A method for determining whether a subject has or is at risk of having central serous chorioretinopathy.

Abstract

Methods for determining whether a subject has or is at risk for having central serous chorioretinopathy Central serous chorioretinopathy (CSCR) is a primarily ophthalmic disease affecting the choroid and retinal pigment epithelium. To date, no systemic biomarkers for CSCR have been identified that correlate the two and aid in the diagnosis of challenging cases. Here, we measured serum levels of NGAL and the NGAL / MMP9 complex in a European cohort of 168 CSCR patients (n=90) with or without epithelial damage (n=78) and 153 control subjects with no history of ocular disease. Serum NGAL (ng / ml) was significantly higher in the control group (108.8±46.8) than in the CSCR cohort (80.4±46.4, p<0.0001). Serum NGAL (ng / ml) was significantly lower in the acute / relapse cohort (n=78, 71.3±32.1) than in the control and chronic cohorts (n=90, 88.3±55, p=0.03). Similarly, serum NGAL / MMP9 (ng / ml) levels were lower in the entire CSCR cohort (44.5±39.6) than in the control group (77.6±47.8, p<0.0001). Serum NGAL / MMP9 (ng / ml) was significantly lower in the acute / relapse cohort (37.6±37.9) than in the control group and in the chronic cohort (50.5±40.3, p=0.002). Thus, serum NGAL and NGAL / MMP9 levels were lower in both CSCR subtypes than in the control group, indicating the biological relevance of both subtypes and their potential susceptibility to oxidative stress and innate immune dysregulation. Systemic LCN2 is also elevated in other retinal diseases, suggesting it may be a specific biomarker for CSCR.

Description

[Technical field]

[0001] The present invention is in the field of medicine, in particular in the field of ophthalmology.

[0002] 2. Background of the Invention Central serous chorioretinopathy is a primarily ophthalmic disease affecting the choroid and retinal pigment epithelium (RPE). 1 There are various types of this disease, but there is no unified nomenclature yet. 2 Although the majority of patients present with localized leakage of the RPE that resolves spontaneously and occasionally recurs with serous detachment, a minority of patients present with extensive pigment epithelial damage, persistent serous detachment, and potential functional and anatomical complications. 1 The exact mechanism and causative factors of the disease remain unknown. Widely recognized risk factors for CSCR include exposure to exogenous or endogenous corticoids, use of psychopharmacological therapy, type A behavior, cardiovascular risk factors such as coronary artery disease and hypertension, sleep disorders, shift work, and Helicobacter pylori infection. 3 Genetic factors include polymorphisms in genes encoding complement factor systems, haplotypes in genes encoding complement factor systems, 4 Haplotypes of the gene encoding the mineralocorticoid receptor 5 , polymorphisms in genes encoding the plasminogen activator system 6 , and polymorphisms in the gene encoding the VIP receptor 7 In typical cases, the diagnosis can be easily made based on clinical symptoms and imaging techniques such as SD-OCT and fluorescein-ICG angiography, but in more complex cases, especially in women with a thin choroid or in the absence of favorable factors, the diagnosis may be difficult. To our knowledge, no systemic biomarkers for CSCR have been identified.

[0003] Lipocalin-2 (LCN2), also known as Neutrophil Gelatinase-Associated Lipocalin (NGAL), siderocalin, uterocalin, or 24p3, is a 21-kD molecule belonging to the lipocalin superfamily. Lipocalins transport small hydrophobic substances such as retinoids, steroids, and fatty acids. Additionally, LCN2 transports iron intracellularly, has an iron-regulatory effect, and functions as an important regulator of innate immunity. 8,9 LCN2 is expressed in many cells and tissues, including innate immune cells, epithelial cells, and brain astrocytes, 10 and is expressed in retinal pigment epithelial cells 11 and retinal glial Müller cells. 12,13 LCN2 is induced by the activation of NF-κB in response to acute injury, infection, and metabolic disorders. 14 However, depending on the course of the disease (acute or chronic) and the organ, LCN2 can exhibit either a pro-inflammatory or anti-inflammatory effect. In metabolic inflammation such as type 2 diabetes and non-alcoholic diseases, LCN2 promotes inflammation through the recruitment of neutrophils and inflammatory cytokines. 15 In transgenic mice lacking lysosomal clearance in RPE cells, LCN2 produced by neutrophils promotes retinal infiltration, leading to phenotypic manifestations of early AMD in the mouse retina and contributing to age-related changes. 16 On the other hand, LCN2 exhibits an anti-inflammatory effect in intestinal inflammation and also shows an anti-inflammatory effect via LCN2 in brain and eye inflammation induced by LPS. 16 An anti-inflammatory effect was shown by inactivating NF-κB. 11 Another function of LCN2 is to promote the activity of MMP-9 by forming a complex with the protease (MMP-9 / NGAL), which is an important mediator of atherosclerotic plaques. 17 It forms a complex with and promotes the activity of MMP-9. In experimental atherosclerosis models, LCN2 plays a dual role of preventing early plaque formation but increasing the activity of MMP-9 and the size of necrotic cores in advanced atherosclerosis. 18

[0004] In the retina, the role of LCN2 has only been incompletely elucidated. After irradiating Abca4− / −Rdh8− / − mice, which are a model of AMD, with light, it is the stress gene that is most rapidly expressed in the RPE and neural retina. Irradiating three Lcn2− / −Abca4− / −Rdh8− / − mice with light promotes glycolysis and microglial activation, indicating that LCN2 is protective in this model. 10,19 Furthermore, LCN2 was able to protect against oxidative stress by increasing the expression of antioxidant enzymes HMOX1 and SOD2 in hiPS-RPE cells. 19 On the other hand, in another study, LCN2 promoted apoptosis of photo-induced photoreceptor cells by increasing the generation of reactive oxygen species and the expression of Bim. 20 。

[0005] LCN2 has been identified as a biomarker for inflammatory and metabolic diseases and is recognized as an optimal biomarker for the diagnosis and prognosis of acute kidney injury. 21 It is recognized as an optimal biomarker for the diagnosis and prognosis of acute kidney injury. 22,23,24,25 In kidney diseases, LCN2 not only serves as a biomarker for the disease but also contributes to the pathogenesis. 26 LCN2 is also a biomarker for arteriosclerosis, myocardial infarction (MI), and heart failure. 27 After myocardial infarction, LCN2 produced by neutrophils induces polarization of macrophages into a phenotype that enables the removal of apoptotic cells and suppresses myocardial fibrosis. Thus, LCN2 is beneficial for cardiac remodeling. 28 。

[0006] In eye diseases, an increase in LCN2 concentration was measured not in the serum but in the aqueous humor of patients with central retinal vein occlusion. 29 In patients with diabetic retinopathy, the plasma LCN2 concentration increased and was correlated with the severity of retinopathy. 30 In AMD, the plasma LCN2 value increased and LCN2 increased in the aqueous humor of wet AMD patients. 31 。

[0007] Summary of the Invention The present invention is defined by the claims. In particular, the present invention relates to a method for determining whether a subject has or is at risk of having central serous chorioretinopathy.

[0008] Detailed Description of the Invention Central serous chorioretinopathy (CSCR) is clinically classified into two types: chronic type and acute type, depending on the presence or absence of an epithelial disorder detected clinically. Whether chronic CSCR is due to the progression of acute CSCR or is an independent entity remains unclear. To date, no systemic biomarkers associated with the chronic type or the acute type have been discovered, which may be helpful in the diagnosis of difficult cases.

[0009] Lipocalin 2 (Lcn2, Neutrophil gelatinase-associated lipocalin, NGAL) is a 25-kD secreted protein with multiple innate immune functions. NGAL also exists as a heterodimer disulfide-bonded to MMP9, stabilizing the activity of MMP9. LCN2 increases in diabetic retinopathy, age-related macular degeneration, and retinitis pigmentosa.

[0010] Measure the serum levels of NGAL and the NGAL / MMP9 complex in a European cohort of CSCR patients (n = 168) with (n = 90) or without (n = 78) epithelioma and control subjects (153) without a history of eye disease. Subjects with CRP > 5 mg / L, creatinine > 100 μmol / L, or urea > 7.5 mmol / L were excluded.

[0011] The mean age of the control group was significantly younger than that of the CSCR group, and there were significantly more females in the control group than in the CSCR group. However, no significant correlation was observed between NGAL and NGAL / MMP9 and age or gender. Serum NGAL (ng / ml) was significantly higher in the control group (108.8 ± 46.8) than in the CSCR cohort (80.4 ± 46.4, p < 0.0001). Serum NGAL (ng / ml) was significantly lower in the acute / recurrent cohort (n = 78, 71.3 ± 32.1) than in the control group and the chronic cohort (n = 90, 88.3 ± 55, p = 0.03). Similarly, the serum NGAL / MMP9 (ng / ml) level was lower in the entire CSCR cohort (44.5 ± 39.6) than in the control group (77.6 ± 47.8, p < 0.0001). Serum NGAL / MMP9 (ng / ml) was significantly lower than the control group in the acute / recurrent cohort (37.6 ± 37.9), and in the chronic cohort it was 50.5 ± 40.3, p = 0.002. The ROC curve shows that the cut-off value of NGAL at 80 ng / mL can distinguish acute / recurrent CSCR from the control group with a sensitivity of 79.5% and a specificity of 74.8%, and the cut-off value of the NGAL / MMP9 complex at 40 ng / mL can distinguish acute / recurrent CSCR from the control group with a sensitivity of 72.7% and a specificity of 76.0%.

[0012] Therefore, in both disease types of CSCR, serum NGAL and NGAL / MMP9 are lower than in the control group, providing the possibility of biological relevance and susceptibility to oxidative stress and innate immune regulation disorders in both disease types. Systemic LCN2 is also elevated in other retinal diseases and is considered a specific biomarker for CSCR.

[0013] Therefore, a first object of the present invention is a method for determining whether a subject has or is at risk of having central serous chorioretinopathy, comprising determining the level of NGAL in a sample obtained from the subject, wherein said level indicates whether the subject has or is at risk of having central serous chorioretinopathy.

[0014] As used herein, the term "central serous chorioretinopathy" or "CSCR" has its ordinary meaning in the art and refers to a disorder characterized by serous retinal detachment and / or retinal pigment epithelium (RPE) detachment, changes most frequently limited to the macula, and leakage of fluid from the RPE into the subretinal space. CSCR is commonly seen in young male patients without systemic disease in most retinal clinics.

[0015] As used herein, the term "risk" in the context of the present invention relates to the probability of an event occurring over a specific period of time and can refer to the "absolute" or "relative" risk of a subject. Absolute risk can be measured by referring to actual post-observation measurements for the relevant time cohort or by referring to index values developed from a statistically valid past cohort followed over the relevant time period. Relative risk refers to the ratio of the absolute risk of a subject compared to the absolute risk of a low-risk cohort or the average population risk and may vary depending on the method of evaluating clinical risk factors. Odds ratio (the ratio of positive to negative events for a certain test result) is also commonly used (odds follow the formula p / (1-p), where p is the probability of an event and (1-p) is the probability of no event) and is used without transformation. "Risk assessment" or "assessment of risk" in the context of the present invention includes predicting the probability, odds, or likelihood that an event or disease state may occur, the incidence of an event, or the conversion from one disease state to another. Risk assessment can also include predicting future clinical parameters, conventional laboratory risk factor values, or other indicators of recurrence from an absolute or relative perspective based on a previously measured population. The methods of the present invention can be used to make continuous or categorical measurements of conversion risk and thus can diagnose and define the risk spectrum of a category of subjects defined as having a risk of conversion. In a categorical scenario, the present invention can be used to distinguish between a normal subject cohort and other subject cohorts at high risk. In some embodiments, the present invention can be used to distinguish those at risk from those that are normal.

[0016] In some embodiments, the methods described herein are applied to a subject presenting symptoms of CSCR without undergoing routine screening to rule out all possible causes of CSCR. The methods described herein can be part of a routine examination performed on a subject presenting symptoms of CSCR, such as blurred vision, distortion, blind spots, faded colors, objects appearing smaller than they actually are, trouble with bright lights, and / or reduced ability to see an object against a background of similar color (contrast sensitivity). The methods of the invention can be performed in addition to other diagnostic tools, including blue fundus autofluorescence imaging, spectral domain optical coherence tomography, and / or fluorescein angiography.

[0017] In some embodiments, the sample is a blood sample. As used herein, the term "blood sample" means any blood sample obtained from a subject. Collection of the blood sample can be performed by methods well known to those of skill in the art. In some embodiments, the blood sample is a serum sample or a plasma sample.

[0018] As used herein, the terms "lipocalin 2", "Lcn2" or "NGAL" have their general meaning in the art and mean Neutrophil Gelatinase-Associated Lipocalin as described in Schmidt-Ott KM. et al. (2007). NGAL can be obtained from any source, but is typically mammalian (e.g., human and non-human primate) NGAL, particularly human NGAL. An exemplary human native NGAL amino acid sequence is provided in the GenPept database under accession number NP_005555. NGAL is a glycoprotein and was originally identified as a neutrophil-specific granule component and as a member of the lipocalin family of proteins. This protein exists as a 25 kDa monomer and a 45 kDa disulfide-linked homodimer, and has also been shown to exist in the form of a 135 kDa heterodimer covalently bound via an intermolecular disulfide bridge to neutrophil gelatinase (also known as matrix metalloprotease 9, MMP-9).

[0019] Methods for determining the expression level of NGAL are well known in the art. For example, any conventional method for determining the level of a protein in a sample can be used. In some embodiments, the method of the invention comprises contacting the sample with a binding partner that can selectively interact with a protein likely to be present in the sample. The binding partner may be an antibody, which may be polyclonal or monoclonal, preferably monoclonal. In some embodiments, the binding partner may be an aptamer. The binding partner of the invention, such as an antibody or aptamer, may be labeled with a detectable molecule or substance such as a fluorescent molecule, a radioactive molecule, or other labels known in the art. Labels are generally known in the art to provide a signal (either directly or indirectly). As used herein, with respect to an antibody, the term "labeled" is intended to include both direct labeling of the antibody or aptamer by coupling (i.e., physically linking) a detectable substance such as a radioactive substance or a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE), indocyanine (Cy5), etc.) to the antibody or aptamer, as well as indirect labeling of a probe or antibody by reactivity with a detectable substance. The antibodies or aptamers of the invention can be labeled with radioactive molecules by any method known in the art. The aforementioned assays generally involve the binding of the binding partner (i.e., antibody or aptamer) to a solid support. Solid supports that can be used in the practice of the invention include nitrocellulose (e.g., in the form of a membrane or microtiter well); polyvinyl chloride (e.g., in the form of a sheet or microtiter well); polystyrene latex (e.g., in the form of beads or microtiter plate); polyvinylidene fluoride; diazotized paper; nylon membrane; activated beads, magnetic responsive beads, and other substrates. The level of the biomarker protein can be measured by using standard immunodiagnostic techniques, including immunoassays such as competitive, direct reaction, or sandwich assays.Such assays include, but are not limited to, agglutination tests; enzyme-labeled and mediated immunoassays such as ELISA; biotin / avidin-type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation. More specifically, the ELISA method can be used. For example, the wells of a microtiter plate are coated with a set of antibodies that recognize the biomarker protein. Then, a sample containing or suspected of containing the biomarker protein is added to the coated wells. After incubating for a period sufficient for the formation of antibody-antigen complexes, the plate is washed to remove unbound portions, and a detectably labeled secondary binding molecule can be added. The secondary binding molecule reacts with the captured sample marker protein, the plate is washed, and the presence of the secondary binding molecule can be detected using methods well known in the art. In certain embodiments, the immunoassay can use two antibodies specific for the protein. Typically, the first antibody is used to "detect" the protein, and the second antibody is used to "capture" the protein. In some embodiments, this method is achieved by i) providing a solid support coating with a specific amount of the first antibody specific for the protein, ii) contacting the sample with the solid support, and iii) adding an amount of the second antibody conjugated to a label. By measuring the amount of the binding partner specific for the label, the amount of protein present in the sample becomes apparent. Usually, the first antibody is directed towards an epitope that does not interfere with the interaction with the second antibody. Usually, after steps ii) and iii), a washing step (using any suitable buffer such as PBS with or without a non-ionic detergent) is performed. Usually, a blocking step is carried out using a buffer containing BSA or milk and / or serum (goat or bovine) to block non-specific binding of the protein.The measurement of the level of a biomarker protein (regardless of whether the method is based on an immunoassay) can include the separation of the compound: centrifugation based on the molecular weight of the compound; electrophoresis based on mass and charge; HPLC based on hydrophobicity; size exclusion chromatography based on size; and solid phase affinity based on the affinity of the compound for the particular solid phase used. Once separated, the biomarker protein can be identified based on its known "separation profile" for the compound, e.g., retention time, and measured using standard techniques. Alternatively, NGAL may be detected and measured, for example, by a mass spectrometer.

[0020] In some embodiments, the level of NGAL is compared to a predetermined reference value. The predetermined reference value is typically a threshold value or a cut-off value. Typically, the "threshold value" or "cut-off value" can be determined experimentally, empirically, or theoretically. Also, the threshold value can be arbitrarily selected based on existing experiments and / or clinical conditions as would be recognized by those skilled in the art. For example, retrospective measurements in appropriately banked past subject samples may be used when setting the predetermined reference value. The threshold value must be determined to obtain optimal sensitivity and specificity according to the function of the test and the benefit / risk balance (clinical impact of false positives and false negatives). Generally, the optimal sensitivity and specificity (and threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. For example, after determining the concentration of NGAL, the concentration determined in the subject is statistically processed by algorithm analysis to obtain a significant classification criterion for classifying the subject. The official name of the ROC curve is the Receiver Operating Characteristic Curve, and it is also called the Receiver Operating Characteristic Curve. It is mainly used in clinical biochemical diagnostic tests. The ROC curve is a comprehensive indicator that reflects the continuous variables of the true positive rate (sensitivity) and the false positive rate (1 - specificity), and clarifies the relationship between sensitivity and the false positive rate. It clarifies the relationship between sensitivity and specificity by the image composition method. A series of different cut-off values (threshold values or critical values, the boundary values between normal and abnormal in diagnostic tests) are set as continuous variables, and a series of sensitivity and specificity values are calculated. Then, a curve is drawn with sensitivity on the vertical axis and specificity on the horizontal axis. The larger the area under the curve (AUC), the higher the diagnostic accuracy. In the ROC curve, the point closest to the upper left end of the coordinate axis is the critical point with both high sensitivity and high specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC > 0.5, the diagnostic result improves as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is medium. When AUC is higher than 0.9, the accuracy is high. This algorithm method is preferably performed by a computer.For drawing the ROC curve, existing software or systems in the relevant technical field can be used. For example, there are the following: MedCalc 9.2.0.1 medical statistics software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR MULTIREADERPOWER.SAS, CREATE-ROC.SAS, GBSTAT VI 0.0 (Dynamic Microsystems, Inc., Silver Spring, Md., USA), etc.

[0021] In some embodiments, the predetermined reference value is the level of NGAL determined in a population of healthy individuals. Typically, when the level of NGAL is lower (at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100 times lower) than the level determined in a population of healthy subjects, the patient is determined to be suffering from CSCR or at risk of recurrence.

[0022] The method of the present invention is particularly suitable for differential diagnosis between CSCR and other eye diseases such as age-related macular degeneration (AMD) where the level of NGAL is typically higher than a predetermined reference value. Accordingly, a further object of the present invention relates to a method for differential diagnosis between CSCR and AMD in a subject, comprising determining the level of NGAL in a sample obtained from the subject, wherein a high level of NGAL indicates that the subject is suffering from AMD, and a low level of NGAL indicates that the subject is suffering from CSCR.

[0023] As used herein, the term "high" means a measurement that is greater than a normal, a reference such as a predetermined reference value or a measurement of a subgroup, or relatively greater than a measurement of another subgroup. For example, a high expression level refers to a level of NGAL that is greater than the normal level of NGAL in a particular set of patient samples. The normal level of NGAL may be determined according to any method available to those skilled in the art. A high level of NGAL may also refer to a level that is equal to or greater than a predetermined reference value such as a predetermined cut-off. A high level of NGAL may also refer to a level of NGAL where a high level subgroup has a relatively greater level of NGAL than another subgroup. For example, but not limited to, according to the present disclosure, two different patient subgroups can be created by dividing the samples around a mathematically determined point such as, but not limited to, the median, and thus, a subgroup with a high (i.e., higher than the median) measurement and another subgroup with a low measurement can be created. In some cases, a "high" level may consist of a range of very high levels and a range of "moderately high" levels, where moderately high is a level that is greater than normal but less than "very high".

[0024] As used herein, the term "low" means a level of NGAL that is lower than normal, lower than a reference such as a predetermined reference value, or relatively lower than the level of another subgroup. For example, a low level of NGAL means a level of NGAL that is lower than the normal level of NGAL in a particular set of patient samples. The normal level of NGAL may be determined according to any method available to those skilled in the art. A low level of NGAL may also mean a level that is less than a predetermined reference value such as a predetermined cut-off. A low level of NGAL may also mean a level at which a low subgroup is relatively lower than another subgroup. For example, but not limited to, according to the present specification, two different patient subgroups can be created by dividing the samples around a mathematically determined point such as the median, and thus, a group with a low measurement value (i.e., less than the median) can be created with respect to another group with a high measurement value (i.e., greater than the median). In some cases, the "low" level may consist of a range of very low levels and a range of "moderate lows", where moderate low is a level that is lower than normal but higher than "very low".

[0025] A further object of the present invention is a method for predicting the risk of recurrence in a subject suffering from CSCR, comprising determining the level of NGAL in a sample obtained from the subject, wherein said level indicates the risk of recurrence.

[0026] As used herein, the term "recurrence" refers to the recurrence of the signs and symptoms of a disease after a subject has enjoyed remission after treatment. Thus, initially, when the target disease is alleviated or cured, or the progression of the disease is stopped or slowed down, and then the disease or one or more characteristics of the disease resume, the subject is said to be "recurring".

[0027] A further object of the present invention is to determine the level of NGAL in a sample obtained from a subject before treatment, ii) compare the level determined in step i) with the level determined in step ii), and iii) conclude that the subject has achieved a response if the level determined in step ii) is higher than the level determined in step i). It relates to a method for determining whether a subject suffering from CSCR achieves a response by treatment, including:

[0028] Therefore, this method is particularly suitable for distinguishing responders from non-responders. As used herein, the term "responder" in the context of the present disclosure refers to a subject who achieves a response, i.e., a subject in a remission state, more specifically, a subject who no longer suffers from CSCR. Non-responder subjects include those in whom the disease does not show a decrease or improvement after treatment.

[0029] According to the present invention, the treatment consists of any method, drug or treatment that may be suitable for the treatment of CSCR. For example, the drug or treatment method consists of an anti-VEGF agent, a carbonic anhydrase inhibitor, a mineralocorticoid antagonist, laser photocoagulation, a diode micropulse laser, Verteporfin photodynamic therapy (PDT) and / or trans-pupillary thermotherapy.

[0030] As used herein, "anti-VEGF agent" means a molecule that inhibits VEGF-mediated angiogenesis. For example, an anti-VEGF therapeutic agent can be an antibody or other antagonist to VEGF. An "anti-VEGF antibody" is an antibody that binds to VEGF with sufficient affinity and specificity to be useful in the methods of the present invention. Anti-VEGF antibodies generally do not bind to other VEGF homologs such as VEGF-B or VEGF-C, or other growth factors such as P1GF, PDGF or bFGF. Preferred anti-VEGF antibodies are monoclonal antibodies that bind to the same epitope as the monoclonal anti-VEGF antibody A4.6.1 produced by hybridoma ATCC RHB10709 and are high-affinity anti-VEGF antibodies. A "high-affinity anti-VEGF antibody" has an affinity for VEGF that is at least 10-fold better than that of the monoclonal anti-VEGF antibody A4.6.1. Preferably, the anti-VEGF antibody is a recombinant humanized anti-VEGF monoclonal antibody fragment produced according to WO98 / 45331 and includes an antibody consisting of the CDR or variable region of Y0317. More preferably, the anti-VEGF antibody is an antibody fragment known as ranibizumab (LUCENTIS®). The anti-VEGF antibody ranibizumab is a humanized affinity-matured anti-human VEGF Fab fragment. Ranibizumab is manufactured by standard recombinant techniques using an E. coli expression vector and bacterial fermentation. Ranibizumab is not glycosylated and has a molecular weight of -48,000 daltons. See W098 / 45331 and US 2003 / 0190317. Anti-VEGF agents include, but are not limited to, bevacizumab (rhuMabVEGF, AVASTIN®, Genentech, South San Francisco Calif.), ranibizumab (rhuFAbV2, LUCENTIS®, Genentech), pegaptanib (Macugen®, Eytech Pharmaceuticals, New York N.Y.), sunitinib malate (Sutent®, Pfizer, Groton Conn.), and the like.In some embodiments, the anti-VEGF agent is a high-affinity dimeric fusion protein that can bind to VEGF and consists of two receptor-Fc fusion proteins in which the extracellular domains of the human VEGFR1 or VEGFR2 receptors are fused to the Fc portion of human IgG1, the major ligand-binding portion (referred to as "VEGF trap"). Specifically, the VEGF trap is composed of the fusion of Ig domain 2 derived from VEGFR1 and Ig domain 3 derived from VEGFR2, which is further fused to the Fc domain of IgG1.

[0031] As used herein, the term "MR antagonist" has its general meaning in the art. The MR antagonist of a compound can be determined using various methods as described by J, Souque A, Wurtz JM, Moras D, Rafestin - Oblin ME. Mol Endocrinol. 2000 Aug;14(8):1210 - 21; Fagart J, Seguin C, Pinon GM, Rafestin - Oblin ME. Mol Pharmacol. 2005 May;67(5):1714 - 22 or Hellal - Levy C, Fagart J, Souque A, Wurtz JM, Moras D, Rafestin - Oblin ME. Mol Endocrinol. 2000 Aug;14(8):1210 - 21. For example, the mineralocorticoid receptor antagonists according to the present invention are generally spironolactone - type steroid compounds. The term "spironolactone - type" is intended to characterize a structure containing a lactone moiety bonded to a steroid nucleus, typically at the steroid "D" ring, via a spiro - bond configuration. Subclasses of spironolactone - type mineralocorticoid receptor antagonist compounds are composed of epoxy - steroid - based mineralocorticoid receptor antagonist compounds such as eplerenone. Another subclass of spironolactone - type antagonist compounds is composed of non - epoxy - steroid - based mineralocorticoid receptor antagonist compounds such as spironolactone. The mineralocorticoid receptor antagonists according to the present invention may also be non - steroidal. For example, the class of non - steroidal MR antagonists has only recently begun to emerge in the past few years (Meyers, Marvin J1; Hu, Xiao Expert Opinion on Therapeutic Patents, Volume 17, Number 1, January 2007, pp. 17 - 23(7) and Piotrowski DW. Mineralocorticoid receptor antagonists for the treatment of hypertension and diabetic nephropathy J. Med. Chem. 2012, 55, 7957 - 7966).For example, dihydropyrimidine has been shown to exhibit MR antagonistic activity (Activation of Mineralocorticoid Receptors by Exogenous Glucocorticoids and the Development of Cardiovascular Inflammatory Responses in Adrenalectomized Rats. Young MJ, Morgan J, Brolin K, Fuller PJ, Funder JW. Endocrinology. 2010 Apr 21). Furthermore, Arhancet et al. have disclosed other classes of non-steroidal MR antagonists (Arhancet GB, Woodard SS, Dietz JD, Garland DJ, Wagner GM, Iyanar K, Collins JT, Blinn JR, Numann RE, Hu X, Huang HC. J. Vol. Stereochemical requirements for mineralocorticoid receptor antagonism of dihydropyridine. J Med Chem. Apr 21, 2010). Other exemplary non-steroidal mineralocorticoid receptor antagonists include, but are not limited to, those described in US20090163472, WO2004052847, WO2008053300, WO2008104306, WO2007025604, WO201264631, WO2008126831. WO2012008435, WO2010104721, WO200985584, WO200978934, WO2008118319, WO200917190, WO200789034, WO2012022121, WO2012022120, WO2011141848 and WO200777961 are hereby incorporated by reference into the present disclosure.

[0032] A further object of the present invention relates to a method of treating CSCR in a subject in need thereof, comprising: i) determining whether the subject has or is at risk of having CSCR according to the method of the present invention; and ii) administering to the subject a therapy or agent as described above if the subject is considered to have or be at risk of having CSCR.

[0033] The present invention will be further described by the following figures and examples. However, these examples and figures should not be construed in any way as limiting the scope of the present invention.

Brief Description of the Drawings

[0034]

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[0035] Examples Method Patients Three cohorts of patients recruited from Jules Gonin Eye Hospital (Lausanne, Switzerland), Ophthalmopole Cochin Hospital (Paris, France), and Rotterdam Eye Hospital (Rotterdam, Netherlands) were included. Serum analysis was planned in Paris before samples were collected at each facility. Case selection was based on the availability of samples from patients with clearly defined phenotypes in multimodal retinal images.

[0036] The diagnostic criteria for CSCR were defined by multimodal images such as blue fundus autofluorescence images, spectral domain optical coherence tomography (SD-OCT, Spectralis, Heidelberg Engineering, Heidelberg, Germany), and fluorescein angiography. Patients were divided into two groups based on the presence of a basic multifocal epitheliopathy characterized by blue autofluorescence and fluorescein angiography. Patients with epithelioma were classified as chronic cases, and patients without epithelioma were classified as acute / recurrent cases.

[0037] Patients with other eye diseases such as age-related macular degeneration (characterized by the presence of drusen), diabetic retinopathy, retinal vein occlusion, high myopia with a refractive error of more than 6D, and glaucoma were excluded from this study.

[0038] Serum of control subjects was obtained from Banque Francaise du Sang (BFS) based on an agreement between BFS and Inserm. Blood was collected from donors without a history of ophthalmic diseases.

[0039] Ethical statement This study was conducted in accordance with the principles of the Declaration of Helsinki and was approved by the institutional review boards of the respective countries in France (CPP Ile de France 1, C16-09 N°DC-2016-2620), Switzerland (CER-VDEyeomics 340 / 15), and the Netherlands. Written informed consent was obtained from each patient and healthy participant.

[0040] Measurement of serum levels of LCN2 and NGAL / MMP9 NGAL and the NGAL / MMP9 complex were measured using the Human Lipocalin-2 / NGAL Quantikine ELISA Kit and the Human MMP-9 / NGAL Complex Quantikine ELISA Kit (R&D Systems®, catalog numbers DLCN20 and DM9L20 respectively, Minneapolis, MN) according to the manufacturer's protocol. All samples were tested in duplicate and required a 20-fold dilution. LCN2 levels were affected by renal function and inflammatory status, so patients with CRP > 5 mg / L, creatinine > 100 μmol / L, and urea > 7.5 mmol / L were excluded (53 and 24 patients were excluded from the CSCR group and the control group, respectively). ELISA analysis was performed in a blinded manner by JC and TJ in Paris in 2019.

[0041] Statistics Descriptive statistics, comparative statistics, and correlation statistics were calculated using GraphPad Prism (version 5.0f, GraphPad Software). Quantitative values were expressed as mean ± standard deviation. The Kolmogorov-Smirnov test was used to evaluate the normal or non-normal distribution of quantitative values. The Mann-Whitney test was used for comparison of quantitative values, and the Spearman correlation coefficient was used for evaluation of correlation. Fisher's exact test or chi-square test was used as needed for comparison of proportions between subgroups. Receiver Operating Characteristic (ROC) curves were plotted and analyzed to evaluate the sensitivity, specificity, and cut-off values of serum marker values. A P-value of 0.05 or less was considered significant.

[0042] Result Demographic characteristics of the cohort Table 1 shows the demographic characteristics of 168 CSCR patients and 153 control subjects. The mean age of the control group was significantly younger than that of the CSCR group (43 ± 12.8 years vs. 50.1 ± 10.7, p = 0.0002). Also, the mean age of chronic-phase CSCR patients (n = 90, 55.2 ± 9.9) was higher than that of acute-phase / recurrent cases (n = 78, 44.1 ± 8.2, p < 0.0001). However, there was no significant difference in age between acute / recurrent CSCR patients and the control group (p = 0.83). There were significantly more females in the control group than in the CSCR cohort, but there was no significant difference compared to the control group because there were more female patients with chronic CSCR than with acute CSCR.

[0043] In CSCR, serum NGAL / LCN2 and NGAL(LCN2) / MMP9 levels are lower than those in the control group.

[0044] Table 2 and Figure 1A show the serum NGAL(LCN2) and LCN2 / MMP9 (NGAL / MMP9) complex concentrations in 168 CSCR patients and 153 control subjects. Serum NGAL (ng / ml) was significantly higher in the control group (108.8 ± 46.8) than in the CSCR cohort (80.4 ± 46.4, p < 0.0001). Serum NGAL (ng / ml) was significantly lower in the acute-phase / recurrent cohort (n = 78, 71.3 ± 32.1) than in the control group and also lower than in the chronic-phase cohort (n = 90, 88.3 ± 55, p = 0.03). Similarly, serum NGAL / MMP9 (ng / ml) levels were lower in the entire CSCR cohort (44.5 ± 39.6) than in the control group (77.6 ± 47.8, p < 0.0001). Serum NGAL / MMP9 (ng / ml) was significantly lower in the acute / recurrent cohort (n = 78, 37.6 ± 37.9) than in the control group and the chronic cohort (n = 90, 50.5 ± 40.3, p = 0.002) (Table 2 and Figure 1B).

[0045] In male CSCR, serum NGAL / LCN2 and NGAL(LCN2) / MMP9 levels are lower than those in the male control group.

[0046] Since there was a significant difference in the sex ratio between the control cohort and the CSCR cohort, to rule out the possibility of sex as a confounding factor, the serum levels of lipocalin (NGAL) and lipocalin / MMP9 complex were also evaluated in 141 male patients with CSCR and 112 control males (Table 3). In the male population, similar to the overall cohort, the level of LCN2 (NGAL, ng / ml) was lower in CSCR patients than in the control group (80.7 ± 47.7 vs 101.5 ± 41.7, p < 0.001), and the level was lower in the acute / recurrent type compared to the chronic type (71.9 ± 33.1 vs 89.1 ± 57.5, p = 0.004). The NGAL / MMP9 level (ng / ml) was also lower in the CSCR cohort than in the control male cohort (43.1 ± 37.7 vs 72.2 ± 42.7, p < 0.0001), and was lower in the acute / recurrent type than in the chronic type (36.8 ± 38.5 vs 49.1 ± 36.4, p = 0.001).

[0047] In CSCR and the control group, the concentrations of NGAL / LCN2 and NGAL / MMP9 in serum were not correlated with age Since the mean age of the control group was lower than that of the CSCR group, and the age of the chronic CSCR group was higher than that of the acute / recurrent group, it was analyzed whether the serum levels of LCN2 (NGAL) and NGAL / MMP9 were correlated with age in both the control group and the CSCR group. As shown in Tables 4 and 5, the level of NGAL was significantly correlated with the level of NGAL / MMP9, but no correlation was found between age and either NGAL or NGAL / MMP9 in both the control group and the CSCR group. Tables 6 and 7 show that there is no correlation between age and the serum levels of NGAL and NGAL / MMP9 in the acute / recurrent CSCR group and the chronic CSCR group, which is the result of excluding age as a confounding factor.

[0048] In CSCR and control subjects, gender did not affect the serum levels of LCN2 (NGAL) and NGAL / MMP9 To ensure that the sex ratio of the control group and the CSCR group did not affect our results, we evaluated whether the levels of LCN2 (NGAL) and NGAL / MMP9 differed between the male and female populations of the CSCR and control groups. Table 8 shows that the levels of NGAL and NGAL / MMP9 were not correlated with sex in either population.

[0049] ROC curve analysis As shown in Figure 2, the ROC curve indicates that for the serum level of NGAL, a cut-off value of 80 ng / mL can distinguish acute / recurrent CSCR (<80 ng / mL) from controls (≥80 ng / mL) with a sensitivity of 79.5% and a specificity of 74.8%. For the serum level of the NGAL / MMP9 complex, a cut-off value of 40 ng / mL can distinguish acute / recurrent CSCR (<40 ng / mL) from controls (≥40 ng / mL) with a sensitivity of 72.7% and a specificity of 76.0% (Figure 3).

[0050] Risk of recurrence Using transgenic rats with NGAL knocked out, we showed that light-induced photoreceptor loss was significantly greater, indicating that NGAL deficiency increases sensitivity to photoinduced oxidative stress (Figure 6). These results suggest that low levels of NGAL are a risk factor for the severity of CSCR and are associated with the risk of recurrence in subjects with CSCR.

[0051] Discussion The results of this study showed that CSCR patients had lower levels of LCN2 and LCN2 / MM9 compared to the control group. The fact that the patients were from three different cohorts reinforces this unexpected finding. The serum levels measured in the control subjects were within the range of other control populations (30-year-old women, 115 ± 86 ng / ml) 32 . 142 subjects, 72 males and 70 females, 56.8 ± 11.57 years old, 122.53 ± 26.15 ng / ml) 33 . Similar to our observations, no significant correlation was found between LCN2 levels and age or sex in other cohorts 34,35Therefore, the weakness of this study lies in the differences in the age and sex ratios between the control group and the CSCR group, although these factors may not have affected our results.

[0052] Metabolism 21 and the heart 36 diseases and acute kidney injury 23,24 In, an increase in LCN2 values is considered as a disease biomarker. Plasma LCN2 has been identified as an early marker for diabetic retinopathy, and an increase in LCN2 levels correlates with the severity of retinopathy. 30 Also, an increase in LCN2 levels has been confirmed in the plasma of patients with Stargardt disease, retinitis pigmentosa, and age-related macular degeneration compared to healthy controls. 10 However, surprisingly, in CSCR, the LCN2 concentration in serum is decreased compared to healthy controls. Furthermore, the NGAL / MMP9 complex is also decreased, suggesting a decrease in the endogenous production of LCN2 in CSCR patients. Additionally, patients without signs of epithelial damage have lower levels of LCN2 and NGAL / MMP9 than patients with epithelial damage, but both forms have lower levels compared to the control group. This finding indicates a biological connection between the acute and chronic forms of this disease, which may be the underlying mechanism. Compared to other organs where LCN2 rather shows a pro-inflammatory effect, in the retina, LCN2 shows anti-inflammatory and antioxidant effects, especially when there are existing RPE lesions such as in light-irradiated Abca4- / -Rdh8- / - mice, and its effect is remarkable. 19 Therefore, the decrease in LCN2 is harmful to the RPE and may contribute to the pathological condition leading to the epithelial damage seen in the chronic form of CSCR. On the other hand, the decrease in NGAL / MMP9 may mean that it protects the retina from leukocyte infiltration due to a decrease in MMP9 activity. Indeed, in the brain, MMP-9 derived from immune cells is required for the initial infiltration of leukocytes through the blood-brain barrier in experimental autoimmune encephalomyelitis. 37 Also, leukocyte infiltration in the retina has been observed in the retina of AMD patients as a result of the AKT2-NFkB-LCN2 axis. 15 .

[0053] Another interesting mechanism is that LCN2 is one of the rare molecules induced by NF-κB. 12 Its expression is increased by glucocorticoids 38 and it exerts an anti-inflammatory effect in endotoxin-induced uveitis by acting as a negative feedback on the activation of NF-κB. 11 Similarly, LCN2 protects the brain from inflammation 16 repairs the blood-brain barrier damaged by ischemic stress, and directly promotes the proper membrane distribution of ZO-1 and VE-Cadherin. 39 The paradoxical effect of glucocorticoids in CSCR patients is a factor that exacerbates rather than reduces retinal edema and RPE barrier disruption, and may result from inappropriate regulation of lipocalin 2 by glucocorticoids.

[0054] Recently, Parmar et al. revealed that LCN2 increases the expression of the antioxidant enzymes heme oxygenase 1 (HMOX1) and superoxide dismutase 2 (SOD2), and exhibits a strong dose-dependent protective effect against H2O2-induced cell death. Furthermore, LCN2 protects hiPS-RPE cells from inflammation-induced apoptosis, and light stress promoted the expression of the LCN2 receptor SLC22A17. This suggests that LCN2 produced by RPE cells or immune cells plays a role in protecting the retina from degeneration due to inflammation and oxidative stress. The decrease in serum LCN2 in CSCR patients may be involved in changes in the RPE barrier and excessive sensitivity to oxidative stress, which is also supported by recent observations that the disulfide / thiol ratio is significantly higher in CSCR patients than in healthy controls. 40 On the other hand, the decrease in the NGAL / MMP9 complex is thought to reduce MMP9 activity and, as a result, protect the blood-retinal barrier, which can explain why only the outer retinal barrier is disrupted in CSCR.

[0055] Whether the serum levels of LCN2 and NGAL / MMP9 in CSCR patients reflect the ocular levels and how these levels change by corticosteroid simulation compared to healthy controls will be clarified in the future.

[0056] In conclusion, the decrease in LCN2 in CSCR with and without epithelial damage indicates the biological relationship between the two types of this disease and suggests its relevance to the disease pathogenesis mechanism. Also, it was suggested that CSCR is not an eye-limited disease but rather a more general LCN2 dysregulation. As far as we know, CSCR is the only eye disease associated with a decrease in LCN2 levels and may be used as a biomarker for this disease, especially when differential diagnosis with AMD is difficult.

[0057] Lipocalin 2 (NGAL) is significantly increased in the plasma of AMD patients compared to an age-matched control group (Plasma level of lipocalin2 is increased in neovascularage-related macular degeneration patients, particularly those with macularfibrosis. Chen M, et al. ImmunAgeing. Nov. 2020. pmid: 33292361). In dry AMD patients, the plasma level of lipocalin 2 has been shown to increase compared to controls (Lipocalin2 Plays an Important Role in Regulating Inflammation in Retinal Degeneration. Parmar T, Parmar VM, Peruse kL, Georges A, Takahashi M, Crabb JW, MaedaA. J Immunol. 2018 May 1;200(9):3128-3141. doi:10.4049 / jimmunol.1701573). The aqueous humor of neovascular AMD patients has elevated lipocalin 2 levels compared to patients who underwent cataract surgery without AMD (The Intraocular Cytokine Profileand Therapeutic Response in Persistent Neovascular Age-Related Macular Degeneration. Rezar-Dreind lS, Sacu S, Eibenberger K, Pollreisz A, Buhl W, Georgopoulos M, Kral lC, Weigert G, Schmidt-Erfurth U. Invest Ophthalmol Vis Sci. 2016 Aug 1;57(10):4144-50). When the NGAL concentration in the serum of wet AMD patients was measured and compared to controls, a significant increase in the NGAL serum concentration was observed (control: n = 88, AMD: n = 46) (Figure 4). Also, the level of NGAL was significantly higher in the serum of wet AMD patients compared to patients with CSCR (Figure 5).From this correlation, it is possible to use the NGAL value for the differential diagnosis of CSCR and AMD.

[0058]

Table 1

[0059]

Table 2

[0060]

Table 3

[0061]

Table 4

[0062]

Table 5

[0063]

Table 6

[0064]

Table 7

[0065]

Table 8

[0066] References Throughout this application, various references describe the state of the art to which the present invention pertains. The disclosures of these documents are hereby incorporated by reference into this disclosure.

[0067]

Table 9

[0068]

Table 10

[0069]

Table 11

[0070]

Table 12

Claims

**Claim 1** A method for providing an indicator for determining whether a subject has or is at risk of having central serous chorioretinopathy (CSCR), the method comprising determining the level of NGAL in a sample obtained from the subject, wherein the level is provided as an indicator indicating whether the subject has or is at risk of having central serous chorioretinopathy. **Claim 2** The method according to claim 1, wherein the sample is a blood sample. **Claim 3** The method according to claim 1, wherein the level of NGAL is compared with a predetermined reference value. **Claim 4** The method according to claim 3, wherein the predetermined reference value is the level of NGAL determined in a population of healthy individuals. **Claim 5** The method according to claim 4, wherein if the level of NGAL is lower than the level determined in a population of healthy individuals, it is concluded that the patient has CSCR or is at risk of recurrence. **Claim 6** A method for providing an indicator for differential diagnosis between CSCR and AMD in a subject, the method comprising determining the level of NGAL in a sample obtained from the subject, wherein a high level of NGAL is provided as an indicator that the subject has AMD, and a low level of NGAL is provided as an indicator that the subject has CSCR. **Claim 7** A method for providing an indicator for predicting the risk of recurrence in a subject having CSCR, the method comprising determining the level of NGAL in a sample obtained from the subject, wherein the level is provided as an indicator for the risk of recurrence. **Claim 8** A method for providing an indicator for determining whether a subject having CSCR achieves a response to treatment, the method comprising: i) determining the level of NGAL in a sample obtained from the subject before treatment; ii) determining the level of NGAL in a sample obtained from the subject after treatment; iii) comparing the level determined in step i) with the level determined in step ii); and iv) providing, when the level determined in step ii) is higher than the level determined in step i), that as an indicator for concluding that the subject has achieved a response. **Claim 9** A method of providing an indicator for treating CSCR, including: i) providing an indicator for determining whether a subject has CSCR or is at risk of having CSCR; and ii) when the indicator indicates that the subject has CSCR or is at risk of having CSCR, providing it as an indicator for treatment or administration of a drug.

10. The method according to claim 9, wherein the drug or treatment is selected from the group consisting of an anti-VEGF agent, a carbonic anhydrase inhibitor, a mineralocorticoid (MR) antagonist, laser photocoagulation, a diode micropulse laser, verteporfin photodynamic therapy (PDT) and / or trans-pupillary thermotherapy.

11. The method according to claim 10, wherein the MR antagonist is spironolactone or eplerenone.

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