Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – The nonhuman animal is a model for human disease
Reexamination Certificate
1997-10-28
2001-01-09
Crouch, Deborah (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
The nonhuman animal is a model for human disease
C800S003000, C800S021000, C800S018000, C424S009200
Reexamination Certificate
active
06172277
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to neurodegenerative disease.
The role of the &bgr;-amyloid peptide (Glenner et al., 1984, Biochem. Biophys. Commun. 120:88-890) in the pathogenesis of Alzheimer's disease is highly controversial. The &bgr;-amyloid protein is derived from the transmembrane amyloid precursor protein (APP; Kang et al., 1987, Nature 325:733-736). While deposition of &bgr;-amyloid is an invariant feature of Alzheimer's disease, research to date has not elucidated the relationship between &bgr;-amyloid plaque deposition and the behavioral and neuropathological features that characterize clinically defined Alzheimer's disease. Studies in vitro have demonstrated that the peptide has both neurotoxic and neurotropic activity, including toxicity in primary cultures of human cortical neurons and dissociated neurons from adult mice. The results of studies in vivo have been less clear.
&bgr;-amyloid synthetic peptides, in a variety of sequence lengths and in a number of vehicles, have been administered to the brains of both primates and rodents resulting in only minimal specific neurodegeneration. When administrated by acute injection into either rodents or primates, &bgr;-amyloid has failed to consistently produce Alzheimer-specific pathological alterations. Damage induced by the delivery system has been extensive, making it difficult to assess whether amyloid caused specific effects.
A chronic infusion paradigm, in which a mixture of &bgr;-amyloid and heparan sulfate proteoglycans are infused, has been reported to produce Congo red positive plaques in rats. Although the &bgr;-amyloid peptide of Alzheimer's disease has been implicated in the disease process, the mechanism by which amyloid may contribute to neurodegeneration is not understood.
SUMMARY OF THE INVENTION
The invention features a nontransgenic animal model (e.g., a rat model) of human Alzheimer's disease and a method useful for evaluating strategies to prevent or treat the development of the disease. In addition to the use of the model in evaluating the efficacy of candidate compounds or other therapeutic interventions, the etiology of the disease may be determined using the model.
A method of inducing amyloid plaque deposition in a mammal is carried out by infusing into the brain of the mammal an amyloid peptide at a basic pH, e.g., a pH greater than 8.0 such as a pH greater than 9.0 but less than 11. Preferably, the pH is 9.5.
An amyloid peptide is a peptide derived from human amyloid &bgr; protein that causes amyloid plaque deposition in mammalian brain tissue. Preferably, the peptide has an amino acid sequence corresponding to the first 40 amino acids of human amyloid &bgr; protein, i.e., the amino acid sequence Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val (SEQ ID NO:1).
Amyloid peptide is continuously infused into the brain for at least one week, preferably two weeks, more preferably four weeks, and most preferably eight weeks. Peptides may be infused continuously for a year or more with regular changes of the pump as the contents becomes depleted. Preferably, the amyloid peptide is infused at a concentration of at least 1 mg/ml (preferably in the range of about 1-10 mg/ml, more preferably in the range of about 1-5 mg/ml, and most preferably at about 2 mg/ml). The rate of infusion is about 0.5 &mgr;l/hour for about 2 weeks. Preferably, the rate is at least 100 mg per week, more preferably the peptide is infused at a rate of about 200 mg per week. Amyloid peptide is administered to an animal e.g., a rat, at a dose within the range of 0.1 mg/kg to 50 mg/kg of body weight. Preferably, the dose is in the range of 1 to 1.5 mg/kg of body weight.
The invention also features a nontransgenic mammalian model for human Alzheimer's disease. Amyloid plaques are induced in the brain tissue of a nontransgenic mammal, e.g., a rodent such as a rat, mouse, hamster, guinea pig, or degu, by infusing into the brain of the mammal an amyloid peptide at a basic pH for an extended period of time, e.g., weeks to months. In animals that are long-lived (e.g., the degu which has an average life span of about 10 years), the amyloid peptide may be infused for a period of months to years to induce or accelerate plaque deposition and/or other Alzheimer's type pathology.
Animals infused with a peptide having the amino acid sequence of SEQ ID NO:1 develop a plurality of amyloid plaques in their brain tissue. Control animals infused with vehicle alone or a non-toxic control peptide, e.g, a peptide which differs from SEQ ID NO:1 by several amino acid substitutions, develop few or no plaques. Preferably, the sequence of the control peptide is Met-His-Phe-Asp-Gly-Val-Glu-Gly-Phe-Gln-Leu-Ala-Ile-Val-Gly-Asn-Ile-Leu-Val-GLy-GLu-Gly-Gly-Ala-Lys-Val-Val-Asp-Ser-Lys-Ala-Tyr-Phe-His-His-Arg-Asp-Val-Ser-Glu (SEQ ID NO:2).
The nontransgenic model is characterized by Alzheimer's associated disrupted sleep and circadian activity patterns compared to a control mammal (receiving vehicle alone or the control peptide). Control mammals typically develop few or no plaques, whereas mammals infused with the amyloid peptide are characterized by at least a 50% increase in the number of amyloid plaques compared to the number in a control mammal. Preferably, the mammal is characterized by at least a 100% increase in the number of amyloid plaques compared to the number in a control mammal. More preferably, the mammal is characterized by at least a 200% or greater increase in the number of amyloid plaques compared to the number in a control mammal.
Also within the invention is an in vivo screening assay to determine whether a compound reduces deposition of amyloid plaques. The screening assay is carried out by (a) providing a first and second nontransgenic mammal, e.g., a rat, each of which is characterized as having amyloid plaques in a brain tissue (the plaques having been induced by infusing into the brain of each mammal an amyloid peptide at a basic pH); (b) contacting the first mammal with a candidate compound; (c) maintaining the second mammal in the absence of the candidate compound; and (d) comparing the degree of amyloid plaque deposition in the brain of the first mammal with the degree of amyloid plaque deposition in the brain of the second mammal. A lesser degree of deposition in the first mammal compared to that in the second mammal is an indication that the candidate compound reduces amyloid plaque deposition associated with Alzheimer's disease pathology. In addition to quantifying plaque deposition, the screening assay may be used to determine whether a compound reduces an Alzheimer's disease-associated disruption in behavioral abnormalities, e.g., sleep and circadian activity disruptions. In the latter case, the last step of the assay requires comparing a sleep and circadian activity pattern of the first mammal with the sleep and circadian activity pattern of the second mammal. A lesser degree of disruption of a normal or control pattern (e.g., a pattern obtained from an animal infused with vehicle alone or control peptide) in the first mammal compared to that in the second mammal is an indication that the candidate compound reduces Alzheimer's disease-associated disruption in sleep and circadian activity.
The methods of the invention have several advantages over known methods of inducing amyloid plaques to simulate Alzheimer's type pathology, e.g., reduced mechanical tissue damage from administration procedures, reduced neurotoxicity of vehicle, increased solubility of the peptide over long periods of time (e.g., over the weeks required for continuous infusion protocols), and increased delivery (total load and efficiency of transfer from infusion apparatus) of peptide to brain tissue.
In contrast to prior art control peptides, the control peptide described herein produces few or no amyloid plaques. In addition, amyloid deposition in amyloid peptide-infused animal
Majocha Ronald
Newton Julie L.
Tate Barbara A.
Crouch Deborah
Mintz Levin Cohn Ferris Glovsky and Popeo P.C.
The Miriam Hospital
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