CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] None.

STATEMENT OF FEDERALLY FUNDED RESEARCH

[0002] This invention was made with government support under AG039389, AG051848 awarded by National Institutes of Health. The government has certain rights in the invention.

TECHNICAL FIELD OF THE INVENTION

[0003] The present invention relates in general to the field of determining which Alzheimer’s disease patients are responsive or stable from NSAID or acetylcholinesterase (AchE) inhibitor treatment, are not likely to respond to NSAID or AChE inhibitor treatment, and those for which the NSAID or AChE inhibitor treatment is contra-indicated.

BACKGROUND OF THE INVENTION

[0004] Without limiting the scope of the invention, its background is described in connection with Alzheimer’s disease.

[0005] Currently, over 5 million Americans suffer from Alzheimer’s disease (AD; the most common form of neurodegenerative dementia)[l] and it is estimated that those numbers will grow exponentially. AD has an annual health care cost similar to that of cardiovascular disease (CVD) and more than cancer [2] While death rates due to CVD and cancer have declined in recent decades, death rates due to AD have steadily increased [1] due to a lack of effective disease-modifying therapeutics. AD is a heterogeneous condition and the identification of specific therapeutically relevant subpopulations could significantly enhance therapeutic options [3]

[0006] Profiling biological pathways associated with neurodegenerative disease has been posited to highlight novel pathways for therapeutics [4, 5], with inflammation being a major implicated pathway [6, 7] Numerous studies link inflammation to AD [8, 9] For example, in the inventor’s prior work it was found that inflammation played a key role in a serum- based proteomic profile for the detection of AD [10-12] Accordingly, a major hypothesis in the field is that anti-inflammatory compounds have the potential for treating AD and other neurodegenerative diseases [13, 16-21] However, none of several randomized clinical trials using NSAIDs has been successful in treating or preventing AD despite promising early phase clinical trial data [22-24] Therefore, a need remains for effective treatments that are customized to a particular patient’s response to AD. There is also a long-standing clinical awareness that cholinesterase inhibitors help some patients whereas they have no benefit or even a detrimental effect on others. However, to date, predicting these outcomes have been unsuccessful. It is also hypothesized that there is a subgroup of patients that will respond maximally to cholinesterase inhibitors and that inflammatory markers may identify these patients.

SUMMARY OF THE INVENTION

[0007] In one embodiment, the present invention includes a method for identifying a patient response to treatment for Alzheimer’s Disease with a non-steroidal anti-inflammatory drug (NSAID) comprising: obtaining a blood or serum sample from the patient; determining the presence of a proinflammatory endophenotype in the blood or serum sample of the patient; using the proinflammatory endophenotype to detect a treatment response (a responder, a stable, a non-responder or an adverse responder); and treating the patient with the NSAID if the patient is in the responder or the stable treatment response phenotype group; or preventing a treatment with the NSAID if the patient is a non-responder or an adverse responder. In one aspect, the proinflammatory endophenotypes is determined using CRP and TNFa. In another aspect, the proinflammatory endophenotype is determined using TNFa, CRP, IL6, and IL10 biomarkers. In another aspect, the proinflammatory endophenotype proteins assayed are: TNFa, CRP, IL5, IL6, IL7, IL10 and IL18. In another aspect, the treatment response is selected from a responder, a stable, a non-responder or an adverse responder. In another aspect, the expression level of the proinflammatory endophenotype proteins is determined by detecting the proteins. In another aspect, the expression level of the proinflammatory endophenotype proteins is determined by detecting nucleic acids. In another aspect, the NSAIDS are selected from at least one of: naproxen, rofecoxib, ibuprofen, ketoprofen, tolmetin, diclofenac, fenoprofen, flurbiprofen, piroxicam, etodolac, ketorolac, imdomethacin, nabumetone, oxaprozin, or mefenamic acid. In another aspect, the method further comprises the step of ranking the expression of the proinflammatory endophenotype proteins and correlating them to the NSAID. In another aspect, the method further comprises the step of ranking the expression of the proinflammatory endophenotype proteins in the following order: TNFa, CRP, IL6, and IL10, and correlating them to response to naproxen. In another aspect, the method further comprises the step of ranking the expression of the proinflammatory endophenotype proteins in the following order: TNFa, CRP, IL6, and IL10, and correlating them to response to rofecoxib. In one aspect, the method further comprises determining the expression level of IL5, IL7, and IL18. In another aspect, the method has an accuracy of greater than 93, 94, 95, 96, 97, 98, 99, or 100% predictive value. In another aspect, the method further comprises the step of excluding patients from treatment with NSAIDs. In another aspect, the method further comprises step of stopping NSAID treatment for patients in which NSAIDs are contraindicated.

[0008] In another embodiment, the present invention includes a method for dividing Alzheimer’s Disease into subgroups that correlate with responsiveness to treatment with a non-steroidal anti-inflammatory (NSAID) drug comprising: obtaining a blood or serum sample from a patient; determining the presence of a proinflammatory endophenotype in the blood or serum sample from an expression of TNFa, CRP, IL6, and IL10; and using the proinflammatory endophenotype to detect a treatment response (a responder, a stable, a non responder or an adverse responder) for treatment with the NSAID. In one aspect, the expression levels of TNFa, CRP, IL6, and IL10, are determined by detecting proteins and/or nucleic acids. In another aspect, the method further comprises detecting the expression levels of IL5, IL7, and IL18 is determined by detecting proteins and/or nucleic acids. In another aspect, the treatment response is selected from a responder, a stable, a non-responder or an adverse responder. In another aspect, the NSAIDS are selected from at least one of: naproxen, Rofecoxib, ibuprofen, ketoprofen, tolmetin, diclofenac, fenoprofen, flurbiprofen, piroxicam, etodolac, ketorolac, imdomethacin, nabumetone, oxaprozin, or mefenamic acid. In another aspect, the method further comprises the step of ranking the expression of the seven proteins and correlating them to a response to the NSAID. In another aspect, the method further comprises the step of ranking the expression of four or more proteins, and in certain cases seven proteins in the following order: CRP, TNFa, IL5, IL6, IL7, IL18 and IL10 are determined, and correlating them to a response to naproxen. In another aspect, the method further comprises the step of ranking the expression of the seven proteins in the following order: IL10, CRP, IL6, IL18, IL7, TNFa, and IL5, and correlating them to a response to rofecoxib. In another aspect, the treatment response is selected from a responder, a stable, a non-responder or an adverse responder. In another aspect, the method has an accuracy of greater than 93, 94, 95, 96, 97, 98, 99, or 100% predictive value. In another aspect, the method further comprises the step of excluding patients from treatment with NSAIDs. In another aspect, the method further comprises the step of stopping NSAID treatment for patients in which NSAIDs are contraindicated.

[0009] In another embodiment, the present invention includes a method for identifying a patient response to treatment for Alzheimer’s Disease with an AChE inhibitor drug (e.g., donepezil, Aricept) comprising: obtaining a blood or serum sample from the patient; determining the presence of a proinflammatory endophenotype in the blood or serum sample of the patient; using the proinflammatory endophenotype to detect treatment response (a responder, a stable, a non-responder or an adverse responder); and treating the patient with AChE inhibitor if the patient is in the responder or the stable treatment response phenotype group; or preventing a treatment with AChE inhibitor if the patient is a non-responder or an adverse responder. In one aspect, the proinflammatory endophenotypes is determined using CRP and TNFa. In another aspect, the proinflammatory endophenotype proteins assayed are: TNFa, CRP, IL5, IL6, IL7, IL10 and IL18. In another aspect, the expression level of the proinflammatory endophenotype proteins is determined by detecting the proteins. In another aspect, the expression level of the proinflammatory endophenotype proteins is determined by detecting nucleic acids. In another aspect, the AChE inhibitor is selected from at least one of: donepezil, Aricept, rivastigmine and galantamine. In another aspect, the method further comprises the step of ranking the expression of the proinflammatory endophenotype proteins and correlating them to a response to an AChE inhibitor. In another aspect, the method further comprises the step of ranking the expression of the proinflammatory endophenotype proteins in the following order: IL7, IL18, CRP, IL6, TNFa, IL10 and IL5, and correlating them to response to donepezil. In another aspect, the method has an accuracy of greater than 93, 94, 95, 96, 97, 98, 99, or 100% predictive value. In another aspect, the method further comprises the step of excluding patients from treatment with AChE inhibitor. In another aspect, the method further comprises step of stopping AChE inhibitor treatment for patients in which an AChE inhibitor is contraindicated.

[0010] In another embodiment, the present invention includes a method for dividing Alzheimer’s Disease into subgroups that correlate with responsiveness to treatment with an AChE inhibitor drug (e.g., donepezil, Aricept) comprising: obtaining a blood or serum sample from a patient; determining the presence of a proinflammatory endophenotype in the blood or serum sample from an expression of TNFa, CRP, IL5, IL6, IL7, IL10, and IL18; and using the proinflammatory endophenotype to detect treatment response (a responder, a stable, a non-responder or an adverse responder) for treatment with the AChE inhibitor. In one aspect, the expression levels of TNFa, CRP, IL5, IL6, IL7, IL10, and IL18 are determined by detecting proteins. In another aspect, the expression levels of TNFa, CRP, IL5, IL6, IL7, IL10, and IL18 are determined by detecting nucleic acids. In another aspect, the AChE inhibitor is selected from at least one of: donepezil, Aricept, rivastigmine and galantamine. In another aspect, the method further comprises the step of ranking the expression of the seven proteins and correlating them to a response to the AChE inhibitor. In another aspect, the method further comprises the step of ranking the expression of the seven proteins in the following order: IL7, IL18, CRP, IL6, TNFa, IL10 and IL5, and correlating them to a response to donepezil. In another aspect, the method has an accuracy of greater than 93, 94, 95, 96, 97, 98, 99, or 100% predictive value. In another aspect, the method further comprises the step of excluding patients from treatment with an AChE inhibitor. In another aspect, the method further comprises the step of stopping AChE inhibitor treatment for patients in which AChE inhibitors are contraindicated.

[0011] In another embodiment, the present invention includes a method of analyzing a response of patients previously treated for Alzheimer’s Disease with an anti -AD drug comprising: obtaining a database of biomarker expression data from patients treated with an anti-AD drug; determining the presence of a proinflammatory endophenotype in the blood or serum sample with a seven protein algorithm; using the proinflammatory endophenotype to detect treatment response (a responder, stable, non-responder or adverse responder); and determining if an effectiveness of the drug correlates with the patient having the proinflammatory endophenotype. In one aspect, the method has an accuracy of greater than 93, 94, 95, 96, 97, 98, 99, or 100% predictive value. In another aspect, the proinflammatory endophenotype in the blood or serum sample is determined from expression of TNFa, CRP, IL5, IL6, IL7, IL10, and IL18. In another aspect, the database is obtained from a clinical trial.

[0012] In another embodiment, the present invention includes a method for excluding patients from recruitment into a clinical study by screening patients for non-responders or adverse responders to NSAID treatment comprising: obtaining a blood or serum sample from a patient; determining the expression levels of TNFa, CRP, IL5, IL6, IL7, IL10, and IL18; determining if the patient has a pro-inflammatory endophenotype; and excluding the patient from recruitment into the clinical study if the patient is ruled out based on a determination that the patient is in a non-responder or adverse responder treatment response phenotype group.

[0013] In another embodiment, the present invention includes a method of determining if an NSAID has an effect on Alzheimer’s Disease or a neurodegenerative disease, wherein the NS AID has previously failed in a clinical trial or has been shown to have no effect on Alzheimer’s Disease or the neurodegenerative disease, comprising: determining a presence of a proinflammatory endophenotype in the blood or serum sample of a patient with Alzheimer’s Disease or the neurodegenerative disease; selecting the patient for treatment with the NSAID if the treatment response is a responder or a stable treatment response proinflammatory endophenotype; and determining if the NSAID has an effect on the Alzheimer’s Disease or a neurodegenerative disease of the patient. In one aspect, the method further comprises treating the patient with the NSAID, e.g., treating the patient prior to determining the proinflammatory endophenotype. In another aspect, the method further comprises selecting a NSAID that has previously failed in a clinical trial or has been shown to have no statistically significant effect on a population with Alzheimer’s Disease or the neurodegenerative disease. In another aspect, the proinflammatory endophenotype is determined using TNFa, CRP, IL6, and IL10 biomarkers. In another aspect, the proinflammatory endophenotype proteins assayed are: TNFa, CRP, IL5, IL6, IL7, IL10 and IL18. In another aspect, the method further comprises the step of excluding patients from treatment with an NSAID if the patient is determined to be in a non-responder or an adverse responder endophenotype group.

[0014] In another embodiment, the present invention includes a method for determining an increased likelihood of pharmacological effectiveness of treatment with an NSAID in a patient diagnosed with Alzheimer’s Disease comprising: obtaining a blood or serum sample from the patient; determining a presence or absence of a proinflammatory endophenotype in the blood or serum sample of the patient; and using the proinflammatory endophenotype to determine the likelihood of pharmacological effectiveness of the NSAID in the patient, wherein a responder or stable proinflammatory endophenotype indicates an increased likelihood of pharmacological effectiveness of treatment by the NSAID in the patient. In one aspect, the proinflammatory endophenotype is determined from a CRP and a TNFa biomarker. In another aspect, the proinflammatory endophenotype is determined using TNFa, CRP, IL6, and IL10 biomarkers. In another aspect, the proinflammatory endophenotype proteins assayed are: TNFa, CRP, IL5, IL6, IL7, IL10 and IL18. In another aspect, a non-responder, or an adverse responder endophenotype indicates a decreased likelihood of pharmacological effectiveness of treatment by the NSAID in the patient. In another aspect, the expression level of the proinflammatory endophenotype is determined by detecting two or more proinflammatory endophenotype proteins. In another aspect, the expression level of the proinflammatory endophenotype is determined by detecting two or more proinflammatory endophenotype nucleic acids. In another aspect, the NSAID is selected from at least one of: naproxen, rofecoxib, ibuprofen, ketoprofen, tolmetin, diclofenac, fenoprofen, flurbiprofen, piroxicam, etodolac, ketorolac, imdomethacin, nabumetone, oxaprozin, or mefenamic acid. In another aspect, the method further comprises the step of ranking the expression of the proinflammatory endophenotype proteins and correlating them to an increased likelihood of pharmacological effectiveness of treatment with NSAID. In another aspect, the method further comprises the step of ranking the expression of the proinflammatory endophenotype proteins in the following order: CRP, TNFa, IL5, IL6, IL7, IL18 and IL10, and correlating them to the likelihood of pharmacological effectiveness of treatment by naproxen in the patient. In another aspect, the method further comprises the step of ranking the expression of the proinflammatory endophenotype proteins in the following order: IL10, CRP, IL6, IL18, IL7, TNFa, and IL5, and correlating them to the likelihood of pharmacological effectiveness of treatment by rofecoxib in the patient. In another aspect, the method has an accuracy of greater than 93, 94, 95, 96, 97, 98, 99, or 100% predictive value. In another aspect, the method further comprises the step of treating the patient with an NSAID if the patient has a responder endophenotype. In another aspect, the method further comprises the step of treating the patient with an NSAID if the patient has a stable endophenotype. In another aspect, the method further comprises the step of excluding patients from treatment with an NSAID if the patient has a non-responder or an adverse responder endophenotype. In another aspect, the method further comprises the step of stopping NSAID treatment for a patient that has a non-responder or an adverse responder endophenotype.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention along with the accompanying figures and in which: [0016] FIG. 1 is a diagram that shows a deconstruction of heart disease, specifically, atherosclerosis into generally disease conditions, and the markers used to identify atherosclerosis.

[0017] FIG. 2 is a diagram that shows a model for deconstructing Alzheimer’s Disease into subgroups based on the findings of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0018] While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.

[0019] To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as“a”,“an” and “the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not limit the invention, except as outlined in the claims.

[0020] Despite billions of dollars expended, no new drugs for Alzheimer’s disease (AD) have passed regulatory hurdles in over 10 years. In the inventor’s prior work, they proposed that a paradigm shift is needed for the creation of a precision medicine model for AD, similar to that in cancer. Specifically, the present inventor has identified subgroups of patients suffering from AD based on biological dysfunctions. The ultimate goal was to provide a guided treatment regime (or a change in the same) using specific medications that address the specific biological dysfunction(s) suffered by a given patient suffering from AD.

[0021] More particularly, the present invention includes methods for the identification of specific sub-populations of patients suffering from AD. One subgroup is based on dysfunction of the inflammatory system (the proinflammatory endophenotype). In order to determine if the proinflammatory endophenotype can guide therapies, the inventor examined baseline (pre-randomization) blood samples from AD patients that participated in a therapeutic trial of NSAIDs in AD (rofecoxib, naproxen; the placebo arm was donepezil, an AChE inhibitor marketed as ARICEPT ^® ). In this work, a drug-specific companion diagnostic was created that specifically, and robustly predicted treatment response to each therapy.

[0022] As used herein, the term“pro-inflammatory endophenotypes” refers to a response detectable at the nucleic acid or protein level that is indicative of an immune response that drives inflammation. When providing treatment for those subjects identified with the pro- inflammatory endophenotypes, the treatment can include the following. Nonsteroidal anti inflammatory drugs (NSAIDs): Non-selective NSAIDs - non-selective NSAIDs would be selected for those patients falling into the high end of the proinflammatory endophenotype. As shown herein, non-selective NSAIDs (naproxen) were the superior treatment to selective NSAIDs (celecoxib). Non-selective NSAIDs can be tested with anyone falling within the high end of the proinflammatory endophenotype.

[0023] To determine a proinflammatory endophenotype, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15 or more of the following biomarkers in a sample selected for analysis. In one specific example, four biomarkers have been shown to be effective to determine the proinflammatory endophenotype, namely, TNFa, CRP, IL6, and IL10, to achieve a predictive value greater than 93, 94, 95, 96, 97, 98, 99, or 100%. Specifically, the biomarkers include: interleukin (IL)-7, tumor necrosis factor-alpha (TNFa), IL-5, IL-6, C-reactive protein (CRP), IL-10, tenascin C (TNC), intracellular adhesion molecule-l (ICAM1), coagulation factor VII (FVII), 1309, tumor necrosis factor receptor- 1 (TNFR1), alpha-2 macroglobulin (A2M), chemokine (C-C motif) ligand 17 (TARC), eotaxin3, vascular cell adhesion molecule-l (VCAM1), thrombopoietin (TPO), fatty acid binding protein (FABP), IL-18, beta-2 microblogulin (B2M), serum amyloid Al cluster (SAA), pancreatic polypeptide (PPY), Parkinson protein 7 (DJ1), beta amyloid (Ab), tau, or a-synuclein, IL7, TNFa, IL5, IL6, CRP, IL10, TNC, ICAM1, FVII, 1309, TNFR1, A2M, TARC, eotaxin3, VCAM1, TPO, FABP, IL18, B2M, SAA, PPY, DJ1, Ab, tau, or a-synuclein. Based on the results from the detection of these biomarkers at the nucleic acid or protein expression level, a high or a low proinflammatory endophenotype profile is determined from the level of expression of the two or more biomarkers. In one particular embodiment of the present invention, the proinflammatory endophenotypes markers are TNFa, CRP, IL6, and IL10, and in another example, the biomarkers are TNFa, CRP, IL5, IL6, IL7, IL10 and IL18.

[0024] As used herein, the terms“markers,”“detectable markers” and“detectable labels” are used interchangeably to refer to compounds and/or elements that can be detected due to their specific functional properties and/or chemical characteristics, the use of which allows the agent to which they are attached to be detected, and/or further quantified if desired, such as, e.g., a nucleic acid, a protein, an antibody, an enzyme, radioisotope, electron dense particles, magnetic particles or chromophore that can be detected directly or indirectly, with or without amplification. There are many types of detectable labels, including fluorescent labels, which are easily handled, inexpensive and nontoxic.

[0025] Data source. Banked plasma samples from the Alzheimer’s Disease Cooperative Studies (ADCS) anti-inflammatory clinical trial [23] were used. A full description of this trial, its endpoints and informed consent has been published elsewhere [23, 25], relevant portions incorporated herein by reference. This was a multicenter, randomized, double-blind, placebo-controlled parallel group trial with l-year exposure to study medications across forty ambulatory treatment centers affiliated with the ADCS. Individuals with a diagnosis of probable AD (n=35l) were recruited from December 1999 to November 2000 and randomized to one of the following treatment arms: rofecoxib (25 mg once daily), naproxen (220 mg twice- daily) or placebo. Inclusion criteria were as follows: age 50 or older, MMSE score of 13-26 and diagnosis of probable AD. Stable use of cholinesterase inhibitors was allowed. Exclusion criteria were as follows: presence of comorbid conditions that increased risk for adverse events associated with NSAID treatment (hypersensitivity to aspirin or NSAIDS, active peptic ulcer disease (5 year), renal insufficiency [serum creatinine level >l.5mg/dL or >l32.6umol/L], clinically significant liver disease, poorly controlled hypertension, congestive heart failure, or bleeding ulcer); comorbid conditions that might respond to NSAIDs (e.g. inflammatory arthritis); history (2 month) of regular use of inflammatory medications (aspirin at a daily dose <=325mg was allowed), neuroleptics, antidepressants, sedatives, anti-Parkinsonian medications, or any investigational treatment for AD. A total of 88 completed (111 randomized) the placebo arm, 90 completed (118 randomized) the naproxen arm and 89 completed (122 randomized) the rofecoxib arm. For the current study, proteomic and requisite clinical data were available for 156 participants who were included into the current analyses (naproxen n=5l, placebo n=5l, rofecoxib n=55).

[0026] Assays. All pre-randomization plasma samples were assayed in duplicate via a multi-plex biomarker assay platform via electrochemiluminescence using the SECTOR Imager 2400A from Meso Scale Discovery (MSD; www.mesoscale.com) per the inventors previously published protocols [8], relevant portions incorporated herein by reference. Blood samples were collected and processed per the original clinical trial methods [23] with samples stored centrally at the ADCS biorepository. The proteins assayed included TNFa, CRP, IL5, IL6, IL7, IL10 and IL18, which have been previously linked to the detection of AD [8, 10]

[0027] Statistical Analyses. Biomarker data were transformed using the Box-Cox transformation per previously published protocols [8, 10], relevant portions incorporated herein by reference. Support vector machine (SVM) analyses were utilized to build the proteomic profiles simultaneously classifying all outcome groups in R per the inventors previously published methods [8, 10-12, 26], relevant portions incorporated herein by reference. SVM analyses are capable of simultaneously taking into account large volume data to generate an overall profile (e.g., over and under-expression of select proteins) that most accurately classifies multiple outcomes rather than only binary outcomes. MMSE change scores were utilized as the outcome variables for the study (i.e. MMSE screening - MMSE 12-month follow-up = MMSE change). Participants were classified into four groups based on change in MMSE scores over l2-months: responders (improved MMSE scores of >=l point over l2mo), stable (no change in MMSE over 12 months), non- responders (MMSE decline of 1-2 points over l2mo) or adverse responders (MMSE decline of 3 or more points over l2mo). To build inflammatory profiles of treatment response, group status (i.e. treatment response) was utilized as the outcome variable in SVM models with pre randomization inflammatory proteins as the predictor variable. In order to determine if the proinflammatory profile of treatment response was drug-specific, analyses were conducted utilizing the total sample as well as within specific study treatment arms.

[0028] Demographic characteristics of the cohort can be found in Table 1. The full characterization of the cohort can be found elsewhere [23, 25], relevant portions incorporated herein by reference. A total of 34 (22%) of participants showed an improved MMSE score over the course of the trail, 19 (12%) remained stable, 30 (19%) declined mildly (1-2 points) whereas 73 (47%) of the participants demonstrated a decline of 3 or more points on the MMSE over the 12-month period. First, an SVM-based proinflammatory profile was generated to predict treatment response amongst the entire cohort; results are in Table 2. The overall accuracy of the proinflammatory profile in predicting treatment response group membership was 93%. With regards to prediction of treatment response, the SVM generated proinflammatory profile was 88% (29 out of 34) accurate in detecting those participants that experienced improved cognitive function, 89% accurate in detecting those who remained stable, 100% (30 out of 30) accurate in detecting mild decline and 89% accurate in detecting those who declined 3 or more points on the MMSE over time (Table 2).

Table 1 Demographic Characteristics of tie Sample Cohort

[0029] Next, an SVM-based proinflammatory profile was generated separately for each treatment arm. Within the naproxen arm, a total of 10 (20%) of the participants demonstrated improvement in MMSE scores over 12-months, 4 (8%) remained stable, 10 (20%) demonstrated mild decline, whereas 26 (51%) declined 3 or more points. The naproxen- specific proinflammatory profile was 100% accurate in predicting all treatment response groups. For the rofecoxib arm, 9 (16%) participants demonstrated an improved MMSE score over time, 8 (15%) remained stable, 14 (25%) declined mildly, and 23 (42%) declined 3 or more points over 12-months. The rofecoxib-specific proinflammatory profile was 100% accurate in predicting all treatment response groups. Within the placebo group (i.e. cholinesterase inhibitors), 14 (27%) demonstrated improvement in MMSE scores, 6 (12%) remained stable, 11 (22%) declined mildly and 20 (39%) declined 3 or more points over 12- months (Table 2). The placebo-specific proinflammatory profile was 100% accurate in predicting all treatment response groups.

Table 2 Treatment Response Prediction Using Proteomic Profiling Analyses

SVM Predicted SVM Predicted SVM Predicted SVM Predicted Adverse Non- Stable Responder

Responder Responder

Total Sample

(93% accurate)

Actual Adverse 65 2 2 4

Responder

Actual Non- 0 30 0 0 Responder

Actual Stable 1 1 17 0

Actual Responder 3 2 0 29

Naproxen Arm

(100% accurate)

Actual Adverse 26 0 0 0

Responder

Actual Non- 0 10 0 0

Responder

Actual Stable 0 0 4 0

Actual Responder 0 0 0 10

Placebo Arm

(100%) accurate

Actual Adverse 20 0 0 0

Responder

Actual Non- 0 11 0 0

Responder

Actual Stable 0 0 6 0

Actual Responder 0 0 0 14

Rofecoxib Arm

(100% accurate)

Actual Adverse 23 0 0 0

Responder

Actual Non- 0 14 0 0

Responder

Actual Stable 0 0 8 0

Actual Responder 0 0 0 9

[0030] Next, the importance plots of the proinflammatory profiles were examined across each study arm (see Table 3). As can be seen in Table 3, the relative importance of each marker within the drug-specific algorithm was different. For example, the three most important markers in predicting treatment response to naproxen were CRP, TNFa and IL5, respectively.

[0031] Alternatively, the top three markers for the placebo (i.e., AChE inhibitor) group and rofecoxib group were IL7, IL18, CRP and IL10, CRP and IL6, respectively. Therefore, the proinflammatory profile that identified treatment response was, as hypothesized, drug- specific.

[0032] Lastly, the inventor applied the relative importance plot data for biomarker cut-scores to predict treatment outcomes within the naproxen arm. The top two markers within this arm were CRP and TNFa. Both CRP and TNFa were overexpressed as a function of treatment response (i.e. higher levels were associated with treatment response to naproxen). Therefore, tertile scores were created for both markers for creation of cut-scores per level of the proinflammatory endophenotype using only these two markers. The cut-scores for inclusion into the naproxen proinflammatory endophenotype were as follows: low proinflammatory endophenotype = CRP <335.64 & TNFa <0.49 (10% of the arm); middle proinflammatory endophenotype = CRP of 335.64 - 1015.51 & TNFa of 0.49 - 0.63 (74% of the naproxen arm); high proinflammatory endophenotype = CRP >1015.52 & TNFa>0.64 (16% of the naproxen arm). When examining those specifically within the high end of the proinflammatory endophenotype, the naproxen group demonstrated significantly reduced decline in MMSE scores over a l2mo period (F[l ,6] = 8.2, p=0.03; partial eta squared = 0.58, observed power =0.7) as compared to the placebo group. Next, MMSE change scores were dichotomized into improvement versus stable or decline over the l2-mo period. The average score was 0.25 for the Placebo group (25% improved) and 0.63 for the Naproxen group (63% improved). The Naproxen group experienced significant improvement in MMSE scores over 12 months as compared to the Placebo group with age, gender, education and APOE4 genotype entered as covariates (F[l,6]=l0.9, p=0.02, Partial Eta Square = 0.65, observed power 0.79)(See Table 4). There was a trend towards those in the high end of the proinflammatory endophenotype who were treated with placebo (i.e., AChE inhibitor) to get significantly worse cognitively over time (p=0.07).

MMSE MMSE MMSE CDR-SB CDR-SB CDR-SB

Arm

Baseline l2mo Change Baseline l2mo Change

Naproxen

Low (n=7) 17.1 (4.3) 16.1 (5.5) 1.5(3.1) 7.9(3.0) 9.8(4.1) -2.5(2.1)

14-26 7-24 -2 - 7 2.5-12.0 3-15 -5 - 0

Middle (n=36) 20.7(3.4) 16.5(6.4) 4.2(4.6) 6.1(2.8) 8.4(4.1) -2.6(2.5)

13-26 4-25 -2 - 19 1-12 2-18 -8 - 2.5

High (n=8) 21.1(3.9) 21.2(5.3) -0.13(3.1) 5.8(2.7) 5.36(3.0) 0.7(2.5)

14-15 15-28 -3 - 5 1-12 2-12 -6.5 - 1 p=0.02*

Placebo

Low (n=5) 18.7(3.6) 19.8(4.4) -0.6(3.5) 5.7(2.4) 7.0(3.1) -1.2(1.0)

15-24 12-23 -4 - 3 3.5-10 3.5-11 -2.5 - p=0.07** 0.0

Middle (n=42) 21.5(3.7) 19.9(4.4) 2.0(4.0) 5.3(2.5) 6.8(3.7) 1.9(2.1)

13-27 12-23 -4 - 12 1.5-13 1.5-16 -7 - 1.5

High (n=4) 20.7(3.8) 16.3(8.4) 4.8(6.4) 6. l(2.4) 7.8(5.0) -2.0(3.7)

15-25 5-24 -2 - 12 2.5-9 3-13 -5 - 2.5

NOTE: sample size includes only those patients with all data available (baseline, l2mo and change schores); *significant difference as compared to placebo high proinflammatory group; **as compared to the low end of the proinflammatory group in the naproxen arm. [0033] These results demonstrate for the first time that NSAID therapy is associated with cognitive stability and benefit for a select subgroup of AD patients as well as adverse response among others.

[0034] Specifically, in the ADCS AD NSAID trial, 22% of participants demonstrated improved MMSE scores over time; however, 43% experienced a worsening of cognition of 3 or more points over time. In the follow-up analyses within the naproxen arm, the change in MMSE scores among the high proinflammatory AD receiving active therapy was significantly better than that observed in the placebo group. Therefore, treatment with NSAID medications is clinically appropriate for only a select subset of patients. These data also show that a significant portion of AD cases are at risk for more rapid progression as a result of taking NSAID drugs, that is, the NSAIDs are contra-indicated.

[0035] This finding explains the failure to meet clinical endpoints for NSAIDs. The current findings, on the other hand, demonstrates that subgroups within AD patients exists and that baseline proinflammatory profiles predict treatment response for a select portion of patients.

[0036] The inventor further conducted analyses based on a novel four-biomarker profile.

The biomarkers selected for this study were TNFa, CRP, IL6, and IL10. For these analyses, patients were classified as follows based on change in MMSE scores over time.

[0037] Table 5. Patient Classification.

[0038] Table 6. Treatment Response Prediction Using Four Biomarker Analyses

[0039] Prediction by arm: Naproxen (68 patients)

[0040] Prediction accuracy: 97.06%

[0041] Table 7. Treatment Response Prediction Using Four Biomarker Analyses

[0042] Prediction by arm: Rofecoxib (55 patients)

[0043] Prediction accuracy: 98.18%

[0044] Table 8. Treatment Response Prediction Using Four Biomarker Analyses [0045] Prediction by arm: Naproxen and Rofecoxib (Combined) (123 patients)

[0046] Prediction accuracy: 88.62%

[0047] Table 9. Inflammatory Profile Variable Importance by Arm (Based on

[0048] Four Biomarker Analyses)

[0049] When examining treatment response within arms, the proinflammatory profiles (1) changed significantly based on drug administered and (2) became more accurate. The variable importance plots explain the significantly improved performance. Specifically, the overall algorithm did not take into account drug-specific effects (but is able to) and the variable importance plots clearly indicate a different pattern per arm. Using only two of the biomarkers from the naproxen-specific algorithm (CRP and TNFa), cut-scores were generated to create subgroups that predict treatment response. Those in the high end of the proinflammatory endophenotype experience significant improvement while there was a trend towards those in the low end to experience significant decline. This two-marker approach provides a more simplistic method for demonstrating the proof-of-concept; however, the multi-marker SVM approach is more accurate and is preferable for generating profiles that may be utilized for enriching into clinical trials.

[0050] When taken within the context of previously conducted clinical trials, the present invention can also be used to evaluate data from these trials and select for subgroups of AD patients for which the treatment was successful. The assays from pre-randomization biobanked samples from other trials can be used for validation purposes and the patients sub divided into the subgroups of the present invention. The proinflammatory biomarker profile can also be utilized for patient selection and stratification for novel clinical trials utilizing NS AID therapy to treat and perhaps prevent AD with the current methods utilized for targeted patient enrichment. That is, only those patients with baseline alterations of the proinflammatory profile would be enrolled into the study. The enrichment of such a targeted population would significantly reduce the overall cost of the trial by: (1) reducing the required number of patients to detect the predicted response, as well as, (2) reduce the time to market as the effect size would be much larger and be detectable in briefer trials. Additionally, the removal of those patients most likely to experience harm from the drug is of tremendous benefit. [0051] Profiling biological pathways, including inflammation, highlights novel pathways relevant to AD. Additionally, biomarkers of inflammation have consistently been important in a blood-based algorithm of disease presence [10-13] Histopathologically, inflammatory markers have been found in association with both neurofibrillary tangles [27] and senile plaques [28] in AD tissue. Longitudinally, studies also support the link between inflammation and AD. For example, Schmidt and colleagues [14] analyzed data from the Honolulu-Aging Study & Honolulu-heart study and found that increased CRP levels at midlife were associated with increased risk for the development of AD, as well as VaD 25- years later.

[0052] There is a large base of epidemiological evidence supporting the notion that anti inflammatory compounds reduce the risk of developing AD. In a prospective, population- based cohort study of nearly 7,000 individuals 55 years of age and older, all of whom were dementia- free at baseline, long-term use of NSAIDs was associated with a reduced risk of developing AD (relative risk = 0.20, 0=0.05-0.83)[l5] When analyzing data from the Cache County Study, Anthony and colleagues [16] found that use of non-aspirin NSAIDs alone reduced the risk of developing AD (Odds Ratio [OR] = 0.43, Cl = 0.23-0.75) and that use of non-aspirin NSAIDs and aspirin reduced that risk even further (OR = 0.17, Cl = 0.04- 0.48). A meta-analysis of nine published studies (pooled sample size = 14,654) further supported the notion of a protective effect of NSAID use in terms of AD development with the relative risk of 0.27 (95% Cl = 0.13- 0.58) associated with long-term use [17]

[0053] Based on these findings, anti-inflammatory compounds have the potential for treating those suffering from AD and other neurodegenerative diseases [13, 16-21], and three clinical trial have been completed, one on AD [18], one on MCI [22], and the Alzheimer’s Disease Anti-inflammatory Prevention Trial (ADAPT)[24, 29] These studies, based on the available literature, utilized a traditional NSAID (naproxen) as well as a COX-2 inhibitor. Naproxen was utilized based on a several reasons. First, several epidemiological studies (see above) suggest a protective effect of non-selective NSAIDs against neurodegeneration[30]

[0054] NSAIDs block microglial activation in vitro [31, 32] and appear to reduce accumulation of activated microglia in the AD brain[33]. On the other hand, non-selective NSAIDS are also associated with toxicity and high drop-out rates [34], which makes them difficult for studies of AD patients. Rofecoxib, a COX-2 inhibitor was utilized for several reasons. First, COX-2 may play a central role in neurodegeneration via excitotoxicity (glutamate and kainic acid) pathways[35]. Thus, the present invention for the first time provides for a detailed analysis and determination of those AD patients that will respond positively to NS AID treatment.

[0055] COX-2 expression (mRNA and protein) has also been found upregulated in human AD brains[36]. At the time, it was also thought that COX-2 inhibitors would be less toxic than non- selective NSAIDs. Despite the significant basic, clinical and epidemiological literature in support of the use of NSAIDs in AD, none of those trials successfully met targeted clinical outcomes. Therefore, the effect of NSAIDs on AD was previously mixed.

[0056] The present invention addresses what none of those trials found (and a possible reason why they failed); specific subsets of patients where inflammation played a prominent role in cognitive loss and whom were most likely to benefit from the trial. The current results demonstrate that targeted treatment with NSAID medications may benefit select subsets of patients with AD, while being contra-indicated for others. Additionally, given that the placebo group was treated with cholinesterase inhibitors, the current approach shows the generation of a proteomic profile of treatment response to such medication.

[0057] The current findings are as follows: (1) the use of NSAIDs (e.g., naproxen) to effectively treat certain individuals with AD and, more importantly, (2) provide a novel method for clinical trials in AD. Specifically, the present invention demonstrates that subgroups of AD patients exist, within the heterogeneous categorization of AD, and that these subgroups are useful in determining which patients will respond best to particular medications. It also serves to demonstrate which patients will not respond, or may respond adversely, to particular medications.

[0058] These results are a paradigm shift in the conceptualization of the therapeutic approach to AD. By way of explanation, and in no way a limitation of the present invention, the inventor proposes a model that targets select patients to specific interventions based on the subgroup(s) to which patient belongs. As an analogy, the approach herein is adopted from that utilized in the successful treatment of cardiovascular disease. For example, FIG. 1 outlines the therapeutic approach to atherosclerotic heart disease, which is only one form of heart disease. In cardiology, one does not treat“atherosclerosis” as a diagnostic category, but rather, specific types of atherosclerotic factors are treated. However, in the prevailing models for the treatment of AD, all patients are enrolled into clinical trials based on a general clinical diagnosis. [0059] In FIG. 2, the inventor proposes a first-step towards a more refined model for treating AD. In the inventor’s prior work several subgroups of AD patients were identified based on proteomic and neuropsychiatric data. When combined with other findings, the specific subgroups proposed currently in the model include: inflammation [10, 12, 13], APOE4 genotype[37], metabolic dysfunction[38], amyloid, oxidative stress[39], and depression[40, 41] The current findings validate a person-centered AD therapeutic model via the inflammatory subgroup. Additionally, as with CVD, the presence of dysfunction in one sub category does not preclude the presence of abnormality in another, and the current model directly provides a conceptual framework for combination therapy. Taken together, these findings show a novel approach to treating specific subsets of AD patients. For example, the present invention can be used to assay existing biorepositories of >20,000 samples across previously conducted clinical trials, which can be the first step towards the generation of a person-centered therapeutic approach to the treatment and prevention of AD and other neurodegenerative diseases.

[0060] As such, the present invention provides: (1) a companion diagnostic to identify specific populations of patients most likely to respond to therapy; (2) a companion diagnostic to identify specific patients most likely to suffer from adverse events (and should not be prescribed treatment); (3) novel technology that can be used to revolutionize clinical trials in AD to only enroll those patients that have a high likelihood of responding to therapy, thereby reducing the sample size (and cost) of trials significantly; (4) technology that has a tremendous impact on clinical trials by reducing the timeframe needed to complete trials by only enrolling those most likely benefit (both #3 and #4 have significant impacts on cost containment); and/or (5) a companion diagnostic that can be used to identify patients that are likely to remain stable or improve from therapy rather than only declining more slowly (i.e. the therapeutic impact of the intervention is much larger).

[0061] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

[0062] It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.

[0063] All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0064] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean“one,” but it is also consistent with the meaning of“one or more,”“at least one,” and“one or more than one.” The use of the term“or” in the claims is used to mean“and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and“and/or.” Throughout this application, the term“about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[0065] As used in this specification and claim(s), the words“comprising” (and any form of comprising, such as“comprise” and“comprises”),“having” (and any form of having, such as“have” and“has”),“including” (and any form of including, such as“includes” and “include”) or“containing” (and any form of containing, such as“contains” and“contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. In embodiments of any of the compositions and methods provided herein, “comprising” may be replaced with“consisting essentially of’ or“consisting of’. As used herein, the phrase“consisting essentially of’ requires the specified integer(s) or steps as well as those that do not materially affect the character or function of the claimed invention. As used herein, the term“consisting” is used to indicate the presence of the recited integer (e.g., a feature, an element, a characteristic, a property, a method/process step or a limitation) or group of integers (e.g., feature(s), element(s), characteristic(s), property(ies), method/process steps or limitation(s)) only.

[0066] The term“or combinations thereof’ as used herein refers to all permutations and combinations of the listed items preceding the term. For example,“A, B, C, or combinations thereof’ is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0067] As used herein, words of approximation such as, without limitation, “about”, "substantial" or "substantially" refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skilled in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion, a numerical value herein that is modified by a word of approximation such as“about” may vary from the stated value by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.

[0068] All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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