Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
  • A61P25/16—Anti-Parkinson drugs
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • 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
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
  • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
  • G01N2400/38—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence, e.g. gluco- or galactomannans, Konjac gum, Locust bean gum or Guar gum
  • G01N2400/40—Glycosaminoglycans, i.e. GAG or mucopolysaccharides, e.g. chondroitin sulfate, dermatan sulfate, hyaluronic acid, heparin, heparan sulfate, and related sulfated polysaccharides
• • 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
  • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

• the invention relates to methods of formation of particular amyloid plaques and screening applications for such plaques in the treatment of Alzheimer's and Parkinson's Diseases.
• Alzheimer's disease is characterized by the accumulation of a 39-43 amino acid peptide termed the beta-amyloid protein or A ⁇ , in a fibrillar form, existing as extracellular amyloid plaques and as amyloid within the walls of cerebral blood vessels. Fibrillar A ⁇ amyloid deposition in Alzheimer's disease is believed to be detrimental to the patient and eventually leads to toxicity and neuronal cell death, characteristic hallmarks of Alzheimer's disease.
• a variety of morphologically distinct types of A ⁇ -containing plaques have been described in the brains of Alzheimer's disease patients including diffuse plaques (which demonstrate A ⁇ immunoreactivity but do not stain for fibrillar amyloid using amyloid stains such as Congo red and Thioflavin S), neuritic plaques (which contain a central amyloid core which stains with Congo red and Thioflavin S, and which is surrounded by dystrophic neurites) and compact, burned-out or "amyloid star” plaques (which usually demonstrate a maltese-cross pattern when stained with Congo red and viewed under polarized light).
• diffuse plaques which demonstrate A ⁇ immunoreactivity but do not stain for fibrillar amyloid using amyloid stains such as Congo red and Thioflavin S
• neuritic plaques which contain a central amyloid core which stains with Congo red and Thioflavin S, and which is surrounded by dystrophic neurites
• compact, burned-out or "amyloid star” plaques which usually demonstrate a
• amyloid star plaques in a human Alzheimer's brain is generally considered in the art to take years to form and accumulate. Once present however, these plaques are then resistant to natural proteases or other clearance mechanisms from the brain. Thus, any agent that would help to dissolve these amyloid star plaques, or modify them towards becoming more sensitive to protease digestion and clearance, would be a good candidate in treating Alzheimer's disease.
• Neuritic plaques are considered more mature 'and contain dystrophic neurites surrounding a spherical amyloid plaque core (Barcikowska et al, Acta Neuropath. 78:225-231, 1989; Ikeda et al, Lab, Invest. 60:113-122, 1989; Masliah et ' al, J. Neuropath. Exp. Neurol. 52:619-632, 1993).
• the amyloid cores within these plaques are A3 immunopositive and stain with Congo red and Thioflavin S.
• amyloid plaque cores within neuritic plaques are usually spherical and resemble a maltese-cross when stained with Congo red and viewed under polarized light (Ikeda et al, Lab. Invest. 60:113-122, 1989; Wisniewski et al, Acta Neuropath. 78:337-347, 1989; Schmidt et al, Am. J.. Path. 147:503-515, 1995).
• the amyloid cores within neuritic plaques resembl,e .amyloid stars when viewed by electron microscopy (Wisniewski et al, Acta Neuropath. 78:337-347, 1989).
• Compact amyloid cores also referred to as burnt-out or core plaques
• Compact amyloid cores also resemble amyloid stars when viewed by electron microscopy (Selkoe et al, J. Neurochem. 46:1820-1834, 1986; Snow et al, Am. J. Path. 133:456-463, 1988), and are generally believed to represent a more mature form of plaque formation (Wisniewski et al, Acta Neuropath. 78:337-347, 1989; Schmidt et al, Am. J. Path. 147:503-515, 1995; Dickson, J. 'Neuropath. Exp.' Neurol. 56:321-339, 1997) .
• These spherical plaques are A ⁇ -immunopositive and stain with Congo red (also resembling a maltese-cross when viewed under polarized fight) and Thioflavin S.
• amyloid star plaques there has been therefore a need to create amyloid star plaques in vitro, and that need has been met, as previously disclosed in the parent appUcation, with many technical difficulties.
• the in vitro amyloid plaques themselves would ideally retain characteristics known to amyloid plaques ' in vivo, such as stability, protease resistance, and the characteristic that when stained with Congo red they display a maltese-cross pattern when viewed under polarized fight.
• in vitro amyloid plaque formation must occur rapidly and in large number, rather than the time it takes in human brain.
• a method of rapid forming of large quantities of congophilic maltese-cross spherical amyloid plaques i.e. "compact plaques” or "amyloid star” in vitro that are virtually identical to congophilic maltese-cross compact plaques present in human Alzheimer's disease brain is disclosed.
• Methods to consistently form such Alzheimer's plaques for use in .a number of different assay techniques and. animal model ' s to identify anti-plaque therapeutics for Alzheimer's and Parkinson's diseases are ' also disclosed.
• Such compact congophilic maltese-cross amyloid plaques were not consistently formed following incubations of A ⁇ 1-40 or 1-42 only (up to a 1 wee at 37°C).
• Preferable in vitro conditions to induce, amyloid plaque formation require A ⁇ 40, not A ⁇ 42, co-incubated with a sulfated GAG (such as heparin, heparan sulfate, keratan sulfate, dermatan sulfate, chondroitin-4-sulfate) or.
• a sulfated GAG such as heparin, heparan sulfate, keratan sulfate, dermatan sulfate, chondroitin-4-sulfate
• plaques include a) spherical or compact shape, b) a maltese-cross pattern (i.e. red color of plaque 90 degrees to green color of plaque) of congophilia following staining with Congo red an when viewed under polarized fight, c) positive staining with Thioflavin S when viewed by fluorescence microscopy, d) spherical and/or "amyloid star” appearance when viewed by transmission electron microscopy, e) spherical or compact in shape (with plaques 10-40 ⁇ m in diameter) when viewed by scanning electron microscopy and or f) resistance to protease degradation.
• Use of such amyloid plaques formed in vitro as screening tools for the identification of Alzheimer's and Parkinson's diseases anti-plaque therapeutics is also disclosed.
• GAGs glycosaminoglycans
• a ⁇ beta- amyloid protein
• variable temperatures (-80°C to 45°C), optimal A ⁇ 40 initial concentration (25, 37.5, 50, 62.5, 75, 87.5, 100, 112.5, 125, 250, and 500 ⁇ M), various sulfated GAGs (heparin, heparan sulfate (from multiple sources), keratan sulfate, dermatan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate) or sulfated GAG-related macromolecules (dextran sulfate) for co-incubation, different ratios of A ⁇ 40:sulfated GAG or macromolecule (1:0.01, 1:0.05, 1:0.1, 1:0.1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:14, 1:16, 1:18, 1:20 wt/wt), with or without s
• Sonication is a term known to those skilled in the cell research art. Optimal conditions within these disclosed ranges for inducing formation of the compact congophilic maltese-cross, "amyloid star" plaques as observed in Alzheimer's Disease brain are disclosed herein. Consistent amyloid plaque formation in vitro is optimized not only by co-incubation of A ⁇ 1-40 with highly sulfated GAG or GAG-related macromolecule, but also by the quality of A ⁇ 1-40 used.
• a ⁇ 1-40 be low fibrillar, such that thioflavin T fluorometry reading at initial solubifization should read between 400 to 4000 FU (ex 444 nm/ em 485 nm), preferably FU ⁇ 2000; A ⁇ 1-40 in lx TBS of relatively neutral pH (pH ⁇ 6.5-7.5); and an A ⁇ 1-40 maximal concentration not to exceed 1 mg/ml or 250 ⁇ M are also preferred.
• GAGs include heparan sulfate, heparin, dermatan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, keratan sulfate, hyaluronic acid, and dextran sulfate.
• such compact amyloid plaque formation is achieved by the co-incubation of A ⁇ 1-40 with sulfated GAGs following incubation at 25°C to 40°C, and preferably about 37°C, and under the appropriate A ⁇ :GAG weight and/or molar ratios as described herein.
• a method of induction of amyloid plaques including the steps of a) immobilizing a quantity of a selected sulfated glycosaminoglycan (SGAG) or a GAG-related macromolecule on a selected medium; b) adding to the immobilized SGAG on the medium a quantity of dissolved low fibrillar A ⁇ 1-40 (LFA ⁇ ).
• SGAG selected sulfated glycosaminoglycan
• LFA ⁇ dissolved low fibrillar A ⁇ 1-40
• the LFA ⁇ is added in a A ⁇ :SGAG weight/weight (w/w) ratio range of between .1:0.01 to 1:20, or in a A ⁇ :SGAG w/w ratio range of between 1:0.1 to 1:10, advantageously in a A ⁇ :SGAG w/w ratio range of between 1:0.5 to 1:2, and preferably in a A ⁇ :SGAG w/w ratio of about 1:1.
• the selected medium is either a slide, a film or a titer well plate, and a preferred titer well plate is an 18 - 96 well Teflon partitioned slide.
• An SGAG is preferably selected from heparin, heparan sulfate, keratan sulfate, dermatan sulfate, chondroitin-4-sulfate andchondroitin-6-sulfate, and the GAG-related macromolecule is preferably dextran sulfate.
• LFA ⁇ is advantageously added to the immobilized SGAG by a bubbling technique involving pipetting small quantity of liquid into the Teflon defined well such that the Teflon partially repels the liquid and holds the liquid in a bubble over the substrate, in this case the immobilized SGAGs.
• a method of screening a selected amyloid therapeutic candidate having the steps of: a) immobilizing a ' quantity of a selected sulfated glycosaminoglycan (SGAG) or a GAG-related macromolecule on a selected medium; b) adding to a quantity of dissolve ⁇ low fibrillar A ⁇ 1-40 (LFA ⁇ ) a selected quantity of the selected amyloid therapeutic candidate to create a test solution; c) adding to the immobilized SGAG on the medium a selected quantity of the test solution; whereby "a percentage inhibition in formation of amyloid plaques, as compared to a test reference prepared as above without the selected amyloid therapeutic candidate, is indicative of a percentage efficacy of the selected amyloid therapeutic candidate.
• SGAG sulfated glycosaminoglycan
• LFA ⁇ dissolve ⁇ low fibrillar A ⁇ 1-40
• An alternate' method of screening a selected amyloid therapeutic candidate includes the steps of: a) • immobilizing a quantity of a selected sulfated .glycosaminoglycan
• SGAG SGAG or a GAG-related macromolecule on a selected medium
• LFA ⁇ dissolved low fibrillar A ⁇ 1-40
• Kits are disclosed for screening a selected amyloid therapeutic candidate, one kit having an immobilized quantity of a sulfated glycosaminoglycan (SGAG) on a medium and a quantity of low fibrillar A ⁇ 1-40 (LFA ⁇ ) with screening instructions as above for testing inhibition, and another kit having a quantity of amyloid plaques preformed on a medium as above disclosed and screening instructions as above for disruption.
• SGAG sulfated glycosaminoglycan
• LFA ⁇ low fibrillar A ⁇ 1-40
• Inhibition and disruption of amyloid plaque formation as discussed herein may advantageously be noted and quantified, as will be appreciated by those skilled in the art, by comparing morphology of plaques, number of plaques, or other measures known to those skilled in the art, and may include after testing with protease or microgfia degradation to confirm that affected plaques are now subject to natural clearance and no longer represent the particular amyloid star plaques discussed herein, both in Alzheimer's brain and synthesized, that resist such degradation.
• congophilic maltese-cross compact amyloid plaques are formed utilizing AI3 1-40 with heparin (either low or high molecular weight).
• AI3 1-40 in a range of 10 ⁇ M to 250 ⁇ M is incubated in distilled water, PBS or Tris-buffered saline (pH 7.4) with heparin at 37°C within a range of AJ3:heparin molar ratios from 1:0.02 to 1:100, and preferably about 1:0.5 ⁇ M.
• congophilic maltese-cross compact amyloid plaques are formed utilizing A ⁇ 1-40 with non-anticoagulant heparins.
• a ⁇ 1-40 at 10 ⁇ M to 250 ⁇ M is incubated in distilled water, PBS or Tris-buffered saline (pH 7.4) with non-anticoagulant heparin, a heparin-like molecule, or fragments thereof, at 37°C within a range of A ⁇ rnon-anticoagulant heparin molar ratios from 1:0.02 to 1:100, and preferably about 1:0.5 ⁇ M.
• congophilic maltese-cross compact amyloid plaques are formed utilizing A ⁇ 1-40 with heparan sulfate.
• a ⁇ 1-40 is incubated in distilled water, PBS or Tris-buffered saline (pH 7.4) with heparan sulfate at 37°C within a range of AJ3:heparan sulfate weight ratios from 1:0.1 to 1:100, and preferably about 1:1.
• This same range and preference also applies to other sulfated GAGs such as dermatan sulfate, keratan sulfate, chondroitin-4-sulfate, . and chondroitin-6-sulfate.
• congophilic maltese-cross compact amyloid plaques are formed utilizing A ⁇ 1-40 with dextran sulfate.
• a ⁇ 1-40 at 10 ⁇ M to 250 ⁇ M is incubated in distilled water, PBS or Tris-buffered saline (pH 7.4) with dextran sulfate at 37°C within a range of AJ3:dextran sulfate molar ratios from 1:0.02 to 1:100, and preferably about 1:0.5 ⁇ M.
• congophilic maltese-cross compact amyloid plaques are formed utilizing A ⁇ 1-40 with pentosan polysulfate.
• a ⁇ 1-40 at 10 ⁇ M to 250 ⁇ M is incubated in- distilled water, PBS or Tris-buffered saline (pH 7.4) with pentosan polysulfate at 37°C within a range of Afi:pentosan polysulfate molar ratios from 1:0.02 to 1:100, and preferably about 1:0.5 ⁇ M.
• congophilic maltese-cross compact amyloid plaques are formed utilizing A ⁇ 1-40 with polyvinyl sulphonate.
• Afi 1-40 is incubated in distilled water, PBS or Tris-buffered saline (pH 7.4) with polyvinyl sulphonate at 37°C within a range of A ⁇ :polyvinyl sulphonate weight ratios from 1:0.1 to 1:100, and preferably about 1:1.
• amyloid plaques produced herein for screening methods to identify anti-plaque therapeutic agents in vitro, agents which inhibit, disrupt or eliminate the congophilic maltese-cross spherical amyloid plaques can be identified utilizing polarization microscopy are disclosed.
• amyloid plaque cores will first be formed in vitro which demonstrate a typical maltese-cross pattern following staining with Congo red and when viewed under polarized light.
• amyloid plaque cores will be viewed under polarization microscopy to determine if a given compound or agent is capable of inhibition, disruption or elimination of the amyloid plaque structure such that there is a loss of congophilia and/or maltese-cross formation.
• Such compounds initially identified by such polarization microscopy techniques can be further .analyzed in secondary or tertiary assays utilizing transmission and/or scanning electron microscopy methods, and/or protease digestion methods, to confirm plaque inhibition, disruption or elimination.
• compact amyloid plaques produced herein for screening methods are used to identify anti-plaque therapeutic agents in vitro, agents which inhibit, disrupt the structure (i.e. size and/or diameter) of the spherical amyloid plaques can be identified using methodologies involving a cell sorter. In such assays, compact spherical amyloid plaques formed in vitro can be placed through a cell sorter to determine the average diameter (and range of diameters) of such plaques.
• plaques can then be incubated with a variety of compounds or agents (at a given dosage and incubation time to be determined empirically) and then be placed through the cell sorter again to determine if the given compound was effective in breaking apart to disrupting the size (and hence diameter) of such plaques.
• FIGURES 1A-1L are photomicrographs of the in vitro formation of congophilic maltese-cross spherical amyloid plaques in one embodiment.
• FIGURES 2A-2I are photomicrographs of the in vitro formation of congophilic and maltese-cross spherical amyloid plaques by another embodiment.
• FIGURES 3A-3I are photomicrographs of the in vitro formation of congophilic and maltese-cross spherical amyloid plaques by another embodiment.
• FIGURES 4A-4B are photomicrographs of the in vitro formation of congophilic maltese-cross compact amyloid plaque formation by another embodiment.
• FIGURES 5A-5B are photomicrographs of in vitro formation of spherical amyloid plaques in alternate embodiment of the inventive method.
• FIGURES 6A-6D are photomicrographs of spherical "amyloid star” formation induced by perlecan which is virtually identical to isolated amyloid plaque cores derived from human Alzheimer's disease brain as viewed by transmission electron microscopy.
• FIGURES 7A-7F are photomicrographs of amyloid plaque core formation induced by perlecan or dextran sulfate and viewed by scanning electron microscopy.
• FIGURE 8 shows in vitro plaque formation with different sulfated GAG or GAG-related macromolecules.
• FIGURE 9 shows examples of amyloid star plaques in human Alzheimer's brain compared to maltese-cross in vitro plaques and corresponding electron micrographs (EM).
• FIGURE 10 shows maltese-cross plaque formation in vitro, with and without sonication.
• FIGURE 11 shows maltese-cross plaque formation in vitro in an immobilized GAG method (2X objective).
• FIGURE 12 shows the maltese-cross plaque formation of Figure 4, but with a 10X objective.
• FIGURE 13 demonstrates an image analysis of congo red stained maltese cross plaques to determine plaque size (diameter in microns - ⁇ m).
• FIGURE 14 shows ThioS stained plaques formed in vitro on immobilized GAGs (and or GAG-related macromolecules). '
• FIGURE 15 demonstrates an image analysis of ThioS stained plaques formed in vitro on immobilized GAGs (and/or GAG-related macromolecules).
• FIGURE 16 shows disruption of plaques upon application of various lead compounds.
• FIGURE 17 shows disruption of plaques with lead compounds after protease digestion.
• diffuse plaques is used herein to refer to amyloid plaques in human Alzheimer's disease brain which are immunoreactive with a variety of different anti-A- ⁇ antibodies but generally do not stain for fibrillar amyloid (i.e. Congo red, Thioflavin S)(Ikeda et al, Lab. Invest. 60:113-122, 1989; Verga et al, Neurosc. Lett. 105:294-299, 1989).
• neuritic plaques is used herein to refer to plaques in human • Alzheimer's disease brain which contain dystrophic neurites surrounding a spherical amyloid plaque core (Barcikowska et al, Acta Neuropath. 78:225-231, 1989; Ikeda et al, Lab. Invest. 60:113-122, 1989; Masliah et al, J. Neuropath. Exp. Neurol. 52:619-632, 1993).
• the amyloid cores within these plaques are A ⁇ immunopositive and stain with Congo red and Thioflavin S.
• amyloid plaque cores within neuritic plaques are usually spherical and resemble a maltese-cross- when stained with Congo red and viewed under polarized fight (Ikeda et al, Lab. Invest.60: 113- 122, 1989; Wisniewski et al, Acta Neuropath. 78:337-347, 1989; Schmidt et al, Am. J. Path. 147:503-515, 1995).
• compact or “burned-out” plaques is used herein to refer to plaques in human Alzheimer's disease or prion disease brain that are generally believed to represent a more mature form of plaque formation (Wisniewski et al, Acta Neuropath. 78:337-347, 1989; Schmidt et al, Am. J. Path. 147:503-515, 1995; Dickson, J. Neuropath. Exp. Neurol. 56:321-339, 1997).
• These spherical plaques are A ⁇ or prion protein-immunopositive and stain ' with Congo red (also resembling a maltese-cross when viewed under polarized light) and Thioflavin S.
• “Compact” or “burned-out” plaques also demonstrate a maltese-cross pattern when stained with Congo red and viewed under polarized light.
• Congophilia is used. herein to describe fibrillar amyloid deposits • which demonstrate a red/apple-green birefringence when stained with Congo red and when viewed under polarized light. Congophilic deposits do not necessarily exhibit a maltese-cross pattern (see below for definition).
• maltese-cross refers to spherical and compact amyloid plaques which when stained with Congo red and viewed under polarized light demonstrate a maltese-cross pattern (i.e. red color is 90 degrees to apple-green color). Upon rotation' of the polarizer, a shift in colors of the plaque occurs such that the red color will change to apple-green, and the apple-green color will change to red (i.e. red green birefringence).
• amyloid plaques formed in vitro as described in the present invention demonstrate a "maltese-cross” pattern, when stained with Congo red and viewed under polarized light.
• amyloid star is used herein to refer to "compact” or "burned-out” amyloid plaques which resemble star-shaped deposits of amyloid when viewed by electron microscopy (Selkoe et al, J. Neurochem. 46:1820-1834, 1986; Snow et al, Am. J. Path. 133:456-463, 1988).
• the "amyloid star”appearance of the plaque is due to bundles of radiating amyloid fibrils appearing to emanate from the center of the plaque.
• induction or “formation” is used herein to refer to compact amyloid plaques that are formed in vitro when incubated under the appropriate conditions. Gentle mixing of the incubation components is optionally included with the scope of the disclosed methods as discussed in terms of induction or formation.
• anti-plaque, therapeutics is used herein to refer to compounds or drugs which are effective in a) directly dissolving, inhibiting or disrupting the architecture, staining characteristics or structure of the compact plaque, and/or b) inhibiting the detrimental ' effects (i.e. neu otoxicity).that the compact plaque may have . on other cells (i.e. neurons), tissues or organs.
• beta-amyloid protein (AU 1-40, also referred to herein as A ⁇ 40) refers to SEQ ID NO: 1, and may include all single or multiple ami ⁇ o acid
• I t ' substitutions that occur in human disease (such as Alzheimer's; where single ami ⁇ o acid substitutions in the A ⁇ 1-40 are known), or in species variation (such as rodent A ⁇ 1-40 which is known to have three amino acid differences in comparison to human AJ3 1-40).
• FIGURE 1 demonstrates the in vitro formation of congophilic maltese.-cross , spherical amyloid plaques by perlecan but not other amyloid plaque associated macromolecules known to be present in human Alzheimer's disease brain.
• 25 ⁇ M of A ⁇ (1-40) was incubated in double distilled water or Tris-buffered saline at 37°C either alone (Fig. 1C), or in the presence of lOOnM of P component (Fig. ID), alphal-antichymotrypsin (Fig, ⁇ E), apqE (Fig. IF), Clq (Fig. 1G), laminin (Fig. 1 1H), fibronectin (Fig. II), type IN collagen (Fig.
• Figs. A, C and H are taken at the same magnification, as are Figs. B, D-G and I-J.
• FIGURE 2 demonstrates the in vitro formation of congophilic and maltese-cross spherical, amyloid plaques by highly sulfated glycosaminoglycans (i.e heparin and heparan sulfate) and related sulfated macromolecules (ie. dextran sulfate, pentosan polysulfate).
• highly sulfated glycosaminoglycans i.e heparin and heparan sulfate
• related sulfated macromolecules ie. dextran sulfate, pentosan polysulfate.
• 25 ⁇ M of A ⁇ 1-40 was incubated in double distilled water or Tris-buffered saline (pH 7.4) at 37°C either alone (Fig. 2B), or in the presence of various amounts of heparin (Fig. 2C), heparan sulfate (Fig.
• FIGURE 3 demonstrates the in vitro formation of congophilic and maltese-cross spherical amyloid plaques, by polyvinyl sulphonate (PNS), and demonstrates how changes in the weight ratio of A ⁇ :PVS influences the potential for compact amyloid plaque formation.
• PNS polyvinyl sulphonate
• FIGURE 4 demonstrates congophilic maltese-cross compact amyloid plaque formation induced by a ⁇ 220 kDa heparan sulfate proteoglycan (HSPG) isolated from Engelbreth-Holm-Swarm tumor. 50 Fg of A ⁇ (1-40) in 100F1 Tris-buffered saline (pH 7.4) was incubated at 37°C either alone or in the presence of 10 Fg of the ⁇ 220 kDa HSPG (A ⁇ :HSPG weight ratio of 5:1).
• Fig. 4A demonstrates irregular congophilic amyloid deposits (arrows) formed following a 1 week incubation of AJ3 alone, with no apparent congophilic maltese-cross amyloid plaques formed.
• Fig. 4B demonstrates congophilic maltese-cross amyloid plaques (arrowheads) formed following a 1 week incubation of A ⁇ 1-40 plus ⁇ - 20 kDa HSPG.
• the amyloid plaques formed were identical to those compact plaques present in human Alzheimer's disease brain (see Fig. 1A and 2A).
• FIGURE 5 demonstrates in vitro formation of spherical amyloid plaques induced by perlecan, as it appears in fixed in plastic.
• 125 ⁇ M of A ⁇ 1-40 was incubated in double distilled water at 37°C in the presence of 0.625 ⁇ M of perlecan (AJ3:perlecan molar ratio of 200:1; AB:perlecan weight ratio of 1:1).
• a 10 Fl aliquot of the incubation mixture was then air-dried for one hour on plastic petri dishes, and then fixed in situ with 3% glutaraldehyde in 0.1M NaPO4 buffer (pH 7.3) for 10 minutes.
• FIGURE 6 demonstrates spherical "amyloid star”formation induced by perlecan which is virtually identical to isolated amyloid plaque cores derived from human Alzheimer's disease brain as viewed by transmission electron microscopy.
• 125 ⁇ M of A ⁇ 1-40 was incubated in double distilled water at 37°C in the presence of 0.625 ⁇ M of perlecan (AJ3:perlecan molar ratio of 200: 1; AJ3:perlecan weight ratio of 1:1).
• Amyloid plaque cores induced by perlecan (Figs. 6C and 6D) formed amyloid stars with radiating bundles of amyloid fibrils appearing to emanate from a central source. Individual amyloid fibril diameters were determined to be 7-10 nm.
• Figs. A and B are of the same magnification as are Figs. C and D.
• FIGURE 7 demonstrates amyloid plaque core formation induced by perlecan or dextran sulfate and viewed by scanning electron microscopy.
• 125 ⁇ M of A ⁇ 1-40 was incubated in double distilled water at 37°C either alone (Fig. 7B), or in the presence of 0.625 ⁇ M of perlecan (A ⁇ : ⁇ erlecan molar ratio of 200: l)(Figs. 7D and 7E) or dextran sulfate (A ⁇ :dextran sulfate molar ratio of 1:5).
• 0.625 ⁇ M of perlecan alone was incubated at 37°C (Fig. 7C).
• Amyloid plaque core formation was not observed following a 1 week incubation of Afi (Fig. 7B) or perlecan (Fig. 7C) alone. However, compact amyloid plaque formation was induced by A ⁇ in the presence of perlecan (Fig. ' 7D and 7E) or dextran sulfate (Fig. 7F).
• the shape and general morphology of the amyloid plaquesinduced by perlecan or dextran sulfate were similar to the shape and general morphology to isolated amyloid plaque cores derived from human Alzheimer's disease brain, as viewed by scanning electron microscopy. Magnifications are. given at the bottom of each figure.
• Method #2 in vitro plaque formation on immobilized sulfated GAGs:
• a ⁇ 40 solution by adding 1 ml of lx TBS directly into 1 mg lyophilized A ⁇ 40 for a final concentration of lmg/ml.
• initial thioT reading is 4000 FU (excitation 444 nm emission 485 nm) or below.
• Example 1 in vitro plaque formation with different sulfated GAG or GAG-related macromolecules.
• Figure 8 demonstrates the in vitro formation of congophilic maltese-cross spherical amyloid plaques induced by several different sulfated GAGs at various weight ratios tested including 1:0.1; 1:1, and 1:10. In these studies, 50 microfiters of
• GAGs (10 mg/ml stock for 1:10 w/w, 1 mg/ml stock for 1:1 w/w, 0.1 mg/ml stock for
• Congophilic maltese-cross amyloid plaques were viewed under polarized light microscopy.
• Sulfated GAGs examined included Heparin, Heparan sulfate (porcine mucosal intestinal; HS (pirn)), Dermatan sulfate (DermS), Chondroitin-4-sulfate
• C-4-S Chondroitin-6-sulfate
• KeratanS Keratan sulfate
• GAG-related macromolecules tested include dextran sulfate (DexS) and polyvinyl sulphonate (not shown).
• Example 2 amyloid star plaques in human Alzheimer's brain compared to maltese-cross in vitro plaques and corresponding electron micrographs (EM).
• Figure 9 demonstrates the similarity in physical characteristics between amyloid star plaques in Alzheimer's disease brain to amyloid plaques formed in vitro.
• D Scanning electron microscopy of a single amyloid star plaque formed in vitro. Note the similarities in the spherical shape and the closeness in size.
• Example 3 maltese-cross plaque formation in vitro, with and without sonication.
• Figure 10 demonstrates the rapid method of sonication for in vitro plaque formation in Tris-Buffered Saline (pH 7.4) solution.
• 200 microfiters of 250 ⁇ M of A ⁇ 40 was solubifized in lx Tris-Buffered saline (pH 7.4) and mixed with or without 200 microfiters of 10 mg/ml dextran sulfate for a final molar ratio of 1:5 or a weight to weight ratio of 1:10.
• This mixture was then either subjected to a brief 5 minute sonication or was not sonicated at all and then incubated at 37°C for 3 to 7 days, preferably 5 days.
• a ⁇ 40 in TBS alone without sonication shows congophilic material but no red/green birefringence or maltese-cross formation.
• Example 4 maltese-cross plaque formation in vitro in an immobilized GAG method.
• Figure 11 demonstrates the rapid method of in vitro plaque formation in Tris-buffered saline (pH 7.4) on immobifized GAGs or GAG-related macromolecule.
• TBS Tris-buffered saline
• Incubation slide was then stained with 5 microfiters per well of 125 ⁇ M congo red solution and viewed under polarized light at 2x objective to show the abundance of maltese-cross plaque formation seen.
• Figure 12 demonstrates congophilic maltese-cross plaque formation in vitro with A ⁇ 40 on immobilized GAGs or GAG-related macromolecules such as Heparin
• Example 5 image analysis of congo red- stained maltese cross plaques to determine plaque size.
• Figure 13 demonstrates congophilic maltese-cross plaques formed in vitro and subjected to image analysis using ' Image ProPlus 4.1 that has the capability to iot only calculate the quantity of maltese-cross plaques formed in vitro but also to align the plaques in order of size.
• Figure 12F showing maltese-cross plaque formed in vitro with A ⁇ 40 on immobilized dextran sulfate (high molecular weight' form) at a weight ratio of 1:10 or molar ratio of 1:5 was used for further image analysis.
• Plaque quantities in this 5 microliter aliquot was 63 plaques for an estimated total of 12,600 plaques formed pe mL used.- he plaque diameter ranges from 1-4 microns to 60-100 microns whereby the average plaque diameter ranges between 10-45 microns.
• Example 6 ThioS stained plaques formed in vitro on immobifized GAGs.
• Figure 14 demonstrates thioS stained plaques formed in vitro and viewed under fluorescence fight.
• 18-well Teflon-coated glass slide was coated with either TBS or 50 microfiters per well of dextran sulfate (10 mg/ml stock in de-ionized water; high molecular weight forms as indicated), polyvinyl sulphonate (10 mg/ml stock in de-ionized water) or dermatan sulfate (10 mg/ml stock in de-ionized water) and allowed to air dry down to a thin transparent film.
• FITC light at 2x objective to show the abundance of thioS-positive plaques seen as spherical bodies.
• Example 7 image analysis of ThioS stained plaques formed in vitro on immobilized GAGs.
• Figure 15 demonstrates thioflavin S-positive plaques formed in vitro and subjected to image analysis using Image ProPlus 4.1 that has the capability to not only calculate the quantity of spherical thioS-positive plaques formed in vitro but also to align the plaques in order of size.
• a ⁇ 40 on immobilized dextran sulfate (high molecular weight form) at a weight ratio of 1: 10 or molar ratio of 1:5 was used for image analysis.
• the count range for image analysis was set as Area ranging between 30-3500 micron squared and Diameter ranging between 4-100 micron. Anything falling below or above this set threshold was not included in the data analysis.
• Plaque quantities in this 8 microliter aliquot example was 323 plaques for an estimated total of 40,375 plaques formed per mL used.
• the plaque diameter ranges from 1-4 microns to 60-100 microns whereby the average plaque diameter ranges between 10-25 microns.
• Example 8 disruption of plaques upon application of various lead compounds.
• Figure 16 demonstrates compounds or agents that were incubated with pre-formed amyloid plaques in vitro to screen for the ability of these compounds to inhibit, decrease or eliminate the congophilic maltese-cross pattern of the plaques identified utilizing polarization light microscopy after congo red staining.
• Dextran sulfate Dextran sulfate
• dermatan sulfate DermS at 1 mg/ml stock in de-ionized water
• heparin (1 mg/ml stock in de-ionized water) or TBS alone were spotted onto 18-well Teflon-coated glass slide and allowed to air dry down to a thin transparent film.
• DC-0004 was able to disrupt maltese-cross plaques in all conditions while DC-0021 was only moderately disruptive to these pre-formed maltese-cross plaques.
• DC-0051 was not as robust as DC-0004, however, but was able to affect maltese-cross plaques in A ⁇ 40 only, A ⁇ 40 + Dextran sulfate and A ⁇ 40 + Dermatan sulfate.
• a ⁇ 40 + Heparin plaques were less affected by DC-0051 than other in vitro formed plaques by other. GAGs or GAG ⁇ related molecules. This type of screening can also take place on 96-well assay plates using thioflavin T as the marker for amyloid plaque quantity for a more high L through put screening.
• Example 9 disruption of plaques with lead compounds after protease digestion.
• Figure 17 demonstrates compounds or agents that were incubated with pre-formed amyloid plaques in vitro 1 and followed by proteinase K treatment for 24 hours to screen for the ability of these compounds to render the plaques sensitive to protease digestion.
• 30 microfiters per well of Dextran sulfate (DexS at lmg/ml stock in de-ionized water), dermatan sulfate (DermS at 1 mg/ml stock in de-ionized water), heparin (1 mg/ml. stock in de-ionized water) or TBS alone were spotted onto 18-welLTeflon-coated glass slide and allowed to air dry down to a ' thin transparent film.
• DC-0021 treatment rendered protease sensitivity in plaques formed in vitro with the combination of A ⁇ 40 + dermatan sulfate and heparan sulfate but not with A ⁇ 40 + dextran sulfate or A ⁇ 40 + Heparin.
• Treatment with DC-0051 was only moderately successful in rendering A ⁇ 40 + dermatan sulfate and A ⁇ 40 + heparan sulfate pre-formed plaques sensitive to protease treatment- whereas it was ineffective with A ⁇ 40 + dextran sulfate and A ⁇ 40 + heparin pre-formed plaques.
• Congophilic maltese-cross compact amyloid plaques formed in vitro as described herein can be utilized for screening methods to identify anti-plaque therapeutics as lead compounds for the treatment of Alzheimer's disease.
• screening methods will utilize amyloid proteins (A ⁇ ) sulfated GAGs, sulfated or anionic macromolecules or fragments thereof, that are radiolabelled.
• a ⁇ amyloid proteins
• the AJ3 1-40 is bound to a radioactive label such as radioactive iodine (i.e. 1251).
• radioactive iodine i.e. 1251
• other appropriate labelling agents and techniques can be used and include, but are not limited to, an enzyme label, a fluorescent label, a chemiluminescent label, or an antigen label.
• any radioactive substance that may be incorporated into the A ⁇ protein or fragments thereof may be used.
• Preferred isotopes include, but are not limited to 1251, 1231, and 1311. 1311 has a shorter half -life and higher energy level.
• Iodine radioisotopes may be incorporated into the protein or protein fragments by oxidative iodination.
• radioactive iodine may be incorporated by use of Bolton-Hunter reagent to add a 3-iodo-4-hydroxyphenylproprionyl or 3,5-diiodo-4-hydroxyproprionyl group to a nucleophile in the pep tide.
• isotopes may also be incorporated by reaction with nucleophile groups or peptides.
• tritium can be incorporated by reaction with propionyl-N-hydroxysuccinimide
• radioactive sulfur 35S
• the labelling of GAGs or sulfated macromolecules using 35S would also allow the amyloid plaque cores formed in vitro to be labelled' and monitored as described below.
• Radioactive phosphorous 32P may be incorporated by enzymatic methods.
• various radioactive metal ions such as 99m technetium, may be incorporated into A ⁇ or fragments thereof, if an appropriate chelating group is added first.
• enzyme labelling is also useful.
• enzyme labels are peroxidases such as horseradish peroxidase (HRP), or phosphatases such as alkaline phosphatase.
• Modifying the peptide or peptide fragment by adding an antigenic group that will bind with an antibody allows direct detection of the peptide or peptide fragment itself.
• the antigen digoxige in can be linked to a peptide, and then visualized with a labelled digoxigenin-specific antibody, or labelled anti-antibody.
• fluorophores may also be incorporated into the A ⁇ , peptide and detected according to known fluorescent detection techniques.
• suitable fluorophores include fluorescein, Texas red, and the like.
• Direct or indir.ect chemiluminescent labels may also be used according to the invention such as dioxetanes,
• the A ⁇ peptide would be modified with a group that is capable -of emitting light, as it decomposes.
• an avidin-biotin system may be used to detect the A ⁇ ' peptide or peptide fragment in an in vitro assay.
• the peptide or ' fragment may be functionalized with biotin, and avidin or streptavidin added to detect' the protein or fragment.
• the A ⁇ is appropriately labelled as described above, it is combined with specific GAGs, sulfated or anionic macromolecules as described herein and incubated at 37°C to form congophilic maltese-cross compact amyloid plaques.
• the labelled plaques will first be tested to ensure that the staining and structural features of the amyloid plaques formed as the same as those formed in the absence of label.
• the parameters to ensure plaque stability following an appropriate labelling technique include: a) a spherical or compact shape of the plaque formed, b) a maltese-cross pattern (i.e.
• red color of plaque 90 degrees to green color of plaque of congophilia following staining with Congo red, and when viewed under polarized light
• labelled plaque cores are seeded onto 96-well plates, and allowed to bind overnight.
• Different methods known to those in the art, will be utilized to determine the optimum for such labelled plaque binding to wells. Once such binding is achieved, a number of compounds or agents in various solutions/buffers (to be determined empirically) will be added to wells containing labelled plaques for various times of incubation (to be determined empirically).
• Agents or compounds able to break apart, disrupt or eliminate the staining characteristics or structure of the compact amyloid plaques are identified by comparing staining and structural characteristics to those wells that do hot contain compound or agents, or those that contain compounds or agents thought not to be an effective in altering plaque architecture.
• Agents or compounds that are able to break apart, disrupt or eliminate the staining or structural composition of the compact amyloid plaques can be identified by a variety of means including:
• radiolabel in the supernatant i.e. liquid phase
• the method of detecting the label such as radioactive isotopes will vary according to the isotope and its corresponding energy level.
• a gamma counter is capable of detecting 1251, but not 3H (tritium) or 35S-sulfate, where a scintillation counter will be required.
• the increase in label in the supernatant are those plaques that have been disrupted or broken apart, demonstrating that the given compound or agent was effective, in breaking apart or disrupted the plaque architecture andis therefore identified as a potential anti-plaque therapeutic.
• Such identified agents or compounds can be further identified by secondary or tertiary screens including, but not limited to: 1) a decrease or elimination of the maltese-cross pattern of congophilia following staining with Congo red, and when viewed under polarized fight indicating that the given compound or agent was effective in decreasing or altering the amyloid fibril structure, and is therefore identified as a potential anti-plaque therapeutic, 2) a decrease or elimination of positive staining with Thioflavin S indicating that the given compound or agent was effective in decreasing or altering the amyloid fibril structure, and is therefore identified as a potential anti-plaq ⁇ e therapeutic 3) a decrease, alteration or elimination of the spherical and or "amyloid star" appearance when viewed by electron microscopy indicating that the given compound or agent was effective in altering the architecture of the amyloid plaque, and is therefore identified as a potential anti-plaque therapeutic, and/or 4) a decrease, alteration or elimination of the spherical or compact shape (with plaques 10-40
• Peptides containing aromatic amino acids can be radiolabelled by oxidative radioiodination using Na 1251 and chloramine-T and separated from free iodine by reverse-phase absorption using the methods of WM Hunter and FC Greenwood, Nature 194:495, 1962; AE Bolton and WM Hunter, Biochem. J. 133:529, 1973; and HP Too and JE Maggio, Meth. Neurosc. 6;232, 1991, the disclosures of which are incorporated by reference therein.
• Another method of in vitro screening to identify anti-plaque therapeutics will utilize unlabelled compact amyloid plaques formed in vitro as described hereinj that demonstrate the maltese-cross pattern when stained with Congo red and viewed under polarized light.
• Compounds or agents, following incubation with the compact amyloid plaque for an appropriate time (to be determined empirically) that are able to inhibit, decrease or eliminate the congophilic maltese-cross pattern of the plaque are identified utilizing polarization microscopy as potential anti-plaque therapeutics.
• compact amyloid plaques will first be formed in vitro which demonstrate a typical maltese-cross pattern following staining with Congo red and when viewed under polarized light (as described herein).
• compact amyloid plaques will be air-dried on gelatin-coated slides (as describedherein), stained with Congo red, and viewed under polarization microscopy to determine if a given compound or agent is capable of inhibition, disruption or elimination of the amyloid plaque structure such that there is a loss of congophilia and or maltese-cross formation.
• Secondary and tertiary screens will include analysis of such plaques following incubation of the given agent or compound by transmission and scanning electron microscopy.
• Another method of in vitro screening to identify anti-plaque therapeutics will utilize compact amyloid plaques formed in vitro as describedherein, that demonstrate positive staining when stained with Thioflavin S and when viewed by fluorescent microscopy. Compounds or agents, following incubation with the compact amyloid. plaques for an appropriate time (to be determined empirically) that are able to decrease or eliminate the positive Thioflavin S fluorescence of the plaque are identified as potential anti-plaque therapeutics. Secondary and tertiary screens will include analysis of such plaques following incubation of the given agent or compound by transmission and scanning electron microscopy.
• Yet another method of in vitro screening to identify anti-plaque therapeutics will utilize compact amyloid plaques formed in vitro as described herein, that demonstrate a spherical or "amyloid star” appearance when viewed by transmission electron microscopy.
• Compounds or agents, following incubation with the compact amyloid plaques for an appropriate time (to be determined empirically) that are able to disrupt or alter the spherical plaque shape or "amyloid star" appearance are identified as potential anti-plaque therapeutics.
• Yet another method of in vitro screening to identify anti-plaque therapeutics will utilize compact amyloid plaques formed in vitro as described herein, that demonstrate a spherical shape with amyloid plaque diameters of 10-40 ⁇ m (average plaque diameter of 25 ⁇ m) when viewed by scanning electron microscopy.
• Compounds or agents, following incubation with the compact amyloid plaques for an appropriate time (to be determined empirically) that are able to disrupt or alter the spherical plaque shape or substantially decrease the diameter of the amyloid plaque are identified as potential anti-plaque therapeutics.
• Yet another method of in vitro screening to identify anti-plaque therapeutics will utilize the size and shape of the compact amyloid plaques formed as described herein.
• Agents or compounds which inhibit, disrupt or eliminate the structure (i.e. size and/or diameter) of the spherical amyloid plaques can be identified using methodologies involving a cell sorter.
• compact spherical amyloid plaques formed in vitro can be placed through a cell sorter to determine the average diameter (and range of diameters) of such plaques.
• amyloid plaque cores formed are loaded on a Coulter EPICS Elite ESP cell sorter (Coulter Corporation,.
• amyloid plaques formed in vitro by methods described herein usually have a range of diameters from 10-40 ⁇ m,' ith an average diameter of 25 ⁇ m.
• plaques formed in the absence of agent or compounds are compared to plaques formed that have been incubated with agents or compounds, by assessment using a cell sorter to determine the average plaque diameter (i.e. size).
• plaques formed in vitro using procedures as described herein are treated with a compound or agent for a specific time (to be determined empirically), and then the average diameter of such treated plaques are determined using a cell sorter and compared to the average diameter of untreated plaques. If a given compound or agent is effective in breaking apart or disrupting the size (and hence diameter) of compact plaques then an increase in the proportion of smaller diameters (i.e.
• amyloid plaques formed in vitro as described herein Another potential. utility of the amyloid plaques formed in vitro as described herein is to identify agents or compounds that are effective in reducing or efiminating the neurotoxic effects of A ⁇ .
• compact amyloid plaques will first be formed in vitro as described herein, and will be placed in petri dishes containing primary neurons (isolated using standard techniques and known to those in the art), or neuronal cell lines. Following prolonged incubation (i.e.
• amyloid plaques with neuronal cultures, levels of neurotoxicity (using standard assays known to those in the art) will be measured and compared to those cultures that do not contain amyloid plaques. If the compact amyloid plaques are able to demonstrate neurotoxicity effects in cell culture, then these amyloid plaques can be further utilized to screen for and identify agents or compounds that are potential anti- neurotoxic therape ⁇ tics. In such a method, compact amyloid plaques formed in vitro will be incubated in primary neuronal cultures, or in neuronal cell lines, for prolonged periods (i.e. 48 or 72 hours), and in the presence or absence of a given test compound or agent. Agents or compounds that, are able to inhibit or decrease neurotoxicity caused by the incubation of amyloid plaques are then identified anti-neurotoxic agents. Research Applications
• Compact amyloid plaques formed in' vitro are expected to be useful for a variety of different research applications.
• pre-formed compact amyloid plaques can be placed in cultures containing other cells (examples: neurons, microgfia, astrocytes, oligodendrocytes) and the response of the cells (i.e. phagocytosis, degradation) to such amyloid plaques in culture can be determined.
• the response of individual macromolecules i.e. other components implicated in amyloidosis such as apofipoprotein E, amyloid P component, complement factors, cytokines, inflammatory factors
• to such compact amyloid plaques in culture can also be assessed using standard techniques to those known in the art.
• compact amyloid plaque deposition, accumulation and persistence on cell architecture and or the metabolism of various macromolecules i.e beta-amyloid precursor protein, specific proteoglycans
• various macromolecules i.e beta-amyloid precursor protein, specific proteoglycans
• kits could be useful for the screening and identification of compounds or agents that have potential as anti-plaque therapeutics.
• a kit could comprise of a) a first container having a low fibrillar A ⁇ 1-40 (preferably A ⁇ 40 from Recombinant Peptides (catalog no.
• A- 1052-2 in solution or lyophilized) at the appropriate amount or concentration needed (described herein) for compact amyloid plaque formation
• a second container containing specific GAGs such as heparin or heparan sulfate
• specific sulfated macromolecules such as dextran sulfate, pentosan polysulfate or polyvinyl sulphonate
• dextran sulfate, pentosan polysulfate or polyvinyl sulphonate in solution or lyophilized, at the appropriate amount or concentration needed (described herein) for compact amyloid plaque formation.
• Such congophilic maltese-cross amyloid .plaque formation would occur following the mixing of the appropriate amounts from each of the two containers.
• the compact amyloid plaques could be pre-formed and then frozen or lyophilized for distribution. Once received by the researcher or individual, the compact amyloid plaques may be re-formed by placing in an appropriate solution such as distilled water or Tris-buffered saline (pH 7.4), and in an appropriate volume of solution. Such kits may be' used for research and/or commercial applications.

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