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Prevention and treatment of amyloid-associated disordersRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test StripPrevention and treatment of amyloid-associated disorders description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060210964, Prevention and treatment of amyloid-associated disorders. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE [0001] This application claims priority to U.S. provisional application Ser. No. 60/142,175, filed Jul. 1, 1999, which application is incorporated herein by reference in its entirety. FIELD OF THE INVENTION [0002] The invention relates generally to the treatment of neurological diseases and specifically to treatment of neurological diseases involving amyloid plaque formation. BACKGROUND OF THE INVENTION [0003] A number of important neurological diseases including Alzheimer's disease (AD), cerebral amyloid angiopathy (CAA), and prion-mediated diseases are characterized by the deposition of aggregated proteins, referred to as amyloid, in the central nervous system (CNS) (for reviews, see Glenner et al. (1989) J. Neurol. Sci. 94:1-28; Haan et al. (1990) Clin. Neurol. Neurosurg. 92(4):305-310. These highly insoluble aggregates are composed of nonbranching, fibrillar proteins with the common characteristic of a .beta.-pleated sheet conformation. In the CNS, amyloid can be present in cerebral and meningeal blood vessels (cerebrovascular deposits) and in brain parenchyma (plaques). Neuropathological studies in human and animal models indicate that cells proximal to amyloid deposits are disturbed in their normal functions (Mandybur (1989) Acta Neuropathol. 78:329-331; Kawai et al. (1993) Brain Res. 623:142-6; Martinet al. (1994) Am. J. Pathol. 145:1348-1381; Kalaria et al. (1995) Neuroreport 6:477-80; Masliah et al. (1996) J. Neurosci. 16:5795-5811). AD studies additionally indicate that amyloid fibrils may actually initiate neurodegeneration (Lendon et al. (1997) J. Am. Med. Assoc. 277:825-31; Yankner (1996) Nat. Med. 2:850-2; Selkoe (1996) J. Biol. Chem. 271:18295-8; Hardy (1997) Trends Neurosci. 20:154-9). [0004] AD and CAA share biochemical and neuropathological markers, but differ somewhat in the extent and location of amyloid deposits as well as in the symptoms exhibited by affected individuals. The neurodegenerative process of AD, the most common cause of progressive intellectual failure in aged humans, is characterized by the progressive and irreversible deafferentation of the limbic system, association neocortex, and basal forebrain accompanied by neuritic plaque and tangle formation (for a review see Terry et al. (1994) "Structural alteration in Alzheimer's disease." In: Alzheimer's disease (Terry et al. eds.), pp. 179-196. Raven Press, New York). Dystrophic neurites, as well as reactive astrocytes and microglia, are associated with these amyloid-associated neurite plaques. Although, the neuritic population in any given plaque is mixed, the plaques generally are composed of spherical neurites that contain synaptic proteins, APP (type I), and fusiform neurites containing cytoskeletal proteins and paired helical filaments (PHF; type II). [0005] CAA patients display various vascular syndromes, of which the most documented is cerebral parenchymal hemorrhage. Cerebral parenchymal hemorrhage is the result of extensive. amyloid deposition within cerebral vessels (Hardy (1997) Trends Neurosci. 20:154-9; Haan et al. (1990) Clin. Neurol. Neurosurg. 92:305-10; Terry et al., supra; Vinters (1987) Stroke 18:211-24; Itoh et al. (1993) J. Neurological Sci. 116:135-41; Yamada et al. (1993) J. Neurol. Neurosurg. Psychiatry 56:543-7; Greenberg et al. (1993) Neurology 43:2073-9; Levy et al. (1990) Science 248:1124-6). In some familial CAA cases, dementia was noted before the onset of hemorrhages, suggesting the possibility that cerebrovascular arnyloid deposits may also interfere with cognitive functions. [0006] The precise mechanisms by which neuritic plaques are formed and the relationship of plaque formation to the AD-associated and CAA-associated neurodegenerative processes are not well-defined. Several factors that increase the likelihood of developing AD have already been identified. The risk of developing AD definitely increases with: (1) age, (2) head injuries, (3) family history of AD or Down syndrome, (4) sex, with a higher prevalence of AD in women, (5) vascular disease, (6) exposure to environmental toxins, (7) infectious processes, or (8) changes in immune function. Recent advances in molecular genetics have suggested that genetic predisposition is one of the most important risk factors in the development of AD. For example, a significant increase in the number of amyloid plaques in AD patients with an apolipoprotein E4 (apoE4) allele has been observed and the results of several genetic studies indicate that the etiology of this neurodegenerative disease is associated with the presence of the apoE4 allele. [0007] In both AD and CAA, the main amyloid component is the amyloid .beta. protein (A.beta.). The A.beta. peptide, which is generated from the amyloid .beta. precursor protein (APP) by two putative secretases, is present at low levels in the normal CNS and blood. Two major variants, A.beta..sub.1-40 and A.beta..sub.1-42, are produced by alternative carboxy-terminal truncation of APP (Selkoe et al.(1988) Proc. Natl. Acad. Sci. USA 85:7341-7345; Selkoe, (1993) Trends Neurosci 16:403-409). A.beta..sub.1-42 is the more fibrillogenic and more abundant of the two peptides in amyloid deposits of both AD and CAA. In addition to the amyloid deposits in AD cases described above, most AD cases are also associated with amyloid deposition in the vascular walls (Hardy (1997), supra; Haan et al. (1990), supra; Terry et al., supra; Vinters (1987), supra; Itoh et al. (1993), supra; Yamada et al. (1993), supra; Greenberg et al. (1993), supra; Levy et al. (1990), supra). These vascular lesions are the hallmark of CAA, which can exist in the absence of AD. [0008] Glial cell activation is believed to play an essential pathogenic role in the development of dementia. A source of damage in the AD brain is an altered response triggered by microglial activation, which is associated with amyloid plaques. For example, a correlation between genetic predisposition and the proliferation and activation of microglial cells was obtained in AD primary in vitro microglial cell cultures (Lombardi et al. (1998) J Neurosci Res 54:539-53). Many studies have shown that microglia secrete both cytokines and cytotoxins and since reactive microglia appears in nearly every type of brain damage, it is likely that their secreted products ultimately help to determine the rate of damaged brain tissue. See e.g., Giulian, et al. (1994) Neurochem Int. 25:227-33. Reactive microglia may also contribute to neuronal damage by the generation of free oxygen radicals and nitric oxide (NO), which forms the particularly aggressive peroxynitrites, and by the release of potentially neurotoxic cytokines such as tumor necrosis factor-.alpha. (TNF-.alpha.) (P. Schubert et al. (1998) Alzheimer Dis Assoc Disord., 12 Suppl 2:S21-8). [0009] Prostaglandins and nitric oxide (NO). are among the numerous substances released by activated microglial cells. Cyclooxygenase-2 (COX-2) and inducible NO synthase (iNOS), the two key enzymes in prostaglandin and NO synthesis, respectively, are rapidly co-induced in rat neonatal microglial cultures activated by bacterial endotoxin (lipopolysaccharide [LPS]). COX-2 expression appears to be under the negative control of endogenous as well as exogenous NO (Minghetti et al. (1997) Eur J Neurosci. 9:934-40). Inhibitors of the inducible form of cyclooxygenase (COX-2) have been examined for the treatment of AD. It is becoming increasingly clear, however, that the products of COX-2 mediate both pro- and anti-inflammatory responses, and that inhibiting all COX-2 products in chronic neuroinflammatory states to reduce neuroinflammation inhibits the anti-inflammatory properties certain COX-2 products. Caggiano, (1998) J Neurochemistry 70:2357-68. For example, PGI.sub.2 and PGF.sub.2.alpha.are associated with anti-inflammatory activity, and regulation using COX-2 inhibitors may reduce their anti-inflammatory effects. [0010] Prostaglandin E2 (PGE.sub.2) is also produced by activated microglial cells, and is known to increase cyclic adenosine monophosphate (cAMP) levels in microglial cells (Minghetti et al., supra). Traditionally, PGE.sub.2 has been considered to be a positive factor in inflammation. More recently, however, PGE.sub.2 has been shown to: 1) protect neurons from cytotoxic injury (Akaike et al. (1994) Brain Research, 663:237-243); 2) inhibit LPS-induced outwardly rectifying potassium current and IL-1.beta. production (Caggiano et al., (1998) J. Neurochemistry, 70:2357-68); 3) downregulate LPS-induced iNOS expression in a dose-dependent manner in cultured rat microglia (Minghetti (1997) Glia, 19:152-60); and 4) reduce nitrous oxide-mediated cell injury by microglia (Thery, (1994) Glia, 11:383-86). In addition, PGE.sub.2 has been shown to modulate macrophage-derived TNF-.alpha. gene expression (S. L. Kunkle et al. (1998) J. Biol. Chem, 263:5380-84). [0011] There is a need in the art for a more specific therapeutic targeting system to control microglial cell activation. In addition, there is a need for a method of inhibiting amyloid plaque formation in patients suffering from neurodegenerative disorders. SUMMARY OF THE INVENTION [0012] The present invention provides assays to identify compounds that affect microglial cell activation, compounds identified in these assays which inhibit A.beta.:PGE.sub.2 activation of microglial cells, and methods of using such compounds in therapeutic intervention. Assays of the invention affect microglia activation through modulation of A.beta.:PGE.sub.2-mediated activity. A.beta.:PGE.sub.2 exposure to microglia activates the microglia to a greater extent than additive exposure to either agent alone. Since this synergistic activation of microglia presents a particularly pathogenic mechanism, methods of identifying compounds using the assays of the invention are particularly useful, since they can identify therapeutic agents that inhibit either or both arms of the synergistic effect. Moreover, the therapeutic agents identified using the assays of the invention may be particularly suited for patient intervention, as they exhibit a specific effect on this synergistic activation process. [0013] The assays of the invention include assays for testing microglial cell activation by contacting microglia with compounds that modulate A.beta. and/or PGE.sub.2-mediated activation. The effect of the candidate compound can be determined by comparing the effect with a control. culture which is not in contact with the compound, for example by measuring secretion of cytokines such as TNF-.alpha. and IL-.alpha. or by comparing the effect with a standardized cytokine profile. [0014] In one preferred embodiment, the invention features an assay to identify compounds which alter, halt or prevent progression of an amyloid-associated disorder by culturing microglial cells with A.beta.:PGE.sub.2 and a compound to be tested. The culture is then examined for synergistic activation by A.beta.:PGE.sub.2 as evidence by a change in cellular activity, for example cytokine secretion, elevation of nitric oxide synthetase (NOS) or its products, reactive oxygen species (ROS) or expression of molecules associated with activation such as LFA-1, VLA-4, or Mac-1. The culture can also be compared to levels prior to exposure with the compound or, alternatively, to a standardized profile for one or more of these cellular activities. The compound may be added to the cells prior to exposure with the A.beta. peptide (e.g., to examine the ability of the compound to prevent plaque formation), simultaneously with the A.beta. peptide, or following incubation with the A.beta. peptide (e.g., to determine the ability of the compound to halt or reverse progression of plaque formation). Preferably, the amyloid-associated disorder is AD or CAA, and the cytokine used in the assay is preferably IL-1.alpha., IL-1.beta., TNF-.alpha. and/or IL-6. [0015] The invention also features a method for determining the particular molecules that are therapeutic targets for modulation of microglia activation. For example, the receptor isoform involved in the A.beta.:PGE.sub.2 synergy was determined by using compounds that affects a particular isoform of PGE.sub.2, and the receptor isoform involved was identified by examining the effect of the compounds on microglia activation. [0016] In another embodiment, the invention provides a method for modulating cytokine secretion in a patient by analyzing microglial cells from the central nervous system of a patient, determining the level of expression. of cytokines from the microglial cells, and administering a, compound in an amount sufficient to reduce cytokine expression. Cytokine secretion may be monitored, e.g., by monitoring soluble factors associated with microglial cell activation from the cerebrospinal fluid of a patient, which can be obtained via a spinal tap. [0017] The invention also provides a method for reducing the level of .beta.-amyloid plaque in the brain tissue of a mammalian host by administering to the host a compound in an amount effective to reduce microglial activation. Preferably, the compound used in treatment reduces the microglial activation by 30 to 80%, and reduces cytokine secretion levels by 20 to 80%. [0018] The invention also provides standardized cellular profiles and methods of using such standards as a positive control in a neurodegenerative disease assay. The assay may be a bioassay which uses transgenic animals or an immunoassay, and can be used for purposes such as diagnosis, prognosis, determination of the efficacy of a therapeutic, etc. The standards function to ensure reproducibility and specificity of an assay by functioning as a reference material with a known and consistent level of microglial activation. The standards also make it possible to determine sensitivity and to adjust selectivity relative to sensitivity as needed. [0019] The invention also features a method of calibrating an assay using the standards of the invention. Calibration can be within a single assay, to determine efficacy at a given level of cytokine concentration, or between assays, to allow comparison of results of different assays by adjusting detection levels between assays. For example, if one assay is more sensitive than another, calibration with a standard can be used to determine the factor for converting measured levels to corrected levels for comparison of results obtained using different assays. [0020] The invention also features a method of determining the quality of reagents used in a diagnostic or prognostic assay by testing the reagents using standards of the invention. The standards provide a consistent level of microglial activation, and preferably a consistent background. Testing reagents against the standard can ensure selectivity and/or reproducibility of a reagent used in an assay. Continue reading about Prevention and treatment of amyloid-associated disorders... 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