WO2004001426A2 - Method for identifying drugs for the treatment of alzheimer's disease - Google Patents
Method for identifying drugs for the treatment of alzheimer's disease Download PDFInfo
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- WO2004001426A2 WO2004001426A2 PCT/US2003/019375 US0319375W WO2004001426A2 WO 2004001426 A2 WO2004001426 A2 WO 2004001426A2 US 0319375 W US0319375 W US 0319375W WO 2004001426 A2 WO2004001426 A2 WO 2004001426A2
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- cerebral
- amyloidosis
- amyloid
- cholesterol
- angiopathy
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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
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
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- 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
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- 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
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- 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
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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
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
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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
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/5432—Liposomes or microcapsules
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
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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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4709—Amyloid plaque core protein
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- 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
- This invention relates to a method and device for rapid screening of drug candidates, especially candidate agents for treatment of Alzheimer's disease and stroke.
- the invention provides a biosensor method and device which is particularly applicable to screening libraries of compounds.
- AD Alzheimer's disease
- a ⁇ amyloid ⁇ protein
- CAA cerebral amyloid angiopathy
- CAA cerebrovascular smooth muscle cells
- SMCs cerebrovascular smooth muscle cells
- Vinters 1987; Ghiso and Frangione, 2001
- amyloid is deposited predominantly as CAA (Ghiso and Frangione, 2001).
- amyloid deposits The major component of the amyloid deposits is a 4 kDa polypeptide known as ⁇ -amyloid protein (A ⁇ ) (Glenner et ⁇ l., 1984; Masters et al, 1985), which is derived from a much larger ⁇ -amyloid protein precursor (APP) (Kang et al., 1987).
- a ⁇ ⁇ -amyloid protein
- APP ⁇ -amyloid protein precursor
- the major form of A ⁇ which is produced in the brain contains 40 amino acid residues. However, minor forms containing 42 or 43 residues are also formed. Production of these minor forms is closely linked to the pathogenesis of AD (Scheuner et al, 1996).
- AD therapy is to inhibit production of A ⁇ in the brain.
- Proteolytic cleavage of APP by BACEi and ⁇ -secretase generates the full-length A ⁇ , which is then released from cells (Nunan and Small, 2000). Therefore inhibitors of either BACEi or ⁇ -secretase maybe of therapeutic value.
- a number of studies have shown that cholesterol can influence A ⁇ release (Simons et al., 1998; Hartmann, 2001; Fassbender et al, 2001; Frears et al, 1999; Friedhoff et al, 2001). Therefore inhibitors of cholesterol biosynthesis, such as statins, may also be of therapeutic value.
- statins have relatively low toxicities, and their mode of action is much better understood than many other compounds currently being investigated as therapeutic agents for AD.
- statins there is some disagreement in the art as to the value of lowering cholesterol levels, and some workers consider that cholesterol is actually beneficial (Ji et al, 2002).
- the mechanism of A ⁇ toxicity is poorly understood (Small et al,
- a ⁇ can bind to lipids (Curtain et ⁇ l., 2001; Valdez-Gonzalez et al., 2001), including gangliosides (Ariga et ⁇ l., 2001), sphingolipids (Mahfoud et al., 2002) and cholesterol (Kremer et al, 2000; Avdulov et al, 1997; Eckbert et al, 2000).
- a ⁇ can bind to membrane lipids, and this interaction may be toxic for cells (Hertel et al, 1997).
- AChE acetylcholinesterase
- amyloid deposits of the A ⁇ type there are a number of other proteins which form amyloid deposits, and of these several are known to be involved in the pathogenesis of clinical conditions.
- immunoglobulin light chains form the amyloid fibrils in primary amyloidosis, and reactive or secondary amyloidosis is caused by a number of different proteins.
- Familial amyloidosis is caused by deposits of mutant forms of transthyretin, apolipoprotein A-I, gelsolin fibrinogen A ⁇ , or lysozyme; of these, the most common form of homelial amyloidosis is that caused by mutant transthyretin.
- These amyloidoses are all systemic, and rarely if ever involve the central nervous system. The systemic amyloidoses have recently been reviewed (Falk et al., 1997).
- Deposition of amyloid-like fibrils may also be important in other neurodegenerative diseases, such as Parkinson's disease and other conditions in which ⁇ -synuclein fibrils are deposited. These include Parkinson's disease itself, dementia with Lewy body formation, multiple system atrophy, Haller convinced-Spatz disease, and diffuse Lewy body disease.
- SPR surface plasmon resonance
- Biosensor methods have the advantage of being performed in real time without any requirement for labelling, and are especially suitable for screening large numbers of samples, and consequently they are extremely useful in high-throughput screening of candidate pharmaceutical agents.
- biosensors have been used extensively for the study of the interactions between peptides and membranes (Mozsolits and Aguilar (in press, 2002).
- sensor chips such as the HPA and Li chips from BIAcore AB and the Hydrophobic Surface cuvette from Affinity Sensors, Inc are useful in the study of lipid interactions. See for example US Patent No. 5,922,594 by Lofang, and the papers by Valdez-Gonzalez et al and by Ariga et ⁇ l. referred to above.
- a biosensor technique could be used for the high- throughput screening of candidate agents for the treatment of any amyloid- related condition.
- the binding of an amyloid protein to isolated membranes from a given cell type correlates very well with the degree of toxicity against that cell type, as measured by a cytotoxicity assay.
- the invention uses the biosensor to measure binding of amyloid protein to isolated membranes from a cell type such as a neuron, as an index of toxicity. In this way, compounds can be rapidly screened for their therapeutic properties. While it was previously known that A ⁇ can bind to synthetic lipids or to cells in culture, this is the first demonstration that actual cell membranes can be used for this purpose in a biosensor.
- the invention provides a device for screening of candidate agents for treatment of a condition involving cerebral amyloidosis, cerebral angiopathy, or systemic amyloidosis, comprising a biosensor membrane coupled to a lipid preparation which comprises cholesterol and phospholipid.
- the lipid preparation comprises about 30% to 80% cholesterol. More preferably the lipid preparation is a plasma membrane- enriched fraction of a cellular homogenate of smooth muscle cells, nerve cells, kidney cells, cardiac myocytes, or hepatocytes, chosen according to the target condition. For a condition involving a cerebral amyloidosis or cerebral angiopathy, the lipid preparation is prepared from smooth muscle cells or nerve cells. Even more preferably the cells are vascular smooth muscle cells or neuronal cells. For the systemic amyloidoses, the major tissues involved are kidney, heart and liver. Therefore for these conditions the cellular homogenate is prepared from kidney cells, cardiac myocytes or hepatocytes.
- the cells may be obtained from primary tissue samples, or may be prepared from cells of a primary or transformed cell line.
- primary tissue samples For neuronal cells, cell lines or primary cells of cortical or hippocampal origin are particularly preferred.
- the invention provides a method of screening of candidate agents for treatment of a condition involving cerebral amyloidosis, cerebral angiopathy, or systemic amyloidosis, comprising the step of assessing the effect of a candidate agent on binding of an amyloid peptide to a lipid preparation which comprises cholesterol and phospholipid, in which inhibition of binding indicates potentially useful activity.
- the invention provides a method of screening of candidate agents for potential toxicity to the brain or to cerebral blood vessels, comprising the step of assessing the effect of a candidate agent on binding of an amyloid peptide to a lipid preparation which comprises cholesterol and phospholipid, in which promotion of binding indicates potentially toxic activity.
- a lipid preparation which comprises cholesterol and phospholipid, in which promotion of binding indicates potentially toxic activity.
- the lipid preparation is coupled to a biosensor membrane.
- the lipid preparation comprises about 30% to 80% cholesterol. More preferably the lipid preparation is a plasma membrane-enriched fraction of muscle cells or nerve cells. Even more preferably the cells are vascular smooth muscle cells or neurons.
- the amyloid peptide is A ⁇
- the method is performed in the presence of acetylcholinesterase (AChE). More preferably AChE is added to the A ⁇ prior to its addition to the lipid preparation.
- the lipid preparation is coupled to a biosensor membrane.
- both AChE and A ⁇ are at a concentration of up to about IOUM.
- the invention provides an agent for the treatment of a condition involving cerebral amyloidosis or cerebral angiopathy, which is a compound which has the ability to inhibit binding of an amyloid peptide to a lipid preparation which comprises cholesterol and phospholipid.
- the compound also has the ability to inhibit cholesterol biosynthesis.
- the invention provides a composition for the treatment of a condition involving cerebral amyloidosis, cerebral angiopathy, or systemic amyloidosis, comprising a compound which has the ability to inhibit binding of an amyloid peptide to a lipid preparation which comprises cholesterol and phospholipid, together with a pharmaceutically acceptable carrier.
- the invention provides an method of treatment of a condition involving cerebral amyloidosis, cerebral angiopathy, or systemic amyloidosis, comprising the step of administering an effective amount of a compound which has the ability to inhibit binding of an amyloid peptide to a lipid preparation which comprises cholesterol and phospholipid to a subject in need of such treatment.
- the amyloid protein will generally be selected from those known to be associated with the target condition.
- the amyloid peptide will preferably be A ⁇ .
- amyloidosis is immunoglobulin light chain-related (AL) amyloidosis
- the protein will be immunoglobulin light chain or a biologically functional fragment thereof, preferably the light chain variable region.
- amyloid protein is preferably amyloid A.
- familial amyloidosis the protein is preferably selected from the group consisting of transthyretin, apolipoprotein A-I, gelsolin, fibrinogen A oc, and lysozyme.
- the condition involving cerebral amyloidosis or cerebral angiopathy may be a sporadic condition such as Alzheimer's disease, amyloidosis associated with Down syndrome, prion-related cerebral amyloidosis, including Creutzfeld-Jacob disease and its new variant associated with "mad cow” disease, or sporadic cerebral angiopathy, or may be a familial condition such as one of the several forms of autosomal dominant forms of familial Alzheimer's disease (reviewed in St George-Hyslop, 2000); hereditary cerebral haemorrhage associated with the Flemish, Arctic, Dutch, or Italian mutations of A ⁇ precursor protein (reviewed in Ghiso and Frangione, 2001); hereditary cerebral haemorrhage with amyloidosis (Icelandic type); meningocerebrovascular and oculoleptomeningeal amyloidosis; familial British dementia; familial Danish dementia; Cystatin C-related cerebral amyloid angiopathy; transth
- the condition involving cerebral amyloidosis or cerebral angiopathy is sporadic or familial Alzheimer's disease, amyloidosis associated with Down syndrome, sporadic cerebral angiopathy, prion-related cerebral amyloidosis, familial British dementia, Cystatin C-related cerebral amyloid angiopathy, transthyretin-related cerebral amyloid angiopathy, or gelsolin-related spinal and cerebral amyloid angiopathy.
- the condition involving systemic amyloidosis may be primary amyloidosis, or may be a reactive amyloidosis or a familial amyloidosis.
- this condition is selected from the group consisting of AL, familial transthyretin-associated amyloidosis, amyloid protein A-associated amyloidosis, or familial amyloidosis associated with apolipoprotein A-I, gelsolin, fibrinogen A ⁇ , or lysozyme.
- the method of treatment and the composition according to the invention may be used in conjunction with other treatments for the relevant condition.
- the condition involves cerebral amyloidosis or cerebral angiopathy, particularly
- Alzheimer's disease they may be used in conjunction with treatment with another agent such as an acetylcholinesterase active site inhibitor, for example phenserine, galantamine, or tacrine; an antioxidant, such as Vitamin E or Vitamin C; an oestrogenic agent such as 17- ⁇ -oestradiol; a chelating agent, such as clioquinol; or AChE peripheral site inhibitor such as propidium or gallamine.
- an acetylcholinesterase active site inhibitor for example phenserine, galantamine, or tacrine
- an antioxidant such as Vitamin E or Vitamin C
- an oestrogenic agent such as 17- ⁇ -oestradiol
- a chelating agent such as clioquinol
- AChE peripheral site inhibitor such as propidium or gallamine.
- the condition is associated with an amyloid-type protein other than A ⁇ , such as synuclein.
- the condition is selected from the group consisting of Parkinson's disease, dementia with Lewy body formation, multiple system atrophy, Haller Camill-Spatz disease, and diffuse Lewy body disease.
- subject refers to any mammal having a disease or condition which requires treatment with a pharmaceutically- active agent.
- the mammal may be a human, or may be a domestic or companion animal. While it is particularly contemplated that the compounds of the invention are suitable for use in medical treatment of humans, they are also applicable to veterinary treatment, including treatment of companion animals such as dogs and cats, and domestic animals such as horses, cattle and sheep, or zoo animals such as apes and monkeys, felids, canids, bovids, and ungulates.
- the compounds and compositions of the invention may be administered by any suitable route, and the person skilled in the art will readily be able to determine the most suitable route and dose for the condition to be treated. Dosage will be at the discretion of the attendant physician or veterinarian, and will depend on the nature and state of the condition to be treated, the age and general state of health of the subject to be treated, the route of administration, and any previous treatment which may have been administered.
- the compound of the invention may optionally be administered in conjunction with one or more other pharmaceutically-active agents suitable for the treatment of the condition, ie it maybe given together with, before, or after one or more such agents.
- the term "therapeutically effective amount” means an amount of a compound of the present invention effective to yield a desired therapeutic response, for example to prevent or treat a disease which is susceptible to treatment by administration of a pharmaceutically-active agent.
- the specific "therapeutically effective amount” will of course vary with such factors as the particular condition being treated, the physical condition and clinical history of the subject, the type of animal being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the compound or its derivatives.
- a “pharmaceutical carrier” is a pharmaceutically acceptable solvent, suspending agent, excipient or vehicle for delivering the compound of the invention and/ or another pharmaceutically-active agent to the subject.
- the carrier may be liquid or solid, and is selected with the planned manner of administration in mind.
- the compound of the invention may be administered orally, topically, or parenterally in dosage unit formulations containing conventional non- toxic pharmaceutically acceptable carriers, adjuvants, and vehicles.
- parenteral as used herein includes subcutaneous, intravenous, intramuscular, intrathecal, intracranial, injection or infusion techniques.
- the terms "treating”, “treatment” and the like are used herein to mean affecting a subject, tissue or cell to obtain a desired pharmacological and/or physiological effect.
- the effect may be prophylactic in terms of completely or partially preventing a disease or sign or symptom thereof, and/or may be therapeutic in terms of a partial or complete cure of a disease.
- Treating covers any treatment of, or prevention of disease in a vertebrate, a mammal, particularly a human, and includes: preventing the disease from occurring in a subject which may be predisposed to the disease, but has not yet been diagnosed as having it; inhibiting the disease, ie., arresting its development; or relieving or ameliorating the effects of the disease, ie., causing regression of the effects of the disease.
- the invention includes various pharmaceutical compositions useful for ameliorating disease.
- the pharmaceutical compositions according to one embodiment of the invention are prepared by bringing a compound of the invention and optionally one or more other pharmaceutically-active agents or combinations of the compound of the invention and one or more other pharmaceutically-active agents into a form suitable for administration to a subject, using carriers, excipients and additives or auxiliaries.
- Frequently used carriers or auxiliaries include magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk protein, gelatin, starch, vitamins, cellulose and its derivatives, animal and vegetable oils, polyethylene glycols and solvents, such as sterile water, alcohols, glycerol and polyhydric alcohols.
- Intravenous vehicles include fluid and nutrient replenishers.
- Preservatives include antimicrobial, anti-oxidants, chelating agents and inert gases.
- Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like, as described, for instance, in Remington's Pharmaceutical Sciences, 20th ed. Williams & Wilkins (2000) and The
- the pharmaceutical compositions are preferably prepared and administered in dosage units. Solid dosage units include tablets, capsules and suppositories.
- the administration of the daily dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administration of subdivided doses at specific intervals.
- Figure l shows the effect of A ⁇ peptides on vascular SMC viability.
- Vascular SMC cultures were incubated with A ⁇ peptides (lo ⁇ M, unaged or aged for 5 days) for 24 h.
- Asterisks show values that are significantly different from the values obtained from the corresponding incubation using unaged peptide (P ⁇ 0.05, Student's t test).
- Figure 2 shows the levels of amyloid fibrils in fresh or aged A ⁇ peptide preparations, as measured by a Congo Red binding assay.
- a ⁇ peptides were allowed to age for 5 days or prepared fresh and then incubated with Congo Red.
- the concentration of amyloid fibrils was determined spectrophotometrically.
- Asterisks show values which are significantly different from the values obtained from the corresponding experiment using unaged peptide (P ⁇ 0.05, Student's t test).
- Figures 3A and B illustrates sensorgrams showing the binding of various concentrations of unaged A ⁇ 1-42 (A) and A ⁇ 1-40 (B) to SUV containing 60% cholesterol and 40% phospholipid. The binding is shown in response units (RU).
- Figure 3C shows the results of Scatchard plot analysis of the binding of A ⁇ i- 42 and A ⁇ -40 to SUV containing 60% cholesterol and 40% phospholipid.
- Req theoretical maximum value of RU for each concentration.
- C ( concentration of A ⁇ in ⁇ M) was used as an approximation of the total amount of unbound A ⁇ .
- Figure 4 shows the results of quantitative analysis of the effect of aging on the binding of A ⁇ peptides to synthetic phospholipid membranes.
- a ⁇ peptides (10 ⁇ M), aged for o or 5 days, were injected over a cholesterol : phospholipid (60:40, w/w) surface. The binding is shown in response units recorded 20 min after addition of the peptide (RU 20). Values are means ⁇ SEM (n 3).
- Asterisks show values which are significantly different from the values obtained from the corresponding experiment using unaged peptide (P ⁇ 0.05, Student's t test).
- Figure 6 shows the effect of lovastatin on the binding of A ⁇ peptides to vascular SMC membranes.
- Unaged A ⁇ peptides (10 ⁇ M) were injected over the lovastatin-treated (10 ⁇ g / ml) and untreated vascular SMC membrane surface, and the binding was measured.
- Asterisks show values which are significantly different from the corresponding incubations without lovastatin (P ⁇ 0.05, Student's t test).
- Figure 7 shows the effect of lovastatin on A ⁇ toxicity.
- Lovastatin-treated and untreated vascular SMC cultures were incubated with unaged A ⁇ peptides (10 ⁇ M).
- Asterisks show values which are significantly different from the corresponding incubations without lovastatin (P ⁇ 0.05, Student's t test).
- DMPE dimyristoyl-L- ⁇ - phosphatidylethanolamine MPG dimyristoyl-L- ⁇ -phosphatidylglycerol
- DMPS dimyristoyl-L- ⁇ -phosphatidylserine MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2(4-sulfophenyl)-2H- tetrazolium SMC smooth muscle cell
- SUV small 100 nm unilamellar vesicles SUV small 100 nm unilamellar vesicles.
- a ⁇ peptides were synthesised using manual solid-phase Boc (N-rerr- butocarbonyl) amino acid synthesis. Peptides were synthesized using manual solid-phase Boc amino acid chemistry with in situ neutralisation.
- Peptide purification was achieved using an acetonitrile/water (0.01% trifluoroacetic acid) gradient on a reversed-phase preparative Zorbax high performance liquid chromatography (HPLC) column heated to 6o°C.
- HPLC high performance liquid chromatography
- the purity (> 95%) and identity of the peptide was analysed by analytical HPLC, electrospray mass spectrometry and amino acid analysis.
- Dulbecco's modified Eagle's Medium (DMEM) and penicillin/streptomycin were purchased from Gibco Life Technologies (Mulgrave, Vic, Australia), and foetal bovine serum (heat inactivated) was purchased from Commonwealth Serum Laboratories (Parkville, Vic, Australia).
- N-octyl- -D-glucopyranoside dimyristoyl-L- ⁇ - phosphatidylcholine (DMPC), dimyristoyl-L- ⁇ -phosphatidyl-DL-glycerol (DMPG), dimyristoyl-L- ⁇ -phosphatidylethanolamine (DMPE), dimyristoyl- L- ⁇ -phosphatidylserine (DMPS) and D-cholesterol were purchased from Sigma (St Louis, MO, USA). Lovastatin was purchased from Calbiochem (Sydney, ⁇ SW, Australia), and activated to its active open-ring form as previously described (Jakobisiak et al, 1991).
- Solubilization and aging of A ⁇ peptides A ⁇ 1-42, A ⁇ 1-40, A ⁇ 1-28, A ⁇ 17-42 and A ⁇ 29-42 were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 2 mM.
- DMSO dimethyl sulfoxide
- the peptide solutions were then sonicated (42 kHz) for 5 minutes, and centrifuged at 5,000 rpm for 1 minute at room temperature using a Hermle Z160M bench microfuge. Solubilised peptides were immediately snap-frozen and mixed on a vortex mixer for 15 seconds.
- the peptides were then diluted into DMEM for cell culture experiments, or in 0.02 M sodium phosphate buffer, pH 6.8 for biosensor experiments, to give a final concentration of 10 ⁇ M.
- a ⁇ peptides To “age” A ⁇ peptides, a process which increases the proportion of fibrillar oligomeric species (Jarrett and Lansbury, 1992), peptides were incubated at 37°C in a humidified atmosphere of 5% CO 2 for 5 days at a concentration of 100 ⁇ M.
- the concentration of amyloid fibrils was measured using the assay of Klunk et al. (1999). A ⁇ peptides were mixed with Congo red (CR) in 0.02 M sodium phosphate buffer, pH 6.8. The final concentration of peptide and CR was 10 ⁇ M. Solutions of CR alone were also prepared in 0.02 M sodium phosphate buffer, pH 6.8. The mixture was vortexed briefly and then incubated at room temperature for 15 min. The absorbances at 403 and 541 nm were measured using a BioRad SmartSpec 3000 spectrophotometer. Background absorbance values of buffer alone were subtracted from the values obtained from each sample. The concentration of A ⁇ fibrils in each preparation was then determined using the formula
- [A fibrils] (A 54 mm A ⁇ :CR solution / 4780) - ( ⁇ nm of A ⁇ :CR solution / 6830) - ( ⁇ nm of CR solution / 8620)
- Vascular SMCs from aortae of Sprague-Dawley rats were cultured in DMEM supplemented with 10% foetal bovine serum and penicillin/streptomycin at 37°C in a humidified atmosphere of 5% CO 2 .
- Vascular SMCs were plated at a density of ⁇ o4 cells / well in a 96-well plate, or at a density of 10 6 cells / 75 cm 2 cell culture flask (Nunc, Denmark) in 20 ml culture medium, and grown to 80% confluence, after which the medium was removed and replaced with fresh, serum-free medium with or without 10 ⁇ M lovastatin. The cells were then incubated for 72 h. The cells were then either used for the preparation of membranes, or incubated with A ⁇ peptides (10 ⁇ M) for cytotoxicity assay. In the latter case, A ⁇ peptides were added to the culture medium and the cells incubated for a further 24 h. In control incubations, vehicle alone, ie lacking peptide, was added. MTS assay of cytotoxicity
- Cytotoxicity was determined using the CellTiter 96 AQueous One Solution Cell Proliferation Assay kit (Promega Corporation, Madison, WI, USA) (Cory et al., 1991).
- the [3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2(4-sulfophenyl)-2H-tetrazolium (MTS) reagent solution was added to the culture medium at a concentration of 10% (by volume).
- the cells were then incubated for a further 2 h at 37 °C, and the absorbance of the samples read at a wavelength of 560 nm using a Wallac Victor 1420 plate reader.
- a crude plasma membrane preparation was prepared from vascular SMCs by differential centrifugation (Hubbard et al, 1983). Cells were scraped off 10 x 75 cm 2 flasks using a cell scraper, and centrifuged in DMEM at 3,000 rpm in a Beckman Coulter Allegra 21R centrifuge at 4 °C for 3 min.
- the pellet was then washed with phosphate-buffered saline (PBS), added to 10 ml of STM buffer (0.25 M sucrose/ 5 mM Tris-HCl, pH 7.4 / 1.0 mM MgCl 2 ), and homogenized on ice using 10 up and down strokes in a 40 ml Dounce-type glass homogenizer with a loose-fitting pestle.
- PBS phosphate-buffered saline
- STM buffer 0.25 M sucrose/ 5 mM Tris-HCl, pH 7.4 / 1.0 mM MgCl 2
- the homogenate was centrifuged at 1,100 rpm for 5 min. The supernatant fraction was saved and the pellet rehomogenized in 5 ml of STM buffer.
- the suspension was again centrifuged, and the first and second supernatant fractions combined, then centrifuged at 40,000 rpm for 2 h (Beckman L8-M Ultracentrifuge, 7 ⁇ Ti rotor, no brake) and the resulting crude plasma membrane fraction resuspended in 1.0 ml of 0.02 M sodium phosphate buffer, pH 7.4.
- Total membrane cholesterol was determined using the Amplex Red cholesterol assay kit (Molecular Probes, Eugene, OR, USA).
- the protein content of the membrane preparations was determined using the bicinchoninic acid (BCA) assay using bovine serum albumin as standard.
- Small 100 nm unilamellar vesicles containing DMPC, DMPG, DMPS and DMPE, and cholesterol, were prepared in 0.02 M phosphate buffer (pH 6.8) by sonication and extraction. Briefly, 1.5 mg of total lipid was dissolved in 1.5 ml of CHC1 3 : MeOH (3:1, v/v). Aliquots (408 ⁇ l) were removed and evaporated under a stream of nitrogen, and the lipids further dried in ⁇ acuo overnight. The lipids were then resuspended in 600 ⁇ l of 0.02 M sodium phosphate buffer, pH 6.8.
- the resulting lipid dispersion was sonicated in a bath type sonicator until clear, and then extruded 17 times through 100 nm pore diameter polycarbonate filters using Liposofast apparatus (Avestin, Ottawa, Canada) to obtain 100 nm SUV.
- the mixed lipid vesicles contained 80% (w/w), 60% (w/w), 40% (w/w), 30% (w/w) or 0% (w/w) cholesterol.
- the remaining lipid comprised a mixture of DMPC : DMPE : DMPS : DMPG in a ratio of 75 : 20 : 2.5 : 2.5 (by weight).
- Binding experiments were carried out with a BIAcore X analytical system (Biacore, Uppsala, Sweden) using an Li sensor chip (Biacore), as supplied by the manufacturer. It will be appreciated that other types of biosensor may also be used, preferably provided that the chip surface is such that the bound lipid material is able to retain a bilayer or monolayer structure.
- the running buffer used for all experiments was 0.02 M sodium phosphate buffer, pH 6.8 (phosphate buffer).
- the washing solution was 40 mM N-octyl ⁇ -D-glucopyranoside.
- the regeneration solution was 10 mM sodium hydroxide. All solutions were freshly prepared, degassed and filtered through a 0.22 ⁇ M filter. The operating temperature was 25°C.
- the alkyl surface of the Li chip was cleaned by an injection of 25 ⁇ l of non-ionic 40 mM octyl glucoside at a flow rate of 5 ⁇ l/min.
- SUV 100 ⁇ l
- vascular SMC membranes 100 ⁇ l containing 0.33 mg protein
- 30 ⁇ l of 10 mM sodium hydroxide was injected at a flow rate of 50 ⁇ l/min, resulting in a stable baseline corresponding to the successful formation of an immobilized layer of SUVs.
- Peptide solutions were prepared at concentrations ranging from 0.5 to 10 ⁇ M. The solutions were injected over the lipid surface at a flow rate of 5 ⁇ l/min for 20 min. The peptide solution was then replaced by phosphate buffer and the peptide- membrane complex allowed to dissociate. The removal of the bound peptide and regeneration of the Li chip surface, without removal of the synthetic lipid or vascular SMC membrane layer, was achieved by an injection of sodium hydroxide (30 ⁇ l, 10 mM) at a flow rate of 50 ⁇ l/min.
- a ⁇ toxicity against vascular SMCs cultures of cells were treated with A ⁇ peptides and analogues (10 ⁇ M) and the amount of toxicity determined using the MTS assay (Cory et al., 1991).
- a ⁇ -40, A ⁇ i-42, A ⁇ 29- 42 and A ⁇ i7 ⁇ 42 were all found to cause significant toxicity, as shown in Figure 1.
- a ⁇ -28 was less toxic, suggesting that the toxicity of A ⁇ may be associated with the more hydrophobic C-terminal region of the peptide. Aging the peptides by incubation for 5 days caused a significant increase in toxicity.
- the dissociation curve of the bound complex followed a similar biphasic pattern.
- the signal fell rapidly at the end of the injection period, since the peptide was no longer present and the buffer flow removed a large amount of weakly bound peptide.
- the peptide sensorgrams did not return to zero until the biosensor was stripped with 10 mM NaOH, indicating that a proportion of the peptide remained bound to the surface.
- a comparison of A ⁇ peptides and analogues for their abilities to bind to the synthetic lipid mixture showed that there was good agreement between the extent of lipid binding, as shown in Figure 4, and the amount of toxicity caused by each peptide as shown in Figure 1.
- the more toxic C-terminal A ⁇ fragments (A ⁇ 29-42 and A ⁇ 17-42) bound more strongly than the less toxic N-terminal fragment (A ⁇ 1-28).
- the ratio of cholesterol to phospholipid in the synthetic lipid mixture was directly related to the extent of high-affinity lipid binding, as illustrated in Figure 5.
- a pure phospholipid mixture was used, very little binding was observed.
- the amount of binding increased.
- This increase in binding was also reflected by an increase in the equilibrium association constants determined after fitting the kinetic data from Figures 3A and 3B to a competing reaction model.
- both A ⁇ 1-42 and A ⁇ 1-40 peptides had a higher binding affinity for synthetic lipid mixtures containing a greater percentage of cholesterol.
- KAI equilibrium association constant for high affinity binding component.
- KA2 equilibrium association constant of low-affinity binding component.
- lovastatin a cholesterol biosynthesis inhibitor
- a plasma membrane-enriched fraction was prepared.
- the protein composition of the membrane fractions was closely similar in the control (3.29 + 0.15 mg/ml) and lovastatin-treated groups (3.24 + 0.03 mg/ml), indicating that lovastatin treatment did not significantly alter the amount of total membrane protein.
- Treatment of the cells with lovastatin was found to strongly decrease binding of both A ⁇ -40 and A ⁇ i-42 to the membrane fraction, as shown in Figures 6A and B).
- KAI equilibrium association constant for high affinity binding component.
- KA2 equilibrium association constant of low-affinity binding component.
- the binding may be biphasic; however, more data may reveal a more complex interaction.
- affinity constants calculated from the competing reaction model should be viewed as indicating an overall affinity of A ⁇ for the lipid membrane, rather than as having any independent significance.
- a ⁇ -membrane binding is involved in A ⁇ toxicity.
- a ⁇ can bind to phospholipids (Waschuk et al., 2001), and inhibition of electrostatic interactions between A ⁇ and the negative phospholipids can inhibit A ⁇ toxicity (Hertel et al., 1997).
- bound A ⁇ was easily removed from lipid membranes with sodium hydroxide, suggesting that electrostatic, rather than hydrophobic forces, are involved. This contrasts with the behaviour of many other peptides, which bind hydrophobically and penetrate deeply into the lipid bilayer (Mozsolits et al, 2001). Indeed, although cholesterol can bind A ⁇ , presumably via hydrophobic interactions, Ji et al.
- a ⁇ -cholesterol binding inhibits fibril formation, and have speculated that cholesterol might be neuroprotective.
- increased cholesterol was clearly associated with increased toxicity. Therefore direct binding of A ⁇ to cholesterol may not be involved in the A ⁇ -membrane interaction in our system.
- a ⁇ can also bind to sphingolipids and gangliosides (Ariga et al., 2001; Valdez-Gonzalez et al., 2001; Mahouf et al., 200).
- KA equilibrium association constant
- a ⁇ binding to the cell membrane causes cell toxicity is still unclear. Free radical production, lipid peroxidation and alterations in ion channel function have been implicated (reviewed by Small and McLean, 1999). If A ⁇ binds directly to the lipid component of the membrane, then alterations in membrane fluidity may occur. Chochina et al. (2001), using synaptic plasma membranes, have reported that A ⁇ 1-40 can increase neuronal membrane fluidity, although in contrast to this Kremer et al. (2001), using synthetic lipids, found that A ⁇ can decrease membrane fluidity. Certainly changes in membrane fluidity could affect the function of a variety of proteins on the cell surface, including ion channels. For example, alterations in fluidity are known to affect the sub-membrane localization and function of the nicotinic receptor (Baenziger et al, 2000).
- a ⁇ aggregation correlates with the vascular SMC toxic response.
- the process of aging increases the number of amyloid fibrils formed from A ⁇ , as assessed using a CR binding assay, this does not prove that fibrils are the major toxic form of A ⁇ .
- a ⁇ is probably secreted as a monome, and subsequently aggregates into soluble oligomers or fibrils (Podlisny et al, 1998). It is likely that the levels of soluble oligomeric species of A ⁇ are also increased by the process of aging.
- AD Alzheimer's disease
- the ⁇ 4 allele of the apolipoprotein E gene is known to be a risk factor for Alzheimer's disease (Bales et al, 1997; Corder etal, 1993), and demented individuals who are homozygous for the ⁇ 4 allele have been reported to have higher plasma cholesterol levels than do normal elderly controls (Czech et al, 1994)-
- This idea is also supported by the observation that AD-like pathology is less severe in APP transgenic mice which have been treated with a cholesterol-lowering drug (Refolo et al, 2001).
- two retrospective studies using cholesterol-lowering statins have reported dramatic decreases in the risk of developing AD (Jick et al, 2000; Wolozin et al, 2000).
- Cholesterol could have more than one role in the pathogenesis of AD.
- a number of studies have shown that cholesterol is also important for the regulation of A ⁇ production (Mizuno et al, 1998).
- High cholesterol uptake can increase A ⁇ deposition in transgenic mice (Refolo et al, 2000; Sparks et al, 1994).
- Cholesterol depletion can inhibit the generation of A ⁇ in hippocampal neurons (Simons et al, 1998), and this effect may be mediated by APP secretases (Kojiro et al, 2001).
- inhibition of cholesterol biosynthesis as a therapeutic strategy for AD may have a dual beneficial role, not only decreasing A ⁇ production in the brain, but also decreasing the toxic consequences of A ⁇ accumulation.
- Amyloid ⁇ -peptide (1-40) increases neuronal membrane fluidity. Role of cholesterol and brain region.
- Alzheimer's disease amyloid ⁇ binds copper and zinc to generate an allosterically ordered membrane-penetrating structure containing superoxide dismutase-like subunits.
- Apolipoprotein E-4 gene dose in clinically disgnosed Alzhiemer's disease prevalence, plasma cholesterol levels and cerebrovascular change.
- Cholesterol modulates the membrane-disordering effects of ⁇ -amyloid peptides in the hippocampus specific changes in Alzheimer's disease. Dement. Geriatr. Cogn. Disord. 11, 181-186
- the systemic amyloidosis The systemic amyloidosis.
- Simvastatin strongly reduces levels of Alzheimer's disease ⁇ -amyloid peptides A ⁇ 42 and A ⁇ 40 in vitro and in vivo. Proc. Natl. Acad. Sci. USA. 98, 5856-5861
- Alzheimers disease initial report of the purification and characterization of a novel cerebrovascular amyloid protein.
- Acetylcholinesterase accelerates assembly of amyloid-beta-peptides into Alzheimer's fibrils: possible role of the peripheral site of the enzyme. Neuron ⁇ 6(4):88 ⁇ -9i
- Cholesterol is an important factor affecting the membrane insertion of ⁇ - amyloid peptide (A ⁇ 1-40), which may potentially inhibit the fibril formation.
- Amyloid plaque core protein in Alzheimer disease and Down syndrome Proc. Natl. Acad. Sci. USA 82, 4245-4249
- Hypercholesterolemia accelerates the Alzheimer's amyloid pathology in a transgenic mouse model.
- a cholesterol-lowering drug reduces ⁇ -amyloid pathology in a transgenic mouse model of Alzheimer's disease. Neurobiol. Dis. 8, 890-899
- Amyloid and lipids in the pathology of Alzheimer's disease Amyloid 6, 136-
- Alzheimer's disease is incorporated in vivo by the presenilin 1 and 2 and APP mutations linked to familial Alzheimer's disease.
- Simvastatin strongly reduces levels of Alzheimer's disease ⁇ -amyloid peptides A ⁇ 42 and A ⁇ 40 in vitro and in vivo. Proc. Natl. Acad. Sci. USA. 95,460-6464
- Alzheimer's disease and the amyloid ⁇ protein What is the role of amyloid? J. Neurochem. 73, 443-449
- Cholesterol protects PC12 cells from ⁇ amyloid induced calcium disordering and cytotoxicity.
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JP2007529545A (en) * | 2004-03-19 | 2007-10-25 | アクソニクス,インコーポレイテッド | Down syndrome treatment |
US7763250B2 (en) | 2005-04-29 | 2010-07-27 | Rinat Neuroscience Corp. | Antibodies directed against amyloid-beta peptide and nucleic acids encoding same |
US7807165B2 (en) | 2004-07-30 | 2010-10-05 | Rinat Neuroscience Corp. | Antibodies directed against amyloid-beta peptide and methods using same |
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JP2007529545A (en) * | 2004-03-19 | 2007-10-25 | アクソニクス,インコーポレイテッド | Down syndrome treatment |
US7807165B2 (en) | 2004-07-30 | 2010-10-05 | Rinat Neuroscience Corp. | Antibodies directed against amyloid-beta peptide and methods using same |
US7927594B2 (en) | 2004-07-30 | 2011-04-19 | Rinat Neuroscience Corp. | Antibodies directed against amyloid-beta peptide |
US7763250B2 (en) | 2005-04-29 | 2010-07-27 | Rinat Neuroscience Corp. | Antibodies directed against amyloid-beta peptide and nucleic acids encoding same |
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