WO2024044687A1

WO2024044687A1 - Biomarker combinations for prognosis and management of diabetic retinopathy

Info

Publication number: WO2024044687A1
Application number: PCT/US2023/072819
Authority: WO
Inventors: Ella T. NKHOMA; Bharath Ramaswamy; Luke MUELLER; Jun Luo; Kunaal PATEL; Joseph KATIGBAK; Mengran WANG; Benjamin ZHU; Genevieve LYONS
Original assignee: Valo Health, Inc.
Priority date: 2022-08-26
Filing date: 2023-08-24
Publication date: 2024-02-29

Abstract

Methods are described for identifying a subject as being at increased risk of having rapid progression of diabetic retinopathy. The methods involve the quantification of various combinations of biomarkers which distinguish patients who are predicted to progress in the disease from patients who are not predicted to progress in the disease. Methods of treating diabetic retinopathy in a subject or preventing progression of diabetic retinopathy in a subject, based on the quantification of the various combinations of biomarker, are also described.

Description

BIOMARKER COMBINATIONS FOR PROGNOSIS AND MANAGEMENT OF DIABETIC RETINOPATHY

FIELD

[0001] This disclosure concerns a method of identifying a subject as being at increased risk of having rapid progression of diabetic retinopathy. The disclosure also concerns methods of treating diabetic retinopathy in a subject or preventing progression of diabetic retinopathy in a subject.

BACKGROUND

[0002] Diabetic retinopathy (DR) is a serious complication of diabetes mellitus (DM) and the leading cause of preventable blindness in the adult working population. DR occurs when blood vessels in the retina leak blood and other fluids causing tissue swelling and cloudy or blurred vision. Patients with any type of diabetes (T1D, T2D, gestational) are at risk for DR, and evidence indicates that after 20 years of living with diabetes, DR is present in almost all patients with T1D and >60% of those with T2D (Coney. Am J Manag Care. 2019 Oct;25(16 Suppl):S311-S316).

[0003] Diabetic retinopathy has 2 types and 4 stages. Stage 1 is mild non-proliferative diabetic retinopathy (NPDR), which is characterised by tiny areas of swelling in the blood vessels of the retina, called microaneurysms, beginning to form. Stage 2 is moderate NPDR, which is characterised by increased swelling interfering with blood flow to the retina, causing an accumulation of fluids in the macula. Stage 3 is severe NPDR, which is characterised by a larger blocked section of blood vessels in the retina causing a significant decrease in blood flow and triggering new growth. Signs of increasing ischemia include venous abnormalities (for example, dilation, beading, loops), intra-retinal microvascular abnormalities (IRMA), and more severe and extensive vascular leakage characterized by increasing retinal hemorrhages and exudation (American Academy of Ophthalmology 2020).

[0004] Stage 4 is proliferative diabetic retinopathy (PDR), which is characterised by new fragile blood vessels that are more prone to fluid leakage form in the retina, which can trigger vision problems such as blurriness, reduced field of vision, and blindness. Depending on PDR severity, it is treated with intravitreal injection of anti-VEGF, panretinal photocoagulation or focal or grid laser (Flaxel, Christina J. et al. Diabetic Retinopathy Preferred Practice Pattern® Ophthalmology, Volume 127, Issue 1, P66 - P145).

[0005] Diabetic Macular Edema (DME) is also a complication of DR and can develop at any stage of DR. Clinically significant macular edema (CSME) is defined as retinal thickening and/or adjacent hard exudates that either involve the center of the macula or threaten to involve it. Patients with CSME should be considered for prompt treatment (intravitreal (IVT) injection of anti-VEGF, panretinal photocoagulation, focal (or grid) laser), particularly when the center of the macula is already involved or if retinal thickening and/or hard exudates are very close to the center (Flaxel, Christina J. et al. Diabetic Retinopathy Preferred Practice Pattern® Ophthalmology, Volume 127, Issue 1, P66 - P145).

[0006] Treatment options

[0007] Eylea® (aflibercept) and Lucentis® (ranibizumab) are FDA approved for the treatment of DR and the treatment of DME. These therapies bind to VEGF through different mechanisms of action turning off the VEGF mediated signalling cascade that leads to angiogenesis and inflammation. Several large trials have demonstrated efficacy of anti-VEGF IVT injections in eyes with center-involved (CI-)DME and visual acuity of 20/32 or worse. Anti-VEGF therapy is recommended by American Guidelines as standard of care for these patients (Flaxel, Christina J. et al. Diabetic Retinopathy Preferred Practice Pattern® Ophthalmology, Volume 127, Issue 1, P66 - P145; Yonekawa Y et al. American Society of Retina Specialists Clinical Practice Guidelines on the Management of Nonproliferative and Proliferative Diabetic Retinopathy without Diabetic Macular Edema. J Vitreoretin Dis. 2020 Mar 1 ;4(2): 125-135).

[0008] The presence of good vision in an eye with CI-DME is a common clinical scenario. In a population-based study in US, among patients with DME, 84% had best corrected visual acuity (BCVA) of 20/40 or better in the eye with DME (Bressler et al, 2014). However, the optimal therapy for these eyes has not been established. NPDR patients without DME are at risk for the development of vision-threatening complications, however the role of IVT anti- VEGF therapy for eyes with NPDR without vision-threatening complications is not clear.

[0009] The Early Treatment of Diabetes Retinopathy Study (ETDRS) Diabetic Retinopathy Severity Scale (DRSS) has become the gold standard for research purposes (Early Treatment Diabetic Retinopathy Study Research Group. Grading Diabetic Retinopathy from Stereoscopic Color Fundus Photographs-An Extension of the Modified Airlie House Classification. ETDRS Report Number 10. Ophthalmology. 1991 May;98 (5):786-806). According to the natural history of DR progression defined in ETDRS, the risk of progression to PDR within 1 year was 26% and 44%-51% for moderately severe and severe NPDR respectively (Early Treatment Diabetic Retinopathy Study Research Group. Fundus photographic risk factors for progression of diabetic retinopathy. ETDRS report number 12. Ophthalmology, 98 (1991), pp. 823-833). [0010] The PANORAMA trial (Brown DM et al. Evaluation of Intravitreal Aflibercept for the Treatment of Severe Nonproliferative Diabetic Retinopathy: Results From the PANORAMA Randomized Clinical Trial. JAMA Ophthalmol. 2021;139(9):946-955) was a phase 3, doublemasked, randomized study of the efficacy and safety of intravitreal aflibercept injection in patients with moderately severe to severe NPDR. The study reported a cumulative risk of 41% of vision-threatening progression within 1 year in moderately severe and severe NPDR (American Society of Retinal Specialists Clinical Practice Guidelines, 2020). NPDR without DME has accumulated strong evidence that anti-VEGF treatment can improve the DRSS level. However, long term data are not available, and intravitreal injections carry risks such as acute bacterial endophthalmitis. Currently there is no definitive answer regarding treating NPDR without DME with anti-VEGF (Yonekawa Y et al. American Society of Retina Specialists Clinical Practice Guidelines on the Management of Nonproliferative and Proliferative Diabetic Retinopathy without Diabetic Macular Edema. J Vitreoretin Dis. 2020 Mar 1 ;4(2): 125-135). [0011] An optimal therapy for moderately severe and severe NPDR patients without CI-DME as well as for patients with CI-DME with good visual acuity has not yet been established.

[0012] The current approach to management of mild and moderate NPDR is regular follow up, with no specific recommended treatment. However, some patients with mild and moderate NPDR can progress quickly and may benefit from early treatment. (Davis, 1998 (ETDRS Study)). Natural history of Type 1 DM patients with mild retinopathy suggests that approximately 16% will progress to proliferative disease within 4 years (Klein R, Klein BE, Moss SE, et al. The Wisconsin Epidemiologic Study of Diabetic Retinopathy. IX. Four- year incidence and progression of diabetic retinopathy when age at diagnosis is less than 30 years. Arch Ophthalmol. 1989;107:237-43). For Type 1 and 2 DM patients with moderate NPDR, the risk increases: 12% patients progress to any PDR within 1 year and 27% progress to high- risk PDR within 5 years (Early Treatment Diabetic Retinopathy Study Research Group. Fundus photographic risk factors for progression of diabetic retinopathy. ETDRS report number 12. Ophthalmology, 98 (1991), pp. 823-833).

[0013] There is therefore a need to establish factors associated with NPDR progression to be able to identify patients that are likely to progress in the disease, so that these patients can receive targeted treatment. SUMMARY

[0014] In a first aspect, there is provided a method of identifying a subject as being at increased risk of having rapid progression of diabetic retinopathy. The method comprises quantifying at least two biomarkers in one or more samples obtained from the subject, and identifying the subject as being at increased risk of having rapid progression of diabetic retinopathy if the quantified values of the at least two biomarkers are statistically different compared to a control. The at least two biomarkers comprise:

(i) hemoglobin Ale and the ratio of albumin to creatinine; and/or

(ii) red cell distribution width and the ratio of albumin to creatinine; and/or

(iii) hemoglobin Ale and alanine aminotransferase, and wherein the subject is not being treated with a 3 -hydroxy-3 -methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor; and/or

(iv) alkaline phosphatase and the ratio of albumin to creatinine.

[0015] In a second aspect, there is provided a method of treating or preventing progression of diabetic retinopathy in a subject. The method comprises identifying the subject as being at increased risk of having rapid progression of diabetic retinopathy if the quantified values of at least two biomarkers are statistically different compared to a control. The at least two biomarkers comprise:

(i) hemoglobin Ale and the ratio of albumin to creatinine; and/or

(ii) red cell distribution width and the ratio of albumin to creatinine; and/or

(iv) alkaline phosphatase and the ratio of albumin to creatinine.

[0016] The method further comprises administering a therapy to the subject, wherein the therapy is an immunomodulatory therapy capable of treating or preventing progression of diabetic retinopathy in the subject. Accordingly, the second aspect includes an active step of identifying the subject as being at increased risk of having rapid progression of diabetic retinopathy.

[0017] In a third aspect, there is provided a method of treating or preventing progression of diabetic retinopathy in a subject. The method comprises selecting a subject having quantified values of at least two biomarkers that are statistically different compared to a control. The at least two biomarkers comprise: (i) hemoglobin Ale and the ratio of albumin to creatinine; and/or

(ii) red cell distribution width and the ratio of albumin to creatinine; and/or

(iii) hemoglobin Ale and alanine aminotransferase, and wherein the subject is not being treated with a 3 -hydroxy-3 -methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor; and/or alkaline phosphatase and the ratio of albumin to creatinine.

[0018] The method further comprises administering a therapy to the subject, wherein the therapy is an immunomodulatory therapy capable of treating or preventing progression of diabetic retinopathy in the subject.

[0019] Accordingly, the third aspect involves selecting a subject that has been previously identified as being at increased risk of having rapid progression of diabetic retinopathy owing to the quantified values of the at least two biomarkers being statistically different compared to a control.

[0020] The foregoing and other objects, features and advantages of the invention will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] 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.

[0022] Figure 1 shows the survival probability of different subpopulations using a Cox-PH model. “acr_only” is the subpopulation with an albumin creatinine ratio of > 60: 1. “hbalc only” is the subpopulation with a hemoglobin Ale value > 7.7. “both_criteria” is the subpopulation with an albumin creatinine ratio of > 60: 1 and a hemoglobin Ale value > 7.7. “no_criteria_met” and “criteria not met” is the subpopulation that do not meet an albumin creatinine ratio of > 60: 1 and also do not meet a hemoglobin Ale value > 7.7.

[0023] Figures 2-5 are stacked bar charts showing the progression of subpopulations of patients to treatment within 1 year of diagnosis (grey bars) and within 2 years of diagnosis (black bars). The far left bar (“None”) shows the percentage of patients who progressed to treatment of the total subpopulation of patients defined by the parameters indicated. The second bar (“VA < 80) shows the percentage of patients who progressed to treatment of the total subpopulation of patients defined by the parameters indicated and having a visual acuity score of < 80. The third bar (“VA < 75) shows the percentage of patients who progressed to treatment of the total subpopulation of patients defined by the parameters indicated and having a visual acuity score of < 75. The fourth bar (“VA < 70) shows the percentage of patients who progressed to treatment of the total subpopulation of patients defined by the parameters indicated and having a visual acuity score of < 70. The fifth bar (“VA < 65) shows the percentage of patients who progressed to treatment of the total subpopulation of patients defined by the parameters indicated and having a visual acuity score of < 65. The cohort size is indicated for each subpopulation, expressed as a percentage of total patients. Figure 1: ACR > 92 and HbAlc > 8.25; Figure 2: RDW < 14 and ACR > 92; Figure 3: No statins and ALT < 24.5 and HbAlc > 7.85; Figure 4: ALKP < 79 and ACR > 20; Figure 5: ACR > 92 and HbAlc > 8.25, or RDW < 14 and ACR > 92, or no statins and ALT < 24.5 and HbAlc > 7.85, or ALKP < 79 and ACR > 20.

DETAILED DESCRIPTION

[0024] I. Abbreviations

[0025] ACR: albumin/creatinine ratio

[0026] ALKP: alkaline phosphatase

[0027] ALT: alanine aminotransferase (also known as alanine transaminase)

[0028] DM: diabetes mellitus

[0029] DME: diabetic macular edema

[0030] DR: diabetic retinopathy

[0031] EMR: electronic medical record

[0032] ETDRS: Early Treatment Diabetic Retinopathy Study

[0033] Hb: hemoglobin

[0034] HbAlc: hemoglobin Ale

[0035] HMG-CoA: 3-hydroxy-3-methyl-glutaryl-coenzyme A

[0036] NPDR: non-proliferative diabetic retinopathy

[0037] PDR: proliferative diabetic retinopathy

[0038] RDW: red cell distribution width

[0039] T1D: type 1 diabetes

[0040] T2D: type 2 diabetes

[0041] VA: visual acuity [0042] II. Terms and Methods

[0043] Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds), The Encyclopaedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081- 569-8).

[0044] In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided:

[0045] 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor:

Refers to statins, and includes, for example, atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin.

[0046] Administer: As used herein, administering a therapy (for example, a rho-associated coiled coil forming kinase (ROCK) inhibitor, or an antibody such as anti-VEGF) to a subject means to give, apply or bring the therapy into contact with the subject. Administration can be accomplished by any of a number of routes depending on the therapeutic agent being administered, and include, for example, intravitreal, conjunctival, intracorneal, intraocular, intravenous, ophthalmic, topical, oral, intranasal, subcutaneous, intramuscular and intraperitoneal.

[0047] Antibody: Immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, for example, molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.

[0048] A naturally occurring antibody (for example, IgG, IgM, IgD) includes four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. However, it has been shown that the antigen-binding function of an antibody can be performed by fragments of a naturally occurring antibody. Thus, these antigen-binding fragments are also intended to be designated by the term “antibody.” Specific, non-limiting examples of binding fragments encompassed within the term antibody include (i) a Fab fragment consisting of the VL, VH, CL and Cm domains; (ii) an Fa fragment consisting of the VH and CHI domains; (iii) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (iv) a dAb fragment (Ward et al., Nature 341:544-546, 1989) which consists of a VH domain; (v) an isolated complementarity determining region (CDR); and (vi) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region. [0049] Immunoglobulins and certain variants thereof are known and many have been prepared in recombinant cell culture (for example, see U.S. Patent Nos. 4,745,055 and 4,444,487; WO 88/03565; EP 256,654; EP 120,694; EP 125,023; Falkner et al., Nature 298:286, 1982; Morrison, J. Immunol. 123:793, 1979; Morrison et al., Ann. Rev. Immunol. 2:239, 1984).

[0050] Biomarker: In the context of the present disclosure, a “biomarker” is a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention. Biomarkers include biomolecules, for example DNA, RNA, proteins, lipids, carbohydrates and metabolites. The biomarker may be a measured amount or concentration of a single biomolecule (for example, alkaline phosphatase, alanine aminotransferase), or, for example, a ratio of the measured amounts of multiple biomolecules (such as a ratio of albumin to creatinine). The biomarker may be a fraction of a subtype of a biomolecule to the total level of the biomolecule (such as the percentage of HbAlc of total hemoglobin). Biomarkers further include measurable biological parameters that serve as indicators of a particular physiological, pathogenic or pharmacologic state, such as cell counts (for example, complete blood cell count, neutrophil- to-lymphocyte ratio, platelet-to-lymphocyte ratio, etc.), cell size heterogeneity (such as red cell distribution width), behaviour, and disease phenotype scores (such as visual acuity scores). In the context of the present disclosure, the biomarkers are quantifiable predictive or prognostic markers.

[0051] Control: A “control” refers to a sample or standard used for comparison with an experimental sample, wherein the experimental sample is a sample obtained from a patient (for example, a blood sample) to be tested for biomarkers (such as alkaline phosphatase).

[0052] The control may be a matched or paired sample (for example, matched on factors such as age, sex, race, or a combination thereof).

[0053] In some embodiments, the control is a sample obtained from a healthy patient or a group of samples obtained from healthy patients.

[0054] In various embodiments, the control is a sample previously obtained from the same patient at a time point (for example, six months previously) where the patient was identified as not being at increased risk of having rapid progression of diabetic retinopathy.

[0055] In particular embodiments, the control is a sample obtained from a patient with diabetic retinopathy that progresses at an average rate, or a group of samples obtained from patients with diabetic retinopathy that progress at an average rate. [0056] In particularly preferred embodiments, the control is substituted with a pre-determined value which functions as a threshold. The predetermined value may be a biomarker threshold value as described herein.

[0057] Control value: As used herein, “control value” is a value of a biomarker measured in a control sample, or an average value of a biomarker measured in multiple control samples. “Control value” also includes historical control values and reference standards (in other words, the quantified value of a biomarker in a previously tested control sample or group of samples that represents baseline or normal values, such as the average value of a particular biomarker in the blood of healthy subjects).

[0058] Hydrate: The term “hydrate” refers to a compound that comprises one molecule of water per n molecule(s) of another molecule, such as an active pharmaceutical ingredient. Depending on the hydrate, n can be any fraction from 0.1 to 0.9 or any number from 1 to 20. For example, a monohydrate of an active pharmaceutical ingredient or pharmaceutically acceptable salt thereof has one molecule of water per molecule of the active pharmaceutical ingredient or pharmaceutically acceptable salt thereof. A dihydrate of an active pharmaceutical ingredient or pharmaceutically acceptable salt thereof has two molecules of water per molecule of the active pharmaceutical ingredient or pharmaceutically acceptable salt thereof (in other words, one molecule of water per half molecule of the active pharmaceutical ingredient or pharmaceutically acceptable salt thereof). A hemihydrate of an active pharmaceutical ingredient or pharmaceutically acceptable salt thereof has one molecule of water per two molecules of the active pharmaceutical ingredient or pharmaceutically acceptable salt thereof. [0059] Quantifying: As used herein, “quantifying” a particular biomarker refers to measuring the property of that biomarker that is used as the indicator for the physiological, pathological or pharmacological state. For example, where the amount of a biomolecule is used as an indicator, “quantifying” refers to measuring the amount of that biomolecule in a sample (or multiple samples) (for example, alkaline phosphatase, alanine aminotransferase). As another example, where the ratio or percentage of amounts of biomolecules is used as an indicator, “quantifying” refers to measuring the amounts of those biomolecules in a sample or multiple samples, and calculating the ratio or percentage. In another example, where a measurable biological parameter is used as an indicator, “quantifying” refers to measuring that biological parameter in a sample (or multiple samples) (for example, red cell distribution width).

[0060] Quantification can be either numerical or relative. Detecting and quantifying the biomarker can be achieved using any method known in the art or described herein. For example, the amount or ratio of biomolecule biomarkers, such as proteins, may be quantified by ELISA or aptamer array. As a further example, the red cell distribution width may be quantified by obtaining a standard complete blood cell count and dividing the standard deviation of the mean corpuscular volume (MCV) by the MCV and multiplying by 100 to yield a percentage value to be on behalf of the red blood cell size heterogeneity.

[0061] Typically, the same method for quantifying the biomarker is used for the control and for the subject sample (in other words, the experimental sample). However, any suitable equivalent methods that obtains the same results can be used.

[0062] Typically, the biomarker is quantified in the same sample type for the subject sample as was used for determining the control value.

[0063] Pharmaceutically acceptable: The term “pharmaceutically acceptable” refers to a non-toxic material that does not interfere with the effectiveness of an active pharmaceutical ingredient(s).

[0064] Pharmaceutically acceptable salt: A salt that is prepared from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, metaphosphoric acid, nitric acid, or sulfuric acid, and/or from organic acids such as formic acid, acetic acid, trifluoroacetic acid, benzenesulfonic acid, benzoic acid, citric acid, ethanesulfonic acid, fumaric acid, gluconic acid, glycolic acid, lactic acid, maleic acid, malic acid, methanesulfonic acid, succinic acid, -tolucncsul Ionic acid, or tartaric acid by methods known in the art.

[0065] Predetermined value: The control may be substituted with a pre-determined value which functions as a threshold. By “threshold”, it is meant that the operator of the method compares the quantified value of a biomarker with the predetermined value, and determines whether the quantified value meets the conditions for exceeding the threshold. For example, if the threshold is >10, a quantified value of 11 exceeds the threshold, whereas a quantified value of 9 or 10 does not exceed the threshold. As a further example, if the threshold is <20, a quantified value of 19 exceeds the threshold, whereas a quantified value of 20 or 21 does not exceed the threshold.

[0066] Prognosis: The term “prognosis” refers to a forecast as to the probable outcome of the disease as well as the prospect of recovery from the disease as indicated by the nature and symptoms of the case. Accordingly, a negative or poor prognosis is defined by a rapid progression of the disease. Conversely, a positive or good prognosis is defined by a slow, minimal or negligible progression of the disease.

[0067] ROCK inhibitor: As used herein, a ROCK inhibitor is an inhibitor of Rho kinase. ROCK inhibitors may target a single Rho kinase (for example, ROCK1 or ROCK2), or multiple Rho kinases (for example, ROCK1 and ROCK2). [0068] Sample or biological sample: As used herein, a “sample” obtained from a subject refers to a cell, fluid or tissue sample. Bodily fluids include, but are not limited to, cerebral spinal fluid, tears, blood, serum, urine and saliva. In the context of the present disclosure “obtaining a biological sample” includes either directly collecting the sample from the subject, or obtaining the sample from a laboratory or service provider that has collected the sample from the subject. A sample “obtained from a subject” is a sample acquired by similar means.

[0069] Subject: The terms “subject” and “patient” are used interchangeably herein, and is meant an animal to be identified or treated in the methods described herein, with human subjects being preferred.

[0070] Statistically different: The value (or average value) of the biomarker quantified in the experimental sample differs from the control value in a statistically significant manner, as determined by a suitable statistical test, which are well known to those skilled in the art.

[0071] Statistically increased: The value (or average value) of the biomarker quantified in the experimental sample is increased relative to the control value in a statistically significant manner, as determined by a suitable statistical test, which are well known to those skilled in the art.

[0072] Statistically decreased: The value (or average value) of the biomarker quantified in the experimental sample is decreased relative to the control value in a statistically significant manner, as determined by a suitable statistical test, which are well known to those skilled in the art.

[0073] Therapeutically effective amount: A dose sufficient to prevent, or at least reduce the rate of, advancement, or to cause regression of the disease, or which is capable of reducing symptoms caused by the disease.

[0074] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. “Comprising A or B” means including A, or B, or A and B. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. [0075] III. Overview of Biomarker Combinations and Methods of Use Thereof

[0076] Diabetic retinopathy is a disease that progresses through various different stages (also referred to as severity levels). These stages are mild non-proliferative, moderate nonproliferative, severe non-proliferative and proliferative. The various stages of the disease are defined according to clinically recognised severity scales, such as the ETDRS Diabetic Retinopathy Severity Scale (DRSS) or the International Clinical Diabetic Retinopathy Disease Severity Scale (Flaxel CJ, Adelman RA, Bailey ST, Fawzi A, Lim JI, Vemulakonda GA, Ying GS. Diabetic Retinopathy Preferred Practice Pattern®. Ophthalmology. 2020 Jan;127(l):P66- P145. doi: 10.1016/j.ophtha.2019.09.025. Epub 2019 Sep 25. Erratum in: Ophthalmology. 2020 Sep; 127(9): 1279. PMID: 31757498; Yonekawa Y, Modi YS, Kim LA, Skondra D, Kim JE, Wykoff CC. American Society of Retina Specialists Clinical Practice Guidelines: Management of Nonproliferative and Proliferative Diabetic Retinopathy Without Diabetic Macular Edema. Journal of VitreoRetinal Diseases. 2020;4(2): 125-135. doi: 10.1177/2474126419893829). In some embodiments, the stages of the disease are defined according to the International Clinical Diabetic Retinopathy Disease Severity Scale. A patient may progress through these stages or may stay in one stage. A patient with disease which moves to the next stage is said to have “progressive disease”, and “having disease progression”. It is understood in the art that a subject may progress through stages of the disease at different speeds. Thus, a patient which progresses through stages of the disease slowly (in other words, at a relatively slow speed compared to average progression) is considered to have slow progression of the disease. In contrast, a patient which progresses through stages of the disease quickly (in other words, at a faster speed compared to average progression) is considered to have rapid progression of the disease. “Rapid” progression is a term used interchangeable with “fast” and “accelerated” progression.

[0077] In some embodiments, rapid progression of diabetic retinopathy is defined as progression of the disease from an initial stage up to a higher stage within 3 years of diagnosis of the initial stage. For example, a patient being diagnosed as having mild non-proliferative retinopathy and being subsequently diagnosed as having moderate non-proliferative retinopathy within three years of the initial diagnosis would be defined as having rapid progression of the disease. In various embodiments, rapid progression of diabetic retinopathy is defined as progression of the disease from an initial stage up to a higher stage within 2 years of diagnosis of the initial stage. In some embodiments, rapid progression of diabetic retinopathy is defined as progression of the disease from an initial stage up to a higher stage within 1 year of diagnosis of the initial stage. [0078] A method described herein is a method of identifying a subject as being at increased risk of having rapid progression of diabetic retinopathy. The term “identifying” is understood to mean that the subject is analysed in some manner to determine that the subject is at increased risk of having rapid progression of diabetic retinopathy. In other words, the method is a method of prognosis of a subject to forecast the likely course of diabetic retinopathy. Identifying a subject as being at increased risk of having rapid progression of diabetic retinopathy typically means that the subject is forecasted to have rapid progression of disease, in other words, the subject has an increased chance of progressing quickly through stages of the disease.

[0079] In some embodiments, the term “ increased risk of having rapid progression” is understood to mean that the subject is predicted to progress to a higher stage of the disease within 3 years of the method being carried out. In various embodiments, the term "increased risk of having rapid progression” is understood to mean that the subject is predicted to progress to a higher stage of the disease within 2 years of the method being carried out. In other embodiments, the term "increased risk of having rapid progression” is understood to mean that the subject is predicted to progress to a higher stage of the disease within 1 year of the method being carried out. “Increased risk” and “predicted to progress” typically mean that the subject is statistically more likely to have rapidly progressing disease compared to a patient who does not have rapidly progressing disease.

[0080] The subject may be any subject. For example, the subject may be a human or a nonhuman animal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal selected from the group consisting of primate, horse, sheep, pig, cow, mouse, rat, rabbit, dog, and cat.

[0081] Typically, the subject has diabetic retinopathy. The subject may have non-proliferative or proliferative diabetic retinopathy. The subject may have any stage of diabetic retinopathy. In some embodiments, the subject has mild or moderate non-proliferative diabetic retinopathy. In some embodiments, the subject has mild non-proliferative diabetic retinopathy.

[0082] The at least two biomarkers may be quantified in any suitable manner. The term “quantifying” is used interchangeably with “measuring”. Methods of quantifying biomarkers are known in the art, and include, for example, immunohistochemistry, flow cytometry, karyotyping, ISH/FISH, PCR, gene sequencing, LC-MS, ligand-binding assays, automated cell counting (and calculation of heterogeneity), immunoassays and aptamer-binding assays.

[0083] The ratio of albumin to creatinine may be measured by using any suitable method that is known in the art, including immuno turbidimetric, fluorometric and/or enzymatic assays for the measurement of amounts of albumin and creatinine, and division of the albumin amount in milligrams by the creatinine amount in grams. An example of a conventional assay is the Albumin Creatinine Ratio Assay Kit from Abeam (ab241018; https://www.abcam.com/ps/products/241/ab241018/documents/Albumin-Creatinine-Ratio- As say-protocol-book-v 1 a-ab241018 %20( website) .pdf) .

[0084] HbAlc percentage of total hemoglobin may be measured by using any suitable method that is known in the art, including chromatography-based HPLC assays, antibody-based immunoassays or enzyme-based enzymatic assays. An example of a conventional assay is the AFINION™ HbAlc assay from Abbott Diagnostics Technologies AS (https://ensur.invmed.com/ensur/contentAction.aspx?key=ensur.477593.S2R4ElA3.2021020 2.9744.4159617.).

[0085] Red cell distribution width may be measured by using any suitable method that is known in the art. Conventionally, red cell distribution width is measured by obtaining a standard complete blood cell count and dividing the standard deviation of the mean corpuscular volume (MCV) by the MCV and multiplying by 100 to yield a percentage value to be on behalf of the red blood cell size heterogeneity. See, for example, Benie T. Constantino, ART, MLT(CSMLS), SH(ASCP)I, Red Cell Distribution Width, Revisited, Laboratory Medicine, Volume 44, Issue 2, May 2013, Pages e2-e9.

[0086] Alkaline phosphatase may be measured by using any suitable method that is known in the art, including colorimetric immunoassays. An example of a conventional assay is the Abeam Alkaline Phosphatase Assay Kit (ab83369; https://www.abcam.com/ps/products/83/ab83369/documents/alkaline-phosphatase-assay-kit- protocol-book-vl2-ab83369%20(website).pdf).

[0087] Alanine aminotransferase may be measured by using any suitable method that is known in the art, including colorimetric assays. An example of a conventional assay is the Sigma- Aldrich Alanine Aminotransferase Activity Assay Kit (MAK052; https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/731/802/ma k052bul.pdf).

[0088] In some embodiments, the at least two biomarkers are measured using the same type of quantification method (for example, one or more immunoassays, or one or more cell counts and parameter analysis, etc.). In other embodiments, the at least two biomarkers are measured using different types of quantification method (for example, a first biomarker is quantified using an immunoassay and a second biomarker is quantified using a cell count and parameter analysis). For example, HbAlc may be quantified using an immunoassay and RDW may be quantified using a complete blood cell count and dividing the standard deviation of the mean corpuscular volume (MCV) by the MCV and multiplying by 100.

[0089] The one or more samples may be any suitable sample(s) obtained from the subject. The number and type of samples being obtained will vary depending on the biomarkers being quantified. For example, if the at least two biomarkers are to be quantified in blood, then a single blood sample is required; whereas if a first biomarker is to be quantified in blood and a second biomarker is to be quantified in urine, then both blood and urine samples are required. [0090] In some embodiments, the one or more samples comprise one sample obtained from the subject. In some embodiments, the one or more samples comprise at least two samples obtained from the subject. In various embodiments, the one or more samples comprise a plurality of samples obtained from the subject. The samples may be the same type of sample or different types of sample. For example, the one or more samples may comprise a blood sample and a urine sample.

[0091] Typically, the one or more samples are fluid biological samples, such as blood, serum, urine, plasma, saliva, sebum, sputum, tears, or a combination thereof. Typically, the one or more samples are selected from a blood sample, a serum sample, a urine sample, another fluid biological sample or a combination thereof. In other embodiments, the one or more samples comprises a tissue sample. More typically, the one or more samples are selected from a blood sample, a serum sample, a urine sample or a combination thereof.

[0092] The one or more samples may be obtained from the subject in any manner. Procedures for obtaining various types of sample are well known and described in the art. Typically, the one or more samples are obtained from the subject using routine clinical procedures. In various embodiments, the method further comprises obtaining the one or more samples from the subject. In some embodiments, the method further comprises the step of processing the one or more samples obtained from the subject. For example, a blood sample may be obtained from a subject, allowed to coagulate and processed by centrifugation to separate the liquid supernatant to provide a serum sample.

[0093] In the method, the subject is identified as being at increased risk of having rapid progression of diabetic retinopathy if the quantified values of the at least two biomarkers are statistically different compared to a control. ” Statistically different” may include statistically increased and statistically decreased. Various methods for computing statistical difference are known in the art and include, among others, t-test, ANOVA, Kruskal-Wallis, Wilcoxon, Mann- Whitney and odds ratio. [0094] The control may be any suitable control. Typically, the control is a reference standard, an average value obtained from healthy subjects, or an average value obtained from subjects with diabetic retinopathy who are not at increased risk of having rapid progression of diabetic retinopathy. In certain embodiments, the control is an average value obtained from subjects with diabetic retinopathy who are not at increased risk of having rapid progression of diabetic retinopathy. In some embodiments, this may be substituted with a predetermined value functioning as a threshold.

[0095] When referring to “a control”, it is typically understood that each biomarker will be compared against its own control. For example, a quantified value of hemoglobin Ale will be statistically different compared to a control value or range of hemoglobin Ale, whereas a quantified value of the ratio of albumin to creatinine will be statistically different compared to a control value or range of the ratio of albumin to creatinine. Example control ranges from healthy subjects for various biomarkers are provided below.

Hemoglobin Ale 4-5.6%

Ratio of albumin to creatinine<30 mg/g

Red cell distribution width 12.2-16.1% in females; 11.8-14.5% in males

Alanine aminotransferase 4-36 U/L

Alkaline phosphatase 44-147 IU/L

[0096] In various embodiments, the method may comprise the step of comparing each of the quantified values of the biomarkers to their respective controls (for example, a quantified value or average value of the biomarker in a control sample or group of control samples), and calculating whether there is a statistical difference.

[0097] In some embodiments, the quantified values of the at least two biomarkers are statistically different compared to a control (in other words, their respective controls) with a p value of less than 0.05, optionally less than 0.01, optionally less than 0.005.

[0098] In some embodiments, the method further comprises determining that a visual acuity score of the subject is below a predetermined value. Visual acuity scores and methods for determining a visual acuity score are known in the art. Common tests for determining visual acuity scores include the Snellen test, the random E test, the Sloan acuity test or the Early Treatment Diabetic Retinopathy Study (ETDRS) test (Kniestedt C, Stamper RL. Visual acuity and its measurement. Ophthalmol Clin North Am. 2003 Jun; 16(2): 155-70, v. doi: 10.1016/s0896-1549(03)00013-0. PMID: 12809155; Ranibizumab (Lucentis): Visual Impairment due to Choroidal Neovascularization Secondary to Pathologic Myopia [Internet]. Ottawa (ON): Canadian Agency for Drugs and Technologies in Health; 2015 Aug. APPENDIX 4, VALIDITY OF OUTCOME MEASURES. Available from: https://www.ncbi.nlm.nih.gov/books/NBK349531/).

[0099] In some embodiments, the pre-determined value of the visual acuity score is 80 using the ETDRS letters system or 20/25 using the Snellen system. In various embodiments, the predetermined value of the visual acuity score is 65 using the ETDRS letters system or 20/50 using the Snellen system.

[0100] A. Hemoglobin Ale (HbAlc) and the ratio of albumin to creatinine (ACR) as a biomarker combination for identifying a subject as being a rapid progressor of diabetic retinopathy

[0101] Provided herein is a method of identifying a subject as being at increased risk of having rapid progression of diabetic retinopathy. The method comprises quantifying at least two biomarkers in one or more samples obtained from the subject, and identifying the subject as being at increased risk of having rapid progression of diabetic retinopathy if the quantified values of the at least two biomarkers are statistically different compared to a control. In some embodiments, the at least two biomarkers are hemoglobin Ale and the ratio of albumin to creatinine. The subject may be identified as being at increased risk of having rapid progression of diabetic retinopathy when the quantified values of hemoglobin Ale and the ratio of albumin to creatinine are statistically increased relative to the control.

[0102] The biomarkers may be quantified in any suitable sample. Typically, hemoglobin Ale is quantified in blood. Typically, the ratio of albumin to creatinine is quantified in urine.

[0103] Hemoglobin Ale percentage of total hemoglobin may be measured using any technique known in the art. Typically, hemoglobin Ale percentage of total hemoglobin is measured using the A1C test which is a common blood test.

[0104] The ratio of albumin to creatinine may be measured using any technique known in the art. Typically, the ratio of albumin to creatinine is measured using the albumin to creatinine ratio test which is well known in the art.

[0105] In various embodiments, the quantified value of hemoglobin Ale is statistically increased relative to the control when the quantified value of hemoglobin Ale is greater than a pre-determined value. The pre-determined value may be any value that provides a statistically significant threshold.

[0106] In some embodiments, the pre-determined value of hemoglobin Ale is 7.0% of total hemoglobin. In many embodiments, the pre-determined value of hemoglobin Ale is 7.25% of total hemoglobin. In some embodiments, the pre-determined value of hemoglobin Ale is 7.65% of total hemoglobin. In various embodiments, the pre-determined value of hemoglobin Ale is 8.25% of total hemoglobin. Typically, the pre-determined value of hemoglobin Ale is 7.7%.

[0107] In various embodiments, the quantified value of the ratio of albumin to creatinine is statistically increased relative to the control when the quantified value of the ratio of albumin to creatinine is greater than a pre-determined value.

[0108] In some embodiments, the pre-determined value of the ratio of albumin to creatinine is 50 mg albumin to 1g creatinine (50: 1 mg/g). In many embodiments, the pre-determined value of the ratio of albumin to creatinine is 60 mg albumin to 1 g creatinine (60: 1 mg/g). In various embodiments, the pre-determined value of the ratio of albumin to creatinine is 70 mg albumin to 1 g creatinine (70: 1 mg/g). In particular embodiments, the pre-determined value of the ratio of albumin to creatinine is 57 mg albumin to 1g creatinine (57: 1 mg/g). In some embodiments, the pre-determined value of the ratio of albumin to creatinine is 92 mg albumin to 1 g creatinine (92: 1 mg/g). Typically, the pre-determined value of the ratio of albumin to creatinine is 60 mg albumin to 1 g creatinine (60: 1 mg/g).

[0109] In particular embodiments, the at least two biomarkers are hemoglobin Ale and the ratio of albumin to creatinine, and the subject is identified as being at increased risk of having rapid progression of diabetic retinopathy when the quantified value of hemoglobin Ale is greater than or equal to 7.65% of total hemoglobin, and the quantified value of the ratio of albumin to creatinine is greater than or equal to 57 mg albumin to 1g creatinine.

[0110] In particular embodiments, the at least two biomarkers are hemoglobin Ale and the ratio of albumin to creatinine, and the subject is identified as being at increased risk of having rapid progression of diabetic retinopathy when the quantified value of hemoglobin Ale is greater than or equal to 8.25% of total hemoglobin, and the quantified value of the ratio of albumin to creatinine is greater than or equal to 92 mg albumin to 1g creatinine.

[0111] In particular embodiments, the at least two biomarkers are hemoglobin Ale and the ratio of albumin to creatinine, and the subject is identified as being at increased risk of having rapid progression of diabetic retinopathy when the quantified value of hemoglobin Ale is greater than or equal to 7.7% of total hemoglobin, and the quantified value of the ratio of albumin to creatinine is greater than or equal to 60 mg albumin to 1g creatinine.

[0112] In particular embodiments, the at least two biomarkers are hemoglobin Ale and the ratio of albumin to creatinine, and the subject is identified as being at increased risk of having rapid progression of diabetic retinopathy when the quantified value of hemoglobin Ale in blood is greater than or equal to 7.7% of total hemoglobin, and the quantified value of the ratio of albumin to creatinine in urine is greater than or equal to 60 mg albumin to 1g creatinine.

[0113] As indicated above, the method may further comprise determining that a visual acuity score of the subject is below a predetermined value. In some embodiments, the predetermined value of the visual acuity score is 80 using the ETDRS letters system or 20/25 using the Snellen system. In various embodiments, the predetermined value of the visual acuity score is 65 using the ETDRS letters system or 20/50 using the Snellen system.

[0114] B. Red cell distribution width (RDW) and the ratio of albumin to creatinine (ACR) as a biomarker combination for identifying a subject as being a rapid progressor of diabetic retinopathy

[0115] Provided herein is a method of identifying a subject as being at increased risk of having rapid progression of diabetic retinopathy. The method comprises quantifying at least two biomarkers in one or more samples obtained from the subject, and identifying the subject as being at increased risk of having rapid progression of diabetic retinopathy if the quantified values of the at least two biomarkers are statistically different compared to a control. In some embodiments, the at least two biomarkers are red cell distribution width and the ratio of albumin to creatinine. The subject may be identified as being at increased risk of having rapid progression of diabetic retinopathy when the quantified value of the red cell distribution width is statistically decreased relative to the control and the quantified value of the ratio of albumin to creatinine is statistically increased relative to the control.

[0116] The biomarkers may be quantified in any suitable sample. Typically, red cell distribution width is quantified in blood. Typically, the ratio of albumin to creatinine is quantified in urine.

[0117] Red cell distribution width is a measure of the range of variation of red blood cell volume. Red cell distribution width may be measured using any technique known in the art. Typically, red cell distribution width is reported as part of a standard complete blood count.

[0118] The ratio of albumin to creatinine may be measured using any technique known in the art. Typically, the ratio of albumin to creatinine is measured using the albumin to creatinine ratio test which is well known in the art.

[0119] In various embodiments, the quantified value of the red cell distribution width is statistically decreased relative to the control when the quantified value of the red cell distribution width is lower than a pre-determined value.

[0120] In some embodiments, the pre-determined value of the red cell distribution width is 14.5%. In many embodiments, the pre-determined value of the red cell distribution width is 13.5%. In particular embodiments, the pre-determined value of the red cell distribution width is 14.0%.

[0121] In various embodiments, the quantified value of the ratio of albumin to creatinine is statistically increased relative to the control when the quantified value of the ratio of albumin to creatinine is greater than a pre-determined value.

[0122] In some embodiments, the pre-determined value of the ratio of albumin to creatinine is 80 mg albumin to 1g creatinine (80: 1 mg/g). In many embodiments, the pre-determined value of the ratio of albumin to creatinine is 90: 1 mg/g. In various embodiments, the pre-determined value of the ratio of albumin to creatinine is 100: 1 mg/g. In particular embodiments, the predetermined value of the ratio of albumin to creatinine is 92 mg albumin to 1g creatinine.

[0123] In particular embodiments, the at least two biomarkers are red cell distribution width and the ratio of albumin to creatinine, and the subject is identified as being at increased risk of having rapid progression of diabetic retinopathy when the quantified value of the red cell distribution width is lower than or equal to 14.5% and the quantified value of the ratio of albumin to creatinine is greater than or equal to 80 mg albumin to 1g creatinine.

[0124] In particular embodiments, the at least two biomarkers are red cell distribution width and the ratio of albumin to creatinine, and the subject is identified as being at increased risk of having rapid progression of diabetic retinopathy when the quantified value of the red cell distribution width is lower than or equal to 14.0% and the quantified value of the ratio of albumin to creatinine is greater than or equal to 92 mg albumin to 1g creatinine.

[0125] In particular embodiments, the at least two biomarkers are red cell distribution width and the ratio of albumin to creatinine, and the subject is identified as being at increased risk of having rapid progression of diabetic retinopathy when the quantified value of the red cell distribution width in blood is lower than or equal to 14.0% and the quantified value of the ratio of albumin to creatinine in urine is greater than or equal to 92 mg albumin to 1g creatinine.

[0126] As indicated above, the method may further comprise determining that a visual acuity score of the subject is below a predetermined value. In some embodiments, the predetermined value of the visual acuity score is 80 using the ETDRS letters system or 20/25 using the Snellen system. In various embodiments, the predetermined value of the visual acuity score is 65 using the ETDRS letters system or 20/50 using the Snellen system.

[0127] C. Hemoglobin Ale (HbAlc), alanine aminotransferase (ALT), and no treatment with a 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor as a biomarker combination for identifying a subject as being a rapid progressor of diabetic retinopathy [0128] Provided herein is a method of identifying a subject as being at increased risk of having rapid progression of diabetic retinopathy. The method comprises quantifying at least two biomarkers in one or more samples obtained from the subject, and identifying the subject as being at increased risk of having rapid progression of diabetic retinopathy if the quantified values of the at least two biomarkers are statistically different compared to a control. In some embodiments, the at least two biomarkers are hemoglobin Ale and alanine aminotransferase, and wherein the subject is not being treated with a 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor. The subject may be identified as being at increased risk of having rapid progression of diabetic retinopathy when the quantified value of hemoglobin Ale is statistically increased relative to the control and the quantified value of alanine aminotransferase is statistically decreased relative to the control.

[0129] The subject may be categorised as not being treated with a 3-hydroxy-3-methyl- glutaryl-coenzyme A (HMG-CoA) reductase inhibitor if they have not been administered a 3- hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor in the 180 days preceding the method being carried out. 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG- CoA) reductase inhibitors include, for example, atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin. The subject may be categorised as not being treated with a 3 -hydroxy-3 -methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor if they have not been administered a 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor in the year preceding the method being carried out.

[0130] The biomarkers may be quantified in any suitable sample. Typically, hemoglobin Ale is quantified in blood. Typically, alanine aminotransferase is quantified in blood.

[0131] Hemoglobin Ale percentage of total hemoglobin may be measured using any technique known in the art. Typically, hemoglobin Ale percentage of total hemoglobin is measured using the A1C test which is a common blood test.

[0132] Alanine aminotransferase may be measured using any technique known in the art. Typically, alanine aminotransferase is measured using the alanine aminotransferase test which is a common blood test.

[0133] In various embodiments, the quantified value of hemoglobin Ale is statistically increased relative to the control when the quantified value of hemoglobin Ale is greater than a pre-determined value.

[0134] In some embodiments, the pre-determined value of hemoglobin Ale is 7.0% of total hemoglobin. In many embodiments, the pre-determined value of hemoglobin Ale is 8.0% of total hemoglobin. In various embodiments, the pre-determined value of hemoglobin Ale is 9.0% of total hemoglobin. In particular embodiments, the pre-determined value of hemoglobin Ale is 7.85% of total hemoglobin.

[0135] In various embodiments, the quantified value of alanine aminotransferase is statistically decreased relative to the control when the quantified value of alanine aminotransferase is lower than a pre-determined value.

[0136] In some embodiments, the pre-determined value of alanine aminotransferase is 28.0 IU/L. In many embodiments, the pre-determined value of alanine aminotransferase is 26.0 IU/L. In various embodiments, the pre-determined value of alanine aminotransferase is 24.0 IU/L. In some embodiments, the pre-determined value of alanine aminotransferase is 22.0 IU/L. In particular embodiments, the pre-determined value of alanine aminotransferase is 24.5 IU/L.

[0137] In particular embodiments, the at least two biomarkers are hemoglobin Ale and alanine aminotransferase, and the subject is not being treated with a 3-hydroxy-3-methyl-glutaryl- coenzyme A (HMG-CoA) reductase inhibitor, and the subject is identified as being at increased risk of having rapid progression of diabetic retinopathy when the quantified value of hemoglobin Ale is greater than or equal to 7.0% of total hemoglobin and the quantified value of alanine aminotransferase is lower than or equal to 28.0 IU/L.

[0138] In particular embodiments, the at least two biomarkers are hemoglobin Ale and alanine aminotransferase, and the subject is not being treated with a 3-hydroxy-3-methyl-glutaryl- coenzyme A (HMG-CoA) reductase inhibitor, and the subject is identified as being at increased risk of having rapid progression of diabetic retinopathy when the quantified value of hemoglobin Ale is greater than or equal to 8.25% of total hemoglobin and the quantified value of alanine aminotransferase is lower than or equal to 24.5 IU/L.

[0139] In particular embodiments, the at least two biomarkers are hemoglobin Ale and alanine aminotransferase, and the subject is not being treated with a 3-hydroxy-3-methyl-glutaryl- coenzyme A (HMG-CoA) reductase inhibitor, and the subject is identified as being at increased risk of having rapid progression of diabetic retinopathy when the quantified value of hemoglobin Ale in blood is greater than or equal to 8.25% of total hemoglobin and the quantified value of alanine aminotransferase in blood is lower than or equal to 24.5 IU/L.

[0140] As indicated above, the method may further comprise determining that a visual acuity score of the subject is below a predetermined value. In some embodiments, the predetermined value of the visual acuity score is 80 using the ETDRS letters system or 20/25 using the Snellen system. In various embodiments, the predetermined value of the visual acuity score is 65 using the ETDRS letters system or 20/50 using the Snellen system.

[0141] D. Alkaline phosphatase (ALKP) and the ratio of albumin to creatinine (ACR) as a biomarker combination for identifying a subject as being a rapid progressor of diabetic retinopathy

[0142] Provided herein is a method of identifying a subject as being at increased risk of having rapid progression of diabetic retinopathy. The method comprises quantifying at least two biomarkers in one or more samples obtained from the subject, and identifying the subject as being at increased risk of having rapid progression of diabetic retinopathy if the quantified values of the at least two biomarkers are statistically different compared to a control. In some embodiments, the at least two biomarkers are alkaline phosphatase and the ratio of albumin to creatinine. The subject may be identified as being at increased risk of having rapid progression of diabetic retinopathy when the quantified values of alkaline phosphatase and the ratio of albumin to creatinine are statistically increased relative to the control.

[0143] The biomarkers may be quantified in any suitable sample. Typically, alkaline phosphatase is quantified in blood. Typically, the ratio of albumin to creatinine is quantified in urine.

[0144] Alkaline phosphatase may be measured using any technique known in the art. Typically, alkaline phosphatase is measured using the alkaline phosphatase (ALP) test, which is a common blood test.

[0145] The ratio of albumin to creatinine may be measured using any technique known in the art. Typically, the ratio of albumin to creatinine is measured using the albumin to creatinine ratio test which is well known in the art.

[0146] In various embodiments, the quantified value of alkaline phosphatase is statistically increased relative to the control when the quantified value of alkaline phosphatase is greater than a pre-determined value.

[0147] In some embodiments, the pre-determined value of alkaline phosphatase is 72.0 IU/L. In many embodiments, the pre-determined value of alkaline phosphatase is 75.0 IU/L. In various embodiments, the pre-determined value of alkaline phosphatase is 80.0 IU/L. In other embodiments, the pre-determined value of alkaline phosphatase is 85.0 IU/L. In particular embodiments, the pre-determined value of alkaline phosphatase is 79.0 IU/L.

[0148] In various embodiments, the quantified value of the ratio of albumin to creatinine is statistically increased relative to the control when the quantified value of the ratio of albumin to creatinine is greater than a pre-determined value. [0149] In some embodiments, the pre-determined value of the ratio of albumin to creatinine is 15 mg albumin to 1g creatinine. In many embodiments, the pre-determined value of the ratio of albumin to creatinine is 25 mg albumin to 1g creatinine. In particular embodiments, the predetermined value of the ratio of albumin to creatinine is 20 mg albumin to 1 g creatinine.

[0150] In particular embodiments, the at least two biomarkers are alkaline phosphatase and the ratio of albumin to creatinine, and the subject is identified as being at increased risk of having rapid progression of diabetic retinopathy when the quantified value of alkaline phosphatase is greater than or equal to 72.0 IU/L and the ratio of albumin to creatinine is greater than or equal to 15 mg albumin to 1 g creatinine.

[0151] In particular embodiments, the at least two biomarkers are alkaline phosphatase and the ratio of albumin to creatinine, and the subject is identified as being at increased risk of having rapid progression of diabetic retinopathy when the quantified value of alkaline phosphatase is greater than or equal to 79.0 IU/L and the ratio of albumin to creatinine is greater than or equal to 20 mg albumin to 1 g creatinine.

[0152] In particular embodiments, the at least two biomarkers are alkaline phosphatase and the ratio of albumin to creatinine, and the subject is identified as being at increased risk of having rapid progression of diabetic retinopathy when the quantified value of alkaline phosphatase in blood is greater than or equal to 79.0 IU/L and the ratio of albumin to creatinine in urine is greater than or equal to 20 mg albumin to 1 g creatinine.

[0153] As indicated above, the method may further comprise determining that a visual acuity score of the subject is below a predetermined value. In some embodiments, the predetermined value of the visual acuity score is 80 using the ETDRS letters system or 20/25 using the Snellen system. In various embodiments, the predetermined value of the visual acuity score is 65 using the ETDRS letters system or 20/50 using the Snellen system.

[0154] E. Further combinations

[0155] All possible combinations of the claimed biomarkers are envisaged, including the following:

1. HbAlc and ACR (both statistically different compared to a control)

2. >7.0% HbAlc and >50: 1 ACR, and optionally <80 VA score, and optionally <65 VA score

3. >7.0% HbAlc and >60: 1 ACR, and optionally <80 VA score, and optionally <65 VA score

4. >7.7% HbAlc and >50: 1 ACR, and optionally <80 VA score, and optionally <65 VA score >7.7% HbAlc and >60: 1 ACR, and optionally <80 VA score, and optionally <65 VA score RDW and ACR (both statistically different compared to a control) <14.5% RDW and >80: 1 ACR, and optionally <80 VA score, and optionally <65 VA score <14.5% RDW and >92: 1 ACR, and optionally <80 VA score, and optionally <65 VA score <14.0% RDW and >80: 1 ACR, and optionally <80 VA score, and optionally <65 VA score <14.0% RDW and >92: 1 ACR, and optionally <80 VA score, and optionally <65 VA score HbAlc and ALT (both statistically different compared to a control) and no treatment with a 3 -hydroxy-3 -methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor >7.0% HbAlc and <28.0 IU/L ALT and no treatment with a 3 -hydroxy- 3 -methyl- glutaryl-coenzyme A (HMG-CoA) reductase inhibitor, and optionally <80 VA score, and optionally <65 VA score >7.0% HbAlc and <24.5 IU/L ALT and no treatment with a 3 -hydroxy- 3 -methyl- glutaryl-coenzyme A (HMG-CoA) reductase inhibitor, and optionally <80 VA score, and optionally <65 VA score >7.85% HbAlc and <28.0 IU/L ALT and no treatment with a 3 -hydroxy-3 -methyl- glutaryl-coenzyme A (HMG-CoA) reductase inhibitor, and optionally <80 VA score, and optionally <65 VA score >7.85% HbAlc and <24.5 IU/L ALT and no treatment with a 3 -hydroxy-3 -methyl- glutaryl-coenzyme A (HMG-CoA) reductase inhibitor, and optionally <80 VA score, and optionally <65 VA score ALKP and ACR (both statistically different compared to a control) >72.0 IU/L ALKP and >15: 1 ACR, and optionally <80 VA score, and optionally <65 VA score >72.0 IU/L ALKP and >20: 1 ACR, and optionally <80 VA score, and optionally <65 VA score >79.0 IU/L ALKP and >15: 1 ACR, and optionally <80 VA score, and optionally <65 VA score >79.0 IU/L ALKP and >20: 1 ACR, and optionally <80 VA score, and optionally <65 VA score 21. Any of combinations 1-5 or 10-20, and RDW, and optionally <14.5% RDW, and optionally <14.0% RDW.

22. Any of combinations 1-10 or 16-20, and ALT, and optionally <24.5 IU/L ALT, and optionally <28.0 IU/L ALT

23. Any of combinations 1-15, and ALKP, and optionally >72.0 IU/L ALKP, and optionally >79.0 IU/L ALKP

24. Any of combinations 1-15, and ACR, and optionally >15: 1 ACR, and optionally >20: 1 ACR.

25. Any of combinations 1-5 and any of combinations 6-20

26. Any of combinations 1-5 and any of combinations 6-10 and any of combinations 11-20

27. Any of combinations 1-5 and any of combinations 11-15 and any of combinations 6-10 or 16-20

28. Any of combinations 1-5 and any of combinations 16-20 and any of combinations 6-15

29. Any of combinations 6-10 and any of combinations 1-5 and 11-20

30. Any of combinations 6-10 and any of combinations 11-15 and any of combinations 1- 5 or 16-20

31. Any of combinations 6-10 and any of combinations 16-20 and any of combinations 1- 5 or 11-15

32. Any of combinations 11-15 and any of combinations 1-10 and 16-20.

33. Any of combinations 1-5 and any of combinations 6-10 and any of combinations 11-15 and any of combinations 16-20.

34. Any of combinations 1-5 OR any of combinations 6-10 OR any of combinations 11-15 OR any of combinations 16-20.

[0156] The above description is applicable for each of the above combinations of combinations.

[0157] In some embodiments, the at least two biomarkers comprise hemoglobin Ale, the ratio of albumin to creatinine, red cell distribution width, alanine aminotransferase, alkaline phosphatase, and wherein the subject is not being treated with a 3-hydroxy-3-methyl-glutaryl- coenzyme A (HMG-CoA) reductase inhibitor.

[0158] In some embodiments, the at least two biomarkers comprise hemoglobin Ale, the ratio of albumin to creatinine, red cell distribution width, alanine aminotransferase, alkaline phosphatase, visual acuity, and wherein the subject is not being treated with a 3-hydroxy-3- methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor. [0159] IV. Overview of Methods of Treatment or Prevention

[0160] Provided herein is a method of treating diabetic retinopathy in a subject or preventing progression of diabetic retinopathy in a subject. The method comprises identifying the subject as being at increased risk of having rapid progression of diabetic retinopathy according to any of the embodiments described herein, and administering a therapy to the subject, thereby treating diabetic retinopathy in the subject or preventing progression of diabetic retinopathy in the subject.

[0161] Also provided herein is a method of treating diabetic retinopathy in a subject or preventing progression of diabetic retinopathy in a subject. The method comprises selecting a subject having quantified values of at least two biomarkers that are statistically different compared to a control. The method further comprises administering a therapy to the subject, thereby treating diabetic retinopathy in the subject or preventing progression of diabetic retinopathy in the subject. The at least two biomarkers comprise:

(i) hemoglobin Ale and the ratio of albumin to creatinine; and/or

(ii) red cell distribution width and the ratio of albumin to creatinine; and/or

(iv) alkaline phosphatase and the ratio of albumin to creatinine.

[0162] The above description in relation to the biomarker combinations and methods of identifying a subject as being at increased risk of having rapid progression of diabetic retinopathy is equally applicable to the methods of treatment or prevention.

[0163] Treating diabetic retinopathy in a subject means that one or more symptoms of diabetic retinopathy are ameliorated. It does not necessarily mean that the symptoms of diabetic retinopathy are completely remedied so that they are no longer present in the patient, although in some methods, this may be the case. Treating diabetic retinopathy in the subject results in one or more of the symptoms of diabetic retinopathy being less severe than before treatment.

[0164] Preventing progression of diabetic retinopathy in a subject means that the subject does not advance, or does not advance as quickly, to a stage of diabetic retinopathy or a related disease which threatens the subject’s vision, for example, DME. In various embodiments, preventing progression of diabetic retinopathy in a subject is preventing the subject progressing, or slowing progression in the subject, to proliferative diabetic retinopathy and/or diabetic macular edema (DME). In some embodiments, preventing progression of diabetic retinopathy in a subject is stabilizing the stage of the diabetic retinopathy such that the disease does not progress, or does not progress as quickly, to a stage of diabetic retinopathy or a related disease which threatens the subject’s vision, for example, DME.

[0165] In certain embodiments, treating diabetic retinopathy in the subject or preventing progression of diabetic retinopathy in the subject results in the prevention of the subject developing a vision threatening disease.

[0166] Administering a therapy to a subject means to give, apply or bring the therapy into contact with the subject. Administration can be accomplished by any of a number of routes depending on the therapeutic agent being administered, and include, for example, intravitreal, conjunctival, intracorneal, intraocular, intravenous, ophthalmic, topical, oral, intranasal, subcutaneous, intramuscular and intraperitoneal.

[0167] Typically, administering the therapy to the subject involves administering a therapeutically effective amount of the therapy to the subject.

[0168] The therapy may be any suitable therapy for treating or preventing diabetic retinopathy. In some embodiments, the therapy is an immunomodulatory therapy. In various embodiments, the immunomodulatory therapy is selected from the group consisting of an inhibitor of Rho- associated kinase, an anti-VEGF therapy, a plasma kallikrein inhibitor, a pan-RGD integrin antagonist, tonabersat, an inhibitor of apurinic/apyrimidinic endonuclease 1/reduction- oxidation (redox) effector factor-1 (APEl/Ref-1; APEX1), runcaciguat, lazucirnon, RG-7774, Emixustat hydrochloride, brolucizumab, EGT022, OTT166, EOM147, ST2666, BSL01, BI764524, MHU650 or a combination thereof. In some embodiments, the immunomodulatory therapy is selected from the group consisting of an anti-VEGF therapy, an inhibitor of Rho- associated kinase, or a combination thereof. In particular embodiments, the immunomodulatory therapy is an inhibitor of Rho-associated kinase. In certain embodiments, the immunomodulatory therapy is a 6-substituted- 1 -(2//)-isoquinolinone compound of formula (I)

wherein n is 1, 2, 3, or 4 and Ri is H, a Ci-Ce alkyl group, or a pharmaceutically acceptable salt thereof. In certain embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof exists in hydrate form. In certain embodiments, the immunomodulatory therapy is 6-(4-piperidinyloxy)-l(2H)-isoquinolinone or a pharmaceutically acceptable salt thereof. In certain embodiments, the 6-(4-piperidinyloxy)-l(2H)-isoquinolinone or a pharmaceutically acceptable salt thereof exists in hydrate form. In certain embodiments, the immunomodulatory therapy is 6-(4-piperidinyloxy)-l(2H)-isoquinolinone hydrochloride dihydrate.

[0169] In some embodiments, the immunomodulatory therapy is a combination of a 6- s ubsti tutcd- l -(2/7)-isoc|ui noli none compound of formula (I) or a pharmaceutically acceptable salt thereof and an anti-VEGF therapy. In certain embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof exists in hydrate form. In certain embodiments, the immunomodulatory therapy is a combination of 6-(4-piperidinyloxy)-l(2H)- isoquinolinone or a pharmaceutically acceptable salt thereof and an anti-VEGF therapy. In certain embodiments, the 6-(4-piperidinyloxy)-l(2H)-isoquinolinone or a pharmaceutically acceptable salt thereof exists in hydrate form. In certain embodiments, the immunomodulatory therapy is a combination of 6-(4-piperidinyloxy)-l(2H)-isoquinolinone hydrochloride dihydrate and an anti-VEGF therapy. The term “anti-VEGF therapy” includes monoclonal antibodies directed to VEGF (for example, bevacizumab), anti-VEGF antibody biopolymer conjugates (such as KSI-301), anti-VEGF antibody derivatives or fragments such as ranibuzumab, small molecules that inhibit the tyrosine kinases stimulated by VEGF (for example, sunitinib, sorafenib, axitinib, pazopanib, vorolanib), and vectors containing a gene encoding for a monoclonal antibody directed to VEGF.

[0170] V. Kits

[0171] Provided herein is a kit for quantifying values of at least two biomarkers in a sample obtained from a subject, wherein the kit comprises assay components for quantifying the values of the least two biomarkers, wherein the at least two biomarkers comprise:

(i) hemoglobin Ale and the ratio of albumin to creatinine; and/or

(ii) red cell distribution width and the ratio of albumin to creatinine; and/or

(iv) alkaline phosphatase and the ratio of albumin to creatinine.

[0172] The kit may additionally include control values or ranges for each of the biomarkers being quantified. Such control values or ranges are as defined above. The kit may additionally include instructions for quantifying the values and for comparing the quantified values with the control values or ranges. [0173] The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the disclosure to the particular features or embodiments described.

EXAMPLES

[0174] The inventors have found that particular combinations of biomarkers are effective in predicting whether a patient with diabetic retinopathy is more likely to progress at an accelerated rate in the disease. While various markers are known for diagnosing diabetic retinopathy, it was unexpected that specific combinations of these biomarkers would be able to identify a cohort of patients that, if left untreated, would likely progress quickly to the next stage of the disease, requiring treatment or further treatment. This cohort of patients has been termed “Fast Progressors” as they progress at an accelerated rate through the disease. Accordingly, these biomarker combinations can be used to identify the fast progressors of diabetic retinopathy at an early stage of the disease, thereby enabling healthcare practitioners to administer earlier treatment to these patients, and specifically target the cohort of patients that will likely progress in the disease. This avoids the need to treat all patients earlier on in the disease if they are less likely to rapidly progress through the disease.

[0175] The analysis was conducted using two large databases: the Maccabi database and, to assess the generalizability of the Maccabi data, a second large database, HealthVerity. The Maccabi database is an electronic medical record (EMR) database from one of the largest health maintenance organizations in Israel, representing about 26% of the total Israeli population and spanning the years 2010-2020. The HealthVerity database is a US database including both insurance claims (medical and pharmacy) as well as EMR data from some specialty visits spanning the years 2016-2021. The database represents multiple payer types, with the largest proportion of patients covered by Medicaid (40%).

[0176] The inventors developed the fast progressor algorithm in an incident NPDR cohort and a prevalent NPDR cohort. The incident cohort consisted of patients newly diagnosed with NPDR, whereas the prevalent cohort captured all existing NPDR patients over the course of the study period, regardless of NPDR disease duration. Among patients with newly diagnosed NPDR, rapid progression was associated with higher thresholds of markers for kidney dysfunction (for example, ACR) and metabolic dysfunction (for example, HbAlc), versus the prevalent NPDR cohort with varying NPDR disease duration. [0177] Fast progression was defined as progression to treatment with anti- Vascular endothelial growth factor (VEGF) or photocoagulation within 1-year post-index or a 10-letter drop in visual acuity (VA) score between 6 months and 2 years post-index. Applying this definition, 1,024 or 15.9% of patients in the Maccabi database and 1,494 or 41.9% in the HealthVerity database were identified as fast progressors.

[0178] Considering the wide recognition of the clinical need for anti- VEGF treatment in the moderately severe and severe NPDR population, the 1-year rate of progression in this population was used as a benchmark. Consequently, using the 1-year progression to proliferative diabetic retinopathy (PDR) in the moderately severe population as proxy, the fast progression mild and moderate NPDR population is defined as a population with approximately 25% likelihood of progression in 1-year (Davis, 1998 (ETDRS study)).

[0179] Analyses were conducted using iterative classification and regression trees (CART) to identify combinations of risk factors and relevant thresholds, where applicable, that predicted the risk of fast progression.

[0180] Around 300 patient characteristics were analysed, with 67 variables interrogated. These variables are listed in Table 1.

TABLE 1

[0181] Of the 67 variables, four combinations of biomarkers were identified as being particularly effective at predicting fast progressors with an increased probability. These combinations are discussed in further detail below.

[0182] Albumin creatinine ratio (ACR) > 60 mg/g and hemoglobin Ale (HbAlc) > 7.7%

[0183] In both databases, the inventors identified patients with a prevalent NPDR diagnosis over the period of available data who were treatment naive, met criteria consistent with a “mild” or “moderate” NPDR diagnosis, and did not have other ophthalmic diagnoses. Applying these criteria yielded 6,434 patients in the Maccabi database and 3,562 patients in the HealthVerity database.

[0184] A set of criteria that resulted in a high empirical probability of progression and covered at least 10% of the patient population in the two databases was an albumin creatinine ratio (ACR) > 60 mg/g and HbAlc > 7.7% (rounded from ACR > 57 mg/g and HbAlc > 7.65%).

[0185] As shown in Figure 1, patients with ACR > 60 mg/g or HbAlc > 7.7% had a lower survival probability compared to patients not meeting either of these criteria. Patients with both criteria (ACR > 60 mg/g and HbAlc > 7.7%) had a significantly lower survival probability compared to patients meeting a single criteria, demonstrating the effectiveness of this particular biomarker combination in identifying fast progressors.

[0186] Albumin creatinine ratio (ACR) > 92 mg/g and hemoglobin Ale (HbAlc) > 8.25% [0187] The inventors assessed the incident cohort consisting of patients newly diagnosed with NPDR and identified the same biomarker combination as above.

[0188] Overall, 5.1% of patients with mild NPDR progressed to treatment within 1 year of NPDR diagnosis, and 7.4% of patients with mild NPDR progressed to treatment within 2 years of NPDR diagnosis. As shown in Figure 2, of the subpopulation of patients with ACR > 92 mg/g and HbAlc > 8.25%, 10.8% of these patients progressed to treatment within 1 year, and 15.2% of these patients progressed to treatment within 2 years. Therefore, this biomarker combination identified a subpopulation of patients who progress ~2x more often than the mild NPDR cohort of patients.

[0189] Figure 2 further shows the empirical probability of progression of patients further defined by a visual acuity (VA) score threshold. Of the subpopulation of patients with ACR > 92 mg/g and HbAlc > 8.25% and a VA score < 65, 25% of these patients progressed within 2 years, compared to 7.4% of patients within the mild NPDR cohort. Therefore, this biomarker combination together with a restriction of VA score identified a subpopulation of patients who progress ~3-4x more often than the mild NPDR cohort of patients.

[0190] Red cell distribution width (RDW) < 14.0% and ACR > 92 mg/g

[0191] A further set of criteria that resulted in a high empirical probability of progression from analysis of the incident cohort was a red cell distribution width (RDW) < 14.0% and ACR > 92 mg/g.

[0192] As shown in Figure 3, of the subpopulation of patients with RDW < 14.0% and ACR > 92 mg/g, 12.2% of these patients progressed to treatment within 1 year, and 15.9% of these patients progressed to treatment within 2 years. Therefore, this biomarker combination identified a subpopulation of patients who progress ~2x more often than the mild NPDR cohort of patients.

[0193] Figure 3 further shows the empirical probability of progression of patients further defined by a visual acuity (VA) score threshold. Of the subpopulation of patients with RDW < 14.0% and ACR > 92 mg/g and a VA score < 80, 13.2% of these patients progressed within 1 year and 21.1% of these patients progressed within 2 years. Further restricting the VA score increased the percentage of patients within the subpopulation that progressed to treatment. For example, of the subpopulation of patients with RDW < 14.0% and ACR > 92 mg/g and a VA score < 65, 30% of these patients progressed to treatment within 2 years, compared to 7.4% of patients with mild NPDR. Therefore, this biomarker combination together with a restriction of VA score identified a subpopulation of patients who progress within two years ~3-4x more often than the mild NPDR cohort of patients.

[0194] Alanine aminotransferase (ALT) < 24.5 and HbAlc > 7.85, and wherein the subject is not being treated with a 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor

[0195] A further set of criteria that resulted in a high empirical probability of progression from analysis of the incident cohort was an alanine aminotransferase (ALT) < 24.5 and HbAlc > 7.85, and wherein the subject is not being treated with a 3-hydroxy-3-methyl-glutaryl- coenzyme A (HMG-CoA) reductase inhibitor.

[0196] As shown in Figure 4, of the subpopulation of patients who were not being treated with statins and had ALT < 24.5 and HbAlc > 7.85, 11.0% of these patients progressed to treatment within 1 year, and 15.9% of these patients progressed to treatment within 2 years. Therefore, this biomarker combination identified a subpopulation of patients who progress ~2x more often than the mild NPDR cohort of patients.

[0197] Figure 4 further shows the empirical probability of progression of patients further defined by a visual acuity (VA) score threshold. Of the subpopulation of patients who were not being treated with statins and had ALT < 24.5 and HbAlc > 7.85 and a VA score of <65, 15% of these patients progressed to treatment within 1 year, and 20% of these patients progressed to treatment within 2 years. Therefore, this biomarker combination together with a restriction of VA score identified a subpopulation of patients who progress within two years ~3x more often than the mild NPDR cohort of patients.

[0198] Alkaline phosphatase (ALKP) < 79 and ACR > 20

[0199] A further set of criteria that resulted in a high empirical probability of progression from analysis of the incident cohort was an alkaline phosphatase (ALKP) < 79 and ACR > 20. [0200] As shown in Figure 5, of the subpopulation of patients who had ALKP < 79 and ACR

> 20, 5.3% of these patients progressed to treatment within 1 year, and 8.7% of these patients progressed to treatment within 2 years.

[0201] Figure 5 further shows the empirical probability of progression of patients further defined by a visual acuity (VA) score threshold. Of the subpopulation of patients who had ALKP < 79 and ACR > 20 and VA <80, 11.9% of these patients progressed to treatment within 1 year and 20.2% of these patients progressed to treatment within 2 years. Of the subpopulation of patients who had ALKP < 79 and ACR > 20 and VA <65, 14.8% of these patients progressed to treatment within 1 year and 25.9% of these patients progressed to treatment within 2 years. Therefore, this biomarker combination together with a restriction of VA score identified a subpopulation of patients who progress within two years ~3-4x more often than the mild NPDR cohort of patients.

[0202] RDW < 14.0% and ACR > 92 mg/g; or ALT < 24.5 and HbAlc > 7.85, and wherein the subject is not being treated with a 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor; or ACR > 92 mg/g and HbAlc > 8.25%; or ALKP < 79 and ACR > 20 [0203] It was subsequently found that combining the four biomarker combinations resulted in a higher sample size with an equivalent probability of progression.

[0204] As shown in Figure 6, of the subpopulation of patients who had: RDW < 14.0% and ACR > 92 mg/g; or no statins and ALT < 24.5 and HbAlc > 7.85; or ACR > 92 mg/g and HbAlc > 8.25%; or ALKP < 79 and ACR > 20, 8.2% of these patients progressed to treatment within 1 year, and 11.8% of these patients progressed to treatment within 2 years. Thus, while Figures 2-5 demonstrate that each individual combination of biomarkers provides a good predictor of whether a patient is a fast progressor of DR, combining the four combinations increases the cohort of patients and provides a good predictor of whether a patient is a fast progressor of DR.

[0205] Figure 6 further shows the empirical probability of progression of patients further defined by a visual acuity (VA) score threshold. Of the population of patients who had a VA score <80, and: RDW < 14.0% and ACR > 92 mg/g; or no statins and ALT < 24.5 and HbAlc

> 7.85; or ACR > 92 mg/g and HbAlc > 8.25%; or ALKP < 79 and ACR > 20, 10.5% of these patients progressed to treatment within 1 year, and 17.5% of these patients progressed to treatment within 2 years. Of the population of patients who had a VA score <65, and: RDW < 14.0% and ACR > 92 mg/g; or no statins and ALT < 24.5 and HbAlc > 7.85; or ACR > 92 mg/g and HbAlc > 8.25%; or ALKP < 79 and ACR > 20, 12.2% of these patients progressed to treatment within 1 year, and 22.4% of these patients progressed to treatment within 2 years. Therefore, this biomarker combination together with a restriction of VA score identified a subpopulation of patients who progress within two years ~3x more often than the mild NPDR cohort of patients.

[0206] In summary, the inventors have identified four combinations of biomarkers that can be used to predict whether a patient with DR will progress rapidly through the disease. Further combining these biomarker combinations with visual acuity scores increases the accuracy in which these patients are identified.

[0207] Non-predictive biomarker combinations

Many combinations of biomarkers did not accurately predict fast progressors. A selection of these combinations is provided below.

MPV = Mean Platelet Volume; T1DM = Type 1 Diabetes Mellitus; PLR = Platelet Lymphocyte

Ratio

[0208] In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognised that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

[0209] The subject-matter of the disclosure may also relate, among others, to the following aspects: [0210] A first aspect relates to a method of identifying a subject as being at increased risk of having rapid progression of diabetic retinopathy, wherein the method comprises: quantifying at least two biomarkers in one or more samples obtained from the subject, and identifying the subject as being at increased risk of having rapid progression of diabetic retinopathy if the quantified values of the at least two biomarkers are statistically different compared to a control, wherein the at least two biomarkers comprise: (i) hemoglobin Ale and the ratio of albumin to creatinine; and/or (ii) red cell distribution width and the ratio of albumin to creatinine; and/or (iii) hemoglobin Ale and alanine aminotransferase, and wherein the subject is not being treated with a 3 -hydroxy-3 -methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor; and/or (iv) alkaline phosphatase and the ratio of albumin to creatinine.

[0211] A second aspect relates to the method of aspect 1, wherein the at least two biomarkers are hemoglobin Ale and the ratio of albumin to creatinine, and wherein the subject is identified as being at increased risk of having rapid progression of diabetic retinopathy when the quantified values of hemoglobin Ale and the ratio of albumin to creatinine are statistically increased relative to the control.

[0212] A third aspect relates to the method of any preceding aspect, wherein the quantified value of hemoglobin Ale is statistically increased relative to the control when the quantified value of hemoglobin Ale is greater than a pre-determined value.

[0213] A fourth aspect relates to the method of any preceding aspect, wherein the predetermined value of hemoglobin Ale is 7.0% of total hemoglobin.

[0214] A fifth aspect relates to the method of aspects 1 to 3, wherein the predetermined value of hemoglobin Ale is 7.65% of total hemoglobin.

[0215] A sixth aspect relates to the method of aspects 1 to 3, wherein the predetermined value of hemoglobin Ale is 7.7% of total hemoglobin.

[0216] A seventh aspect relates to the method of aspects 1 to 3, wherein the pre-determined value of hemoglobin Ale is 8.25% of total hemoglobin.

[0217] An eighth aspect relates to the method of any preceding aspect, wherein the quantified value of the ratio of albumin to creatinine is statistically increased relative to the control when the quantified value of the ratio of albumin to creatinine is greater than a pre-determined value. [0218] A ninth aspect relates to the method of aspect 8, wherein the pre-determined value of the ratio of albumin to creatinine is 50 mg albumin to 1g creatinine.

[0219] A tenth aspect relates to the method of aspect 8, wherein the pre-determined value of the ratio of albumin to creatinine is 57 mg albumin to 1g creatinine. [0220] An eleventh aspect relates to the method of aspect 8, wherein the pre-determined value of the ratio of albumin to creatinine is 60 mg albumin to 1g creatinine.

[0221] A twelfth aspect relates to the method of aspect 8, wherein the pre-determined value of the ratio of albumin to creatinine is 92 mg albumin to 1g creatinine.

[0222] A thirteenth aspect relates to the method of any preceding aspect, wherein the at least two biomarkers are red cell distribution width and the ratio of albumin to creatinine, and wherein the subject is identified as being at increased risk of having rapid progression of diabetic retinopathy when the quantified value of the red cell distribution width is statistically decreased relative to the control and the quantified value of the ratio of albumin to creatinine is statistically increased relative to the control.

[0223] A fourteenth aspect relates to the method of any preceding aspect, wherein the quantified value of the red cell distribution width is statistically decreased relative to the control when the quantified value of the red cell distribution width is lower than a pre-determined value.

[0224] A fifteenth aspect relates to the method of aspect 14, wherein the pre-determined value of the red cell distribution with is 14.5%.

[0225] A sixteenth aspect relates to the method of aspect 14, wherein the pre-determined value of the red cell distribution width is 14.0%.

[0226] A seventeenth aspect relates to the method of any preceding aspect, wherein the quantified value of the ratio of albumin to creatinine is statistically increased relative to the control when the quantified value of the ratio of albumin to creatinine is greater than a predetermined value.

[0227] An eighteenth aspect relates to the method of aspect 17, wherein the pre-determined value of the ratio of albumin to creatinine is 80 mg albumin to 1g creatinine.

[0228] A nineteenth aspect relates to the method of aspect 17, wherein the pre-determined value of the ratio of albumin to creatinine is 92 mg albumin to 1g creatinine.

[0229] A twentieth aspect relates to the method of any preceding aspect, wherein the at least two biomarkers are hemoglobin Ale and alanine aminotransferase, and wherein the subject is not being treated with a 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor, and wherein the subject is identified as being at increased risk of having rapid progression of diabetic retinopathy when the quantified value of hemoglobin Ale is statistically increased relative to the control and the quantified value of alanine aminotransferase is statistically decreased relative to the control. [0230] A twenty-first aspect relates to the method of any preceding aspect, wherein the quantified value of hemoglobin Ale is statistically increased relative to the control when the quantified value of hemoglobin Ale is greater than a pre-determined value.

[0231] A twenty-second aspect relates to the method of aspect 21, wherein the pre-determined value of hemoglobin Ale is 7% of total hemoglobin.

[0232] A twenty-third aspect relates to the method of aspect 21, wherein the pre-determined value of hemoglobin Ale is 7.85% of total hemoglobin.

[0233] A twenty-fourth aspect relates to the method of any preceding aspect, wherein the quantified value of alanine aminotransferase is statistically decreased relative to the control when the quantified value of alanine aminotransferase is lower than a pre-determined value.

[0234] A twenty-fifth aspect relates to the method of aspect 24, wherein the pre-determined value of alanine aminotransferase is 28.0 IU/L.

[0235] A twenty-sixth aspect relates to the method of aspect 24, wherein the pre-determined value of alanine aminotransferase is 24.5 IU/L.

[0236] A twenty- seventh aspect relates to the method of any preceding aspect, wherein the at least two biomarkers are alkaline phosphatase and the ratio of albumin to creatinine, and wherein the subject is identified as being at increased risk of having rapid progression of diabetic retinopathy when the quantified values of alkaline phosphatase and the ratio of albumin to creatinine are statistically increased relative to the control.

[0237] A twenty-eighth aspect relates to the method of any preceding aspect, wherein the quantified value of alkaline phosphatase is statistically increased relative to the control when the quantified value of alkaline phosphatase is greater than a pre-determined value.

[0238] A twenty-ninth aspect relates to the method of aspect 28, wherein the pre-determined value of alkaline phosphatase is 72.0 IU/L.

[0239] A thirtieth aspect relates to the method of aspect 28, wherein the pre-determined value of alkaline phosphatase is 79.0 IU/L.

[0240] A thirty-first aspect relates to the method of any preceding aspect, wherein the quantified value of the ratio of albumin to creatinine is statistically increased relative to the control when the quantified value of the ratio of albumin to creatinine is greater than a predetermined value.

[0241] A thirty-second aspect relates to the method of aspect 31, wherein the pre-determined value of the ratio of albumin to creatinine is 20 mg albumin to 1g creatinine. [0242] A thirty-third aspect relates to the method of any preceding aspect, wherein the method further comprises: determining that a visual acuity score of the subject is below a predetermined value.

[0243] A thirty-fourth aspect relates to the method of aspect 33, wherein the predetermined value of the visual acuity score is 80 using the ETDRS letters system or 20/25 using the Snellen system.

[0244] A thirty-fifth aspect relates to the method of aspect 34, wherein the predetermined value of the visual acuity score is 65 using the ETDRS letters system or 20/50 using the Snellen system.

[0245] A thirty-sixth aspect relates to the method of any preceding aspect, wherein the control is a reference standard, an average value obtained from healthy subjects, or an average value obtained from subjects with diabetic retinopathy who are not at increased risk of having rapid progression of diabetic retinopathy.

[0246] A thirty- seventh aspect relates to the method of any preceding aspect, further comprising: obtaining the one or more samples from the subject.

[0247] A thirty-eighth aspect relates to the method of aspect 37, wherein the one or more samples are selected from a blood sample, a serum sample, a urine sample, another fluid biological sample or a combination thereof.

[0248] A thirty-ninth aspect relates to the method of any preceding aspect, wherein the subject is a human.

[0249] A fortieth aspect relates to the method of aspects 1 to 39, wherein the subject is a nonhuman mammal.

[0250] A forty-first aspect relates to the method of any preceding aspect, wherein the subject has mild or moderate non-proliferative diabetic retinopathy.

[0251] A forty-second aspect relates to a method of treating diabetic retinopathy in a subject of preventing progression of diabetic retinopathy in a subject, wherein the method comprises:

(1) identifying the subject as being at increased risk of having rapid progression of diabetic retinopathy if the quantified values of at least two biomarkers are statistically different compared to a control, wherein the at least two biomarkers comprise: (i) hemoglobin Ale and the ratio of albumin to creatinine; and/or (ii) red cell distribution width and the ratio of albumin to creatinine; and/or (iii) hemoglobin Ale and alanine aminotransferase, and wherein the subject is not being treated with a 3 -hydroxy-3 -methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor; and/or (iv) alkaline phosphatase and the ratio of albumin to creatinine; and

(2) administering a therapy to the subject, wherein the therapy is an immunomodulatory therapy capable of treating diabetic retinopathy in the subject or preventing progression of diabetic retinopathy in the subject.

[0252] A forty-third aspect relates to the method of aspect 42, wherein the immunomodulatory therapy is selected from the group consisting of an inhibitor of Rho-associated kinase, an anti- VEGF therapy, a plasma kallikrein inhibitor, a pan-RGD integrin antagonist, tonabersat, an inhibitor of apurinic/apyrimidinic endonuclease 1 /reduction-oxidation (redox) effector factor- 1 (APEl/Ref-1; APEX1), runcaciguat, lazucirnon, RG-7774, Emixustate hydrochloride, brolucizumab, EGT022, OTT166, EOM147, ST2666, BSL01, BI764524, MHU650, and any combination thereof.

[0253] A forty-fourth aspect relates to the method of aspect 42 or 43, wherein the immunomodulatory therapy is an inhibitor of Rho-associated kinase.

[0254] A forty-fifth aspect relates to the method of aspects 42 to 44, wherein the immunomodulatory therapy is a combination of an inhibitor of Rho-associated kinase and an anti-VGF therapy.

[0255] A forty-sixth aspect relates to the method of aspects 42 to 45, wherein the control is a reference standard, an average value obtained from healthy subjects, or an average value obtained form subjects with diabetic retinopathy who are not at increased risk of having rapid progression of diabetic retinopathy.

[0256] A forty-seventh aspect relates to the method of aspects 42 to 46, the method further comprising: obtaining the one or more samples from the subject.

[0257] A forty-eighth aspect relates to the method of aspect 47, wherein the one or more samples are selected from a blood sample, a serum sample, a urine sample, another fluid biological sample, or a combination thereof.

[0258] A forty-ninth aspect relates to the method of aspects 42 to 48, wherein the subject is a human.

[0259] A fiftieth aspect relates to the method of aspects 42 to 48, wherein the subject is a nonhuman mammal.

[0260] A fifty-first aspect relates to the method of aspects 42 to 50, wherein the subject has mild or moderate non-proliferative diabetic retinopathy.

[0261] A fifty-second aspect relates to the method of aspects 42 to 51, wherein administration of the immunomodulatory therapy to the subject is intravitreal, conjunctival, intracorneal, intraocular, intravenous, ophthalmic, topical, oral, intranasal, subcutaneous, intramuscular, or intraperitoneal. [0262] A fifth-third aspect relates to the method of aspects 42 to 52, wherein administration of the immunomodulatory therapy to the subject is intravenous, ophthalmic, topical, oral, intranasal, subcutaneous, intramuscular, or intraperitoneal.

[0263] A fifty-fourth aspect relates to the method of aspects 42 to 53, wherein administration of the immunomodulatory therapy to the subject is topical or oral.

[0264] A fifty-fifth aspect relates to the method of aspects 42 to 54, wherein administration of the immunomodulatory therapy to the subject is oral.

[0265] A fifty-sixth aspect relates to the method of aspects 42 to 55, wherein the at least two biomarkers are hemoglobin Ale and the ratio of albumin to creatinine, and the subject is identified as being at increased risk of having rapid progression of diabetic retinopathy when the quantified value of hemoglobin Ale is greater than or equal to 7.65% of total hemoglobin, and the quantified value of the ratio of albumin to creatinine is greater than or equal to 57 mg albumin to 1g creatinine.

[0266] A fifty-seventh aspect relates to the method of aspects 42 to 55, wherein the at least two biomarkers are hemoglobin Ale and the ratio of albumin to creatinine, and the subject is identified as being at increased risk of having rapid progression of diabetic retinopathy when the quantified value of hemoglobin Ale is greater than or equal to 8.25% of total hemoglobin, and the quantified value of the ratio of albumin to creatinine is greater than or equal to 92 mg albumin to 1g creatinine.

[0267] A fifty-eighth aspect relates to the method of aspects 42 to 55, wherein the at least two biomarkers are hemoglobin Ale and the ratio of albumin to creatinine, and the subject is identified as being at increased risk of having rapid progression of diabetic retinopathy when the quantified value of hemoglobin Ale is greater than or equal to 7.7% of total hemoglobin, and the quantified value of the ratio of albumin to creatinine is greater than or equal to 60 mg albumin to 1g creatinine.

[0268] A fifty-ninth aspect relates to the method of aspects 42 to 55, wherein the at least two biomarkers are hemoglobin Ale and the ratio of albumin to creatinine, and the subject is identified as being at increased risk of having rapid progression of diabetic retinopathy when the quantified value of hemoglobin Ale in blood is greater than or equal to 7.7% of total hemoglobin, and the quantified value of the ratio of albumin to creatinine in urine is greater than or equal to 60 mg albumin to 1g creatinine.

[0269] A sixtieth aspect relates to the method of aspects 42 to 59, wherein the method prevents progression of diabetic retinopathy in the subject. [0270] A sixty-first aspect relates to the method of aspect 60, wherein the method stabilizes the stage of the diabetic retinopathy such that the disease does not progress, or does not progress as quickly, to a stage of diabetic retinopathy or a related disease which threatens the subject’s vision.

[0271] A sixty-second aspect relates to a method of treating diabetic retinopathy in a subject or preventing progression of diabetic retinopathy in a subject, wherein the method comprises: (1) selecting a subject having quantified values of at least two biomarkers that are statistically different compared to a control, wherein the at least two biomarkers comprise: (i) hemoglobin Ale and the ratio of albumin to creatinine; and/or (ii) red cell distribution width and the ratio of albumin to creatinine; and/or (iii) hemoglobin Ale and alanine aminotransferase, and wherein the subject is not being treated with a 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor; and/or (iv) alkaline phosphatase and the ratio of albumin to creatinine; and (2) administering a therapy to the subject, wherein the therapy is an immunomodulatory therapy capable of treating diabetic retinopathy in the subject or preventing progression of diabetic retinopathy in the subject.

[0272] A sixty-third aspect relates to the method of aspect 62, wherein the immunomodulatory therapy is selected from the group consisting of an inhibitor of Rho-associated kinase, an anti- VEGF therapy, a plasma kallikrein inhibitor, a pan-RGD integrin antagonist, tonabersat, an inhibitor of apurinic/apyrimidinic endonuclease 1 /reduction-oxidation (redox) effector factor- 1 (APEl/Ref-1; APEX1), runcaciguat, lazucimon, RG-7774, Emixustat hydrochloride, brolucizumab, EGT022, OTT166, EOM147, ST2666, BSL01, BI764524, MHU650, and any combination thereof.

[0273] A sixty-fourth aspect relates to the method of aspect 62 or 63, wherein the immunomodulatory therapy is an inhibitor of Rho-associated kinase.

[0274] A sixty-fifth aspect relates to the method of aspects 62 to 64, wherein the immunomodulatory therapy is a combination of an inhibitor of Rho-associated kinase and an anti-VEGF therapy.

[0275] A sixty-sixth aspect relates to the method of aspects 62 to 65, wherein the control is a reference standard, an average value obtained from healthy subjects, or an average value obtained from subjects with diabetic retinopathy who are not at increased risk of having rapid progression of diabetic retinopathy.

[0276] A sixty-seventh aspect relates to the method of aspects 62 to 66, the method further comprising: obtaining the one or more samples from the subject. [0277] A sixty-eighth aspect relates to the method of aspect 67, wherein the one or more samples are selected from a blood sample, a serum sample, a urine sample, another fluid biological sample, or a combination thereof.

[0278] A sixty-ninth aspect relates to the method of aspects 62 to 68, wherein the subject is a human.

[0279] A seventieth aspect relates to the method of aspects 62 to 68, wherein the subject is a non-human mammal.

[0280] A seventy-first aspect relates to the method of aspects 62 to 70, wherein the subject has mild or moderate non-proliferative diabetic retinopathy.

[0281] A seventy- second aspect relates to the method of aspects 62 to 71, wherein administration of the immunomodulatory therapy to the subject is intravitreal, conjunctival, intracorneal, intraocular, intravenous, ophthalmic, topical, oral, intranasal, subcutaneous, intramuscular, or intraperitoneal.

[0282] A seventy-third aspect relates to the method of aspects 62 to 72, wherein administration of the immunomodulatory therapy to the subject is intravenous, ophthalmic, topical, oral, intranasal, subcutaneous, intramuscular, or intraperitoneal.

[0283] A seventy-fourth aspect relates to the method of aspects 62 to 73, wherein administration of the immunomodulatory therapy to the subject is topical or oral.

[0284] A seventy-fifth aspect relates to the method of aspects 62 to 74, wherein administration of the immunomodulatory therapy to the subject is oral.

[0285] A seventy-sixth aspect relates to the method of aspects 62 to 75, wherein the at least two biomarkers are hemoglobin Ale and the ratio of albumin to creatinine, and the quantified value of hemoglobin Ale is greater than or equal to 7.65% of total hemoglobin, and the quantified value of the ratio of albumin to creatinine is greater than or equal to 57 mg albumin to 1g creatinine.

[0286] A seventy- seventh aspect relates to the method of aspects 62 to 75, wherein the at least two biomarkers are hemoglobin Ale and the ratio of albumin to creatinine, and the quantified value of hemoglobin Ale is greater than or equal to 8.25% of total hemoglobin, and the quantified value of the ratio of albumin to creatinine is greater than or equal to 92 mg albumin to 1g creatinine.

[0287] A seventy-eighth aspect relates to the method of aspects 62 to 75, wherein the at least two biomarkers are hemoglobin Ale and the ratio of albumin to creatinine, and the quantified value of hemoglobin Ale is greater than or equal to 7.7% of total hemoglobin, and the quantified value of the ratio of albumin to creatinine is greater than or equal to 60 mg albumin to 1g creatinine.

[0288] A seventy-ninth aspect relates to the method of aspects 62 to 75, wherein the at least two biomarkers are hemoglobin Ale and the ratio of albumin to creatinine, and the quantified value of hemoglobin Ale in blood is greater than or equal to 7.7% of total hemoglobin, and the quantified value of the ratio of albumin to creatinine in urine is greater than or equal to 60 mg albumin to 1g creatinine.

[0289] An eightieth aspect relates to the method of aspects 62 to 79, wherein the method prevents progression of diabetic retinopathy in the subject.

[0290] An eighty-first aspect relates to the method of aspects 62 to 80, wherein the method stabilizes the stage of diabetic retinopathy such that the disease does not progress, or does not progress as quickly, to a stage of diabetic retinopathy or a related disease which threatens the subject’s vision.

[0291] An eighty- second aspect relates to a kit for quantifying values of at least two biomarkers in a sample obtained from a subject, wherein the kit comprises assay components for quantifying the values of the at least two biomarkers, wherein the at least two biomarkers comprise: (i) hemoglobin Ale and the ratio of albumin to creatinine; and/or (ii) red cell distribution width and the ratio of albumin to creatinine; and/or (iii) hemoglobin Ale and alanine aminotransferase, and wherein the subject is not being treated with a 3-hydroxy-3- methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor; and/or (iv) alkaline phosphatase and the ratio of albumin to creatinine.

[0292] An eighty-third aspect relates to the kit of aspect 82, wherein the kit includes control values or ranges for each of the biomarkers being quantified.

[0293] An eighty-fourth aspect relates to the kit of aspect 83, wherein the kit includes instructions for quantifying the values and for comparing the quantified values with the control values or ranges.

[0294] An eighty-fifth aspect relates to an immunomodulatory therapeutic agent for use in the treatment or prevention of progression of diabetic retinopathy in a subject, wherein the subject has been identified as being at increased risk of having rapid progression according to aspects 1 to 41.

[0295] An eighty-sixth aspect relates to the immunomodulatory therapeutic agent of aspect 85, wherein the immunomodulatory therapeutic agent is selected from the group consisting of an inhibitor of Rho-associated kinase, an anti-VEGF therapy, a plasma kallikrein inhibitor, a pan- RGD integrin antagonist, tonabersat, an inhibitor of apurinic/apyrimidinic endonuclease 1/reduction-oxidation (redox) effector factor-1 (APEl/Ref-1; APEX1), runcaciguat, lazucirnon, RG-7774, Emixustat hydrochloride, brolucizumab, EGT022, OTT166, EOM147, ST2666, BSL01, BI764524, MHU650, and any combination thereof.

[0296] An eighty- seventh aspect relates to the therapeutic agent for use according to aspect 85 or 86, wherein the immunomodulatory therapeutic agent is an inhibitor of Rho-associated kinase.

[0297] An eighty-eighth aspect relates to the immunomodulatory therapeutic agent for use according to aspects 85 to 87, wherein the immunomodulatory therapeutic agent is a combination of an inhibitor of Rho-associated kinase and an anti-VEGF therapy.

[0298] An eighty-ninth aspect relates to the immunomodulatory therapeutic agent for use according to aspects 85 to 88, wherein the subject is a human.

[0299] A ninetieth aspect relates to the immunomodulatory therapeutic agent for use according to aspects 85 to 88, wherein the subject is a non-human mammal.

[0300] A ninety-first aspect relates to the immunomodulatory therapeutic agent for use according to aspects 85 to 90, wherein the subject has mild or moderate non-proliferative diabetic retinopathy.

[0301] A ninety-second aspect relates to an immunomodulatory therapeutic agent for use in the treatment of prevention of progression of diabetic retinopathy in a subject, wherein the subject has quantified values of at least two biomarkers that are statistically different compared to a control, wherein the at least two biomarkers comprise: (i) hemoglobin Ale and the ratio of albumin to creatinine; and/or (ii) red cell distribution width and the ratio of albumin to creatinine; and/or (iii) hemoglobin Ale and alanine aminotransferase, and wherein the subject is not being treated with a 3 -hydroxy-3 -methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor; and/or (iv) alkaline phosphatase and the ratio of albumin to creatinine.

[0302] A ninety-third aspect relates to the immunomodulatory therapeutic agent for use according to aspect 92, wherein the immunomodulatory therapy is selected from the group consisting of an inhibitor of Rho-associated kinase, an anti-VEGF therapy, a plasma kallikrein inhibitor, a pan-RGD integrin antagonist, tonabersat, an inhibitor of apurinic/apyrimidinic endonuclease 1/reduction-oxidation (redox) effector factor-1 (APEl/Ref-1; APEX1), runcaciguat, lazucirnon, RG-7774, Emixustat hydrochloride, brolucizumab, EGT022, OTT166, EOM147, ST2666, BSL01, BI764524, MHU650, and any combination thereof.

[0303] A ninety-fourth aspect relates to the immunomodulatory therapeutic agent for use according to aspect 92 or 93, wherein the immunomodulatory therapy is an inhibitor of Rho- associated kinase. [0304] A ninety-fifth aspect relates to the immunomodulatory therapeutic agent for use according to aspects 92 to 94, wherein the immunomodulatory therapy is a combination of an inhibitor of Rho-associated kinase and an anti-VEGF therapy.

[0305] A ninety-sixth aspect relates to the immunomodulatory therapeutic agent for use according to aspects 92 to 95, wherein the control is a reference standard, an average value obtained from healthy subjects, or an average value obtained from subjects with diabetic retinopathy who are not at increased risk of having rapid progression of diabetic retinopathy.

[0306] A ninety-seventh aspect relates to the immunomodulatory therapeutic agent for use according to aspects 92 to 96, wherein the subject is a human.

[0307] A ninety-eighth aspect relates to the immunomodulatory therapeutic agent for use according to aspects 92 to 97, wherein the subject is a non-human mammal.

[0308] A ninety-ninth aspect relates to the immunomodulatory therapeutic agent for use according to aspects 92 to 98, wherein the subject has mild or moderate non-proliferative diabetic retinopathy.

[0309] In addition to the features mentioned in each of the independent aspects enumerated above, some examples may show, alone or in combination, the optional features mentioned in the dependent aspects and/or as disclosed in the description above and shown in the figures.

Claims

CLAIMS A method of identifying a subject as being at increased risk of having rapid progression of diabetic retinopathy, wherein the method comprises: quantifying at least two biomarkers in one or more samples obtained from the subject, and identifying the subject as being at increased risk of having rapid progression of diabetic retinopathy if the quantified values of the at least two biomarkers are statistically different compared to a control, wherein the at least two biomarkers comprise: (i) hemoglobin Ale and the ratio of albumin to creatinine; and/or (ii) red cell distribution width and the ratio of albumin to creatinine; and/or (iii) hemoglobin Ale and alanine aminotransferase, and wherein the subject is not being treated with a 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor; and/or (iv) alkaline phosphatase and the ratio of albumin to creatinine. The method of claim 1, wherein the at least two biomarkers are hemoglobin Ale and the ratio of albumin to creatinine, and wherein the subject is identified as being at increased risk of having rapid progression of diabetic retinopathy when the quantified values of hemoglobin Ale and the ratio of albumin to creatinine are statistically increased relative to the control. The method of claim 2, wherein the quantified value of hemoglobin Ale is statistically increased relative to the control when the quantified value of hemoglobin Ale is greater than a pre-determined value. The method of claim 3, wherein the pre-determined value of hemoglobin Ale is 7.0% of total hemoglobin. The method of claim 3, wherein the pre-determined value of hemoglobin Ale is 7.65% of total hemoglobin. The method of claim 3, wherein the pre-determined value of hemoglobin Ale is 7.7% of total hemoglobin. The method of claim 3, wherein the pre-determined value of hemoglobin Ale is 8.25% of total hemoglobin. The method of claim 2, wherein the quantified value of the ratio of albumin to creatinine is statistically increased relative to the control when the quantified value of the ratio of albumin to creatinine is greater than a pre-determined value. The method of claim 8, wherein the pre-determined value of the ratio of albumin to creatinine is 50 mg albumin to 1g creatinine. The method of claim 8, wherein the pre-determined value of the ratio of albumin to creatinine is 57 mg albumin to 1g creatinine. The method of claim 8, wherein the pre-determined value of the ratio of albumin to creatinine is 60 mg albumin to 1g creatinine. The method of claim 8, wherein the pre-determined value of the ratio of albumin to creatinine is 92 mg albumin to 1g creatinine. The method of claim 1 , wherein the at least two bio markers are red cell distribution width and the ratio of albumin to creatinine, and wherein the subject is identified as being at increased risk of having rapid progression of diabetic retinopathy when the quantified value of the red cell distribution width is statistically decreased relative to the control and the quantified value of the ratio of albumin to creatinine is statistically increased relative to the control. The method of claim 13, wherein the quantified value of the red cell distribution width is statistically decreased relative to the control when the quantified value of the red cell distribution width is lower than a pre-determined value. The method of claim 14, wherein the pre-determined value of the red cell distribution width is 14.5%. The method of claim 14, wherein the pre-determined value of the red cell distribution width is 14.0%. The method of claim 13, wherein the quantified value of the ratio of albumin to creatinine is statistically increased relative to the control when the quantified value of the ratio of albumin to creatinine is greater than a pre-determined value. The method of claim 17, wherein the pre-determined value of the ratio of albumin to creatinine is 80 mg albumin to 1g creatinine. The method of claim 17, wherein the pre-determined value of the ratio of albumin to creatinine is 92 mg albumin to 1g creatinine. The method of claim 1, wherein the at least two biomarkers are hemoglobin Ale and alanine aminotransferase, and wherein the subject is not being treated with a 3-hydroxy- 3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor, and wherein the subject is identified as being at increased risk of having rapid progression of diabetic retinopathy when the quantified value of hemoglobin Ale is statistically increased relative to the control and the quantified value of alanine aminotransferase is statistically decreased relative to the control. The method of claim 20, wherein the quantified value of hemoglobin Ale is statistically increased relative to the control when the quantified value of hemoglobin Ale is greater than a pre-determined value. The method of claim 21, wherein the pre-determined value of hemoglobin Ale is 7% of total hemoglobin. The method of claim 21, wherein the pre-determined value of hemoglobin Ale is 7.85% of total hemoglobin. The method of claim 20, wherein the quantified value of alanine aminotransferase is statistically decreased relative to the control when the quantified value of alanine aminotransferase is lower than a pre-determined value. The method of claim 24, wherein the pre-determined value of alanine aminotransferase is 28.0 IU/L. The method of claim 24, wherein the pre-determined value of alanine aminotransferase is 24.5 IU/L. The method of claim 1, wherein the at least two biomarkers are alkaline phosphatase and the ratio of albumin to creatinine, and wherein the subject is identified as being at increased risk of having rapid progression of diabetic retinopathy when the quantified values of alkaline phosphatase and the ratio of albumin to creatinine are statistically increased relative to the control. The method of claim 27, wherein the quantified value of alkaline phosphatase is statistically increased relative to the control when the quantified value of alkaline phosphatase is greater than a pre-determined value. The method of claim 28, wherein the pre-determined value of alkaline phosphatase is 72.0 IU/L. The method of claim 28, wherein the pre-determined value of alkaline phosphatase is 79.0 IU/L. The method of claim 27, wherein the quantified value of the ratio of albumin to creatinine is statistically increased relative to the control when the quantified value of the ratio of albumin to creatinine is greater than a pre-determined value. The method of claim 31, wherein the pre-determined value of the ratio of albumin to creatinine is 20 mg albumin to 1g creatinine. The method of any of claim 1, wherein the method further comprises: determining that a visual acuity score of the subject is below a predetermined value. The method of claim 33, wherein the predetermined value of the visual acuity score is 80 using the ETDRS letters system or 20/25 using the Snellen system. The method of claim 33, wherein the predetermined value of the visual acuity score is 65 using the ETDRS letters system or 20/50 using the Snellen system. The method of claim 1, wherein the control is a reference standard, an average value obtained from healthy subjects, or an average value obtained from subjects with diabetic retinopathy who are not at increased risk of having rapid progression of diabetic retinopathy. The method of claim 1, further comprising: obtaining the one or more samples from the subject. The method of claim 1, wherein the one or more samples are selected from a blood sample, a serum sample, a urine sample, another fluid biological sample or a combination thereof. The method of claim 1, wherein the subject is a human. The method of claim 1, wherein the subject is a non-human mammal. The method of claim 1, wherein the subject has mild or moderate non-proliferative diabetic retinopathy. A method of treating diabetic retinopathy in a subject or preventing progression of diabetic retinopathy in a subject, wherein the method comprises:

(1) identifying the subject as being at increased risk of having rapid progression of diabetic retinopathy if the quantified values of at least two biomarkers are statistically different compared to a control, wherein the at least two biomarkers comprise:

(i) hemoglobin Ale and the ratio of albumin to creatinine; and/or

(ii) red cell distribution width and the ratio of albumin to creatinine; and/or (iii) hemoglobin Ale and alanine aminotransferase, and wherein the subject is not being treated with a 3 -hydroxy-3 -methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor; and/or

(iv) alkaline phosphatase and the ratio of albumin to creatinine, and

(2) administering a therapy to the subject, wherein the therapy is an immunomodulatory therapy capable of treating diabetic retinopathy in the subject or preventing progression of diabetic retinopathy in the subject. The method of claim 42, wherein the immunomodulatory therapy is selected from the group consisting of an inhibitor of Rho-associated kinase, an anti-VEGF therapy, a plasma kallikrein inhibitor, a pan-RGD integrin antagonist, tonabersat, an inhibitor of apurinic/apyrimidinic endonuclease 1/reduction-oxidation (redox) effector factor- 1 (APEl/Ref-1; APEX1), runcaciguat, lazucirnon, RG-7774, Emixustat hydrochloride, brolucizumab, EGT022, OTT166, EOM147, ST2666, BSL01, BI764524, MHU650, and any combination thereof. The method of claim 43, wherein the immunomodulatory therapy is an inhibitor of Rho- associated kinase. The method of claim 43, wherein the immunomodulatory therapy is a combination of an inhibitor of Rho-associated kinase and an anti-VEGF therapy. The method of claim 42, wherein the control is a reference standard, an average value obtained from healthy subjects, or an average value obtained from subjects with diabetic retinopathy who are not at increased risk of having rapid progression of diabetic retinopathy. The method of claim 42, the method further comprising: obtaining the one or more samples from the subject. The method of claim 42, wherein the one or more samples are selected from a blood sample, a serum sample, a urine sample, another fluid biological sample or a combination thereof. The method of claim 42, wherein the subject is a human. The method of claim 42, wherein the subject is a non-human mammal. The method of claim 42, wherein the subject has mild or moderate non-proliferative diabetic retinopathy. The method of claim 42, wherein administration of the immunomodulatory therapy to the subject is intravitreal, conjunctival, intracorneal, intraocular, intravenous, ophthalmic, topical, oral, intranasal, subcutaneous, intramuscular, or intraperitoneal. The method of claim 52, wherein administration of the immunomodulatory therapy to the subject is intravenous, ophthalmic, topical, oral, intranasal, subcutaneous, intramuscular, or intraperitoneal. The method of claim 52, wherein administration of the immunomodulatory therapy to the subject is topical or oral. The method of claim 52, wherein administration of the immunomodulatory therapy to the subject is oral. The method of claim 42, wherein the at least two biomarkers are hemoglobin Ale and the ratio of albumin to creatinine, and the subject is identified as being at increased risk of having rapid progression of diabetic retinopathy when the quantified value of hemoglobin Ale is greater than or equal to 7.65% of total hemoglobin, and the quantified value of the ratio of albumin to creatinine is greater than or equal to 57 mg albumin to 1g creatinine. The method of claim 42, wherein the at least two biomarkers are hemoglobin Ale and the ratio of albumin to creatinine, and the subject is identified as being at increased risk of having rapid progression of diabetic retinopathy when the quantified value of hemoglobin Ale is greater than or equal to 8.25% of total hemoglobin, and the quantified value of the ratio of albumin to creatinine is greater than or equal to 92 mg albumin to 1g creatinine. The method of claim 42, wherein the at least two biomarkers are hemoglobin Ale and the ratio of albumin to creatinine, and the subject is identified as being at increased risk of having rapid progression of diabetic retinopathy when the quantified value of hemoglobin Ale is greater than or equal to 7.7% of total hemoglobin, and the quantified value of the ratio of albumin to creatinine is greater than or equal to 60 mg albumin to 1g creatinine. The method of claim 42, wherein the at least two biomarkers are hemoglobin Ale and the ratio of albumin to creatinine, and the subject is identified as being at increased risk of having rapid progression of diabetic retinopathy when the quantified value of hemoglobin Ale in blood is greater than or equal to 7.7% of total hemoglobin, and the quantified value of the ratio of albumin to creatinine in urine is greater than or equal to 60 mg albumin to 1g creatinine. The method of claim 42, wherein the method prevents progression of diabetic retinopathy in the subject. The method of claim 60, wherein the method stabilizes the stage of the diabetic retinopathy such that the disease does not progress, or does not progress as quickly, to a stage of diabetic retinopathy or a related disease which threatens the subject’s vision. A method of treating diabetic retinopathy in a subject or preventing progression of diabetic retinopathy in a subject, wherein the method comprises:

(1) selecting a subject having quantified values of at least two biomarkers that are statistically different compared to a control, wherein the at least two biomarkers comprise:

(i) hemoglobin Ale and the ratio of albumin to creatinine; and/or

(ii) red cell distribution width and the ratio of albumin to creatinine; and/or

(iii) hemoglobin Ale and alanine aminotransferase, and wherein the subject is not being treated with a 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitor; and/or

(iv) alkaline phosphatase and the ratio of albumin to creatinine; and (2) administering a therapy to the subject, wherein the therapy is an immunomodulatory therapy capable of treating diabetic retinopathy in the subject or preventing progression of diabetic retinopathy in the subject. The method of claim 62, wherein the immunomodulatory therapy is selected from the group consisting of an inhibitor of Rho-associated kinase, an anti-VEGF therapy, a plasma kallikrein inhibitor, a pan-RGD integrin antagonist, tonabersat, an inhibitor of apurinic/apyrimidinic endonuclease 1/reduction-oxidation (redox) effector factor- 1 (APEl/Ref-1; APEX1), runcaciguat, lazucirnon, RG-7774, Emixustat hydrochloride, brolucizumab, EGT022, OTT166, EOM147, ST2666, BSL01, BI764524, MHU650, and any combination thereof. The method of claim 62, wherein the immunomodulatory therapy is an inhibitor of Rho- associated kinase. The method of claim 62, wherein the immunomodulatory therapy is a combination of an inhibitor of Rho-associated kinase and an anti-VEGF therapy. The method of claim 62, wherein the control is a reference standard, an average value obtained from healthy subjects, or an average value obtained from subjects with diabetic retinopathy who are not at increased risk of having rapid progression of diabetic retinopathy. The method of claim 62, the method further comprising: obtaining the one or more samples from the subject. The method of claim 62, wherein the one or more samples are selected from a blood sample, a serum sample, a urine sample, another fluid biological sample or a combination thereof. The method of claim 62, wherein the subject is a human. The method of claim 62, wherein the subject is a non-human mammal. The method of claim 62, wherein the subject has mild or moderate non-proliferative diabetic retinopathy. The method of claim 62, wherein administration of the immunomodulatory therapy to the subject is intravitreal, conjunctival, intracorneal, intraocular, intravenous, ophthalmic, topical, oral, intranasal, subcutaneous, intramuscular, or intraperitoneal. The method of claim 62, wherein administration of the immunomodulatory therapy to the subject is intravenous, ophthalmic, topical, oral, intranasal, subcutaneous, intramuscular, or intraperitoneal. The method of claim 62, wherein administration of the immunomodulatory therapy to the subject is topical or oral. The method of claim 62, wherein administration of the immunomodulatory therapy to the subject is oral. The method of claim 62, wherein the at least two biomarkers are hemoglobin Ale and the ratio of albumin to creatinine, and the quantified value of hemoglobin Ale is greater than or equal to 7.65% of total hemoglobin, and the quantified value of the ratio of albumin to creatinine is greater than or equal to 57 mg albumin to 1g creatinine. The method of claim 62, wherein the at least two biomarkers are hemoglobin Ale and the ratio of albumin to creatinine, and the quantified value of hemoglobin Ale is greater than or equal to 8.25% of total hemoglobin, and the quantified value of the ratio of albumin to creatinine is greater than or equal to 92 mg albumin to 1g creatinine. The method of claim 62, wherein the at least two biomarkers are hemoglobin Ale and the ratio of albumin to creatinine, and the quantified value of hemoglobin Ale is greater than or equal to 7.7% of total hemoglobin, and the quantified value of the ratio of albumin to creatinine is greater than or equal to 60 mg albumin to 1g creatinine. The method of claim 62, wherein the at least two biomarkers are hemoglobin Ale and the ratio of albumin to creatinine, and the quantified value of hemoglobin Ale in blood is greater than or equal to 7.7% of total hemoglobin, and the quantified value of the ratio of albumin to creatinine in urine is greater than or equal to 60 mg albumin to 1g creatinine. The method of claim 62, wherein the method prevents progression of diabetic retinopathy in the subject. The method of claim 80, wherein the method stabilizes the stage of the diabetic retinopathy such that the disease does not progress, or does not progress as quickly, to a stage of diabetic retinopathy or a related disease which threatens the subject’s vision. A kit for quantifying values of at least two biomarkers in a sample obtained from a subject, wherein the kit comprises assay components for quantifying the values of the least two biomarkers, wherein the at least two biomarkers comprise:

(i) hemoglobin Ale and the ratio of albumin to creatinine; and/or

(ii) red cell distribution width and the ratio of albumin to creatinine; and/or

(iv) alkaline phosphatase and the ratio of albumin to creatinine. The kit of claim 82, wherein the kit includes control values or ranges for each of the biomarkers being quantified. The kit of claim 83, wherein the kit includes instructions for quantifying the values and for comparing the quantified values with the control values or ranges. An immunomodulatory therapeutic agent for use in the treatment or prevention of progression of diabetic retinopathy in a subject, wherein the subject has been identified as being at increased risk of having rapid progression according to claim 1. The immunomodulatory therapeutic agent for use according to claim 85, wherein the immunomodulatory therapeutic agent is selected from the group consisting of an inhibitor of Rho-associated kinase, an anti-VEGF therapy, a plasma kallikrein inhibitor, a pan-RGD integrin antagonist, tonabersat, an inhibitor of apurinic/apyrimidinic endonuclease 1/reduction-oxidation (redox) effector factor- 1 (APEl/Ref-1; APEX1), runcaciguat, lazucimon, RG-7774, Emixustat hydrochloride, brolucizumab, EGT022, OTT166, EOM147, ST2666, BSL01, BI764524, MHU650, and any combination thereof. The therapeutic agent for use according to claim 86, wherein the immunomodulatory therapeutic agent is an inhibitor of Rho-associated kinase. The immunomodulatory therapeutic agent for use according to claim 86, wherein the immunomodulatory therapeutic agent is a combination of an inhibitor of Rho-associated kinase and an anti-VEGF therapy. The immunomodulatory therapeutic agent for use according to claim 85, wherein the subject is a human. The immunomodulatory therapeutic agent for use according to claim 85, wherein the subject is a non-human mammal. The immunomodulatory therapeutic agent for use according to claim 85, wherein the subject has mild or moderate non-proliferative diabetic retinopathy. An immunomodulatory therapeutic agent for use in the treatment of prevention of progression of diabetic retinopathy in a subject, wherein the subject has quantified values of at least two biomarkers that are statistically different compared to a control, wherein the at least two biomarkers comprise:

(i) hemoglobin Ale and the ratio of albumin to creatinine; and/or

(ii) red cell distribution width and the ratio of albumin to creatinine; and/or

(iv) alkaline phosphatase and the ratio of albumin to creatinine. The immunomodulatory therapeutic agent for use according to claim 92, wherein the immunomodulatory therapy is selected from the group consisting of an inhibitor of Rho- associated kinase, an anti-VEGF therapy, a plasma kallikrein inhibitor, a pan-RGD integrin antagonist, tonabersat, an inhibitor of apurinic/apyrimidinic endonuclease 1/reduction-oxidation (redox) effector factor-1 (APEl/Ref-1; APEX1), runcaciguat, lazucirnon, RG-7774, Emixustat hydrochloride, brolucizumab, EGT022, OTT166, EOM147, ST2666, BSL01, BI764524, MHU650, and any combination thereof. The immunomodulatory therapeutic agent for use according to claim 93, wherein the immunomodulatory therapy is an inhibitor of Rho-associated kinase. The immunomodulatory therapeutic agent for use according to claim 93, wherein the immunomodulatory therapy is a combination of an inhibitor of Rho-associated kinase and an anti-VEGF therapy. The immunomodulatory therapeutic agent for use according to claim 92, wherein the control is a reference standard, an average value obtained from healthy subjects, or an average value obtained from subjects with diabetic retinopathy who are not at increased risk of having rapid progression of diabetic retinopathy. The immunomodulatory therapeutic agent for use according to claim 92, wherein the subject is a human. The immunomodulatory therapeutic agent for use according to claim 92, wherein the subject is a non-human mammal. The immunomodulatory therapeutic agent for use according to claim 92, wherein the subject has mild or moderate non-proliferative diabetic retinopathy.