Priority Claim:

[0001] This application claims priority from the provisional application numbered 202041030776 filed with Indian Patent Office, Chennai on 19 ^th July 2020 entitled “A formulation of combination of genistein and calcitriol for management of inflammatory ocular surface conditions the entirety of which is expressly incorporated herein by reference

DESCRIPTION OF THE INVENTION Technical field of the invention

[0001] The present invention discloses a formulation with a combination of genistein and calcitriol (vitamin D) for treatment of the Dry Eye Disease (DED). More particularly, the invention discloses the anti-inflammatory formulation with a combination of genistein and calcitriol for topical administration to mitigate hyperosmotic stress-induced Tonicity -responsive enhancer binding protein (TonEBP) and Cystic Fibrosis Transmembrane Conductance Regulator CFTR dysfunction, Vitamin D Receptor (VDR) degradation and inflammation in dry eye disease.

Background of the invention [0002] Dry eye disease is a common condition that occurs when tears are not able to provide adequate lubrication for the eyes. It is a chronic multifactorial condition associated with pain and discomfort. The volume and the stability of tears can be inadequate for multiple reasons. Dry eye disease is caused by a chronic lack of sufficient lubrication and moisture on the surface of the eye. The consequences of dry eyes range from subtle but constant eye irritation to significant inflammation and even scarring of the front surface of the eye.

[0003] Dry eye disease symptoms are associated with increased ocular surface (OS) inflammation. The other common symptoms include burning sensation, itchy eyes, dryness sensation, photophobia, blurred vision. There are a group of patients who have severe symptoms but with no observable signs of disease or in whom the symptoms continue to persist despite resolution of clinical signs.

[0004] It is observed that the worldwide prevalence of dry eye disease is ranging up to 50%. Dry eye syndrome can occur at any age and in people who are otherwise healthy. However, it is more common with older age, when the individual produces lesser tears. It is also more common in women than in men.

[0005] Dry eye disease is characterized by aberrant ocular surface inflammation and increased tear osmolarity. The studies also show that the induction of inflammatory factors such as Tumor Necrosis Factor-a (TNFa), Matrix metalloproteinase 9 (MMP9), Interleukin-6 (IL-6), Interleukin-8 (IL-8), macrophage chemoattractant protein 1 (MCP-1), as well as cellular stress and death following exposure to hyperosmotic stress. The hyperosmotic stress-induced inflammation has been mediated through an osmoresponsive factor Tonicity- responsive enhancer binding protein/nuclear factor of activated T cells -5 (TonEBP/NFAT5) in ocular surface cells.

[0006] Osmotic stress is a physiologic dysfunction caused by a sudden change in the solute concentration around a cell, which causes a rapid change in the movement of water across its cell membrane. Hyperosmotic stress stimulates inflammation at the ocular surface and is thought to be a central mechanism leading to dry eye disease. The evidence shows that the hyperosmotic stress as a potent inflammatory stimulus by triggering proinflammatory cytokine release and inflammation.

[0007] It is observed that the active ion transport by the corneal and conjunctival epithelium contributes to basal tear film status on the ocular surface. The transport of chloride in ocular surface cells is crucial in establishing high osmotic gradient for fluid to enter the tear film. Cystic fibrosis transmembrane conductance regulator is a major channel protein that regulates chloride transport and balance both in the lacrimal gland and ocular surface cells, thus aiding in the maintenance of ocular surface integrity and the accumulation of chloride is associated with inflammation. Hence, determining the rate of chloride transport and restoring chloride balance may be beneficial in the management of dry eye disease.

[0008] Vitamin D is a fat soluble and naturally produced steroid hormone that is present in very few food supplements. Vitamin D is also produced endogenously when ultraviolet rays from sunlight strike the skin and trigger vitamin D synthesis. Vitamin D obtained from sun exposure, food, and supplements is biologically inert and must undergo two hydroxylations in the body for activation. The first occurs in the liver and converts vitamin D to 25 -hydroxy vitamin D [25(OH)D], also known as calcidiol. The second occurs primarily in the kidney and forms the physiologically active 1,25-dihydroxy vitamin D [l,25(OH)2D] also known as calcitriol. Vitamin D is known for its role in regulation of inflammation, calcium homeostasis and cell fate. Vitamin D also supports the immune system. There is also evidence that low levels of vitamin D are linked with higher levels of inflammation. The effect of vitamin D on inflammation modulation and protection of barrier function is achieved by binding to vitamin D receptor.

[0009] Hence, it is required to analyze the status of VDR and chloride channel function on the ocular surface of patients with dry eye disease and human comeal epithelial cells exposed to hyperosmotic stress and also the role of CFTR and VDR activation in mitigating hyperosmotic stress-associated response in human comeal epithelial cells.

[0010] Genistein is a polyphenolic isoflavone that belongs to the flavonoid group and a phytoestrogen primarily found in soybeans and soybean-enriched products, which has selective estrogen receptor modulator properties. Genistein binds to and inhibits protein-tyrosine kinase, thereby disrupting signal transduction. [0011] The available formulations may not be effective in reducing the hyperosmotic stress and inflammation. Hence, there is a need for a formulation, which is useful for reducing hyperosmotic stress and inflammation for the management of dry eye disease and to know the association of hyperosmotic stress and inflammation in dry eye disease.

Summary of the Invention

[0012] The invention overcomes the drawbacks of the existing technology by providing a formulation with a combination of genistein and calcitriol (vitamin D) for the management of dry eye disease. The formulation of the present invention comprises genistein at a concentration in the range between 10 mM to 100 pM and 1,25-dihydroxy vitamin D3 or calcitriol at a concentration in the range between 20 nM to 60 nM.

[0013] The ingredients of the formulation genistein and calcitriol acts together to reduce the inflammation and the hyperosmotic stress thus managing the dry eye disease. The present invention discloses the association of hyperosmotic stress and inflammation with dry eye disease and is confirmed by analyzing the expression of different hyperosmotic stress and inflammation markers in patients with dry eye disease and in Human Corneal Epithelial Cells (HCECs).

[0014] The status of hyperosmotic stress and inflammation in the ocular surface cells is determined by using the expression of the markers in order to analyze the association of hyperosmotic stress and inflammation in dry eye disease. The inflammatory markers such as Interleukin-6 (IL-6), Interleukin-8 (IL-8), Interleukin- 17 A (IL-17A), Interleukin- 17F (IL-17F), TNFa, MMP9, Monocyte Chemoattractant Protein- 1 (MCP1) and hyperosmotic stress maker, TonEBP/NFAT5 are increased in ocular surface cells from patients with dry eye disease than the control group without disease. The altered expression of the inflammatory markers and hyperosmotic stress maker are restored in HCECs exposed to hyperosmotic stress in vitro, by the treatment with the formulation of the present invention. [0015] The combination is also effective in restoring the hyperosmotic stress- induced changes in the expression of VDR in HCECs exposed to hyperosmotic stress in vitro. Reduced VDR protein expression was observed in ocular surface cells of patients with dry eye disease than the control group without disease.

The hyperosmotic stress resulted in altered morphology and viability of human corneal epithelial cells and the treatment of the formulation retained the cell morphology and improved viability. The accumulation of intracellular chloride ion is analyzed by the expression of GLRX5, a chloride-dependent gene that is increased in the presence of intracellular chloride. The accumulation of chloride ion is high in the HCECs exposed to hyperosmotic stress and in ocular surface cells of patients with dry eye disease. The treatment of genistein reduced the expression of GLRX5 which is indicative of reduced intracellular accumulation of chloride ion.

[0016] The formulation comprising the combination of genistein and calcitriol is effective in mitigating the deleterious effects of hyperosmotic stress on human corneal epithelial cells. As a result of mitigation of hyperosmotic stress and inflammation by altering the expression of different markers, the formulation is effective in management of dry eye disease.

[0017] The formulation is useful for topical administration, which improves ocular surface health thus reducing the severity in dry eye disease.

Brief description of the drawings

[0018] The foregoing and other features of embodiments will become more apparent from the following detailed description of embodiments, when read in conjunction with the accompanying drawings. In the drawings, like reference numerals refer to like elements.

[0019] FIG 1 illustrates the expression of inflammatory markers in controls and cells from patients with dry eye disease.

[0020] FIG 2 illustrates the effect of the formulation on hyperosmotic stress induced inflammatory factors expression in human corneal epithelial cells. Detailed description of the invention

[0021] In order to more clearly and concisely describe and point out the subject matter of the claimed invention, the following definitions are provided for specific terms, which are used in the following written description.

[0022] The term "CalcitrioF refers to the active form of vitamin D, normally formed in the kidney.

[0023] The term " Inflammation ” refers to a body's natural reaction against injury, infection and/or stress stimuli.

[0024] The invention discloses a formulation with a combination of genistein and calcitriol in the management of dry eye disease. The formulation comprises genistein and calcitriol to mitigate the hyperosmotic stress and inflammation through different factors, thus alleviating dry eye disease.

[0025] The formulation comprises genistein at a concentration in the range between 10 mM to 100 pM and 1,25-dihydroxy vitamin D3 or calcitriol at a concentration in the range between 20 nM to 60 nM. The formulation of the present invention is unique in nature in the specific concentration of genistein and calcitriol and the combination exhibit synergistic effect.

[0026] Vitamin D mediates its immunomodulatory and anti-inflammatory effects through the interaction in its activated form with vitamin D receptor.

[0027] Dry eye disease is associated with increased hyperosmotic stress and inflammation in the ocular surface. The present invention discloses the association of hyperosmotic stress and inflammation with dry eye disease. In order to analyze the relationship, the expression of different hyperosmotic stress and inflammation markers are quantified in patients with dry eye disease and in Human Comeal Epithelial Cells (HCECs). [0028] The formulation of the present invention is analysed for its efficacy in reducing the hyperosmotic stress and inflammation in the ocular surface by analysing the markers in human comeal epithelial cells.

[0029] The following examples are offered to illustrate various aspects of the invention. However, the examples are not intended to limit or define the scope of the invention in any manner.

Example 1: Efficacy of the formulation on the expression of inflammatory markers in human corneal epithelial cells exposed to hyperosmotic stress which is an in vitro model for dry eye disease

[0030] The expression of inflammatory markers is analyzed in ocular surface cells from human subjects with and without dry eye disease. The mRNA expression of the inflammatory factors is determined by quantitative real-time PCR.

[0031] FIG 1 illustrates the expression of inflammatory markers in ocular surface cells from human subjects with and without dry eye disease. The inflammatory markers such as Interleukin-6 (IL-6), Interleukin-8 (IL-8), Interleukin- 17 A (IL- 17A), Interleukin- 17F (IL-17F), TNFa, MMP9, Monocyte Chemoattractant Protein- 1 (MCP1) are increased in ocular surface cells from patients with dry eye disease than the control group without disease.

[0032] The formulation was effective in decreasing the expression of the IL-6, IL- 8, IL-17A, IL-17F, TNFa, MMP9, MCP1 in HCE Cells exposed to hyperosmotic stress, which is an in vitro model for dry eye disease.

[0033] In addition, a positive association is observed between TonEBP and most of the inflammatory genes in the cells from patients with DED but not in controls. This interprets the association between hyperosmotic stress and inflammation in dry eye disease.

Example 2: Efficacy of the formulation on the expression of inflammatory markers in human corneal epithelial cells exposed to hyperosmotic stress which is an in vitro model for dry eye disease [0034] The formulation is analysed for its activity against hyperosmotic stress- induced changes in HCECs. SV40-immortalized HCECs are cultured in a suitable culture media and in the presence of antibiotics. HCECs are cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) media without serum supplementation for 12 hours followed by the replacement of serum free hyperosmotic stress media for another 6 hours or 24 hours. The formulation of the present invention is added to the culture media. Hyperosmotic media is prepared by increasing concentration of sodium chloride in serum free DMEM/F-12 media by addition of 25mM, 50mM or lOOmM of NaCl to obtain culture media with increased osmolarity of +50 mOsm, +100mOsm or +200mOsm, respectively.

[0035] The mRNA and/or protein expression of IL-6, IL-8, IL-17A, IL-17F, TNFa, MMP9, MCP1, TonEB P/NFAT 5 , VDR, Glutaredoxin 5 (GLRX5) and phosphorylated p38 are analyzed as markers for inflammation and hyperosmotic stress. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Tubulin and/or Actin are used as loading or housing keeping control. The results interpreted that the increased expression of IL-6, IL-8, IL-17A, IL-17F, TNFa, MMP9, MCP1, TonEBP/NFAT5 and decreased VDR in response to hyperosmotic stress was prevented following addition of formulation in HCECs exposed to hyperosmotic stress. The hyperosmotic stress is also associated with change in the cell morphology and reduction of viability in HCECs

[0036] FIG 2 illustrates the effect of the formulation on hyperosmotic stress induced inflammatory factors expression in human corneal epithelial cells. The human corneal epithelial cells are under hyperosmotic stress (+200 mOsm) with or without genistein and calcitriol for 6 hours. Human corneal epithelial cells are categorized into untreated cells as control, cells under hyperosmotic stress (+200 mOsm), cells under hyperosmotic stress in presence of genistein (+200 mOsm + Gen.), cells under hyperosmotic stress in presence of calcitriol (+200 mOsm + Cal.) and cells under hyperosmotic stress in presence of formulation of the present invention i.e., genistein and calcitriol combination (+200 mOsm + Gen.+Cak). The results interpreted that the expression of IL-6, IL-8, IL-17A, IL-17F, TNFa, MMP9, MCP1 are greatly reduced in cells exposed to hyperosmotic stress in presence of genistein and calcitriol combination compared to the control cells without treatment.

[0037] The hyperosmotic stress is also associated with change in the cell morphology and reduction of viability in HCECs. The formulation is effective in mitigating the effect of hyperosmotic stress by retaining the normal cell morphology and viability in HCECs.

[0038] The intracellular ion such as chloride ion is a secondary messenger that mediates the expression of different genes including that are associated the inflammation. The accumulation of intracellular chloride ion is analysed by the expression of GLRX5, a chloride-dependent gene that is increased in the presence of intracellular chloride. The accumulation of chloride ion is high in the HCECs exposed to hyperosmotic stress. The treatment of genistein reduced the expression of GLRX5 which is indicative of reduced intracellular accumulation of chloride ion.

Example 3: Effect of the formulation on the expression of VDR and CFTR in HCECs.

[0039] The combination of genistein and calcitriol is analyzed for the expression of VDR and CFTR. Generally, VDR is induced by vitamin D whereas CFTR channel function is augmented by genistein. Hence, the effect of the formulation of the invention is analyzed on the channel function of CFTR and VDR expression in HCECs. The addition of the formulation exhibited rescue of hyperosmotic stress induced VDR degradation and facilitated induction of VDR, compared to single use of either genistein or calcitriol.

[0040] The formulation is also effective in reducing hyperosmotic stress-induced expression of TonEBP and phosphorylated p38 in HCECs.

[0041] The formulation is also effective in restoring the altered morphology and normalizing the viability reduced in HCECs exposed to hyperosmotic stress. Thus, the formulation comprising the combination of genistein and calcitriol is effective in mitigating the deleterious effects of hyperosmotic stress on human corneal epithelial cells. As a result of mitigation of hyperosmotic stress and inflammation by altering the expression of different markers, the formulation is effective in management of dry eye disease.

[0042] The formulation of the present invention is used to mitigate hyperosmotic stress-mediated inflammation and re-establish homeostatic status of intracellular chloride ion balance and vitamin D receptor in dry eye disease.

[0043] The present invention also discloses the role of VDR and the function of chloride ion channel on the ocular surface of patients with dry eye disease and also in human corneal epithelial cells exposed to hyperosmotic stress. The present invention also implies the significance of therapeutic effects of CFTR and VDR activation by the combination of genistein and calcitriol in mitigating hyperosmotic stress-associated response in human comeal epithelial cells. [0044] The formulation of the present invention is suitable for topical administration. The combination of genistein and calcitriol exhibits synergistic effect in mitigating the inflammation and hyperosmotic stress-associated response in human corneal epithelial cells.