WO2005084188A2

WO2005084188A2 - Systemic inflammatory markers and age-related macular degeneration (amd)

Info

Publication number: WO2005084188A2
Application number: PCT/US2004/005626
Authority: WO
Inventors: Johanna M. Seddon
Original assignee: Massachusetts Eye & Ear Infirmary
Priority date: 2004-02-25
Filing date: 2004-02-25
Publication date: 2005-09-15
Also published as: WO2005084188A3

Abstract

Provided are methods of using levels of markers of systemic inflammation, e.g., CRP, to predict a subject’s risk of development or progression of Age-Related Macular Degeneration (AMD), and methods of treating, delaying or preventing the development or progression of AMD.

Description

SYSTEMIC INFLAMMATORY MARKERS AND AGE-RELATED MACULAR DEGENERATION (AMD)

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under Grant No. RO1EY13982, NO1EY02117, and NO1EY02126 awarded by the National Institutes of Health. The Government has certain rights in the invention.

TECHNICAL FIELD This invention relates to methods of using a systemic inflammatory marker as a diagnostic and prognostic tool for age-related macular degeneration (AMD), and methods for treating, preventing or delaying the development or progression of AMD. BACKGROUND Age-related macular degeneration (AMD) is a sudden worsening and distortion of central vision that progresses rapidly, typically with a course of only weeks or months. AMD is characterized by abnormalities in the macular area. The central area (or fovea) of the macula contains the highest density of cone photoreceptors in the retina and mediates high-acuity vision. The disease typically has a preclinical, asymptomatic phase, in which extracellular waste material accumulates in the space between the basement membrane (Bruch's membrane) and the epithelial layer, forming yellow-white spots known as drusen. Advanced forms of AMD includes both dry and wet (or "neovascular") AMD. The dry form of AMD is far more common, but the wet form occurs simultaneously with the dry form in about 30% of cases. Dry AMD is characterized by progressive apoptosis of cells in the epithelial layer, in the overlying photoreceptor cells and in the underlying cells in the choroidal capillary layer. Wet AMD is characterized by choroidal neovascularization with vascular leakage into subretinal spaces. AMD impairs central vision that is required for reading, driving, face recognition, and fine visual tasks. Neurosensory detachment, retinal hemorrhages, and retinal scarring gradually result in decreased visual function of photoreceptors in the central vision, eventually resulting in legal blindness, with preservation of peripheral vision. AMD is the most common cause of blindness among the elderly. Subjects with a family history of AMD and those who smoke have a higher risk than non-smokers and those with no family history, however, subjects who have favorable risk profiles also develop the disease. Current therapeutic efforts and clinical trials are aimed at halting the growth of the neovascular membrane in wet AMD, e.g., using angio genesis inhibitors, laser photocoagulation, and/or photodynamic therapy. Antioxidants can retard the progression of the disease. Despite advances in treatment, AMD is still the most common cause of visual impairment in the developed world.

SUMMARY The present invention is based, in part, on the discovery of a relationship between C-Reactive Protein (CRP) and Age-Related Macular Degeneration (AMD). Thus in one aspect, the invention provides new diagnostic methods that determine the magnitude of systemic inflammation, and use that information to predict the risk of developing new or progressing to more advanced AMD. These methods can be used to predict risk of development or progression of age-related maculopathy, and/or to determine the likelihood that specific subjects will benefit from certain treatments (e.g., the administration of anti-inflammatory agents) for non-neovascular as well as neovascular AMD. The invention also provides methods for treating, preventing, and/or delaying the development or progression of AMD. This invention describes new diagnostic and prognostic methods that determine and utilize the magnitude of systemic inflammation. These new methods broadly include the prediction of risk of development and/or progression of AMD; and the determination of the likelihood that certain subjects will benefit from the use of certain treatments designed to prevent development or progression of AMD and/or treat AMD. These new methods are based in part upon the discoveries described herein. As described herein, elevated levels of markers of systemic inflammation, e.g., CRP, are predictive of development or progression of AMD. Elevated levels of markers of systemic inflammation in otherwise healthy subjects, regardless of whether they have ever smoked, are predictive of development or progression of AMD. Therefore, the likelihood that a specific subject will benefit to a greater or a lesser extent from the use of certain therapeutic agents (e.g., anti-inflammatory agents) for reducing the risk of development or progression of AMD can be determined by evaluating levels of markers of systemic inflammation, e.g., levels of CRP, IL-6 TNF-alpha RII, ICAM, or NCAM, in the subject, and comparing the levels to a reference for the marker. The predictive value of markers of systemic inflammation for risk of development or progression of AMD is independent of other predictors. In fact, risk prediction based on levels of markers of systemic inflammation is additive with some other risk factors, and does not simply duplicate the information derivable from evaluating those other factors (e.g., smoking, obesity, body mass index, fat intake, antioxidant vitamin and mineral intake, age, sex, family history of AMD, and physical exercise levels, inter alia). In one aspect, the invention provides methods for characterizing a subject's risk of development or progression of AMD. The methods include obtaining a level of a marker of systemic inflammation in the subject, e.g., levels of CRP, IL-6, TΝF- alpha RII, ICAM or NCAM. The level of the marker is compared to a reference, and the subject's risk of development or progression of AMD is characterized based upon the level of the marker in comparison to the reference. In some embodiments, the level of the marker of systemic inflammation is elevated as compared to the reference, and it indicates that the subject has an increased risk of development or progression of AMD, e.g., increased relative to a control or other reference (including a previous level obtained from the same subject). In some embodiments, the level of the marker of systemic inflammation is reduced as compared to the reference, and it indicates that the subject has a reduced risk of development or progression of AMD, e.g., reduced relative to a control or other reference (including a previous level obtained from the same subject). In some embodiments, the method also includes selecting a treatment option based on the level of the marker of systemic inflammation; in one embodiment, the level of the marker of systemic inflammation is high, and a treatment option including administering an anti-inflammatory agent is selected. As used herein, progression of AMD refers to an increase in severity of the disease, e.g., an objective worsening in one or more parameters or symptoms associated with the disease. In some embodiments, the subject is re-evaluated, e.g., the level of the marker of systemic inflammation is obtained again after or during administration of a treatment (e.g., after administration of one or more doses of an anti-inflammatory agent), and the level is compared to a reference, e.g., the level previously obtained, to evaluate the efficacy of the treatment. In some embodiments, after administration of the treatment, the level of a marker of systemic inflammation is reduced as compared to a previously-obtained level, and indicates that the subject's risk or development or progression of AMD is reduced. According to yet another aspect of the invention, a method is provided that uses levels of an inflammatory marker, e.g., levels of CRP, TNF-alpha RII, ICAM or NCAM, together with one or more second risk factors, e.g., a second risk factor as described herein, to characterize a subject's risk profile of development or progression of AMD. The method includes obtaining a level of a marker of systemic inflammation in the subject. The level of the marker is compared to a reference to establish a first risk value. At least one second risk factor is also evaluated. The level of the second risk factor in the subject is compared to a second reference to establish a second risk value. The subject's risk profile for development or progression of AMD is characterized based upon the combination of the first risk value and the second risk value, wherein the combination of the first risk value and second risk value establishes a third risk value different from the first and second risk values, hi some embodiments, the third risk value is greater than either of the first and second risk values, e.g., the first and second risk values are additive. In another aspect of the invention, a method is provided for evaluating the likelihood that a subject will benefit from treatment with an agent, e.g., an anti- inflammatory agent, to reduce the risk of development or progression of AMD. The method includes obtaining a level of a marker of systemic inflammation in a subject. This level then is compared to a reference, wherein the level of the marker of systemic inflammation in comparison to the reference is indicative of the likelihood that the subject will benefit from treatment with the agent. The subject then can be characterized in terms of the net benefit likely to be obtained by treatment with the . agent. hi some embodiments, the subject is apparently healthy, i.e., has no or few overt clinical signs of AMD (e.g., is in the first maculopathy quartile as described herein); has minimal or early AMD (e.g., is in the second maculopathy quartile); has intermediate AMD (e.g., is in the third maculopathy quartile); or has advanced AMD (e.g., is in the fourth maculopathy quartile). In some embodiments, the subject is a non-smoker, e.g., has never smoked, or is a smoker, e.g., a current or past smoker. A non-smoker is a subject who, at the time of the evaluation, has never smoked. Smokers include subjects who currently smoke, as well as subjects who have smoked at some time in the past but presently no longer smoke. In some embodiments, the subject has no second risk factors as described herein. In some embodiments, the subject has one or more second risk factors as described herein. hi some embodiments, characterizing a subject's risk of development or progression of AMD includes characterizing the subject's risk of developing advanced AMD. In some embodiments, characterizing the subject's risk of future development or progression of AMD includes characterizing the subject's risk of developing neovascular AMD. The reference can be a single value, multiple values, a single range or multiple ranges. Thus, in one embodiment, the reference is a plurality of marker level ranges, and the comparing step comprises determining in which marker level range the subject's level falls. In some embodiments, the subject is apparently healthy. In some embodiments, the subject is a smoker, hi some embodiments the marker of systemic inflammation is C-reactive protein. In some embodiments, the marker of systemic inflammation is an interleukin, e.g., interleukin-6 (IL-6), tissue necrosis factor alpha receptor- 11 (TNF-alpha Rl 1), intracellular adhesion molecule-(ICAM), or vascular adhesion molecule (NCAM). In some embodiments, levels of multiple markers of systemic inflammation are obtained concurrently. In some embodiments, levels of one or more lipid biomarkers are obtained in place of or in addition to a marker of systemic inflammations. In some embodiments, the lipid biomarkers are apolipoprotein B (ApoB) or lipoprotein (a) (Lp(a)). In some embodiments, when the marker of systemic inflammation is C- reactive protein, then the is about 1.7 mg/1 of blood, about 2.7 mg/1 of blood; about 4.5 mg/L of blood; or about 6.5 mg/1 of blood. In some embodiments, when ranges are employed, one of the plurality of ranges can be below about 2.7 mg/1 of blood, another of the ranges can be above about 2.7 mg/1; another of the ranges can be between about 2.7 mg/1 and about 6.5 mg/1 of blood; and another of the ranges can be above about 6.5 mg/1 of blood. In some embodiments, when ranges are employed, one of the plurality of ranges can be below about 1.7 mg/1 of blood, another of the ranges can be above about 1.7 mg/1; another of the ranges can be between about 1.7 mg/1 and about 4.5 mg/1 of blood; and another of the ranges can be above 4.5 mg/1 of blood. One of skill in the art will appreciate that the reference will typically depend on the particular marker selected and even upon the characteristics of the patient population in which the subject lies, described in greater detail below. As mentioned above, the methods described herein can be adapted to determining which subjects are most likely to benefit from treatment with an agent for reducing the risk in the development or progression of AMD. The methods can also be used to select candidate subjects and/or populations for clinical trials and for treatment with candidate drugs, by identifying, for example, subjects most likely to benefit from a new treatment or from a known treatment with a high risk profile of adverse side effects. Thus, the methods described herein can provide information for evaluating the likely net benefit of certain treatments for candidate subj ects. The invention also includes kits comprising a package including an assay for a marker of systemic inflammation (e.g., CRP) and instructions for use in a method described herein, and optionally related materials such as charts, e.g., numeric or color charts, for correlating the level of the marker as determined by the assay with a risk of development or progression of AMD. In some embodiments, the instructions include information for determining the subject's risk of development or progression of AMD, by correlating the level of the marker determined by the assay and one or more second risk factors with a risk of development or progression of AMD. The invention also involves a method for treating subjects with anti- inflammatory therapies, to treat, prevent, and/or delay the development or progression of AMD. In some embodiments, a non-aspirin anti-inflammatory agent is administered to a subject who has an above-normal level of a marker of systemic inflammation, but who is otherwise free of symptoms calling for an anti-inflammatory agent. The anti-inflammatory agent is administered in an amount effective to treat, prevent, and/or delay the development or progression of AMD. In some embodiments, the anti-inflammatory agent is administered in an amount effective to reduce the subject's CRP levels, e.g., to below a reference , e.g., below about

1.7 mg/1 of blood, below about 2.7 mg/1 of blood; below about 4.5 mg/1 of blood, or below about 6.5 mg/1 of blood. In some embodiments, the subjects are apparently healthy subjects who are essentially free of current need for anti-inflammatory treatment, such as free of symptoms of rheumatoid arthritis, chronic back pain, autoimmune diseases (e.g., amyotrophic lateral sclerosis, multiple sclerosis, type I diabetes, graft-versus-host disease, rheumatoid arthritis, inflammatory bowel disease, uveitis, and thyroiditis), and the like. In addition, the invention includes packages including an anti-inflammatory agent and instructions for administering the anti- inflammatory agent to a subject in order to treat, prevent, and/or delay the development or progression of AMD. In some embodiments, the anti-inflammatory agent is in a therapeutic composition also including a pharmacologically acceptable carrier. In some embodiments, the anti-inflammatory agent is in a form suitable for local delivery to the macular area. As used herein, "age-related macular degeneration" or "AMD" includes both early, intermediate, and advanced AMD. "Advanced AMD" includes both dry AMD and wet AMD (wet AMD is also referred to as neovascular AMD). As used herein, "subject" is a mammal, e.g., canine, feline, ovine, primate, equine, porcine, caprine, camelid, avian, bovine, and murine organisms. Typically, the subjects are human. As used herein, "apparently healthy" means subjects who do not have clinical signs of AMD, e.g., are in the first maculopathy quartile as described herein. Apparently healthy subjects also do not otherwise exhibit symptoms of disease. In other words, such subjects, if examined by a medical professional, would be characterized as healthy and generally free of symptoms of disease. As used herein, a "second risk factor" means a risk factor that differs from a first risk factor. In some embodiments, the second risk factor is family history of AMD, age, sex, smoking history, obesity, weight change since age 20, dietary fat intake, linoleic acid intake, and/or elevated cholesterol levels. In some embodiments, the second risk factor is also a marker of systemic inflammation, hi some embodiments, the second risk factor is elevated levels of a lipid biomarker. In some embodiments, the second risk factor is the presence or absence of a genetic marker. For example, in some embodiments the genetic marker is a four-marker single- nucleotide polymorphism (SNP) haplotype in the locus spanning the gene ALOX5 AP encoding 5-lipoxygenase activating protein (FLAP) (see Helgadottir et al., The gene encoding 5-lipoxygenase activating protein confers risk of myocardial infarction and stroke. Nat Genet, (published online Feb 8, 2004)). In some embodiments, the genetic marker is on chromosome lq (236-140 cM in the Marshfield genetic map), 2p (10 cM), 5p (40-50 cM), 9q (111 cM), and/or 22q (25 cM) (see Abecasis et al, Am. J. Hum. Genet. 74:482-494, 2004).

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety, hi case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

DESCRIPTION OF DRAWINGS FIG. 1 is a bar graph illustrating the adjusted odds ratios for prevalence of maculopathy according to CRP level and smoking status as shown in Table 5.

DETAILED DESCRIPTION As described herein, it has been discovered that elevated levels of CRP, a marker of systemic inflammation, are predictive of AMD. This association is present for both smokers and non-smokers. It has been fiirther discovered that elevated levels of CRP predict risk of AMD independent of other risk factors, including dietary fat and higher body mass index. Thus, information about this biomarker does not duplicate, and is likely to be independent of, the information provided by other known or presumed risk factors. Even when numerous other known risk factors are controlled for (see Examples, below), the inflammatory markers improve prediction. Furthermore, using a marker of systemic inflammation as an assay for AMD risk can provide a simpler method of evaluating a subject's risk that does not require information about a subject's personal medical history. The results described herein are believed to be the first to implicate CRP as a systemic inflammatory marker for the development of AMD. Higher CRP values were found to be significantly related to AMD independent of established risk factors including smoking and obesity. Among smokers and non-smokers, higher baseline CRP was associated with an increased risk of AMD. These results shed light on the mechanisms and patho genesis of AMD development and prognosis. Moreover, CRP levels add clinically relevant predictive information concerning risk of AMD in addition to known risk factors. Anti-inflammatory agents thus may have a role in preventing and/or treating AMD, and inflammatory biomarkers such as CRP provide a method of identifying people for whom these agents would be more or less effective. As described herein, elevated levels of markers of systemic inflammation

(e.g., IL-6, TNF-alpha RII, ICAM and VCAM) are associated with AMD. After adjustment for age, sex, and other variables including smoking and body mass index, CRP levels were significantly higher among subjects with intermediate and advanced stages of AMD compared with controls (e.g., subjects in the first and/or second maculopathy quartiles, as described herein). The magnitude of effect ranged from an odds ratio (OR) of 1.65 to 2.16 for the highest levels of CRP. Similar results were observed for other markers of systemic inflammation, e.g., IL-6, TNF-alpha RU, ICAM and NCAM, as well as lipid biomarkers, apolipoprotein B (ApoB) and lipoprotein a (LP(a). The odds ratio refers to the odds of getting a disease, if a selected factor is present. For example, an OR of 2 refers to a two-fold increase in risk of getting the disease. Risk of AMD was lowest among those with low CRP values who never smoked. In contrast, risk tended to be highest among smokers who also had higher levels of CRP. Even among those who never smoked, the risk of AMD was increased two-fold among those with the highest category of CRP, compared with the lowest level of CRP as the referent category. The findings described herein demonstrate that inflammation is associated with the pathogenesis of AMD. Several mechanisms that could lead to inflammatory responses may be involved including oxidative stress caused by risk factors for AMD, such as smoking (Seddon et al., JAMA 1996;276:1141-1146; Smith et al., Ophthalmology 2001; 108: 697-704), insufficient antioxidants in the diet (Seddon et al., JAMA 1994;272:1413-1420; Cho et al., Am J Clin Nutr 2001; 73: 209-218), dietary fat (Seddon et al., Arch Ophthalmol 2001;119:1191-1199; Cho et al., Am J Clin Nutr 2001; 73: 209-218), or obesity (Seddon et al., Arch Ophthalmol 2003;121:785-792). Smoking is one of the most consistent risk factors for AMD, yet many subjects who have never smoked develop AMD. As described herein, higher CRP values were associated with increased risk of AMD among smokers as well as those among who never smoked, independent of the other risk factors in the model. Therefore, it is likely that factors other than smoking in these subjects create an adverse milieu or damage the RPE-retina-choroidal complex in some way, which in turn leads to an inflammatory stimulus and increased CRP values. The study described herein evaluated a systemic biomarker for inflammation in a large and well-characterized population of subjects with and without maculopathy from two geographical areas in the United States. Standardized collection of risk factor information including direct measurements of blood pressure and body mass index, as well as classification of maculopathy by means of standardized ophthalmological examinations and fundus photography, was employed. Misclassification was unlikely because CRP values were quantified using objective laboratory methods without knowledge of the subjects' maculopathy status, and AMD grade was assigned without knowledge of CRP status. Residual confounding is a concern in many epidemiological studies. Known AMD risk factors and those associated with AMD in this study cohort were controlled for. For example, obesity and cigarette smoking are related to AMD, and are also related to increased levels of CRP and other systemic inflammatory markers (Visser et al., JAMA 1999;282:2131-2135). As described herein, increased CRP levels were significantly and independently related to AMD in this study after adjustment for these confounding factors. Although some unmeasured and therefore uncontrolled factors might still be confounding this relationship, they would have to be both highly associated with CRP and a strong risk factor for AMD to explain these results. The study population consisted of subjects with a range of maculopathy and some subjects without AMD who participated in a randomized trial of nutritional supplements. Results were not altered after adjustment for assignment to antioxidants within the randomized trial. Controls were more likely to be female, non-smokers, and with more education. However, these analyses were adjusted statistically for these differences, and previous case-control analyses of the entire Age-Related Eye Disease Study (AREDS) cohort, as well as this subset at two centers, demonstrated an association with known risk factors for AMD similar to other study populations. Although the study population was a selected population, these cases likely represent the typical subject with AMD. In addition, the study population overall was comparable to the general population in this age range in terms of smoking status and prevalence of obesity. Measures of CRP were taken from single fasting blood specimens that were stored in a repository at -140°C until analyzed. These are standardized methods that are in use in several large-scale epidemiologic studies throughout the country (Ridker et al., JAMA 2001;285:2481-2485; Ridker, Circulation 2003;107:363-369). The medians and ranges of CRP in the various quartiles in this study are similar to other published studies of CRP and cardiovascular diseases (Id.).

Markers of Systemic Inflammation In some embodiments, the methods described herein include determining a level of a marker of systemic inflammation in a subject. Markers of systemic inflammation are known to those in the art. In some embodiments, the markers of systemic inflammation are selected from the group consisting of C-reactive protein, cytokines, tissue necrosis factor alpha receptor-11 (TNF-alpha Rll), and cellular adhesion molecules. Cytokines are known to those in the art and include human interleukins 1-17 (IL-1 through IL-17); in some embodiments, the marker is interleukin-6 (IL-6). Cellular adhesion molecules are known to those in the art and include integrins, intracellular adhesion molecules (e.g., ICAM-1, ICAM-3), B- lymphocyte cell adhesion molecule (BL-CAM), lymphocyte function-associated adhesion molecules (e.g., LFA-2), vascular cell adhesion molecules (e.g., NCAM-1), neural cell adhesion molecule (ΝCAM), platelet endothelial cell adhesion molecule (PECAM), and soluble intercellular adhesion molecule (sICAM-1). In some embodiments, the marker is an intracellular adhesion molecule (ICAM) or a vascular adhesion molecule (VCAM).

Level of a Marker of Systemic Inflammation The level of the marker of systemic inflammation for the subject can be obtained by any art recognized method. Typically, the level is obtained by measuring the level of the marker in a body fluid, for example, blood, lymph, saliva, urine, and the like. The level can be determined by immunoassays, e.g., enzyme-linked immunoassays (EIA) or enzyme-linked immunosorbent assays (ELISA); particle agglutination or flocculation tests (e.g., rapid latex agglutination); laser and rate nephelometry; turbidometry; or other known techniques for determining the presence of the marker. The methods can include obtaining a level of a marker of systemic inflammation in a subject by sending one or more samples of the subject's body fluid to a laboratory, e.g., a commercial laboratory, for measurement of levels. Thus, in some embodiments, the methods include measuring the level of the marker in a body fluid from a subject, and providing information regarding the level of the marker, e.g., to the subject or a caregiver, e.g., a clinical entity such as a physician, nurse, hospital, clinical practice, or third-party payor, e.g., an insurance company. In some embodiments, the methods described herein also include comparing the level of a marker for the subject with a reference. The reference can take a variety of forms. It can be single cut-off value, such as a median or mean. It can be established based upon comparative groups, such as where the risk in one defined group is double the risk in another defined group. It can be a range, for example, where the tested population is divided equally (or unequally) into groups, such as-a low-risk group, a medium-risk group and a high-risk group, or into quadrants, the lowest quadrant being subjects with the lowest risk and the highest quadrant being subjects with the highest risk. The reference can depend upon the particular population selected. For example, an apparently healthy, nonsmoker population (no detectable disease and no prior history of AMD) will have a different "normal" range of markers of systemic inflammation than will a smoking population or a population the members of which have some stage of AMD. Accordingly, the reference selected may take into account the category in which a subject falls. Appropriate ranges and categories can be selected with no more than routine experimentation by those of ordinary skill in the art. In some embodiments, the reference is a predetermined value. In some embodiments, when the marker of systemic inflammation is C-reactive protein, then the predetermined value is about 1.7 mg/1 of blood, about 2.7 mg/1 of blood; about 4.5 mg/L of blood; or about 6.5 mg/1 of blood. In some embodiments, when ranges are employed, one of the plurality of ranges can be below about 2.7 mg/1 of blood, another of the ranges can be above about 2.7 mg/1; another of the ranges can be between about 2.7 mg/1 and about 6.5 mg/1 of blood; and another of the ranges can be above about 6.5 mg/1 of blood. In some embodiments, when ranges are employed, one of the plurality of ranges can be below about 1.7 mg/1 of blood, another of the ranges can be above about 1.7 mg/1; another of the ranges can be between about 1.7 mg/1 and about 4.5 mg/1 of blood; and another of the ranges can be above about 4.5 mg/1 of blood. In one embodiment, the body fluid is blood and the marker is C-reactive protein. For

C-reactive protein, one important reference for a population of apparently healthy, nonsmokers is 2.7 mg/liter (median). Another important reference for C-reactive protein is 6.5 mg/liter (highest quartile of risk). For smokers, important references include 1.7 mg/L (second fertile) and 4.5 mg/L (third fertile). In characterizing risk, numerous references can be established. Commercially available assays and reagents can be used for measuring levels of C-reactive protein. Commercial sources for these reagents and assays include, but are not limited to, Denka Seiken (Niigata, Japan), Abbott Pharmaceuticals (Abbott Park, 111.), Dade Behring, Inc. (Newark, DE), Randox Laboratories (Crumlin, Co. Antrim, UK), The Binding Site (Santa Monica, CA), Alfa Biotech S.p.A. (Pomezia, Rome, Italy), CalBiochem (San Diego, Calif.), Roche Diagnostics Corp. (Indianapolis, IN), Orion Diagnostics, Div. Orion Corp. (Espoo, Finland), Ortho- Clinical Diagnostics, Inc. (Rochester, NY), Johnson & Johnson Clinical Diagnostics, Inc. (Rochester, NY), and Behringwerke (Marburg, Germany), among many others. The U.S. Food and Drug Administration (FDA) regulates such diagnostic tests as medical devices, typically as Class II medical devices. CRP immunoreactive tests are categorized under section 866.5270; assays, reagents, and devices listed thereunder can be used in the methods described herein to obtain the CRP level of a subject. A number of assays for levels of inflammatory cytokines and cellular adhesion molecules are known in the art. Commercial sources for inflammatory cytokine and cellular adhesion molecule measurements, include, but are not limited to, R&D Systems (Minneapolis, Minn.), Genzyme (Cambridge, Mass.), and Immunotech (Westbrook, Me.). Also provided are novel kits or assays that are specific for, and have appropriate sensitivity with respect to, reference s selected on the basis of the present invention. In some embodiments, therefore, the kits or assays would differ from those presently commercially available, by including, for example, different cut-offs, different sensitivities at particular cut-offs as well as instructions or other printed material for characterizing risk based upon the outcome of the assay.

Methods for Predicting and Evaluating the Efficacy of a Treatment Also provided herein are methods for evaluating the likelihood that a subject will benefit from treatment with an anti-inflammatory agent for reducing risk of development or progression of AMD. The method includes determining the level of a marker of a systemic inflammation (e.g., CRP) in the subject; if the level of the marker is high, then the subject is likely to benefit from the administration of an anti- inflammatory agent. In some embodiments, the method can further include administering an anti-inflammatory agent to the subject. The methods described herein can also be used to evaluate the efficacy of a treatment for reducing the risk of development or progression of AMD. For example, the method can include determining the level of a marker of systemic inflammation (e.g., AMD) before, concurrently with, and/or after the administration of the treatment. In some embodiments, the subject receives multiple treatments, e.g., a treatment is administered in multiple doses, e.g., one or more doses per day for one or more days, weeks, months, or years, and the level of a marker of systemic inflammation (e.g., AMD) is determined, e.g., before any treatment, and after one or more treatments. In some embodiments, the treatment is the administration of an anti- inflammatory agent, e.g., as described herein. In some embodiments, the methods described herein are performed as part of a clinical trial of a treatment to reduce the risk of the development or progression of AMD. These methods have important implications for subject treatment and also for clinical development of new therapeutics. Physicians typically select therapeutic regimens for subject treatment based upon the expected net benefit to the subject. The net benefit is derived from the risk to benefit ratio. The present methods permit selection of subjects who are more likely to benefit by intervention, thereby aiding the physician in selecting a therapeutic regimen. This might include using drugs with a higher risk profile where the likelihood of expected benefit has increased. Likewise, clinical investigators may desire to select for clinical trials a population with a high or low likelihood of obtaining a net benefit with a particular protocol. The methods described herein can be used by clinical investigators select such a population. Thus, in some embodiments, the methods provide entry criteria and methods for selecting subjects for clinical trials, e.g., trials of AMD therapeutics, by selecting subjects having a given CRP level, e.g., having a CRP above or below a reference . As described herein, it has been discovered that markers of systemic inflammation (e.g., CRP) have predictive value independent of other known predictors of development or progression of AMD. Thus, the methods described herein do not involve simply duplicating a measurement that previously could be made using other predictors. Instead, the markers of systemic inflammation provide information that is additive to previously known predictors. This is illustrated in Table 5, wherein the data were analyzed to characterize the risk profiles of subjects, taking into account both smoking history and levels of C-reactive protein. These data are illustrated in FIG. 1, which shows the relative risk of developing AMD associated with low, middle and high tertiles of total C reactive protein, and smoking. As is discussed in more detail below (see Example 1), the risk is additive.

Methods of Treatment Also provided herein are methods for treating subjects with anti-inflammatory therapies to treat, prevent, or delay the development or progression of AMD. In some embodiments, an anti-inflammatory agent is administered to a subject who has an above-normal level of a marker of systemic inflammation, but who is otherwise free of symptoms calling for an anti-inflammatory agent. In some embodiments, the anti- inflammatory agent is administered in conjunction with another modality for treating, preventing or delaying the development AMD, e.g., photodynamic therapy or laser photocoagulation to treat wet AMD, and/or vitamin supplements, e.g., as described in AREDS Research Group, Arch Ophthalmol 2001:119, 1417-1436; Seddon et al, JAMA, 1994; 272: 1413-1420, and U.S. Pat. No. 6,660,297. In some embodiments, the methods include administering to the subject an anti-angiogenesis agent, e.g., ^" agents that inhibit vascular endothelial growth factor ( anti-NEGF treatments) such as Macugen, a VEGF inhibitor, (Pfizer) and Lucentis, a similar molecule (Genetech), as well as anecortave acetate (Alcon), which reduces matrix metalloproteinase production, a key agent in the growth of neovascular membranes. Finally, triamcinolone acetate is another drug, currently in trials for the treatment of the neovascular stage of AMD, which can act as an anti-inflammatory agent and is administered by an intravitreal injection. In some embodiments, the methods include using CRP levels to predict which subjects will be most likely to be responsive to treatment. In some embodiments, the methods include selecting (and, in some embodiments, administering) a particular treatment depending on the level of CRP or the other inflammatory markers in the subject. Anti-inflammatory agents that can be used in the methods described herein include, but are not limited to, Alclofenac; Alclometasone Dipropionate; Algestone Acetonide; Alpha Arnylase; Amcinafal; Amcinafide; Amfenac Sodium; Amiprilose Hydrochloride; Anakinra; Anirolac; Anitrazafen; Apazone; Balsalazide Disodium; Bendazac; Benoxaprofen; Benzydamine Hydrochloride; Bromelains; Broperamole; Budesonide; Carprofen; Cicloprofen; Cintazone; Cliprofen; Clobetasol Propionate; Clobetasone Butyrate; Clopirac; Cloticasone Propionate; Cormethasone Acetate;

Cortodoxone; Deflazacort; Desonide; Desoximetasone; Dexamethasone Dipropionate; Diclofenac Potassium; Diclofenac Sodium; Diflorasone Diacetate; Diflumidone Sodium; Diflunisal; Difluprednate; Diftalone; Dimethyl Sulfoxide; Drocinonide; Endrysone; Enlimomab; Enolicam Sodium; Epirizole; Etodolac; Etofenamate; Felbinac; Fenamole; Fenbufen; Fenclofenac; Fenclorac; Fendosal; Fenpipalone; Fentiazac; Flazalone; Fluazacort; Flufenamic Acid; Flumizole; Flunisolide Acetate; Flunixin; Flunixin Meglumine; Fluocortin Butyl; Fluorometholone Acetate; Fluquazone; Flurbiprofen; Fluretofen; Fluticasone Propionate; Furaprofen; Furobufen; Halcinonide; Halobetasol Propionate; Halopredone Acetate; Ibufenac; Ibuprofen; Ibuprofen Aluminum; Ibuprofen Piconol; Ilonidap; Indomethacin; Indomethacin

Sodium; Indoprofen; Indoxole; Intrazole; Isoflupredone Acetate; Isoxepac; Isoxicam; Ketoprofen; Lofemizole Hydrochloride; Lornoxicam; Loteprednol Etabonate; Meclofenamate Sodium; Meclofenamic Acid; Meclorisone Dibutyrate; Mefenamic Acid; Mesalamine; Meseclazone; Methylprednisolone Suleptanate; Morniflumate; Nabumetone; Naproxen; Naproxen Sodium; Naproxol; Nimazone; Olsalazine Sodium; Orgotein; Orpanoxin; Oxaprozin; Oxyphenbutazone; Paranyline Hydrochloride; Pentosan Polysulfate Sodium; Phenbutazone Sodium Glycerate; Pirfenidone; Piroxicam; Piroxicam Cinnamate; Piroxicam Olamine; Pirprofen; Prednazate; Prifelone; Prodolic Acid; Proquazone; Proxazole; Proxazole Citrate; Rimexolone; Romazarit; Salcolex; Salnacedin; Salsalate; Salycilates; Sanguinarium Chloride; Seclazone; Sermetacin; Sudoxicam; Sulindac; Suprofen; Talmetacin; Talniflumate; Talosalate; Tebufelone; Tenidap; Tenidap Sodium; Tenoxicam; Tesicam; Tesimide; Tetrydamine; Tiopinac; Tixocortol Pivalate; Tolmetin; Tolmetin Sodium; Triclonide; Triflumidate; Zidometacin; Glucocorticoids; Zomepirac Sodium. Statins (HMG-CoA reductase inhibitors) are also considered anti- inflammatory agents (see, e.g., Curr Control Trials Cardiovasc Med 2000, 1:161-165) and can be used in the methods described herein. Statins include Pravachol (pravastatin, Bristol-Myers Squibb); Mevacor (Lovastatin, Merck); Zocor (simvastatin, Merck); Lescol (fluvastatin, Novartis); Lipitor (atorvastatin, Parke- Davis); Baycol (cerivastatin, Bayer); Crestor (rosuvastatin, Astra-Zeneca); and Advicor (lovastatin plus extended release Niacin, Kos Pharmaceutical). In some embodiments, the anti-inflammatory agent is not a statin. In some embodiments, the anti-inflammatory agent is aspirin. The invention further provides kits including an anti-inflammatory agent and instructions (e.g., on a label or package insert such as instructions to the subject or to the clinician) for administering the anti-inflammatory agent to a subject in order to treat, prevent, and/or delay the development or progression of AMD. The anti- inflammatory agent can be in a pharmaceutical composition also including a pharmacologically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. A pharmaceutical composition is typically formulated to be compatible with its intended route of administration, e.g., oral, mucosal, topical, transdermal, or parenteral. Supplementary active compounds can also be incorporated into the compositions. In some embodiments, the anti- inflammatory agent is in a form suitable for local delivery to the macular area, e.g., an implantable form. An effective amount is a dosage of the anti-inflammatory agent sufficient to provide a medically desirable result. The effective amount will vary with the particular condition being treated, the age and physical condition of the subject being treated, the severity of the condition, the duration of the treatment, the nature of the concurrent therapy (if any), the specific route of administration and the like factors within the knowledge and expertise of the health practitioner. For example, an effective amount can depend upon the degree to which a subject has abnormally elevated levels of markers of systemic inflammation. In some embodiments, the anti- inflammatory agents of the invention are used to prevent the development or progression of AMD, that is, they are used prophylactically in subjects at risk of developing AMD, or in subjects that already have AMD but whose AMD is likely to progress, e.g., to a more severe form of the disease. Thus, an effective amount is that amount which can lower the risk of, slow or perhaps prevent altogether the development or progression of AMD. In some embodiments, the anti-inflammatory agent is administered in an effective amount, e.g., in an amount effective to reduce levels of the marker of systemic inflammation, e.g., to reduce the levels of the marker to place the subject in a lower risk category. For example, where the marker of systemic inflammation is CRP, an amount effective to treat or prevent the development or progression of AMD would be an amount sufficient to reduce CRP levels, e.g., to below 6.5 mg/L, below 4.5 mg/L, below 2.7 mg/L or below 1.7 mg/L. Generally, doses of active compounds can be from about 0.01 mg/kg per day to 1000 mg/kg per day. It is expected that doses ranging from 50-500 mg/kg will be suitable, typically administered orally, and in one to three (or more) administrations per day. Lower doses may result from other forms of administration, such as intravenous administration. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that subject tolerance permits. Multiple doses per day are contemplated to achieve appropriate systemic levels of compounds. The dosage and schedule will depend on the anti-inflammatory agent selected; a skilled practitioner would be able to select a regimen appropriate for the particular agent and subject. A number of anti-inflammatory agents are known in the art, and can be used in the methods described herein. A variety of administration routes are available. The particular mode selected will depend upon the particular drug selected, the severity of the condition being treated and the dosage required for therapeutic efficacy. The methods described herein, generally speaking, can be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of the active compounds without causing clinically unacceptable adverse effects. Such modes of administration include oral, rectal, topical, nasal, transdermal, or parenteral routes. The term "parenteral" includes subcutaneous, intravenous, intramuscular, or infusion. Local administration to the macular area can also be used, hi some embodiments, the invention includes the use of implantable formulations, e.g., anti-inflammatory agents that are contained in a slow-release formula that can be implanted at or near the site of inflammation. Oral administration will typically be used for prophylactic treatment because of the convenience to the subject as well as the dosing schedule. A number of oral compositions are known in the art and can be used in the methods described herein. The delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the anti-inflammatory agent, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Pat. No. 5,075,109. Delivery systems can also include non-polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-, di- and tri-glycerides; hydro gel release systems; sylastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include, but are not limited to erosional systems in which the anti-inflammatory agent is contained in a form within a matrix such as those described in U.S. Pat. Nos. 4,452,775, 4,667,014, 4,748,034 and 5,239,660, and diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Pat. Nos. 3,832,253, and 3,854,480. Pump-based hardware delivery systems can be used, some of which are adapted for implantation, i addition, U.S. Pat. No. 6,331 ,313 describes a biocompatible ocular drug delivery implant device that can be used to deliver anti-inflammatory agents directly to the macular region. Use of a long-term sustained release implant may be particularly suitable for treatment of chronic conditions. Long-term release means that the implant is constructed and arranged to delivery therapeutic levels of the active ingredient for at least 30 days, e.g., 60 days. Long-term sustained release implants are known to those in the art and include some of the release systems described herein. Thus, the invention includes the use of an anti-inflammatory compound to treat, delay or prevent the development or progression of AMD. The invention further includes the use of an anti-inflammatory compound to modulate CRP levels, thereby treating, delaying or preventing the development or progression of AMD. Finally, the invention includes the use of an anti-inflammatory agent in the preparation of a medicament for use in the treatment of AMD. The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES Example 1 : Association Between Markers of Systemic Inflammatioii and Age- Related Macular Degeneration (AMD) Study Population The data described herein are a result of an ancillary study to the Age-Related Eye Disease Study (AREDS). AREDS is a prospective cohort study designed to assess the incidence, clinical course, prognosis and risk factors for AMD and cataract Details of the AREDS design have been elsewhere (16). For the present study, the Massachusetts Eye and Ear Infirmary (MEEI) and Devers Eye Institute (Devers) enrolled 1,026 subjects (517 and 509 respectively) into the AREDS clinical trial. Between January 1996 and April 1997, 930 subjects (91%) had a blood specimen drawn after randomization for this ancillary study, 465 at each clinic. All but 1.3% of the specimens were obtained after fasting by the subject for at least 8 hours. Blood samples were processed immediately and then frozen in liquid nitrogen freezers at minus 140 degrees Centigrade. The study was approved by the Human Subjects' Committees of the two clinical centers and all subjects signed an informed consent statement. Subjects were aged 55 to 80 at the time of enrollment. They were required to be in overall good health and were excluded if they had diseases with a poor seven- year survival prognosis (e.g., end stage cancer, advanced heart disease); hemochromatosis or Wilson's disease; oxalate kidney stone, alcoholism or drug abuse; or were unwilling or unable to discontinue non-study antioxidant vitamin or zinc supplementation. Subjects were also excluded if they had visual acuity of less than 20/32 in both eyes, advanced AMD, laser photocoagulation for AMD in both eyes, bilateral cataract extraction without signs of AMD, other eye diseases that would potentially compromise the evaluation of study outcomes, or if they used medications known to be toxic to the lens or retina. Subjects were examined at six-month intervals, at which time information was collected on changes in visual acuity, disease incidence and progression, and risk factors from a visual acuity test, dilated lens and fundus examination, and a clinical interview. Baseline levels were established at the first visit. In addition, at the annual visit (occurring 12, 24, 36, etc. months after randomization), serum was drawn for specified AREDS tests, fundus and lens photographs were taken (except at the first annual visit), and a refraction was completed. Since the end of the clinical trial in April 2001, subjects have been examined annually.

Case-control Definitions Case-control definitions were adopted from a previous AREDS publication

(8). According to reading center grading of fundus photographs at the visit most closely associated with the specimen draw, subjects in this ancillary study were divided into four maculopathy groups according to the size and extent of drusen in each eye, presence of geographic atrophy, and neovascular disease. These groups, numbered serially and based on increasing severity of drusen or type of AMD, were defined as follows. Group 1 (No Drusen). (n=183) Each eye had no drusen or nonextensive small drusen, no pigment abnormalities, no advanced AMD, and no disqualifying ocular conditions. Most subjects had visual acuity of 20/32 or better in both eyes. Group 2 (Intermediate Drusen). (n=200) At least one eye had one or more intermediate-size drusen, extensive small drusen, or pigment abnormalities associated with AMD. Neither eye had large drusen, advanced AMD, or a disqualifying ocular condition. Most subjects had visual acuity of 20/32 or better in both eyes. Group 3 (Large Drusen or Intermediate AMD). (n=325) At least one eye had either one or more large sized drusen, about 20 intermediate-size soft drusen, or about 65 intermediate-size hard drusen. Neither eye had advanced AMD, a disqualifying ocular condition, or presence of geographic atrophy with diameter at least one eighth of that of the average disc, and most subjects had visual acuity of 20/32 or better in both eyes. Also included were subjects in whom one eye met these criteria and the fellow eye had either a disqualifying ocular condition or visual acuity of 20/32 or less not due to AMD. Group 4 (Geographic Atrophy or Neovascular AMD - advanced AMD).

(n=222) At least one eye had geographic atrophy definitely present (with diameter at least one eighth of that of the average disc; n=58) or neovascular AMD (further defined below; n=164). In most cases, there was a fellow eye with visual acuity of 20/32 or better with no evidence of advanced AMD or a disqualifying ocular condition. Neovascular AMD included choroidal neovascularization or retinal pigment epithelial (RPE) detachment in one eye (non-drusenoid RPE detachment, serous sensory or hemorrhagic retinal detachment), subretinal hemorrhage, subretinal pigment epithelial hemorrhage, subretinal fϊbrosis, or evidence of confluent photocoagulation for neovascular AMD. The term "neovascular" is used as a summary term for this group of subjects because most subjects in this group have direct evidence of choroidal neovascularization based on the assessment of fundus photographs. A few subjects in this group have serous RPE detachments. The AREDS clinical trial (Seddon et al., Arch Ophthalmol. 2003;121 :1728- 1737) showed that rates of progression to advanced AMD in Groups 1 and 2 were very low (5-year rates of 0.5% and 1.3%, respectively), and they were therefore combined here into one larger control group. For regression analyses, to enhance statistical power, Group 3 (5-year rate of about 18%) was combined with Group 4 (5- year rate of about 43%) to form the case group. CRP Analysis Serum samples were thawed and assayed for CRP. C-reactive protein was measured with a high-sensitivity assay as in studies of cardiovascular disease (Ridker et al., JAMA 2001;285:2481-2485; Ridker, Circulation 2003;107:363-369). The concentration of CRP was determined using an immunoturbidimetric assay on the Hitachi 911 analyzer (Roche Diagnostics, Indianapolis, IN), using reagents and calibrators from Denka Seiken (Niigata, Japan). In this assay, an antigen-antibody reaction occurs between CRP in the sample and an anti-CRP antibody that has been sensitized to latex particles, and agglutination results. The resulting antigen-antibody complex causes an increase in light scattering, which was detected specfrophotometrically, with the magnitude of the change being proportional to the concentration of CRP in the sample. This assay had a sensitivity of 0.003 mg/dL. The coefficients of variation of the assay at concentrations of 0.91, 3.07, and 1.338 mg/L were 2.81, 1.61 and 1.1%, respectively.

Apolipoprotein B Analysis This assay was performed by an immunoturbidimetric technique on the Hitachi 911 analyzer (Roche Diagnostics - Indianapolis, IN), using reagents and calibrators from Wako (Wako Chemicals USA - Richmond, VA). Polyclonal anti-apo B antibodies agglutinate with apo B present in the serum sample and form a complex. This agglutination was detected specfrophotometrically, with the magnitude of the change being proportional to the concentration of apoB in the sample. The day-to-day variabilities at apoB concenfrations of 42.6, 88.3 and 132.8 mg/dL were 5.1, 3.9 and 4:0%), respectively.

Interleukin-6 (IL-6) Analysis IL-6 was measured by an ultra-sensitive ELISA assay from R & D Systems. The assay employed the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for IL-6 was been pre-coated onto a microtitre plate. After the addition of samples, standards, controls and conjugates to the wells, IL-6 was sandwiched between the immobilized antibody and the enzyme-linked antibody specific to IL-6. Upon the addition of substrate, a color was generated that is proportional to the amount of IL-6 present in the sample. The minimum required volume for this assay was 200 uL. The assay had a sensitivity of 0.094 pg/mL, and the day-to-day variabilities of the assay at concentrations of 0.66, 1.97 and 8.16 pg/mL were 12.2, 7.6 and 9.9%, respectively. Lipoprotein (a) Analysis The concentration of Lp(a) was determined using a turbidimetric assay on the Hitachi 911 analyzer (Roche Diagnostics - Indianapolis, IN), using reagents and calibrators from Denka Seiken (Niigata, Japan). In this assay, an antigen-antibody reaction occurs between Lp(a) in the sample and an anti-Lp(a) antibody adsorbed to latex particles, and an agglutination results. This agglutination was detected specfrophotometrically, with the magnitude of the change being proportional to the concentration of Lp(a) in the sample. This method is the only commercial assay that is not affected by the Kringle Type 2 repeats (Marcovina SM et al. Use of a Reference Material Proposed by the International Federation of Clinical Chemistry and Laboratory Medicine to Evaluate Analytical Methods for the Determination of Plasma Lipoprotein(a). Clin Chem 2000;46:1956-67). The day-to-day variabilities at Lp(a) concentrations of 17.6 and 58.1 mg/dL were 3.6 and 1.5%, respectively.

Soluble intercellular adhesion molecule- 1 (sICAM-1 Analysis sICAM was measured by an ELIS A assay (R & D Systems, Minneapolis, MN). The assay employed the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for sICAM-1 was pre-coated onto a microtitre plate. After the addition of samples, standards, controls and conjugates to the wells, sICAM was sandwiched between the immobilized antibody and the enzyme-linked antibody specific to sICAM. Upon the addition of substrate, a color was generated that is proportional to the amount of sICAM present in the sample. The minimum required volume for this assay was 25 uL. The assay had a sensitivity of 0.35 ng/mL and the day-to-day variabilities of the assay at concentrations of 64.2, 117, 290 and 453 ng/mL were 10.1, 7.4, 6.0 and 6.1%, respectively.

Soluble vascular cell adhesion molecule- 1 (sNCAM-1 Analysis sNCAM was measured by an ELISA assay (R & D Systems, Minneapolis,

MΝ). The assay employed the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for sNCAM-1 was pre-coated onto a microtitre plate. After the addition of samples, standards, controls and conjugates to the wells, sNCAM was sandwiched between the immobilized antibody and the enzyme-linked antibody specific to sNCAM. Upon the addition of substrate, a color was generated that was proportional to the amount of sNCAM present in the sample. The minimum required volume for this assay was 25 uL. The assay had a sensitivity of 2.0 ng/mL. The day-to-day variabilities of the assay at concentrations of 9.8, 24.9 and 49.6 ng/mL were 10.2, 8.5 and 8.9%, respectively.

TΝF-alpha-receptor II Analysis TNF-alpha RII was measured by an ELISA assay from R & D Systems. The assay employed the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for TNF-alpha RII was pre-coated onto a microtitre plate. After the addition of samples, standards, controls and conjugates to the wells, TNF-RII was sandwiched between the immobilized antibody and the enzyme-linked antibody specific to TNF-RII. Upon the addition of substrate, a color was generated that was proportional to the amount of TNF-RII present in the sample. The minimum required volume for this assay was 50 uL. The day-to-day variabilities of the assay at concentrations of 89.9, 197, and 444 pg/mL were 5.1, 3.5, and 3.6%, respectively. Risk Factor Definitions Interviews to determine general risk factor and dietary information were conducted at the baseline (first) visit, and slitlamp biomicroscopy and ophthalmoscopy were performed at the time of the blood drawing. The baseline risk factor variables that were considered in the analyses fall into five classes: demographic, medical, dietary/supplementation, use of medication, and ocular factors. For analysis, continuous variables (body-mass index, weight change from age 20 and sunlight exposure) were categorized by quartiles or tertiles based on the group without drusen (Group 1). Demographic. The demographic variables included age, sex, race, education, and sunlight exposure (adult lifetime average annual ocular ultraviolet B exposure), adapted from McCarty et al (McCarty et al., Bull World Health Organ 1996;74:353- 360). Medical. Medical variables included history of smoking, body mass index (BMI), weight change (increase or decrease) since age 20, hypertension (systolic > 160 mmHg, diastolic > 90 mmHg, or current use of antihypertensive medication), history of cardiovascular disease (at least one of the following: newly-developed heart disease after enrollment but prior to blood draw; occurrence of a stroke or myocardial infarction ( developed after enrollment, but prior to blood draw); history of angina and taking an angina medication (e.g., dipyridamole, propranolol, beta-blocker, calcium- channel blocker, nitro glycerin, or isobide dinifrate); taking a heart disease medication (e.g., furosemide, ACE inhibitor, digoxin, blood thinning medication, cholesterol- lowering medication]), diabetes ( e.g., under treatment for diabetes), and arthritis. Dietary/Supplementation. The dietary/supplement variables included an antioxidant index and use of study treatment containing antioxidants. The antioxidant index was based on dietary results from a modified Block Food Frequency questionnaire (AREDS Manual of Operations) completed at the subj ect' s baseline visit. Three measures were assessed: carotenoid intake (alpha-carotene, beta- carotene, lutein, lycopene, and beta-cryptoxanthin), vitamin C intake, and vitamin E intake. Subjects were grouped as having high antioxidant intake (above the highest quartile of intake for two out of the three measurements), low antioxidant intake (below the lowest quartile of intake for two out of the three measurements), or mixed antioxidant intake. Subjects randomized to receive the study supplements containing high-dose antioxidants or high-dose antioxidants and zinc comprised the antioxidant treatment group. Subjects randomized to receive the study supplements containing zinc or placebo comprised those not in the antioxidant treatment group. Use of Medication. Use of medication was defined as current use with five or more lifetime years of regular use. These medications included use of hydrochlorothiazide, diuretics (other than hydrochlorothiazide), aspirin, antacids, nonsteroidal anti-inflammatory drugs, thyroid hormones, beta-blockers, and estrogen and progesterone use (women). Ocular. Ocular variables included iris color and refractive error. Iris color was graded at the reading center by comparing photographs of each eye with standards on a scale from 1 (light or blue) to 4 (dark or brown); a subject was 'light' if both eyes were code 1, 'dark' if both eyes were code 4, 'mixed' if at least one eye was code 2 or code 3 or eyes were not of the same code. A subject was 'myopic' if both eyes were myopic by -1.0 diopters spherical equivalent refractive error or more, 'hyperopic' if both eyes had +1.0 diopters spherical equivalent refractive error or more, or else 'other' which includes emmetropes and mixed cases. Statistical Modeling and Analysis The median values and interquartile ranges of CRP were calculated for each maculopathy group, and the most advanced AMD grade was compared with Group 1 using a non-parametric test of all p values. Conditional logistic regression analysis (SAS procedure LOGISTIC; version 8.02, SAS Institute, Inc. Cary, NC) was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) after the population was divided into quartile groups according to the quartile cut points of CRP values for the disease-free Group 1 (Ridker et al., JAMA 2001;285:2481-2485; Ridker, Circulation 2003;107:363-369). Prevalence odds ratios, which describe the association between disease and CRP (comparing cases in Groups 3 and 4 with controls in Groups 1 and 2), were computed for each CRP quartile group relative to the lowest quartile group. A test for linear trend was calculated based on the median levels of CRP within each quartile group. Multivariate ORs were estimated from conditional logistic regression models, and were adjusted for age (57-65, 66-70, 71-83), sex, race, (white vs. other), smoking (ever smoked vs. never smoked), education (never completed high school, high school graduate, some college, or college graduate), body mass index (measured a weight in kilograms divided by height in meters squared; <23.9, 23.9-29.9, >29.9), antioxidant index (low, mixed, high), diabetes, history of cardiovascular disease (CVD), hypertension, and antioxidant treatment (taking study supplement containing antioxidants vs. taking study supplement containing no antioxidants). History of CVD and hypertension were not correlated. Other risk factors were evaluated as potential covariates but did not reach statistical significance in this ancillary study population (including weight change from age 20, sunlight exposure, arthritis, anti- inflammatory drugs, thyroid hormones, beta-blocker use, hormone use (women), iris color, and refractive error). To evaluate a possible threshold effect, additional analyses were performed comparing cases with controls according to levels of CRP for Group 1 above and below the 90^th percentile, and above and below the mean CRP plus 2 standard deviations. Finally, to evaluate effect modification by cigarette smoking, additional logistic regression analyses were conducted to determine the ORs for AMD in 6 subgroups defined by never and ever smoking, and low, intermediate, and high tertiles of CRP. Baseline characteristics Of the 930 subjects in this study, 61% were female and 39% were male, with a mean age of 69 years. The most of subjects (71%) had some college or higher education. Forty-one percent of subjects had never smoked, 51%₀ were former smokers, and 8% were current smokers. The median CRP value for all subjects was 2.7 mg/L, with a range of 0.2 to 117 mg/L, and the 90^th percentile range was 0.2 to 10.6 mg/1. CRP values did not differ according to age groups (55-65, 66-70, and 71+). Table 1 displays the relationships between baseline characteristics and maculopathy groups, unadjusted for other variables. Significant differences (p<0.05) between subjects in maculopathy Groups 3 and 4 as compared to control subjects in Groups 1 and 2 included sex (lower proportion of females), smoking status (lower proportion of never smokers), and education (lower proportion with a college degree).

Data are No. (%) unless otherwise indicated. ** Adult lifetime average annual ocular UV-B exposure (for reference, 1.0 = ocular exposure from model is equivalent to 1 day outdoors in temperate months in the United States, not over water, and not wearing hat, glasses, or sunglasses) . §Νumbers reported use data from the "Female" row as denominators to accurately report the percentages. ^t Antioxidant index based on dietary results from a modified Block Food Frequency questionnaire (AREDS Manual of Operations). Three measures were assessed: carotenoid intake (alpha-carotene, beta-carotene, lutein, lycopene and beta-cryptoxanthin), vitamin C intake, and vitamin E intake. Subjects were grouped as having high antioxidant intake (above the highest quartile of intake for two out of the three measurements), low antioxidant intake (below the lowest quartile of intake for two out of the three measurements), or mixed antioxidant intake. * Study treatment containing antioxidant formulation (vitamin C, vitamin E, beta-carotene). ^§ History of cardiovascular disease defined by at least one of the following: Newly-developed heart disease after enrollment, but prior to blood draw; occurrence of a stroke or myocardial infarction after enrollment, but prior to blood draw; history of angina and taking an angina medication (dipyridamole, propranolol, beta-blocker, calcium-channel blocker, nitroglycerin, or isobide dinitrate); taking a heart disease medication (furosemide, ACE inhibitor, digoxin, blood thinning medication, cholesterol- lowering medication). " Iris color was graded on a scale of 1 (light or blue) to 4 (dark or brown); a subject was grouped as either light (if both eyes were code 1), dark (if both eyes were code 4) or mixed (at least one eye was • code 2 or code 3 or eyes were not of the same code). ¹¹ A subject who was myopic by -1.0 diopters spherical equivalent refractive error or more was considered myopic; if both eyes had +1.0 diopters spherical equivalent refractive error, a subject was hyperopic; emmetropes and mixed cases are grouped as other.

Example 2: Association between Maculopathy and C-Reactive Protein (CRP ) Median baseline plasma levels of the inflammatory marker CRP were higher among subjects who had more severe maculopathy (Table 2). The difference between the median value for the most advanced maculopathy Group 4 ( 3.4 mg/L) and the median for maculopathy group 1 (2.7 mg/L) was statistically significant (p=0.02).

*Non-ρarametric test of all CRP values.

Table 3 displays the odds ratios for risk of AMD according to the quartile of CRP, for maculopathy case Groups 3 and 4 as compared with Groups 1 and 2, after adjustment for various other known and potential factors associated with AMD. In an age- and sex-adjusted model, subjects above the highest quartile of CRP had higher risk of AMD (OR, 1.53; 95% confidence interval (CI), 1.03-2.28). The trend for an increase in risk of maculopathy with increase in CRP was statistically significant (p=0.02). After adjustment for additional covariates, the significant trend for an increase in risk remained for the highest quartile of CRP (OR, 1.65;95% CI, 1.07-2.55, p for trend = 0.02). In a separate analysis, Group 4 maculopathy was compared with that of Group 1. The effect estimate was similar for the highest level of CRP (OR, 1.72;9% CI, 0.88-3.38 ) but was not significant, possibly due to the reduced sample size (p for trend= 0.09).

Table 3. Sample Sizes, CRP Levels, and ORs* for Prevalence of Age-Related Macular De eneration Accordin to Quartile of CRP*

CI, confidence interval; CRP, C-reactive protein; OR, odds ratio. *ORs: comparison between maculopathy groups 3 and 4 (cases) and groups 1 and 2 (controls). **P = 0.02 for trend across quartiles. ^§ Adjusted for age (57-65, 66-70, and 71-83 years), sex, race (white vs. other), smoking (ever smoked vs. never smoked), education (never completed high school, high school graduate, some college, or college graduate), body mass index (<23.9, 23.9-29.9, >29.9), antioxidant index (low, mixed, high), diabetes, history of cardiovascular disease, hypertension, and antioxidant treatment (taking study supplement containing antioxidants vs. taking study supplement containing no antioxidants).

Table 4 displays the association between CRP and maculopathy, using different outpoints for values of CRP. Subjects with CRP levels above the 90th percentile had a significantly increased risk, with an OR of 1.75 (95% CI, 1.12, 2.75) for the age- and sex-adjusted model and an OR of 1.92 (95% CI, 1.20-3.06) for the full multivariate model. Subjects with CRP values more than two standard deviations above the mean were also at increased risk with an OR of 1.89 (95% CI, 0.98-3.66) for the age- and sex-adjusted model and an OR of 2.03 (95% CI, 1.03-4.00) for the full multivariate model. Table 4. Sample Sizes, CRP Levels, and ORs* for Prevalence of Age-Related Macular De eneration Accordin to Cut Points of CRP

CI, confidence interval; CRP, C-reactive protein; OR, odds ratio. *ORs: comparison between maculopathy groups 3 and 4 (cases) and groups 1 and 2 (controls). **Adjusted for age (57-65, 66-70, and 71-83 years), sex, race (white vs. other), smoking (ever smoked vs. never smoked), education (never completed high school, high school graduate, some college, or college graduate), body mass index (<23.9, 23.9-29.9, >29.9), antioxidant index (low, mixed, high), diabetes, history of cardiovascular disease, hypertension, and antioxidant treatment (taking study supplement containing antioxidants vs. taking study supplement containing no antioxidants).

These results demonstrate that elevated CRP levels are associated with risk of developing AMD. Thus, CRP levels can be used to predict a subject's risk of developing AMD. In addition, lowering levels of systemic inflammation, e.g., by administering an anti-inflammatory agent, can be used to treat, prevent, or delay progression or development of AMD.

Example 3: Effect Modification by Smoking To determine whether the effect of CRP was modified by cigarette smoking, a consistently strong risk factor for AMD, OR's were computed for AMD in analyses in which subjects were stratified into six groups according to smoking (ever or never) and fertile of CRP, as shown in Table 5. For smokers and never-smokers, higher levels of CRP were associated with higher risk of AMD. Subjects in the high-risk group (current and past smokers with the highest level of CRP) had a statistically significant 2.16-fold higher risk (95% CI, 1.33-3.49) of maculopathy compared with the low risk group (those who never smoked and had the lowest CRP level), after adjustment for other factors. In light of the clinical observation that many subjects who do not smoke nonetheless develop maculopathy, it is interesting to note that among never smokers, the odds of developing AMD were 2.03 in the highest fertile of CRP (95% CI 1.19, 3.46), compared to those in the lowest quartile of CRP, after adjustment for other factors. To evaluate this relationship further, the effect of smoking (ever or never) stratified by tertile of CRP was analyzed. Cigarette smoking increased risk of AMD more than 1.7 fold in the lower two tertiles of CRP ORs 11.79 (95% CI, 1.06-3.00) and 1.90 (95% CI, 1.12-3.22),), but there was no association between smoking and CRP in the highest level of CRP (OR 1.02).

CI, confidence interval; CRP, C-reactive protein; OR, odds ratio. *Adjusted for age (57-65, 66-70, and 71-83 years), sex, race (white vs. other), education (never completed high school, high school graduate, some college, or college graduate), body mass index (<23.9, 23.9-29.9, >29.9), antioxidant index (low, mixed, high), diabetes, history of cardiovascular disease, hypertension, and antioxidant treatment (taking study supplement containing antioxidants vs. taking study supplement containing no antioxidants). These results demonstrate that the highest levels of CRP appear to increase risk of developing AMD, independent of smoking. Thus, CRP levels can be used to predict a subject's risk of developing AMD. In addition, lowering levels of systemic inflammation, e.g., by administering an anti-inflammatory agent, can be used to treat, prevent, or delay progression of AMD.

Example 4: Association between Inflammatory and Lipid Biomarkers and Advanced AMD In the prospective study described in this Example, the relative risk of development of more advanced AMD was evaluated in a cohort of about 261 subjects ( in the Progression of Age-Related Macular Degeneration Study, or "Progression Study") with early or intermediate stages of AMD, followed prospectively to determine risk and preventive factors for development of advanced AMD. The average follow-up time was 4.6 years. The methodology for this study was described previously; see Seddon et al., Arch Ophthalmol. 2003 Jun; 121(6):785-92 and Seddon et al., Arch Ophthalmol. 2003 Dec; 121(12):1728-37. Tables 6-8 illustrate the characteristics of the study population by quartile of levels of inflammatory biomarkers CRP, IL-6, TNF-alpha Receptor II, ICAM, and NCAM, and lipid biomarkers ApoB and LP(a).

Table 6. Characteristics of Progression Study Population by Quartiles of CRP,

Physica ctivity = Mean number o t mes wee vigorous act v ty. Table 7. Characteristics of Study Population by Quartiles of ICAM, VCAM, and LP(a) Biomarker ICAM VCAM LP(a) QUARTILE 1 2 3 4 1 2 3 4 1 2 3 4 61 62 63 63 60 63 63 63 61 64 64 62 Age (y) 72 72 71 73 71 72 72 74 73 71.5 73 72 Male (%) 36 39 38 38 63 62 62 62 38 39 37.5 39 Education (%) 95 84 83 89 93 87 87 82.5 88.5 97 87.5 77 Initial Worse Eye Grade 2 Grade 3 8 10 7 9 11 3 10 10 6 7 15 7 Grade 4 23 23 31 18 26 29 26 14 20 28 21 25 Grade 5 10 7 10 11 7 11 6 14 15 12 1 10 20 22 15 25 16 20 21 25 20 17 27 20 Physical Activity 1.6 1.9 1.4 1.9 1.8 1.8 1.9 1.3 1.6 1.6 2.2 1.2 (mean) Current smoker 2 10 16 10 8 10 14 5 10 12.5 3 11 (%)

Past Smoker (%) 59 52 51 65 57 51 54 65 57 52 64 52 Systolic BP (mmHg) (mean) 138 136 139 142 133 139 139 144 142 137 137 138 Cardiovascular Disease 11.5 21 17.5 29 10 24 19 25 13 19 25 23 (Yes) (%) ¹ Fish Intake (%) < l/wk 31 39 36.5 38 40 35 36.5 33 34 41 23 47 > 1/wk, <2/wk 31 29 38 30 25 33 33 36.5 38 27 34 31 >2/wk 38 32 25 32 35 32 30 30 28 33 42 23 BMI, Kg/m² 27 28 28 28 26 27 28 29 28 27 27 28 Alcohol, g/day 8 7.5 7 7 10.6 7 7 5 7.28 11 5.8 4.9 Calories 1374 1360 1375 1426 1413 1347 1323 1456 1338 1348 1411 1466 . Beta carotene 3115 4229 3444 3255 3928 3531 3225 3323 3701 3649 3310 3160 intake* (IU) Zinc* (mg) 17 14 15 17 15 15 15 17 13 17 16 16

Vitamin C* (mg) 210 242 219 219 238 195 238 221 208 215 241 223

Vitamin E* (mg) 30 31 16 23 28 21 21 28 21 29 23 23

* Geometric mean after sex-specific calorie-adjustment; other values are means or percents. Education = Percent with at least high school education. Physical Activity = Mean number of times/week vigorous activity.

Table 8. Characteristics of Study Population by Quartiles of ApoB Biomarker ApoB QUARTILES 1 2 3 4 N 62 64 62 63 Age (y) 73 71 72 72 Male (%) 37 37.5 40 38 Education (%) 92 86 87 86 Initial Worse Eye Grade 2 8 7 10 10 Grade 3 16 29 30 19 Grade 4 13 8 10 7 Grade 5 25 20 12 27 Physical Activity (mean) 1.4 2.2 1.8 1.3 Current smoker (%) 8 3 13 13 Past Smoker (%) 60 48 56 60 Systolic BP (mmHg) (mean) 137 138 140 140 Cardiovascular Disease (Yes)(%) 16 22 21 21 Fish Intake < l/wk 47 31 29 38 > 1/wk, <2/wk 24 36 35.5 33 >2/wk 29 33 35.5 29 BMI, Kg/m² 27 27 29 28 Alcohol, g/day 7 8 4 10 Calories 1375 1443 1451 1296 Beta carotene intake* (IU) 3688 3584 2937 3629 Zinc* (mg) 18 15 15 14 Vitamin C* (mg) 249 233 193 216 Vitamin E* (mg) 33 20 22 23 * Geometric mean after sex-specific calorie-adjustment; other values are means or percents. Education = Percent with at least high school education. Physical Activity = Mean number of times/week vigorous activity.

Table 9 illustrates the relative risk (RR, the risk of developing advanced AMD relative to the first, lowest quartile) for progression to advanced AMD by quartile of a number of biomarkers. Table 9. Relative Risks for Progression to Advanced AMD by Quartiles of biomarkers.

* adjusted for age-sex group (60-69Male/70-79Male/80+Male/60-69Female/70- 79Female/80+Female), log calories (continuous), and protein intake (quartile) ** adjusted for age-sex group (60-69Male/70-79Male/80+Male/60-69Female/70-

79Female/80+Female), education ( ≥high school vs. < high school), smoking (current/past/never), BMI (<25/25-29.9/30+), systolic blood pressure, cardiovascular disease, log calories (continuous), protein intake (quartile), calorie-adjusted beta carotene intake (continuous), alcohol intake (continuous), physical activity (continuous-times/wk vigorous), and initial AMD grade (categorical). t adjusted for variables in model 1 plus total intake of zinc, vitamin C and vitamin E. Table 10 illustrates the relative risk (RR) for progression to advanced AMD by quartile of two lipid biomarkers.

Table 10. Relative Risks for Progression to Advanced AMD by Lipid Biomarkers

* adjusted for age-sex group (60-69Male/70-79Male/80+Male/60-69Female/70- 79Female/80+Female), log calories (continuous), and protein intake (quartile) ** adjusted for age-sex group (60-69Male/70-79Male/80+Male/60-69Female/70- 79Female/80+Female), education ( ≥high school vs. < high school), smoking (current past never), BMI (<25/25-29.9/30+), systolic blood pressure, cardiovascular disease, log calories (continuous), protein intake (quartile), calorie-adjusted Beta carotene intake (continuous), alcohol intake (continuous), physical activity (continuous-times/wk vigorous), and initial AMD grade (categorical), f adjusted for variables in model 1 plus total intake of zinc, vitamin C and vitamin E.

These data demonstrate that there is a relationship between inflammatory markers and development of more advanced AMD, in particular CRP and IL-6, and to some extent V-CAM, and that there is a slightly increased risk with the lipid biomarker ApoB. Thus, these makers can be used to determine a subject's risk of progressing to advanced AMD. In addition, lowering levels of systemic inflammation, e.g., by administering an anti-inflammatory agent, can be used to prevent or delay progression to advanced AMD. Example 4: Effect Modification by Intake of Linoleic Acid ^■ In this study, the effect of levels of intake of linoleic acid was evaluated. Table 11 illustrates a modification of the effect of biomarkers depending on the level of linoleic acid intake, which is an omega-6 fatty acid. Higher levels of linoleic acid in combination with elevated levels of CRP are associated with an even greater risk of developing AMD than for elevated levels of CRP alone (relative risks are 3.80 and 2.30 in different statistical models). Previous reports on dietary fat and AMD show that omega-3 fatty acids (e.g., in foods and in fish) have a protective effect only when linoleic acid intake levels are below the median, e.g., in the lower two quartiles (Seddon et al, Arch Ophthalmol 2001;119:1191-1199; Seddon et al., Arch Ophthalmol. 2003;121:1728-1737).

Table 11. Relative Risks for Progression to Advanced AMD by Quartile of CRP Within Strata of Linoleic Acid Intake

* adjusted for age-sex group (60-69Male/70-79Male/80+Male/60-69Female/70- 79Female/80+Female), log calories (continuous), and protein intake (quartile). ** adjusted for age-sex group (60-69Male/70-79Male/80+Male/60-69Female.70- 79Female/80+Female), education ( ≥high school vs. < high school), smoking (current/past never), BMI (<25/25-29.9/30+), systolic blood pressure, cardiovascular disease, log calories (continuous), protein intake (quartile), calorie-adjusted Beta carotene intake (continuous), alcohol intake (continuous), physical activity (continuous-times/wk vigorous), and initial AMD grade (categorical). f adjusted for variables in model 1 plus total intake of zinc, vitamin C and vitamin E. RR's (or p-values) in boldface are significant at p=.05 level These data demonstrate that the adverse effect associated with high CRP levels is enhanced in the higher quartiles of linoleic acid intake. Thus, CRP levels can also be used in combination with linoleic acid intake levels to predict a subject's risk of progressing to advanced AMD. In addition, reducing the subject's levels of linoleic acid intake can be used to prevent or delay the progression to advanced AMD. Furthermore, lowering intake of linoleic acid in combination with lowering CRP levels, e.g., by administering an anti-inflammatory agent, can be used to prevent or delay progression to advanced AMD.

Example 5: Association between CRP Levels and Onset of Advanced AMD Prospective analysis of CRP levels was performed using the same population of subjects and methodology described in Example 1 (the AREDS study). Tables 12- 14 show an increased relative risk (1.34 overall, 1.28 for neovascular disease, and 1.68 for geographic atrophy) for onset of advanced AMD. Table 15 summarizes the unadjusted risk ratios for AMD events by CRP quartile. Table 12: Unadjusted Risk Ratios for AMD Events by CRP Quartile and Presence of Advanced AMD at the Time of Blood Draw: Neovascular Events

Table 13: Unadjusted Risk Ratios for AMD Events by CRP Quartile and Presence of Advanced AMD at the Time of Blood Draw: CGA Events

Table 14: Unadjusted Risk Ratios for AMD Events by CRP Quartile and Presence of Advanced AMD at the Time of Blood Draw: All Events

Table 15: Summary of Unadjusted Risk Ratios for AMD Events By CRP Quartile

These data demonstrate that assays of CRP levels can be used to determine a subject's risk of progressing to advanced AMD. In addition, lowering CRP levels, e.g., by administering an anti-inflammatory agent, can be used to prevent or delay progression to advanced AMD. Example 6: Association between Maculopathy and Inflammatory and Lipid Biomarkers The case control study described in this example evaluated the odds of developing AMD in the AREDS population as described in Example 1. These results show an increased risk of AMD for a number of inflammatory biomarkers, including N-CAM (OR 1.6, Q4 vs. Ql), TΝF-alpha receptor II (OR 1.8, Q4 vs. Ql), and possibly IL-6 (1.78, mean ± 2 standard deviations cut-off).

Table 16: Association between Maculopathy and Inflammatory and Lipid Biomarkers

Multivariate*+ CRP I 1.17 I 1.66 I 1.87 I 1.1 I 1.35 I 1.02

OR's (or p-values) in boldface are significant at p=.05 level

* Multivariate includes all variables in CRP manuscript plus thyroid hormones

These data demonstrate that there is a relationship between inflammatory markers and AMD, in particular CRP, TNF-alpha-RII, IL-6, and N-CAM, and that there is a slightly increased risk with the lipid biomarker lipoprotein (a) (Lp(a)). Thus, these makers can be used to determine a subject's risk of progressing to advanced AMD. In addition, lowering levels of systemic inflammation, e.g., by administering an anti-inflammatory agent, can be used to treat, prevent or delay progression or development of AMD .

OTHER EMBODIMENTS It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS :

1. A method for determining a subject's risk of developing age-related macular degeneration (AMD), the method comprising obtaining a level of a marker of systemic inflammation in the subject, comparing the level of the marker to a reference, and determining the subj ect's risk of developing AMD based upon the level of the marker in comparison to the reference.

2. A method for determining a subject's risk of progression of age-related macular degeneration (AMD), the method comprising obtaining a level of a marker of systemic inflammation in the subject, comparing the level of the marker to a reference, and determining the subject's risk of progression of AMD, based upon the level of the marker in comparison to the reference.

3. The method of claim 2, wherein the progression of AMD is progression to severe AMD.

4. A method for evaluating the likelihood that a subject will benefit from treatment with an anti-inflammatory agent for reducing the risk of development or progression of age-related macular degeneration, the method comprising obtaining a level of a marker of systemic inflammation in the subject, and comparing the level of the marker to a reference, wherein the level of the marker of systemic inflammation in comparison to the reference is indicative of whether the subject will benefit from treatment with the agent.

5. The method of any of claims 1 -4, wherein the marker of systemic inflammation is selected from the group consisting of C-reactive protein, IL-6, TNF-alpha receptor II, ICAM, and NCAM.

6. The method of any of claims 1 -4, wherein the marker of systemic inflammation is C-reactive protein.

7. The method of any of claims 1 -4, wherein the reference is a predetermined value.

8. The method of claim 7, wherein the predetermined value is selected from the group consisting of about 1.7 mg/1 of blood, about 2.7 mg/1 of blood, about 4.5 mg/1 of blood, and about 6.5 mg/1 of blood.

9. The method of claim 7, wherein the reference is a plurality of marker level ranges and the comparing step comprises determining in which of the marker level ranges the subjects level falls.

10. The method of claim 9, wherein one of the plurality of marker level ranges is below about 2.7 mg/1 blood, and another of the ranges is above about 2.7 mg/1 blood.

11. The method of claim 9, wherein one of the plurality of marker level ranges is below about 1.7 mg/1 blood, and another of the ranges is above about 1.7 mg/1 blood.

12. The method of claim 9, wherein one of the plurality of marker level ranges is below about 4.5 mg/1 blood and another of the ranges is above about 4.5 mg/1 blood..

13. The method of claim 9, wherein one of the plurality of marker level ranges is below about 6.5 mg/1 blood and another of the ranges is above about 6.5 mg/1 blood.

14. The method of claim 9, wherein one of the plurality of marker level ranges is ^" below about 2.7 mg/1 blood, another of the ranges is between about 2.7 mg/1 blood and about 6.5 mg/1 blood, and another of the ranges is above about 6.5 mg/1 blood.

15. The method of claim 9, wherein one of the plurality of marker level ranges is below about 1.7 mg/1 blood, another of the ranges is between about 1.7 mg/1 blood and about 4.5 mg/1 blood, and another of the ranges being above about 4.5 mg/1 blood.

16. The method of any of claims 1-15, wherein the subject is apparently healthy.

17. The method of any of claims 1-15, wherein the subject is a non-smoking subject.

18. The method of any of claims 1-15, wherein the subject is a smoker.

19. The method of any of claims 1 -9, wherein the level of the marker of systemic inflammation in the subject is elevated as compared to the reference.

20. The method of claim 19, wherein the elevated level indicates that the subject has an increased risk.

21. The method of any of claims 1-9, wherein the level of the marker of systemic inflammation in the subject is reduced as compared to the reference.

22. The method of claim 21, wherein the reduced level indicates that the subject has a reduced risk.

23. The method of claim 19, further comprising administering to the subject a therapeutically effective amount of an anti-inflammatory agent.

24. The method of claim 23, further comprising obtaining a second level of a marker of systemic inflammation in the subject, comparing the second level of the marker to a reference, and determining the subject's risk of developing AMD based upon the level of the marker in comparison to the reference.

25. The method of claim 24, wherein the reference is a level of a marker of systemic inflammation in the subject obtained previously.

26. A method of treating or preventing the development or progression of age-related macular degeneration (AMD) in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-inflammatory agent.

27. A method for determining a subject's risk of developing AMD or progression of AMD, the method comprising: obtaining a level of a marker of systemic inflammation in the subject, comparing the level of the marker to a first reference to establish a first risk value, obtaining a level of a second risk factor in the subject, comparing the level of the second risk factor to a second reference to establish a second risk value, and determining the subject's risk of developing AMD or progression of AMD based upon the combination of the first risk value and the second risk value.

28. The method of claim 27, wherein the combination of the first risk value and second risk value establishes a third risk value.

29. The method of claim 28, wherein the third risk value is greater than either of the first and second risk values.

30. The method of claim 27, wherein the marker of systemic inflammation is selected from the group consisting of C-reactive protein, IL-6, TNF-alpha receptor II, ICAM, and VCAM.

31. The method of claim 27, wherein the second risk factor is selected from the group consisting of family history of AMD, age, sex, smoking history, obesity, weight change since age 20, dietary fat intake, linoleic acid intake, and elevated cholesterol levels.

32. The method of claim 27, wherein the second risk factor is smoking history or linoleic acid intake.