Title:  Assay for compounds which affect conformationally altered proteins

United States Patent:  6,419,916

Inventors:  Prusiner; Stanley B. (San Francisco, CA); Supattapone; Surachai (San Francisco, CA); Scott; Michael R. (San Francisco, CA)

Assignee:  The Regents of the University of California (Oakland, CA)

Appl. No.:  406972

Filed:  September 28, 1999

An assay comprises contacting cells containing a conformationally altered protein with test compound and determining if the altered protein is cleared. The cells may be scrapie-infected neuroblastoma cells. Another assay comprises contacting organ or tissue homogenate (at pH 5.0 or less) with test compound to determine if altered protein in the homogenate is 10 cleared. The homogenate may be brain homogenate from a transgenic mouse infected with human prions. Compounds which are found to clear the altered protein are useful in preventing, arresting and/or reversing (i.e. treating) a disease associated with the conformationally altered protein.

SUMMARY OF THE INVENTION

An assay is provided whereby compounds are tested to determine their potential efficacy as therapeutics for the treatment of disorders associated with conformationally altered protein, e.g. prion diseases associated with the PrP^Sc conformation of a PrP protein. The assay comprises contacting scrapie-infected neuroblastoma (ScN2a) cells in culture with a test compound to determine if the test compound reduces levels of PrP^Sc. Preferably the assay includes a plurality of tests wherein different concentrations of the test compounds are separately contacted with different portions of the same cell culture and further wherein different cell cultures are contacted with the test compound for a plurality of different exposure times prior to testing for PrP^Sc levels.

In another embodiment of the assay of the invention an organ homogenate (e.g. a brain homogenate) is provided which homogenate comprises conformationally altered proteins, i.e. comprises PrP^Sc particles. The pH of the homogenate is then reduced to a pH of about 4.0.+-.1.0 and a test compound is added to determine if the test compound reduces levels of the conformationally altered protein (e.g., PrP^Sc) in the homogenate. The assay preferably comprises a plurality of tests wherein different concentrations of the test compound are separately contacted with different portions of the same homogenate and farther wherein the test compound is brought into contact with the different portions of a homogenate for different exposure times prior to testing for conformationally altered protein levels.

In any assay of the invention the results obtained in terms of reduced levels of conformationally altered protein (e.g., PrP^Sc) obtained using a test compound can be compared to negative and positive controls with the positive control being a highly-branched polycation.

In addition to assays the present invention provides methods of arresting, preventing and/or reversing the impairment of physiologic systems, the methods comprising reducing the burden of insoluble protein deposits by the administration of branched polycationic agents or pharmaceutical compositions containing such branched polycationic agents. The agents used in the preferred method of the invention are highly-branched polycations, e.g. dendritic polycations.

In one embodiment, the invention provides pharmaceutical compositions for the treatment of protein deposit formation in an animal which compositions contain branched polycations agents, preferably highly-branched polycations. Branched polycations for use in the invention include, but are not limited to, polypropylene imine, polyethyleneimine (PEI) poly(4'-aza-4'-methylheptamethylene D-glucaramide), polyamidoamines and suitable fragments and/or variants of these compounds. The pharmaceutical compositions can also contain other active ingredients, either separate or complexed to the branched polycations.

The invention also provides methods for reducing the burden of insoluble protein deposits in various host tissues by administering a highly-branched polycationic agent to the host. Preferably the highly-branched polycation is administered over a period of time, either continuously or in multiple dosage units. The animal treated may be suffering from any degenerative disorder associated with insoluble protein deposits. For example, the animal may be a human suffering from Alzheimer's Disease or a cow suffering from BSE.

The invention also features a method for reversing protein deposits in degenerative diseases of a subject by administration of a polycationic compound. The compound is preferably a highly-branched polycation, and the subject may be suffering from degenerative disorder.

The invention also features a method for preventing the formation of protein deposits in animals or humans at risk for a degenerative disease by administration of a highly-branched polycationic compound in an amount sufficient to suppress formation of the protein deposits. The compound used in this method is preferably a highly-branched polycation. Subjects for treatment with this method may be genetically predisposed to developing degenerative disease, such as humans genetically at risk for AD, Parkinson's disease, ALS, FTD, Pick's disease, Huntington's disease or CJD. Subjects may also be determined to be at risk due to exposure to infectious agents causing amyloid-associated disorders, e.g. cattle exposed to bovine prions from a BSE contaminated source.

An object of the invention is to provide an assay for identifying compounds which affect conformationally altered proteins and particularly which aid in reducing levels of such proteins in a low pH environment.

An aspect of the invention is an assay whereby a scrapie-infected neuroblastoma (ScN2a) cell culture is contacted with a test compound to determine if the test compound can reduce the PrP^Sc level in the cell culture.

Another aspect of the invention is an assay whereby organ (e.g. brain) homogenate is reduced to a pH of less than 5 and contacted with a test compound to determine if the test compound can reduce the level of conformationally altered protein (e.g. PrP^Sc) in the homogenate.

An advantage of the invention is that the basic methodology is applicable to assaying for compounds with potential therapeutic utility for a wide range of diseases associated with conformationally altered proteins.

A feature of the compounds of the present invention is their ability to mediate the clearance of PrP^Sc from cultured cells under non-cytotoxic conditions.

An advantage of the pharmaceutical compositions of the invention is that the highly-branched polycation administered is non-toxic to the mammalian host at a dosage of 0.001 mg to 1 mg/kg body weight per day.

Another advantage is that subjects treated with the methods of invention remain free of insoluble protein deposits after clearance.

Claim 1 of 8 Claims

That which is claimed is:

1. A method of enhancing clearance of a conformationally altered protein from cells, comprising the steps of:

contacting cells with a polycationic dendrimer compound which enhances clearance of PrP^Sc ; and

allowing the compound to remain in contact with the cells for a time and under conditions sufficient to allow for clearance of the PrP^Sc from the cells wherein the compound and conditions are non-cytotoxic to the cells.
 

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