BACKGROUND

All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art. Refractive surgery, such as Lasik or PRK is a very common procedure. However, approximately 10% of those undergoing this procedure are affected by mild to severe transitory to chronic adverse reactions. A principal adverse reaction is the development of a post-surgical dry eye syndrome which can have a significant impact on visual function. A less common but also serious problem is the development of corneal epitheliopathy. In some of these individuals, the dry eye and epitheliopathy may be a serious and chronic condition which can be visually impairing, At present there is no method for predicting whether a given individual will develop a severe dry eye or epitheliopathy secondary to surgery.

Thus, there is a need in the art for novel methods of prescreening and eliminating from surgery, including corneal surgery, those individuals at risk of developing adverse reactions.

SUMMARY OF THE INVENTION

Various embodiments include a method of diagnosing an increased likelihood of developing dry eye syndrome post corneal surgery relative to a normal subject in an individual, comprising obtaining a sample from the individual, assaying the sample to determine the presence or absence of a high ratio of matrix metal loproteinase (MMP) levels to tissue inhibitor of metalloproteinase (TIMP) levels relative to a normal subject, and diagnosing an increased likelihood of developing dry eye syndrome post corneal surgery relative to a normal subject in the individual based on the presence of a high ratio of MMP levels to TIMP levels, In another embodiment, MMP comprises MMP-I, MMP-2, MMP-3, MMP-8, MMP-9, MMP-10 and/or MMP-13. In another embodiment, TIMP comprises TIMP-I and/or TIMP-2. In another embodiment, the sample comprises a pre-surgical tear sample. In another embodiment, the assay comprises a protein and/or microarray assay. In another embodiment, the high ratio of MMP levels to TIMP levels comprise about 0.8 or higher. In another embodiment, the high ratio of MMP levels to TIMP levels comprise about 0.7 or higher. In another embodiment, the high ratio of MMP levels to TIMP levels comprise about 0.51 or higher. In another embodiment, the corneal surgery comprises laser-assisted in situ keratomileusis (Lasik), photorefractive keratectomy (Prk), and/or corneal transplants.

Other embodiments include a method of treating an individual for dry eye syndrome post corneal surgery, comprising determining the presence of a depressed level of tissue inhibitor of metalloproteinase (TIMP) relative to a normal subject and/or a high ratio of matrix metalloproteinase (MMP) levels to TIMP levels relative to a normal subject, and administering a treatment to the individual to address the dry eye syndrome post corneal surgery. In another embodiment, the treatment comprises a therapeutically effective amount of MMP inhibitor. In another embodiment, the treatment is administered prior to corneal surgery, In another embodiment, the treatment is administered post corneal surgery. In another embodiment, the treatment comprises a therapeutically effective amount of TIMP and/or chelating agent. In another embodiment, the high ratio of MMP levels to TIMP levels comprise about 0.8 or higher. In another embodiment, the high ratio of MMP levels to TIMP levels comprise about 0.7 or higher. In another embodiment, the high ratio of MMP levels to TIMP levels comprise about 0.51 or higher.

Other embodiments include a method of diagnosing dry eye syndrome post corneal surgery in an individual, comprising determining the presence or absence of a low level of tissue inhibitor of metalloproteinase (TIMP) relative to a normal subject, and diagnosing dry eye syndrome post corneal surgery based upon the presence of the low level of TIMP relative to a normal subject. In another embodiment, TIMP comprises TIMP-I and/or TIMP-2.

Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various embodiments of the invention.

BRIEF DESCRIPTION OF THE FIGURES

Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.

Figure I depicts, in accordance with an embodiment described herein, the basic steps involved in a protein array assay. In this instance the arrays consist of a three by three set of nine capture antibodies for MMP-I, MMP-2, MMP-3, MMP-8, MMP-9, MMP-IO, MMP- 13 and TlMPs 1 and 2 as shown in figure 3 herein. Copies of these arrays are imprinted in individual wells of a 96 micro well plate. Tears are added to the array and a series of steps similar to classical sandwich ELISA assays are carried out including a streptavidin-biotin amplification step. Quantification is based upon a peroxidase reporter enzyme with parallel wells containing known amounts of each of the 9 proteins with detection based upon chemo luminescence.

Figure 2 depicts, in accordance with an embodiment described herein, the volume of tears collected on Schirmer strips (length in mm from the bulb) in an atypical lasik surgical subject (lasik-4) before and after (at set periods of time) surgery. The rapid onset of a dry eye syndrome is apparent one day after surgery and is still present 3 months after surgery. Data includes Lasik-4 atypical subject who developed post surgical dry eye syndrome.

Figure 3 depicts, in accordance with an embodiment described herein, the results of MMP protein array assays of identical size extracts from Schirmer strips recovered from patient lasik-4 with sampling carried out before and after surgery. Note that in samples recovered from the left and right eyes immediately before surgery, the signal for TIMP-I was virtually absent. Tear samples 2 of 5 of the patients who developed post-surgical dry eye syndrome contained negligible levels of TIMP-I in the pre-surgical tear samples. 3/5 exhibited negligible levels of TIMPl in 1 or more samples.

Figure 4 depicts, in accordance with an embodiment described herein, the results of MMP and TIMP assays from similar sets of Schirmer strip extracts from individuals who did not develop post surgical dry eye syndrome with high levels of TIMP-I present in all of the extracts. Normals: Pre and Post Surgical Samples (none of these patents developed post surgical dry eye syndrome). None of these samples exhibited low levels of TIMPs.

Figure 5 depicts, in accordance with an embodiment described herein, the results of the assays of typical groups of normal and pathological tear samples using the MMP array. High levels of TIMP-I were common to all of these samples. Dozens of other tear samples have been assayed from normals and individuals with a wide range of pathologies. None of these samples had a low level of TIMP-I or 2.

Figure 6 depicts, in accordance with an embodiment described herein, the results of the assays of typical groups of normal and pathological tear samples using the MMP array. High levels of TIMP-I and a high MMP-9/TIMP-1 ratio were common to all of these samples. Typical Results Assay Chronic and Acute Allergic Reactions. Note that all samples exhibit strong signals for TIMP-I and TIMP-2. Figure 7 depicts, in accordance with an embodiment described herein, the results of array assays of the pre surgical tear samples from the single patient who developed a post surgical epitheliopathy and dry eye. Note that TIMPs 1 and 2 were absent in these samples. Pre surgical tear samples from subjects who developed dry eye or epithelial opathy. Figure 8 depicts, in accordance with an embodiment described herein, results depicting a clear association between MMP-I O/TIMP-1 ratio of the pre-surgicai tear samples and a likelihood of developing clinical defined post-surgical dry eye syndrome. Measured unit is in pg/ml. The first four columns indicate the sex (male or female) of the test subject, (OD/OS-eye) and the type of refractive surgery employed upon each of the patients, as well as their respective age. The next three additional columns indicate the concentrations of MMP-10, TIMP-I , and the MMP-IO to TIMP-I ratio, respectively, as obtained on the assay of the pre-surgical tear sample, as determined by an MMP and TlMP array of the Schirmer extract with the ratio presented in descending order. Finally, the last few columns indicate with a "#1 " those eyes which have been diagnosed with dry eye syndrome at least once. The columns indicate the exact times when the diagnosis was made. "~9" is indicative of no data obtained. Diagnosis is based upon clinical examination encompassing a variety of measurements. Based on the data depicted in Figure 8 and herein, the inventors are able to conclude that for at least a MMP-10 to TIMP-I ratio of 0.8 or higher, one could significantly reduce the incidence of surgically induced dry eye syndrome. Thus, for example, the overall quality of surgery would be improved and a significant fraction of patients who are at risk for an adverse reaction would be identified.

DESCRIPTION OF THE INVENTION

All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 3 ^r ed., J. Wiley & Sons (New York, NY 2001); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 5 ^)h ed. , 3. Wiley & Sons (New York, NY 2001); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 3rd ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2001), provide one skilled in the art with a general guide to many of the terms used in the present application. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described.

As used herein, the term "MMP" means matrix metal loproteinase. As used herein, the term "TlMP" means tissue inhibitor of metalloproteinase.

As used herein, the term "Lasik" means laser-assisted in situ keratomileusis and is a type of refractive laser eye surgery that may be performed for correcting a condition such as myopia, hyperopia or astigmatism.

As used herein, the term "PRK" means photorefractive keratectomy, or also called advanced surface ablation, and is a type of laser eye surgery that may be used to correct an individual's vision.

As used herein, the term ''MMP inhibitor" means a compound or molecule capable of inhibiting the activity or signaling capacity of matrix metalloproteases.

As used herein, "dry eye syndrome" is a condition in which the eyes do not produce enough tears, causing them to become dry and irritated and is frequently associated with inflammation.

As used herein, the term "normal subject" refers to a population when taken as a whole or average, with the average amount of incidence.

As disclosed herein, data is presented showing that an imbalance in the down regulatory capacity of the tear film to inhibit matrix metalloproteases (a specific group of proteases involved in wound healing and inflammation) in the form of decreased levels of TlMP-I and a high MMP-9 to TIMP-I ratio is common to the pre surgical tear fluid in -40 % of those individuals who go on to develop severe post surgical dry eye syndrome and the near absence of TIMP-I and T1MP-2 in the pre-ocular tear film is the single patient who developed epitheliopathy. Similarly, as disclosed in Figure 8 herein, the inventors are able to conclude that for at least a MMP-IO to TIMP-I ratio of 0.8 or higher, one could significantly reduce the incidence of surgically induced dry eye syndrome. These inhibitors and the corresponding MMPs that are down regulated by these inhibitors are well known factors critical to wound healing and tissue remodeling. These factors as shown herein can be readily assayed using a microwell based array using minute tear samples and other forms of assay of TIMP-I and 2 and/or the corresponding affected MMPs should have wide spread applicability as a pre-screening agent identifying those subjects who are poor candidates for a positive outcome from this elective form of surgery. The general nature of this apparent biochemical defect in this small population is also a diagnostic indicator of individuals who might be at risk of serious consequences of other forms of corneal surgery in which wound healing is a critical factor, which includes corneal transplants where -10% of the transplants are rejected. The results also demonstrate that the prophylactic treatment or pre-treatment prior to and after surgery of individuals who might be at risk with TIMP-I and or mixtures of TIMPl and TIMP2 as well as other MMP inhibitors (such as chelating agents) could reduce the risk and severity of developing these complications.

Thus, for example, the overall quality of surgery would be improved and a significant fraction of patients who are at risk for an adverse reaction would be identified. These patients could be counseled to considering avoiding surgery or more closely followed after surgery with earlier intervention.

In one embodiment, the present invention provides a method of diagnosing susceptibility to post surgical dry eye syndrome in an individual by determining the presence or absence of a low level of TIMP and/or a high MMP/TIMP ratio, where the presence of the low level of TIMP and/or high MMP/TIMP ratio is indicative of susceptibility to post surgical dry eye syndrome in the individual. In another embodiment, TIMP is TIMP-I and/or T1MP-2. In another embodiment, MMP is MMP-2, MMP-9, and/or MMP-IO. In another embodiment, the TIMP and/or MMP is taken from a tear sample. In another embodiment, the tear sample is used in conjunction with a 4-plex assay for TIMP-I, TIMP-2 and MMP-2 and MMP-9 with the ratios of MMPs to associated inhibitors determining risk. In another embodiment, post surgical dry eye syndrome includes epitheliopathy.

In one embodiment, the present invention provides a method of diagnosing dry eye syndrome in an individual by determining the presence or absence of a low level of TIMP and/or a high MMP/TIMP ratio, where the presence of the low level of TIMP and/or high MMP/TIMP ratio is indicative of post surgical dry eye syndrome in the individual. In another embodiment, TIMP is TIMP-I and/or TIMP-2. In another embodiment, MMP is MMP-2, MMP-9 and/or MMP-IO. In another embodiment, the TIMP and/or MMP is taken from a tear sample. In another embodiment, post surgical dry eye syndrome includes epitheliopathy. In another embodiment, the dry eye syndrome is secondary to surgery. In another embodiment, the surgery is Lasik or PRK and variants of refractive surgery. In one embodiment, the present invention provides a method of treating post surgical dry eye syndrome and/or epitheliopathy by determining the presence of a low level of TIMP- 1 and/or TIMP-2 and administering a therapeutically effective amount of an MMP inhibitor to the individual. In another embodiment, the MMP inhibitor is TIMP-I and/or TIMP-2. In another embodiment, the MMP inhibitor is a chelating agent. The present invention is also directed to a kit to prepare an assay for determining the presence or absence of TIMP and/or MMP, as well as the delivery of the MMP inhibitors to an individual, and may include a pipette, pipette solution, tear sample solution, T1MP/MMP assay solution, and combinations thereof. The kit is an assemblage of materials or components, including at least one of the inventive compositions. Thus, in some embodiments the kit contains a composition including a therapeutically effective dosage of MMP inhibitor, as described above.

The exact nature of the components configured in the inventive kit depends on its intended purpose. For example, some embodiments are configured for the purpose of delivering a therapeutically effective dosage of MMP inhibitor to a subject, such as, but not limited to, human subjects, farm animals, domestic animals, and laboratory animals. Other embodiments, for example, are configured for preparing a therapeutically effective dosage of MMP inhibitor to a subject, such as, but not limited to, human subjects, farm animals, domestic animals, and laboratory animals. Instructions for use may be included in the kit. "Instructions for use" typically include a tangible expression describing the technique to be employed in using the components of the kit to effect a desired outcome, such as to prepare a solution for determining the presence of TIMP or MMP and/or deliver a therapeutically effective dosage of MMP inhibitor either pre or post surgery. Optionally, the kit also contains other useful components, such as, diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, bandaging materials or other useful paraphernalia as will be readily recognized by those of skill in the art.

The materials or components assembled in the kit can be provided to the practitioner stored in any convenient and suitable ways that preserve their operability and utility. For example the components can be in dissolved, dehydrated, or lyophilized form; they can be provided at room, refrigerated or frozen temperatures. The components are typically contained in suitable packaging material(s). As employed herein, the phrase "packaging material ^' ' refers to one or more physical structures used to house the contents of the kit, such as inventive compositions and the like. The packaging material is constructed by well known methods, preferably to provide a sterile, contaminant-free environment. As used herein, the term "package" refers to a suitable solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding the individual kit components. Thus, for example, a package can be a glass vial used to contain suitable quantities of an inventive composition containing a solution of MMP inhibitor or components thereof. The packaging material generally has an external label which indicates the contents and/or purpose of the kit and/or its components.

In various embodiments, the present invention provides pharmaceutical compositions including a pharmaceutically acceptable excipient along with a therapeutically effective amount of MMP inhibitor. '"Pharmaceutically acceptable excipient" means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients may be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous. In various embodiments, the pharmaceutical compositions according to the invention may be formulated for delivery via any route of administration. "Route of administration" may refer to any administration pathway known in the art, including but not limited to pipette, intravenous injection, aerosol, nasal, oral, transmucosal, transdermal or parenteral. "Parenteral" refers to a route of administration that is generally associated with injection, including intraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. Via the parenteral route, the compositions may be in the form of solutions or suspensions for infusion or for injection, or as lyophilized powders. The MMP inhibitor according to the invention can also contain any pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" as used herein refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. Each component of the carrier must be "pharmaceutically acceptable" in that it must be compatible with the other ingredients of the formulation. It must also be suitable for use in contact with any tissues or organs with which it may come in contact, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.

The MMP inhibitor according to the invention can also be encapsulated, tableted or prepared in an emulsion or syrup for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition. Liquid carriers include syrup, peanut oil, olive oil, glycerin, saline, alcohols and water. Solid carriers include starch, lactose, calcium sulfate, dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax. The preparations of the MMP inhibitor are made following the conventional techniques of pharmacy involving milling, mixing, granulation, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms. When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous suspension. Such a liquid formulation may be administered directly p.o. or filled into a soft gelatin capsule.

The MMP inhibitor according to the invention may be delivered in a therapeutically effective amount. The precise therapeutically effective amount is that amount of the composition that will yield the most effective results in terms of efficacy of treatment in a given subject. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, for instance, by monitoring a subject's response to administration of a compound and adjusting the dosage accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy (Gennaro ed. 20th edition, Williams & Wilkins PA, USA) (2000). Typical dosages of MMP inhibitors can be in the ranges recommended by the manufacturer where known therapeutic compounds are used, and also as indicated to the skilled artisan by the in vitro responses or responses in animal models. Such dosages typically can be reduced by up to about one order of magnitude in concentration or amount without losing the relevant biological activity. Thus, the actual dosage will depend upon the judgment of the physician, the condition of the patient, and the effectiveness of the therapeutic method based, for example, on the in vitro responsiveness of the relevant primary cultured cells or histocultured tissue sample, such as the responses observed in the appropriate animal model. There are many techniques readily available in the field for detecting the presence or absence of MMP and TlMP polypeptides or other biomarkers, including protein microarrays. For example, some of the detection paradigms that can be employed to this end include optical methods, electrochemical methods (voltametry and amperometry techniques), atomic force microscopy, and radio frequency methods, e.g., multipolar resonance spectroscopy. Illustrative of optical methods, in addition to microscopy, both confocal and non-confocal, are detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, and birefringence or refractive index (e.g., surface plasmon resonance, ellipsometry, a resonant mirror method, a grating coupler waveguide method or interferometry).

Similarly, there are any number of techniques that may be employed to isolate and/or fractionate biomarkers. For example, a MMP and TIMP biomarkers may be captured using biospecific capture reagents, such as antibodies, aptamers or antibodies that recognize the biomarker and modified forms of it. This method could also result in the capture of protein interactors that are bound to the proteins or that are otherwise recognized by antibodies and that, themselves, can be biomarkers. The biospecific capture reagents may also be bound to a solid phase. Then, the captured proteins can be detected by SELDI mass spectrometry or by eluting the proteins from the capture reagent and detecting the eluted proteins by traditional MALDΪ or by SELDL One example of SELDI is called "affinity capture mass spectrometry," or "Surface-Enhanced Affinity Capture" or "SEAC," which involves the use of probes that have a material on the probe surface that captures analytes through a non- covalent affinity interaction (adsorption) between the material and the analyte. Some examples of mass spectrometers are time-of- flight, magnetic sector, quadrupole filter, ion trap, ion cyclotron resonance, electrostatic sector analyzer and hybrids of these. Alternatively, for example, the presence of biomarkers such as polypeptides maybe detected using traditional immunoassay techniques. Immunoassay requires biospecific capture reagents, such as antibodies, to capture the analytes. The assay may also be designed to specifically distinguish protein and modified forms of protein, which can be done by employing a sandwich assay in which one antibody captures more than one form and second, distinctly labeled antibodies, specifically bind, and provide distinct detection of, the various forms. Antibodies can be produced by immunizing animals with the biomolecules. Traditional immunoassays may also include sandwich immunoassays including ELISA or fluorescence -based immunoassays, as well as other enzyme immunoassays. Prior to detection, MMP and TIMP biomarkers may also be fractionated to isolate them from other components in a solution or of blood that may interfere with detection. Fractionation may include platelet isolation from other blood components, sub-cellular fractionation of platelet components and/or fractionation of the desired biomarkers from other biomolecules found in platelets using techniques such as chromatography, affinity purification, 1 D and 2D mapping, and other methodologies for purification known to those of skill in the art. In one embodiment, a sample is analyzed by means of a biochip. Biochips generally comprise solid substrates and have a generally planar surface, to which a capture reagent (also called an adsorbent or affinity reagent) is attached. Frequently, the surface of a biochip comprises a plurality of addressable locations, each of which has the capture reagent bound there.

As apparent to one of skill in the art, any number of assays and mediums may be used in conjunction with various embodiments described herein. For example, methods of determining the presence or absence of levels of TIMP, MMP, and lheir respective ratio levels may include the use of a strip for rapid diagnosis, a chip, "lab on a chip," a micro fluidic device, a protein array, or any other number of assays or mediums readily available and known in the art. Similarly, the invention is not limited to only be used in conjunction with Lasik or PRK surgery. As readily apparent to one of skill in the art, there are any number of surgical and refractive procedures that the various embodiments described herein may be used in conjunction with, such as corneal transplants or other forms of corneal surgery in which wound healing is a critical factor. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.

EXAMPLES

The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.

Example I Array analysis

At present these is no method for predicting whether a given individual will develop a severe dry eye or epitheliopathy secondary to surgery. As described herein, the data demonstrates that an imbalance in the down regulatory capacity of the tear film to inhibit matrix metal! o proteases (a specific group of proteases involved in wound healing and inflammation) in the form of decreased levels of TIMP-I is common to the pre surgical tear fluid in ~40 % of those individuals who go on to develop severe post surgical dry eye syndrome and the near absence of TIMP-I and TIMP-2 in the pre-ocular tear film is the single patient who developed epitheliopathy,

Example 2

TIMP and MMP assays and treatment

These inhibitors, such as TIMP- 1 and -2 and the corresponding MMPs that are down regulated by these inhibitors are well known factors critical to wound healing and tissue remodeling. These factors as shown here can be readily assayed using a microwell based array using minute tear samples. That and other forms of assay of TIMP-I and -2 and/or also the corresponding affected MMPs should have wide spread applicability as a pre-screening agent identifying those subjects who are poor candidates for a positive outcome from this elective form of surgery. The general nature of this apparent biochemical defect in this small population can also act as a good diagnostic indicator of individuals who might be at risk of serious consequences of other forms of corneal surgery in which wound healing is a critical factor, such as corneal transplants where -10% of the transplants are rejected. The results also demonstrate that the prophylactic treatment or pre-treatment prior to and after surgery of individuals who might be at risk with TIMP-I and or mixtures of TIMPl and TIMP2 as well as other MMP inhibitors (chelating agents etc) can reduce the risk and severity of developing these complications. As described in Figures 1-7 herein, one type of testing protocol for measuring TIMPl and related molecules in tear fluid is depicted for a possible assay. Another possible use could be in the form of a 4-plex assay for TIMP-I, TIMP-2 and MMP-2 and MMP-9 with the ratios of MMPs to associated inhibitors determining the risk. Additionally, a protein array for differential diagnosis of ocular diseases, or an assay on a chip or strip for rapid diagnosis could also be used.

Example 3 Generally MMPs are known to be proteases involved in wound healing and tissue remodeling that are inhibited by TIMPs, with TIMP-I inhibiting MMP-9 and TIMP-2 inhibiting MMP-2.

These proteases are involved in the control of apoptosis and tissue remodeling. LASIK and PRK surgeries involve destruction of regions of the corneal epithelium and removal of some stroma. The absence of a significant reserve of the associated inhibitor TIMP-I and/or

TIMP-2 results in excess inflammation and tissue destruction immediately after lasik or PRK surgery. All subjects described herein who had depressed levels of TIMPl developed serious aqueous deficiency dry eye syndrome. This deficiency was observed in about 50% of the subjects who developed aqueous deficiency dry eye syndrome secondary to surgery.

By prescreening screening tear samples for MMP/TIMP-1 ratios and TIMP-2, at least

40% of the individuals who will develop serious dry eye syndrome can be identified. By excluding these individuals from surgery, the efficacy of the procedure can be greatly enhanced. This population is estimated to represent -2-3% of all individuals.

Example 4 MMP to TIMP ratios The inventors examined clinical data of subjects who had undergone refractive surgery and compared it to data of MMP and TIMP levels of pre- surgical samples taken from those same subjects. The result is that a clear relationship is shown between MMP and TIMP ratios of pre -surgical tear samples and the likelihood of developing clinical defined postsurgical dry eye syndrome. As depicted in Figure 8 herein, the first four columns indicate the sex (male or female) of the test subject, and the type of refractive surgery employed upon each of the patients, as well as their respective age. The next three additional columns indicate the concentrations of MMP-IO, TIMP-I, and the MMP-10 to TIMP-I ratio, respectively, as obtained on the assay of the pre-surgical tear sample, as determined by an MMP and TlMP array of the Schirmer extract with the ratio presented in descending order. Finally, the last few columns indicate with a "#1" those eyes which have been diagnosed with dry eye syndrome at least once. The columns indicate the exact times when the diagnosis was made. Based on the data depicted in Figure 8 and herein, the inventors are able to conclude that for at least a MMP-10 to TIMP-I ratio of 0.8 or higher, one could significantly reduce the incidence of surgically induced dry eye syndrome. Thus, for example, the overall quality of surgery would be improved and a significant fraction of patients who are at risk for an adverse reaction would be identified.

While the description above refers to particular embodiments of the present invention, it should be readily apparent Io people of ordinary skill in the art that a number of modifications may be made without departing from the spirit thereof. The presently disclosed embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Various embodiments of the invention are described above in the Description of the Invention. While these descriptions directly describe the above embodiments, it is understood that those skilled in the art may conceive modifications and/or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the purview of this description are intended to be included therein as well. Unless specifically noted, it is the intention of the inventor that the words and phrases in the specification and claims be given the ordinary and accustomed meanings to those of ordinary skill in the applicable art(s).

The foregoing description of various embodiments of the invention known to the applicant at this time of filing the application has been presented and is intended for the purposes of illustration and description. The present description is not intended to be exhaustive nor limit the invention to the precise form disclosed and many modifications and variations are possible in the light of the above teachings. The embodiments described serve to explain the principles of the invention and its practical application and to enable others skilled in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed for carrying out the invention.

While particular embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that, based upon the teachings herein, changes and modifications may be made without departing from this invention and its broader aspects and, therefore, the appended claims are to encompass within their scope all such changes and modifications as are within the true spirit and scope of this invention. Furthermore, it is to be understood that the invention is solely defined by the appended claims. It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims {e.g., bodies of the appended claims) are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term '"having" should be interpreted as "having at least," the term "includes" should be interpreted as "includes but is not limited to," etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles "a" or "an" limits any particular claim containing such introduced claim recitation to inventions containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and indefinite articles such as "a" or "an" (e.g., "a" and/or "an" should typically be interpreted to mean "at least one" or "one or more"); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should typically be interpreted to mean at least the recited number (e.g., the bare recitation of "two recitations," without other modifiers, typically means at least two recitations, or two or more recitations).

Accordingly, the invention is not limited except as by the appended claims.