WO2008156752A2 - Vitreous biomarkers and related diagnostics and therapeutics - Google Patents

Abstract

The present invention relates to the analysis and monitoring of ocular fluids for determining the physiological state of an organism, to monitor drug efficacy and dynamics, for early disease detection, as well as to certain molecular markers and fingerprints of identified molecules or molecule fragments in such analysis. An ophthalmic aspirating device is provided that allows the relatively non-invasive removal of small volumes of ocular vitreous fluid for diagnostic and other purposes.

Description

VITREOUS BIOMARKERS AND RELATED DIAGNOSTICS AND

THERAPEUTICS

[0001]This application claims priority to U.S. Provisional Patent Application No. 60/929,200, filed June 18, 2007. This application incorporates by reference each of U.S. provisional patent application 60/762,499, filed January 27, 2006, U.S. serial no. 11/698,998, filed January 29, 2007, and PCT/US2007/002452, in their entirety, including specification, claims, figures and drawings and abstracts. BACKGROUND OF THE INVENTION

[0002]Diabetic retinopathy (DR) is the most prevalent cause of vision loss in working adults. Most patients with type 1 diabetes mellitus and over 60% of those with type 2 diabetes eventually develop retinal vascular abnormalities. 20% to 30% of these patients advance to active proliferative diabetic retinopathy (PDR) and/or diabetic macular edema. Increased retinal vascular permeability (RVP) is a primary cause of diabetic macular edema and a characteristic finding in PDR. Cystoid Macular Edema (CME) is a type of edema of the macula that causes retinal damage and occurs in a wide variety of ocular disorders. Age-related macular degeneration (AMD) is the leading cause of vision loss and blindness in individuals over age 60 in the developed world. The disorder is characterized by the loss of central vision caused by pathologic aging of the macula. The manifestations of the disease are classified into two forms: non-exudative (dry) and exudative (wet or neovascular). Early diagnosis and preventative treatments for these disorders remain a major unmet clinical need. [0003]Efforts have been made to identify biomarkers from ocular vitreous fluid in an attempt to diagnosis ocular diseases. However, diagnostic sampling of vitreous fluid has invariably involved invasive procedures such as pars plana vitrectomy that pose risks such as damage to the crystalline lens, accelerated cataract formation, retinal detachment, and vitreous hemorrhage. Vitrectomies have also traditionally involved the removal of a volume of fluid that can cause at least partial collapse of the eye and can necessitate introduction of air or fluid to compensate for the volume removed. These limitations of surgical vitrectomy point to the need for materials and methods for obtaining vitreous fluid in a less invasive, more convenient, and relatively pain free manner. SUMMARY OF THE INVENTION

[0004]Embodiments of the present invention solve these problems, as well as others, by providing the ability to quickly and easily remove small quantities of liquid from the eye of a patient without the need for irrigating the eye and replenishing the eye with an equivalent amount of fluid. Thus, among other things, the patient does not experience the discomfort and pain of having large syringes inserted into the eye, with the inherent danger of infection during the aspiration and irrigation cycle, and the procedure can be performed as a routine out patient surgical technique. [0005] The present invention provides methods and materials for a non-surgical method of predicting or monitoring the physiological state of the eye. The method comprises aspirating a sample of vitreous fluid from the eye of a living subject, wherein the aspirating is not concurrent to eye surgery. A level of a biomarker in the vitreous fluid is detected, wherein the biomarker is associated with at least one of a susceptibility to an ocular condition, presence or absence of an ocular condition, and an efficacy of treatment of an ocular condition. At least one of the susceptibility to the ocular condition, the presence or absence of the ocular condition, or the efficacy of treatment of an ocular condition based on the level of the biomarker is identified. [0006]In accordance with another aspect of the invention, an ophthalmic aspirating device is provided. In one embodiment, an ophthalmic aspirating device, comprises: a negative pressure module; a conduit having first and second ends, wherein the respective ends each comprises an aperture, and wherein the second conduit end is operatively associated with the negative pressure module. In another embodiment, the ophthalmic aspirating device comprises a housing; a tube operatively associated with the housing, the tube having first and second ends; a conduit having first and second ends, wherein the first conduit end comprises an aperture, and wherein the second conduit end is operatively associated with the first tube end; and a negative pressure module.

[0007]Kits for analyzing vitreous fluid samples are provided. A vitreous fluid analysis kit can comprise a vitreous fluid receptacle comprising at least one reservoir, wherein the reservoir comprises at least one chemical to protect polypeptide integrity. [0008] A method of remote vitreous fluid analysis is provided. The method comprises obtaining a vitreous fluid sample from a living subject; storing the vitreous fluid sample in a receptacle comprising a reservoir, wherein the reservoir comprises at least one chemical to protect polypeptide integrity; and sending the vitreous fluid sample and/or data collected therefrom to a laboratory or equivalent facility for analysis. Once received, the vitreous fluid sample can be analyzed. In addition or in the alternative, data collected from the vitreous fluid sample can be sent for analysis. [0009]The present invention also provides a proteomic fingerprint, comprising at least one vitreous fluid biomarker associated with an ocular condition. The biomarker of the fingerprint can be a polypeptide or a unique fragment thereof. [001O]A method of identifying biomarkers of an ocular condition is provided. A vitreous fluid polypeptide spectrum of a subject having an ocular condition is compared to a vitreous polypeptide spectrum of a subject not having the ocular condition, wherein the spectra are of vitreous fluid aspirated from living patients. A difference is determined in the polypeptide spectra in terms of identity or amount of the at least one polypeptide or a fragment thereof. The presence of the condition is correlated with the difference to identify a biomarker.

[001I]A method of identifying biomarkers associated with the development of an ocular condition is provided. Vitreous fluid polypeptide spectra of at least one vitreous fluid sample from a subject prior to the onset of the ocular condition and at least one vitreous fluid sample after the onset of the ocular condition. At least one difference in the polypeptide spectra in terms of identity and amount of a polypeptide among the samples is determined. The presence of the condition is correlated with the difference to identify a biomarker.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012JFIG. 1 illustrates an ophthalmic aspiration device, according to one exemplary embodiment of the present invention;

[0013]FIG. 2 illustrates an ophthalmic aspiration device, according to another exemplary embodiment of the present invention; and

[0014]FIG. 3 illustrates an ophthalmic aspiration device, according to yet another exemplary embodiment of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS [0015] The present invention provides methods and materials for a non-surgical method of predicting or monitoring the physiological state of the eye. The method comprises aspirating a sample of vitreous fluid from the eye of a living subject, wherein the aspirating is not concurrent to eye surgery; detecting a level of a biomarker in the vitreous fluid, wherein the biomarker is associated with at least one of a susceptibility to an ocular condition, presence or absence of an ocular condition, and an efficacy of treatment of an ocular condition; and identifying at least one of the susceptibility to the ocular condition, the presence or absence of the ocular condition, or the efficacy of treatment of an ocular condition based on the level of the biomarker. In some embodiments, the method further comprises treating the ocular condition or treating the susceptibility to the ocular condition.

[0016]The level detected can comprise the presence or absence of the biomarker associated with at least one of the susceptibility to the ocular condition, the presence or absence of the ocular condition, or the efficacy of treatment of an ocular condition. The level detected can be a relative or quantitative measure of the biomarker associated with at least one of the susceptibility to the ocular condition, the presence or absence of the ocular condition, or the efficacy of treatment of an ocular condition. [0017]The ocular condition can be any ocular condition of interest or combination of conditions of interest. The condition can be a disease or a state or stage thereof. Examples of ocular conditions include diabetic retinopathy, age-related macular degeneration, branch vein occlusion, central vein occlusion, ocular histoplasmosis, choroidal neovascularization, retinal neovascularization, retinal edema, a retinopathy, a choroidopathy, retinitis, choroiditis, retinal vasculitis, choroidal vasculitis, a retinal vascular abnormality, a choroidal vascular abnormality, retinal vascular leakage, choroidal leakage, an ocular dystrophy, myopic degeneration, pre-cataract, cataract, glaucoma, an ocular infectious disease, an ocular tumor, an ocular malignancy, an ocular degenerative disorder, an ocular hereditary disorder, and any combination thereof.

[0018]In some embodiments, the detecting and identifying is of the presence or absence of the ocular condition so as to diagnose an ocular condition. In some embodiments, the detecting and identifying is of the susceptibility to the ocular condition, the presence or absence of the ocular condition so as to determine the risk of an ocular condition. In some embodiments, the detecting and identifying is of the efficacy of treatment of an ocular condition so as to monitor the effect of a drug. The subject can have been administered a treatment for an ocular condition. In some embodiments, the treatment comprises administration of a tyrosine kinase inhibitor. The tyrosine kinase inhibitor can inhibit the phosphorylation of a tyrosine-kinase receptor or enzyme. A subject can have been administered a tyrosine kinase inhibitor, drug, or a biological, chemical, protein, antibody, or other therapeutic agent. [0019]The methods of the present invention provide a means for characterizing the identity and/or content of vitreous fluid with respect to the levels or amounts of particular peptides which will be indicative of disease. Peptides that are unique to a disease, wherein the presence of any amount of such peptides can indicate the likelihood of the disease being present are described. In some embodiments, existing peptide biomarkers which correlate with a particular disease or a physiological state, and screen for said peptide(s) using the method described by the present invention. In some cases the presence or absence of the molecule above background can be diagnostic of the disease, because that molecule may not be expected otherwise. An example is molecules associated with vascular leakage during wet macular degeneration. In some embodiments, samples are analyzed to assess other molecules and/or compounds that indicate responsiveness to anti-VEGF agents injected in the eye or delivered by other means. Samples can be analyzed to assess other molecules and/or compounds that indicate other agents that might be injected in the eye or delivered by other means that prevent, slow, stop or reverse neovascularization. [002O]A biomarker can comprise one or more molecules. In some embodiments, the biomarker comprises at least one of a polypeptide and a unique fragment thereof. In some embodiments, the polypeptide is a phosphorylated polypeptide or a unique fragment thereof. In some embodiments, the phosphorylated polypeptide is a receptor polypeptide or a unique fragment thereof. Phosphorylated receptors can be a G- protein coupled receptor and/or a hormone activated receptor. Any appropriate phosphorylated receptor can be detected in a non-phosphorylated, a phosphorlyated, or partially phosphorylated state. Examples of phosphorylated receptors include VEGFR-2 and PDGFR-beta. [0021]Biomarkers identified using the biomarker identification methods of the present invention can be employed with the diagnostic methods of the present invention. Other biomarkers can be employed. Known biomarkers can be employed. Biomarkers identified using other methodologies can be employed. Tables 2-13 of U.S. Patent Application Publication 20070224644 provide a representative list of peptides which are present in the vitreous fluid of the eye. Representative examples of such peptides in relation to ocular diseases (for e.g., macular hole, retinal degeneration, or a combination of macular hole and retinal degeneration) are provided in the tables 5-13 of U.S. Patent Application Publication 20070224644). [0022] Biomarkers can be used to determine the risk of cataracts (e.g., when crystalline are elevated); the integrity of the blood-ocular barrier; and other retinal conditions and diseases. This can be especially useful in patients who are at risk for an ocular disease, e.g., subjects with diabetes, aging subjects, subjects who have been identified as a carrier of a gene defect associated with an ocular disorder, or subjects who have been exposed to agents or conditions that have a known potential for inducing ocular conditions. Such agents include sunlight, chemicals, and biological agents. An example of a biological agent is a histoplasmotic agent such as Histoplasma capsulatum.

[0023]Any appropriate means of detection of biomarkers can be employed in accordance with the methods of the present invention. For example, detection can comprise at least one of a protein microarray, an immunoassay, a ligand binding assay, electrophoresis, and mass spectroscopy of the vitreous fluid sample. Immunologic techniques, antibody diagnostics, radioimmunoassays, mass spectrometry, microarrays, western blotting, gel electrophoresis, and labeled or enzyme amplified diagnostic technologies can be employed. In some embodiments, the detecting comprises a proteomic fingerprint comprising a vitreous fluid polypeptide or unique fragment thereof. Examples of biomarkers that have been isolated from ocular fluids are shown in Table 2 to Table 13 of U.S. Patent Application Publication 20070224644. These can be obtained by running a sample of ocular fluid on an SDS-PAGE gel, and then digesting the entire gel lane from high to low protein molecular weight with trypsin, followed by MS/MS analysis. Examples of biomarkers include retinol binding protein-4 (RBP4), Secreted Protein Acidic and Rich in Cysteine (SPARC), Akt., VEGFR, EGFR, Bcr-Abl, Her2-Neu (erbB2), TGFR, etc.

[0024] Diagnostic and prognostic techniques utilizing micro-aspirates of fluid vitreous are provided. These techniques are minimally invasive and can augment conventional imaging and provide a method to greatly improve the diagnosis, staging and understanding of the pathobiology of retinal and vitreous disorders. Sensitive and precise reverse phase protein array (RPA) proteomic technology that can measure the post-translationally modified state of several hundred low abundance proteins and peptides in 20 microliters of vitreous fluid is provided. Reverse phase protein microarray is a technique that can be used for efficient and accurate detection of proteins in a sample. The proteins extracted from a single sample are immobilized on the substratum. The captured analytes are detected with a primary antibody directed toward the protein/polypeptide of interest and a second tagged molecule is incorporated for detection. Each spot on the array corresponds to a different sample. Total lysates of different samples are immobilized on the array and incubated with one antibody. Each spot on the array corresponds to a different sample (up to 640 lysates per array). Reverse phase microarray allows for probing into the networking and cross-talk between proteins involved in intracellular signaling. Uses of reverse phase microarray techniques in, for example, microarray printing, protein detection, and/or protein quantification are all commensurate with the scope of the present invention. A non-limiting list of antibodies that can be employed to detect proteins is provided in Table A.

Table A: Examples of Antibodies for Biomarker Detection

Antibody Company Cat. # Species Pathway

PAK 1/2 Serl 99/204 Cell Signaling 2605 Rabbit Adhesion/Cytoskeleton

C-Kit Y703 Zymed 34-9300 Rabbit Angiogenesis

C-Kit Y719 Cell Signaling 3391 Rabbit Angiogenesis

BCL-2 Thr56 Cell Signaling 2875 Rabbit Apoptosis

FADD Serl 94 Cell Signaling 2781 Rabbit Apoptosis

CC9 D330 Cell Signaling 9501 Rabbit Apoptosis

BAD Serl 12 Cell Signaling 9291 Rabbit Apoptosis

Complement Factor H Assay Designs Genetic Marker of AMD

Glucose/Insulin

GSK3 a/b Y279/216 BioSource 44-604G Rabbit Metabolism

Glucose/Insulin

GSK3 ab S21/9 Cell Signaling 9331 Rabbit Metabolism p-FGFR Cell Signaling 3471 Rabbit Growth/Survival PDGFR Y716 Upstate 07-021 Rabbit Growth/Survival PDGFR Y751 Cell Signaling 3161 Rabbit Growth/Survival VEGFR2 Cell Signaling 2472 Rabbit Growth/Survival VEGFR2 Y996 Cell Signaling 2474 Rabbit Growth/Survival VEGFR2 Y1175 Cell Signaling 2478 Rabbit Growth/Survival VEGFR2 Y951 Cell Signaling 4491 Rabbit Growth/Survival VEGF -A Santa Cruz Rabbit Growth/Survival VEGF -B Cell Signaling Rabbit Growth/Survival VEGF -C Cell Signaling Rabbit Growth/Survival AKT Thr 308 Cell Signaling 9275 Rabbit Growth/Survival

AMPKaI S485 Cell Signaling 4184 Rabbit Hypoxia/Ischemia eNos Serl 177 Cell Signaling 9571 Rabbit Hypoxia/Ischem ia

Heme Oxygenase 1 Assay Designs SPA-894 Rabbit Inflammation

TNF-alpha Cell Signaling 3707 Rabbit Inflammation

IL-I Cell Signaling 2022 Rabbit Inflammation

IL-6 Inflammation

IL-6R Inflammation

IL-8 Inflammation

IL-10 Inflammation

Alpha B Crystallin Assay Designs SPA-222 Mouse Normal Vitreous Protein

Phosphatase in AKT

Shipl Y1020 Cell Signaling 3941 Rabbit Survival Pathway

S6 Kinase Ser 240/244 Cell Signaling 2215 Rabbit Protein Translation EIF4G S1108 Cell Signaling 2441 Rabbit Protein Translation

Mushashi Cell Signaling 2154 Rabbit Stem Cell Signaling c-Abl Thr 735 Cell Signaling 2864 Rabbit Transcription Factor

[0025] Detection of polypeptides or other biomarkers can be made by any suitable technique. Polypeptide backbone can be detected, as well as post-translational modifications of it, such as glycosylation and phosphorylation. Antibodies can be used routinely, e.g., which are generated to amino acid epitopes of the target polypeptide; phosphorylated amino acids, etc. Reverse phase assay can be used to detect ocular polypeptides, where the array is comprised of ocular fluid immobilized to a substrate such as nitrocellulose, and binding partners (such as antibodies) are applied that specifically bind the target of interest. These can be rapidly used to characterize the contents of the fluid and generate disease biomarkers, including proteomic fingerprints. See, e.g., Grubb et al., Proteomics, 3:2142-2146, 2003. Mass spectroscopy and other conventional proteomic methods can also be used. [0026] Examples of methods for the identification and qualification of proteins in complex mixtures include those described in Aebersold et al., Nature. 422, 198-207, 2003; de Hoog et al., Annu Rev Genomics Hum Genet. 5, 267-93, 2004. Proteome analyses can employ online chromatographic/tandem mass spectrometric (LC/MS- MS) methods in attempts to fractionate and identify large numbers of peptides from enzyme-digested proteins.

[0027]Molecules employed for the detection of biomarkers can be produced or derivatized to provide ionic groups (such as carboxylate, protonated amine, quaternary ammonium, and sulfate groups), hydrogen-bond acceptors or hydrogen- bond donors, electron donors or electron acceptors, polar groups (such as amino, hydroxyl, ester, sulfhydryl and nitrile groups), hydrophobic groups (such as alkyl, alkenyl and alkynyl groups or groups with specific partition coefficients), peptides, proteins, nucleic acids, carbohydrates, lipids or any combination thereof, on their surfaces or in their interiors. A "carrier protein" can be used to collect and concentrate biomarkers from biological fluids. Examples of carrier proteins include albumin, iron binding proteins (such as transferrin), fibrinogen, alpha-2- macroglobulin, immunoglobulins (such as IgA, IgE and IgG), complement, haptoglobulin, lipoproteins, prealbumin, alpha-1-acid glycoprotein, fibronectin, and ceruloplasmin, and fragments, combinations and chemical derivatives thereof. Albumin, proteoglycans, glucosaminglycans, and heparan sulfates, are examples of molecules that can be use to detect biomarkers.

[0028]The present invention also provides a proteomic fingerprint, comprising at least one vitreous fluid biomarker associated with an ocular condition. The biomarker of the fingerprint can be a polypeptide or a unique fragment thereof. In some embodiments, the fingerprint is obtained and analyzed using the methods of the present invention. The present invention provides a proteomic fingerprint of an ocular fluid sample, comprising at least one polypeptide or other molecule present in the sample. Biomarkers can be determined using any suitable technology. A proteomic fingerprint can comprise as few as one polypeptide, or it can comprise more than one polypeptide (i.e., a plurality).

[0029] Detection of the presence, absence, or variable quantitative level of proteins and other biomarker in the vitreous fluid of the eye can comprise isolation of the protein, enzymatic hydrolysis (for e.g., using trypsin), HPLC separation, resolved using mass spectrometric analysis, and the retrieved fragments are searched a database of candidate polypeptides. Routine methods for HPLC analysis of peptides are known in the art, and can involve utilization of separation columns and/or buffers of interest (for e.g., modified C-18 column). Techniques for mass-spectrometric analysis of peptides are also known, and can involve, for e.g., nano-spray/linear Ion Trap mass spectrometric analysis.

[003O]In some embodiments, the set of polypeptides detected and/or identified in accordance with the present invention is comprised by a "fingerprint" in that they are a distinctive pattern of polypeptides present in the ocular fluid. Fingerprints can be prepared using any suitable technologies or purification processes, e.g., characterizing polypeptides present in the ocular fluids (See Table 2 to Table 13 of U.S. Patent Application Publication 20070224644 for a representative example of such polypeptides). Methods of isolating biomarker attractant-associated biomolecules are described in WO05036180, which is hereby incorporated by reference in its entirety. [003I]A set of polypeptides can be used as a unique identifier to characterize the fluid, as well as the physiological status of the subject. The ocular fingerprint can be viewed as a snapshot of the elements (e.g., polypeptides) that are involved in, or a product of, the physiological processes that are occurring in the body. Examples of physiological states that can be characterized in accordance with the present invention include without limitation, diseases states (e.g., cancer, retinopathy, diabetes, macular degeneration, venous occlusive disease, cataracts, and other disorders mentioned herein); therapeutic states (e.g., for monitoring drug efficacy and adverse events); organ function (e.g., to monitor normal organ function, such as brain, kidney, and liver functions); toxicological states (e.g., to detect toxins or perturbations caused by toxins); etc. Thus, an ocular fluid fingerprint can be used for a variety of medical, diagnostic, and therapeutic purposes, including, for example: to detect the risk of cataract formation (see below); to monitor blood-ocular breakdown; to detect age- related macular degeneration; to detect therapeutic efficacy of kinase inhibitors and other drugs; etc.

[0032] In addition to the ocular disorders, the ocular fluids can also be used generally to monitor a subject's health and physiological status. The ocular fluid is in communication with other body compartments, and thus is useful to monitor extraocular compartments, including the brain, kidney, liver, etc. Because developmentally the eye is an extension of the brain the state of the molecular composition of ocular fluids can provide information about diseases in the brain. With regard to more distant organs, molecules derived from these organs can enter the ocular fluids through the circulation, or the ocular fluid markers can reflect a systemic body-wide process that affects the distant organ.

[0033]Any ocular or eye-related fluid can be analyzed in accordance with the present invention, including, e.g., vitreous fluids; aqueous fluids; retinal blood, such as blood present in the choroid; and tears, including tears extracted from the lacrimal sac. In some cases the state of specific diseases as reflected in ocular fluids can be measured by fluorescent, magnetic, or radio nucleotide imaging.

[0034] Any molecule with sufficiently specific affinity to a particular biomarker can be employed with the materials and methods of the present invention to detect a biomarker. In some embodiments, an antibody or an antigen binding fragment thereof is used to detect a biomarker. Examples of molecules that can be used to detect biomarkers include chimeric proteins, proteins with modified amino acid composition, proteins modified postranslationally, nucleic acids, carbohydrate decorated molecules, and organic polymers), dendrimers and particles (such as microparticles and nanoparticles, including silica, metal, ceramic and carbohydrate microparticles and nanoparticles), and cellular microparticles (see, for example, Diamant et. al., Eur J Clin Invest. 34: 392-401, 2004). The polypeptides can be present as intact proteins, or as fragments. Such fragments can be naturally- occurring, or can be produced during processing of a sample, either by inadvertent or deliberate proteolysis (e.g., contacting a sample with a proteolytic enzyme or a chemical cleavage agent).

[0035]In addition to substantially full-length polypeptides expressed by genes or coding segments thereof, the present invention includes use of biologically active fragments of the polypeptides, or analogs thereof, including organic molecules that simulate the interactions of the peptides. Biologically active fragments include any portion of the full-length polypeptide that confers a biological function on the expressed product, including ligand binding and antibody binding. Ligand binding includes binding by nucleic acids, proteins or polypeptides, small biologically active molecules or large cellular structures. In some embodiments, the polypeptide is at least 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 22, 25, 30, 35, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 1000, 10,000, 50,000, 100,000 or more amino acids in length, or 100,000, 75,000, 50,000, 10,000, 5,000, 1000, 750, 500, 250, 200, 100, 50, 40, 30, 25, 22, 20, 17, 15, 12, 10, 9, 8, 7, 6, 5, or fewer amino acids in length. A polypeptide can have a length in a range from any one of the above lengths to any other of the above lengths including endpoints. A polypeptide in accordance with the present invention can be 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% identical to reference sequence provided herein.

[0036]Detection of a biomarker can comprise the use of an aptamer. In some embodiments, the aptamer comprises a nucleic acid. The aptamer can comprise RNA. The aptamer can comprise DNA. Aptamers comprising nucleic acids can comprise natural and/or modified (non-natural) nucleotides. The aptamer can comprise an amino acid. Aptamers can be selected and produced using any suitable technique or protocol. In some embodiments, the biomarker comprises a nucleic acid. Detection of a biomarker can be through the complementary pairing of a sufficient number and unique sequence between a nucleic acid biomarker and probe.

[0037JA nucleic acid or nucleotide sequence thereof includes one or more nucleotides. Exemplary nucleic acids include RNA, DNA, any combination thereof. Nucleic acids can include both naturally occurring as well non-naturally occurring nucleotides, and encompass ribonucleic acid nucleotides, as well as deoxyribonucleic acid nucleotides. When a nucleic acid is recited it refers generically to DNA and RNA unless the recitation explicitly states that the nucleic acid is a specific one, e.g., DNA or RNA. If a nucleic acid refers to a sequence that contains thymine (t), that does not necessarily indicate that the nucleic acid is DNA; in some embodiments the nucleic acid is RNA and/or DNA. Similarly, if a nucleic acid refers to a sequence that contains uracil (u) that does not necessarily indicate that the nucleic acid is RNA; in some embodiments the nucleic acid is DNA and/or RNA.

[0038]The nucleic acid molecules relevant to the present invention can readily be obtained in a variety of ways, including, without limitation, chemical synthesis, cDNA or genomic library screening, expression library screening, and/or PCR amplification of cDNA. These methods and others useful for isolating such DNA are set forth, for example, by Sambrook et al., "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (1989), by Ausubel, et al., eds., "Current Protocols In Molecular Biology," Current Protocols Press (1994), and by Berger and Kimmel, "Methods In Enzymology: Guide To Molecular Cloning Techniques," vol. 152, Academic Press, Inc., San Diego, Calif. (1987).

[0039]The present invention provides for the use of isolated, purified or enriched nucleic acid sequences of any length, In some embodiments, the nucleic acid is from 15 to 500 nucleotides in length, 15 to 100 nucleotides in length, 15 to 50 nucleotides in length, 15 to 30 nucleotides in length, 30 to 300 nucleotides in length, or 45 to 200 nucleotides in length, or 45 to 100 nucleotides in length, which have sequence that corresponds to a portion of one of the nucleic acids or nucleotide sequences described herein. The nucleic acid can be at least 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 22, 25, 30, 35, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 1000, 10,000, 50,000, 100,000 or more nucleotides in length, or 100,000, 75,000, 50,000, 10,000, 5,000, 1000, 750, 500, 250, 200, 100, 50, 40, 30, 25, 22, 20, 17, 15, 12, 10, 9, 8, 7, 6, 5, or fewer nucleotides in length. The nucleic acid can have a length in a range from any one of the above lengths to any other of the above lengths including endpoints. [004O]A nucleic acid in accordance with the present invention can be 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any reference sequences provided herein. A nucleic acid that hybridizes under stringent conditions to a nucleotide described herein can be employed. Unless otherwise specified, percent identities for nucleic acids and amino acid sequences are determined as follows: Percent identity of two nucleic acid sequences or two amino acid sequences is determined using the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 87:2264-2268 (2002), modified as in Karlin and Altschul et al., Proc. Nat. Acad. Sci. USA, 90:5873-5877 (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., J. MoI. Biol. 215:403-410 (1990). BLAST nucleotide searches are performed with the NBLAST program, score = 100, wordlength = 1, to obtain nucleotide sequences with a percent identity to a nucleic acid employed in the present invention. BLAST protein searches are performed with the XBLAST program, score = 50, wordlength = 3, to obtain amino acid sequences with a percent identity to a reference polypeptide. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul et al., Nucleic Acids Res., 25:3389-3402 (1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used. See <www.ncbi.nih.gov>. [0041]Unless otherwise specified, a nucleic acid and nucleic acid probe can include one or more nucleotide analogs, labels or other substituents or moieties so long as the base-pairing function is retained. The nucleic acid probe can comprise a detectable label, such as a radioactive or fluorescent label. A variety of other detectable labels are known to those skilled in the art. Unless otherwise specified, where the sequence for a given strand is provided, the present invention also includes its complement in addition or in the alternative.

[0042] In connection with nucleic acid hybridization, the term "specifically hybridizes" indicates that the probe hybridizes to a sufficiently greater degree to the target sequence than to a non-target sequence, e.g., at a level which allows ready identification of probe/target sequence hybridization under selective hybridization conditions. "Selective hybridization conditions" refer to conditions that allow such differential binding. Similarly, the terms "specifically binds" and "selective binding conditions" refer to such differential binding of any type of probe, and to the conditions that allow such differential binding. [0043]Variables can be adjusted to optimize the specificity of a nucleic acid probe, including changes in salt concentration, temperature, pH and addition of various compounds that affect the differential affinity of GC vs. AT base pairs, such as tetramethyl ammonium chloride. [See Current Protocols in Molecular Biology, Ausubel et al. (Editors), John Wiley & Sons.] Hybridization conditions should be sufficiently stringent such that there is a significant difference in hybridization intensity between alleles, and preferably an essentially binary response, whereby a probe hybridizes to only one of the alleles. Hybridizations can be performed under stringent conditions that allow for specific binding between an oligonucleotide and a target nucleic acid. Stringent conditions are defined as any suitable buffer concentrations and temperatures that allow specific hybridization of the oligonucleotide and any washing conditions that remove non-specific binding of the oligonucleotide. For example, conditions of 5xSSPE (750 mM NaCl, 50 mM Na Phosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-30⁰C are suitable for allele- specific probe hybridizations. The washing conditions can range from room temperature to 60⁰C.

[0044]A vitreous sample to be analyzed in accordance with the present invention can be stored by any suitable means for the detecting step. For example, the sample can be extracted into a reservoir comprising at least one chemical to protect the polypeptide integrity. In some embodiments, the chemical is a protease inhibitor or a phosphatase inhibitor.

[0045] Samples (preferably small samples, for example, 50 to 100 microliters) of fluid vitreous (without cells) of human eyes can be collected and analyzed in a minimally invasive manner. The methods of the present invention can be used to assess the status of neovascularization in eyes with age-related macular degeneration (AMD), the status of neovascularization in eyes with diabetic retinopathy, the status of neovascularization in eyes with venous occlusive disorders, and/or the cell receptor status that indicates responsiveness to anti-VEGF agents injected in the eye or delivered by other means. In some embodiments, samples (preferably small, samples e.g., 50 to 100 microliters) of fluid vitreous (without cells) of human eyes are repeatedly collected and analyzed to assess the status of macular edema in eyes with age-related macular degeneration (AMD), the status of macular edema in eyes with diabetic retinopathy, and/or the status of macular edema in human eyes. Samples can be analyzed to assess other molecules and/or compounds for injecting in the eye or delivered by other means for preventing, slowing, stopping or reversing macular edema. In some embodiments, small samples (e.g., 50 to 100 microliters) of fluid vitreous (without cells) of human eyes are repeatedly collected and analyzed to assess the status of pathobiology in human eyes. In some embodiments, samples are analyzed to assess and identify other molecules and/or compounds to inject in the eye or delivered by other means or procedures that prevents, slows, stops or reverses. [0046]Technology for the separation and concentration of the low abundance phosphorylated peptides and proteins away from the abundant proteins in the vitreous including albumin and proteoglycans and sequence the isolated peptides by mass spectrometry is provided. Phosphorylated forms of VEGF receptors, PDGF receptors, and a variety of other signal pathway proteins, can be identified and detected. The levels of the phosphorylated forms of receptors such as VEGFR, FGFR and PDGFR are correlated with clinically-responsive, compared to refractory disease or controls (non-neovascular diseases represented by macular hole and epiretinal membrane), and which appear to change following anti-VEGF therapy.

[0047] In the present invention, there is provided a method for detecting macular diseases, retinal detachment, inflammation of the eye, diabetic retinopathy and many other diseases comprising comparing a profile of shed receptors or signal transduction molecules and/or their phosphorylated forms, for e.g., VEGFR, PDGFR, EGFR, RBP4 in a healthy subject with that of a patient. These receptor proteins can be used as drug targets for drugs such as Gleevec™, Iressa™, and Avastin™, demonstrating that this information could be used to tailor therapy for the patient. A series of crystallins in vitreous samples of patients who have had a vitrectomy for retinal detachment can be sampled for susceptibility for immediate development of cataracts. With regard to macular hole or macular detachment therapy or macular vascular leak, therapy can include administration of natural autologous protein such as platelet extracts.

[0048] In some embodiments, the level of the molecule concentration or the level of the phosphorylated molecules (phosphorylation on one or more specific residues) can be quantitatively related to the severity of the disease or the amount of disease suppression produced by a drug administered to the patient. An example is a method for detecting the phosphorylation status of the VEGFR, (which may have no correlation with the amount of total receptor protein) as a predictor of (a) requirement for an angiogenesis inhibitor, and (b) whether or not an angiogenesis inhibitor is working to suppress the VEGF ligand from triggering its receptor. If the receptor is active or engaged with ligand then and only then will it be phosphorylated. [0049]The invention also relates to methods of monitoring the physiological status of a subject, comprising: measuring the presence of a post-translationally modified polypeptide (e.g., phosphorylation) in a vitreous fluid sample extracted from a subject. Signaling pathways can be monitored. Signaling pathways include any pathway in the body that involves generating a chemical event (e.g., phosphorylation) that modulates a cellular activity (e.g., indicating receptor occupancy, site-directed protein-protein binding, and triggering a cascade of enzymatic reactions that culminates in gene expression). For example, phosphorylation is a key post- translational modification event in many biological pathways involved in cell growth, cell death, gene expression, and cellular responses to stimuli. In addition, aberrant phosphorylation patterns can be associated with diseases, such as cancer and other hyper-proliferation disorders.

[0050]Further examples include, e.g., G-protein receptor mediated pathways, especially receptors for tyrosine kinases, such as vascular growth factor receptors (e.g., VEGFR-I, VEGFR-2), epidermal growth factor receptors (EGFR), HER2, adrenergic receptors (e.g., alpha- and beta-types); hormone mediated receptors; etc. Examples of receptors include, VEGFR-2 (e.g., including phosphorylation sites Y951, Y996, Y1054, Y1059, Yl 175, Y1214); PDGFR-beta (e.g., including phosphorylation sites Y740, Y751, and Y771), and EGFR (e.g., including phosphorylation sites Yl 173, Yl 148, Y1068, Y845, and Y992).

[0051]These methods can also be utilized to measure or monitor the efficacy of a drug, especially a drug which is utilized to modulate a kinase, such as a tyrosine kinase, or a biological based therapeutic, such as an autologous platelet concentrate. A variety of therapeutic agents are being used to treat diseases or disorders associated with aberrant or increased kinase activity, including cancers and angiogenesis. Targets include, but are not limited to, e.g., raf, PDGFR-alpha, PDGFR-beta, EGFR, VEGFR, VEGFRl, VEGFR2, VEGFR3, HER-2, KIT, FLT3, c-MET, FGFR, FGFRl, FGFR3, c-FMS, RET, ABL, ALK, ARG, NTRKl, NTRK3, JAK2, ROS, etc. Other signaling targets include, e.g., ERK, AKT, PYK2, etc.

[0052]Examples of kinase effecting drugs, include, but are not limited to, e.g., Avastin™ (bevacizumab), cetuximab, erlotinib (tarceva or OSI774), everolimus (RADOOOl), fasudil, FK506, gefitinib (ZD1839), imatinib mesylate (STI57 or Gleevec™), lapatinib ditosylate (GSK572016), rapamycin, sorafinib, sirolimus, sunitinib (sutent), trastuzumab (Herceptin™), serafanib, and wortmannin. [0053] One goal of such drug therapy is to reduce the amount of phosphorylation of a target polypeptide. For example, several anti-cancer drugs are being utilized to block angiogenesis by blocking the phosphorylation of VEGFR-2. The efficacy of such drugs can be monitored by detecting the appearance of shed phosphorylated receptor into the vitreous fluid. As shown in the examples, phosphorylated VEGFR-2 and PDGF-R polypeptide fragments are detected in vitreous fluid using reverse phase assays.

[0054]The proteome of the human vitreous contains soluble phosphorylated proteins or protein fragments derived from signal pathway receptors, kinases and kinase substrates, particularly those involved in angiogenesis, apoptosis, and inflammation. The identity of sequenced and post translationally modified proteins that fluctuate over the course of disease remission or recurrence provide useful data for predicting response to treatment, timing of disease progression, and clues for therapeutic targets. [0055]Therapies can be employed to suppress the phosphorylated state of the kinase receptor target. Thus if the treatment is suppressing the targeted kinase pathway it would be expected to alter or block the phosphorylation of the receptor land to in turn block the phosphorylation of proteins downstream of the receptor kinase. In the case of anti-VEGF therapy, efficacy will be reflected in the suppression of the phosphorylation of the VEGF receptor which is normally stimulated by VEGF. This is because phosphorylation of the VEGF receptor is induced by its engagement with the VEGF ligand. Consequently if a therapeutic antibody blocks the binding of VEGF to its receptor, then the receptor protein will not autophosphorylate. In this way the phosphorylated form of the receptor can provide a functional read-out of whether the therapy is working or not. [0056]Proteomic analysis of phosphorylated proteins or phosphoproteomics can be utilized. The targeting of phosphoproteins for analysis can yield not only a smaller set of proteins but also those with high biological relevance. For example, such phosphoproteins can be indicative of cell signaling, which allows the research to be directed by biological hypotheses. When enriching phosphorylated proteins on a proteome-wide scale (probing a complex mixture of a large number of proteins as opposed to a smaller number of proteins from an antibody-based immunoprecipitation (IP)), can involve at least one strategy. Examples of strategies include those that are based on affinity-based enrichment using (1) immobilized metals (e.g., Fe3+ and Ga3+; immobilized metal affinity chromatography, IMAC) and (2) titanium dioxide. For the former, methyl esterification derivatization to minimize non-specific binding (non-phosphorylated peptides) (Ficarro et al., Nat Biotechnol. 20, 301-5, 2002.) can be employed. The latter can be more conducive to speed and simplicity (Thingholm et al., Nat Protoc. 1, 1929-35, 2006.). Both methods have been used successfully to identify large numbers of phosphorylated peptides, and therefore proteins, in a wide variety of biological studies, although mostly of cell lysates (Pandey et al., Sci STKE. 2000,PLI, 2000., Gruhler et al., MoI Cell Proteomics. 4, 31 0-27, 2005., Chi et al., J Proteome Res. 5, 3135-44, 2006.). Anti-phosphotyrosine (pY) antibody-based IP can be combined with subsequent IMAC enrichment to allow the identification by LC/MS-MS of 100+ pY phosphorylation sites (Zhang et al., MoI Cell Proteomics. 4, 1240-50, 2005.).

[0057] In some embodiments ocular fluids can be removed from a patient using a whole-bore vitrectomy cannula or cutter containing agents that inhibit polypeptide degradation, and then subjecting the fluid to analysis for the presence of biomarkers. However, less invasive methods and instrumentation are preferred and are an object of the present invention.

[0058] In accordance with another aspect of the invention, an ophthalmic aspirating device is provided. In one embodiment, an ophthalmic aspirating device, comprises: a negative pressure module; a conduit having first and second ends, wherein the respective ends each comprises an aperture, and wherein the second conduit end is operatively associated with the negative pressure module. The ophthalmic aspirating device can further comprise a housing. In another embodiment, the ophthalmic aspirating device comprises a housing; a tube operatively associated with the housing, the tube having first and second ends; a conduit having first and second ends, wherein the first conduit end comprises an aperture, and wherein the second conduit end is operatively associated with the first tube end; and a negative pressure module. In some embodiments, the negative pressure module comprises a reservoir operably associated with the housing, wherein the reservoir comprises at least one chemical to protect polypeptide integrity. In some embodiments, the reservoir is comprised by an attachable/detachable receptacle. A negative pressure module can be or can comprise the reservoir or receptacle. The negative pressure module can be configured for at least one of attachment and deattachment from a housing. Chemicals that can be included in the reservoir include, e.g., protease inhibitors; phosphatase inhibitors; various preservatives, etc. Specific examples include, serine protease inhibitors, cysteine protease inhibitors, aspartic protease inhibitors, and metalloprotease inhibitors. Examples of these include, AEBSF, aprotinin, E-64, EDTA, leupeptin, bestatin, O-phenanthroline, cathepsin, etc. In some embodiments, the negative pressure module comprises a plunger assembly comprising at least a plunger. The plunger assembly can be configured for accuracy, a pre-set amount, and to minimize the amount of vitreous fluid extracted. In some embodiments, the plunger assembly comprises a biasing device connected to the plunger, wherein the biasing device is under the operational control of an actuator. The conduit can comprise at least one of a needle and a cannula. The ophthalmic aspirating device can be employed with the methods of the present invention.

[0059]One embodiment of this invention comprises a syringe-like device that holds a needle, cannula or other conduit of a very small diameter and that can aspirate liquid from inside the eye of a patient. Further, the syringe-like device can be operated by a medical practitioner using only one hand and using a fairly rapid motion. [0060]According to one embodiment of the present invention, a medical device includes a tube with two needles, cannula or conduits, one connected to each end of the tube. At both ends of the tube, the needles, cannula, or conduits can be of a very small size, small enough to aspirate very small amounts of liquid, such as, on the order of 20 to 100 microliters. At one end of the tube, a first needle, cannula, or other conduit, can be inserted into a low pressure chamber, and a valve can control the communication between the low pressure chamber and the tube. At the other end of the tube, a second needle, cannula or other conduit can be inserted into the eye of a patient.

[006I]In operation, once the second needle, cannula, or other conduit is inserted into the eye of the patient, the valve at the other end of the tube can be opened so that the tube is able to communicate with the reduced pressure inside the low pressure chamber via the first needle, cannula or other conduit, thus creating an aspiration force inside the tube. As a result, liquid from inside the eye can be aspirated through the second needle, cannula or conduit into the tube at a fast rate. The aspiration rate of the liquid is commensurate with the pressure of the vacuum inside the low pressure chamber. In this case, the aspiration rate is fast, enough to aspirate the liquid from the eye and leave any biological gel that can exist in the vitreous fluid inside the eye. Once the liquid is aspirated into the tube, the second needle, cannula or other conduit which was inserted into the eye, can be removed from the eye. The size and/or pressure of the low pressure chamber can be adjusted so as to aspirate a precise and desired amount of liquid.

[0062]According to another embodiment of the present invention, a syringe can be provided that includes a needle, cannula or other conduit of a very small size at at least one end, for example that would allow the aspiration of about 20 to 200 microliters of liquid, of 50 to 100 microliters, and a plunger is provided at the end of the syringe opposite the needle, cannula or other conduit. The plunger, when pulled away from the needle, cannula or other conduit creates a vacuum inside the syringe that aspirates fluid in the vicinity of the needle. In one embodiment, the syringe includes a biasing device connected to the plunger, and an actuator controls the operation of the biasing device. Accordingly, once the actuator is engaged by a user (e.g., a medical practitioner), the biasing device is activated and urges the plunger of the syringe in a direction away from the eye of the patient so that an aspiration force is created. In operation, the actuator can be arranged so that, once urged, the biasing device pulls the plunger of the syringe away from the eye at a rate sufficient to allow the fast extraction of liquid from the eye, without extracting any of the gel present in the vitreous fluid from the eye. As a result, only liquid from the eye is aspirated into the syringe, not any of the biological gel. [0063]According to various exemplary embodiments, the needle, cannula or other conduit, or the syringe tube can have markings indicating the amount of fluid being aspirated into the syringe tube, and the user can disengage the actuator once the needle, cannula or other conduit, or the syringe tube indicates that a desired amount of fluid has been aspirated. Once the actuator is disengaged, the biasing device is prevented from further aspirating fluid. Needles and other conduits can have apertures at their ends. One or more of these ends can be sharp to allow puncture of the eye and/or a container such a as a low pressure chamber. A secondary housing can be employed to hold at least one of the conduit and the low pressure chamber. [0064] Before, after, or concurrent to the removal of fluid from the eye, one or more therapeutic agents can be administered to the eye. The therapeutic agent can be administered using the same conduit used for fluid removal. The therapeutic agent can be housed with the ophthalmic aspiration device. In some embodiments the therapeutic agent is housed within a chamber operatively associated with at least one end of the ophthalmic aspiration device. The therapeutic agent can be housed in a container separate from the ophthalmic aspiration device. A chamber or container housing the therapeutic agent can comprise one or more segments. The segments can be separated by one or more septa. A septum can allow passage, e.g., by puncture, by a conduit. Segments can house same or different therapeutic agents. In some embodiments, puncturing of one or more septum allows mixing of ingredients for the therapeutic agent. A secondary housing employed to hold at least one of the conduit and the low pressure chamber can in addition or in the alternative be used with the therapeutic agent container. In some embodiments, the low pressure chamber and therapeutic agent container are used in succession, in either order, by removing one and then inserting the other. In some embodiments, the therapeutic agent container is an ampoule. The therapeutic agent can be flexible such that when squeezed, the therapeutic agent is released.

[0065]Advantages of this invention include that a very small amount of fluid is extracted from the eye of the patient, and that the user can use one hand to extract the fluid, thus making extraction easier and more efficient than existing art devices and methods. Accordingly, such fluid extractions can be performed multiple times on a patient without significant adverse effects, such as once every other week if needed, and can be performed as an outpatient procedure in a doctor's office. [0066]FIG. 1 illustrates an ophthalmic aspiration device 1, according to one exemplary embodiment of the invention. In Fig. 1, a conduit 20, such as a needle or cannula, has first and second ends with apertures at the respective ends. The conduit can be operatively associated with at least one of an eye 40 and a low pressure chamber 50. Such operative association allow for transfer, e.g., aspiration, of fluid 60 (e.g., vitreous fluid) from the eye 40 to the low pressure chamber 50. The ophthalmic aspiration device can be operated in a manner similar to that used for drawing blood from veins. That is the needle has two sharp ends, one for entering the eye and the opposite end for puncturing a stopper in a low pressure chamber. By applying pressure to the low pressure chamber, the stopper presses against the end of the needle opposite the end in the eye, thereby puncturing the stopper and causing the negative pressure in the chamber to withdraw vitreous fluid from the eye into the chamber. As in the other embodiments, the low pressure chamber can contain preservatives such as a protease inhibitor. Once the fluid has been removed from the eye, the needle with the tube can be removed from the eye. Alternatively, the low pressure chamber can be removed with needle in place and a non-vacuum container or ampoule can replace the low pressure chamber on the needle. The ampoule can house a drug to be released into the eye. The ampoule can be made out of a soft material that can be squeezed to push the drug through the needle into the eye. Once the drug is released, the needle and the ampoule can be removed from the eye. In some embodiments, a Vacutainer® tube or system is used.

[0067]FIG. 2 illustrates an ophthalmic aspiration device 100, according to one exemplary embodiment of the invention. In Fig. 2, a tube 110 is provided with two needles, cannula or other conduits 120 and 130, are at each end of the tube 110. A first needle, cannula or other conduit 120 is connected to the tube 100 at one end and can be inserted inside the eye 140 of a patient via the end opposite the connection to the tube. A second needle, cannula or other conduit 130, located at an end opposite to the first needle, cannula or other conduit 120, communicates with a low pressure chamber 150. In operation, when the needle, cannula or other conduit 120 is inserted into the eye 140, and the needle, cannula or other conduit 130 is inserted into the low pressure chamber 150, the reduced pressure in the low pressure chamber 150 generates an aspirating action that is transmitted through the tube 150 and via the needle, cannula or other conduit 120 to the interior of the eye 140 or other location to be aspirated. As a result, fluid 160 (e.g., vitreous fluid) present inside the eye 140 is aspirated through the needle 120 and into the tube 110. Alternatively, the fluid 160 can also be aspirated directly into the low pressure chamber 150. A certain amount of fluid 160 can be aspirated from the eye 140 of a patient, for example, and into either the tube 110, or into both the tube 110 and the low pressure chamber 150. In some embodiments, a Vacutainer® tube or system is used.

[0068] As an alternative option, a valve 170 can be provided for controlling communication between the low pressure chamber 150 and the needle, cannula or other conduit 130, so that once the needle, cannula or other conduit 120 is inserted into the eye 140 of a patient, for example, no aspiration occurs until the valve 170 is opened. Once the valve 170 is opened, the low pressure chamber 150 communicates with the tube 110, and via the tube 110 with the needle, cannula or other conduit 120 and the interior of the eye 140. Furthermore, indicators 180 can be present on the side of the tube 110 to allow measurement of the volume of fluid aspirated into the tube 110. The valve 170 can be closed by a user once the amount of fluid 160 aspirated reaches a desired volume. Thus, a better control of the volume of fluid 160 that is aspirated can be achieved.

[0069] According to an exemplary embodiment of this invention, the tube 110 and the needles, cannulae or other conduits 120 and 130 can be reusable together or separately. Once the user has extracted fluid 160 from a patient's eye, and once the fluid 160 has been transferred to a storage area or has been analyzed, the tube and needle, cannula or other conduit can be cleaned, sterilized and reused with another patient. For the tube 110 and the needles, cannulae or other conduits 120 and 130 to be reusable, additional components, such as a hub and a ferrule, can be used with the needles, cannulae or other conduits 120 and 130. Accordingly, when the user activates the valve 170 to discontinue the aspiration of fluid 160, the valve 170 can incur a small delay, during which an additional volume of fluid 160 can be aspirated beyond the desired amount. For example, the additional volume of fluid 160 can correspond to the volume of fluid present inside the hub and the ferrule of the needles, cannulae or other conduits 120 and 130. Any losses that would have been due to the existence of the hub and the ferrule on the syringe can be compensated, and a precise amount of fluid 160 can be extracted from the inside of the eye 140 of the patient, into the tube 110.

[0070]One aspect of various embodiments of this invention comprises the aspiration of fluid 160 from the eye 140 very quickly as the aspirating action is caused by communication with the reduced pressure inside the low pressure chamber 150. Because the vitreous fluid inside the eye 140 can contain both fluid and gel, and because of the quick aspiration action created by communication with the low pressure chamber 150, only the fluid portion 160 of the vitreous fluid inside the eye 140 can be aspirated into the tube 110 and/or into the low pressure chamber 150. [0071] The amount of fluid aspirated from the inside of the eye 140 can be minimal if the low pressure chamber used is relatively small. For example, volumes of about 20 to 100 microliters can be aspirated, or volumes of about 50 to 100 microliters. Also, because very small amounts of fluids can be aspirated from the inside of the eye 140, the procedure can be performed without irrigating the eye or replenishing the aspirated fluid with antibiotics and/or other fluids, as is generally the case for conventional techniques.

[0072]Another advantage of some embodiments of the present invention is that a medical practitioner can extract liquid from a patient's eye in a routine outpatient procedure using one hand and in a fairly short amount of time.

[0073]FIG. 3 illustrates an ophthalmic aspiration device, according to another exemplary embodiment of the present invention. In Fig. 3, a tube 210 is provided with a needle, cannula or other conduit 220 at one end, and a plunger 230 at the other end. The plunger 230 can be activated via a biasing mechanism 270 such as a spring or other energy storing mechanical or electrical device. The biasing mechanism 270 can be controlled by an engagement mechanism 280 that urges the biasing mechanism 270. As such, when the engagement mechanism 280 is engaged by a user, the biasing mechanism 270 is activated, and the plunger 230 travels away from the needle 220, thus creating an aspirating force. When the engaging mechanism 280 is released, operation of the biasing mechanism 270 is arrested, and the plunger 230 is placed in a stationary state. The tube 210 can include indicators 250 indicating the volume of fluid that has been aspirated into the tube 210.

[0074] In operation, the needle, cannula or other conduit 220 can be inserted inside the eye 240 of a patient. If the plunger 230 is in a stationary state, then no aspiration of the fluid 260 from the eye 240 can occur. However, when a user engages the urging mechanism 280 to engage the biasing mechanism 270, the plunger 230 is pulled away at a high rate from the needle 220, and an aspiration force is created. Accordingly, the fluid 260 in the eye 240 of a patient can be aspirated inside the tube 210. Once the aspiration of the fluid 260 inside the tube 210 has started, the user can have several options. As a first option, the user can simply arrest the urging mechanism 280 to deactivate the spring biasing mechanism 270 when the aspirated fluid has filled the tube 210. As a second option, the user can monitor the volume of fluid that is being aspirated from the eye 240 of the patient via the indicators 250. Once the fluid reaches a desired volume, the user can then disengage the urging mechanism 280 to arrest the biasing mechanism 270 and to render the plunger 230 stationary, thus terminating the aspirating action. Accordingly, the user is able to aspirate very small amounts of fluid, for example in the range of 20 to 200 microliters, or in the range of 50 to 100 microliters.

[0075] Because the vitreous fluid inside of the eye 240 can contain not only fluid 260, but also gel-like substances, the fluid 260 from the inside of the eye 240 can be aspirated by having a high rate of aspiration from the plunger 230. The biasing mechanism 270 can pull away the plunger 230 at a high rate to allow the aspiration of the fluid 260 in a rapid burst of suction, and to leave any gel-like substance inside the eye 240. Once the plunger 230 is pulled away rapidly, the fluid 260 fills the tube 210 more slowly than the rate at which the plunger 230 is pulled, and the user can monitor the fluid 260 and disengage the urging mechanism 280 to stop the plunger 230 when the fluid 260 reaches a desired volume. Because very small amounts of fluids can be aspirated from the eye 240, irrigating the eye or replenishing the aspirated fluid with antibiotics and other fluids can be avoided. Another advantage of some embodiments of the present invention is that a medical practitioner can extract fluid from a patient's eye in a routine outpatient procedure using one hand and in a fairly short amount of time. [0076] According to an exemplary embodiment of this invention, the tube 210 and the needle, cannula or other conduit 220 can be reusable together or separately. Thus, once the user has extracted fluid 260 from a patient's eye 240, and once the fluid 260 has been transferred to a storage area or has been analyzed, the tube and needle, cannula or other conduit can be cleaned, sterilized and reused with another patient. For the tube 210 and the needle, cannula or other conduit 220 to be reusable, additional pieces such as a hub and a ferrule can be added between the needle, cannula or other conduit 220 and the tube 210. Accordingly, when the user engages the biasing mechanism 270 via the engaging mechanism 280 to discontinue pulling the plunger 230 away from the needle, and thus to terminate the aspiration of the fluid 260, the urging mechanism 280 can include a small delay during which the biasing mechanism 270 is still pulling the plunger 230 to aspirate an additional volume of fluid 260, wherein the additional volume of fluid 260 corresponds to the volume of fluid 260 that is present inside the hub and the ferrule. Thus, any losses that may be due to fluid 260 remaining inside the hub and the ferrule can be compensated, and precise amounts of fluid 260 can be extracted into the tube 210. [0077]Kits including one or more material of the present invention are provided that can be used to carry out the methods of the invention in whole or part. Kits for analyzing vitreous fluid samples are provided. A vitreous fluid analysis kit can comprise a vitreous fluid receptacle comprising a reservoir, wherein the reservoir comprises at least one chemical to protect polypeptide integrity. In some embodiments, the vitreous fluid analysis kit further comprises an ophthalmic aspirating device, comprising a housing to which the receptacle can be operatively attached and detached. The vitreous fluid analysis kit can further comprise at least one vitreous fluid biomarker detector. In some embodiments, the detector comprises a primary antibody specific to a biomarker polypeptide or a unique fragment thereof. In some embodiments, the detector comprises a secondary antibody coupled to a label, such as a radioactive or fluorescent label. A variety of other detectable labels are known to those skilled in the art.

[0078] A method of remote vitreous fluid analysis is provided. The method comprises obtaining a vitreous fluid sample from a living subject; storing the vitreous fluid sample in a receptacle comprising a reservoir, wherein the reservoir comprises at least one chemical to protect polypeptide integrity; and sending the vitreous fluid sample to a laboratory for analysis. Once received, the vitreous fluid sample can be analyzed. In some embodiments, the analyzing comprises detecting a level of a biomarker in the vitreous fluid, wherein the biomarker is associated with at least one of a susceptibility to an ocular condition, presence or absence of an ocular condition, and an efficacy of treatment of an ocular condition; and identifying at least one of the susceptibility to the ocular condition, the presence or absence of the ocular condition, or the efficacy of treatment of an ocular condition based on the level of the biomarker. An analysis report can be returned to the sender or a third party. In some embodiments, the method comprises selling and/or purchasing a kit of the present invention. [0079]The method of remote vitreous fluid analysis can comprise establishing a contractual relationship between a vitreous fluid analysis organization and an ophthalmology clinic, wherein the clinic performs the obtaining step, and wherein the clinic is contractually obligated to send the sample or data collected therefrom to a laboratory designated by the vitreous fluid analysis organization for analysis. In some embodiments, the contractual relationship establishes a fee for each sample analyzed. The contractual relationship can require the ophthalmology clinic to buy at least one class of articles from the vitreous fluid analysis organization or a supplier designated by the organization. In some embodiments, the class of articles is a vitreous fluid analysis kit comprising a vitreous fluid receptacle comprising a reservoir, wherein the reservoir comprises at least one chemical to protect polypeptide integrity. [0080]The contractual relationship can require the ophthalmology clinic to have an employee or contractor complete an education program directed to vitreous fluid aspiration, wherein the education program is provided by a trainer designated by the vitreous fluid analysis organization, and wherein the employee or contractor performs at least one of the obtaining step and supervising of the obtaining step. [0081]Any appropriate treatment can be used in conjunction with the methods of the present invention. Examples of anti-VEGF therapies in use are: pegaptanib, ranibizumab, and bevacizumab. Ranibizumab (a recombinant, humanized, monoclonal antibody Fab) and bevacizumab (a full-length recombinant, humanized, monoclonal antibody) bind and inhibit all forms of VEGF-A. [0082]Human VEGF exists as at least six isoforms (VEGF 121, VEGF 145, VEGF 183, VEGF 189, and VEGF 206) that arise from alternative splicing from mRNA of a single gene (Ferrara et al., Retina. 26, 859-70, 2006.). VEGF 165, the most abundant isoform, is the target for pegaptanib. Pegaptanib is an aptamer (synthetic short strand of RNA) that selectively inhibits extracellular VEGF 165. The VEGF Inhibition Study in Ocular Vascularization (VISION) studies were two concurrent, randomized clinical controlled trial (RCT) that evaluated the safety and efficacy of intravitreal pegaptanib injections every six weeks in neovascular macular degeneration patients (Chakravarthy et al., Ophthalmology. 113, 1508 el-25, 2006.). In these studies, reduction in the proportion of patients who lost 15 letters or more ranged from 3% for 0.3 mg pegaptanib and 19% for the control group in one RCT, to 12% for 0.3% pegaptanib and 9% for the controls in the other RCT (Takeda, A.L., et al., Pegaptanib and ranibizumab for neovascular age-related macular degeneration: a systematic review, Br J Ophthalmol. 2007.).

[0083]Ranibizumab (a recombinant, humanized, monoclonal antibody Fab) and bevacizumab (a full-length recombinant, humanized, monoclonal antibody) bind and inhibit all forms of VEGF-A. The Minimally Classic/Occult Trial of the Anti-VEGF Antibody Ranibizumab in the Treatment of Necvascular Age-Related Macular Degeneration (MARINA) trial was a 2 year, multi-center, randomized clinical control trial comparing 24 monthly intravitreal ranibizumab injections versus sham injection. At 12 months, 94.6% of those given 0.5 mg ranibizumab lost fewer than 15 letters, as compared with 62.2% of patients receiving sham injections (Rosenfeld et al., N Engl J Med. 355, 141 9-31,2006.). Visual acuity improved by 15 or more letters in 33.8% of the 0.5-mg group, as compared with 5.0% of the sham-injection group. Mean increases in visual acuity was 7.2 letters in the 0.5-mg group, as compared with a decrease of 10.4 letters in the sham-injection group.

[0084]Methods of identifying biomarkers are also provided by the present invention. A method of identifying biomarkers of an ocular condition is provided. A vitreous fluid polypeptide spectrum of a subject having an ocular condition is compared to a vitreous polypeptide spectrum of a subject not having the ocular condition, wherein the spectra are of vitreous fluid aspirated from living patients. A difference is determined in the polypeptide spectra in terms of identity or amount of the at least one polypeptide or a fragment thereof. The presence of the condition is correlated with the difference to identify a biomarker. In some embodiments, the method comprises aspirating vitreous fluid from a living subject having an ocular condition and a living subject not having the ocular condition.

[0085]A method of identifying biomarkers associated with the development of an ocular condition is provided. Vitreous fluid polypeptide spectra of at least one vitreous fluid sample from a subject prior to the onset of the ocular condition and at least one vitreous fluid sample after the onset of the ocular condition. At least one difference in the polypeptide spectra in terms of identity and amount of a polypeptide among the samples is determined. The presence of the condition is correlated with the difference to identify a biomarker. The method can further comprise aspirating the vitreous fluid samples before and after development of the ocular condition. In some embodiments, the vitreous fluid samples are aspirated at periodic intervals before and after development of the ocular condition. The correlating can comprise correlating polypeptide spectra from at least two different stages of an ocular disease, wherein said differences are biomarkers for a given stage of said ocular disease. [0086]The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope. In the forgoing and in the following examples, all temperatures are set forth uncorrected in degrees Celsius (⁰C) and, all parts and percentages are by weight, unless otherwise indicated. While some surgical methods of vitreous fluid sampling are described herein, the methods and examples can be performed in the absence of surgery in the alternative. The examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions of this invention for those used in the preceding examples.

EXAMPLE l

[0087]This example and those that follow demonstrate that biomarkers, such as phosphorylated forms of protein receptors and other signal pathway proteins, are shed into the vitreous of patients with age-related wet macular degeneration and other ocular conditions that can be assayed using the materials and methods of the present invention. These examples demonstrate that these protein markers can be correlated with response to therapy. Further demonstrated is the use of phospho-protein affinity capture technology as a general method to discover and sequence by mass spectrometry (MS) other biomarker classes of phosphorylated proteins in the ocular vitreous fluid.

[0088]This example and those that follow addresses the hypothesis that phosphorylated forms of the activated VEGFR receptors, and phosphorylated forms of related downstream or interconnected signaling proteins associated with neovascularization, vascular permeability, apoptosis, and inflammation, will be shed into the vitreous during angiogenesis and remodeling of the retina in wet macular degeneration. With increased vascular and retinal permeability in wet macular degeneration, there is accompanied shedding of protein receptors and downstream cell signaling proteins into the vitreous. These molecules constitute a recording of the disease state of ocular tissues including the retina and the choroid. This hypothesis is supported by the evidence of intraretinal and subretinal therapeutic response after an intravitreal injection of bevacizumab, a large

(149 kD), full-length, recombinant, humanized, monoclonal antibody. [0089]A signal pathway phospho-protein biomarker analysis is conducted in vitreous samples procured from a representative group of patients with wet AMD and other retinal diseases. The patient characteristics are summarized in Table 1. Vitreous collection is conducted in accordance with the teachings of the present invention. Starting with a volume of 20 μL of vitreous, forty-seven analytes are measured. The analytes span phosphorylated proteins, receptors, and signal pathway nodes involved in angiogenesis (e.g. VEGFR, FGFR, PDGFR), and the upstream or downstream pathways associated with hypoxia, inflammation, prosurvival/apoptosis, and adhesion. The stem cell markers CDl 33 and Mushashi are also included. Unsupervised Bayesian clustering of human vitreous sample data from reverse phase protein microarrays. AMD=wet age related macular degeneration, DR= diabetic retinopathy, RD=retinal detachment, CNV=choroidal neovascularization, ERM=epiretinal membrane, VH=vitreous hemorrhage, MH=macular hole. Analytes in human vitreous fluid evaluated by reverse phase protein microarray for display on the heat map: Caspase 8; mTOR ser2481; Heme oxygenase-1; MMP-9; PDGFRβ Y716; C-KIT Y703; ERK T202N204; Cofilin; STAT3 Y727; VEGFR Y996; JNK T183/185; IRS-I ser612; p90RSK ser380; PDGFRβ Y751; AMPKαl ser485; CC9 Asp 330; AMPKβl ser308; CC3 Aspl75; BAX; CC6 Aspl 62; PAK 1/2 serl99/204; S6 kinase ser240/244; AKT Thr308; C-KIT Y719; GSK3α/β ser21/9; Shipl Y1020; BAD serl 12; AKT ser473; elF4G serl lO8; eNOS serl l77; IGF-IR Yl 131; Src Y527; CD133; VEGFR Y 1175; BCL-2 Thr56; BCL-2 ser70; FADD serl94; GSK3α/β Y279/216; β -Catenin ser32/22; Mushashi; Smac Diablo; CREB serl33; c- ABL Thr735; FAK Y576/577; CC7 Aspl98; Annexin II; FAK Y397. List of the angiogenic associated analytes for display on heat map (unsupervised clustering program) for patients with wet Age Related macular Degeneration: C-KIT Y703: VEGFR Y996; Heme oxygenase- 1; PDGFRβ Y716; FAK Y397; PDGFRβ Y751; VEGFR Yl 175; Shipl Y1020; AKT ser473; C-KIT Y719; GSK3α/β ser21/9; AMPKαl ser485; AMPKβl ser308; β -Catenin ser32/22.

[0090]The specificity of all analyte assays are validated as a single band on western blotting in accordance with methods described in Sheehan et al., MoI Cell Proteomics. 4, 346-55, 2005; Petricoin et al., Cancer Res. 67, 3431-40, 2007. Each array slide contained built-in calibrator dilution curves, positive and negative controls, and each specimen is printed in duplicate in a dilution curve in accordance with methods described in Sheehan et al., MoI Cell Proteomics. 4, 346-55, 2005; Petricoin et al., Cancer Res. 67, 3431-40, 2007; Liotta et al., Cancer Cell. 3, 317-25, 2003. The calibrators and controls included lysates of a) human microvascular endothelial cells, +/-treatment with vanadate, b) HeLa cells +/-pervanadate, c) CHO T cells +/-insulin and d) A431 cells +1- EGF. The phosphatase inhibitor vanadate treatment can maximize the levels of kinase driven protein phosphorylation.

[009I]AIl patients are provided written informed consent under an approved IRB protocol prior to vitreous sampling. Vitreous samples are collected from 24 patients. 15 patients undergo vitreous sampling in the office prior to intravitreal bevacizumab injection. Two patients have vitreous samples taken at the time of intravitreal injection and one month later. A subset of AMD patients are characterized as either non-responders or responders to treatment: Response to therapy is defined as: 1) improvement in visual acuity of 15 letters or more, 2) decrease in intraretinal and/or subretinal fluid on OCT, 3) decreased leakage on fluorescein angiography by 50%, 4) attenuation/closure of retinal angiomatous proliferation (RAP) lesion or feeder vessel, or decreased activity of choroidal neovascularization brush border by high-speed ICG angiography. Two control samples are collected from surgical patients prior to pars plana vitrectomy. All specimens are frozen at -20⁰C or at -80⁰C for storage until subsequent analysis by reverse phase protein microarray. At least 100 μL of vitreous is procured for all patients.

[0092]Of the fifteen patients who underwent office vitreous sampling, 13 have wet age-related macular degeneration and 1 has idiopathic choroidal neovascularization, all of whom required treatment with intravitreal bevacizumab. The eyes are characterized as either responders or non-responders based on their clinical response to therapy. Parameters such as visual acuity, leakage on fluorescein angiography, and intraretinal and/or subretinal fluid on ocular coherence tomography are used to determine response. Four eyes are characterized as non-responders, and five eyes are determined to be responders. Two vitreous samples are collected prior to pars plana vitrectomy. One case has a diagnosis of senile macular hole and one eye has an idiopathic epiretinal membrane. For patients with active neovascular disease, the phosphorylated forms of VEGFR (VEGFR Yl 175) and PDGFRβ (PDGFRβ Y716) are present in the vitreous along with other cell signaling proteins. Among the AMD patients there are two major clusters of signaling activity. This is keeping with the concept that the molecular basis of AMD is heterogeneous with a subset highly sensitive to anti-VEGF therapy while others are not responsive.

[0093JA linkage analysis is conducted to map the signal proteins that appear to be interrelated in the group of patients defined as neovascular disease versus those with non neovascular disease. This procedure is done by conducting a Spearman Rho correlation analysis of paired phosphoproteins. The following pathway protein linkages of phosphorylated proteins emerge as highly significantly different between the neovascular and non-neovascular disease sets (Table 2). A high degree of significance can indicate that the phosphorylation state of the paired proteins change coordinately among the patients in the disease group.

Table 2 Spearman Rho correlation analysis of selected signal pathway proteins.

Table 2A: Additional Spearman's Rho non-parametric correlations of protein linkages in vitreous from patients with neovascular ocular disease compared to patients with non-neovascular ocular disease.

Table 2B: Additional Spearman's Rho non-parametric correlations of protein linkages in vitreous from patients with neovascular ocular disease compared to patients with non-neovascular ocular disease (Continuation of Table 2A).

[0094]Even though the sample size is relatively small, the vitreous samples from wet macular degeneration patients characterized as non- responders have lower levels of VEGFR Yl 175 compared to those judged to be responders. In two eyes, VEGFR Yl 175 is measured from vitreous samples taken just prior to and one month following intravitreal injection of bevacizumab. For example, patient 9, characterized as a responder, shows a decrease in leakage on fluorescein angiography, decreased intraretinal edema on OCT, and closure of feeder vessel. This patient demonstrates a five fold drop in VEGFR-Yl 175 levels before and after treatment with bevacizumab. In contrast, two of the surgical non-angiogenic control eyes (macular hole or epiretinal membrane) exhibit very low levels of VEGFR Yl 175 (less than 10% of the mean levels (n=24)).

Table 3. Vitreous Phosphor-Protein Endpoints Judged as Different among the 50 Test Analytes:

[0095]The following downstream activated (phosphorylated) signal pathway proteins are found to differ between the neovascular and non neovascular disease groups (Table 4).

Table 4. Comparison of vitreous analytes in disease classes (Wilcoxon Text).

[0096]These preliminary studies support the hypothesis of phosphoproteomic vitreous analysis and demonstrate the technical feasibility of the following examples. Vitreous samples can be safely collected before and after anti-VEGF therapy. The yield of sample is four times greater than used (20 μL) for measuring fifty vitreous protein analytes and controls. A large number of signal pathway phosphoproteins can be detected in the vitreous. A high level of active signal pathway activity exists in neovascular versus non-neovascular disease. This includes upstream and downstream activation of known interconnected signal proteins in vascular permeability, apoptosis, prosurvival, and inflammation (Table 4). This activation supports the hypothesis that a high level of active downstream signaling events is reflected in the vitreous proteome in the case of ongoing active angiogenesis. This finding challenges existing paradigms and supports the clinical relevance of this class of markers that appear to reflect ongoing in vivo retinal pathophysiology. The activated signal pathways represented in Tables 2-4 can provide strategies for therapies for use in combination with anti-VEGF therapy, or therapies that may be effective in patients who are non responders. The levels of phosphorylated (e.g. activated) VEGF Receptor changed (along with other selected signal molecules) in a small test set of Responders versus Non-Responders. This supports the rationale for the existence of VEGF treatment-related phosphoprotein vitreous biomarkers.

EXAMPLE 2

[0097]Building on Example 1, this example demonstrates that phosphorylated forms of protein receptors (e.g. VEGFR) and other signal pathway proteins are shed into the vitreous of patients with age-related wet macular degeneration. Vitreous specimen collection is performed from eyes with and without wet AMD. Fresh frozen vitreous samples are collected from 50 subjects with wet age-related macular degeneration before and after 3 to 6 serial ocular injections of anti-VEGF therapy over a period of at least one year. 30 samples will also be collected from non-AMD patients to serve as controls. Using high sensitivity Reverse Phase Protein Microarray (RPA) technology, in 20 microliters of vitreous, 50 validated phosphorylated proteins and signal pathway analytes involved in angiogenesis (e.g. VEGFR, PDGFR), and the upstream or downstream pathways associated with vascular permeability, hypoxia, inflammation, prosurvival/apoptosis, and adhesion are measured. The levels of these analytes in disease versus control samples are evaluated as well as before and after treatment.

[0098]This example further demonstrates the correlation of protein markers identified in the vitreous of wet macular degeneration patients with response to therapy. Vitreous specimen collection is performed before and during the course of anti-VEGF therapy for wet AMD. The clinical response with levels of activated signal proteins before and after therapy will be correlated over 2-6 cycles of therapy in the same patient for 50 patients over a period of one year.

[0099]Two classes of innovative proteomics technology are used to identify and quantitatively analyze phosphorylated proteins, a class of functional biomarkers in the vitreous. These technologies are applied to wet macular degeneration. The study set consists of 50 subjects undergoing anti-VEGF therapy for wet macular degeneration versus 30 controls. Over the course of a year, phosphoproteins and signal pathway protein levels are correlated in vitreous with the therapeutic efficacy in each subject. This experimental design provides the opportunity to test two hypotheses a) vitreous phosphoproteins change as a functional consequence of disease state and anti- angiogenesis therapy, and b) a subset of vitreous phosphoprotein biomarkers correlate with therapeutic outcome.

[OlOOJThe proteomic analysis proceeds in two phases. In the first phase, reverse phase protein microarray technology is applied to measure 50 phosphoprotein and signal pathway analytes in a small volume of 20 microliters of vitreous. The specific 50 analytes, associated with the VEGF Receptor signaling pathway, are selected from a panel of known proteins involved in angiogenesis, inflammation, apoptosis, and motility/adhesion signaling (Table 5). The analysis is conducted in a proteomics lab following CAP/CLIA guidelines so that any findings can be readily translated to larger validation trials.

[010I]A longitudinal series of vitreous samples are collected from subjects with wet macular degeneration with complete clinical follow-up. 50 subjects are followed for one year and collect approximately 50-100 microliters of vitreous fluid before and after anti-VEGF therapy. 30 control samples are collected as described above. The samples are grouped as follows: A) Before therapy, or B) After Therapy, or C) Controls. In Groups A and B, the subjects are further subdivided into Responders and Non Responders. In some embodiments, at least half the subjects are responders. Analysis is done between and among the four groups with the goal of identifying subsets of markers which a) show significant (p<0.05) linkage within a group by Spearman Rho correlation analysis and unsupervised cluster analysis, b) show a significant (p<0.05) mean difference between two groups, and c) show a significant change before and after therapy in the same subject. The subset of analytes identified in this group comparison are then evaluated for significant (p<0.05) correlation with response category. These results are further compared for those obtained from 30 control subjects with macular hole or epiretinal membrane that do not have neovascular retinal disease.

[0102] Statistical Power Calculation: Based on the between run and within run precision of the assay and the experience with the vitreous samples described under the Preliminary Studies section, an n of 50 subjects achieves a greater than 95% power for detecting a 15% change using a Satterthwaite t test with alpha=0.05. [0103] A second class of proteomics technology can be employed for the purpose of a) globally characterizing the vitreous phosphoproteome and b) identification of phosphorylated proteins and peptides in the vitreous that can serve as therapeutic targets. This technology constitutes a general and versatile platform for the eye research community and generates additional candidate biomarkers that can be measured using protein microarray technology. Thus the two-phase technologic approach provides a means to both identify and measure vitreous biomarkers. [0104]After obtaining informed consent, 50 subjects with wet macular degeneration requiring anti-VEGF therapy undergo vitreous sampling prior to a series of 3 monthly intravitreal injections with anti- VEGF therapy. An intravitreal injection of anti- VEGF therapy, either pegaptanib (0.3 mg), ranibizumab (0.5 mg), or bevacizumab (1.25 mg), are administered to all patients at baseline, month 1, and month 2. Following the series of 3 injections, if subsequent re-treatment is required, a repeat vitreous sample is collected prior to intravitreal injection. This procedure yields a longitudinal series of pre- and post-treatment samples for 50 subjects with wet macular degeneration. 30 control vitreous samples are also collected from subjects undergoing vitrectomy surgery for epiretinal membrane or macular hole. Specimens of vitreous are evaluated for 50 phosphoprotein endpoints and total proteome. Based on clinical parameters, response to therapy is characterized. As described in Example 1, response versus non-response is correlated with cell signaling proteins in the vitreous.

[0105]Baseline clinical examination: A history, including duration of symptoms is obtained by the physician. Best corrected visual acuity is determined using the ETDRS visual acuity chart by a certified technician. A full ophthalmic exam including slit-lamp biomicroscopy, intraocular pressure, and extended dilated ophthalmoscopy is performed. The presence or absence of drusen, geographic atrophy, intra- and/or sub- retinal hemorrhage, retinal edema, subretinal fluid, subretinal fibrosis, lipid, epiretinal membrane, retinal angiomatous proliferation, retino-choroidal anastomoses, pigment epithelial detachment, angioid streaks, RPE rip, and polypoidal choroidal neovascularization is documented. Diagnostic imaging is obtained including: Fundus Photography, Fluorescein Angiography, Static and High-speed indocyanine green angiography (ICG), Ocular Coherence Tomography (OCT) and Macular Microperimetry (MP).

[0106]Patients meeting the inclusion criteria and the informed consent are enrolled consecutively. (Table 6: 1. AEs should be recorded from the time the informed consent has been signed until completion/withdrawal. Verify any changes in concomitant medications. 2. Measure IOP pre-dose and 15 minutes post-injection. 3. If re-treat, complete subsequent 10 day+4 day f/u visit.)

Table 6 Inclusion Criteria

Table 6 Inclusion Criteria (Continued)

Table 6 Inclusion Criteria (Continued)

[0107]Vitreous Sampling Wet Macular Degeneration Patients: Anesthesia is given in the form of a topical anesthetic followed by additional anesthetic with a cotton- tipped applicator pre-soaked in lidocaine 4% and applied to the pars plana. A sterile eyelid speculum is placed to expose the conjunctiva out to the region of the pars plana. Betadine 5% is applied to the pars plana and fornix to achieve sterility. A Ice syringe with a 25 gauge needle is used to obtain a small quantity (50 to 100 μL) of vitreous, avoiding any aspiration of any subconjunctival or surface fluid while withdrawing the needle from the eye. At this point, one proceeds with routine clinical management with intravitreal injection of anti-VEGF therapy. All subjects begin a 5-day course of topical antimicrobial eye drops as prophylaxis against infection. [0108]Controls: The subject are prepped and draped in the usual sterile fashion. Prior to the vitrectomy portion of the surgery, a minute quantity of vitreous (100 μL) is obtained with a sterile TB syringe through the pars plana. The surgery then proceeds as per routine. The vitreous sample are stored at -20°C for subsequent analysis.

[0109]Risks of procedure: The most serious adverse event is an iatrogenic infection known as endophthalmitis. The risk of this sight-threatening occurrence is about 0.1 %. Strict adherence to sterile technique and the use of topical antibiotics further minimizes this risk. Other rare complications include hypotony due to wound leakage; however, the risk of this is very minimal and the needle is similar to instruments utilized in sutureless 25-gauge pars plana vitrectomy. Pain from the procedure is typically at the level of mild irritation that is transient and is not be greater than that experienced from the injection of medication alone. [0110]Inclusion Criteria for Wet Age-related Macular Degeneration subjects include the following: Ability to provide informed consent and comply with study assessments for the full duration of the study; Age 50 years or older; Active primary or recurrent neovascularization secondary to age-related macular degeneration with clinical indications for treatment with anti-VEGF therapy; Best corrected visual acuity, using ETDRS charts, of 20/40 to 20/400 (Snellen equivalent) in the study eye. [0111]Exclusion Criteria for Macular Degeneration subjects include the following: Patients with other co-morbid ocular diseases (e.g. diabetic retinopathy, retinal vascular occlusion, inflammatory disease, retinal detachment, significant cataract requiring surgery in the next 24 months); Treatment with intravitreal or systemic anti- VEGF treatment within two (2) months of enrollment; History of intravitreal triamcinolone; History of photodynamic therapy, external beam radiation therapy, or transpupillary thermotherapy in the study eye; Aphakia or absence of the posterior capsule in the study eye; Previous violation of the posterior capsule in the study eye is also excluded unless it occurred as a result of YAG posterior capsulotomy in association with prior posterior chamber intraocular lens implantation; History of any intraocular surgery in the study eye within one (1) month of enrollment; History of vitrectomy surgery in the study eye; Uncontrolled glaucoma in the study eye (defined as intraocular pressure >30 mm Hg despite treatment with anti-glaucoma medication). [0112]Inclusion Criteria for Controls include the following: Age 50 years or older; Undergoing vitrectomy for idiopathic epiretinal membrane or macular hole. Exclusion Criteria for Controls include the following: Any history of age-related macular degeneration; Patients with other co-morbid ocular diseases involving neovascularization (i.e. diabetic retinopathy, retinal vascular occlusion); Treatment with intravitreal or systemic anti-VEGF treatment; History of intravitreal injections; History of any ocular surgery in the study eye within one (1) month of enrollment; History of vitrectomy surgery in the study eye.

[0113] Study Ophthalmologic Assessments: The evaluating physician(s) or other site personnel perform the following study procedures and assessments at the screening and each visit: Ocular Assessments: best corrected visual acuity, slit-lamp biomicroscopy, IOP, dilated extended ophthalmoscopy, fundus photography/ angiography, OCT and/or Macular Microperimetry. Best corrected ETDRS visual acuity by certified technician: VA testing can begin at 4 meters. If subject reads 3 or fewer letters on the first line then will proceed with 1 -meter testing. If the visual acuity is not measurable, will proceed with low vision testing (Counting Fingers, Hand Motion, Light perception). OCT measurements: Quantitative assessment: Central 1 mm retinal thickness and volume. Qualitative assessment can include the following: Presence of fluid in the macula. Identify intraretinal fluid (cysts), subretinal fluid. Fluid-free macula: absence of retinal cysts and subretinal fluid by OCT. Presence of fluid under the RPE, or pigment epithelial detachment (PED) is recorded, if present on the OCT but does not serve as a re-treatment criterion. Review of Case Report Forms (CRPs). Determination whether additional re-injection with anti-VEGF therapy is required based on indications for re-treatment. [0114]Indications for Re-treatment: The decision for repeat treatment with anti- VEGF therapy are based on the following clinical parameters or left to the discretion of the treating physician. Visual Acuity: A loss of at least 5 letters by ETDRS visual acuity with OCT evidence of fluid in the macula. An increase in OCT central retinal thickness of at least 100 microns. Evidence of persistent fluid on OCT at least one month after the previous injection. New macular hemorrhage. Persistent or increased leakage on fluorescein angiography. Evidence of retinal angiomatous proliferation (RAP) lesions, feeder vessel, or increased activity of choroidal neovascularization brush border by high-speed ICG angiography. If a patient is determined to require re- treatment, a repeat vitreous sample is obtained prior to intravitreal injection of anti- VEGF therapy.

[0115] Efficacy outcome measures of anti-VEGF treatment can include primary and secondary outcome measures. Primacy efficacy outcome measure is visual acuity, the proportion of patients who gain at least 15 letters by best corrected ETDRS visual acuity compared to baseline. Secondary efficacy outcome measures include the following: Assessing the impact of anti-VEGF therapy on time to improvement of retinal thickness by OCT. Assessing the impact of anti-VEGF therapy on decreasing area of leakage on fluorescein angiography by 50%. Assessing the impact of Anti- VEGF therapy on closure of retinal angiomatous proliferation (RAP) lesion or feeder vessel, or decreasing the area of choroidal neovascularization active brush border by 50% on high-speed ICG angiography. Assessing the impact of anti-VEGF therapy on development of subretinal fibrosis as determined by clinical examination. Number of intravitreal injections required over one year is also a secondary efficacy outcome measure.

[0116]Data Collection, Management and Storage: Each patient's eye is given a unique identifier. Specimens taken under this protocol are each labeled with a unique identifier at the time a sample is obtained using pre-printed labels with a bar code and/or alphanumeric code. No other markings are placed on the specimen or its container that contain personally identifying information. A physical log sheet and computerized data entry system containing appropriate study identifiers is completed at the time of sample collection. The log sheet or data system contain the following information: unique identifier for patient's eye; unique specimen identifier; date, time volume (μL), and type of specimen collection; relevant qualifying protocol code in which the subject was enrolled; qualifying protocol subject identifier(s); code of person collecting the specimen. The length of follow-up is 1 year [0117]Proteomics Technology: Reverse-phase protein microarrays quantitative analysis of phosphoprotein and signal pathway proteins in vitreous is performed. Protein arrays are used for quantitation of phosphoproteins in vitreous. Reverse Phase Protein Array (RPA) is used to collect quantitative data (Sheehan et al., MoI Cell Proteomics. 4, 346-55, 2005; Petricoin et al., Cancer Res. 67, 3431-40, 2007; Liotta, L.A., et al., Cancer Cell. 3, 317-25, 2003; Paweletz, C.P., et al., Oncogene.20, 1981-9, 2001; Espina et al., Proteomics. 3, 2091 -1 00, :2003). This technology is well suited to measure phosphorylated peptide and protein vitreous biomarkers. The RPA technology involves one antibody per analyte and can utilize the denatured analyte form. RPA protein array technology, applied has the following significant advantages for routine measurement of phosphoproteins in comparison with existing technology a) requirement for only one class of antibodies: a sandwich format is not required; b) high sensitivity at the level of one-tenth of a cellular equivalent 2:SDs above background and less than 5000 molecules; c) high dynamic range: the array contains a built in analyte dilution curve; d) parallel calibrators and controls printed on each array; e) proven applicability for small numbers of cells from tissue biopsies and small volumes (< 10 μL); f) applicable to standard equipment available to any hospital pathology lab; g) <10.0% CVs achieved between run and within run. [0118] An individual test sample is immobilized in a miniature dilution curve such that an array is comprised of hundreds of different patient samples, treatments, or time points. Each array is incubated with one detection protein (e.g. anti-peptide antibody), and a single analyte endpoint is measured and directly compared across multiple samples (Sheehan et al., MoI Cell Proteomics. 4, 346-55, 2005; Petricoin et al., Cancer Res. 67, 3431-40, 2007; Liotta, L.A., et al., Cancer Cell. 3, 317-25, 2003; Paweletz, C.P., et al., Oncogene.20, 1981-9, 2001; Espina et al., Proteomics. 3, 2091 - 1 00, :2003). The RPA format achieves detection levels approaching attogram amounts of a given analyte (Paweletz et al., Oncogene.20, 1981-9, 2001; Rapkiewicz et al., Cancer. 111,173-1 84, 2007.). Third-generation amplification chemistries now available can be used for highly sensitive detection. In summary, the analyte mixture (e.g. vitreous fluid) is immobilized on a solid phase to capture the protein or peptide of interest. The sample is then incubated with a primary antibody, followed by a labeled secondary antibody and Tyramide/HRP enzyme amplification of the signal. [0119]The current antibody repertoire encompasses over 150 validated antibodies recognizing a wide variety of analytes. The teachings of Example 1 can be used to select 50 vitreous proteome analytes for the proposed study (Table 5). The analytes span phosphorylated proteins and signal pathway nodes involved in angiogenesis (e.g. VEGFR, PDGFR), and the upstream or downstream pathways associated with vascular permeability, hypoxia, inflammation, prosurvival/apoptosis, and adhesion. One hundred fifty phosphoprotein antibodies are validated by Western blotting using a heterogenous tissue sample and a series of cell lines as the input. Validation involves a single band at the expected of the antibodies listed in Table 5. Cell lysates prepared from cell lines, whole tissue sections, or microdissected tissue are used as positive controls. Cells are lysed with protein extraction buffer to generate 2.0 μg/μL of protein. Reference phosphopeptides containing the antigen recognition epitope are used for specificity and sensitivity testing.

[0120] Calibration. The optimal dilution for each antibody is determined in an array format that includes positive and negative controls as well as reference antigen. Positive controls are cultured cell lines expressing the cognate phospho-antigen. Reference antigens are phosphopeptides with a purity greater than 95%. Acceptable calibration is a sensitivity level equal to 10 cellular equivalents for the antigen of interest and/or a sensitivity of at least 5000 molecules 2 SD above background. Background and negative control are determined by developing the array signal in the absence of a primary antigen. All values are normalized to total protein. All analysis is done in duplicate dilution curves at 5 levels of concentration. Image analysis and data collection are done using MicroVigene software (VigeneTech). [0121]Selection of endpoints. From an initial 100 validated antibodies, 50 endpoints are chosen to span a wide range of cellular compartments and pathways (Table 5). These endpoints represent angiogenic, stress, growth, proliferation, migration, adhesion, and stem cell pathways. [0122JA RPA calibration dose response curve can be generated comparing within run and between run precision for VEGF Receptor Phosphorylation Site Y95I. Highly satisfactory between run precision is observed for the dose range of analyte discovered to be present in human vitreous. The calibrator source is pervanadate- treated human microvascular endothelial cells that express IxIO⁵ VEGFW-I. The sensitivity is 5000 molecules 2SDs above background. This is equivalent to a fraction the receptor content of a single endothelial cell. The range of receptor molecules (or fragments) detectable in the vitreous is equivalent to that shed by muck less than 100 endothelial cells.

[0123]Microarray analyte immobilization: Vitreous is diluted in protein extraction buffer and denatured by heating for 8 minutes at 100⁰C prior to dilution in the microtiter plate. Serial two-fold dilutions of the lysates are printed in duplicate on glass backed nitrocellulose array slides (FAST Slides, Whatman) using an Aushon 2470 arrayer equipped with 350μm pins. Each spot is printed with approximately 30.OnL of lysate/spot. The slides are either stored with desiccant at -20⁰C or immediately processed for immunostaining.

[0124]Protein Microarray Immunostaining: The microarray slides are pretreated with ReblotTM mild solution rinsed twice, and blocked with I-block blocking solution. After blocking, the slides are placed on an automated slide stainer (Autostainer Dako) and blocked with 3% hydrogen peroxide, avidin, biotin and protein block per manufacturer's instructions (CSA kit Dako). Each slide is incubated with a single primary antibody. Subsequent protein detection is via horseradish peroxidase mediated biotinyl tyramide amplification reaction (CSA kit). Air dried slides are scanned on an Epson flatbed scanner at 1200 dpi (Adobe Photoshop software). [0125]Quality control and array design: Incorporation of a lysate of known performance characteristics enhances the quality control aspects and potential clinical utility of each array. The control lysate typically consists of a pool of cell culture protein lysates with known cognate antigens. The use of multiple sample dilutions ensures that the protein of interest is within the dynamic range of the assay, based on the antibody sensitivity and affinity.

[0126]Bioinformatic method for microarray analysis: Spot intensity is integrated over a fixed area (MicroVigene software). This area is fixed for each spot, minimizing intensity variation in the interrogation area. Each array is scanned, spot intensity analyzed, data normalized, and a standardized, single data value is generated for each sample on the array (Liotta et al., Cancer Cell. 3, 317-25, 2003; Carlisle et al., Carcinog. 28, 12-22, 2000.). This single data point is used for comparison to every other spot on the array using Ward's method two-way hierarchical clustering analysis (JMP software ver 50)

Key for Table 5:

A=Apoptosis

B=Trascription/Cytokine

Production/Apoptosis

C=Cycle Cycle Control

D=Migration/Adhesion

E=Transcription/proliferation/Differentiation

F=Growth/Differentiation

G=Pro-Survival/Proliferation/Apoptosis

H=Growth/Translation/Protein Synthesis

I=Angiogenesis/Nitric Oxide synthesis

J=Motility/Adhesion/Cytoskeletal structure

M=Membrane trafficking

N=Oxidative stress

O=stem cell marker

P=Tissue remodeling/angiogenesis

Table 5A Selected Analytically Validated Vitreous Phosphoprotein Analytes.

Antibody/Location Code Function Nucleus

Annexin OO M Member Trafficking

Catenin(beta) D Migration/Adhesion

(Ser33/37/Thr4

CREB (S 122) E Transcription

Factor/Proliferation/Differentiation

Cytoplasm

Akt (S473) H Survival/Proliferation/Glycogen

Metabolism

Akt (T308) H Survival/Proliferation/Glycogen

Metabolism

AMPKaI (S485) H Cell Growth/Protein Synthesis

AMPKbI (S308) H Cell Growth Protein Synthesis c-Abl (T735) G Cell

Proliferation/differentiation/apoptosis

Bad (S 112) G Apoptosis/Survival

Caspase-3, cleaved A Apoptosis

(D 175)

Caspase-6, cleaved A Apoptosis

(D 162)

Caspase-7, Cleaved A Apoptosis

(D 198)

Caspase 8 A Apoptosis

Caspase-9, cleaved A Apoptosis

(D330)

Cofilin J Cell-Motility/Cell

Adhesion/Phagocytosis

Cu/Zn N Oxidative stress

SuperOxideDismutase eIF4G (S1108) H Translational Control eNOS (S1177) I Nitric oxide synthesis/angiogenesis

ERK 1/2 (T202/Y204) F Growth and Differentiation

FADD (S 194) A Apoptosis

FAK (Y576/577) J Cytoskeletal Signaling

GSK-3alpha (Y279)/beta H Cell Survival/glycogen synthesis

(Y21

GSK-3alpha/beta (S21/9) H Cell Survival/glycogen synthesis

Table 5B Selected Analytically Validated Vitreous Phosphoprotein Analytes

Antibody/Location Cytoplasm Code Function

Heme oxygenase- 1 N Oxidative damage

MMP-9 J Cell Motility/Cell

Adhesion/Phagocytosis

MMP-9 P Tissue remodeling/angiogenesis

Mtor (s2481) H Cell Growth/Protein

Synthesis

Mushashi O Stem cell marker

P90RSK (S380) F Growth/Differentiation/Cell

Cycle Contri

PAKl (S199/204)/PAK2 J Cytoskeletal Reorganization

(S 192/197)

S6 kinase (S240/244) H Cell Growth/Protein

Synthesis

SAPK/JNK (T183/Y185) A Apoptosis regulation/Transcription

Ship 1 (Y 1020) G Apoptosis/Survival

Src (Y527) F Growth/Differentiation

STAT3 (Ser727) B Transcription

Membrane c-Kit (Y703) F Hematopoiesis, gametogenesis c-Kit (Y719 F Hematopoiesis, gametogenesis

E-Cadherin D Migration/Adhesion

EGFR (Yl 148) F Growth/Differentiation

Fibroblast Growth Factor F Growth/Differentiation

IGF-I Rec (Yl l 31)/Insulin H Glucose/Energy Metabolism rec (Y1148)

IRS-I (S612) H Glucose/Energy Metabolism

PDGF Receptor beta I Angiogenesis

(Y718)

PDGF Receptor beta I Angiogenesis

(Y751)

VEGF Receptor- 1 I Angiogenesis

VEGF Receptor-2 (Yl 175) I Angiogenesis

VEGFR 2 (Y996) I Angiogenesis

Mitochondria

Bax A Apoptosis

Bcl-2 (S70) G Pro-survival

Bcl-2 (T56) G Pro-survival [0127]The number of patients seen by the NRI group is greater than 22,000 per year. The expected number of eligible patients is three times greater than the projected number of 50. If accrual is slower than expected, the enrollment is into the second year. All vitreous are frozen immediately at collection. In parallel, full stability studies are conducted and the addition of phosphatase and proteinase inhibitors in the collection vial is investigated. Assay volume requirement is less than 20 μL. Greater than 50 μL can be collected. Samples are evaluated as adequate or inadequate for analysis; prior to experimental processing, to achieve only adequate samples in the test series. All new batches of antibodies for all analytes are full validated by Western Blotting. All relevant statistically different analytes are re-validated by western blotting of vitreous.

EXAMPLE 3

[0128]This example demonstrates the phospho-protein affinity capture technology as a general method to identify and sequence by mass spectrometry (MS) phosphorylated proteins with utility as biomarkers. Three methods for affinity capture of general phosphorylated residues in ocular vitreous fluid are compared. Anti phospho-tyrosine residue antibodies are used as an affinity ligand. Additional phosphorylated residues of selected signal pathway proteins VEGFR-I and VEGFR-2 and PDGFR involved in angiogenesis, and vascular maturation in a pilot series of vitreous samples collected in patients with wet macular degeneration and controls are identified. Immunoassay or multiple reaction ion monitoring (MRM) are employed to validate the existence of candidate phosphoproteins predicted to exist by the phosphopeptide capture and MS sequencing. Final validation are done employing the protein array technology described in Example 2. These steps confirm the feasibility of using vitreous phosphoprotein capture technology as a general discovery tool for the field of eye proteomics.

[0129]Mass Spectrometry phosphoprotein affinity enrichment discovery technology: The core mass spectrometry platform is an LCIMS system consisting of an Agilent 1100 HF'LC and a Thermo Linear Trap Quadrupole (LTQ) mass spectrometer, which represents state-of-the-art ion trap technology providing unique performance capabilities including high sensitivity, rapid data acquisition rate, and efficient multiple MSn experiments. An electron transfer dissociation (ETD) device that fragmentation and subsequent sequence identification of large protein fragments and post-translational modifications (phosphorylation) can be added to the instrument. In addition to the LTQ/ETD system, a Thermo LTQ-Orbitrap hybrid mass spectrometer that provides unsurpassed high mass resolution and mass accuracy measurements can be utilized. The MS-MS data are searched against public protein databases using multiple computer algorithms (e.g., SEQUEST, Mascot), and the search results are compiled, visualized and filtered using the Scaffold program (Proteome Software). [0130]Global and targeted phosphoproteomics can be employed. For global phosphoproteomics, modifications can be used to increase the analytical specificity (detect more phosphopeptides relative to fewer non- phosphorylated peptides) and sensitivity (detect more and lower level phosphopeptides).

[0131]First, the enzymatically-generated peptides can be derivatized to convert the free carboxylic acid groups (aspartic acid, glutamic acid and the peptide c-terminus) to methyl esters, and thereby decrease non-specific binding to the IMAC column (Ficarro et al., Nat Biotechnol. 20, 301-5, 2002; Ndassa, Y.M., et al., J Proteome Res. 5, 2789-99,2006.). This method allows differential labeling whereby peptides from one sample type (control or healthy) are derivatized using an undeuterated methanol reagent (-OCH3 or do methyl esters) and the other sample type (disease, AMD) are derivatized using deuterated methanol (-OCD3 or d3 methyl esters). The d0 and d3 derivatized samples are mixed and then subjected to IMAC-based enrichment. The mixed dθ/d3 (control/disease) samples are loaded onto an IMAC column fabricated using fused silica and POROS 20MC (Applied Biosystems) beads. The IMAC column preparation, sample loading and sample elution are described in Ndassa, Y.M., et al., J Proteome Res. 5, 2789-99,2006. The IMAC column is prepared by rinsing with EDTA, water, iron chloride (which charges the beads with Fe3+) and finally acetic acid solutions. Then the sample (dθ/d3 derivatized peptides) is loaded onto the IMAC column followed by a wash step using a mixture of organic and aqueous solvents to remove non-phosphorylated peptides. The enriched phosphopeptides are eluted from the IMAC column with a sodium phosphate solution directly onto a homemade Cl 8 fused-silica column, which then are used in an LCIMS-MS analysis as described herein. [0132]The second method modification is to substitute titanium dioxide (TiO2) for the Fe3+/IMAC in the phosphopeptide enrichment step. Commercial material (e.g., Titansphere, GL Sciences) and published methods (Thingholm et al., Nat Protoc. 1, 1929-35, 2006; Larsen et al., MoI Cell Proteomics. 4, 873-86, 2005.) that have been demonstrated to yield high-quality results are employed. Peptide samples are loaded onto a homemade or commercial TiO2 column in an acidic aqueous solvent, the TiO2 beads washed to remove non-phosphorylated peptides, the phosphopeptides eluted with a strongly basic aqueous solution, and then analyzed by LC/MS-MS. Addition of dihydroxybenzoic acid to the sample loading buffer significantly reduces nonspecific binding. This method can provide several advantages, including (l)an enrichment that is simpler (fewer steps), faster and more tolerant of contaminants relative to IMAC, (2) a process that can be made higher throughput (plate format) and possibly automated, and (3) identification of a different set of phosphopeptides compared with the IMAC method see Bodenmiller et al., Nat Methods. 4, 231-7, 2007.

[0133]The third method modification is to use electron transfer dissociation (ETD) to produce phosphopeptide fragmentation in the MS-MS analysis. An ETD device is added to the LTQ mass spectrometer that allows easier interpretable sequence data from peptides with post-translational modifications, such as phosphorylation. (Chi et al., Proc Natl Acad Sci U S A. 104, 2193-8, 2007; Syka et al., Proc Batl Acad Sci U S A. 101, 9528-33, 2004). Energy is transferred to mass-selected peptide ions via capture of an electron, which yields fragmentation at different bonds along the peptide backbone (vs. collisions with helium atoms in CID); and does not cause the loss of the phosphate group, which is a facile fragmentation in CID and yields spectra that are more difficult to interpret. Vitreous protein samples prepared for ETD analysis are digested with endoproteinase Lys-C, which cleaves proteins only at the c-terminus of lysine. This digestion yields fewer and larger protein fragments, compared to trypsin, which cleaves at Lys, Arg and sometimes other residues, which connects to the second strength of ETD that is the analysis of higher charge state (>=3+), therefore larger, peptides (Coon et al., J Am Soc Mass Spectrom. 16, 880-2, 2005.). The result is a greater likelihood of generating and identifying more phosphorylation sites, which might be missed on smaller tryptic fragments that are not detected. [0134] Targeted phosphoproteomics: For a second phosphoprotein enrichment strategy, phosphotyrosine (pTyr or pY)-containing peptides are targeted for analysis by selective enrichment. A method based on published procedures (Zhang et al., MoI Cell Proteomics. 4, 1240-50, 2005; Zhang, Y., et al., Methods MoI Biol. 359, 203-1 2, 2007) in which a combination of pY-specific antibodies is used to immunoprecipitate (IP) pY-peptides from vitreous protein digests. Lys-C is used to generate larger peptides for IP and analysis. A subsequent IMAC or TiO2 enrichment step can be used depending orf the amount non-phosphorylated peptides (non-specific binding) obtained from the IP. Phosphopeptides enriched by pY-IP are analyzed by LC/MS- MS using sequential CID and ETD fragmentation on the LTQ instrument. The advantages of CID for lower charge state peptides (<=+3) and the ETD for higher charge ions can be realized in each analysis, which can yield greater sequence coverage of vitreous proteins and a greater likelihood of identifying phosphorylation sites.

[0135JMS data analysis: Phosphorylated peptides are identified by searching the MS- MS (CID and ETD) spectra against public human protein databases using one or more search algorithms (SEQUEST, Mascot, OMSSA

(http://pubchem.ncbi.nlm.nih.gov/omssa/). The peptide and protein sequence-to-data matches obtained from the searches are analyzed to extract high-confidence assignments using filters based on several match scoring parameters (as described above) and manual inspection of the spectra, facilitated by the Scaffold software program. Comparative analysis of phosphopeptide abundances in control vs. diseased samples, or treated versus untreated are accomplished by one or more methods. [0136]First, test analyses using the dθ/d3 method described above demonstrate the viability of this approach. Chemical derivatization steps such as this are not ideal because of losses and non-completeness, and this strategy may not be appropriate for the pY approach because of expected low levels of pY-proteins. Therefore, peptide ion signal intensities can be used as a first-step, qualitative measure of relative abundances of peptides across multiple analyses and samples. This approach is augmented by spiking commercially-available phosphorylated proteins (e.g., alpha- and beta-caseins) and peptides (e.g., angiotensin II phosphate) into all vitreous samples to provide internal standards that serve as the bases for comparing vitreous phosphopeptide intensities in different samples and analyses (i.e., control/healthy v. AMD). A differential phosphopeptide abundance ratio (control v. AMD) of at least 2- 3 and measured in replicate analyses of multiple biological samples and preparations is used for classification as statistically-significant differentially abundant proteins, and therefore candidate biomarkers. The abundances of the majority of phosphopeptides/proteins do not differ from control v. AMD samples (1:1 ratio), and these serve as the basis for determination of statistically significant differential ratios. [0137]Clinical vitreous sample analysis to determine candidate biomarkers: The analyses, started in year one, of vitreous samples collected from controls and AMD subjects are continued. Within the first 3-4 months of the second year at least 10 control: AMD sample pairs are analyzed to demonstrate and evaluate the two phosphoproteomic strategies. Based on number of phosphopeptides identified and of candidate differentially abundant phosphopeptides one of the two methods may be evaluated to be superior. For example, considering the above metrics and the facts that pY peptides could be enriched more easily and selectively (pY-mAbs) and are directly relevant to the disease drug target (VEGFR tyrosine kinase) (versus what likely can be primarily pSer and pThr peptides identified in the global approach), the targeted strategy can be chosen for application to a larger number of clinical samples throughout this second year to generate a larger set of data that can yield greater statistical confidence in the results.

[0138]Validation of candidate phosphoprotein biomarkers: The first validation analyses are Western blot assays of vitreous samples using commercially available antibodies specific for the candidate phosphoproteins. These assays are performed with samples from controls and AMD subjects that are analyzed in the discovery phase and with a different set of samples to provide further validation of differential abundances.

[0139]An alternative validation strategy based on triple quadrupole mass spectrometry (TQMS) technology can be employed. TQMS instruments and methods have been used successfully for environmental and pharmaceutical analyses that involve quantitative analyses of low-level analytes in complex mixtures. The technique that allows this analysis is called multiple reaction monitoring (MRM) and it consists of 1) detection and selection of molecular ions with the first quadrupole, 2) fragmentation of these ions in the second quadrupole, and 3) detection of a small number of known fragment ions in the third quadrupole. The analysis yields an analyte molecular weight, relative abundances of fragment ions that are characteristic of the analyte structure and a chromatographic elution time (LC/MS). Modern TQMS instruments provide advanced MRM performance with higher resolution and accuracy mass measurement, fast electronics for switching between a large number of selected analyte and fragmentation masses monitored, and ease of use. Inherent advantages of LC/TQMS include high detection sensitivity, large dynamic range of detection response, and the ability to incorporate stable isotope labeled synthetic analogs of the targeted analytes, which allows unmatched quantitative analytical performance. Examples of applicable MRM methods, capabilities and applications include those described in (Wolf-Yadlin et al., Proc Natl Acad Sci U S A. 104, 5860-5, 2007; Unwin, et al., MoI Cell Proteomic: 4, 1134-44, 2005; Anderson et al., Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins, MoI Cell Proteomics. 5, 573-88, 2006).

[014O]A MRM-based method for high-throughput, multiplexed and quantitative analyses of candidate phosphopeptides from vitreous samples can be employed, for example, using a thermo Quantum triple quadrupole mass spectrometer. HPLC materials and methods are incorporated onto this platform. Phosphopeptide elution times and precursor and fragment ion masses can be used to facilitate MRM data acquisition methods. These studies can be augmented with spiked internal standards, as in the discovery phase, and with isotopically-labeled synthetic analogs of phosphopeptide biomarkers. An autosampler and other methods can be added to increase the throughput and automated as possible of the platform, e.g., plate-based sample peptide enrichment and cleanup prior to LC/MS). The MRM methodology can be validated by comparison of MRM analyses of known biomarker peptides from physiological fluids with results from approved clinical assays for these analytes (e.g., gastrin in serum by immunoassay).

[014I]If the yield of isolated phosphopeptide candidates is low, the following can be performed: a) directly verify the presence of phosphoproteins in the sample by 4G10 anti-phospho tyrosine western blotting, b) sequentially conduct the three phosphoprotein capture strategies on the same sample to increase the yield, and c) increase the starting sample volume by pooling cases or selecting patient samples with an initial greater volume.

[0142]The typical vitreous sample amount collected is lOOμL, and protein assay measurements yielded a typical total protein concentration of 0.5μg/μL, which is approximately 100-fold lower than a serum protein concentration. Samples of vitreous proteins are concentrated to a small volume, reduced and alkylated (to remove cysteine disulfide bonds), reconstituted in an enzyme digestion buffer (ammonium bicarbonate), digested with trypsin overnight at 37°C, and finally desalted to yield 50-100μg total digested vitreous protein (peptide fragments) ready for online liquid chromatography/tandem mass spectrometry (LC/MS-MS) analysis. [0143]Aliquots of l-10μg of digested vitreous protein samples are loaded onto a reverse-phase LC column that is prepared by packing 5μm, 200A Cl 8 resin into a 10- cm piece of 360-μm O.d. x 100-μm i.d. fused silica, which has an integrated laser- pulled tip. The peptides are eluted with a linear binary solvent gradient of 100% solvent A (0.1 % formic acid) to 100% solvent B (0.1% formic acid, 80% acetonitrile) in approximately 1 hour with a flow rate of 200 μL/min. The LC column eluate is electrosprayed directly from the column tip into a Thermo LTQ linear two- dimensional quadrupole ion trap mass spectrometer for peptide molecular weight and sequence analysis. The LTQ is operated in a data-dependent mode, in which each full mass spectral (MS) scan (peptide molecular ions in the range of m/z 400-2000) is followed by five tandem MS scans (MS-MS) in which each of the top five most abundant ions in the MS scan are sequentially selected, fragmented by collision- induced dissociation (CID) with helium atoms in the trap, and the fragment ions detected. The data acquisition cycle of 1 MS and 5 MS-MS scans is repeated (1 cycle/3-4 seconds) over the course of the LC gradient (ca. 40-60 minutes of peptide elution), yielding 3,000 to 5,000 MS-MS spectra per sample analysis. [0144]The MS-MS spectra is searched against a public human protein database (NCBI) using the SEQUEST search algorithm to obtain matches of the experimentally measured peptide molecular and fragment ion masses with those of known peptide/protein sequences. High confidence identifications are obtained by filtering the search results based on several match scores, including the rank of the match (RSp=I), the cross-correlation score as function of the peptide molecular ion charge state (XCorr > 1.9 (1+), 2.2 (2+) and 3.5 (3+)), the difference between the first and second ranked match (Cn>0.1) and the probability of a random match (p<0.01). In addition, the Scaffold software program (Proteome Software Inc.) is used to facilitate visualization, filtering, sorting, and manual confirmation of the identifications. An assessment of the relative amount of a protein in different samples is obtained by counting the number of MS-MS spectra matched to peptides corresponding to that protein. This is based on a greater amount of a protein yielding larger abundances of enzymatically-generated peptides that result in a larger number of MS-MS spectra obtained for these peptides in the analysis.

[0145]Mass spectrometry-phosphoproteomics: A global proteomic analysis is performed to identify as many vitreous phosphoproteins as possible with minimal bias. To accomplish this, IMAC (homemade (Ndassa 2006) and commercial (Qiagen) materials and methods) is used to enrich for phoshopeptides generated by a trypsin digest of vitreous proteins. The phosphopeptides bound to the IMAC beads/column (by interaction of the peptide phosphate moiety with Fe3+) are eluted with a sodium phosphate buffer solution and then loaded onto a C18 LC column for LCIMS-MS analysis as described herein. These analyses yield identification of approximately 15 phosphorylated peptides, which correspond to approximately 10 proteins (a selection is presented in Table 6). Included in these are specific phosphorylation sites identified in secreted phosphoprotein isoforms a and b, chromogranin B, kininogen 1 and, of particular note, two proteins associated specifically with the eye -opticin and interphotoreceptor retinoid-binding protein precursor. With the protein microarray data, these findings demonstrate that phosphorylated proteins can be affinity captured in the vitreous.

Table 6 Selected identified Vitreous Phosphorylated Peptides (Many of these peptides can be detected in multiple analyses of several vitreous protein tryptic digest samples using TiO2 and IMAC methods.)

EXAMPLE 4 [0146]This example demonstrates that measured levels of phosphorylated VEGF receptor in eyes with either acute wet AMD, chronic wet AMD, or a macular hole and/or epiretinal membrane levels can be correlated to disease status. [0147]Growth factors such as VEGF interact with their target cells, most likely vascular endothelial cells in the case of wet AMD, by activating cellular receptors. Receptors are activated when they become phosphorylated. The phosphorylation status of VEGF receptor can be detected and measured using specific antiphosphoprotein antibodies. Applying these antibodies to reverse phase protein microarrays provides a technique to measure levels of phosphorylated VEGF receptor in small volumes. Cells of various tissues, including tumors, have been shown to release phosphorylated receptor into surrounding fluid and the release of these phosphorylated receptors is used to measure activity of the associated cellular pathways. As the vitreous is constantly bathing the retina and its supporting tissues, it can accumulate shed receptors that could serve as indicators of cellular activity in diseases such as AMD.

[0148]Levels of phosphorylated VEGF receptor in eyes with either acute wet AMD, chronic wet AMD, or a macular hole and/or epiretinal membrane are measured and these levels are correlated to disease status. In two of the eyes, VEGF receptor is measured just prior to and one month following intravitreal injection of bevacizumab. [0149] Patients are provided written informed consent under an approved IRB protocol prior to vitreous sampling. Vitreous samples are collected from 14 patients. Eleven patients undergo vitreous sampling in the office prior to intravitreal bevacizumab injection. Two patients have vitreous samples taken at the time of intravitreal injection and one month later. Patients are characterized as either non-responders or responders to treatment. Response to therapy is defined as: 1) improvement in visual acuity of 15 letters or more 2) decrease in intraretinal and/or subretinal fluid on OCT 3) decreased leakage on fluorescein angiography by 50 % 4) attenution/closure of retinal angiomatous proliferation (RAP) lesion or feeder vessel, or decreased activity of choroidal neovascularization brush border by high-speed ICG angiography. Three samples are collected from surgical patients prior to pars plana vitrectomy. All specimens are frozen at -20⁰C or at -80⁰C for storage until subsequent analysis by reverse phase protein microarrays. [015O]Of the eleven patients who underwent office vitreous sampling, 10 have wet age-related macular degeneration and 1 has idiopathic choroidal neovascularization, all of whom required treatment with intravitreal bevacizumab. The eyes are characterized as either responders or non-responders based on their clinical response to therapy. Parameters such as visual acuity, leakage on fluorescein angiography, and intraretinal and/or subretinal fluid on ocular coherence tomography are used to determine response. Six eyes are characterized as nonresponders, and five eyes are determined to be responders. Three vitreous samples are collected prior to pars plana vitrectomy. Two samples have a senile macular hole and one eye has an idiopathic epiretinal membrane.

[0151]The phosphorylated form of vascular endothelial growth factor receptor (VEGFR-Yl 175) is present in the vitreous along with other cell signaling proteins. There is a difference in VEGFR-Yl 175 between the samples (Table 1). The vitreous samples from patients characterized as non-responders has lower levels of VEGFR- Yl 175 and the responder vitreous samples has higher levels of VEGFR. In two eyes (patients 2 and 9), VEGFR-Yl 175 is measured from vitreous samples taken just prior to and one month following intravitreal injection of bevacizumab. Patient 9, characterized as a responder, shows a decrease in leakage on fluorescein angiography, decreased intraretinal edema on OCT, and closure of feeder vessel. This patient has a significant decrease in VEGFR-Yl 175 levels before and after treatment with bevacizumab. Patient 2, characterized as a non-responder, showed minimal decrease in leakage on FA, retinal thickness on OCT, and no improvement in vision. This patient has a low level of VEGFR-Y 1175 prior to treatment. There is a decrease in VEGFR one month later. The three non-angiogenic, control eyes reveal minimal VEGFR-Yl 175.

Table 7. Level of VEGFR-Yl 175

[0152]In this study, significant differences in levels of VEGFR-Yl 175 between AMD patients are shown, which can correlate with the disease state and response to therapy with bevacizumab. The control samples have minimal VEGFR-Yl 175, which correlates to the fact that the underlying disease states are not angiogenic. Measurement of specific phosphorylation patterns of targets such as VEGFR in the vitreous can become the basis for prognosis and timing of therapy for AMD patients as well as identify other potential therapeutic targets.

EXAMPLE 5

[0153]This example demonstrates that the level of phosphorylated vascular endothelial growth factor receptor (VEGFR-Yl 175) can be correlated with disease activity and can predict response to bevacizumab.

[0154]Phosphorylated receptors of several growth factors can be detected and quantified in the vitreous of wet age-related macular degeneration patients.

Phosphorylation indicates activity of the receptor ,and resultant cell signaling. A study is performed to demonstrate the prediction of response to anti-VEGF therapy based on levels of phosphorylated VEGF receptor in the vitreous of wet AMD patients.

[0155]A prospective, consecutive case series of 8 patients with wet age-related macular degeneration (AMD) undergoing treatment with intravitreal bevacizumab and

3 control surgical patients (2 macular hole, 1 epiretinal membrane) is performed. Patients either undergo vitreous sampling prior to intravitreal injection or prior to vitrectomy. The vitreous is analyzed by reverse-phase protein microarrays for phosphoproteins involved in the angiogenesis cell signaling pathway. [0156]Vitreous from control eyes show minimal to no VEGFR-Yl 175. Of the AMD patients, four patients show similar low levels of VEGFR-Yl 175. Clinically these patients have chronic low grade activity and showed no significant response to treatment with intravitreal bevacizumab. The four patients with acute active disease have 3.5 fold higher levels of VEGFR-Yl 175 compared to controls. These patients have a good clinical response to treatment with bevacizumab with marked decrease in cystoid macular edema (CME) on ocular coherence tomography (OCT) and leakage on fluorescein angiography (FA). Two patients have vitreous samples taken immediately prior to intravitreal injection and then one month later. One patient has a good clinical response with a decrease in leakage on FA and cystoid macular edema on OCT. The pre-treatment sample has high levels of VEGFR-Yl 175. The repeat sample one month later shows a 1.5 fold decrease in the amount of VEGFR-Y 1175 levels, down to the level of controls. The patient with no improvement by visual acuity, fluorescein leakage, or retinal thickness on OCT has VEGFR-Yl 175 similar to controls in both vitreous samples.

[0157]In this study, intravitreal levels of phosphorylated VEGFR-Yl 175 in AMD patients are shown to correlate to the disease state and response to therapy with bevacizumab. These findings demonstrate the utility of a diagnostic tool that allows the clinician to determine the need for anti-VEGF treatment prior to the development of macular edema and vision loss. The availability of such a diagnostic tool can result in improved visual outcomes and reduced costs by allowing optimization of the dosage regime using parameters that are predictive rather than the existing parameters that are reactive to progression of pathology.

* * ♦

[0158JA11 references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. [0159]The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non- claimed element as essential to the practice of the invention.

[0160] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

ClaimsWhat is claimed is:

1. A non-surgical method of predicting or monitoring the physiological state of the eye, comprising: aspirating a sample of vitreous fluid from the eye of a living subject, wherein the aspirating is not concurrent to eye surgery; detecting a level of a biomarker in the vitreous fluid, wherein the biomarker is associated with at least one of a susceptibility to an ocular condition, presence or absence of an ocular condition, and an efficacy of treatment of an ocular condition; and identifying at least one of the susceptibility to the ocular condition, the presence or absence of the ocular condition, or the efficacy of treatment of an ocular condition based on the level of the biomarker.

2. The method of claim 1, wherein the biomarker is a polypeptide or a unique fragment thereof.

3. The method of claim 2, wherein the polypeptide is a phosphorylated polypeptide or a unique fragment thereof.

4. The method of claim 3, wherein the phosphorylated polypeptide is a receptor polypeptide or a unique fragment thereof.

5. The method of claim 4, wherein the phosphorylated receptor is at least one of a G-protein coupled receptor and a hormone activated receptor, or a unique fragment thereof.

6. The method of claim 4 or 5, wherein the phosphorylated receptor is VEGFR-2 or PDGFR-beta or a unique fragment thereof.

7. The method of any one of claims 1-6, wherein the level comprises the presence or absence of the biomarker associated with at least one of the susceptibility to the ocular condition, the presence or absence of the ocular condition, or the efficacy of treatment of an ocular condition.

8. The method of any one claims 1-7, wherein the ocular condition is selected from the group consisting of diabetic retinopathy, age-related macular degeneration, branch vein occlusion, central vein occlusion, retinal vein occlusion, ocular histoplasmosis, choroidal neovascularization, retinal neovascularization, retinal edema, a retinopathy, a choroidopathy, retinitis, choroiditis, retinal vasculitis, choroidal vasculitis, a retinal vascular abnormality, a choroidal vascular abnormality, retinal vascular leakage, choroidal leakage, an ocular dystrophy, myopic degeneration, pre-cataract, cataract, glaucoma, an ocular infectious disease, an ocular tumor, an ocular malignancy, an ocular degenerative disorder, an ocular hereditary disorder, and any combination thereof.

9. The method of any one of claims 1-8, wherein the detecting and identifying is of the presence or absence of the ocular condition so as to diagnose an ocular condition.

10. The method of any one of claims 1-8, wherein the detecting and identifying is of the susceptibility to the ocular condition, the presence or absence of the ocular condition so as to determine the risk of an ocular condition.

11. The method of claim 1, wherein the detecting and identifying is of the efficacy of treatment of an ocular condition so as to monitor the effect of a drug.

12. The method of claim 11, wherein the subject has been administered a treatment for an ocular condition.

13. The method of claim 12, wherein the treatment comprises administration of a tyrosine kinase inhibitor.

14. The method of claim 13, wherein the tyrosine kinase inhibitor inhibits the phosphorylation of a tyrosine-kinase receptor or enzyme.

15. The method of any one of claims 1-14, wherein the detecting comprises at least one of a protein microarray, an immunoassay, a ligand binding assay, electrophoresis, and mass spectroscopy of the vitreous fluid sample.

16. The method of any one of claims 1-15, wherein the detecting comprises a proteomic fingerprint comprising a vitreous fluid polypeptide or unique fragment thereof.

17. The method of any one of claims 1-16, wherein the sample is extracted into a reservoir comprising at least one chemical to protect the polypeptide integrity.

18. The method of claim 17, wherein the chemical is a protease inhibitor or a phosphatase inhibitor.

19. The method of any one of claims 1-10, and 15-17, further comprising treating the ocular condition or susceptibility thereto identified.

20. An ophthalmic aspirating device, comprising: a negative pressure module; and a conduit having first and second ends, wherein the respective ends each comprises an aperture, and wherein the second conduit end is operatively associated with the negative pressure module.

21. The ophthalmic aspirating device of claim 20, further comprising a housing operatively associated with the negative pressure module and conduit.

22. An ophthalmic aspirating device, comprising: a housing; a tube operatively associated with the housing, the tube having first and second ends; a conduit having first and second ends, wherein the first conduit end comprises an aperture, and wherein the second conduit end is operatively associated with the first tube end; and a negative pressure module.

23. The ophthalmic aspirating device of claim 21 or 22, wherein the negative pressure module comprises at least one chemical to protect polypeptide integrity.

24. The ophthalmic aspirating device of any of claims 20-23, wherein the negative pressure module is configured for at least one of attachment and deattachment from the housing.

25. The ophthalmic aspirating device of claim 22, wherein the negative pressure module comprises a plunger assembly comprises a plunger.

26. The ophthalmic aspirating device of claim 25, wherein the plunger assembly comprises a biasing device connected to the plunger, wherein the biasing device is under the operational control of an actuator.

27. The ophthalmic aspirating device of any one of claims 22-26, wherein the conduit comprises at least one of a needle and a cannula.

28. A vitreous fluid analysis kit comprising a vitreous fluid receptacle comprising a reservoir, wherein the reservoir comprises at least one chemical to protect polypeptide integrity.

29. The vitreous fluid analysis kit of claim 28, further comprising an ophthalmic aspirating device, comprising a housing to which the receptacle can be operatively attached and detached.

30. The vitreous fluid analysis kit of claim 28 or 29, further comprising a vitreous fluid biomarker detector.

31. The vitreous fluid analysis kit of claim 30, wherein the detector comprises a primary antibody specific to a biomarker polypeptide or a unique fragment thereof.

32. The vitreous fluid analysis kit of claim 31, wherein the detector comprises a secondary antibody coupled to a label.

33. A method of remote vitreous fluid analysis comprising: obtaining a vitreous fluid sample from a living subject; storing the vitreous fluid sample in a receptacle comprising a reservoir, wherein the reservoir comprises at least one chemical to protect polypeptide integrity; and sending at least one of the vitreous fluid sample and data collected therefrom to a laboratory for analysis.

34. The method of claim 33, further comprising analyzing the vitreous fluid sample.

35. The method of claim 34, wherein the analyzing comprises: detecting a level of a biomarker in the vitreous fluid, wherein the biomarker is associated with at least one of a susceptibility to an ocular condition, presence or absence of an ocular condition, and an efficacy of treatment of an ocular condition; and identifying at least one of the susceptibility to the ocular condition, the presence or absence of the ocular condition, or the efficacy of treatment of an ocular condition based on the level of the biomarker.

36. The method of any one of claims 33-35, further comprising returning an analysis report to the sender or a third party.

37. The method of any one of claims 33-36, further comprising selling the kit of any one of claims 28-32.

38. The method of any one claims 33-37, further comprising: establishing a contractual relationship between a vitreous fluid analysis organization and an ophthalmology clinic, wherein the clinic performs the obtaining step, and wherein the clinic is contractually obligated to send the sample or data collected therefrom to a laboratory designated by the vitreous fluid analysis organization for analysis.

39. The method of claim 38, wherein the contractual relationship establishes a fee for each sample analyzed.

40. The method of claim 38 or 39, wherein the contractual relationship requires the ophthalmology clinic to buy at least one class of articles from the vitreous fluid analysis organization or a supplier designated by the organization.

41. The method of claim 40, wherein the class of articles is a vitreous fluid analysis kit comprising a vitreous fluid receptacle comprising a reservoir, wherein the reservoir comprises at least one chemical to protect polypeptide integrity.

42. The method of any one of claims 38-41, wherein the contractual relationship requires the ophthalmology clinic to have an employee or contractor complete an education program directed to vitreous fluid aspiration, wherein the education program is provided by a trainer designated by the vitreous fluid analysis organization, and wherein the employee or contractor performs at least one of the obtaining step and supervising of the obtaining step.

43. A proteomic fingerprint, comprising at least one vitreous fluid biomarker associated with an ocular condition.

44. The proteomic fingerprint of claim 43, wherein the biomarker is a polypeptide or a unique fragment thereof.

45. A method of identifying biomarkers of an ocular condition, comprising: comparing a vitreous fluid polypeptide spectrum of a subject having an ocular condition to a vitreous polypeptide spectrum of a subject not having the ocular condition, wherein the spectra are of vitreous fluid aspirated from living patients; determining a difference in the polypeptide spectra in terms of identity or amount of the at least one polypeptide or a fragment thereof; and correlating the presence of the condition with the difference to identify a biomarker.

46. The method of claim 45, further comprising: aspirating vitreous fluid from a living subject having an ocular condition and a living subject not having the ocular condition.

47. A method of identifying biomarkers associated with the development of an ocular condition, the method comprising: comparing vitreous fluid polypeptide spectra of at least one vitreous fluid sample from a subject prior to the onset of the ocular condition and at least one vitreous fluid sample after the onset of the ocular condition; determining at least one difference in the polypeptide spectra in terms of identity and amount of a polypeptide among the samples; and correlating the presence of the condition with the difference to identify a biomarker.

48. The method of claim 47, further comprising: aspirating the vitreous fluid samples before and after development of the ocular condition.

49. The method of claim 48, wherein the samples are aspirated at periodic intervals before and after development of the ocular condition.

50. The method of any one of claim 47-49, wherein the correlating comprises correlating polypeptide spectra from at least two different stages of an ocular disease, wherein each difference is the biomarker for a given stage of the ocular disease.