Login| Sign Up| Help| Contact|

Patent Searching and Data


Title:
ANGIOGENIN AS A DIAGNOSTIC OR PROGNOSTIC BIOMARKER AND DRUG TARGET IN AGE-RELATED MACULAR DEGENERATION
Document Type and Number:
WIPO Patent Application WO/2017/175181
Kind Code:
A1
Abstract:
The present invention relates to the quantification of various soluble proteins, including angiogenin in aqueous humor and plasma of patients with AMD. The invention further describes the role of angiogenin in the pathogenesis, progression of AMD. Hence, the level of angiogenin serves as a diagnostic and/or prognostic marker in AMD. Angiogenin modulators administered in the form of either intra-vitreal injection or topical solution to patients suffering from AMD can cause the required changes in the levels of angiogenin along with improved prognosis. The invention discloses the quantification of soluble proteins, which is useful for simultaneous detection and quantification of angiogenin along with other molecular factors associated with AMD. The invention also highlights that plausibility of modulating angiogenin function in the management of AMD.

Inventors:
GHOSH, Arkasubhra (Narayana Nethralaya, Narayana Health City# 258/A, Bommasandra, Hosur Road, Bangalore 9, 560 099, IN)
SETHU, Swaminathan (Narayana Nethralaya, Narayana Health City# 258/A, Bommasandra, Hosur Road, Bangalore 9, 560 099, IN)
KRISHNA, Santosh Gopi (Narayana Nethralaya, Narayana Health City# 258/A, Bommasandra, Hosur Road, Bangalore 9, 560 099, IN)
YADAV, Naresh (Narayana Nethralaya, Narayana Health City# 258/A, Bommasandra, Hosur Road, Bangalore 9, 560 099, IN)
CHEVOUR, Priyanka (Narayana Nethralaya, Narayana Health City# 258/A, Bommasandra, Hosur Road, Bangalore 9, 560 099, IN)
PINDIPAPANAHALLI, Narendra (Narayana Nethralaya, Narayana Health City# 258/A, Bommasandra, Hosur Road, Bangalore 9, 560 099, IN)
SHETTY, Katkeri Bhujang (Narayana Nethralaya, Narayana Health City# 258/A, Bommasandra, Hosur Road, Bangalore 9, 560 099, IN)
Application Number:
IB2017/051998
Publication Date:
October 12, 2017
Filing Date:
April 07, 2017
Export Citation:
Click for automatic bibliography generation   Help
Assignee:
NARAYANA NETHRALAYA FOUNDATION (Narayana Nethralaya, Narayana Health City# 258/A, Bommasandra, Hosur Road, Bangalore 9, 560 099, IN)
International Classes:
C07K14/00; A61P21/02; C07K14/515; C07K14/54
Domestic Patent References:
WO2006055739A22006-05-26
Foreign References:
KR101337046B12013-12-06
US20140370087A12014-12-18
EP1743031A22007-01-17
Other References:
NAGINENI, CHANDRASEKHARAM N. ET AL.: "Regulation of VEGF expression in human retinal cells by cytokines: Implications for the role of inflammation in agerelated macular degeneration", JOURNAL OF CELLULAR PHYSIOLOGY, vol. 227, no. 1, 2012, pages 116 - 126
SKEIE, JESSICA M. ET AL.: "Angiogenin in age-related macular degeneration", MOLECULAR VISION, vol. 17, 2011, pages 580 - 581
Attorney, Agent or Firm:
KALIA, Anita et al. (#213, Sobha AquamarineSarjapur Outer Ring Road, Bellandur, Bangalore - Karnataka 3, 560103, IN)
Download PDF:
Claims:
[0037] Claims:

We Claim:

1. A biomarker to identify the risk of ocular conditions, wherein:

a. the biomarker is a soluble protein;

b. the biomarker quantified to be used to diagnose and prognosticate disease including eye disease; and

c. the biomarker is quantified in a fluid of a human.

The biomarker as claimed in claim 1, wherein the biomarker is selected from a group comprising VEGF (Vascular Endothelial Growth Factor) VEGF, angiogenin, fractalkine, ICAM-1 (Intercellular Adhesion Molecule- 1), VCAM-1 (Vascular Cell Adhesion Molecule), L-selectin, IL- 2 (Interleukin-2), IP10 (Inflammatory Protein-10), MIP-1 beta (Macrophage Inflammatory Protein- lbeta) and MCP-1 (Monocyte Chemotactic Protein- 1).

The biomarker as claimed in claim 1, wherein the fluid is selected from a group comprising aqueous humor, vitreous humor, serum and plasma.

The biomarker as claimed in claim 1, wherein the ocular conditions are selected from a group comprising of dry and wet Age-Related Macular Degeneration (AMD), Diabetic Retinopathy (DR), Central Retinal Vein Occlusion (CRVO), Branch Retinal Vein Occlusion (BRVO) and pterygium.

The biomarker as claimed in claim 1, wherein the levels of angiogenin is high in aqueous humor of patient with age-related macular degeneration.

The biomarker as claimed in claim 1, wherein the levels of angiogenin in plasma patient with dry age-related macular degeneration is high compared to control such that angiogenin is suitable as a drug target for treatment of AMD.

Description:
TITLE OF THE INVENTION

Angiogenin as a diagnostic or prognostic biomarker and drug target in age-related macular degeneration

[0001] DESCRIPTION OF THE INVENTION [0002] Technical field of the invention

[0003] The present invention relates to the association observed between soluble proteins and ocular condition. The invention, in particular, relates to the association between angiogenin and ocular conditions such as Age-Related Macular Degeneration (AMD), Diabetic Retinopathy (DR), Central Retinal Vein Occlusion (CRVO), Branch Retinal Vein Occlusion (BRVO) and pterygium. The invention further relates to the use of angiogenin as a diagnostic and prognostic biomarker and also the use of angiogenin and modulation of its function as a therapeutic target in the management of AMD.

[0004] Background of the invention

[0005] Angiogenesis is a process, which helps in the formation of new blood vessels in the body. Angiogenin is a protein with ribonuclease activity and is made up of single chain amino acids consisting of 123 amino acids and induces cell migration, proliferation, invasion and angiogenesis.

[0006] AMD is the leading cause of vision loss and blindness in people, which results due to degeneration of the macula, a part of the retina which is responsible for the sharp vision. Degeneration of macula in addition to the formation of new blood vessels is one of the causes for loss of central vision in case of patients affected with AMD. [0007] There are two types of AMD namely dry AMD and wet AMD. Dry AMD is an early stage of the disease and results from aging and thinning of macular tissues, depositing yellow colored pigmented spots in the macula. This aging and thinning of macular tissues causes inflammation and results in vision loss. Dry AMD further advances into a stage where growth of newer blood vessels below the retina is observed, with leakage of blood and other fluids. This condition is called as wet AMD. The leakage also leads to permanent damage of the retinal cells, which creates blind spots in the central vision in later stage.

[0008] Various proteins including cytokines (Interleukin-6; Interleukin-8), chemokines (ΜΙΡ-Ιβ, Macrophage Inflammatory Protein 1 beta; MCP-1, Monocyte Chemotactic Protein- 1; RANTES, Regulated on Activation, Normal T cell Expressed and Secreted; Fractalkine) IFNs (Interferons), TGF (Transforming Growth Factors), E-selectin, L-selectin, VEGF (Vascular Endothelial Growth Factor) and angiogenin are molecular factors which are present in the eye. The increase in the levels of these proteins are associated with the formation of AMD.

[0009] Various clinical studies have been carried out for detecting angiogenin levels in patients with dry and wet AMD. A German study suggests that there is no significant difference in the angiogenin levels of aqueous humor between control and various stages of AMD. Whereas, a Japanese study states that aqueous humor angiogenin levels are significantly higher in patients with AMD as compared to control group.

[0010] There are various therapies, which are used in the treatment of AMD. Intra-vitreal bevacizumab injection decreases the levels of aqueous VEGF and increases the levels of inflammatory mediators namely IL-6, IL-8, MlP-lbeta and MCP-1 levels but without changes in Angiogenin levels.

[0011] Serum angiogenin levels are also monitored in various diseased conditions such as Behcet's disease wherein serum angiogenin levels are significantly higher in the diseased group than the control group. Angiogenin levels are also highly expressed in cultured fibroblasts of pterygia with severe phenotype, compared to those with normal conjunctiva. Angiogenin levels in diabetic retinopathy (DR) are found to be lower in the vitreous fluid as compared to the diabetic individuals without complications. Patients without diabetic complications have high local and systemic concentration of angiogenin in the serum. Also, a decrease in tyrosine phosphorylation of many vitreous proteins including angiogenin is observed in DR, which may indicate an alternate disease specific function. Furthermore, a significant elevation of IL-8, VEGF and angiogenin in the vitreous but not in serum samples is observed in diabetic individuals compared to control subjects. A significant increase in the vitreous angiogenin levels in eyes with proliferative diabetic retinopathy (PDR) and proliferative vitreo retinopathy (PVR) is observed.

[0012] The US Patent Application US20140370087 titled "Novel use of angiogenin " discloses a pharmaceutical composition that includes angiogenin or a fragment as an active ingredient, which is useful for the prevention and treatment of glaucoma. Angiogenin or its fragments suppresses the inflammatory response by activating the aqueous humor outflow due to which there is an increase in the nitrous oxide (NO) generation which in turn reduces the intraocular pressure. However, the invention is silent with respect to the means by which angiogenin levels are decreased in AMD.

[0013] The Patent Application WO1998040487 titled "Angiogenin receptor, compositions and methods related thereto " discloses the use of nucleic acids, which are used as probes to detect an angiogenin receptor-encoding nucleic acid, and to inhibit expression of the receptor. The invention also provides antibodies for drug screening assays such as CAM assay, blotting techniques, which help in detecting the angiogenin levels and therapeutic and diagnostic methods and compositions related thereto. However, the invention does not describe angiogenin use as diagnostic and/or prognostic marker for AMD. [0014] The Patent Application AU2015252112A1 titled "Use of Complement Pathway Inhibitors to Treat Ocular Diseases " discloses the treatment of ocular diseases including age-related macular degeneration (ARMD), diabetic retinopathy (DR) and ocular angiogenesis (OA) with the administration of Complement Pathway inhibitors such as anti-Factor D antibody, Factor H or inhibitors that block the action of properdin, factor B, factor Ba, factor Bb, C2, C2a, C3a, C5, C5a, C5b, C6, C7, C8, C9, or C5b-9. However, the invention does not disclose the mechanism of targeting angiogenin for the management of AMD. [0015] There are different methods available for the quantification of soluble proteins. The quantification of soluble proteins is crucial in understanding the relationship between the biomolecules in the biological fluids and disease pathogenesis. Hence, quantification of soluble proteins determines the relationship between the levels of soluble proteins and stage/progression of disease. It can also serve as diagnostic and prognostic markers for various conditions including ocular diseases.

[0016] Summary of the invention [0017] The present invention relates to quantification of various soluble proteins such as inflammatory cytokines, chemokines, secreted cell adhesion molecules and pro-angiogenic factors in aqueous humor and plasma, which helps in diagnosis and also in determining the prognosis of AMD. The invention relates to identification of soluble proteins as diagnostic or prognostic markers in analysis of progression of the ocular diseases. The soluble proteins are analyzed in fluids such as aqueous humor, vitreous humor, serum and plasma of patients with AMD.

[0018] The invention utilizes a standard method for measuring or analyzing the levels of various soluble proteins such as inflammatory cytokines, chemokines, growth factors, secreted cell adhesion molecules and pro-angiogenic factors in aqueous humor and plasma using cytometric bead array, which is useful for simultaneous detection and quantification of various soluble factors. The assay is performed and the capture beads and analyte signals are acquired by a flow cytometer. A comparative study is performed using analyte signal intensities with reference standards to determine absolute concentrations of individual analytes using BD FCAP Array Software. The statistical analysis and graphical representation of the results are performed using GraphPad.

[0019] The comparative levels of VEGF, angiogenin, fractalkine, ICAM-1 (Intercellular Adhesion Molecule- 1), VCAM-1 (Vascular Cell Adhesion Molecule), L-selectin, IL-2, IP10, MIP-1 beta and MCP-1 are quantified by cytometric bead array in the aqueous humor of control and AMD patients. The control group comprises aqueous humor from patients with diabetes undergoing cataract surgery with no other associated ocular pathologies. The results indicate that the levels of pro-angiogenic factors, cytokines, chemokines and secreted cell adhesion molecules are higher in the aqueous humor of patients with wet and dry AMD as compared to that of the control group.

[0020] The invention indicates the higher level of angiogenin in aqueous humor in patients with AMD compared to controls. The levels of angiogenin in related to the progression of the disease. Angiogenin can act as a diagnostic and prognostic biomarker for diagnosis or prognosis of the ocular diseases such as AMD.

[0021] The invention thus describes that higher levels of angiogenin are found in aqueous humor and plasma in patients with AMD. Angiogenin levels are higher in the plasma of patients with dry AMD. Thus, suggesting angiogenin as a potential drug target in the management of AMD.

[0022] Brief description of the drawings

[0023] 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. [0024] FIG 1 illustrates the levels of pro-angiogenic factors, cytokines, chemokines and secreted cell adhesion molecules in the aqueous humor of patients with wet AMD. [0025] FIG 2 illustrates the levels of pro-angiogenic factors, cytokines, chemokines and secreted cell adhesion molecules in the plasma of patients with dry AMD.

[0026] Detailed description of the invention

[0027] In order to make the matter of the invention clear and concise, the following definitions are provided for specific terms used in the following description. [0028] The term "Diagnostic Biomarkef refers to a chemical/biological/physical entity which by means of specific reactions assists in qualitative and quantitative analysis of the disease.

[0029] The term "Prognostic Biomarkef refers to a chemical/biological/physical entity that provides information about the status of a specific disease in either untreated or treated individuals.

[0030] The present invention relates to quantification of various soluble proteins such as inflammatory cytokines, chemokines, secreted cell adhesion molecules and pro-angiogenic factors in aqueous humor and plasma, which helps in diagnosis and also in determining the prognosis of AMD.

[0031] The invention relates to identification of soluble proteins as diagnostic or prognostic markers in analysis of progression of the ocular diseases. The soluble proteins are analyzed in fluids such as aqueous humor, vitreous humor, serum and plasma of patients with AMD. [0032] The invention utilizes a method for measuring or analyzing the levels of various soluble proteins such as inflammatory cytokines, chemokines, growth factors, secreted cell adhesion molecules and pro-angiogenic factors in aqueous humor and plasma using cytometric bead array, which is useful for simultaneous detection and quantification of various soluble factors. The assay for the aforementioned soluble proteins is performed as per manufacturer's instruction. The capture beads and analyte signals are acquired by a flow cytometer (BD FACSCalibur™) using BD Cell Quest Pro Software. A comparative study is performed using analyte signal intensities with reference standards to determine absolute concentrations of individual analytes using BD FCAP Array Software. The statistical analysis and graphical representation of the results are performed using GraphPad.

[0033] FIG 1 illustrates the levels of pro-angiogenic factors, cytokines, chemokines and secreted cell adhesion molecules in aqueous humor of patients with wet AMD. The comparative levels of VEGF, angiogenin, fractalkine, ICAM- 1 (Intercellular Adhesion Molecule- 1), VCAM-1 (Vascular Cell Adhesion Molecule), L-selectin, IL-2, IP10, MIP-1 beta and MCP-1 are quantified by cytometric bead array using a flow cytometer in the aqueous humor of control and wet AMD patients. Aqueous humor is obtained for analysis immediately following intra-vitreal injection of anti-VEGF. The control group comprises aqueous humor from patients with diabetes undergoing cataract surgery with no other associated ocular pathologies. The results indicate that the levels of pro- angiogenic factors, cytokines, chemokines and secreted cell adhesion molecules are higher in the aqueous humor of patients with wet AMD as compared to that of the control group.

[0034] FIG 2 illustrates the levels of pro-angiogenic factors, cytokines, chemokines and secreted cell adhesion molecules in plasma of patients with dry AMD. The comparative levels of VEGF, angiogenin, fractalkine, ICAM-1, VCAM-1, L-Selectin, IL-2, IP10, MIP-lbeta and MCP-1 are quantified by cytometric bead array in the plasma of healthy controls and dry AMD patients. The control group comprises plasma from patients with diabetes undergoing cataract surgery with no other associated ocular pathologies. The results indicate that the level of pro-angiogenic factors, cytokines, chemokines and secreted cell adhesion molecules are similar in the plasma of patients with dry AMD as compared to control group. However, there is a significant increase in angiogenin level in plasma of patients with dry AMD compared to other factors that were quantified. [0035] The increase in the levels of angiogenin in the plasma and aqueous humor of dry and wet AMD, respectively indicates the use of angiogenin as a diagnostic and/or prognostic biomarker, which can be detected in the aqueous humor, vitreous humor, serum or plasma of the patients with AMD. Thus, suggesting angiogenin as a potential drug target in the management of AMD.

[0036] The invention thus describes that higher levels of angiogenin are found in aqueous humor and plasma of patients with AMD. Angiogenin levels are higher in the plasma of patients with dry AMD. Hence, indicating a vital role for angiogenin in the pathogenesis and progression of AMD.