Patent Application
20220193006
The invention in general relates to a method and compositions for use in inhibiting proliferation, migration of Fibroblast-like Synoviocytes (FLS) in a subject. The invention also discloses a composition and method for regulating imbalance in T-helper 17 (Th17), T-cell. Still more specifically, the invention discloses the composition comprising 20-50% BDMC, 10-25% w/w DMC and 30-50% w/w curcumin, further comprising boswellic acids and polysaccharides from Boswellia serrata.
The clinical manifestation of Rheumatoid arthritis (RA), an autoimmune synovial disease caused by a complex interaction between genetic and environmental factors, caused by synovial inflammation and joint damage. Fibroblast-like synoviocytes (FLS) are mesenchymal cells located inside joints of the synovium, soft tissue lining the space of diarthrodial joints, 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 intimal lining plays a key role in producing cytokines that perpetuate inflammation and proteases contributing for cartilage destruction. The intimal lining layer is composed to two cell types, type A or macrophage-like synovial cells and type B or FLS. The role of FLS, which resides in the intimal lining layer of the synovium, is to control the composition of extracellular matrix (ECM) and the synovial fluid for lubricating and nourishing cartilage surfaces. Various studies have shown that in RA, type A cells predominates to produce pro-inflammatory cytokines, chemokines, and growth factors, which in turn activates local FLS and induces the production of IL-6, prostanoids, and matrix metallo-proteinases (MMPs). This process causes the destruction of the extracellular matrix actuated by the autocrine/paracrine network to perpetuate synovitis. It is also documented that RA promotes survival of FLS preventing deletion through apoptosis (Bartok B, Firestein G S: Fibroblast-like synoviocytes: key effector cells in rheumatoid arthritis. Immunol Rev. 2010: 233:233-255). The balance between regulatory T-cell (Treg) T-helper 17 (Th17) cell is implicated in the progression of RA. The Th17 cells mediate a pro-inflammatory response by secreting IL-17A and TNF-α, which are instrumental in tissue destruction and articular cartilage damage, while the Tregs mediate the anti-inflammatory response and maintain a state of autoimmune tolerance. This imbalance seems to be the underlying cause of several autoimmune diseases including multiple sclerosis, atherosclerosis, SLE to name a few (Li, C. et al. (Arsenic trioxide improves Treg and Th17 balance by modulating STAT3 in treatment-naïve rheumatoid arthritis patients. Int. Immunopharmacol. 73, 539-551, 2019) Further, current treatment of RA using synthetic Disease-modifying antirheumatic drugs (DMARDs) and biological DMARDs either individually or in combination offers therapeutic strategies but show significant side effects including hypersensitivity, especially immune related. There is considerable research efforts in this area to find a therapeutic target that are effective and non-immunomodulating (Kohler et al. Current Therapeutic Options in the Treatment of Rheumatoid Arthritis, J. Clin. Med. 8, 1-15 (2019).
Rizaldy et al. have shown that the extracts of Curcuma longa and Boswellia serrate having therapeutic benefits for 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 article does not shed light on the pathway and the agents responsible for this activity. And moreover, targeting isolated joint in osteoarthritis and several joints in RA are entirely different.
Svensson et al. have reassured the combination therapy worked effectively against arthritis with better efficacy and lesser side effects through non-immunomodulatory anti-FLS approach comprising TNF inhibitor, and Fc-Ig1 and Ig2 (Svensson et al. Synoviocyte-targeted therapy synergizes with TNF inhibition in arthritis reversal. Sci. Adv. 6. eaba4353, Pgs. 1-17 (2020)).
Shaikh et al. have shown that curcumin with greater anti-inflammatory action compared to other curcuminoids in rheumatoid arthritis treatment. Further they have shown that DMC and BDMC hindered 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 for RA but revealed that mechanism of action and its effect on cell populations needs further study (The Immunomodulatory and Anti-Inflammatory Effect of Curcumin on Immune Cell Populations, Cytokines, and In Vivo Models of Rheumatoid Arthritis, Pharmaceuticals, 14, Pgs. 1-18, 2021). Kloesch et al. showed treatment of FLS with high concentrations of curcumin led to a dramatic decrease in cell viability and induced apoptosis. But the molecular mechanism of curcumin was unclear. (kloesch et al. Anti-Inflammatory and Apoptotic Effects of the Polyphenol Curcumin on Human Fibroblast-like Synoviocytes, 15, Pgs. 400-405 (2013)).
WO 2021/090154 A1 includes combination treatment for osteoarthritis covering withanolide enriched ashwagandha, BDMC enriched curcumin and AKBA enriched Boswellia. But it does not cover the FLS pathway for non-immunomodulatory effects.
Similarly, AU 2010/345338 B2 covers synergistic composition comprising Boswellia extract and species of curcuma for use in inflammatory conditions including arthritis, but fails to cover any specific pathway.
In essence, there is an unmet need for a treatment that is safe, lesser toxic and effective for RA, which can act along with current approaches and improve disease control without suppressing the immune system. Moreover, if the treatment is based out of natural herbs, many of the side effects associated with immunomodulatory effects of RA treatment can be overcome.
It is the main objective of the invention to disclose a method and composition for use in inhibiting proliferation and migration of Fibroblast-like synoviocytes (FLS) in a subject, comprising individual concentration of BDMC, DMC, Curcumin, curcuminoids, Boswellic acids (B), or polysaccharides (PS). Another objective is to disclose a method and composition for use in inhibiting proliferation and migration of Fibroblast-like synoviocytes in a subject comprising composition of enriched BDMC curcuminoids cand BPS.
In yet another main objective of the invention to disclose a method and composition for use in regulating imbalance in T-helper 17 (Th17) and, T-cell (Treg) in the spleen of a mammal comprising administering composition comprising individual concentration of BDMC, DMC, Curcumin, curcuminoids, Boswellic acids or polysaccharides. Another objective is to disclose a method and composition for use in moderating an imbalance in T-helper 17 (Th17), T-cell (Treg) in a mammal using a composition comprising combination of enriched BDMC curcuminoids and BPS.
In another main objective of the invention to disclose a method and composition for use in therapeutic management of rheumatoid arthritis in a mammal, said method comprising administering a composition comprising individual concentration of BDMC, DMC, Curcumin, curcuminoids, Boswellic acids, or polysaccharides. Another objective is to disclose a method and composition for use in therapeutic management of rheumatoid arthritis in a mammal, said method comprising administering a composition comprising combination enriched BDMC curcuminoids and BPS.
The invention broadly solves the aforementioned problems mentioned in the background by covering a method and composition for use in inhibiting proliferation, migration of FLS in a subject, and regulating imbalance in T-helper 17 (Th17), T-cell (Treg) in the spleen of a mammal either individually of BDMC, DMC, Curcumin, curcuminoids, Boswellic acids, or polysaccharides. or combination of enriched BDMC curcuminoids composition and Boswellic acids polysaccharides.
The first aspect of the invention relates to a composition for use in inhibiting proliferation and migration of Fibroblast-like synoviocytes (FLS) in a subject, wherein said composition comprises individual concentration of BDMC, DMC, Curcumin, curcuminoids, Boswellic acids, or polysaccharides. Also covers a composition comprising combination of enriched BDMC curcuminoids and BPS.
In yet another aspect of the invention covers a composition for use in regulating imbalance in T-helper 17 (Th17) and, T-cell (Treg) in the spleen of a mammal, wherein the composition comprises individual concentration of BDMC, DMC, Curcumin, curcuminoids, Boswellic acids, or polysaccharides. Also covers administering a combination of enriched BDMC curcuminoids and BPS.
In another aspect of the invention covers a composition for use in therapeutic management of rheumatoid arthritis in a mammal, wherein the composition comprises individual concentration of BDMC, DMC, Curcumin, curcuminoids, Boswellic acids, or polysaccharides. Also covers administering a combination of enriched BDMC curcuminoids and BPS.
In another aspect of the invention covers a method for inhibiting the proliferation and migration of Fibroblast-like synoviocytes (FLS) in a mammal, said method comprising step of bringing into contact mammalian FLS with a composition comprising individual concentration of BDMC, DMC, Curcumin, curcuminoids, Boswellic acids, or polysaccharides. Also covers composition comprising combination of enriched BDMC curcuminoids and BPS.
In yet another aspect of the invention covers a method for regulating imbalance in T-helper 17 (Th17), T-cell (Treg) in the spleen of a mammal, said method comprising step of a) identifying the mammal with T-helper 17 (Th17), Treg imbalance and b) administering a composition comprising individual concentration of BDMC, DMC, Curcumin, curcuminoids, Boswellic acids, polysaccharides. Also covers composition comprising combination of enriched BDMC curcuminoids and BPS.
In yet another aspect of the invention covers a method for therapeutic management of rheumatoid arthritis in a subject, said method comprising step of a) identifying the subject with rheumatoid arthritis and b) administering a composition comprising individual concentration of BDMC, DMC, Curcumin, curcuminoids, Boswellic acids, or polysaccharides. Also covers composition comprising combination of enriched BDMC curcuminoids and BPS.
The broader scope of applicability of the present invention will be apparent from the detailed description below. However, it should be understood that the detailed description and specific examples below, while indicating preferred embodiments of the invention, should not be construed as the limitations to the invention, and it is within the scope of those skilled in the art to make various changes and modifications, such as changing the concentration range of samples used, derivatives/analogs of curcuminoids, BPS, experimental conditions, choice of mammals, are well within the spirit and scope of the invention from this detailed description.
The patent or 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.
All the terms used in this application carry ordinary meaning as known in the prior art unless otherwise specified. Few other specific definitions used in this invention are explained below, which applies throughout this specification. Claims provide broader definition unless and otherwise specified.
In this application, any reference to sample refers to either one or combination of the following agents, which brings about therapeutic effect. The agents include, curcuminoids in general refers to either of curcumin, bisdemethoxycurcumin, and demethoxycurcumin or their combination wherever appropriately mentioned. Enriched BDMC composition refers to curcuminoids composition comprising at least 20% w/w of BDMC. More specifically, AC3 refers to a composition, which is 20-50% w/w bisdemethoxycurcumin, 10-25% w/w demethoxycurcumin and 30-50% w/w curcumin. C3 complex is enriched with curcumin, which is 75-81% curcumin, 15-19% demethoxycurcumin, and 2.2-6.5% bisdemethoxycurcumin. Boswellic acid, a natural extract isolated from Boswellia serrata as disclosed in U.S. 60/268,713, and PS is polysaccharide from the gum resin of Boswellia serrata. Specifically, wherever Boswellic acids (B) are mentioned it includes a total Boswellic acids content of 35-50% w/w standardized to contain β-Boswellic acids 20-30% w/w, 3-acetyl-11-keto-β-boswellic acid, 12% w/w (AKBA). Wherever polysaccharides (PS) are mentioned, it includes 35-45% w/w polysaccharides (PS) to contain neutral sugars, consisting of galactose, arabinose and D-glucuronic acid. (The Boswellia serrata composition is commercially available from Sami-Sabinsa Group Limited, Bangalore, India as Boswellin® PS)
Therapeutically managing or management refers to a condition of effectively ameliorating conditions disclosed in the invention. Any reference to control in this specification refers to either untreated, RA control, or celecoxib control depending on the experiment, examples covered and control details are mentioned wherever appropriate.
The invention in general covers a method and composition for use in inhibiting proliferation, migration of Fibroblast-like synoviocytes, and regulating an imbalance in T-helper 17 (Th17) and, T-cell (Treg) comprising individual concentration of BDMC, DMC, Curcumin, curcuminoids, Boswellic acids (B), or polysaccharides (PS). Also covers composition comprising combination of enriched BDMC curcuminoids and BPS. The invention also covers a method for therapeutic management of rheumatoid arthritis in a subject using the aforementioned composition.
In the most preferred embodiment, the invention discloses a composition for use in inhibiting proliferation and migration of Fibroblast-like synoviocytes (FLS) in a subject, wherein said composition comprises enriched Bisdemethoxycurcumin (BDMC) present not less than 20% w/w.
In another preferred embodiment of the invention, the invention discloses a composition for use in regulating imbalance in in T-helper 17 (Th17), T-cell (Treg) in the spleen of a mammal, wherein the composition comprises not less than 20% w/w Bisdemethoxycurcumin (BDMC). In a related aspect of this embodiment, the imbalance is regulated by reducing Th17 cells and increasing T-cell (Treg) (
In yet another preferred embodiment of the invention, the invention discloses a composition for use in therapeutic management of rheumatoid arthritis in a mammal, wherein the composition comprises enriched Bisdemethoxycurcumin (BDMC) present not less than 20% w/w. In a related aspect of this embodiment, wherein the management of rheumatoid arthritis in a mammal is brought by inhibiting the proliferation and migration of fibroblast like synoviocytes, regulating imbalance in T-helper 17 (Th17) and, T-cell (Treg) in the spleen of the mammal, decreasing the levels of inflammatory markers, and promoting apoptosis. In a related aspect of this embodiment, results in improved arthritis score. The scope and details of inhibition and migration of fibroblast like synoviocytes, regulating imbalance in T-helper 17 (Th17), T-cell (Treg), decreasing the levels of inflammatory markers, promoting apoptosis, and improved arthritis score are explained in the related embodiments.
In another most preferred embodiment of the invention, the invention discloses a method for inhibiting the proliferation and migration of Fibroblast-like synoviocytes (FLS) in a mammal, said method comprising step of bringing into contact mammalian FLS with a composition comprising enriched Bisdemethoxycurcumin (BDMC) present not less than 20% w/w.
In another most preferred embodiment of the invention, the invention discloses a method of regulating imbalance in T-helper 17 (Th17), T-cell (Treg) in the spleen of the mammal, said method comprising step of a) identifying the mammal with T-helper 17 (Th17) and, Treg imbalance and b) administering a composition comprising enriched Bisdemethoxycurcumin (BDMC) present not less than 20% w/w. In a related aspect of this embodiment, the imbalance is regulated by reducing Th17 cells and increasing T-cell (Treg) (
In yet another most preferred embodiment of the invention, discloses a method for therapeutic management of rheumatoid arthritis in a subject, said method comprising step of a) identifying the subject with rheumatoid arthritis and b) administering a composition comprising enriched Bisdemethoxycurcumin (BDMC) present not less than 20% w/w. In a related aspect of this embodiment, wherein the management of rheumatoid arthritis in a mammal is brought by inhibiting the proliferation and migration of fibroblast like synoviocytes, regulating imbalance in in T-helper 17 (Th17) and, T-cell (Treg) in the spleen of the mammal, decreasing the levels of inflammatory markers, and promoting apoptosis. In a related aspect of this embodiment, results in improved arthritis score. The scope and details of inhibition and migration of fibroblast like synoviocytes, regulating an imbalance in in T-helper 17 (Th17), T-cell (Treg), decreasing the levels of inflammatory markers, promoting apoptosis, and improved arthritis score are explained in the related embodiments.
In the embodiments related to proliferation and inhibition of FLS, wherein the composition comprises of 20-50% w/w BDMC, 10-25% w/w demethoxycurcumin (DMC) and 30-50% w/w curcumin, with the total curcuminoids in the composition are in the range of 20-95% w/w. In the related aspect of this embodiment of the invention, the composition further comprises Boswellic acids (B) and polysaccharide (PS) from Boswellia serrata, wherein the boswellic acids and polysaccharides (BPS) are present in the range of 35-50% w/w and 35-45% w/w respectively. In another related aspect of this and other embodiments of the invention, when used individually the concentration of BDMC, DMC, Curcumin, curcuminoids, Boswellic acids, polysaccharides in a composition are selected from the range consisting of 10 to 100 μg/mL. In this aspect and other related embodiments of the invention, the composition comprises a composition enriched BDMC curcuminoids and BPS are present in a combination at a ratio of 1:1, or preferably 1:2, or preferably 1:3, or preferably 1:4 or preferably 4:1, or preferably 3:1, or preferably 2:1 respectively. In a related aspect of this embodiment of the invention, C3 and BPS are used as a combination as the above specified ranges. It is a common knowledge and to those skilled in the art to find combinations within the specified range or finding a suitable range. Further in this aspect and other embodiments of the invention, more preferably AC3 is used alone or in combination with BPS. In further aspect of this and other embodiments of the invention when used individually the concentration of BDMC, curcumin, DMC, BPS, AC3, C3 are used in the range between 10 to 100 μg/mL, or preferably in the range 20 to 80 μg/mL, or preferably between 40 to 60 μg/mL.
In a related aspect of this and other aforementioned embodiments of the invention, inhibiting Fibroblast-like synoviocytes (FLS) is brought about by cell cycle inhibition, decreasing the levels of inflammatory markers, promoting apoptosis, and improved arthritis score. In a related aspect of this embodiment, after treatment with the sample the inhibition of FLS proliferation is in the range of 10-90%, or more preferably in the range between 50-90%, or most preferably in the range of 60-90% compared to the untreated control (
In embodiments related to regulating imbalance in T-helper 17 (Th17), T-cell (Treg) in the spleen of the mammal and therapeutic management of RA in a mammal, the composition when used individually the concentration of BDMC, DMC, Curcumin, curcuminoids, Boswellic acids, or polysaccharides in a composition is preferably selected from the range consisting of 40 mg/kg to 100 mg/kg, or preferably 50-100 mg/kg of the bodyweight of the mammal. In a related embodiment wherein the enriched BDMC curcuminoids composition and BPS are present in a combination at a ratio of 1:1, or preferably 1:2, or preferably 1:3, or preferably 1:4 or preferably 4:1, or preferably 3:1, or preferably 2:1 respectively.
In another related embodiment of the invention, wherein the composition further comprises of stabilizing agents, bioavailability enhancers and antioxidants, pharmaceutically or nutraceutically or cosmeceutically accepted excipients and enhancers and suitably formulated to be administered orally in the form of tablets, capsules, syrups, gummies, powders, suspensions, emulsions, chewables, candies or eatables (Example 10). It is well within the scope of a person skilled in the art to come up with a suitable formulation for administration.
Synovial tissues were obtained from collagen induced arthritis rats under sterile conditions, according to the method followed by (Jinjun Zhao, Qingqing Onyang, 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 tissues by enzymatic digestion. In short, the tissues were separated into 1-mm3 blocks with microsurgery scissors. After which the tissues were incubated with 0.1% collagenase type II enzyme in DMEM supplemented with 10% FBS for enzymatic digestion at 37° C. for 45 min in an orbital shaker incubator (200 rpm).
After the incubation time, the tubes were vortexed vigorously for 1-2 min to release the cells and filtered using a 100 μm mesh strainer. The filtrate was centrifuged for 5 min at 1200 rpm, the cells were resuspended in DMEM supplemented with 10% FBS and 100 μg/ml streptomycin and 100 U/ml penicillin and cultured in a humidified incubator at 37° C. under 5% CO2. After FLS identification based on cell morphology and immunocytochemical staining with anti-vimentin antibody (PA5-27231, Thermo Fisher Scientific), (
WST-1 assay was used to measure the effect of different samples on FLS proliferation. FLS were seeded in 96-well plates at a density of 5×103 cells/well and incubated overnight at 37° C. under 5% CO2. Then, the cells were treated with different concentrations of the sample (AC3, C3, Cur, BDMC, DMC, BPS) as shown in the
FLS cells (8-104 cells/well) were seeded in 24-well plates and incubated overnight at 37° C. under 5% CO2. After synchronization in serum-free medium for 24 h, cells were treated with or without the presence of samples in DMEM 10% FBS for 24 h. After the treatment period, the cells were collected, suspended in ice cold PBS. After centrifugation, the cells were fixed by adding 70% ice cold ethanol dropwise to the pellet while vortexing and stored at 4° C. for 30 min. The cells were stained with propidium iodide (PI) solution (50 μg/ml) for 30 min at 37° C. in the dark. DNA content was analyzed by flow cytometry (BD FACS Celesta flow cytometer). AC3, BDMC among curcuminoids showed maximum inhibition of FLS replication (
FLS cells (8×104 cells/well) were seeded in 24-well plates and incubated overnight at 37° C. under 5% CO2. Cells were induced with 10 ng/ml recombinant rat TNF-α (Cat No. 400-14, Peprotech, NJ, USA) with or without the presence of different concentrations of sample in DMEM 10% FBS for 6 hr and 24 hr to evaluate the anti-inflammatory effect of the sample. Curcumin showed better anti-inflammatory activity by suppressing the inflammatory markers (IFN γ, CCL-5, MMP-3 and Cathepsin, Table 2), whereas BDMC was most effective in TNF-α and most importantly the combination of AC3 and BPS was the most effective of all (Table 2).
ELISA: After the incubation period of 24 hr, culture supernatants were collected and evaluated for inflammatory marker IL6 by ELISA method. For the concentrations of 10 μg/mL (AC3) and 40 μg/mL (BPS), when used as a combination the inhibition was 20% compared to 3 and 1% when used AC3 and BPS individually.
Kit used—Rat IL-6 DuoSet ELISA R&D Systems, DY506-05.
RT-PCR expression study: After 6 hrs of incubation, total cellular RNA from untreated and treated cells were 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 Revert-aid First Strand cDNA synthesis kit (ThermoFisher Scientific), according to the manufacturer's instructions, and stored at −80° C. until use.
Then 20 μl of the reaction mixture was subjected to PCR using SYBR green qPCR master mix for amplification of cDNAs using specifically designed primers procured from Eurofins India, as an internal control, the house keeping gene § actin was co-amplified with each reaction (Table 2). PCR was carried out in Light cycler 96 (Roche Life Science) and PCR conditions for genes were initial denaturation at 95° C. for 10 min followed by 35 cycles consisting of denaturation at 95° C. for 30 seconds, annealing of primers at 60° C. for 30 seconds, extension at 72° C. for 30 seconds and final cooling at 72° C. for 30 seconds (Table 1).
FLS apoptosis was assessed with RT-PCR expression study. In short, FLS cells (8×104 cells/well) were seeded in 24-well plates and incubated overnight at 37° C. under 5% CO2. After synchronization in serum-free medium for 24 h, cells were treated with or without the presence of samples in DMEM 10% FBS for 24 h. After the treatment period, the cells were collected, total cellular RNA was isolated and processed for RT-PCR analysis (Table 3) as mentioned before. BCl-2 and Bax protein family play a central role in the regulation of apoptosis. Bax/Bcl-2 ratio can act as a rheostat, which determines the cell susceptibility to apoptosis, cells undergoing apoptosis have higher ratio of Bax/Bcl-2 (
The migration and invasion of FLS plays an important role in synovitis and bone destruction. FLS migrate locally, and can also invade distant areas and joints through the bloodstream. The RA-FLSs secrete MMPs that further aggravate the matrix degradation of cartilage, ultimately resulting in bone erosion.
The migration potential of FLS can be studied by a simple scratch assay and assessing the closure of the wound by FLS migration. The untreated FLS migrate and close the wound while the treated ones the closure is delayed (J Immunol Mar. 1, 2014, 192 (5) 2063-2070). FLS cells (5×104 cells/well) were seeded in 24-well plates, incubated at 37° C. under 5% CO2 to reach a confluence of about 80-90%, and then wounded by scratching with a 200 μl pipette tip. The cells were washed with PBS to ensure the complete removal of debris and floating cells. The cells were then incubated with different concentration of sample in DMEM medium containing 2% FBS. The control sample contained the cells and the culture medium without any sample. The cell migration was assessed by evaluating the monolayer gap closure using Magvision software. The percentage inhibition of wound closure was calculated in comparison to the control wells. Amongst the curcuminoids each at 15 μg/mL, BDMC (
The 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 titers of antibody specific for both the immunogen (heterologous CII) and the autoantigen (mouse or rat CII).
Arthritis induced by administering type-2 collagen (chicken sternal cartilage, Sigma catalog no: C9301) which will be emulsified with incomplete Freund's adjuvant (FIA) at 1:1 ratio. On Day 0, Rats were intradermally injected with 200 μL (200 μg/animal) of collagen-FIA emulsion at the base of the tail (First Immunization). For booster injection the emulsion was prepared same as above and injected with 100 μL (100 μg/animal) of collagen-FIA emulsion given on day 7. Test sample administered from day 0 to day 20.
A standardized method of arthritis scoring was used to evaluate the degree of swelling and erythema of all four paws 0—No sign; 1—Redness without edema; 2—Redness with mild edema; 3—Redness with severe edema; 4—Redness, severe edema and stiffness in movement. The combination of AC3 and BPS(
Spleen was obtained from collagen induced arthritis rats under sterile conditions for the lymphocyte isolation. The spleen was washed twice with PBS containing 100 μg/ml streptomycin and 100 U/ml penicillin and cut into small pieces with microsurgery scissors, homogenized with plunger end of the syringe and filtered using a 100 μm mesh strainer. The filtrate was centrifuged for 5 min at 1200 rpm, washed 1-2 times with PBS, after which 1×106 cells/ml were suspended in RPMI media supplemented with 10% FBS. The imbalance is effected by reducing Th17 cells and increasing T-cell (Treg) (
Flow Cytometry (FCM) Analysis:
To assess Th17 and Treg cells, the isolated lymphocytes were stimulated for 4 h with 20 ng/mL phorbol myristate acetate (P8139, Sigma) and 500 ng/mL ionomycin along with BD GolgiStop, a Protein Transport Inhibitor containing Monensin (554724, BD) at a concentration as suggested by the manufacturer.
After the stimulation, cells were collected and 2×105 cells were aliquoted into different tubes for evaluating Th17 and Treg population. The antibody dilutions used for the analysis were as recommended by the respective manufacturer. To evaluate Th17 population, cells were initially stained with FITC Mouse Anti-Rat CD3 (559975, BD pharmingen) and APC Mouse Anti-Rat CD4 (550057, BD pharmingen) antibodies. To evaluate Treg population, cells were initially stained with APC Mouse Anti-Rat CD4 (550057, BD pharmingen) and BV421 Mouse Anti-Rat CD25 (565608, BD pharmingen) antibodies. Subsequent to the addition of respective surface staining antibodies, tubes were incubated for 30 min at 4° C. in the dark. After surface staining, the cells were fixed and permeabilized using fixation and permeabilization solution (BD Cytofix/Cytoperm, 554722) as per the manufacturer's instructions and resuspended in a 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 min at 4° C. in the dark. Cells were then washed two times with saponin containing buffer and resuspended in a staining buffer prior to flow cytometric analysis. FCM was performed on a BD FACSCelesta system (BD Biosciences, Franklin Lakes, N.J., USA) and analyzed using FlowJo software.
The composition is formulated along with pharmaceutically/nutraceutically acceptable excipients, adjuvants, diluents, stabilizing agents, dispersible gums, bioavailability enhancers or carriers and administered orally in the form of tablets, capsules, syrups, gummies, powders, suspensions, emulsions, chewables, candies or eatables.
In a related aspect the bioavailability enhancer is selected from the group of piperine (BioPerine®), quercetin, garlic extract, ginger extract, and naringin. In another related aspect, the stabilizing agent is selected from the group consisting rosmarinic acid, butylated hydroxyanisole, butylated hydroxytoluene, sodium metabisulfite, propyl gallate, cysteine, ascorbic acid and tocopherols. In yet another related aspect, the dispersible gums are 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 nutraceutical formulations containing bisdemethoxycurcumin
The above formulations are merely illustrative examples, any formulation containing the above active ingredient intended for the said purpose will be considered equivalent.
Other modifications and variations of the invention will be apparent to those skilled in the art from the foregoing disclosure and teachings. Thus, while only certain embodiments of the 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 and is to be interpreted only in conjunction with the appended claims.
This is a non-provisional application claiming priority from U.S. provisional application No. 63/126,920 filed on 17 Dec. 2020, the contents of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 63126920 | Dec 2020 | US |
