WO2007043998A1 - Modifications specifiques de la maladie d'alzheimer du rapport de phosphorylation erk1/erk2 - Google Patents

Modifications specifiques de la maladie d'alzheimer du rapport de phosphorylation erk1/erk2 Download PDF

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Publication number
WO2007043998A1
WO2007043998A1 PCT/US2005/036014 US2005036014W WO2007043998A1 WO 2007043998 A1 WO2007043998 A1 WO 2007043998A1 US 2005036014 W US2005036014 W US 2005036014W WO 2007043998 A1 WO2007043998 A1 WO 2007043998A1
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Prior art keywords
cells
disease
alzheimer
protein
phosphorylated
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PCT/US2005/036014
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English (en)
Inventor
Tapan Kumar Khan
Daniel L. Alkon
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Blanchette Rockefeller Neurosciences Institute
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Application filed by Blanchette Rockefeller Neurosciences Institute filed Critical Blanchette Rockefeller Neurosciences Institute
Priority to PCT/US2005/036014 priority Critical patent/WO2007043998A1/fr
Priority to PCT/US2006/022156 priority patent/WO2007044094A1/fr
Priority to ES10012836.2T priority patent/ES2477284T3/es
Priority to US12/083,056 priority patent/US20090029873A1/en
Priority to ES08020258T priority patent/ES2412268T3/es
Priority to EP06825096A priority patent/EP1934618B1/fr
Priority to ES10011288.7T priority patent/ES2588376T3/es
Priority to JP2008535546A priority patent/JP4908514B2/ja
Priority to PCT/US2006/037186 priority patent/WO2007047029A2/fr
Priority to EP10011289.5A priority patent/EP2322934B1/fr
Priority to DE602006006855T priority patent/DE602006006855D1/de
Priority to CA2625300A priority patent/CA2625300C/fr
Priority to KR1020137031223A priority patent/KR20140002073A/ko
Priority to EP08020258A priority patent/EP2031398B1/fr
Priority to AT06825096T priority patent/ATE431558T1/de
Priority to EP10012836.2A priority patent/EP2317321B1/fr
Priority to EP10011290A priority patent/EP2322936A1/fr
Priority to ES10011289.5T priority patent/ES2596881T3/es
Priority to ES06825096T priority patent/ES2323813T3/es
Priority to EP10011288.7A priority patent/EP2339349B1/fr
Priority to TW102146766A priority patent/TW201413246A/zh
Priority to TW095137389A priority patent/TWI448688B/zh
Publication of WO2007043998A1 publication Critical patent/WO2007043998A1/fr
Priority to US12/078,857 priority patent/US20080221042A1/en
Priority to KR1020087011105A priority patent/KR101375552B1/ko
Priority to US13/774,049 priority patent/US9797913B2/en
Priority to US15/719,714 priority patent/US20180024146A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer

Definitions

  • the present invention relates to methods of diagnosing
  • Alzheimer's disease in a subject by detecting alterations in the ratio of specific phosphorylated MAP kinase proteins in cells after stimulation with a protein kinase C activator.
  • the Alzheimer's disease-specific molecular biomarkers disclosed herein are useful for the diagnosis of Alzheimer's disease, for monitoring disease progression and in screening methods for the identification of compounds for treating or preventing Alzheimer's disease.
  • AD Alzheimer's disease
  • bradykinin As a potent inflammation mediator, bradykinin (BK) is produced by brain and peripheral cells under patho-physiological conditions such as trauma, stroke, pain ischemia, and asthma (Regoli et al, 1993; Bockmann & Paegelow, 2000; Ellis et al, 1989; Kamiya et al, 1993).
  • BK2bR B2 bradykinin receptor
  • PLC phospholipase C
  • IP3 inositol 1,4,5-trisphospate
  • BK also triggers production of other proinflammatory cytokines through activation of mitogen-activated protein (MAP) kinases (Hayashi et al, 2000; Schwaninger et al, 1999; Phagoo et al, 2001).
  • MAP mitogen-activated protein
  • Enhanced elevation of intracellular Ca 2+ levels has been found in AD brains as well as in AD peripheral cells in response to stimulation of bradykinin and inactivation of K + channels (Etcheberrigaray et al, 1993, 1998; Hirashima et al., 1996; Gibson et al, 1996(a)).
  • AP-I AP-I, NF- ⁇ B, and cyclic AMP-responsive element binding protein (CREB).
  • Mitogen- activated protein kinases (such as Erkl and Erk2) regulate phosphorylation of the microtubule associated protein tau and processing of
  • amyloid protein ⁇ both events critical to the pathophysiology of Azheimer's
  • the present inventors have identified, for the first time, unique Alzheimer's disease-specific molecular biomarkers useful for the diagnosis of Alzheimer's disease in a highly sensitive and highly specific manner compared to previously known diagnostic tests.
  • the unique Alzheimer's disease-specific molecular biomarkers disclosed herein serve as the basis for diagnostic methods having a high degree of sensitivity and specificity for the detection and diagnosis of Alzheimer's disease.
  • the unique Alzheimer's disease- specific molecular biomarkers of the present invention are also useful in screening methods to identify compounds which may be used as therapeutic agents in the treatment and prevention of Alzheimer's disease.
  • the present invention is directed, in certain embodiments, to methods of diagnosing Alzheimer's disease in a subject comprising the steps of contacting cells obtained from a subject with an agent that is a protein kinase C activator and measuring the ratio of specific phosphorylated MAP kinase proteins in the cells to diagnose Alzheimer's disease in the subject.
  • the ratio of specific phosphorylted MAP kinase proteins is a ratio between two phosphorylated MAP kinase proteins.
  • the diagnostic methods of the invention comprise an in vitro assay.
  • the specific phosphorylated MAP kinase proteins are sequence variants of each other and belong to the same family of proteins.
  • the ratio of specific phosphorylated MAP kinase proteins is the ratio of phosphorylated Erkl to phosphorylated Erk2 and is calculated by dividing the normalized amount of phosphorylated Erkl by the normalized amount of phosphorylated Erk2.
  • the protein kinase C activator is selected from the group consisting of bradykinin, bryostatin, bombesin, cholecystokinin, thrombin,
  • the cells that are used in the diagnostic assays are peripheral cells, hi preferred embodiments, the cells may be skin cells, skin fibroblast cells, blood cells or buccal mucosa cells. In certain embodiments, the cells are not isolated from cerebral spinal fluid. In other preferred embodiments, the cells do not comprise cerebral spinal fluid. In still other preferred embodiments, the cells are not obtained by a spinal tap or lumbar puncture.
  • a protein kinase C activator is contacted with cells in media comprising serum.
  • a protein kinase C activator is contacted with said cells in serum-free media.
  • phosphorylated MAP kinase proteins are detected by immunoassay.
  • the immunoassay may be a radioimmunoassay, a Western blot assay, an immunofluoresence assay, an enzyme immunoassay, an immunoprecipitation assay, a chemiluminescence assay, an immunohistochemical assay, an Immunoelectrophoresis assay, a dot blot assay, or a slot blot assay.
  • protein arrays or peptide arrays or protein micro arrays may be employed in the diagnostic methods.
  • Alzheimer's disease may be diagnosed in a subject by contacting cells from the subject with an agent that is a protein kinase C activator and then measuring the ratio of a phosphorylated first MAP kinase protein to a phosphorylated second MAP kinase protein, wherein the phosphorylated first and phosphorylated second MAP kinase proteins are obtained from the cells after they have been contacted with the protein kinase C activator.
  • an agent that is a protein kinase C activator
  • the ratio of phosphorylated first MAP kinase protein to phosphorylated second MAP kinase protein in cells from the subject that have not been contacted with the protein kinase C activator is determined and this ratio is subtracted from the ratio of phosphorylated first and second MAP kinase proteins obtained from cells after they have been contacted with the protein kinase C activator to diagnose the presence of Alzheimer's disease in the subject based on the difference in the ratios.
  • the difference in the ratios is diagnostic of Alzheimer's disease in the subject if the difference is a positive value.
  • the difference is diagnostic of the absence of Alzheimer's disease in the subject if the difference is a negative value or zero.
  • kits for diagnosing Alzheimer's disease in a subject may contain an agent that is a protein kinase C activator; in further embodiments of the invention, the kits may contain an antibody specific for a phosphorylated first MAP kinase protein. In still further embodiments of the invention, the kits may contain an antibody specific for a phosphorylated second MAP kinase protein, hi preferred embodiments, the kits may contain any combination of the foregoing.
  • kits for diagnosing are provided. [0014] In certain embodiments of the invention, the kits for diagnosing
  • Alzheimer's disease in a subject may contain one or more protein kinase C activators selected from the group consisting of bradykinin, bryostatin, bombesin,
  • kits for diagnosing are provided.
  • Alzheimer's disease in a subject may contain antibodies specific for phosphorylated Erkl or phosphorylated Erk2 or both.
  • the kit may contain anti-phospho-Erkl antibody.
  • the kit may contain an anti-phospho-Erk2 antibody.
  • Certain embodiments of the invention are directed to methods for screening a test compound (or a lead compound) useful for the treatment or prevention of Alzheimer's disease comprising: contacting cells isolated from a subject diagnosed with Alzheimer's disease with an agent that is a protein kinase C activator, wherein the contacting is done in the presence of the test compound (or a lead compound); measuring the ratio of a phosphorylated first MAP kinase protein to a phosphorylated second MAP kinase protein, wherein the phosphorylated first and phosphorylated second MAP kinase proteins are obtained from the cells after the contacting; measuring the ratio of phosphorylated first MAP kinase protein to phosphorylated second MAP kinase protein in cells from the subject that have not been contacted with the test compound (or a lead compound); subtracting the ratio obtained from the contacting step done in the absence of the test compound (or a lead compound) from the ratio obtained from the contacting step done in the presence of the test
  • the calculated difference in the ratios is indicative of a test compound (or a lead compound) useful for the treatment of Alzheimer's disease if the difference is a negative value or zero.
  • the invention is directed to methods for screening a test compound (or a lead compound) useful for the treatment or prevention of Alzheimer's disease wherein the methods comprise an in vitro assay.
  • the first MAP kinase protein is Erkl and the second MAP kinase protein is Erk2.
  • the ratios are calculated by dividing the normalized amount of phosphorylated Erkl by the normalized amount of phosphorylated Erk2.
  • the protein kinase C activator is selected from the group consisting of bradykinin, bryostatin, bombesin,
  • the cells are peripheral cells.
  • the peripheral cells are selected from the group consisting of skin cells, skin fibroblast cells, blood cells and buccal mucosa cells.
  • the cells are not isolated from cerebral spinal fluid.
  • the cells do not comprise cerebral spinal fluid.
  • the cells are not obtained by a spinal tap or lumbar puncture.
  • the protein kinase C activator is contacted with said cells in media comprising serum.
  • the protein kinase C activator is contacted with said cells in serum-free media.
  • the phosphorylated MAP kinase proteins are detected by immunoassay
  • the immunoassay is a radioimmunoassay, a Western blot assay, an immunofluoresence assay, an enzyme immunoassay, an immunoprecipitation assay, a chemiluminescence assay, an immunohistochemical assay, an Immunoelectrophoresis assay, a dot blot assay, or a slot blot assay.
  • the measuring is done using a protein array, a peptide array, or a protein micro array.
  • the invention is directed to methods of monitoring Alzheimer's disease progression in a subject comprising measuring an Alzheimer's disease-specific molecular biomarker.
  • the method comprises measuring the Alzheimer's disease-specific molecular biomarker at more than one time point.
  • the Alzheimer's disease-specific molecular biomarker is measured at time points separated by at least six months, more preferably, at time points separated by at least 12 months.
  • a decrease in the numerical value of the Alzheimer's disease-specific molecular biomarker is indicative of Alzheimer's disease progression in said subject.
  • the invention is directed to compositions useful for the treatment of Alzheimer's disease comprising a compound identified by any of the methods for screening compounds disclosed herein.
  • the compositions of the invention comprise a pharmaceutical composition for treating Alzheimer's disease in a subject in need thereof comprising a therapeutically effective amount of a compound identified by any of the methods for screening compounds disclosed herein.
  • the present invention is directed to methods of treating Alzheimer's disease comprising administering a therapeutically effective amount of any of the pharmaceutical compositions disclosed herein to a subject in need thereof.
  • FIG 1 shows the results of determinations of Alzheimer's disease-specific molecular biomarkers in banked skin fibroblast cells obtained from the Coriell Cell Repository and in punch skin biopsy samples that were immediately placed in tissue culture and which were obtained from Autopsy confirmed subjects.
  • AD refers to Alzheimer's disease cells
  • Mixed AD/PD/LBD refers to cells taken from patients with mixed pathologies of Alzheimer's disease, Parkinson's Disease and/or Loewi Body disease
  • AC refers to non-dementia age-matched control cells
  • Non-ADD refers to cells taken from subjects diagnosed with non- Alzheimer's disease dementia (e.g. Huntington's disease or Parkinsons's disease or Clinical Schizophrenia).
  • the Alzheimer's disease-specific molecular biomarkers in AD cells tested was a positive value falling between greater than about 0.02 and less than about 0.4.
  • the Alzheimer's disease-specific molecular biomarkers in non-dementia age-matched control cells were negative or very low positive values, i.e. less than about 0.01.
  • the Alzheimer's disease-specific molecular biomarkers in non- ADD cells were negative values.
  • Alzheimer's disease-specific molecular biomarkers were measured by determining the ratio of phosphorylated Erkl to phosphorylated Erk2 in cells that had been stimulated with bradykinin minus the ratio of phosphorylated Erkl to phosphorylated Erk2 in cells that were stimulated with media lacking bradykinin. This is expressed as the following: Alzheimer's disease-specific molecular biomarker
  • Figure 2 shows a linear regression analysis of Alzheimer's disease-specific molecular biomarkers as a function of years of dementia.
  • the linear regression shows a negative slope of approximately -0.01 indicating an inverse correlation between years of dementia and positive magnitude of the Alzheimer's disease-specific molecular biomarker.
  • the Alzheimer's disease-specific molecular biomarker becomes a less positive numerical value. Measurement of the Alzheimer's disease-specific molecular biomarker allows for early diagnosis of Alzheimer's disease because a more highly positive value is indicative of early stages of the disease.
  • Alzheimer's disease-specific molecular biomarkers were measured by determining the ratio of phosphorylated Erkl to phosphorylated Erk2 in cells that had been stimulated with bradykinin minus the ratio of phosphorylated Erkl to phosphorylated Erk2 in cells that were stimulated with media lacking bradykinin. This is expressed as the following: Alzheimer's disease- specific molecular biomarker
  • the present invention relates, in certain aspects, to methods of diagnosing Alzheimer's disease in human cells taken from subjects that have been identified for testing and diagnosis.
  • the diagnosis is based upon the discovery of unique Alzheimer's disease-specific molecular biomarkers.
  • the invention is directed to methods of monitoring Alzheimer's disease progression and to screening methods for the identification of lead compounds for treating or preventing Alzheimer's disease.
  • Alzheimer's disease-specific molecular biomarkers provide highly practical, highly specific and highly selective tests for early diagnosis of Alzheimer's disease.
  • the Alzheimer's disease-specific molecular biomarkers described herein provide a basis for following disease progression and for identifying therapeutic agents for drug development targeted to the treatment and prevention of Alzheimer's disease.
  • Alzheimer's disease using peripheral (non-CNS) tissue that is useful in diagnostic assays that are highly sensitive and highly specific for the diagnosis of Alzheimer's disease.
  • a great advantage of the instant invention is that the tissue used in the assays and methods disclosed herein may be obtained from subjects using minimally invasive procedures, i.e., without the use of a spinal tap.
  • one aspect of the invention is directed to an assay or test for the early detection of Alzheimer's disease in a subject in which an internally controlled ratio of Erkl phosphorylation to Erk2 phosphorylation, which is induced by a protein kinase C activator (such as bradykinin), is measured with specific antibodies using a baseline normalization response to growth media in human cells, such as skin fibroblasts, or other peripheral cells such as blood cells.
  • a protein kinase C activator such as bradykinin
  • the cells that are taken from the individual or patient can be any viable cells.
  • they are skin fibroblasts, but any other peripheral tissue cell (i.e. outside of the central nervous system) may be used in the tests of this invention if such cells are more convenient to obtain or process.
  • Other suitable cells include, but are not limited to, blood cells such as erythrocytes and lymphocytes, buccal mucosal cells, nerve cells such as olfactory neurons, cerebrospinal fluid, urine and any other peripheral cell type.
  • the cells used for purposes of comparison do not necessarily have to be from healthy donors.
  • the cells may be fresh or may be cultured (see, U.S. Patent No.
  • a punch skin biopsy can be used to obtain skin fibroblasts from a subject. These fibroblasts are analyzed directly using the techniques described herein or introduced into cell culture conditions. The resulting cultured fibroblasts are then analyzed as described in the examples and throughout the specification. Other steps may be required to prepare other types of cells which might be used for analysis such as buccal mucosal cells, nerve cells such as olfactory cells, blood cells such as erythrocytes and lymphocytes, etc. For example, blood cells can be easily obtained by drawing blood from peripheral veins. Cells can then be separated by standard procedures (e.g. using a cell sorter, centrifugation, etc.) and later analyzed.
  • the present invention relates, in certain aspects, to methods for the diagnosis and treatment of Alzheimer's disease in a subject.
  • the diagnostic methods of the invention are based on measuring the ratio of two specific and distinct phosphorylated MAP kinase proteins in cells taken from a subject which have been stimulated with an agent that is a protein kinase C activator.
  • the invention is also directed, in certain embodiments, to kits containing reagents useful for the detection or diagnosis of Alzheimer's disease.
  • the invention is directed to methods for screening to identify lead compounds useful for treating Alzheimer's disease as well as to methods of using these compounds or chemical derivatives of the lead compounds in pharmaceutical formulations to treat or prevent Alzheimer's disease in subjects in need thereof.
  • sensitivity in the context of medical screening and diagnosis, means the proportion of affected individuals who give a positive test result for the disease that the test is intended to reveal, i.e., true positive results divided by total true positive and false negative results, usually expressed as a percentage.
  • the term "specificity" in the context of medical screening and diagnosis means the proportion of individuals with negative test results for the disease that the test is intended to reveal, i.e., true negative results as a proportion of the total of true negative and false-positive results, usually expressed as a percentage.
  • the term "highly sensitive” means a diagnostic method that is greater than or equal to about 50% sensitive, or about 55% sensitive, or about 60% sensitive, or about 65% sensitive, or about 70% sensitive, or about 75% sensitive, or about 80% sensitive, or about 85% sensitive, or about 90% sensitive, or about 95% sensitive, or about 96% sensitive, or about 97% sensitive, or about 98% sensitive, or about 99% sensitive or about 100% sensitive.
  • the term "highly specific” means a diagnostic method that is greater than or equal to about 50% specific, or about 55% specific, or about 60% specific, or about 65% specific, or about 70% specific, or about 75% specific, or about 80% specific, or about 85% specific, or about 90% specific, or about 95% specific, or about 96% specific, or about 97% specific, or about 98% specific, or about 99% specific or about 100% specific.
  • lead compounds are compounds identified using the methods of screening compounds disclosed herein. Lead compounds may have activity in shifting the Alzheimer's disease-specific molecular biomarkers disclosed herein to values corresponding to those values calculated for Alzheimer's disease-specific molecular biomarkers determined using normal healthy cells in the assays described herein. Lead compounds may be subsequently chemically modified to optimize or enhance their activity for use in pharmaceutical compositions for the treatment or prevention of Alzheimer's disease.
  • sequence variants are proteins that are related to each other both structurally and functionally.
  • sequence variants are proteins that share structural similarity at the level of amino acid sequence and share functional attributes at the level of enzymatic activity.
  • sequence variants are MAP kinase proteins that catalyze the phosphorylation of other proteins.
  • the present invention is directed, in certain embodiments, to methods of diagnosing Alzheimer's disease.
  • the diagnostic methods involve the steps of obtaining a cell sample from a subject, contacting the cell sample with an agent that is a protein kinase C activator and measuring the ratio of specific phosphorylated MAP kinase proteins in said cell sample to diagnose Alzheimer's disease in said subject.
  • the diagnostic assays disclosed herein may be carried out in vitro or in vivo.
  • the protein kinase C activator is bradykinin.
  • the ratio of specific phosphorylated MAP kinase proteins is the ratio of phosphorylated Erkl to phosphorylated Erk2, which is calculated by dividing the relative or normalized amount of phosphorylated Erkl by the relative or normalized amount of phosphorylated Erk2.
  • the diagnostic methods and methods of screening compounds useful for treating Alzheimer's disease which are disclosed herein are based upon the discovery by the inventors of a unique molecular biomarker for Alzheimer's disease.
  • the numerical value of the Alzheimer's disease-specific molecular biomarker will depend on certain variables, such as, for example, the cells used in the assay, the protein kinase C activator used in the assay and the specific MAP kinase proteins that are targeted for measurement of phosphorylation ratios.
  • the Alzheimer's disease-specific molecular biomarker may be measured by determining the ratio of phosphorylated Erkl to phosphorylated Erk2 in cells that have been stimulated by a protein kinase C activator and subtracting from this the ratio of phosphorylated Erkl to phosphorylated Erk2 in cells that have been stimulated with a control solution (vehicle) that lacks the protein kinase C activator.
  • a control solution that lacks the protein kinase C activator.
  • the difference is greater than zero, i.e. a positive value, this is diagnostic of Alzheimer's disease.
  • the difference is less than or equal to zero, this is indicative of the absence of Alzheimer's disease.
  • the Alzheimer's disease-specific molecular biomarkers of the present invention are measured by determining the ratio of two phosphorylated MAP kinase proteins after stimulation of cells with a protein kinase C activator.
  • the molecular biomarker may be measured by determining the ratio of a first phosphorylated MAP kinase protein to a phosphorylated second MAP kinase protein in cells that have been stimulated by a protein kinase C activator and subtracting from this the ratio of phosphorylated first MAP kinase protein to phosphorylated second MAP kinase protein in cells that have been stimulated with a control solution (vehicle) that lacks the protein kinase C activator.
  • a control solution that lacks the protein kinase C activator.
  • the Alzheimer's disease-specific molecular biomarker is a positive numerical value in cell samples taken from patients diagnosed with Alzheimer's disease (AD cells).
  • AD cells Alzheimer's disease
  • the positive numerical values for the Alzheimer's disease-specific molecular biomarker in AD cells may range from about zero to about 0.5.
  • the Alzheimer's disease-specific molecular biomarker is a negative numerical value when measured in cells taken from subjects diagnosed with non- Alzheimer's disease dementia (non-ADD cells), such as, for example, Parkinson's disease or Huntington's disease or Clinical Schizophrenia.
  • non-ADD cells non- Alzheimer's disease dementia
  • the negative numerical values may range from about zero to about -0.2 or about -0.3.
  • the Alzheimer's disease-specific molecular biomarker may be a negative numerical value, zero or very low positive numerical value in cell samples from age-matched control cells (AC cells) taken from patients who do not have Alzheimer's disease.
  • AC cells age-matched control cells
  • the Alzheimer's disease-specific molecular biomarker in AC cells may range from less than about 0.05 to about -0.2.
  • Protein kinase C activators that are specifically contemplated for use in the diagnostic methods, kits and methods of screening to identify
  • compounds of the instant invention include, but are not limited to: Bradykinin; ⁇ -APP
  • A Micromonospora sp.; Oleic acid; l-Oleoyl-2-acetyl-sn-glycerol; 4 ⁇ -Phorbol;
  • Bryologues are described, for example, in Wender et al. Organic letters ( United States ) May 12, 2005 ,7 (10) pl995-8; Wender et al.
  • a protein kinase C activator may be used alone or in combination with any other protein kinase C activator in the diagnostic methods, kits and methods of screening compounds disclosed herein.
  • Bradykinin is a potent vasoactive nonapeptide that is generated in the course of various inflammatory conditions. Bradykinin binds to and activates specific cell membrane bradykinin receptor(s), thereby triggering a cascade of intracellular events leading to the phosphorylation of proteins known as "mitogen activated protein kinase” (MAPK).
  • MAPK mitogen activated protein kinase
  • Phosphorylation of protein the addition of a phosphate group to a Ser, Thr, or Tyr residue, is mediated by a large number of enzymes known collectively as protein kinases. Phosphorylation normally modifies the function of, and usually activates, a protein.
  • phosphorylation be a transient process, which is reversed by phosphatase enzymes that dephosphorylate the substrate. Any aberration in phosphorylation or dephosphorylation may disrupt biochemical pathways and cellular functions. Such disruptions may be the basis for certain brain diseases. Measuring or Detecting Levels of Phosphorylated Proteins
  • the methods of diagnosing Alzheimer's disease and methods of screening compounds to identify agents useful for the treatment or prevention of Alzheimer's disease herein disclosed depend on measuring the Alzheimer's disease-specific molecular biomarkers of the present invention.
  • the level of phosphorylated protein present in cells is detected by Western blotting.
  • Protein levels of phosphorylated Erkl or phosphorylated Erk2 can be measured in fibroblasts using anti-Erkl, anti-Erk2, anti-phospho-Erkl and anti-phospho-Erk2 antibodies (Cell Signaling Technology).
  • Levels of a different protein may also be measured in the same sample as a reference protein for normalization. Examples of possible reference proteins include, but are not limited to, annexin-II or actin.
  • ELISA is performed according to the following procedures: 1) Add fibroblast cell lysates after treatment in duplicates or triplicates to a 96-well microplate that is previously coated with an anti-Erk antibody. 2) Incubate samples in microplate wells at room temperature for about 2 hours. 3) Aspirate samples and wash wells with a phosphate buffered saline (PBS)-based washing buffer. 4) Add working dilution of an anti-phospho-Erkl/2, or an anti- regular Erkl/2 antibody to each well, and incubate at room temperature for about 1 hour. 5) Aspirate and wash well with washing buffer.
  • PBS phosphate buffered saline
  • NR A P /A R .
  • NR the normalized ratio
  • Ap absorbance values for phospho-Erkl/2
  • a R absorbance for the total (regular) Erkl/2.
  • phosphorylation of Erkl/2 is assayed on Western blots using an anti-phospho-Erkl/2 antibody.
  • Levels of the immunoreactive signals for phosphorylated Erkl/2 are quantified via densitometric scan.
  • the mean density of the phospho-Erkl/2 signals are normalized with the mean density of total Erkl/2 signals that are detected from the same cell lysate samples with an anti-regular Erkl/2 antibody on a separate Western blot.
  • Immunoassays of the present invention for the detection of proteins may be immunofluorescent assays, radioimmunoassays, Western blot assays, enzyme immunoassay, immuno-precipitation, chemiluminescent assay, immunohistochemical assay, dot or slot blot assay and the like.
  • immunofluorescent assays may be immunofluorescent assays, radioimmunoassays, Western blot assays, enzyme immunoassay, immuno-precipitation, chemiluminescent assay, immunohistochemical assay, dot or slot blot assay and the like.
  • Detection may be by colorometric or radioactive methods or any other conventional methods known to those having skill in the art. Standard techniques known in the art for ELISA are described in Methods in Immunodiagnosis, 2 nd Edition, Rose and Bigazzi, eds., John Wiley and Sons, New York 1980 and Campbell et al., Methods of Immunology x W. A. Benjamin, Inc., 1964, both of which are incorporated herein by reference. Such assays may be direct, indirect, competitive, or noncompetitive immunoassays as described in the art (In "Principles and Practice of Immunoassay” (1991) Christopher P. Price and David J. Neoman (eds), Stockton Pres, NY, NY; Oellirich, M. 1984. J. Clin. Chem. Clin. Biochem. 22: 895-904 Ausubel, et al. (eds) 1987 in Current Protocols in Molecular Biology, John Wiley and Sons, New York, New York.
  • the cells taken from the patient being diagnosed may be any cell.
  • examples of cells that may be used include, but are not limited to, skin cells, skin fibroblasts, buccal mucosal cells, blood cells, such as erythrocytes, lymphocytes and lymphoblastoid cells, and nerve cells and any other cell expressing the Erkl/2 protein.
  • Necropsy samples and pathology samples may also be used. Tissues comprising these cells may also be used, including brain tissue or brain cells.
  • the cells may be fresh, cultured or frozen. Protein samples isolated from the cells or tissues may be used immediately in the diagnostic assay or methods for screening compounds or frozen for later use. In a preferred embodiment fibroblast cells are used. Fibroblast cells may be obtained by a skin punch biopsy.
  • Proteins may be isolated from the cells by conventional methods known to one of skill in the art.
  • cells isolated from a patient are washed and pelleted in phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the protein extract may be used fresh or stored at -8O 0 C for later analysis.
  • the antibodies used in the disclosed immunoassays may be monoclonal or polyclonal in origin.
  • the phosphorylated and non-phosphorylated Erkl/2 protein or portions thereof used to generate the antibodies may be from natural or recombinant sources or generated by chemical synthesis. Natural Erkl/2 proteins can be isolated from biological samples by conventional methods.
  • Examples of biological samples that may be used to isolate the Erkl/2 protein include, but are not limited to, skin cells, such as, fibroblasts, fibroblast cell lines, such as Alzheimer's disease fibroblast cell lines and control fibroblast cell lines which are commercially available through Coriell Cell Repositories, (Camden, NJ.) and listed in the National Institute of Aging 1991 Catalog of Cell Lines, National Institute of General Medical Sciences 1992/1993 Catalog of Cell Lines [(NIH Publication 92-2011 (1992)].
  • kits which may be utilized in performing any of the diagnostic tests described above.
  • the kits may contain a single diagnostic test or any combination of the tests described herein.
  • the kits may comprise antibodies which recognize regular Erkl/2 (unphosporylated Erkl or unphosphorylated Erk2) or phosphorylated Erkl/2 (phosphorylated Erkl or phosphorylated Erk2).
  • the kits may contain antibodies that recognize regular MAP kinase proteins as well as phosphorylated MAP kinase proteins.
  • the kits may also contain any one or more of the protein kinase C activators disclosed herein (such as, for example, bradykinin or bryostatin).
  • Antibodies may be polyclonal or monoclonal.
  • kits may contain instruments, buffers and storage containers necessary to perform one or more biopsies, such as punch skin biopsies.
  • the kits may also contain instructions relating to the determination of the ratios used to identify the Alzheimer's disease-specific molecular biomarkers of the instant invention as well as the use of the antibodies or other constituents in the diagnostic tests.
  • the instructions may also describe the procedures for performing a biopsy, such as a punch skin biopsy.
  • the kits may also contain other reagents for carrying out the diagnostic tests such as antibodies for the detection of reference proteins used for normalization. Examples of antibodies that recognize possible reference proteins include, but are not limited to, antibodies that recognize annexin-II or actin.
  • the kits may also include buffers, secondary antibodies, control cells, and the like.
  • the present invention is also directed to methods to screen and identify substances useful for the treatment or prevention of Alzheimer's disease.
  • substances which reverse or improve the Alzheimer's disease-specific molecular biomarkers described herein i.e. back to levels found in normal cells
  • one such method of screening to identify therapeutic substances would involve the steps of contacting sample cells from an Alzheimer's disease patient with a substance being screened in the presence of any of the protein kinase C activators disclosed herein and then measuring any of the Alzheimer's disease- specific molecular biomarkers disclosed herein.
  • An agent that reverses or improves the Alzheimer's disease-specific molecular biomarker back to levels found in normal cells i.e. cells taken from a subject without Alzheimer's disease
  • Alzheimer's disease-specific molecular biomarker is an agent that causes a reduction of a positive value and/or a movement towards more negative values for an Alzheimer's disease-specific molecular biomarker.
  • the present invention is also directed to methods of monitoring the progression of Alzheimer's disease in a subject.
  • Figure 2 provides a linear regression analysis of Alzheimer's disease-specific molecular biomarkers as a function of years of duration of dementia. The linear regression shows a negative slope of approximately
  • Alzheimer's disease-specific molecular biomarker As the years of dementia increases (i.e. as Alzheimer's disease progresses) the Alzheimer's disease-specific molecular biomarker becomes a less positive numerical value. Measurement of the Alzheimer's disease-specific molecular biomarker allows for early diagnosis of Alzheimer's disease because, in certain embodiments, a more highly positive value is indicative of early stages of the disease. In certain embodiments, as Alzheimer's disease progresses in a subject, the Alzheimer's disease specific molecular biomarker becomes a less positive value.
  • the present invention is also directed to compositions useful for the treatment or prevention of Alzheimer's disease.
  • Compounds identified using the screening methods described herein may be formulated as pharmaceutical compositions for administration to subjects in need thereof.
  • a pharmaceutical composition of the present invention or a compound (or a chemical derivative of a lead compound) identified using the screening methods disclosed herein can be administered safely by admixing with, for example, a pharmacologically acceptable carrier according to known methods to give a pharmaceutical composition, such as tablets (inclusive of sugar-coated tablets and film-coated tablets), powders, granules, capsules, (inclusive of soft capsules), liquids, injections, suppositories, sustained release agents and the like, for oral, subcutaneous, transdermal, transcutaneous or parenteral (e.g., topical, rectal or intravenous) administration.
  • a pharmaceutical composition such as tablets (inclusive of sugar-coated tablets and film-coated tablets), powders, granules, capsules, (inclusive of soft capsules), liquids, injections, suppositories, sustained release agents and the like, for oral, subcutaneous, transdermal, transcutaneous or parenteral (e.g., topical, rectal or intravenous
  • Examples of pharmacologically acceptable carriers for use in the pharmaceutical compositions of the invention include, but are not limited to various conventional organic or inorganic carriers, including excipients, lubricants, binders and disintegrators for solid preparations, and solvents, solubilizers, suspending agents, isotonic agents, buffers, soothing agents, and the like for liquid preparations.
  • conventional additives such as antiseptics, antioxidants, coloring agents, sweeteners, absorbents, moistening agents and the like can be used appropriately in suitable amounts.
  • excipients for use in the pharmaceutical compositions of the invention include, but are not limited to agents such as lactose, sucrose, D-mannitol, starch, corn starch, crystalline cellulose, light anhydrous silicic acid, polysaccharides, disaccharides, carbohydrates, trehalose and the like.
  • binders for use in the pharmaceutical compositions of the invention include, but are not limited to crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropylcellulose. hydroxypropylmethylcellulose, polyvinylpyrrolidone, starch, sucrose, gelatin, methylcellulose, carboxymethylcellulose sodium and the like.
  • disintegrators for use in the pharmaceutical compositions of the invention include, but are not limited to starch, carboxymethylcellulose, carboxymethylcellulose calcium, sodium carboxymethyl starch, L-hydroxypropylcellulose and the like.
  • solvents for use in the pharmaceutical compositions of the invention include, but are not limited to water for injection, alcohol, propylene glycol, Macrogol, sesame oil, corn oil, olive oil and the like.
  • solubilizers for use in the pharmaceutical compositions of the invention include, but are not limited to polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, tris-aminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate and the like.
  • surfactants such as stearyl triethanolamine, sodium lauryl sulfate, lauryl aminopropionate, lecithin, benzalkonium chloride, benzethonium chloride, glyceryl monostearate and the like
  • hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, carboxymethylcellulose sodium, methylcellulose, hydroxymethylcellulose, hydroxyeth
  • isotonic agents for use in the pharmaceutical compositions of the invention include, but are not limited to glucose, D-sorbitol, sodium chloride, glycerine, D-mannitol and the like.
  • buffers for use in the pharmaceutical compositions of the invention include, but are not limited to phosphate, acetate, carbonate, citrate etc., and the like.
  • Examples of soothing agents for use in the pharmaceutical compositions of the invention include, but are not limited to benzyl alcohol and the like.
  • antiseptics for use in the pharmaceutical compositions of the invention include, but are not limited to p-oxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like.
  • antioxidants for use in the pharmaceutical compositions of the invention include, but are not limited to sulfite, ascorbic acid, ⁇ - tocopherol and the like.
  • a carrier for injection to be used may include any or all of the following: a solvent, a solubilizer, a suspending agent, an isotonic agent, a buffer, a soothing agent and the like.
  • a solvent include, but are not limited to water for injection, physiological saline, Ringer's solution and the like.
  • the solubilizer include, but are not limited to polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate and the like.
  • Examples of the isotonic agent include but are not limited to glucose, D- sorbitol, sodium chloride, glycerin, D-mannitol and the like.
  • Examples of the buffer include but are not limited to buffers such as phosphate, acetate, carbonate, citrate and the like, and the like.
  • Examples of the soothing agent include but are not limited to benzyl alcohol and the like.
  • Examples of the pH adjusting agent include but are not limited to hydrochloric acid, phosphoric acid, citric acid, sodium hydroxide, sodium bicarbonate, sodium carbonate and the like.
  • the composition for injection of the present invention may be freeze-dried in an aseptically treated freeze dryer and preserved in a powder state, or can be sealed in a container for injection (e.g., ampoule) and preserved.
  • composition of the present invention may be diluted with the aforementioned carrier for injection when in use.
  • the content of an active compound in the pharmaceutical composition of the present invention may vary depending on the form of the preparation, but it is generally about 0.01 -about 99 wt %, preferably about 0.1 -about 50 wt %, more preferably about 0.5-about 20 wt %, of the whole preparation.
  • the content of nonionic surfactant in the pharmaceutical composition of the present invention may vary depending on the form of the preparation, but it is generally about 1 to about 99.99 wt %, preferably about 10 to about 90 wt %, more preferably about 10 to about 70 wt %, of the whole preparation.
  • the content of ethanol, benzyl alcohol or dimethylacetamide in the pharmaceutical compositions of the present invention may vary depending on the form of the preparation, but it is generally about 1 to about 99.99 wt %, preferably about 10 to about 90 wt %, more preferably about 30 to about 90 wt %, of the whole preparation.
  • the content of cyclodextrin derivative readily soluble in water in the pharmaceutical composition of the present invention varies depending on the form of the preparation, but it is generally about 1 to about 99.99 wt %, preferably about 10 to about 99.99 wt %, more preferably about 20 to about 97 wt %, particularly preferably about 50 to about 97 wt %, of the whole preparation.
  • the content of other additives in the pharmaceutical composition of the present invention may vary depending on the form of the preparation, but it is generally about 1 to about 99.99 wt %, preferably about 10 to about 90 wt %, more preferably about 10 to about 70 wt %, of the whole preparation.
  • the pharmaceutical compositions of the present invention may be a pharmaceutical composition comprising an active compound, a nonionic surfactant and a cyclodextrin derivative readily soluble in water.
  • the content of each component, i.e. the active compound, the nonionic surfactant and the cyclodextrin derivative readily soluble in water is the same as in the aforementioned ranges.
  • the present invention is also directed to methods of treating or preventing Alzheimer's disease using the pharmaceutical compositions disclosed herein.
  • the compounds of the present invention may be administered by oral, parenteral (for example, intramuscular, intraperitoneal, intravenous, ICV, intracisternal injection or infusion, subcutaneous injection, or implant), by inhalation spray, nasal, vaginal, rectal, sublingual, or topical routes of administration and may be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles appropriate for each route of administration.
  • the pharmaceutical compositions and method of the present invention may further comprise other therapeutically active compounds which are usually applied in the treatment of Alzheimer's disease.
  • an appropriate dosage level will generally be about 0.001 to 100 mg per kg patient body weight per day which can be administered in single or multiple doses.
  • the dosage level will be about 0.01 to about 25 mg/kg per day; more preferably about 0.05 to about 10 mg/kg per day.
  • a suitable dosage level may be about 0.01 to 25 mg/kg per day, about 0.05 to 10 mg/kg per day, or about 0.1 to 5 mg/kg per day. Within this range the dosage may be about 0.005 to about 0.05, 0.05 to 0.5 or 0.5 to 5 mg/kg per day.
  • compositions are preferably provided in the form of tablets containing about 1 to 1000 milligrams of the active ingredient, particularly about 1, 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900, and 1000 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated.
  • the compounds may be administered on a regimen of 1 to 4 times per day, preferably once or twice per day.
  • EXAMPLE 1 In Vitro Assay of Skin Fibroblasts to Determine Ratio of Phosphorylated Erkl to Phosphorylated Erk2
  • AD dementia non- ADD
  • AC age- matched control cells
  • DMEM serum-free medium
  • BK Bradykinin
  • lysis buffer 10 mM Tris, pH 7.4, 150 mM NaCl, 1 mM
  • EDTA 1 mM EGTA, 0.5% NP-40, 1% Triton X-100, 1% protease inhibitor cocktail, 1% ser/thr/tyrosine phosphatase inhibitor cocktails) was added into each flask.
  • Flasks were kept on an end-to-end shaker in a cold room (4 0 C) for 30 min and cells were collected from each flask with a cell scraper. Cells were sonicated and then centrifuged at 14000 rpm for 15 min, and the supernatant was used for Western blotting after total protein assay.
  • Erk2 (p-Erkl, p-Erk2) were determined using specific antibodies: anti-regular Erkl/2 and anti-phospho ERK 1/2.
  • fibroblasts from Huntington' s disease are from Huntington' s disease (HD) patients, with dementia accompanying typical Huntington' s disease symptoms. Fibroblasts from normal age- and gender- matched individuals are used as controls.
  • DMEM is purchased from Gibco BRL. Fetal bovine serum is purchased from Bio Fluids. Bradykinin, diphenylboric acid 2-aminoethyl ester (2ABP), protease, and phosphatase inhibitor cocktails are purchased from Sigma; bisindolylmaleimide-1 and LY294002 are purchased from Alexis; PD98059 is purchased from Cell Signaling Technology. Anti-phospho-Erkl/2 antibodies are purchased from Cell Signaling Technology. Anti-regular Erkl/2 is purchased from Upstate Biotechnology. SDS minigels (4-20%) are purchased from Invertrogene- Novex. Nitrocellulose membranes are purchased form Schleicher & Schuell (Keene, NH). All the SDS electrophoresis reagents are purchased from Bio-Rad. The SuperS ignal chemilumines-cence substrate kit is purchased from Pierce.
  • Samples are placed in IxPBS and transported in transfer medium to the laboratory for propagation. After the transfer medium is removed, the skin tissues are rinsed with PBS and finely chopped into 1-mm-sized explants. The explants are transferred one by one onto the growth surface of vented T25 flasks with 3 ml of biopsy medium containing 45% FBS and 100 U/ml penicillin and 100 U/ml streptomycin (Pen/Strep). The tissues are cultured at 37 C for 24 h before addition of 2 ml of biopsy medium containing 10% FBS. The medium is replaced after 48 h with 5 ml of regular culture medium containing 10% FBS and 100 U/ml Pen/Strep. The cells are then passaged and maintained according to a regular procedure given above.
  • Bradykinin or different specific protein kinase C activators are used to treat fibroblasts.
  • Banked AC and AD skin fibroblasts are cultured to 80-100% confluence before they are "starved” in serum-free DMEM overnight.
  • Cells are treated with 10 nM protein kinase C activator at 37 C for different lengths of time to establish a time course for the protein kinase C acivator-induced effects.
  • the time point at which reactions are terminated immediately after application of protein kinase C activator is defined as "0 min" post-protein kinase C activator treatment.
  • a control flask of cells for each cell line at each treatment time point is added with the identical volume of PBS.
  • the reaction is terminated by removing the culture medium, rapidly rinsing the cells with precooled PBS, pH 7.4, and transferring the flask onto dry ice/ethanol.
  • a concentration of 0.1 nM protein kinase C activator may be used for cells obtained and cultured from fresh biopsy tissues.
  • the treatment time is about 10 min at 37 C.
  • Cell lysates are treated with an equal volume of 2X SDS- sample buffer and boiled for 10 min. Proteins from each sample are resolved on a A- 20% mini-gradient gel and transferred onto a nitrocellulose membrane. Phosphorylated Erkl/2 is detected with an anti-phospho-Erkl/2 antibody using the SuperSignal ECL detection kit.
  • Fibroblast cells are grown on the surface of 2.5-cm-diameter glass coverslips coated with 0.02 mg polylysine.
  • bradykinin or another protein kinase C activator as described above, cells are rapidly rinsed with cold PBS, pH 7.4, and fixed with 4% formaldehyde in PBS, pH 7.4, at room temperature for 15 min.
  • PBS, pH 7.4, three times, each lasting 5 min cells are penetrated with 0.1% Triton x-100 in PBS, pH 7.4, at room temperature for 30 min.
  • cells are incubated with anti-phospho-Erkl/2
  • a monoclonal anti-BK B 2 antibody, or anti protein kinase C activator antibody is applied to the normal fibroblasts, followed by incubation with Cy5-conjugated anti- mouse IgG.
  • the resulting immunoreactive signals are imaged with a fluorescence microscope.

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Abstract

La présente invention concerne des techniques de diagnostic de la maladie d'Alzheimer chez un sujet par détection de modifications dans le rapport de protéines kinase MAP phosphorilée spécifiques dans des cellules après stimulation avec un activateur C de protéine kinase. Des biomarqueurs moléculaires de l'invention spécifiques de la maladie d'Alzheimer conviennent pour le diagnostic de la maladie d'Alzheimer, la surveillance de la progression de cette maladie chez un sujet et dans des techniques de recherche destinées à identifier des composés permettant de traiter ou de prévenir la maladie d'Alzheimer. Cette invention concerne aussi des kits contenant des réactifs destinés à la détection et au diagnostic de la maladie d'Alzheimer utilisant ces biomarqueurs moléculaires spécifiques de la maladie d'Alzheimer.
PCT/US2005/036014 2005-10-11 2005-10-11 Modifications specifiques de la maladie d'alzheimer du rapport de phosphorylation erk1/erk2 WO2007043998A1 (fr)

Priority Applications (26)

Application Number Priority Date Filing Date Title
PCT/US2005/036014 WO2007043998A1 (fr) 2005-10-11 2005-10-11 Modifications specifiques de la maladie d'alzheimer du rapport de phosphorylation erk1/erk2
PCT/US2006/022156 WO2007044094A1 (fr) 2005-10-11 2006-06-07 Modifications specifique de la maladie d'alzheimer du rapport de phosphorylation erk1/erk2 utilise comme biomarqueurs moleculaires specifiques de la maladie d'alzheimer (adsmb)
EP08020258A EP2031398B1 (fr) 2005-10-11 2006-09-25 Altérations spécifiques à la maladie d'Alzheimer des biomarqueurs moléculaires spécifiques à la maladie d'Alzheimer du rapport de phosphorylation erk1/erk2
EP10012836.2A EP2317321B1 (fr) 2005-10-11 2006-09-25 Méthodes de suivi de la progression d'Alzheimer en mesurant un biomarqueur
ES08020258T ES2412268T3 (es) 2005-10-11 2006-09-25 Alteraciones específicas de la Enfermedad de Alzheimer de la relación de fosforilación ERK1/ERK2 como biomarcadores moleculares específicos de la Enfermedad de Alzheimer (ADSMB)
EP06825096A EP1934618B1 (fr) 2005-10-11 2006-09-25 Modifications du taux de phosphorylation d'erk1/erk2 specifiques de la maladie d'alzheimer relativement aux biomarqueurs moleculaires specifiques de la maladie d'alzheimer
ES10011288.7T ES2588376T3 (es) 2005-10-11 2006-09-25 Procedimientos para la identificación de tratamientos de la enfermedad de Alzheimer
JP2008535546A JP4908514B2 (ja) 2005-10-11 2006-09-25 アルツハイマー病に特異的なｅｒｋ１／ｅｒｋ２リン酸化比の変化−アルツハイマー病に特異的な分子生物マーカー（ａｄｓｍｂ）
PCT/US2006/037186 WO2007047029A2 (fr) 2005-10-11 2006-09-25 Modifications du taux de phosphorylation d'erk1/erk2 specifiques de la maladie d'alzheimer relativement aux biomarqueurs moleculaires specifiques de la maladie d'alzheimer
EP10011289.5A EP2322934B1 (fr) 2005-10-11 2006-09-25 Kit pour le diagnostic de la maladie d'Alzheimer
DE602006006855T DE602006006855D1 (de) 2005-10-11 2006-09-25 Morbus alzheimer-spezifische veränderungen des erkheimer-spezifische molekulare biomarker (adsmb)
CA2625300A CA2625300C (fr) 2005-10-11 2006-09-25 Modifications du taux de phosphorylation d'erk1/erk2 specifiques de la maladie d'alzheimer relativement aux biomarqueurs moleculaires specifiques de la maladie d'alzheimer
KR1020137031223A KR20140002073A (ko) 2005-10-11 2006-09-25 알츠하이머병-특이적 분자 생체 지표(ａｄｓｍｂ)로서 ｅｒｋ1/ｅｒｋ2 포스포릴화비의 알츠하이머병-특이적 변화
ES10012836.2T ES2477284T3 (es) 2005-10-11 2006-09-25 Método de monitorización de la progresión de la enfermedad de Alzheimer por medición de un biomarcador
AT06825096T ATE431558T1 (de) 2005-10-11 2006-09-25 Morbus alzheimer-spezifische veränderungen des erk1/erk2-phosphorylierungsverhältnisses - morbus alzheimer-spezifische molekulare biomarker (adsmb)
US12/083,056 US20090029873A1 (en) 2005-10-11 2006-09-25 Alzheimer's Disease-Specific Alterations of the Erk1/Erk2 Phosphorylation Ratio-Alzheimer's Disease-Specific Molecular Biomarkers (Adsmb)
EP10011290A EP2322936A1 (fr) 2005-10-11 2006-09-25 Méthodes d'identification d'un composé principal pour le traitement de la maladie d'Alzheimer
ES10011289.5T ES2596881T3 (es) 2005-10-11 2006-09-25 Kit para el diagnóstico de la Enfermedad de Alzheimer
ES06825096T ES2323813T3 (es) 2005-10-11 2006-09-25 Alteraciones especificas de la enfermedad de alzheimer de la proporcion de fosforilacion erk1/erk2 - biomarcadores moleculares especificos de la enfermedad de alzheimer (adsmb).
EP10011288.7A EP2339349B1 (fr) 2005-10-11 2006-09-25 Méthodes d'identification des traitements d'Alzheimer
TW102146766A TW201413246A (zh) 2005-10-11 2006-10-11 含有診斷阿茲海默症之試劑的套組
TW095137389A TWI448688B (zh) 2005-10-11 2006-10-11 以阿茲海默症特異性之ｅｒｋ１／ｅｒｋ２磷酸化比率的變化作為阿茲海默症特異性的分子生物標記
US12/078,857 US20080221042A1 (en) 2005-10-11 2008-04-07 Alzheimer's disease-specific alterations of the ERK1/ERK2 Phosphorylation ratio-Alzheimer's disease-specific molecular biomarkers (ADSMB)
KR1020087011105A KR101375552B1 (ko) 2005-10-11 2008-05-08 알츠하이머병-특이적 분자 생체 지표(ａｄｓｍｂ)로서 ｅｒｋ1/ｅｒｋ2 포스포릴화비의 알츠하이머병-특이적 변화
US13/774,049 US9797913B2 (en) 2005-10-11 2013-02-22 Alzheimer's disease-specific alterations of the ERK1/ERK2 phosphorylation ratio-alzheimer's disease-specific molecular biomarkers (ADSMB)
US15/719,714 US20180024146A1 (en) 2005-10-11 2017-09-29 Alzheimer's disease-specific alterations of the erk1/erk2 phosphorylation ratio-alzheimer's disease-specific molecular biomarkers (adsmb)

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US9797913B2 (en) 2005-10-11 2017-10-24 Blanchette Rockefeller Neuroscienses Inc. Alzheimer's disease-specific alterations of the ERK1/ERK2 phosphorylation ratio-alzheimer's disease-specific molecular biomarkers (ADSMB)
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