Method for assessing future onset or progression of ocular health conditions

Abstract

Provided are methods of assessing future onset or progression of an ocular health condition in a subject, comprising obtaining a tear fluid sample from the subject; measuring, in the tear fluid sample, concentrations of a plurality of biomarkers of a biomarker panel, wherein the biomarker panel comprises at least two or more biomarkers from the group consisting of Nerve Growth Factor (NGF), Interleukin-8 (IL-8), Tumor Necrosis Factor-Alpha (TNF-α), and Interleukin- 13 (IL-13); comparing the measured biomarker concentrations to condition-specific signature criteria; and based on the comparison, identifying the subject as having proclivity to future onset or progression of: (i) Contact Lens Discomfort (CLD) when the measured NGF concentration and the measured IL-8 concentration meet a first condition-specific signature criterion, or (ii) Dry Eye Disease (DED) when the measured NGF concentration, the measured TNF-α concentration, and the measured IL-13 concentration meet a second condition-specific signature criterion.

Description

[0001] METHOD FOR ASSESSING FUTURE ONSET OR PROGRESSION OF OCULAR HEALTH CONDITIONS

[0002] The present disclosure claims priority to U.S. Provisional Application No. 63 / 734,275, filed December 16, 2024, the disclosure of which is herein incorporated by reference.

[0003] TECHNICAL FIELD

[0004] The present disclosure relates to the field of in vitro medical diagnostics and personalized medicine. More specifically, the disclosure relates to methods for predicting predisposition to, and assessing future onset or progression of, ocular health conditions including Contact Lens Discomfort (CLD) and Dry Eye Disease (DED) through analysis of tear fluid biomarkers. The methods enable objective, biomarker-based stratification of patients for personalized therapeutic interventions prior to manifestation of clinical symptoms.

[0005] BACKGROUND

[0006] Contact Lens Discomfort

[0007] Contact lens (CL) wear is a widely adopted option for vision correction, with approximately 90% of the global contact lens market served by soft lenses, three-quarters of which are silicone hydrogels. However, Contact Lens Discomfort (CLD) affects almost half of all CL wearers, with prevalence rates ranging from 50-90% in eye care practices. Severe CLD is recognized as the primary reason for complete discontinuation of CL wear (dropout), affecting an estimated 12- 51% of CL users and representing a major concern for the industry.

[0008] The Tear Film & Ocular Surface (TFOS) Society defines CLD as "a condition characterized by episodic or persistent adverse ocular sensations related to lens wear, either with or without visual disturbance, resulting from reduced compatibility between the contact lens and the ocular environment." End of day discomfort and ocular dryness are the most common complaints.

[0009] The etiology of CLD is multifactorial, involving complex interactions between lens-specific factors (material properties such as water content and lubricity, design, and fit), care solution chemistry, wearing schedule, as well as patient-related, environmental, and compliance factors. This complexity makes objective prediction and diagnosis of CLD extremely challenging.

[0010] Current Limitations

[0011] Currently, the primary tools for assessing CLD are subjective patient-reported questionnaires, such as the Contact Lens Dry Eye Questionnaire-8 (CLDEQ-8). While useful clinically, these tools are inherently subjective and can vary based on the patient's current psychological state, making them unreliable for predicting future intolerance to contact lens wear. Research has suggested a link between CLD and inflammation, with reports of changes in inflammatory biomarker concentrations in tear fluid, such as lnterleukin-6 (IL-6) and Tumor Necrosis Factor-Alpha (TNF-ot). However, prior studies that analyzed tear samples during or immediately after CL wear concluded that biomarker fluctuations merely reflect an acute inflammatory response to the physical stimulus of CL wear. In one study, there was no statistically significant difference in most biomarker concentrations between a CLD group and a non-CLD group immediately after CL wear.

[0012] Dry Eye Disease

[0013] Dry Eye Disease (DED) is one of the most common ocular surface diseases, affecting up to 75% of the population in some studies and having significant impact on quality of life and productivity. The TFOS Dry Eye Workshop II (DEWS II) defines DED as "a multifactorial disease of the ocular surface characterized by a loss of homeostasis of the tear film, and accompanied by ocular symptoms, in which tear film instability and hyperosmolarity, ocular surface inflammation and damage, and neurosensory abnormalities play etiological roles."

[0014] Current DED diagnosis is based on evaluation of clinical signs and patient-reported symptoms, but this approach faces challenges including poor correlation between signs and symptoms and the subjective nature of many tests. For example, clinical testing procedures may correlate poorly with symptoms and may be influenced by the subjective interpretation or the skill of the technician. Further, symptoms may not be specific to DED and may be under attributed or over attributed to DED.

[0015] Point-of-care (POC) tests based on single biomarkers have been developed, including the following: TearLab (tear osmolarity measurement), which has shown high variability even in healthy individuals and issues with repeatability; InflammaDry (MMP-9 detection), which lacks specificity, as MMP-9 is elevated not only in DED but also in allergic conjunctivitis, corneal infections, and other inflammatory ocular surface diseases; and Lactoferrin tests, which may be useful for detecting aqueous deficient DED, but are not universal for all DED forms.

[0016] Accordingly, the limitations of conventional methods include high variability, lack of specificity, and the inherent subjectivity of clinical evaluation or questionnaires.

[0017] Multi-Pathway Assessment

[0018] DED is a multifactorial disease involving multiple pathophysiological pathways, including inflammation, nerve damage / pain, and epithelial / goblet cell dysfunction. A marker reflecting only a single pathological pathway is not sufficient to accurately capture this complex disease. Similarly, for CLD, the underlying mechanisms involve persistent ocular immune dysfunction that transcends simple acute inflammatory responses. There exists a strong need for methods that can: (1) objectively predict predisposition to ocular health conditions before symptoms manifest or worsen, (2) distinguish between acute inflammatory responses and persistent immune dysfunction, (3) assess multiple mechanistically distinct pathways simultaneously, and (4) enable personalized, preventative therapeutic approaches.

[0019] SUMMARY

[0020] The present inventor has discovered that specific tear fluid biomarker signatures, measured under defined temporal conditions, can predict future onset or progression of ocular health conditions with high accuracy.

[0021] In one embodiment, provided is a method of assessing future onset or progression of an ocular health condition in a subject, each ocular health condition defined by distinct biomarker panels and clinical purposes, yet complementary for ocular surface management. The ocular health conditions for assessment may include prediction of Contact Lens Discomfort or diagnosis of Dry Eye Disease.

[0022] Contact Lens Discomfort: In subjects prone to CLD, the tear fluid concentrations of Nerve Growth Factor (NGF) and lnterleukin-8 (IL-8) remain at statistically significantly elevated levels even after a contact lens-free "washout period" compared to subjects not prone to CLD. After a 14-day washout period, NGF was elevated and IL-8 was elevated in the CLD group versus non- CLD group.

[0023] This finding demonstrates that predisposition to CLD is rooted not in a transient, acute inflammatory response to CL wear, but in a persistent, sub-clinical inflammatory state where the ocular surface fails to return to normal homeostasis even after removal of the external stressor. A logistic regression model combining NGF and IL-8 achieved an Area Under the Curve (AUC) of 0.76 for predicting CLD predisposition.

[0024] Dry Eye Disease: In the tear fluid of DED patients, three specific biomarkers, Tumor Necrosis Factor-Alpha (TNF-ot), Nerve Growth Factor (NGF), and Interleukin-13 (IL-13), have simultaneous and statistically significant elevation compared to healthy controls.

[0025] Critically, these three biomarkers represent mechanistically distinct pathological pathways. TNF- ot represents the inflammatory cascade central to immune response on the ocular surface. NGF represents the pain / neurogenic component involving corneal nerve response and hyperalgesia. IL-13 represents the epithelial / goblet cell response related to conjunctival epithelium homeostasis and mucin production. A logistic regression model combining these three biomarkers achieved an AUC of 0.79 for DED diagnosis, demonstrating superior diagnostic accuracy compared to single-marker tests. In a first aspect, there are provided methods of assessing future onset or progression of an ocular health condition in a subject, comprising obtaining a tear fluid sample from the subject; measuring, in the tear fluid sample, concentrations of a plurality of biomarkers of a biomarker panel, wherein the biomarker panel comprises at least two or more biomarkers from the group consisting of Nerve Growth Factor (NGF), lnterleukin-8 (IL-8), Tumor Necrosis Factor-Alpha (TNF- ot), and Interleukin-13 (IL-13); comparing the measured biomarker concentrations to conditionspecific signature criteria; and based on the comparison, identifying the subject as having proclivity to future onset or progression of: (i) Contact Lens Discomfort (CLD) when the measured NGF concentration and the measured IL-8 concentration meet a first conditionspecific signature criterion, or (ii) Dry Eye Disease (DED) when the measured NGF concentration, the measured TNF-ot concentration, and the measured IL-13 concentration meet a second condition-specific signature criterion.

[0026] In a second aspect, there are provided methods incorporating a washout period following contact lens wear to assess persistent ocular immune dysfunction indicative of CLD predisposition. There are provided methods of assessing future onset or progression of an ocular health condition, further comprising exposing the subject to contact lens wear conditions constituting a potential inflammatory stimulus; obtaining the tear fluid sample at the conclusion of a predetermined washout period in which the subject abstains from the potentially inflammatory stimulus; and identifying the subject as having persistent ocular immune dysfunction when the measured biomarker concentrations remain elevated above a threshold value of the first condition-specific signature criterion despite removal of the potential inflammatory stimulus during the predetermined washout period, wherein the identified persistent ocular immune dysfunction indicates proclivity to future onset or progression of CLD.

[0027] There is provided a method wherein the washout period is at least 7 days.

[0028] There is provided a method wherein the washout period is at least 14 days.

[0029] There is provided a method wherein the comparing step further comprises calculating a probability score for the first condition-specific signature criterion using a predictive model that combines the concentration of NGF and the concentration of IL-8.

[0030] There is provided a method wherein the predictive model is a logistic regression model.

[0031] There is provided a method wherein the first condition-specific signature criterion is based on the concentration of NGF and the concentration of IL-8 in tears obtained from a population of subjects who do not have contact lens discomfort.

[0032] There is provided a method wherein the biomarker panel further comprises at least one additional biomarker selected from the group consisting of Growth-Related Oncogene-Alpha (GRO-ot), Interferon-gamma (IFN-y), and Interleukin-10 (IL-10). In a third aspect, there are provided methods utilizing multi-pathway signatures that provide improved diagnostic specificity by simultaneously assessing mechanistically distinct physiological pathways. There are provided methods of assessing future onset or progression of an ocular health condition, further comprising generating from the measured biomarker concentrations a multi-pathway signature of the tear fluid sample representing mechanistically distinct pathways of (a) a first physiological pathway related to pain involving corneal nerve response, (b) a second physiological pathway related to ocular surface inflammatory cascade, and (c) a third physiological pathway related to goblet cell involvement in homeostasis of conjunctival epithelium, wherein the measured NGF concentration corresponds to the first physiological pathway, the measured TNF-ot concentration corresponds to the second physiological pathway, and the measured IL-13 concentration corresponds to the third physiological pathway; comparing the multi-pathway signature to the second condition-specific signature criterion; and identifying the subject as having proclivity to future onset or progression of DED when the multi-pathway signature meets the second condition-specific signature criterion, wherein the multi-pathway signature provides improved diagnostic specificity compared to assessment of individual biomarkers.

[0033] There is provided a method wherein the identifying step is performed using a single diagnostic score calculated based on the concentrations of the biomarker panel.

[0034] There is provided a method wherein the single diagnostic score is calculated using a logistic regression model.

[0035] There is provided a method wherein the second condition-specific signature criterion is based on the concentration of NGF, the concentration of TNF-ot and the concentration of IL-13 in tears obtained from a population of subjects without DED.

[0036] There is provided a method wherein the biomarker panel further comprises at least one additional biomarker selected from the group consisting of Growth-Related Oncogene-Alpha (GRO-ot), Monocyte Chemoattractant Protein-1 (MCP-1), and lnterleukin-1 beta (IL-ip).

[0037] There is provided a method wherein the measuring employs at least one selected from the group consisting of Enzyme-Linked Immunosorbent Assay (ELISA), a multiplex immunobeadbased array assay, a lateral-flow assay (LFA), and an electrochemical sensor system.

[0038] In a fourth aspect, there are provided methods enabling therapeutic interventions based on the assessment of future onset or progression of an ocular health condition in a subject.

[0039] There is provided a method of assessing future onset or progression of an ocular health condition, further comprising administering a therapeutic intervention to the subject prior to manifestation of clinical symptoms of CLD, based on the identified proclivity to future onset or progression of CLD, wherein the therapeutic intervention is selected from one or more of alternative contact lens material, modified contact lens use protocol, modified CLD monitoring protocol, modified CLD treatment protocol, and pharmaceutical intervention.

[0040] There is provided a method of assessing future onset or progression of an ocular health condition, further comprising administering a therapeutic intervention to the subject based on the indicated proclivity to future onset or progression of DED, wherein the therapeutic intervention is selected from one or more of alternative contact lens material, modified contact lens use protocol, modified DED monitoring protocol, modified DED treatment protocol, and pharmaceutical intervention.

[0041] There is provided a method of assessing future onset or progression of an ocular health condition, further comprising identifying which of the three physiological pathways shows predominant dysregulation based on relative contributions of the measured NGF concentration, the measured TNF-ot concentration, and the measured IL-13 concentration to the multi-pathway signature; and administering a pathway-targeted therapeutic intervention selected based on which physiological pathway shows predominant dysregulation.

[0042] BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 is a bar chart showing the distribution of contact lens materials (silicone hydrogel (Si Hy) vs. hydrogel (Hy)) worn by participants in the non-CLD group (Group A) and the CLD group (Group B) in the clinical study employed in the Examples herein.

[0044] Figure 2 is a pair of box plots illustrating the statistically significant difference in the tear fluid concentrations of NGF (p=0.009) and IL-8 (p=0.032) between the non-CLD group (Group A) and the CLD group (Group B) at Visit 2 (after a two-week washout period).

[0045] Figure 3 is a series of box plots comparing the concentrations of key biomarkers for both groups at both Visit 1 (immediately post-wear) and Visit 2 (post-washout), visually demonstrating that the significant group separation for NGF and IL-8 is most pronounced at Visit 2.

[0046] Figure 4 is a Receiver Operating Characteristic (ROC) curve for the combined NGF and IL-8 biomarker panel, showing an Area Under the Curve (AUC) of 0.76, which indicates high predictive accuracy.

[0047] Figure 5 shows generalized linear model (GLM) analysis for the combined NGF and IL-8 biomarker panel.

[0048] Figure 6 is a series of box plots comparing the tear fluid concentrations of key inflammatory proteins between a DED group and a non-DED group in the clinical study employed in the Examples herein, specifically showing that TNF-ot, NGF, and IL-13 are statistically significantly or near-significantly elevated in the DED group. Figure 7 shows the results of a Generalized Linear Model (GLM) analysis (left) and a Receiver Operating Characteristic (ROC) curve (right) for the 3-biomarker panel (TNF-ot, NGF, and IL-13). The ROC curve demonstrates an Area Under the Curve (AUC) of 0.79, indicating the high clinical utility of this panel.

[0049] DETAILED DESCRIPTION

[0050] Definitions

[0051] As used herein, the following terms are intended to have the following meanings.

[0052] "Contact Lens Discomfort (CLD)" refers to a condition characterized by episodic or persistent adverse ocular sensations related to lens wear, either with or without visual disturbance, resulting from reduced compatibility between the contact lens and the ocular environment, as defined by the TFOS International Workshop on Contact Lens Discomfort.

[0053] "Dry Eye Disease (DED)" is a multifactorial disease of the ocular surface characterized by a loss of homeostasis of the tear film, and accompanied by ocular symptoms, in which tear film instability and hyperosmolarity, ocular surface inflammation and damage, and neurosensory abnormalities play etiological roles, consistent with the TFOS DEWS II definition.

[0054] "Predisposition" or "proclivity" refers to a biological tendency or vulnerability of a subject to develop an ocular health condition or experience symptoms thereof in the future with higher probability than the general population.

[0055] "Future onset or progression" encompasses both (i) the initial development of a condition in a subject not currently exhibiting clinical symptoms, and (ii) the worsening or advancement of an existing condition to a more severe state.

[0056] "Washout period" refers to a period of abstention from contact lens wear that is sufficient for the subject's ocular surface to recover from acute effects of CL wear and return toward the subject's baseline state. The washout period allows discrimination between transient acute inflammatory responses and persistent immune dysfunction. Suitable washout periods include, for example, at least 3 days, preferably at least 7 days, more preferably at least 14 days, and most preferably 14-21 days.

[0057] "Tear fluid sample" refers to tear fluid collected from a subject's eye by any suitable method, including but not limited to sterile glass capillary micropipettes, Schirmer strips, microcapillary tubes, or other collection devices capable of obtaining tear fluid from the tear meniscus at the lid margin or other ocular surface locations.

[0058] "Biomarker panel" refers to a set of two or more biomarkers measured to assess ocular health conditions. For CLD assessment, the panel comprises at least NGF and IL-8. For DED assessment, the panel comprises at least TN F-ot, NGF, and IL-13. Extended panels may include additional biomarkers as specified herein.

[0059] "Condition-specific signature criterion" or "condition-specific signature criteria" refers to predetermined thresholds, patterns, or combinations of biomarker concentrations that distinguish subjects with proclivity to a specific ocular health condition from those without such proclivity.

[0060] "Elevated concentration" means that the concentration of a biomarker in a subject's tear fluid sample exceeds a predetermined control level or threshold. The control level is established based on biomarker concentrations in tear fluid samples from a reference population without the condition of interest (e.g., non-CLD or non-DED populations). The threshold may be defined as the mean, median, or a specific percentile (e.g., 75th, 80th, or 90th percentile) of the reference population.

[0061] "Persistent ocular immune dysfunction" refers to a state where inflammatory biomarkers remain elevated above threshold values despite removal of an external inflammatory stimulus (such as contact lenses) during a washout period, indicating an underlying biological predisposition rather than a transient acute response.

[0062] "Multi-pathway signature" refers to a biomarker pattern that represents activity across multiple mechanistically distinct physiological pathways involved in ocular surface disease pathology.

[0063] Biomarker Selection

[0064] The inventor herein has discovered that the following biomarkers have significance with respect to assessing future onset or progression of an ocular health condition, as detailed herein.

[0065] Nerve Growth Factor (NGF)

[0066] NGF is one of the most common neurotrophic factors. NGF is involved in corneal wound healing, but also is an endogenous mediator for pain and hyperalgesia. Its increased level in tears has been associated with CLD. NGF in tears can be increased by various underlying ocular surface diseases, which undiagnosed and unmanaged may lead to the CLD and ocular pain. As an example, the increase of NGF in tear samples has been reported in DED patients, Sjogren's syndrome, corneal damage and ulceration.

[0067] NGF is a neurotrophic factor first discovered by Rita Levi-Montalcini in the 1950s (Nobel Prize awarded in the 1980s). In ocular health, NGF has a role in corneal nerve response. The cornea is one of the most innervated tissues in the human body. NGF plays crucial roles in corneal wound healing through promotion of epithelial cell migration and proliferation, modulation of corneal nerve function and density, mediation of pain and hyperalgesia through sensitization of nociceptors, and neurogenic inflammation associated with ocular surface diseases. The cornea is one of the most innervated tissues in the body. Functionality of corneal nerves is crucial for ocular surface homeostasis. Impairment of the tear film and epithelial damage, which is caused by the DED inflammatory state, results in chronic stimulation of corneal nerve ending, promotion neurogenic stress, and ocular pain.

[0068] In response to DED inflammation, increased levels of NGF, a neurotrophic factor with a crucial role in modulation function of the peripheral nervous system, were reported in DED patients. Interestingly, the administration of recombinant NGF has been shown as safe and effective treatment for DED (clinical trials phase II), improving ocular surface impairment and epithelial damage, as well as increase in tear production, possibly due to the increase of corneal sensitivity. NGF is statistically significant in the distinction of DED from non-DED group and is a significant biomarker for DED.

[0069] In relation to DED, elevated NGF in DED patients represents the pain component involving corneal nerve response and neurogenic inflammation. NGF elevation correlates with DED severity and has been reported in Sjogren's syndrome, corneal damage, and ulceration.

[0070] Interleukin-8 (IL-8)

[0071] IL-8 is a chemokine associated with the induction of ocular inflammation and angiogenesis. IL-8, together with TNF-a, is responsible for the persistent nociceptor hypersensitivity and therefore pain sensation. An elevation of IL-8 in the tear samples correlated to contact lens wear has been reported in literature, corneal refractive therapy as well as in ocular surface diseases such as DED (desiccation stress), corneal injury, and allergic conjunctivitis. The increase of IL-8 inflammatory protein was not appreciated immediately after contact lens removal.

[0072] In relation to CLD, IL-8 is statistically significantly elevated in the CLD group after the 14-day washout period, but not immediately after lens wear. This pattern indicates that IL-8 elevation represents persistent immune activation rather than acute response to mechanical stimulus. IL- 8 elevation has been associated with contact lens wear, corneal refractive therapy, desiccation stress, corneal injury, and allergic conjunctivitis.

[0073] Together with TNF-ot, IL-8 is responsible for persistent nociceptor hypersensitivity and pain sensation, linking inflammatory processes to symptomatic discomfort.

[0074] Tumor Necrosis Factor-Alpha (TNF-a)

[0075] TNF-ot is an inflammatory biomarker consistently elevated in DED studies, and it correlates with disease severity. TNF-a is a pro-inflammatory cytokine that amplifies inflammatory response by the stimulation of production of other cytokines and the maturation of antigen-presenting cells (APCs) during the innate immune response on the ocular surface. Also, it is involved in the apoptosis of corneal epithelial cells due to inflammatory epithelial dysfunction. TNF-ot is not only a biomarker for the DED disease, its reduction or suppression is also a therapeutic target. As an example, the topical anti TNF-ot drug Licaminlimab (Novartis) was shown to reduce ocular discomfort in patients with severe DED in clinical studies. TNF-ot is a prototypical pro-inflammatory cytokine central to immune and inflammatory responses.

[0076] In relation to DED, TNF-ot is statistically significantly elevated in DED patients. It represents the inflammatory cascade pathway that is central to the immune response on the ocular surface. TNF-ot levels correlate with DED severity and serve as both a diagnostic marker and therapeutic target.

[0077] Interleukin-13 (IL-13)

[0078] The conjunctival epithelium is a mucous membrane, covering the sclera and inside of eye lids. It is responsible for smooth eye movements, immune response, and is a source of mucin produced by the conjunctival goblet cells. The secreted mucins (highly glycosylated glycoproteins) are essential for ocular surface tear film homeostasis because of their water binding capacity. In Aqueous tear Deficient Dry Eye (ADDE), goblet cell loss and related mucin deficiency are often reported. The increase of pro-inflammatory cytokines due to a persistent inflammatory dry eye condition may lead to cell apoptosis.

[0079] IL-13, a cytokine, has a homeostatic role on the conjunctival epithelium and is related to goblet cells proliferation and mucin secretion stimulation as a response to DED inflammatory stimulus. Accordingly, it has been reported as increased in DED patient tears.

[0080] In relation to DED, IL-13 is elevated in DED patients. The conjunctival epithelium is a mucous membrane responsible for smooth eye movements, immune response, and mucin production. Goblet cells produce mucins essential for tear film homeostasis through water-binding capacity. In aqueous deficient dry eye, goblet cell loss and mucin deficiency are common. In evaporative dry eye, pro-inflammatory cytokines lead to cell apoptosis.

[0081] IL-13 represents a mechanistically distinct pathway from inflammation (TNF-ot) and neurogenic pain (NGF), capturing the epithelial and mucin production aspects of DED pathology.

[0082] Additional Optional Biomarkers

[0083] Growth- Related Oncogene-Alpha (GRO-ot / CXCL-1) is a CXC chemokine that serves as a chemoattractant for neutrophils and T lymphocytes. GRO-ot shows a trend toward elevation in CLD. GRO-ot has been reported elevated in Sjogren's syndrome and contact lens-induced dry eye. It may be relevant to inflammatory grading in DED with correlation to tear stability.

[0084] Interferon -gam ma (IFN-y) is a cytokine with antiproliferative and apoptosis effects, and it is involved in macrophage activation and antiviral / antibacterial immunity. It shows a trend toward elevation in CLD. I FN-y increase correlates with tear hyperosmolarity in DED and goblet cell apoptosis, leading to mucin production imbalance.

[0085] Interleukin-10 (IL-10) is an anti-inflammatory cytokine that halts synthesis of pro-inflammatory proteins. It shows a trend toward elevation in CLD. IL-10 may have protective and analgesic functions, and it may increase following refractive surgery and during contact lens wear.

[0086] Monocyte Chemoattractant Protein-1 (MCP-l / CCL-2) is a chemokine important for monocyte attraction to inflammation sites.

[0087] Interleukin-1 beta (I L-ip) is a pro-inflammatory cytokine with synergistic effects with TNF-ot for promoting inflammatory cytokine / chemokine production in the conjunctiva. IL-1|3 is elevated in DED patients.

[0088] The Washout Period and Persistent Immune Dysfunction

[0089] Biological Basis

[0090] Even "non-problematic" contact lens wear is recognized as an insult to the ocular surface, resulting in sub-symptomatic inflammatory reactions. Asymptomatic CL wear may provoke a low-grade sub-clinical inflammatory status sometimes termed "para-inflammation."

[0091] One aspect of assessing future onset or progression of an ocular health condition lies in the distinction between acute inflammatory response and persistent immune dysfunction.

[0092] Acute inflammatory response is transient elevation of inflammatory mediators in direct response to the mechanical and physiological stress of contact lens wear. This response occurs in essentially all contact lens wearers and resolves quickly upon lens removal (typically within 24 hours to 6 days based on prior literature).

[0093] Persistent immune dysfunction is a sustained elevation of specific inflammatory mediators that persists even after removal of the inflammatory stimulus and completion of a washout period. This persistent elevation reflects an underlying biological predisposition to develop CLD, representing a failure of the ocular surface to return to normal homeostasis.

[0094] Implementation Embodiments

[0095] The measurement of biomarkers can be practically implemented using a method capable of specifically and quantitatively detecting the biomarker proteins. Formats for the measurement of biomarkers may include laboratory, point-of-care, and low-cost formats.

[0096] Suitable methods include, but are not limited to, Enzyme-Linked Immunosorbent Assay (ELISA), Western bloting, and mass spectrometry. Also, multiplex or Point-of-Care (POC) immunoassay formats, such as multiplex immunobead-based arrays, lateral-flow assays (LFA), or electrochemical sensor systems allow for the simultaneous, sensitive, and efficient measurement of multiple biomarkers from a small volume of tear fluid. Further, the measurement may be configured for a multiplex assay (e.g., antibodies immobilized on beads with different fluorescent codes) or for individual ELISA performed for each biomarker.

[0097] In an embodiment, the measurement of biomarkers may employ ELISA-based biomarker quantification. For example, a measurement method may apply to a tear sample fluid one or more reagents for capture and detection of an antibody or antigen specific for a corresponding biomarker protein. The reagents may include, for example, an antigen or antibody, a blocking agent, an enzyme-conjugated secondary antibody, a chromogenic substrate, or a stop solution.

[0098] For example, in an embodiment, wells of a microtiter plate are coated with a capture antibody or antigen. A blocking buffer may be added to prevent non-specific binding. Then, a sample may be added to allow a target biomarker protein to bind. An enzyme-linked detection antibody may be added to bind to the target biomarker protein. A chromogenic material may be added that yields a detectable signal when converted by the enzyme linked to the detection antibody. At each step, appropriate washing and incubation may be performed. The absorbance at a specific wavelength may be measured and plotted to determine the sample concentration.

[0099] In another embodiment, the measurement of biomarkers may employ POC formats. For example, a rapid microfluidic cartridge system may be employed that enables rapid, portable, multiplexed, and low cost measurement of biomarker proteins using a low sample volume. A rapid microfluidic cartridge system may move a sample fluid or reagents through miniaturized channels using capillary action, pressure, or electrokinetics. Biomarkers may be measured by specific binding in conjunction with optical or electrical detection, within a timeframe of approximately 10-15 minutes.

[0100] In another embodiment, the measurement of biomarkers may employ a lateral-flow assay (LFA). For example, biomarkers such as NGF and IL-8 may be measured by applying a sample fluid to a substrate that moves the sample across the substrate by capillary action to antibodies or probes, resulting in a test line and a control line to indicate the presence or absence of a target biomarker protein. The lateral-flow assay may include a sample pad, a conjugate pad, a reaction area such as a nitrocellulose membrane, and an absorbent pad.

[0101] In another embodiment, the measurement of biomarkers may electrochemical sensing. For example, an electrochemical sensor system may employ a receptor unit for molecular recognition of a biomarker that causes physical or chemical changes at the surface of a sensitive layer. For example, a receptor unit may employ antigens, antibodies, aptamers, or molecularly imprinted polymers. An electrochemical sensor system may further employ a transducer unit for converting the recognition signals to electrical signals, such as potential, current, or impedance, that correspond to the concentration of the biomarker protein. For example, a transducer unit may employ a three-electrode system or a field effect transistor.

[0102] Evidence from Clinical Studies

[0103] The inventor conducted a clinical study designed as a prospective, exploratory, single-center pilot study with two arms (Group A: asymptomatic CL wearers without CLD, and Group B: symptomatic CL wearers with CLD; n~20 in each group). Recruitment was based on scores from the CLDEQ-8 questionnaire, with Group A having a score of < 8 points and Group B having a score of > 14 points.

[0104] Subjects enrolled had a visual acuity of at least 0.80 with CL in situ and were healthy without any ocular or systemic disease that concerns ocular health (e.g. diabetes). The recruitment was carried out based on CL comfort levels on habitual soft contact lens wearers (daily disposables, weekly or monthly CL wear schedule) wearing CL at least five days per week for at least eight hours per day showing good CL fitting characteristics. Acceptable fitting parameters were defined as post blink movement in up gaze (B: 0.20 - 0.50 mm), horizontal lag (L: 50-100% change in overlap of the lens onto the limbus) and push-up recovery speed (P). All three variables B, L and P were graded on a 3 point scale: + (more) and - (less).

[0024] , CL comfort evaluation was determined by using the Contact Lens Discomfort Eye Questionnaire, CLDEQ-8.

[0105] Group A: Asymptomatic CL wearers had CLDEQ-8 questionnaire scores < 8 points, whereas Group B Symptomatic CL wearers suffering from CLD had CLDEQ-8 questionnaire with a score of > 14 points. In between scores were excluded.

[0106] In this study, a large variety of various monthly, two-weekly and daily disposable hydrogel (Hy) and silicon hydrogel (Si Hy) contact lenses were worn by the participants. Overall, 14 different materials were worn, 6 Hy and 8 Si Hy. In this study, 70% of the CL used in the study were Si Hy and only 30% Hy (Fig. 1).

[0107] Considering the study groups A: Non-CLD and B: CLD, a large majority 85% of participants not experiencing CLD wore SiHy materials, whereas in the group B experiencing CLD, the percentage of SiHy was 55%.

[0108] The study procedure was scheduled in 2 visits following the enrolment evaluation via CLDEQ-8 questionnaire and CL fitting evaluation.

[0109] Visit 1, CL wear: all participants wore a fresh pair of their habitual CL for a minimum of 6h at the day of measurement. Participants completed the Ocular Surface Disease Index (OSDI) questionnaire. Fitting characteristics, Ocular Surface Temperature (OST), Non-invasive Tear film break up time (NIBUT), and topography was recorded before the CL were removed. Following CL removal Tear Meniscus Height (TMH), bulbar conjunctival hyperaemia, corneal sensitivity, Lidparallel Conjunctival Folds (LIPCOF), tear sampling from both eyes, Lid Wiper Epitheliopathy (LWE), and meibomian gland morphology were performed.

[0110] In the 40 tear samples no statistically significant difference in between the tear protein concentration and the study Groups A (asymptomatic group / Non-CLD) and Group B (symptomatic group / CLD) were found. The only tear inflammatory protein showing a distinctive trend for Group A and Group B was NGF, which was found with a p value of 0.09 in Visit 1 between the two groups.

[0111] Visit 2 was scheduled following 2 weeks of Visit 1, between which the participants refrained from any CL wear in a "washout period." Participants completed the OSDI questionnaire and were evaluated as in Visit 1.

[0112] In the tear samples of Visit 2, two tear inflammatory proteins were found statistically significant different in the study groups (Group A, asymptomatic group / Non-CLD and Group B: symptomatic group / CLD): NGF and IL-8, p value of 0.009 and 0.032. From those data a different tear protein pattern could be detected following 2 weeks after CL wear depending on the participants' experience of CLD or not.

[0113] Fig. 2 shows Visit 2 box plots of the two inflammatory tear proteins NGF and IL-8, which showed statistically significant differences between the study groups. The concentrations were shown to be increased in participants prone to experience CLD (Group B) compared to the asymptomatic participants (Group A).

[0114] All data collection was executed on the right eye except for tear sampling taking place bilaterally. Tear samples were collected using sterile glass capillary micropipettes (Drummond Scientific co, Broomall, PA, USA). Upon sample collection the samples were immediately frozen at -80°C in cryotubes until their analysis.

[0115] At Visit 1, immediately after CL wear, no statistically significant differences in tear protein concentrations were found between the two groups, although NGF showed a trend toward elevation in the CLD group (p=0.090).

[0116] In contrast, at Visit 2, after the 14-day washout period, two proteins showed a statistically significant difference between the groups. As shown in Figure 2, the CLD group (Group B) had significantly higher concentrations of NGF (mean 44.66 pg / ml vs. 27.62 pg / ml, p=0.009) and IL-8 (mean 1125.05 pg / ml vs. 814.68 pg / ml, p=0.032) compared to the non-CLD group (Group A). These results are summarized in Table 1 below. Table 1: Tear Fluid Concentrations of Key Biomarkers at Visit 2

[0117] As an example, based on the clinical data, a condition-specific signature criteria for CLD may include biomarker concentrations for both NGF and IL-8 falling within one standard deviation of the respective mean concentrations for Group B. For example, a CLD-specific signature criteria may include NGF concentration within 20.23-69.09 pg / ml and IL-8 concentration within 659.13- 1590.97 pg / ml.

[0118] As another example, a condition-specific signature criteria for CLD may include biomarker concentrations for both NGF and IL-8 that are higher than a predetermined control level or threshold, such as a specific percentile (e.g., 75th percentile) of the biomarker concentrations in tear fluid samples obtained from a population of healthy subjects who do not experience CLD (a non-CLD population). For example, a CLD-specific signature criteria may include NGF concentration greater than 41.62 pg / ml and IL-8 concentrations greater than 953.51 pg / ml.

[0119] This data demonstrates that a predisposition to CLD is strongly associated with a state of elevated NGF and IL-8 that persists even after a washout period.

[0120] A two-way crossed ANOVA taking into account both the visits and groups A and B was performed and visualized in the boxplots (Fig. 3) illustrating the tear protein concentration across the four subgroups for the relevant tear inflammatory proteins NGF, IL-8, GRO-ot, INFGAMMA, and IL-10.

[0121] Analysis of the boxplots of the protein concentrations shows that for 22 (only 5 shown) of the 28 analyzed proteins, statistically significant differences between the tear protein concentrations of Visit 1 and Visit 2 were found independent of the study groups.

[0122] Fig. 3 shows box plots of NGF, IL-8, GRO-ot, IFN-GAMMA and IL-10 for both visits divided in Group A: asymptomatic group / Non-CLD and Group B: symptomatic group / CLD. The tear protein concentrations for visit 2 were superior to visit 1, therefore the P value of the visits is highly significant. Whereas for the groups when considering Group A visit 1 and visit 2 combined and Group B visit 1 and 2 combined, only biomarker NGF and IL-8 showed statistically significant differences, in line with the t-test results. No significant interaction between groups and visits were found.

[0123] A logistic regression analysis using a Generalized Linear Model (GLM) was performed on the NGF and IL-8 concentration data obtained at Visit 2.

[0124] Grouping tear proteins into an optimized biomarker panel may offer superior accuracy compared to single biomarkers as revealed by the Receiver Operating Characteristic (ROC) curve analysis which plots true positive (sensitivity) and false positive (specificity) rates respectively on y-and x-axes. The Area Under the upward rising Curve (AUC) is a measure of test accuracy, with a perfect test having an AUC value of 1.

[0125] As shown in Fig. 4, the Area Under the Receiver Operating Characteristic (ROC) Curve (AUC) for the panel combining these two biomarkers was 0.76. This value represents the degree to which the combined tear proteins concentrations correctly predict group membership, with its maximum possible value being 1. This value indicates that the biomarker panel has a high capacity to accurately classify subjects into the CLD and non-CLD groups, demonstrating its clinical utility as a tool for predicting predisposition to CLD.

[0126] A predictive model that combines the concentration values of NGF and IL-8 can be used. For example, a logistic regression analysis can be used to calculate a probability score for the predisposition to CLD from the concentrations of both markers. As shown in Fig. 5, generalized linear model (GLM) analysis for the 2-biomarker panel for the 40 study participants in visit 2 was conducted. The analysis shows a separation between the Non-CLD group (group A, asymptomatic CL wearers) and the CLD group (group B, symptomatic CL wearers) when considering the combination of the 2 tear proteins NGF and IL-8.

[0127] In another embodiment, the concentration values of the biomarkers are input into a multivariate analysis algorithm to calculate a single diagnostic score or a probability for assessing predisposition to CLD. Multi-Pathway Signature Approach for DED

[0128] Rationale for Multi-Marker Panels

[0129] DED is a multifactorial disease involving multiple interconnected but mechanistically distinct pathways.

[0130] Pathway 1 - Inflammation: One pathway includes central immune response involving cytokines like TNF-ot, IL-1(3, and IL-6. This pathway drives amplification of inflammatory responses, epithelial damage, and recruitment of immune cells.

[0131] Pathway 2 - Neurogenic / Pain: One pathway involves neurotrophic factors like NGF and neuropeptides like Substance P. This pathway mediates pain sensation, corneal nerve dysfunction, and neurogenic inflammation.

[0132] Pathway 3 - Epithelia l / Goblet Cell Response: One pathway involves cytokines like IL-13 that regulate mucin production, goblet cell function, and epithelial homeostasis.

[0133] Single biomarkers capture only one aspect of this complex pathology and suffer from limited specificity (e.g., MMP-9 elevated in many inflammatory conditions), high inter-individual variability, and inability to distinguish DED from other ocular surface diseases.

[0134] The described multi-pathway panels overcome these limitations by capturing the disease "signature" across multiple mechanistic pathways, providing improved specificity through pattern recognition, and enabling identification of predominant dysregulated pathways for targeted therapy.

[0135] TNF-a / NGF / IL-13 Triad

[0136] Each biomarker represents a distinct mechanistic pathway (inflammation, neurogenic, epithelial), providing comprehensive disease assessment. Further, all three markers showed significant or near-significant individual elevation in DED. The markers provide non-redundant, complementary information about disease state, as they reflect different aspects of pathophysiology. Further, a logistic regression model combining these three markers achieved AUC=0.79, which is considered clinically useful.

[0137] Evidence from Clinical Studies

[0138] The inventor conducted a clinical study designed as a prospective, controlled clinical study comprising 20 DED patients and 20 age- and sex-matched non-DED healthy controls. A total of 40 participants were enrolled, 20 DED participants were age and sex matched to 20 Non-DED controls. All participants were non-contact lens wearers and healthy and free of ocular disease (with the exception of DED). Inclusion criteria determining the DED group were based on the 2017 Tear Film & Ocular Surface Society Dry Eye Workshop II (TFOS DEWS II) diagnostic criteria such as OSDI score of > 13 or DEQ-5 score of > 6. The DED group participants met at least one of the following criteria: Tear film osmolarity > 308 mOsm / L in at least one eye or interocular difference > 8 mOsm / L, Non-invasive tear film break-up time of <5 seconds in at least one eye, more than 5 spots of corneal staining, or > 9 conjunctiva spots or lid margin staining (> 2 mm length & > 25% width) in at least one eye. In order to minimize any effect of DED treatments in the disease group, only subjects on a stable treatment regimen (e.g. lid scrubs; lubricants) were enrolled and kept their treatments unchanged throughout the study.

[0139] Inclusion criteria for the healthy control group were: OSDI score of<13 and DEQ-5 <6. Also, all of the following criteria were met: Tear film osmolarity < 308 mOsm / L in at least one eye, interocular difference < 8 mOsm / L, non-invasive tear film break-up time of >5 seconds in at least one eye, and <5 spots of corneal staining or < 9 conjunctiva spots or lid margin staining (< 2 mm length & < 25% width) in at least one eye.

[0140] Following the screening and recruitment visit, the participants were scheduled for the study visit on the next day to have their basal tear film collected using a sterile glass microcapillary tubes (Drummond Scientific Co., Broomall, PA, USA). Tear samples (2-25 pl) were collected for protein biomarker analysis in cryotubes and immediately frozen at -80°C.

[0141] A comparison of tear protein concentrations between the DED and non-DED groups revealed that three biomarkers showed a statistically significant difference. As shown in Figure 5, the DED group had significantly higher concentrations of TNF-ot (mean 75.75 pg / ml vs. 48.40 pg / ml, p-0.023), NGF (mean 29.84 pg / ml vs. 14.95 pg / ml, p-0.027), and IL-13 (mean 546.91 pg / ml vs. 391.63 pg / ml, p-0.053) compared to the non-DED group. These results are summarized in Table 2 below.

[0142] Table 2: Tear Fluid Concentrations of Key Biomarkers

[0143] As an example, based on the clinical data, a condition-specific signature criteria for DED may include biomarker concentrations for TNF-ot, NGF, and IL-13 each falling within one standard deviation of the respective mean concentrations for the DED Group. For example, a DED-specific signature criteria may include TNF-ot concentration within 31.73-119.77 pg / ml, NGF concentration within 3.19-56.49 pg / ml, and IL-13 concentration within 225.97-546.91 pg / ml.

[0144] As another example, a condition-specific signature criteria for DED may include biomarker concentrations for TNF-ot, NGF, and IL-13 each being higher than a predetermined control level or threshold, such as a specific percentile (e.g., 75th percentile) of the biomarker concentrations in tear fluid samples obtained from a population of healthy subjects who do not have DED (a non-DED population). For example, a DED-specific signature criteria may include TNF-ot concentration greater than 66.21 pg / ml, NGF concentration greater than 21.05 pg / ml, and IL-13 concentration greater than 517.26 pg / ml.

[0145] This data supports that the pathology of DED is associated with the activation of multiple pathways, including inflammation (TNF-ot), nerve response (NGF), and epithelial response (IL- 13).

[0146] The comparison of tear protein concentrations in the two study groups was analyzed performing a Welch t-test from the averaged concentration for each patient. In Figure 5, nine inflammatory proteins which showed the most difference between the study groups (DED and Non-DED) are shown along with their p values. For TNF-ot, NGF and IL-13, statistically significant difference (p 0.05) was detected in between the study groups.

[0147] In a 9-biomarker panel, the tear proteins TNF-ot, NGF, IL-13, GRO-ot, MCP-1, 1 L-l (3, M-CSF, Substance P, IL-2, having a p-value less than 0.09 in the t-test were used in the logistic regression analysis. Logistic regression analysis permits the determination of the usefulness of the biomarkers to predict the patient's classification in DED or Non-DED groups. Grouping biomarkers into an optimized panel may offer superior sensitivity and sensitivity compared to single biomarkers as revealed by the Receiver Operating Characteristic (ROC) curve analysis which plots true positive (sensitivity) and false positive (specificity) rates respectively on y-and x-axes. The Area Under the upward rising Curve (AUC) is a measure of test accuracy, with a perfect test having an AUC value of 1. With the current data and the panel of 9 biomarker, the inventor has achieved an accuracy of 0.85.

[0148] In a 3-biomarker panel, the tear proteins TNF-ot, NGF, and IL-13 were used in the logistic regression analysis. In Fig. 6, the left side shows the GLM analysis for the 3-biomarker panel (TNF-ot, NGF and IL-13) for the 40 study participants, "Dry Eye" group and "Non-Dry Eye", which are statistically significantly separated in the two groups (p:0.0014). The right side of Fig. 6 shows the ROC Curve for 3-biomarker panel TNF-ot, NGF, and IL-13, for the classification of DED and Non-DED patients, with AUC of 0.79.

[0149] By grouping the tear proteins TNF-ot, NGF, and IL-13 in the biomarker panel, the classification capacity between the two study groups DED and Non-DED was reduced compared to the 9- biomarker panel. However, the 3-biomarker panel reached a p value of 0.0014 (Wilcoxon test) in between the two study groups. Accuracy for predicting the participants' classification into one of the two study groups by using the 3 biomarker panel combined was AUC: 0.79, demonstrating the predictive distinction of the two study groups.

[0150] Therapeutic Implications

[0151] There are provided methods enabling therapeutic interventions based on the assessment of future onset or progression of an ocular health condition in a subject.

[0152] There is provided a method of assessing future onset or progression of an ocular health condition, further comprising administering a therapeutic intervention to the subject prior to manifestation of clinical symptoms of CLD, based on the identified proclivity to future onset or progression of CLD, wherein the therapeutic intervention is selected from one or more of alternative contact lens material, modified contact lens use protocol, modified CLD monitoring protocol, modified CLD treatment protocol, and pharmaceutical intervention.

[0153] In an embodiment, preventative interventions for CLD may be applied to a subject based on the assessment described above. For example, when a subject is identified as having proclivity to future CLD, therapeutic interventions can be administered prior to manifestation of clinical symptoms. Alternative contact lens materials may be used by the subject, such as switching from hydrogel to silicone hydrogel materials, selecting high-oxygen-transmissibility lenses, using daily disposable lenses to minimize deposit accumulation, or selecting lenses with enhanced surface treatments for improved wettability. In another embodiment, modified contact lens use protocols may be applied to a subject based on the assessment described above. For example, when a subject is identified as having proclivity to future CLD, a clinician may order reduced wearing time (e.g., 6-8 hours rather than all-day wear), order mandatory lens-free days per week, order avoidance of overnight wear, or order environmental modifications (humidification, reduced screen time).

[0154] In another embodiment, modified CLD monitoring protocols may be applied to a subject based on the assessment described above. For example, when a subject is identified as having proclivity to future CLD, a clinician may require an alternative frequency for follow-up visits for early symptom detection, order objective biomarker monitoring to track inflammatory status, plan for proactive intervention at first signs of increased biomarker levels, or initiate patient education on early warning signs.

[0155] In another embodiment, modified CLD treatment protocol may be applied to a subject based on the assessment described above. For example, when a subject is identified as having proclivity to future CLD, a clinician may order prophylactic use of lubricating drops during lens wear, concurrent tear supplementation therapy, lid hygiene protocols to optimize meibomian gland function, or order nutritional supplementation such as omega-3 fatty acids.

[0156] In another embodiment, pharmaceutical interventions may be applied to a subject based on the assessment described above. For example, when a subject is identified as having proclivity to future CLD, a clinician may prescribe anti-inflammatory agents (topical cyclosporine, lifitegrast), tear supplementation with specific osmolarity / composition, prophylactic corticosteroids for high-risk situations, or autologous serum eye drops for severe cases.

[0157] There is provided a method of assessing future onset or progression of an ocular health condition, further comprising administering a therapeutic intervention to the subject based on the indicated proclivity to future onset or progression of DED, wherein the therapeutic intervention is selected from one or more of alternative contact lens material, modified contact lens use protocol, modified DED monitoring protocol, modified DED treatment protocol, and pharmaceutical intervention.

[0158] In an embodiment, when a subject is identified as having proclivity to future DED onset or progression, interventions may include similar approaches adapted to DED pathophysiology. For example, when a subject is identified as having proclivity DED onset or progression, a clinician may select therapeutic intervention one or more of alternative contact lens material, modified contact lens use protocol, modified DED monitoring protocol, modified DED treatment protocol, and pharmaceutical intervention. For further example, a clinician may select environmental and behavioral modifications such as humidification of work / home environments, reduced screen time, implementation of the 20-20-20 rule, protective eyewear in adverse environments, or dietary modifications.

[0159] There is provided a method of assessing future onset or progression of an ocular health condition, further comprising identifying which of the three physiological pathways shows predominant dysregulation based on relative contributions of the measured NGF concentration, the measured TNF-ot concentration, and the measured IL-13 concentration to the multi-pathway signature; and administering a pathway-targeted therapeutic intervention selected based on which physiological pathway shows predominant dysregulation.

[0160] In an embodiment, pathway-specific findings may guide pathway-specific therapeutic intervention.

[0161] When TNF-ot (Inflammatory Pathway) predominates, anti-inflammatory therapy may be prescribed, such as cyclosporine, corticosteroids, or tetracyclines. These therapies may modulate immune cells, block inflammatory signals like IL-1, and reduce inflammation around meibomian glands in order to restore tear film stability. For example, a clinician may designate a graduated range of therapies including topical cyclosporine 0.05% or 0.09%, topical lifitegrast 5%, topical corticosteroids, or anti-TNF-a biologies. Other therapies may include intense pulsed light (IPL) therapy and autologous serum tears.

[0162] When NGF (Pain / Neurogenic Pathway) predominates, neuropathic pain management may be prescribed. For example, intranasal neurostimulation may be used to activate the trigeminal nerve pathway by electrical or sonic stimuli; or cholinergic agents such as Pilocarpine may be used to stimulate tear glands directly. Suitable neuropathic pain medications may include gabapentin, pregabalin, or duloxetine. For further example, a clinician may prescribe recombinant human NGF (cenegermin) eye drops, neuropathic pain management strategies, autologous serum eye drops containing endogenous growth factors, nerve regeneration support therapies, or avoidance of medications that may damage corneal nerves

[0163] When IL-13 (Epithelial / Goblet Cell Pathway) predominates, therapeutic strategies may be prescribed to protect and repair the ocular surface and restore cellular function. For example, trehalose may be prescribed to protect cells from dehydration and osmotic stress by activating autophagy pathways. Epidermal Growth Factor (EGF), Vitamin A, and fibronectin may promote epithelial cell repair and migration. Fibroblast Growth Factor 10 (FGF-10) may protect corneal cells from oxidative stress and prevent apoptosis by enhancing antioxidant defenses. For further example, a clinician may designate mucin secretagogues such as diquafosol or rebamipide, therapies supporting goblet cell function, optimization of tear film mucin layer, treatment of meibomian gland dysfunction, or nutritional support for epithelial health. Where combined pathways are indicated, multi-modal therapeutic approaches may be used. For example, in such case, a clinician may employ combination therapy addressing multiple pathways, sequential therapy starting with the most dysregulated pathway, periodic biomarker monitoring to assess pathway-specific treatment responses, or adjustment of therapy based on the relevant biomarker trajectory.

[0164] In an embodiment, the methods enable objective monitoring of therapeutic efficacy. For example, objective monitoring may include baseline biomarker assessment before treatment; periodic reassessment (e.g., 4-8 weeks post-treatment initiation); identifying changes in individual pathway markers; if specific pathways remain elevated, intensify pathway-specific therapy; if all pathways normalize, maintaining or de-escalating therapy.

[0165] Clinical Advantages

[0166] The methods provide objective assessment, which eliminates subjectivity of questionnaires, provides quantitative and reproducible measurements, and enables standardized evaluation across clinicians and institutions. Further, the methods provide predictive capability, which identifies at-risk individuals before symptoms develop, enables preventative interventions, reduces dropout rates, and improves patient satisfaction. The methods provide personalized medicine, which matches interventions to individual biological predisposition, optimizes therapeutic selection based on pathway analysis, and improves treatment efficacy and outcomes.

[0167] What has been described above includes examples of the disclosed methods. One of ordinary skill in the art may recognize that further combinations and permutations are possible.

[0168] Accordingly, the present disclosure is intended to embrace all such alterations, modifications and variations.

[0169] The foregoing descriptions of specific embodiments of the present invention have been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the present invention to the precise forms disclosed, and modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the present invention and its practical application, to thereby enable others skilled in the art to best utilize the present invention and various embodiments with various modifications as are suited to the particular use contemplated.

[0170] All such variations are considered within the scope of the present invention as defined by the appended claims.

Claims

What is claimed is:

1. A method of assessing future onset or progression of an ocular health condition in a subject, comprising obtaining a tear fluid sample from the subject; measuring, in the tear fluid sample, concentrations of a plurality of biomarkers of a biomarker panel, wherein the biomarker panel comprises at least two or more biomarkers from the group consisting of Nerve Growth Factor (NGF), lnterleukin-8 (IL-8), Tumor Necrosis Factor- Alpha (TNF-ot), and Interleukin-13 (IL-13); comparing the measured biomarker concentrations to condition-specific signature criteria; and based on the comparison, identifying the subject as having proclivity to future onset or progression of:(i) Contact Lens Discomfort (CLD) when the measured NGF concentration and the measured IL-8 concentration meet a first condition-specific signature criterion, or(ii) Dry Eye Disease (DED) when the measured NGF concentration, the measured TNF-ot concentration, and the measured IL-13 concentration meet a second condition-specific signature criterion.

2. The method of assessing future onset or progression of an ocular health condition according to claim 1, further comprising exposing the subject to contact lens wear conditions constituting a potential inflammatory stimulus; obtaining the tear fluid sample at the conclusion of a predetermined washout period in which the subject abstains from the potentially inflammatory stimulus; and identifying the subject as having persistent ocular immune dysfunction when the measured biomarker concentrations remain elevated above a threshold value of the first condition-specific signature criterion despite removal of the potential inflammatory stimulus during the predetermined washout period,wherein the identified persistent ocular immune dysfunction indicates proclivity to future onset or progression of CLD.

3. The method of assessing future onset or progression of an ocular health condition according to claim 2, wherein the washout period is at least 7 days.

4. The method of assessing future onset or progression of an ocular health condition according to claim 2, wherein the washout period is at least 14 days.

5. The method of assessing future onset or progression of an ocular health condition according to claim 2, wherein the comparing step further comprises calculating a probability score for the first condition-specific signature criterion using a predictive model that combines the concentration of NGF and the concentration of IL-8.

6. The method of assessing future onset or progression of an ocular health condition according to claim 2, wherein the predictive model is a logistic regression model.

7. The method of assessing future onset or progression of an ocular health condition according to claim 2, wherein the first condition-specific signature criterion is based on the concentration of NGF and the concentration of IL-8 in tears obtained from a population of subjects who do not have contact lens discomfort.

8. The method of assessing future onset or progression of an ocular health condition according to claim 2, wherein the biomarker panel further comprises at least one additional biomarker selected from the group consisting of Growth-Related Oncogene-Alpha (GRO-ot), Interferon-gamma (IFN-y), and Interleukin-10 (IL-10).

9. The method of assessing future onset or progression of an ocular health condition according to claim 1, further comprisinggenerating from the measured biomarker concentrations a multi-pathway signature of the tear fluid sample representing mechanistically distinct pathways of(a) a first physiological pathway related to pain involving corneal nerve response,(b) a second physiological pathway related to ocular surface inflammatory cascade, and(c) a third physiological pathway related to goblet cell involvement in homeostasis of conjunctival epithelium, wherein the measured NGF concentration corresponds to the first physiological pathway, the measured TNF-ot concentration corresponds to the second physiological pathway, and the measured IL-13 concentration corresponds to the third physiological pathway; comparing the multi-pathway signature to the second condition-specific signature criterion; and identifying the subject as having proclivity to future onset or progression of DED when the multi-pathway signature meets the second condition-specific signature criterion, wherein the multi-pathway signature provides improved diagnostic specificity compared to assessment of individual biomarkers.

10. The method of assessing future onset or progression of an ocular health condition according to claim 9, wherein the identifying step is performed using a single diagnostic score calculated based on the concentrations of the biomarker panel.

11. The method of assessing future onset or progression of an ocular health condition according to claim 10, wherein the single diagnostic score is calculated using a logistic regression model.

12. The method of assessing future onset or progression of an ocular health condition according to claim 9, wherein the second condition-specific signature criterion is based on the concentration of NGF, the concentration of TNF-ot and the concentration of IL-13 in tears obtained from a population of subjects without DED.

13. The method of assessing future onset or progression of an ocular health condition according to claim 9, wherein the biomarker panel further comprises at least one additional biomarker selected from the group consisting of Growth-Related Oncogene-Alpha (GRO-ot), Monocyte Chemoattractant Protein-1 (MCP-1), and lnterleukin-1 beta (IL-1P).

14. The method of assessing future onset or progression of an ocular health condition according to claim 1, wherein the measuring employs at least one selected from the group consisting of Enzyme-Linked Immunosorbent Assay (ELISA), a multiplex immunobeadbased array assay, a lateral-flow assay (LFA), and an electrochemical sensor system.

15. The method of assessing future onset or progression of an ocular health condition according to claim 1, further comprising administering a therapeutic intervention to the subject prior to manifestation of clinical symptoms of CLD, based on the identified proclivity to future onset or progression of CLD, wherein the therapeutic intervention is selected from one or more of alternative contact lens material, modified contact lens use protocol, modified CLD monitoring protocol, modified CLD treatment protocol, and pharmaceutical intervention.

16. The method of assessing future onset or progression of an ocular health condition according to claim 1, further comprising administering a therapeutic intervention to the subject based on the indicated proclivity to future onset or progression of DED, wherein the therapeutic intervention is selected from one or more of alternative contact lens material, modified contact lens use protocol, modified DED monitoring protocol, 1modified DED treatment protocol, and pharmaceutical intervention.

17. The method of assessing future onset or progression of an ocular health condition according to claim 9, further comprising identifying which of the three physiological pathways shows predominant dysregulation based on relative contributions of the measured NGF concentration, the measured TNF-ot concentration, and the measured IL-13 concentration to the multi-pathway signature; and administering a pathway-targeted therapeutic intervention selected based on which physiological pathway shows predominant dysregulation.

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