected from the range of 40 mg / kg (body weight of a mammal) to 100 mg / kg (body weight of a mammal) when used individually.

17. 13. The composition for use according to any one of claims 1, 10 or 12, wherein the concentrated BDMC curcuminoid composition and BPS are present in combination in a ratio of 1-4:4-1.

18. Management of rheumatoid arthritis in mammals is achieved by inhibiting proliferation and migration of fibroblast-like synoviocytes, T-helper 17 (T h 17) A composition for use according to claim 10 or 12, which is brought about by regulating the imbalance in T cells (Treg), reducing the levels of inflammatory markers, promoting apoptosis and improving arthritis scores.

19. 13. The composition for use according to claim 10 or 12, wherein the composition further comprises stabilizers, bioavailability enhancers and antioxidants, pharma- ceutically or nutraceutical or cosmeceutical acceptable excipients and enhancers, and is orally administered in the form of a tablet, capsule, syrup, gummy, powder, suspension, emulsion, chewable, candy, or foodstuff.

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