COMPOSITIONS FOR THE MANAGEMENT OF DEMYELINATING DISORDERS

Abstract

The invention discloses a composition and method for therapeutically managing a demyelinated disorder through remyelination. The invention includes a composition comprising Withaferin A and Bisdemethoxycurcumin (BDMC), and a pharmaceutically or nutraceutically acceptable excipients, which can be effectively formulated as tablets and capsules. The invention also covers use of composition for effectively managing a demyelinated disorder through remyelination.

Claims

1. A composition comprising Withaferin A and Bisdemethoxycurcumin (BDMC).

2. The composition as claimed in claim 1, wherein Withaferin A and BDMC are in 3:1 w/w: 1-3 w/w.

3. The composition as claimed in claim 1, the composition further comprises stabilizing agents, bioavailability enhancers and antioxidants, pharmaceutically or nutraceutically or cosmeceutically accepted excipients and enhancers, administered orally in the form of tablets, capsules, syrups, gummies, powders, suspensions, emulsions, chewables, candies or eatables.

4. A method for therapeutic management of a demyelinating disorder in a mammal, said method comprising steps of: (a) identifying a mammal showing symptoms of demyelination; and (b) administering to the mammal, an effective dose of a composition comprising Bisdemethoxycurcumin (BDMC), to bring about the effect of promoting remyelination.

5. The method as in claim 4, wherein the composition further comprises withaferin A.

6. The method as in claim 4, wherein the effective dose of BDMC and withaferin A is individually selected from 3 mg/kg to 50 mg/kg bodyweight individually of the said mammal.

7. The method as in claim 4, wherein the total effective dose of the composition is selected from 35 to 40 mg/kg bodyweight, wherein the dose of Withaferin A is 25 mg/kg and BDMC is 12.5 mg/kg.

8. The method as in claim 4, wherein the demyelinating disorder is selected from the group consisting of Multiple Sclerosis, Acute Disseminated Encephalomyelitis (ADEM), Balo's Disease (Concentric Sclerosis), Charcot-Marie-Tooth Disease (CMT), Guillain-Barre Syndrome (GBS), HTLV-I Associated Myelopathy (HAM), Neuromyelitis Optica (Devic's Disease), Schilder's disease, and Transverse Myelitis.

9. The method as in claim 4, wherein management of the demyelinating disorder in the mammal is brought about by promoting remyelination, by increasing proliferation of glycoproteins involved in myelin formation, upregulation of growth factors, down regulation of inflammatory markers and increasing neuromuscular coordination.

10. The method as in claim 9, wherein the glycoproteins are selected from the group consisting of Myelin basic protein (MBP) and Myelin Oligodendrocyte Glycoprotein (MOG).

11. The method as in claim 9, wherein the growth factors are selected from the group consisting of Platelet Derived Growth Factor Receptor alpha (PDGFRa) and Chondroitin Sulfate Proteoglycan 4 (NG2).

12. The method as in claim 9, wherein the inflammatory markers are selected from the group consisting of Transient receptor potential ankyrin 1 (TRPA1), Amphoterin-induced protein 3 (AMIGO3), C-reactive Protein (CRP), and CXC chemokine receptor 2 (CXCR2).

13. The method as in claim 9, wherein the neuro-muscular coordination is selected from the group consisting of grip strength, motor coordination, learning and locomotor activity.

14. The method as in claim 4, wherein the composition further comprises stabilizing agents, bioavailability enhancers and antioxidants, pharmaceutically or nutraceutically or cosmeceutically accepted excipients and enhancers and administered orally in the form of tablets, capsules, syrups, gummies, powders, suspensions, emulsions, chewables, candies or eatables.

15. A method of promoting remyelination of a mammalian neuron, said method comprising: bringing into contact an effective dose of a composition comprising Bisdemethoxycurcumin (BDMC) to the said mammalian neuron, showing characteristic of demyelination, to bring about the effect of promoting remyelination.

16. The method as in claim 15, wherein the neuron is selected from the group consisting of central and peripheral nervous system.

17. The method as in claim 15, wherein the composition further comprises withaferin A.

18. The method as in claim 15, wherein the effective dose of BDMC and withaferin A is individually selected from 3 mg/kg to 50 mg/kg individually of the said neuron.

19. The method as in claim 15, wherein the total effective dose of the composition is selected from 35 to 40 mg/kg of the said neuron, wherein the dose of Withaferin A is 25 mg/kg and BDMC is 12.5 mg/kg.

20. The method as in claim 15, wherein promoting remyelination is effective in management of demyelinating disorders are selected from the group consisting of Multiple Sclerosis, Acute Disseminated Encephalomyelitis (ADEM), Balo's Disease (Concentric Sclerosis), Charcot-Marie-Tooth Disease (CMT), Guillain-Barre Syndrome (GBS), HTLV-I Associated Myelopathy (HAM), Neuromyelitis Optica (Devic's Disease), Schilder's disease, and Transverse Myelitis.

21. The method as in claim 15, wherein remyelination is promoted by increasing proliferation of glycoproteins involved in myelin formation, upregulation of growth factors, down regulation of inflammatory markers and increasing neuromuscular coordination.

22. The method as in claim 21, wherein the glycoproteins are selected from the group consisting of Myelin basic protein (MBP) and Myelin Oligodendrocyte Glycoprotein (MOG).

23. The method as in claim 21, wherein the growth factors are selected from the group consisting of Platelet Derived Growth Factor Receptor alpha (PDGFRa) and Chondroitin Sulfate Proteoglycan 4 (NG2).

24. The method as in claim 21, wherein the inflammatory markers are selected from the group consisting of Transient receptor potential ankyrin 1 (TRPA1), Amphoterin-induced protein 3 (AMIGO3), C-reactive Protein (CRP), and CXC chemokine receptor 2 (CXCR2).

25. The method as in claim 21, wherein the neuro-muscular coordination are selected from the group consisting of grip strength, motor coordination, learning and locomotor activity.

26. The method as in claim 15, wherein the composition further comprises stabilizing agents, bioavailability enhancers and antioxidants, pharmaceutically or nutraceutically or cosmeceutically accepted excipients and enhancers and administered orally in the form of tablets, capsules, syrups, gummies, powders, suspensions, emulsions, chewables, candies or eatables.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0030] 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.

[0031] FIG. 1 is a dose response curve of Withaferin A and BDMC, for measure of Myelin Oligodendrocyte Glycoprotein (MOG).

[0032] FIG. 2 is a dose response curve Withaferin A and BDMC, for measure of Brain Derived Neurotrophic Factor (BDNF).

[0033] FIG. 3 shows measure of proliferation of Glycoprotein biomarker, Myelin basic protein (MBP), in the presence of Withaferin A, BDMC and combination thereof. Values are expressed as Mean±S.E.Number of animals in each group: 10 p value: *<0.01; **<0.001

[0034] FIG. 4 shows upregulation of growth factors biomarker, Platelet Derived Growth Factor Receptor alpha (PDGFRa), Values are expressed as Mean±S.E. Number of animals in each group: 10, p value: *<0.01; **<0.001 and FIG. 5 shows upregulation of growth factors biomarker Chondroitin Sulfate Proteoglycan 4 (NG2), in the presence of Withaferin A, BDMC and combination thereof. Values are expressed as Mean±S. E. Number of animals in each group: 10, p value: *<0.01:

[0035] FIG. 6 shows downregulation of inflammatory biomarker, Transient receptor potential ankyrin 1 (TRPA1), (Values are expressed as Mean±S.E. Number of animals in each group: 10, p value: *<0.01)

[0036] FIG. 7 shows downregulation of inflammatory biomarker, Amphoterin-induced protein 3 (AMIGO3)(Values are expressed as Mean±S.E. Number of animals in each group: 10 p value: *<0.05: **<0.01; **<0.001), FIG. 8 shows downregulation of inflammatory biomarker, C-reactive Protein (CRP)(Values are expressed as Mean±S.E. Number of animals in each group: 10 p value: *<0.01: *<0.001), and FIG. 9 shows downregulation of inflammatory biomarker CXC chemokine receptor 2(CXCR2) in the presence of Withaferin A, BDMC and combination thereof (Values are expressed as Mean±S.E. Number of animals in each group: 10 p value: *<0.05; **<0.01; ***<0.001).

[0037] FIG. 10 shows effect of in Withaferin A, BDMC and combination thereof neuromuscular coordination, grip strength. (Values are expressed as Mean±S.E. Number of animals in each group: 10, p value: *<0.05.

[0038] Immunohistochemistry of cortical layer of Normal control (FIG. 11), demyelinated control (FIG. 12), in the presence of Withaferin A (FIG. 13), BDMC (FIG. 14) and the combinations thereof (FIG. 15).

DESCRIPTION OF PREFERRED EMBODIMENTS

Selected Definitions

[0039] 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.

[0040] Withaferin A used in this invention can be isolated from Solanaceae family, Physalis longifolia, Vassobia breviflora, and Withania somnifera. Bisdemethoxycurcumin (BDMC) used in this invention can be isolated from Curcuma longa.

[0041] Therapeutically managing or management refers to a condition of effectively ameliorating disorders disclosed in the invention. An effective dose refers to positive or modulatory effects of remyelination in mammals covered under this invention.

[0042] The invention covers a composition comprising Withaferin A and Bisdemethoxycurcumin (BDMC), wherein Withaferin A and BDMC are in 3:1 w/w. The invention further covers a method, use of composition for treating demyelinating disorders in a mammal and a mammalian neuron comprising Withaferin A and at Bisdemethoxycurcumin (BDMC).

[0043] In the most preferred embodiment, the invention discloses a composition comprising Withaferin A and Bisdemethoxycurcumin (BDMC). In a related embodiment, wherein Withaferin A and BDMC are in 1:1 w/w, or more preferably 1:2 w/w, or more preferably 1:3 w/w, or more preferably 2:1 w/w, or most preferably 3:1 w/w.

[0044] In a related aspect of this and other embodiments of the invention, the composition further comprises stabilizing agents, bioavailability enhancers and antioxidants, pharmaceutically or nutraceutically or cosmeceutically accepted excipients and enhancers, administered orally in the form of tablets, capsules, syrups, gummies, powders, suspensions, emulsions, chewables, candies or eatables.

[0045] In another most preferred embodiment of the invention, the invention discloses a method for therapeutic management of a demyelinating disorder in a mammal, said method comprising steps of: [0046] (a) identifying a mammal showing symptoms of demyelination; and [0047] (b) administering to the mammal, an effective dose of a composition comprising Bisdemethoxycurcumin (BDMC), to bring about the effect of promoting remyelination.

[0048] In a related aspect of this and other embodiments of the invention, wherein the composition further comprises withaferin A. In a related aspect of this and other embodiments of the invention, the effective dose of withaferin A or BDMC is individually selected from 3 mg/kg to 55 mg/kg of bodyweight of a mammal, more preferably between 10 to 30 mg/kg, and most preferably between 10 to 25 mg/kg of the said mammal. In a related aspect of this and other embodiments of the invention the total effective dose of the composition is selected from 30 to 40 mg/kg bodyweight, and most preferably between 35 to 40 mg/kg bodyweight of the said mammal. In further aspect of this and other embodiments, wherein the dose of Withaferin A is 10 mg/kg and BDMC is 27.5 mg/kg in the total composition of the said mammal, or more preferably the dose of Withaferin A is 12.5 mg/kg and BDMC is 25 mg/kg in the total composition of the said mammal, or more preferably the dose of Withaferin A is 15 mg/kg and BDMC is 12.5 mg/kg in the total composition of the said mammal, or more preferably the dose of Withaferin A is 20 mg/kg and BDMC is 17.5 mg/kg in the total composition of the said mammal, or more preferably the dose of Withaferin A is 17.5 mg/kg and BDMC is 20 mg/kg in the total composition of the said mammal, or more preferably the dose of Withaferin A is 20 mg/kg and BDMC is 20 mg/kg in the total composition of the said mammal, or most preferably the dose of Withaferin A is 25 mg/kg and BDMC is 12.5 mg/kg in the total composition of the said mammal.

[0049] In a related aspect of this and other embodiments of the invention, Withaferin A was isolated from the source, using alcohol as the solvent and concentrating the extract after multiple extractions from the alcohol. Further, treating the concentrated extract with water, and extracting the aqueous layer with organic solvents, non-polar and polar, to yield Withaferin A.

[0050] In a related aspect of this and other embodiments of the invention, the total curcuminoids in the composition are in the range of not less than 20% w/w. In a further aspect, the composition is isolated and formulated using a process well known in scientific literature:

[0051] Further aspect of this and other embodiments of the invention, wherein demyelinating disorders are selected from the group consisting of multiple sclerosis, Acute Disseminated Encephalomyelitis (ADEM), Balo's Disease (Concentric Sclerosis), Charcot-Marie-Tooth Disease (CMT), Guillain-Barre Syndrome (GBS), HTLV-I Associated Myelopathy (HAM), Neuromyelitis Optica (Devic's Disease), Schilder's disease, and Transverse Myelitis. In further aspect of this and other embodiments of the invention, demyelinating disorder is preferably Acute Disseminated Encephalomyelitis (ADEM), or preferably Balo's Disease (Concentric Sclerosis), or preferably Charcot-Marie-Tooth Disease (CMT), or preferably Guillain-Barre Syndrome (GBS), or preferably HTLV-I Associated Myelopathy (HAM), or preferably Neuromyelitis Optica (Devic's Disease), or preferably Schilder's disease, or preferably Transverse Myelitis or most preferably Multiple Sclerosis (MS). In a related aspect of this and other embodiments of the invention, wherein the cause of demyelination is selected from the group consisting of anti-inflammatory, auto-immune, metabolic, damage to blood vessels in the brain, viral infections, and chemically induced. In a related aspect of this and other embodiments of the invention, the symptoms of demyelination disorders are selected from the group consisting of vision loss, lack of neuromuscular coordination, muscle weakness, non-rhythmic heartbeat or palpitations, loss of bladder and bowel control, sensory changes, numbness, loss of reflexes, poorly controlled blood pressure, dizziness, memory problems, pain, and fatigue.

[0052] In further aspect of this and other embodiments of the invention, wherein management of the demyelinating disorder in the mammal is brought about by promoting remyelination, by increasing proliferation of glycoproteins involved in myelin formation, upregulation of growth factors, down regulation of inflammatory markers and increasing neuromuscular coordination.

[0053] In related aspect to this and other related embodiments of the invention, wherein glycoproteins are selected from the group consisting of Myelin basic protein (MBP) and Myelin Oligodendrocyte Glycoprotein (MOG). Further, in this and other related aspect of this embodiment, Withaferin A or BDMC resulted in proliferation of MOG by at least 125%, preferably by 150%, or preferably by 175%, or more preferably by 188%, or most preferably by 225% (FIG. 1). Further, in this and other related aspect of this embodiment, Withaferin A or BDMC resulted in proliferation of Myelin basic protein (MBP) by at least 50%, or most preferably by 60%, or more preferably by 100%. The combination of Withaferin A and BDMC resulted in proliferation of MBP by at least 100%, or most preferably by 175%, compared to the demyelination control (FIG. 3).

[0054] In related aspect to this and other related embodiments of the invention, wherein the growth factors are selected from the group consisting of Platelet Derived Growth Factor Receptor alpha (PDGFRa) and Chondroitin Sulfate Proteoglycan 4 (NG2). Further, in this and other related aspect of this embodiment, Withaferin A or BDMC resulted in upregulation of PDGFRa by at least 20%, or by at least 30%, or more preferably by at least 50% or most preferably by 70%. The combination of Withaferin A and BDMC resulted in upregulation of PDGFRa by at least 50%, or more preferably by at least 100%, or most preferably by at least 125%, compared to the demyelination control (FIG. 4). Further, in this and other related embodiments of the invention, Withaferin A or BDMC resulted in upregulation of NG2 by at least by 30%, or more preferably by at least 50% or most preferably by 90%. The combination of Withaferin A and BDMC resulted in upregulation of NG2 by at least 50%, or more preferably by at least 125%, or most preferably by at least 150% (FIG. 5).

[0055] In related aspect to this and other related embodiments of the invention, wherein the inflammatory markers are selected from the group consisting of Transient receptor potential ankyrin 1 (TRPA1), Amphoterin-induced protein 3 (AMIGO3), C-reactive Protein (CRP), and CXC chemokine receptor 2 (CXCR2). Further in this and other related aspect of the embodiment of this invention, Withaferin A or BDMC resulted in down regulation of Transient receptor potential ankyrin 1 (TRPA1) inflammatory marker, by at least 15%, or by at least 20%, or more preferably between 40 to 50%. The combination of Withaferin A and BDMC resulted in down regulation of Transient receptor potential ankyrin 1 (TRPA1) inflammatory marker, by at least 25%, or by at least 30%, or most preferably by 45% (FIG. 6). Further in this and other related aspect of the embodiment of this invention, Withaferin A or BDMC resulted in down regulation of Amphoterin-induced protein 3 (AMIGO3) inflammatory marker by at least 25%, or by at least 30%, or more preferably by 35%. The combination of Withaferin A and BDMC resulted in down regulation of AMIGO3 inflammatory marker by at least 30%, and most preferably by 40% (FIG. 7). Further in this and other related aspect of the embodiment of this invention, Withaferin A or BDMC resulted in down regulation of C-reactive Protein (CRP) inflammatory marker by at least 30%, or most preferably by 35%. The combination of Withaferin A and BDMC resulted in down regulation of CRP inflammatory marker by at least 50%, or most preferably by 60% (FIG. 8). Further in this and other related aspect of the embodiment of this invention, Withaferin A or BDMC resulted in down regulation of CXC chemokine receptor 2 (CXCR2) inflammatory marker by at least 15%, or more preferably by 20%, or most preferably by at least 25%. The combination of Withaferin A and BDMC resulted in down regulation of CXCR2 inflammatory marker by at least 50%, or preferably by at least 55%, or most preferably by 65% (FIG. 9).

[0056] In related aspect to this and other related embodiments of the invention, wherein neuromuscular coordination is selected from the group consisting of grip strength, motor coordination, learning and locomotor activity. Further in this and other related aspect of the embodiment of this invention, Withaferin A or BDMC resulted in improved grip strength by at least 20%, or more preferably by at least 25%, or most preferably by 30%. The combination of Withaferin A and BDMC resulted in improved grip strength by at least 50%, or most preferably by 55% (FIG. 10). Further in this and other related aspect of the embodiment of this invention, Withaferin A or BDMC resulted in improved motor co-ordination in 5 out of 10 mammals, whereas the combination of Withaferin A and BDMC resulted in improved motor co-ordination in 7 out of 10 mammals, compared to 3 out of 10 in demyelinated control mammals. Further in this and other related aspect of the embodiment of this invention, Withaferin A showed 30 to 40% improvement in the spontaneous or induced or locomotor activity compared to the demyelinated control, BDMC showed 25 to 30% improvement, and the combination showed 60 to 65% improvement compared to the demyelinated control.

[0057] In another most preferred embodiment of the invention, a method for therapeutic management of a demyelinating disorder in a mammal, said method comprising steps of: [0058] (a) identifying a mammal showing symptoms of demyelination; and [0059] (b) administering to the mammal, an effective dose of a composition comprising Bisdemethoxycurcumin (BDMC), to bring about the effect of promoting remyelination.

[0060] In related aspect of this and other embodiments of the invention, wherein the composition further comprises withaferin A. The concentration range of doses, choice of mammals, selection of demyelination disorders, markers associated with promotion of remyelination, formulation for administration, explained in the previous embodiments are applicable to this embodiment

[0061] In yet another most preferred embodiment, the invention discloses Use of a composition comprising Bisdemethoxycurcumin (BDMC) for therapeutic management of a demyelinating disorder in a mammal.

[0062] In related aspect of this and other embodiments of the invention, wherein the composition further comprises withaferin A. The concentration range of doses, choice of mammals, selection of demyelination disorders, markers associated with promotion of remyelination, formulation for administration, explained in the previous embodiments are applicable to this embodiment

[0063] In yet another most preferred embodiment of the invention discloses a method of promoting remyelination of a mammalian neuron, said method comprising: bringing into contact an effective dose of a composition comprising Bisdemethoxycurcumin (BDMC) to the said mammalian neuron, showing characteristic of demyelination, to bring about the effect of promoting remyelination. In further aspect of this and other embodiments of the invention embodiment, wherein the composition further comprises withaferin A. The concentration range of doses, choice of mammals, selection of demyelination disorders, markers associated with promotion of remyelination, formulation for administration, explained in the previous embodiments are applicable to this embodiment

[0064] In yet another most preferred embodiment of the invention discloses use of a composition comprising Bisdemethoxycurcumin (BDMC) for promoting remyelination of a mammalian neuron. In further aspect of this and other embodiments of the invention embodiment, wherein the composition further comprises withaferin A. The concentration range of doses, choice of mammals, selection of demyelination disorders, markers associated with promotion of remyelination, formulation for administration, explained in the previous embodiments are applicable to this embodiment.

[0065] The preferred embodiments of the invention are further described in the following illustrative examples.

EXAMPLES

[0066] Combination of Cuprizone and Rapamycin-Mediated

Demyelination/Remyelination

[0067] The Eight-week-old male C56BL/6 J mice were selected and randomized into ten animals in each group (Table 1). Demyelination was induced by 0.3% cuprizone in combination with rapamycin (Hilary H S, Kathryn K B, Daniela C P, Priya N, Wendy B M. A New Model of Cuprizone-Mediated Demyelination/Remyelination. Am Society Neurochem. 2014: 1-16). Briefly, groups undergo demyelination by feeding on a diet containing 0.3% cuprizone mixed into Rodent Diet. These mice received additionally intra-peritoneal rapamycin injections 5 days a week with a vehicle solution.

[0068] After 8 weeks of treatment, all cuprizone feedings and all injections of rapamycin was discontinued. The animals were subjected to behavioral analysis and at end of study, brain samples and serum samples were collected for estimation of Molecular and protein biomarkers by ELISA and Immunohistochemistry of brain was assessed for Demyelination/Remyelination.

TABLE-US-00001 TABLE 1 Animal groups for study Groups Number of Animals Group I- Normal Control 10 Group II- Demyelinated Control 10 Group III- Withaferin A 10 Group IV- BDMC 10 Group V- Withaferin A + BDMC 10

[0069] Dose Response Study

[0070] Brain-derived neurotrophic factor (BDNF) plays a role in both normal developmental myelination and in remyelination after some forms of demyelinating injury through regulation of proliferation and differentiation of oligodendrocyte proliferation cells (OPCs). A large body of neuropathological, experimental and clinical evidence shows that BDNF may play an important role in neuroinflammation modulation, neuroprotection and neurorepair. Myelin oligodendrocyte glycoprotein (MOG) is a glycoprotein important in the myelination of nerves in the central nervous system (CNS). In humans this protein is encoded by the MOG gene. It serves as a necessary “adhesion molecule” to provide structural integrity to the myelin sheath.

[0071] Withaferin A or BDMC were administered orally once per day to mouse, for 8 weeks after treatment with Cuprizone and Rapamycin (Table 1). The dose response of BDMC or Withaferin was assessed by measuring concentration of biomarkers, Myelin Oligodendrocyte Glycoprotein (MOG) (FIG. 1) Brain Derived Neurotrophic Factor (BDNF) (FIG. 2), in serum. This was used as a measure to find the optimum effective dose of Withaferin A (25 mg/kg) and BDMC (12.5 mg/kg) and effectively, the total concentration of 37.5 mg/kg (Withaferin A (25 mg/kg)+BDMC (12.5 mg/kg) was preferred for testing potencies for remyelination in assays. It must be noted that these concentrations are by no means a limitation rather embodiments cover the scope of concentration ranges.

[0072] Brain Tissue Preparation for Estimation of Molecular Protein Markers

[0073] Before homogenization for each assay (Magari, K., Miyata, S., Ohkubo, Y., Mutoh, S., 2004. Inflammatory cytokine levels in paw tissues during development of rat collagen-induced arthritis: effect of FK506, an inhibitor of T cell activation. Inflamm. Res. 53, 469-474), the brain of the animals was removed and rinsed with ice-cold isotonic saline and were added to 4 ml/g tissue of extraction buffer containing 1 mM phenyl methyl sulfonyl fluoride, 1 mg/ml aprotinin, and 0.05% Tween 20 in phosphate buffered saline. Tissues were homogenized on ice with a polytron, and homogenate was centrifuged at 5000 g for 15 min. Aliquots of the supernatant were separated and used for biochemical analysis. Supernatants were stored at −80° C. until cytokine analysis.

[0074] Estimation of Biomarkers in Brain Tissue Homogenate

[0075] The supernatant from brain tissue homogenate were separated and used for biomarker analysis. Myelin Oligodendrocyte Glycoprotein (FIG. 1), Platelet derived growth factor receptor alpha (FIG. 4), chondroitin sulfate proteoglycan 4 (FIG. 5), Myelin basic protein (FIG. 3), Transient receptor potential ankyrin 1 (FIG. 6), Amphoterin-induced protein 3 (FIG. 7), C-reactive protein (FIG. 8) and CXC chemokine receptor 2 (FIG. 9), were estimated using commercially available kits based on sandwich and competitive ELISA technique according to the manufacturers' instructions. All cytokine concentrations were carried out by means of colorimetric measurement at 450 nm on an ELISA plate reader by interpolation from a standard curve.

[0076] Proliferation of Glycoproteins:

[0077] Myelin oligodendrocyte glycoprotein (MOG) is a glycoprotein important in the myelination of nerves in the central nervous system (CNS). In humans this protein is encoded by the MOG gene. It serves as a necessary “adhesion molecule” to provide structural integrity to the myelin sheath. Differentiation of OPCs into pre-myelinating oligodendrocytes is associated with an increase in cellular complexity and the expression of myelin associated proteins and lipids, including myelin basic protein (MBP), myelin associated glycoprotein (MOG). During this stage, cells are highly susceptible to the induction of cell death, as 50% of the pre-myelinating oligodendrocytes undergo apoptotic cell death. If pre-myelinating oligodendrocytes each associate with an appropriate population of axons they mature into myelinating oligodendrocytes. Animal groups carrying Withaferin A (25 mg/kg) or BDMC (12.5 mg/kg) resulted in proliferation of Myelin basic protein (MBP) by 64 and 122% respectively. The animal group with combination (37.5 mg/kg) of Withaferin A and BDMC resulted in proliferation of Myelin basic protein (MBP) by 176%, compared to the demyelination control (FIG. 3), which is in the absence of Withaferin A or BDMC or the combination.

[0078] Upregulation of Growth Factors:

[0079] At the early stages of oligodendrocyte lineage commitment, oligodendrocyte precursor cell (OPCs) expresses a characteristic molecular phenotype that includes expression of the platelet derived growth factor receptor alpha(PDGFRa). OPCs are highly proliferative in response to growth factors PDGFa and fibroblast growth factor (FGF) and disperse widely throughout the developing CNS. Animal groups carrying Withaferin A (25 mg/kg) or BDMC (12.5 mg/kg) resulted in upregulation of PDGFRa by 22 and 73% respectively. The animal group with combination (37.5 mg/kg) of Withaferin A and BDMC resulted in upregulation of PDGFRa by 138%, compared to the demyelination control (FIG. 4). Similarly, during developmental stages oligodendrocyte precursor cells expressing the proteoglycan Chondroitin Sulfate Proteoglycan 4 (NG2) are expressed in the adult CNS, indicating remyelination in demyelinating diseases. NG2 was upregulated by 30 and 90% for Withaferin A (25 mg/kg) or BDMC (12.5 mg/kg) respectively. The combination (37.5 mg/kg) of Withaferin A and BDMC resulted in upregulation of PDGFRa by 159%, compared to the demyelination control (FIG. 5), which is in the absence of Withaferin A or BDMC or the combination.

[0080] Downregulation of Inflammatory Biomarkers:

[0081] Transient receptor potential ankyrin 1 (TRPA1) is a non-selective cation channel that plays a role in inflammatory pain sensation and nociception and a potential regulator in emotion, cognition and social behaviour. It is involved in the myelin Ca2+ accumulation process activating NADPH oxidase 1 (NOX1), which acts by releasing oxidant molecules and increasing their expression, to produce more ROS, eventually exacerbating oxidative stress. Inhibition of TRPA1 receptors might successfully diminish the degenerative pathology in multiple sclerosis and could be a promising therapeutic target to limit central nervous system damage in demyelinating diseases. Animal groups carrying withaferin A (25 mg/kg) or BDMC (12.5 mg/kg) resulted in down regulation of Transient receptor potential ankyrin 1 (TRPA1) inflammatory marker, by 43% and 17% respectively. The animal group with combination (37.5 mg/kg) of Withaferin A and BDMC resulted in down regulation of (TRPA1) inflammatory marker, by 46% compared to the demyelination control (FIG. 6), which is in the absence of Withaferin A or BDMC or the combination.

[0082] AMIGO3 exerts inhibitory effect on the axonal growth of central nervous system neurons. The increase in spinal cord AMIGO3 expression in MS suggests that this protein plays a role in the acute stages of oligodendrocyte injury. In this context, therapies targeted against AMIGO3 provides benefit in terms of promoting OPC differentiation and remyelination in the early stages of demyelinating diseases (Neural Regeneration Research 2017 August; 12(8): 1247-1251). Animal groups carrying Withaferin A (25 mg/kg) or BDMC (12.5 mg/kg) resulted in down regulation of Amphoterin-induced protein 3 (AMIGO3) inflammatory marker by 36% and 27% respectively. The animal group with combination (37.5 mg/kg) of Withaferin A and BDMC resulted in down regulation of AMIGO3 by 43%, compared to the demyelination control (FIG. 7), which is in the absence of Withaferin A or BDMC or the combination.

[0083] The serum components which are acute phase reactants that become elevated during inflammation and tissue destruction, C-reactive protein was measured in patients with multiple sclerosis (MS) hospitalized with acute exacerbations. Significantly elevated levels of CRP was found in 12 of 13 patients with clinically active MS. Serial studies in two patients revealed that clinical improvement was accompanied by a decline in the serum levels of CRP. These findings suggest that measurement of these serum proteins may be of value in assessing progress of disease activity in MS patients (P C Dowling, S D Cook, Disease markers in acute multiple sclerosis, 1976 October; 33(10):668-7). Animal groups carrying Withaferin A (25 mg/kg) or BDMC (12.5 mg/kg) resulted in down regulation of C-reactive Protein (CRP) inflammatory marker by 32 and 35% respectively. The animal group with combination (37.5 mg/kg) of Withaferin A and BDMC resulted in down regulation of CRP inflammatory marker by 61%, compared to the demyelination control (FIG. 8), which is in the absence of Withaferin A or BDMC or the combination.

[0084] CXCR2 is the inflammation-inducible receptor of CXCL 5 and CXCL 8 expressed by human brain endothelium. Chemokines ligand (CXCL) 5 and 8 are overexpressed in patients with multiple sclerosis. CXCR2 is weakly expressed in resting brain endothelium but highly inducible by inflammatory stimuli. CXCR2 is closely involved in the chemotaxis of neutrophils to sites of injury through high-affinity binding with a gradient of its cognate ligands. Indeed, several preclinical mouse models of MS have shown that CXCR2-mediated recruitment of neutrophils influences the severity of demyelination (Carlson et al., 2008; Simmons et al., 2014; Liu el al., 2015a; Marro el al., 2016, Gris el al., 2018). Animal groups carrying Withaferin A (25 mg/kg) or BDMC (12.5 mg/kg) resulted in down regulation of CXC chemokine receptor 2 (CXCR2) inflammatory marker by 32 and 35% respectively. The animal group with combination (37.5 mg/kg) of Withaferin A and BDMC resulted in down regulation of CXC chemokine receptor 2 (CXCR2) inflammatory marker by at least 50%, or preferably by at least 55%, or most preferably by 65%, compared to the demyelination control (FIG. 9), which is in the absence of Withaferin A or BDMC or the combination.

[0085] Estimation of Biomarkers in Serum:

[0086] Blood was collected from the retro-orbital plexus of the experimental animals, no anti-coagulant was added to the blood, and it was made to stand at room temperature for 1 h. The blood was then centrifuged, and clear serum was separated and stored for analysis.

[0087] Samples from different groups of animals were prepared for the analysis of cytokines and mediators at the termination of the experiment. Brain Derived Neurotrophic Factor (BDNF), C-Reactive Protein were estimated using commercially available kits based on sandwich and competitive ELISA technique according to the manufacturers' instructions. All cytokine concentrations were carried out by means of colorimetric measurement at 450 nm on an ELISA plate reader by interpolation from a standard curve

[0088] Motor Coordination and Learning: Rotarod Test

[0089] Motor coordination and balance were evaluated in a Rotarod apparatus which consists of a motor-driven rotating rod whose speed can be adjusted. All mice were evaluated on the Rotarod three times a day for two consecutive days, with the rotation set at 15 to 16 revolutions per minute (rpm) (Rozas, G.; Guerra, M. J.; Labandeira Garcia, J. L. An automated Rotarod method for quantitative drug-free evaluation of overall motor deficits in rat models of Parkinsonism. Brain Res. Protoc. 1997, 2, 75-84). To test the performance, the mice were placed on the rotating cylinder at an angle of 45 with an initial rotation speed of 16 rpm, and were allowed to run for 60 s. The trial was repeated after 5-10 min. The falls and flips (when the animal hangs on to the cylinder and continues all the way around) were recorded within 60 s of each trial. Animal groups carrying Withaferin A (25 mg/kg) or BDMC (12.5 mg/kg) resulted in improved motor co-ordination in 5 out of 10 mammals, whereas the animal group with combination (37.5 mg/kg) of Withaferin A and BDMC resulted in improved motor co-ordination in 7 out of 10 mammals, compared to 3 out of 10 in demyelinated control mammals (Table 2).

TABLE-US-00002 TABLE 2 Results from Rotarod Test No. of mice which stayed on Treatment rota rod/total no. of animals Normal Control 8/10 Demyelinated Control 3/10 Withaferin 25 mg/kg 5/10 BDMC 12.5 mg/kg 5/10 Withaferin + BDMC 25 + 12.5 mg/kg 7/10

[0090] Grip Strength Test:

[0091] The grip strength test allows for the assessment of neuromuscular functions by determining the maximal peak force developed by a rodent when the operator tries to pull it out of a specially designed grid, available for both the fore and hind limbs. In this context, changes in grip strength peak values of the rodents are interpreted as evidence of motor neurotoxicity. Forelimb strength (g force) was measured with a grip strength tester to detect contralateral paw weakness (Seung Ro Han et al. Differential Expression of miRNAs and Behavioral Change in the Cuprizone-Induced Demyelination Mouse Model. International Journal of Molecular Sciences. 2020, 21, 646). After both forelimbs of the mouse were loosened by pulling the tail, the maximal force was recorded. Each mouse was subjected to three trials of each test, and the mean values (g) were calculated. Animal groups carrying Withaferin A (25 mg/kg) or BDMC (12.5 mg/kg) and animal group with the combination (37.5 mg/kg) of two showed 25%, 37.5%, and 58% improvement in the grip strength respectively compared to the demyelinated control (FIG. 10), which is in the absence of Withaferin A or BDMC or the combination.

[0092] Locomotor Activity Test:

[0093] Actophotometer (Activity cage) is designed to study the spontaneous or induced or locomotor activity in small animals like mice or rats. In this apparatus, the Optical sensors and emitters are present to record the horizontal movements of the animals on a six-digit digital counter display. Each animal was placed in Actophotometer for 5 minutes and basal activity score was recorded for all animals. Each animal was treated with respective drugs and activity score was recorded after 1, 2, and 3 hours. Animal group carrying Withaferin A (25 mg/kg) showed 30 to 40% improvement compared to the demyelinated control, and the animal group with BDMC (12.5 mg/kg) showed 25 to 30% improvement, and the combination (37.5 mg/kg) showed 60 to 65% improvement compared to the demyelinated control (Table 3), which is in the absence of Withaferin A or BDMC or the combination.

TABLE-US-00003 TABLE 3 Results from Locomotor Activity Test, Doses 1 hr after 2 hr after 3 hr after (mg/kg p.o.) treatment treatment treatment Normal Control 342 ± 22.66  330 ± 17.22  310 ± 10.12 Demyelinated 170 ± 20.18  158 ± 14.02  148 ± 14.48 Control Withaferin A  240 ± 17.22** 210 ± 17.23* 200 ± 9.01* 25 mg/kg BDMC 12.5 mg/kg 220 ± 21.11* 200 ± 10.70* 190 ± 7.35* Withaferin A + 275 ± 9.12** 260 ± 8.56**  245 ± 2.35** BDMC 37.5 mg/kg Values are expressed as Mean ± S.E.; Number of animals in each group: 10; p value: *<0.01; **<0.001

[0094] Immunohistochemistry

[0095] For visualization of mature neurons, sections were boiled in citrate buffer solution (10 mM, pH 6.0) for antigen retrieval, followed by 1 h blocking in PBS solution containing 10% goat serum and 0.3% Triton and incubated overnight with glutathione S-transferase Pi (GST-π) antibody (Enzo Life Sciences, Farmingdale, N.Y.) at 4° C. Sections were then rinsed in PBS for washing and further incubated for 3 h in fluorescent secondary antibody (Alexa Fluor Goat anti Rabbit 543, ThermoFisher, 1:200) and to-pro-3 (1:1000, Thermo Fisher, T3605) for nuclear stain (FIG. 7)(Qili Yu, Ryan Hui, Jiyoung Park, Yangyang Huang, Alexander W. Kusnecov et al. Strain differences in cuprizone induced demyelination. Cell Biosci (2017) 7:59). Normal control showed architecture of neurons with intact myelin fibers. The cortex of cerebrum showed no remarkable changes with respect to neurons in the normal control (FIG. 11), whereas the demyelinated control (FIG. 12) showed degenerated Purkinje cells with pyknotic cell bodies and inconspicuous dendritic processes with multifocal vacuolations, demyelination and focal purkinje cell degeneration. In contrast, Withaferin A (25 mg/kg, FIG. 13) showed the histoarchitectural presentation of the cerebellar cortical layer, which showed few degenerated Purkinje cells with pyknotic cell bodies and inconspicuous dendritic processes, whereas BDMC (12.5 mg/kg, FIG. 14) showed Purkinje cells with the Purkinje layer to be better stained with fewer degenerative properties when compared to the untreated animals, the given tissue also showed multifocal neuronal degeneration with moderate demyelination. The combination of Withaferin A+BDMC (37.5 mg/kg, FIG. 15) showed significantly lesser degenerated Purkinje cells and the granular layer was less dense than that of the demyelinated control slides and also showed for remyelination in certain areas and proliferation and granular cell layer.

[0096] Formulations Containing Withaferin a and BDMC

[0097] 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.

[0098] 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.

[0099] Tables 4-8 provide illustrative examples of nutraceutical formulations containing bisdemethoxycurcumin

TABLE-US-00004 TABLE 4 Tablet Active Ingredients Withaferin A Bisdemethoxycurcumin (BDMC) Excipients Microcrystalline cellulose, Colloidal silicon dioxide, Magnesium stearate, BioPerine ®, Polyvinylpyrrolidone/starch/Hydroxy propyl methyl cellulose, Hydroxy propyl cellulose

TABLE-US-00005 TABLE 5 Capsule Active Ingredients Withaferin A Bisdemethoxycurcumin (BDMC) Excipients Microcrystalline cellulose, BioPerine ®

TABLE-US-00006 TABLE 6 Powder Active Ingredients Withaferin A Bisdemethoxycurcumin (BDMC) Excipients BioPerine ®,

TABLE-US-00007 TABLE 7 Gummy formulation Active Ingredients Withaferin A Bisdemethoxycurcumin (BDMC) Excipients BioPerine ®, Gelatin (270 Bloom Mesh 10), Refined Sugar, Glucose Corn Syrup, Citric Acid, Lactic Acid, Water, Natural Mango Flavor M38630, Tartaric Acid, Refined Sugar

TABLE-US-00008 TABLE 8 Candy formulation Active Ingredients Withaferin A Bisdemethoxycurcumin (BDMC) Excipients BioPerine ®, Sucrose, Liquid Glucose, Flavoring agent, Menthol, Acidulants (Citric acid/Tartaric Acid/Maleic Acid), Purified water

[0100] 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.