MXPA01012357A - Method of sterilizing. - Google Patents

Abstract

A method of sterilizing objects as well as the sterilized objects obtained from the method are disclosed. The method involves contacting an object such as a medical device to be reused with polycationic dendrimer under conditions which result in rendering a conformationally altered protein (e.g. a prion) non-infectious. A disinfecting agent or surgical scrub composition which comprises the dendrimers is also disclosed as are gelatin capsules treated with polycationic dendrimers.

Description

# ~ - 's ** METHODS TO STERILIZE GOVERNMENT SUPPORT This work was supported, in part, by the National Institutes of Health grants NS14069, AG08967, AG02132, AG10770 and K08 NS02048-02. The government has certain rights in this work.

FIELD OF THE INVENTION The present invention relates in general to methods for sterilizing materials, and in particular to a method for inactivating infectious prions.

BACKGROUND OF THE INVENTION 15 There are large numbers of known methods for sterilizing materials. Many methods involve heating a material to a temperature at which pathogens are killed or inactivated. Other methods involve exposing the material to compounds that kill or inactivate the pathogens that 20 make contact with the compounds. Still other methods involve irradiating a material with a sufficient amount of a particular type of radiation for a period of time sufficient to inactivate, alter, or annihilate the pathogens in the material. These methods are generally directed towards 25 annihilation of bacteria and the activation of viruses present inside or on the material. Although sterilization methods can be very effective in killing bacteria or inactivating viruses, in general they do not inactivate pathogenic proteins, such as prions, which may be responsible for a number of fatal diseases. There are a considerable number of diseases associated with a conformationally altered protein. For example, Alzheimer's disease is associated with APP, the Aβ peptide, anti-chymotrypsin al, tau and the non-Aß component. Many of these diseases are neurological diseases. However, type II diabetes is associated with amylin, and multiple myeloma-plasma cell dyscrasias are associated with the IgGL chain. The reaction between the establishment of the disease and the transition from the normal protein to the conformationally altered protein has been examined very closely in some cases, such as with the association between prion diseases and PrPSc 'Prion diseases are a group of fatal neurodegenerative disorders that may occur in hereditary, sporadic, and infectious forms (Prusiner, SB Scrapie prions, Annu Rev. Microbiol 43, 345-374 (1989)). These diseases occur in humans and in a variety of other animals (Prusiner, S.B. Prions, Proc.Nat.Acid Sci USA 95.13363-13383 (1998)). Prio- nes are infectious proteins. The normal cellular form of the prion protein (PrP) designated as PrPc, contains three helices a, and has little ß sheet; in contrast, the protein of prions denoted as PrPSc is rich in the ß sheet structure. The accumulation of PrPSc in the central nervous system (CNS) precedes the neurological dysfunction accompanied by neuronal va-culolation and astrocytic gliosis. The spectrum of human prion diseases includes kuru (Gajdusek, DC, Gibbs, CJ, Jr. and Alpers, M. Ex-perimental transmission of a kuru-like syndrome to chimpan-zees.) Na t ure 209, 794-796 (1966) ), Creutzfeldt-Jakob disease (CJD) (Gibbs, CJ, Jr., et al, Creutzfeldt-Jakob disease (spongiform encephalopathy): transmission to the chimpanzee, Science 161, 388-389 (1968)), Gerstmann-Stráussler-Scheinker (GSS) and fatal familial insomnia (FFI) (Goldfarb, LG et al., Fatal familial insomnia and familial Creutzfeldt-Jakob disease: disease phenotype determined by DNA polymorphism.) Sci en 258, 806-808 (1992 ), Medori, R. et al, Fatal familial insomnia: a second kindred with mutation of prion protein gene at codon 178. Neurology 42, 669-670 (1992)), and a new form of human prion disease, the new variant CJD (nvCJD), which emerged in Great Britain and France (Will, RG, and collaborators, A new variant of Creutzf eldt-Jakob disease in the UK. Lancet 347, 921-925 (1996); Cousens, S.N., Vynnycky, E., Zeidler, M., Will, R.G. and Smith, P.G. Predicting the CJD epidemic in humans. Na ture 385, 197-198 (1997); Will, R.G., and collaborators Deaths from variant Creutzfeldt-Jacob disease. Lancet 353, 979 (1999)). Several lines of evidence have suggested a link between the presentation of nvCJD and a preceding epidemic of bovine spongiform encephalopathy (BSE) (Will, RG, et al., A new variant of Creutzfeldt-Jakob disease in the UK.) Lancet 347, 921-925 (1996), Bruce, ME et al, Transmission to mice indicate that 'new variant1 CJD in produced by the BSE agent, Na ture 389, 498-501 (1997); Hill, AF et al.

The same prion strain causes vCJD and BSE. Na ture 389, 448-450 (1997); Lasmézas, C.I., and collaborators. BSE transmission to macaques. Na ture 381, 743-744 (1996)). Although it is too early to predict the number of nvCJD cases that could eventually occur in Britain and elsewhere (Cousens, SN, Vynnycky E., Zeidler, M., Will, RG and Smith, PG Predicting the CJD epidemic in humans, Na ture 385, 197-198 (1997)), it is clear that effective therapies for prion diseases are urgently needed. Unfortunately, although a number of compounds have been reported, including amphotericins, sulfated polyanions, Congo red dye, and anthracycline antibiotics as prospective therapeutic agents (Ingrosso, L., Ladogana, A. and Poc-chiari, M. Congo red prolongations The incubation period in sera-foot-infected hamsters, J. Virol 69, 506-508 (1995), Tagliavini, F. et al, Effectiveness of anthracy-cline against experimental prion disease in Syrian hamsters, Science 276, 119-1122. (1997), Masullo, C, Macchi, G., Xi YG and Pocchiari, M., Failure to ameliorate Creutzfeldt-Jakob disease with amphotericin B therapy, J. Infect. Dis. 165, 784-785 (1992); Ladogana, A., et al, Sulphate poly-anions prolong the incubation period of scrapie-infected hamsters, J. Gen. Virol. 73, 661-665 (1992)), all have shown only a modest potential to prevent the spread of prions, and none have shown to effect the removal of previously existing prions s of an infected host. The PrP gene of mammals expresses a protein that can be the non-sick soluble form PrPc, or can be converted to the insoluble disease form PrPSc. PrPc is encoded by a single copy host gene [Basler, Oesch et al. (1986) CeJl 46: 417-428] and when PrPc is expressed, it is usually found on the outer surface of neurons. Many lines of evidence indicate that prion diseases result from the transformation of the normal form of the prion protein (PrPc) into the abnormal form (PrPSc). There is no detectable difference in the amino acid sequence of the two forms. However, the PrPSc when combined with the PrPc has a conformation with a higher ß sheet content and a lower helix content (Pan, Baldwin et al. (1993) Proc Na ti Acad Sci USA 90: 10962-10966 Safar, Roller et al. (1993) J. Biol Chem 268: 20276-20284). The presence of the abnormal PrPSc form in the brains of humans or infected animals is the only specific diagnostic marker of the disease in prion diseases. PrP? C plays a key role in both the transmission and pathogenesis of prion diseases (spongiform encephalopathies), and is a critical factor in neuronal degeneration (Prusiner (1997) The Molecular and Genetic Basis of Neurological Disease, 2nd edition : 103-143). The most common prion diseases in animals are scrapie of sheep and goats, and bovine spongiform encephalopathy (BSE) of cattle (Wilesmith and Wells (1991) Curr Top Microbiol Immunol 172: 21-38). Four prion diseases in humans have been identified: (1) kuru, (2) Creutzfeldt-Jakob disease (CJD), (3) Gerstmann-Stráussler-Scheinker disease (GSS), and (4) fatal familial insomnia (FFI) ) [Gajdusek (1977) Science 197: 943-960; Medori, Trits-chler et al. (1992) N. Engl J Med 326: 444-449]. Initially, the presentation of inherited human prion diseases presented a conundrum that has since been explained by the cellular genetic origin of PrP. The assembly and mis-assembly of proteins normally soluble in conformationally altered proteins is thought to be a causative process in a variety of other diseases. Structural conformation changes are required for the conversion of a normally soluble and functional protein into a defined insoluble state. Examples of this insoluble protein include: the Aβ peptide in the amyloid plaques of Alzheimer's disease and cerebral amyloid angiopathy (CAA); deposits of synuclein a in the Lewy bodies of Parkinson's disease, tau in neurofibrillary recesses in frontal temporal dementia and in Pick's disease; superoxide dismutase in amyotrophic lateral sclerosis; Huntingtin in Huntington's disease; and prions in Creutzfeldt-Jakob disease (CJD); (for reviews, see Glenner et al., (1989) J. Neu-rol, Sci. 94: 1-28; Haan et al. (1990) Clin. Neurol. Neurosurg., 92 (4): 305-310). Frequently, these highly insoluble proteins form aggregates composed of unbranched fibrils with the common feature of a p-plys-leaf conformation. In the central nervous system, amyloid may be present in the cerebral and meningeal blood vessels (cerebrovascular deposits), and in the parenchyma of the brain (plaques). Neuropathological studies in human and animal models indicate that cells proximal to amyloid deposits are altered in their normal functions (Mandybur (1989) Acta Neuropathol 78: 329-331; Kawai et al. (1993) Brain Res. 623: 142- 6; Martin et al. (1994) Am. J. Pa thol. 145: 1348-1381; Kalaria et al. (1995) Neuroreport 6: 477-80; Masliah et al. (1996) J. Neu-rosci. 16: 5795- 5811). Other studies further indicate that amyloid fibrils can actually initiate neurodegeneration (Lendon et al. (1997) J. Am. Med. Assoc. 227: 825-31; Yankner (1996) Na t. Med. 2: 850-2; Selkoe (1996) J. Biol. Chem. 271: 18295-8; Hardy (1997) Trends Neu-rosci 20: 154-9). In both Alzheimer's disease and cerebral amyloid angiopathy, the primary amyloid component is β-amyloid protein (Aß). The Aβ peptide, which is generated from the β amyloid precursor protein (APP) by two putative secretases, is present at low levels in the normal central nervous system and in the blood. Two main variants, Aβ? _o and Aβ? -2, are produced by the alternative carboxy-terminal truncation of APP (Selkoe et al. (1988) Proc. Na ti. Acad. Sci USA 85: 7341-7345; Selkoe, ( 1993) Trends Neurosci 16: 403-409). Aß? _42 is the most fibrillogenic and most abundant of the two peptides in the amyloid deposits of both Alzheimer's disease and cerebral amyloid angiopathy. In addition to the amyloid deposits in the cases of Alzheimer's disease described above, most cases of Alzheimer's disease are also associated with the amyloid deposit in the vascular walls (Hardy (1997), supra; Haan et al. 1990), supra, Terry et al., Supra, Vinters (1987), supra, Itoh et al. (1993), supra, Yamada et al. (1993), supra, Greenberg et al. (1993), supra, Levy et al. (1990). ), supra). These vascular lesions are the hallmark of cerebral amyloid angiopathy, which may exist in the absence of Alzheimer's disease. Human transthyretin (TTR) is a normal plasma protein composed of four identical units predominantly structured in ß sheet, and serves as a transporter of the hormone thyroxine. Abnormal self-assembly of the TTR in the amyloid fibrils causes two forms of human diseases, namely, systemic senile amyloidosis (SSA), and familial amyloid polyneuropathy (FAP) (Kelly (1996) Curr Opin Strut Biol 6 (l ): ll-7). The cause of amyloid formation in familial amyloid polyneuropathy are point mutations in the TTR gene; The cause of senile systemic amyloidosis is unknown. The clinical diagnosis is established histologically by detecting amyloid deposits in itself in the bioptic material. To date, little is known about the mechanism of the conversion of TTR to amyloid in vivo. However, several laboratories have shown that amyloid conversion can be simulated in vitro by partial denaturation of normal human TTR [McCutchen, Colon et al. (1993) Biochemistry 32 (45): 12119-27; McCutchen and Kelly (1993) Biochem Biophys Res Commun 197 (2) 415-21]. The conformational transition mechanism involves a monomeric forming intermediate that is polymerized in structured linear amyloid fibrils in ß sheet form [Lai, Colon et al. (1996) Biochemistry 35 (20): 6470-82]. The process can be mitigated by binding with stabilizing molecules, such as thyroxine or triiodophenol (Miroy, Lai et al. (1996) Proc Na ti Acad Sci USA 93 (26): 15051-6). The precise mechanisms by which neuritic plaques form, and the relationship of plaque formation to the neurodegenerative processes associated with the disease are not well defined. Amyloid fibrils in the brains of patients with Alzheimer's disease and prion disease result in the inflammatory activation of certain cells. For example, the primary microglyral cultures and the THP-1 monocytic cell line are stimulated by the amyloid-β and prionic fibrillar peptides to activate identical cascades of tyrosine kinase-dependent inflammatory signal transduction. The signaling response caused by amyloid-ß and prion fibrils leads to the production of neurotoxic products, which are partly responsible for neurodegeneration. C.K.

Combs et al. J. Neurosci 19: 928-39 (1999). Although research efforts related to conformationally altered proteins are advancing efforts to sterilize materials to prevent infections with these proteins, they are not keeping pace. The present invention provides a means for sterilizing materials containing conformationally altered proteins, such as prions.

BRIEF DESCRIPTION OF THE DRAWING Figure 1 is a schematic drawing of a dendrimer molecule showing the defined "generations" of homodisperse structure created using a repetitive divergent growth technique. The specific diagram is from PAMAM, generation 2.0 (ethylene diamine nucleus).

SUMMARY OF THE INVENTION A method is disclosed by which any type of object can be sterilized by combining normal sterilization procedures with the use of a polycationic dendrimer that is capable of making a conformationally altered protein, such as a prion. , be non-infectious. The method is particularly useful in the sterilization of medical devices, such as surgical instruments and catheters, which have been used and brought into contact with blood or brain tissue. Objects sterilized by the method are also part of the invention, and include capsules that are made from gelatin extracted from cattle, whose cattle may be infected with prions, that is, they may have undiagnosed BSE, known as "heart disease". crazy cows". Polycationic dendrimers can be combined with conventional antibacterial and antiviral agents in aqueous or alcoholic solutions to produce disinfecting agents or surgical scrubbers. Branched polycations for use in the invention include, but are not limited to, polypropylene imine, polyethylene imine (PEI) poly (4 '-aza-4' -methylheptamethylene D-glucaramide), polyamidoamines and suitable fragments and / or variants of these compounds. One aspect of the invention is a method for treating objects with a composition characterized by its ability to make the proteins associated with the diseases non-infectious. An advantage of the invention is that proteins, such as prions, can be made non-infectious without the need for extreme conditions, such as exposure to heat for long periods of time, for example from 1 to 10 hours at 100 ° C-200. ° C. A feature of the invention is that the compositions can be useful as long as they contain only very low concentrations of polycytonic dendrimers, for example from 1 percent to 0.001 percent. Another aspect of the invention is that capsules made with bovine gelatin can be certified as prion free. Another aspect of the invention is that drugs produced from cell cultures treated with polycationic dendrimers can be certified as prion free. Yet another aspect of the invention is that medical devices that are reused after exposure to blood or brain tissue can be certified as prion-free. Yet another aspect of the invention is that hospitals, operating rooms, and devices and equipment within them can be certified as prion-free, by contacting them with polycationic dendrimers at standard temperatures and pressures. A pharmaceutical composition for the treatment of the formation of the insoluble protein deposit in an animal, said composition comprising a therapeutically effective amount of a branched polycation; and a pharmaceutically acceptable excipient. In one embodiment, the branched polycation is a branched polymer, and wherein at least one branch is positively charged, and the branched polymer can have multiple charged branches. Branches can have the same chemical structure, or the branches may vary in structure within a single molecule. Examples of the polymers that can be used in this pharmaceutical composition include propyleneimine, polyethylenimine (PEI) poly ('-aza-4' -methylheptamethylene D-glucaramide), polyamidoamines, and pharmaceutically effective variants or fragments thereof. In one embodiment of the pharmaceutical composition, the composition also contains a second therapeutic agent, such as an analgesic agent, an antimicrobial agent, an anti-inflammatory agent, an antioncogenic agent, an antiviral agent, and the like. The present invention also provides a method for improving the release of a disease-related conformation of a protein from the cells, by contacting the cells with a branched polycation for a sufficient time to improve the rate of release of a conformation. related to disease of a protein from cells. This branched polycation can be administered in vivo or ex vivo to a subject, including a human, cow, sheep, deer, dog, cat, goat, chicken, and turkey. Examples of these disease-related proteins include PrPSc, APP, Aβ peptide, anti-chymotrypsin a-1. The method, therefore, is useful for subjects suffering from disorders such as bovine-pongiform encephalopathy, Creutzfeldt-Jakob disease, fatal familial insomnia, GSS due to Gerstmann-Straussler-Scheinker disease, kuru, scrapie, Alzheimer's disease , frontal temporal dementia, Huntington's disease, ALS, Pick disease, Parkinson's disease, Type II diabetes, multiple myeloma, familial amyloidotic polyneuropathy, medullary thyroid carcinoma, chronic renal failure, congestive heart failure, cardiac and systemic senile amyloidosis, inflammation chronic, atherosclerosis, and familial amyloidosis. The branched polycation can be administered to a subject in a non-toxic amount for the subject, for example in a dosage of 0.001 milligrams to 1 milligram / kilogram of body weight per day. The polycation can be administered in a single dosage form, or it can be administered repeatedly to the subject. The branched polycation can also be administered prophylactically to prevent the formation of the disease conformation of these proteins. The invention also provides a food composition for preventing the formation of insoluble protein deposition in an animal, wherein the food contains a therapeutically effective amount of a branched cation that enhances the release of the conformationally altered protein. , The food can be any food product, including solid foods such as meat (for example, beef or lamb), and liquid foods such as vinegar, oil, and seasonings such as meat sauce and ketchup.

The branched polycation is left in contact with the food before it is ingested for a sufficient time to allow the release of the conformationally altered proteins. The present invention also provides a method for preventing a farm animal from acquiring a disease associated with a conformationally altered protein, by feeding the animals with animal feed containing a branched polycation. The food containing the branched polycation can be produced synthetically, and fed directly to the animals, or the branched polycation can be introduced into a natural food source, eg, the animal's feed is straw, and the branched polycation It is sprayed on straw or introduced into the straw through a plant fertilizer. One aspect of this embodiment of the invention is a method for preventing disease caused by the ingestion of contaminated food products, by feeding the animals with foods containing branched polycations. The present invention also provides a method for improving the release of a protein-related conformation of a protein from a meat food product by contacting the meat with a compound that improves the release of the conformationally altered protein to a pH of 5 or less, for a sufficient time to allow the destruction of the conformationally altered protein. An advantage of the invention is that the conformationally altered protein, such as prions, can be made non-infectious with a method that only needs to be to apply a polycationic dendrimer preferably maintained at a pH of 5.0 or less. Another aspect of the invention is soaps, surgical scrubbers, detergents, and the like, with polycationic dendrimers therein. An advantage of the invention is that compositions containing polycationic dendrimers can be used to inactivate prions that could be present in surgical instruments, knives and / or other tools or equipment used by butchers, in particular those used in cutting reces or other animals that could be infected with prions. A feature of the invention is that the compositions of the invention can be effective to activate the prions when the polycationic dendrimers are present in very low concentrations, for example from 1 percent to 0.001 percent or less. These and other aspects, advantages, and features of the invention will become clearer to the skilled artisan after a reading of the de-talles of the compounds, and the assay method more fully described below. .

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES Before the present methods, objects, and compositions are described, it should be understood that this invention is not limited to the particular steps, devices, or components described, and as such, may of course vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, because the scope of the present invention will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in relation to which the publications are cited.

The publications discussed herein are provided exclusively by their disclosure prior to the filing date of the present application. Nothing contained herein should be construed as an admission that the present invention is not entitled to pre-date such publication by virtue of a prior invention. In addition, the publication dates provided may be different from the actual publication dates that may need to be confirmed independently.

DEFINITIONS The term "detergent" is used to mean any substance that reduces the surface tension of water. The detergent can be an agent of superficial activity that concentrates in the interfaces of oil-water, that exerts an emulsifying action, and in this way helps to remove the dirt, for example, the sodium soaps of common fatty acids. A detergent can be anionic, cationic, or non-ionic, depending on its mode of chemical action. Detergents include linear alkyl sulfonates (LAS) often aided by "detergency builders". A linear alkyl sulfonate is preferably an alkyl benzene sulfonate ABS which is readily decomposed by microorganisms (biodegradable). The linear alkyl sulfate is generally a straight chain alkyl comprising from 10 to 30 carbon atoms. The detergent can be in liquid or solid form. The term "conformationally altered protein" is used herein to describe any protein that has a three-dimensional conformation associated with a disease. The conformationally altered protein can cause the disease, it can be a factor in a symptom of the disease, or it can appear as a result of other factors. The conformationally altered protein appears in another conformation that has the same amino acid sequence. In general, the conformationally altered protein has a "narrow" conformation compared to the other "relaxed" conformation that is not associated with the disease. The following is a non-limiting list of diseases with associated proteins that assemble two or more different conformations, wherein at least one conformation is an example of a conformationally altered protein. Disease Insoluble Proteins APP Alzheimer's disease, Aβ peptide, anti-chymotrypsin, tau, non-Aß component, presenilin 1, presenilin 2, apoE Prion diseases, Creutzfeldt-Jakob disease, scrapie and bovine spongiform encephalopathy prpSc ALS SOD and neurofilament Pick disease Pick Parkinson's disease synuclein-a in Lewy bodies Frontotemporal dementia tau in fibrils Diabetes Type II Amylin Multiple myeloma dyscrasias-plasma cell IgGI chain Familial amyloidotic polyneuropathy Transthyretin Medullary thyroid carcinoma Procalcitonin Chronic renal failure ß2 microglobulin Congestive heart failure Natriuretic factor atrial cardiac and systemic senile amyloidosis Transthyretin Chronic inflation Amyloid A serum Atherosclerosis ApoAl Familial amyloidosis Gelsolin Huntington's disease The term "acid" is used to describe any compound or group of compounds having one or more characteristics of: (a) sour taste; (b) converts the red of the litmus dye; (c) reacts with certain metals to form a salt; (d) it reacts with bases or alkalines to form a salt. An acid comprises hydrogen, and in water, it undergoes ionization, in such a way that ions of H30 + are formed - also written as H + and referred to as hydronium ions, or simply as hydrogen ions. Weak acids may be used, such as acetic acid or carbonic acid, as well as strong acids, such as hydrochloric acid, nitric acid, and sulfuric acid. In the compositions of the invention, the acid is preferably present in a concentration to obtain a pH of 5 or less, more preferably of 4 or less, and still more preferably of 3.5 + 1. The terms "sterilize", "render sterile", and the like, are used herein to mean doing non-infectious al-go, or doing something incapable of cause a disease In a specific manner, it refers to making a protein non-infectious or incapable of causing a disease or the symptoms of a disease. Still in a more specific manner, it refers to making a conformationally altered protein (e.g., PrPSc known as prio-nes) be incapable of causing a disease or the symptoms of a disease. "Effective dose" or "effective amount" means an amount of a compound sufficient to provide the desired sterilization result. This will vary depending on factors such as the type of object or material being sterilized, and the amount or concentration of infectious proteins that may be present. The polycations of the invention, or more specifically the polycationic dendrimer compounds of the invention, could be mixed with a material in an amount ranging from 1 to 500 micrograms of dendrimer per milliliter or milligram of material to be sterilized. . The concentration is sufficient if the resulting composition is effective to reduce the in-activity of the conformationally altered proteins, such that the material treated over time does not result in infection. Because (1) some materials will have higher concentrations of altered protein than others, (2) some materials are contacted more frequently than others, and (3) individual proteins have different degrees of infectivity, the dose or the Effective concentration range necessary to sterilize can vary considerably. It is also pointed out that the dose necessary to treat a quantity of material can vary a little, based on the pH at which the treatment is carried out, and in the amount of time in which the compound is kept in contact with the material at the pH level. desired low (for example, 5.0 or less). The term "LD50" as used herein, is the dose of an active substance that will result in a 50 percent lethality in all treated experimental animals. Although this normally refers to invasive administration, such as oral, parenteral, and the like, it can also be applied to toxicity using less invasive administration methods, such as topical applications of the active substance. The term "amine-terminated" includes primary, secondary, and tertiary amines. The terms "PrP protein", "PrP", and the like, are used interchangeably herein, and will mean both the infectious particle form PrPc known to cause the diseases (spongiform encephalopathies) in humans and animals, and the non-infectious form PrPc which, under appropriate conditions, is converted to the infectious form PrPSc. The terms "prion", "prion proteins", "PrPSc protein", and the like, are used interchangeably herein to refer to the infectious PrPSc form of a PrP protein, and it is a contraction of the pala-bras. "protein" and "infection". The particles are largely, if not exclusively, comprised of PrPSc molecules encoded by a PrP gene. Prions are different from bacteria, viruses, and viroids. Known prions infect animals to cause scrapie, a transmissible degenerative disease of the nervous system of sheep and goats, as well as bovine spongiform encephalopathy (BSE), or "mad cow disease," and feline spongiform encephalopathy of cats. . Four prion diseases that are known to affect humans are: (1) kuru, (2) Creutzfeldt-Jakob disease (CJD), (3) Gerstmann-Stráussler-Scheinker disease (GSS), and (4) insomnia fatal family (FFI). As used in this, "prion" includes all forms of prions that cause all or any of these diseases or others in any animals used - and in particular in humans and in domestic farm animals. The term "PrP gene" is used herein to describe the genetic material that expresses the proteins, including known polymorphisms and pathogenic mutations. The term "PrP gene" refers generally to any gene of any species that encodes any form of a prion protein. Some commonly known PrP sequences are described in Gabriel et al., Proc. Na ti. Acad. Sci. USA 89: 9097-9101 (1992) and in U.S. Patent Number 5,565,186 incorporated herein by reference to disclose and describe these sequences. The PrP gene can be from any animal, including the "host" and "test" animals described herein, and any and all polymorphisms and mutations thereof, recognizing that the terms include other PrP genes that are still going to discover. The protein expressed by this gene can assume a PrPc (no disease) or PrPSc (disease) form. The terms "standardized prion preparation", "prion preparation", "preparation", and the like, are used interchangeably herein to describe a composition (e.g., brain homogenate) obtained from the brain tissue of mammals, which exhibits signs of prion disease: the mammal can (1) include a transgene as described herein; (2) having an ablated endogenous prion protein gene; (3) have a high number of prion protein genes from a genetically diverse species; and / or (4) being a hybrid with an ablated endogenous prion pro-gene gene and a prion protein gene from a genetically diverse species. Different combinations of 1 to 4 are possible, for example, 1 and 2. The mammals from which the standardized prion preparations are obtained exhibit clinical signs of central nervous system dysfunction as a result of inoculation with prions, and / or due to the development of the age of their genetically modified formation, for example high copy number of prion protein genes. Standardized prions preparations and methods for making them are described and disclosed in U.S. Patent Nos. 5,908,969 issued June 1, 1999, and in Application Serial Number 09 / 199,523, filed on November 25, 1998, both of which are incorporated herein by reference in their entirety to disclose and describe standardized prion preparations. The term "Alzheimer's disease" (abbreviated herein as "AD"), as used herein, refers to a condition associated with the formation of neuritic plaques comprising β-amyloid protein, primarily in the hippocampus and in the cerebral cortex, as well as the deterioration of both learning and memory. The "Alzheimer's disease", as used herein, encompasses both Alzheimer's disease and Alzheimer's disease type pathologies. The term "Alzheimer's disease type pathology", as used herein, refers to a combination of central nervous system disorders, including, but not limited to, formation of neuritic plaques containing the β-amyloid protein in the hippocampus and in the cerebral cortex. These Alzheimer's disease-type pathologies may include, but are not necessarily limited to, disorders associated with aberrant expression and / or PPP deposition, overexpression of APP, expression of aberrant APP gene products, and other phenomena associated with the disease. of Alzheimer's Exemplary Alzheimer's disease type pathologies include, but are not necessarily limited to, the Alzheimer's disease type pathologies associated with Down syndrome that is associated with overexpression of APP. The term "phenomenon associated with Alzheimer's disease", as used herein, refers to a structural, molecular, or functional event associated with Alzheimer's disease, in particular an event that can be easily evaluated in an animal model. These events include, but are not limited to, amyloid deposition, neuropathological developments, learning and memory deficits, and other features associated with Alzheimer's disease. The term "cerebral amyloid angiopathy" (abbreviated herein as CAA), as used herein, refers to a condition associated with the formation of amyloid deposition within cerebral vessels, which may be complicated by cerebral parenchymal hemorrhage. Cerebral amyloid angiopathy is also associated with the increased risk of embolism, as well as the development of cerebellar and subarachnoid hemorrhages (Vinters (1987) Stroke 18: 311-324; Haan et al. (1994) Dementia 5: 210-213; Itoh and collaborators (1993) J. Neurol, Sci. 116: 135-414). Cerebral amyloid angiopathy may also be associated with dementia before the establishment of hemorrhages. Vascular amyloid deposits associated with cerebral amyloid angiopathy may exist in the absence of Alzheimer's disease, but are more often associated with Alzheimer's disease. The term "phenomenon associated with cerebral amyloid angiopathy", as used herein, refers to a molecular, structural, or functional event associated with cerebral amyloid angiopathy, in particular such an event that can be easily evaluated in an animal model. These events include, but are not limited to, amyloid deposition, cerebral parenchymal hemorrhage, and other features associated with cerebral amyloid angiopathy. The term "β-amyloid deposit", as used herein, refers to a deposit in the brain composed of Aβ, as well as other substances.

Abbreviations used herein include: CNS for central nervous system; BSE for bovine spongiform encephalopathy; CJD for Creutzfeldt-Jakob disease; FFI for fatal familial insomnia; GSS for Gerstmann-Stráussler-Scheinker disease; AD for Alzheimer's disease; CAA for cerebral amyloid angiopathy; Hu for human; HuPrP for human prion protein; Mo for mouse; MoPrP for mouse prion protein; SHa for Syrian Hamster; SHaPrP for a Syrian hamster prion protein; PAMAM for polyamidoamide dendrimers; PEI for polyethyleneimine; PPI for polypropylene imine; PrP sc for the scrapie isoform of the prion protein; PrPL for the common normal isoform contained in the prion protein cell; PrP 27-30 or PrPSc 27-30 for the treatment or the protease resistant form of PrPSc; MoPrPsc for the scrapie isoform of the mouse prion protein; N2a for a neuroblastoma cell line established in the present studies; ScN2a for a cell line of neuroblastoma chronically infected with scrapie; ALS for amyotrophic lateral sclerosis; HD for Huntington's disease; FTD for frontotemporal dementia; SOD for superoxide dismutase; GENERAL ASPECTS OF THE INVENTION The invention comprises compositions of compounds found to be effective in making the conformationally altered proteins non-infectious. The compositions are preferably low pH solutions comprised of a non-toxic weak acid, such as acetic acid, which has a branched polycation dissolved therein. Preferred compositions of the invention are in the form of aqueous or alcoholic solutions which are comprised of a branched polycation, an antibacterial compound, an antifungal compound, and an antiviral compound. The compositions are coated on, mixed with, injected into, or otherwise contacted with, a material to be sterilized. The composition is applied in such a way that the branched polycation is maintained at a low pH (eg, 5 or less, and preferably 3.5 + 1) in an amount of 1 microgram or more polycation per milliliter or mi-ligrame of material that is going to be sterilized. The composition is maintained in the desired pH range at a normal temperature (for example, 15 ° C to 30 ° C) for a sufficient period of time (for example 1 hour to a week) to make the protein conformationally altered present on or within the material is destroyed (eg, hydrolyzed) or becomes noninfectious. Preferred compositions of the invention are useful in cleaning and sterilization, and may be comprised of polycationic dendrimers, a detergent, and an acid having a pH of about 3.5 + 1.

COMPONENTS OF DENDRIMERS THAT CLEAN THE PRIONS Dendrimers are branched compounds also known as "star explosion" or "star" polymers due to a characteristic star-type structure (see Figure 1). The dendrimers of the invention are polymers with structures constructed from monomers ABn, with n > 2, and preferably n = 2 or 3. These dendrimers are highly branched, and have three distinct structural features: 1) a core, 2) multiple peripheral end groups, and 3) branching units that link the two. The dendrimers can be cationic (full-generation dendrimers) or anionic (half-generation dendrimers). For a review on the general synthesis, physical properties, and applications of dendrimers, see, for example, Tomalia et al., Angew. Chem. Int. Ed. Engl. , 29: 138-175, (1990), Y. Kim and C. Zimmerman, Curr Opin Chem Biol, 2: 733-7421 (1997). In a preferred embodiment, the sterilizing compositions of the invention comprise a cationic dendrimer preferably dissolved in a low pH solvent, such as acetic acid. Examples of suitable dendrimers are disclosed in U.S. Patents Nos. 4,507,466; 4,558,120; 4,568,737; 4,587,329; 4,631,337; 4,694,064; 4,713,975; 4,737,550; 4,871,779 and 4,857,599 to D.A. Tomalia et al., Which are incorporated herein by reference to make known and described such compounds. Dendrimers usually have tertiary amines that have a pKa of 5.7. The dendrimers can optionally be treated chemically or with heat to remove some of the tertiary amines. Other suitable cations include polypropylene imine, polyethyleneimine (PEI), having tertiary amines with a pKa of 5.9, and poly (4'-aza-4 '-methylheptamethylene D-glucaramide), having tertiary amines with a pKa of 6.0. The cationic dendrimer is preferably dissolved in the low pH solvent, such as vinegar, in a concentration of 0.0001 percent or more, preferably 0.01 percent or more, and more preferably about 1 percent. Preferably, the dendrimers for use in the invention are polyamidoamines (hereinafter referred to as "PAMAM"). PAMAM dendrimers are particularly biocompatible, because the polyamidoamine groups resemble the peptide bonds of the proteins. The dendrimers are prepared in rows called generations (see generations 0, 1, and 2 in Figure 1), and therefore, have specific molecular weights. The full-generation PAMAM dendrimers have amine end groups, and are cationic, while the half-generation dendrimers are carboxyl terminated. Accordingly, full generation PAMAM dendrimers are preferred for use in the present invention. PAMAM dendrimers can be prepared with different molecular weights, and have specific values, as described in the following Table 1 for generations 0 to 10.

TABLE A LIST OF DENDRIMEROS PAMAM AND ITS MOLECULAR WEIGHTS (Ethylene-diamine nucleus, finished in amine). Generation End groups Molecular weight, g / mol 0 4 517 1 8 1430 2 216 3256 3 32 6909 4 64 14,215 5 128 28,795 6 256 58,048 7 512 116,493 8 1024 233,383 9 2048 467,162 10 4096 934,720 As shown in Table A the number of terminal amine groups for generations 0 to 10 of the PAMAM dendrimers is from 4 to 4,096, with molecular weights from 517 to 934,720. PAMAM dendrimers are commercially available from Aldrich or Dendritech. The polyethyleneimine or polypropylene dendrimers, or the quaternized forms of the amine terminated dendrimers, can be prepared as described by Tomalia et al., Angew, Chem. In t. Ed. Engl. , 29: 138-175 (1990), incorporated as reference to describe and make known the methods to make dendrimers.

STERILIZING COMPOSITIONS The examples provided herein show that highly branched polycations, for example dendrimer compounds, affect the extent and distribution of PrPSc protein deposits in scrapie infected cells. The presence of dendrimers in a low pH environment and at relatively low non-cytotoxic levels results in a significant reduction in the detectable PrPSc in brain cells and homogenates. Accordingly, the present invention encompasses compositions for reducing, inhibiting, or otherwise mitigating the degree of infectivity of a protein. A composition of the invention is comprised of any compound capable of destroying conformationally altered proteins when they are in a low pH environment (eg, a polycationic dendrimer) in solution, suspension, or mixture.

STERILIZING FORMULATIONS The sterilizing compositions of the invention preferably contain highly branched polycations, for example polycationic dendrimer, in a concentration of 0.0001 to 10 percent of the formulation. The following methods and excipients are purely exemplary, and are in no way limiting. In addition to including the compound, such as a highly branched cationic compound, in formation, it is important to maintain that compound in a low pH environment. Any number of known acids or mixtures of acids with the invention could be used. Non-limiting examples of the commercially available products that could be used to supplement the cationic compounds are described below. In these formulations, the percentage amount of each ingredient can vary. In general, a solvent ingredient (for example water or alcohol) is present in amounts of 40 percent to 100 percent, and the last ingredient listed is present in a range of 0.5 percent to 5 percent. The other ingredients are present in an amount ranging from 1 percent to 60 percent, and more generally from 5 percent to 20 percent. In each case, the polycationic compounds of the invention are added in amounts of about 0.01 percent to 5 percent, and preferably 0.1 percent to 2 percent, and more preferably about 1 percent. The amount added is the amount needed to obtain the desired effect.

By utilizing the disclosure provided herein and other information, as taught in U.S. Patent Nos. 5,767,054; 6,007,831; 5,830,488; 5,968,539; 5,416,075; 5,296,158; and in the patents and publications cited therein, those skilled in the art can produce other in-countable formulations of the invention. In addition, these formulations can be used as described in those publications, and can be packaged in any suitable container or dosing device, for example as taught in U.S. Patent No. 5,992,698.

The formulations of the invention used with a cell culture have the advantage that they are non-toxic. For example, parenteral administration of a solution of the formulations of the invention is preferably non-toxic at a dosage of 0.1 milligrams / mouse, which is an LD50 less than 1 to 40 milligrams / kilogram. Different formulations of nutrients and / or injectable formulations of a type known to those skilled in the art can be used to prepare the formulations for the treatment of cell cultures. Those skilled in the art will understand that, in some situations, it may be desirable to further reduce the pH environment to obtain the desired results. This can be done by the addition of any desired acid. If desired, the pH can be raised to a normal level after finishing the treatment, i.e., after a sufficient amount of any conformationally altered protein present is destroyed. The effective compounds in the sterilizing compositions containing conformationally altered proteins are determined by means of a cell culture assay and an organ homogenate assay, each of which is described in detail below.

SCN2a-based cell assay Efforts were made to optimize the transfection of ScN2a cells with pSPOX expression plasmids (Scott, MR, Kohier, R., Foster, D. and Prusiner, Chimeric prion protein expression in cultured cells and transgenic mice, Protein Sci. 1, 986-997 (1992)). In relation to these effects, an evaluation of a transfection protocol was made using the SuperFect reagent (QIAGEN®). It was found that epitope-tagged PrPSc (MHM2) could not be detected (Scott, MR, Kohier, R., Foster, D. and Prusiner, SB Chimeric prion protein expression in cultured cells and transgenic mice, Protein Sci. 1, 986 -997 (1992)) in ScN2a cells following SuperFect-mediated transfection, whereas PrPSc MHM2 was efficiently formed when a cationic liposome method was used for DNA delivery. Close scrutiny revealed that, before protease digestion, samples transfected with SuperFect expressed MHM2 bands, which are not seen in the background pattern of a non-transfected sample. The monoclonal antibody 3F4 does not react with MoPrP, but exhibits a high background staining in Western blots of mouse ScN2a cells. An increase in immunostaining was observed in the 20-30 kDa region, compared to the untransfected sample. These observations led us to conclude that PrP MHM2 was expressed successfully using the SuperFect transfection reagent, but that the conversion of PrPc MHM2 to the protease resistant PrPSc MHM2 was inhibited by SuperFect. To investigate this apparent inhibition, a Western blot was re-probed with R073 polyclonal antiserum to detect endogenous MoPrPSc, whose presence is diagnostic for prion infection in ScN2a cells (Butler, DA, et al., Scrapie-infected murine neuroblastoma. cells produce protease resistant prion proteins, J. Virol. 62, 1558-1564 (1988)). Surprisingly, it was found that ScN2a cells treated with SuperFect no longer contained detectable amounts of MoPrPSc - also confirmed in Western blots. To investigate the mechanism by which the SuperFect reduced the level of 1 PrPSc previously existing in chronically infected ScN2a cells, endogenous PrPSc measurements were made in ScN2a cells exposed to different concentrations of SuperFect in the absence of the plasmid DNA. The results showed that treatment with SuperFect (a branched polycation) caused the disappearance of PrPSc from ScN2a cells in a dose-dependent manner. It was found that the concentration of SuperFect required to eliminate the > 95 percent of the previously existing PrPSc with a 3-hour exposure, is approximately 150 micrograms / milliliter. The duration of the treatment also influenced the ability of the SuperFect to remove the PrPSc from the ScN2a cells: exposure to 150 mi-crograms / milliliter of SuperFect for 10 minutes did not affect the PrPSc levels, while 7.5 micrograms / milliliter of SuperFect eliminated all detectable PrPSc with a tl / 2 = 8 hours. SuperFect is a mixture of branched polyamines derived from the heat-induced degradation of a PAMAM dendrimer (Tang, MX, Redemann, CT and Szoka, FCJ In vitro gene delivery by degraded polyamidoamine dendrimers, Bioconjug Chem 7, 703-714 (nineteen ninety six)). Knowing this structure, we can see the capacity of other different branched and unbranched polymers to eliminate PrPSc from ScN2a cells (Table 1). The investigated branched polymers include different preparations of PEI, as well as intact PAMAM and PPI dendrimers. Dendrimers are manufactured by a repetitive divergent growth technique, which allows the synthesis of well-defined successive "generations" of homodisperse structures (Figure 1). The potency of both PAMAM and PPI dendrimers to remove PrPSc from ScN2a cells increased as the generation level increased. The most potent compounds with respect to PrPSc elimination were PAMAM generation 4.0, and PPI generation 4.0, while PAMAM generation 1.0 showed very little ability to eliminate PrPSc (Table 1). In a similar manner, a high molecular weight fraction of PEI was more potent than the low molecular weight PEI.

From the above data, it is clear that, for the three branched polyamines tested, the increase in molecular size corresponded to a higher power to eliminate the PrPSc. To determine if this trend could be directly attributed to the higher surface density of the amino groups on the larger molecules, PAMAM-OH generation 4.0 was tested. This is a dendrimer that looks like PAMAM generation 4.0, except that hydroxyl substitutes amino groups on its surface. Unlike generation 4.0 PAMAM, generation 4.0 PAMAM-OH did not cause a reduction in PrPSc levels, even at the highest tested concentration (10 milligrams / milliliter), establishing that amino groups are required for the elimination of PrPSc by PAMAM (Table 1). In an effort to evaluate the contribution of the branched architecture to the release capacity of the polyamines for PrPSc, the linear molecules of poly- (L) lysine in linear PEI were also tested. Both of these linear compounds were less potent than a branched PEI preparation with a similar average molecular weight (Table 1), establishing that a branched molecular architecture optimizes the ability of polyamines to eliminate PrPSc, presumably because the branched structures reach a higher density of the super-ficial amino groups.

Kinetics of the elimination of PrPSc by polyamines. The above results demonstrate the potent ability of the branched polyamines to release PrPSc from ScN2a cells within a few hours of treatment. The utility of these compounds to act as therapeutics for the treatment of prion disease was tested by determining whether they were cytotoxic for ScN2a cells, using the criteria of cell growth, morphology, and viability, measured by dyeing with blue trypan. None of the compounds was cytotoxic to ScN2a cells after exposure for one week at concentrations up to 7.5 micrograms / milliliter. To determine whether the branched polyamines can cure ScN2a cells from scrapie infection without affecting cell viability, the prion release kinetics were examined in the presence of a non-cytotoxic concentration (7.5 micrograms / milliliter) of 3 different branched polyamines . The ScN2a cells were exposed to SuperFect, PEI, or PAMAM generation 4.0 for different periods of time. The kinetics of elimination of PrPSc was evaluated by Western blot. All three compounds caused a substantial reduction in PrPSc levels after 8 to 16 hours of treatment, and of the three compounds, PEI appeared to remove PrPSc more rapidly, with tl / 2 = 4 hours.

Curing neuroblastoma cells of scrapie infection The above results show that it is possible to reverse the accumulation of PrPSc in ScN2a cells under non-cytotoxic conditions. It was also found that prolonged exposure to even lower levels of the branched polyamines (1.5 micrograms / milliliter) was insufficient to eliminate the PrPSc. Based on these findings, this protocol was used to determine if the severe reduction in PrPSc levels would persist following exposure to branched polyamines after the removal of the compounds. Following the exposure of the ScN2a cells at 1.5 micrograms / milliliter of SuperFect for one week, the PrPSc was reduced to < 1 percent of the baseline level, but then increased back to approximately 5 percent of the baseline level after 3 additional weeks in culture in the absence of polyamine. You found, after exposure to 1.5 micrograms / milliliter of PEI or PAMAM generation 4.0 for one week, the PrPSc was completely eliminated, and did not return even after 3 weeks in cultures without polyamines. A more intensive course of treatment with 1.8 micrograms / milliliter of SuperFect for 9 days also cured ScN2a cells from scrapie infection completely, manifested by the absence of PrPSc 1 month after removing the SuperFect.

Evidence of polyamines acting within an acidic compartment The above results showed the potent activity of the branched polyamines to rapidly release the scrapie prions from the cultured ScN2a cells. Based on these results, the mechanism by which these compounds act was investigated. All compounds that effect the removal of PrPSc from ScN2a cells are known to be trafficked through endosomes (Boussif, 0., and co-workers, A versatile vector for gene and oligonucleotide transfer into cells in culture and in vi tro : polyethyleneimi-ne. Proc. Na ti. Acad. Sci. USA 92, 7297-7301 (1995); Haens-ler, J. and Szoka, FCJ Polyamidoamine cascade polymers by efficient transfection of cells in culture Bioconjug Chem. 4, 372-379 (1993)). Because PrPc becomes PrPSc in the caveolar-like domains (CLDs) or rafts (Go-rodinsky, A. and Harris, DA Glycolipid-anchored proteins in neuroblastoma cells form detergent-resistant complexes without caveolin J. CeJl Biol 129, 619-627 (1995); Taraboulos, A., et al Cholesterol depletion and modification of COOH-terminal targeting sequence of the prion protein inhibits formation of the scrapie isoform J. Cell Biol. 129, 121-132 (1995); Vey, M. et al Subcellular colocali-zation of the cellular and scrapie prion proteins in caveo-lae-like membranous domains Proc. Na ti.Acid Sci. USA 93, 14945-14949 (1996); Kaneko, K. and collaborators, COOH-terminal sequence of the cellular prion protein directs subcellular trafficking and controls conversion into the scrapie isoform, Proc. Na ti, Acad. Sci. USA 94, 2333-2338 (1997)), and then internalized through the endocytic trajectory (Caughey, B., Raymond, GJ, Ernst, D and Race, RE N-terminal trunca-tion of the scrapie-associated form of PrP by lysosomal protease (s): implications regarding the site of conversion of PrP to the protease-resistant state. J. Virol 65, 6597-6603 (1991); Borchelt, D.R., Taraboulos, A. and Prusiner, S.B. Evidence for synthesis of scrapie proteins in the endo-cytic pathway. J. Biol. Chem. 267, 16188-16199 (1992)), it was deduced that the polyamines act on the PrPSc in the endosomes or lysosomes. This deduction was investigated by determining the effect of previous treatment with lysosomotropic agents chloroquine and NH4C1 on the ability of polyamines to eliminate PrPSc. These lysosomotropic agents alkalize the endosomes and have no effect on the levels of PrPSc when administered to ScN2a cells (Tarabou-los, A., Raeber, AJ, Borchelt, DR, Serban, D and Prusiner, SB Synthesis and trafficking of prion proteins in cultured cells, Mol. Biol. Cell 3, 851-863 (1992)). The experimental results obtained show that 100 μM of chloroquine, but not 30 μM of NH4C1, blocked the ability of the PEI to eliminate the PrPSc. Similar results were obtained with Su-perFect and PAMAM generation 4.0. Although the failure of NH4C1 to affect PrPSc is not easily explained, the ability of chloroquine to attenuate the ability of branched polyamines to remove PrPSc is consistent with the notion that these agents act on endosomes or lysosomes.

TESTING OF ORGAN HOMOGENATE The previous results with cell cultures promoted the investigation of the possibility that, in an acidic environment, the branched polyamines, either through their indirect interaction with the PrPSc or with another cellular component, could cause PrPSc become susceptible to the hydrolases present in the endosome / lysozyme. An in vi tro degradation assay was developed to evaluate the effect of pH on the ability of the polyamines to make the PrPSc sensitive to the protease. Crude homogenates of scrapie-infected mouse brain were exposed to a wide range of pH values in the presence or absence of SuperFect, and then treated with proteinase K before the Wes-tern spot. Although PrPSc remained resistant to protease hydrolysis throughout the pH range (3.6-9.6) in the absence of SuperFect, the addition of the branched polyamine at a pH of 4.0 or less caused the PrPSc to become almost completely degraded by the protease. The addition of polyamine showed a dramatic effect on in vitro release, which was optimized at a pH of 4 or less. These results show that the polyamines act on the PrPSc in an acid compartment. To establish that the in vitro degradation assay is a valid approximation of the mechanism by which the branched polyamines improve the release of PrP? A from the cultured cells, an analysis of structure activity was performed with several of the compounds tested in the culture cells. An excellent correlation was found between the release of PrPSc in the cultured ScN2a cells (Table 1) and the ability to render PrPSc susceptible to the protease at an acidic pH in vi tro. Notably, the PAMAM-OH generation 4.0 failed to make the PrPSc susceptible to the protease, while the PAMAM generation 4.0 and the PPI generation 4.0 exhibited an even stronger activity than the SuperFect in vi tro, as expected by its observed power in the cultured ScN2a cells (Table 1).

MECHANISM OF ACTION The results discussed herein show that certain branched polyamines cause rapid elimination of PrPSc from ScN2a cells in a dose and time dependent manner. These compounds demonstrate a potent ability to remove prions from cells grown in concentrations that are completely non-cytotoxic. The cells can be maintained indefinitely in culture in the presence of therapeutic levels of branched polyamines. In addition, when the ScN2a cells were exposed to these compounds for about a week, PrPSc was reduced to undetectable levels, and remained so for at least one month after removing the polyamine. The clarification of the exact mechanism of elimination of PrPSc by branched polyamines is an important objective. Although there are a number of possible scenarios, several possibilities can already be excluded. One possibility that was eliminated was that the polyamines act by induction of chaperones, such as heat shock proteins that mediate the retraction of the prion protein because the previous results show that it was possible to reproduce the phenomenon in vitro. In addition, polyamines seem to offer advantages over other supposed therapies that seek to promote retraction: in very high concentrations, in dimethyl sulfoxide (DMSO) and glycerol act as direct "chemical chaperones", and inhibit the formation of new PrPSc ( Tatzelt, J., Prusiner, SB and Welch, WJ Chemical chaperones interfere with the formation of scrapie prion protein EMBO J. 15, 6363-6373 (1996)), but these compounds can not reduce the previously existing levels of PrPSc. In addition, polyamines inhibit the formation of PrPSc at concentrations much lower than these agents. The ability of the polyamines to effect the rapid release of PrPSc also contrasts with the activity of other potential prion therapeutics. Sulphated polyanions can inhibit the accumulation of PrPSc in ScN2a cells by direct binding to PrPc (Gabizon, R., Meiner, Z., Halimi, M. and Ben-Sasson, SA Heparin-like molecules bind differentially to prion -proteins and change their intracellular metabolic fate, J. Cell, Physiol, 157, (1993), Caughey, B., Brown, K., Raymond, GJ, Katzenstein, GE and Thresher, W. Binding of the protease-sensitive form of PrP (prion protein) to sulfa-ted glycosaminoglycan and Congo red J. Virol 68, 2135-2141 (1994)), but because the branched polyamines are capable of releasing the previously existing PrPSc, its mechanism of action can not simply involve the link with PrPc and the inhibition of de novo synthesis. Another possible mechanism that can be excluded is the endosomal break. The branched polyamines that were effective in releasing the PrPSc from ScN2a cells in our experiments, PEI, SuperFect and PAMAM, are also potent osmotic lysomotropic agents, which can swell in acid environments and break endosomes (Boussif, O., and collaborators, A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: po-lyethyleneimine, Proc. Na ti.Acid Sci. USA, 92, 7297-7301 (1995); Haensler, J. and Szoka, FCJ Polyamidoamine cascade polymers mediate efficient transfection of cells in culture, Bioconjug Chem. 4, 372-379 (1993)). This could suggest that the branched polyamines release the PrPSc from the ScN2a cells breaking the endosomes and exposing the PrPSc to the processes of cytosolic degradation. However, it is known that endosome cleavage lysomotropic agents NH4C1, chloroquine, and monensin do not interfere with the formation of PrPSc in ScN2a cells (Taraboulos, A., Raeber, AJ, Borchelt, DR, Serban, D. Prusiner, SB Synthesis and trafficking of prion proteins in cultured cells, Mol. Biol. Cell 3, 851-863 (1992)). In addition, the results also show that chloroquine interferes with the ability of branched polyamines to release PrPSc, and that polyamines can release PrPSc in vi tro at an acidic pH in the absence of cell membranes. Together, these observations eliminate endosome disruption as the mechanism by which branched polyamines remove PrPSc. Without committing to any particular mechanism of action, it seems possible that the branched polyamines require the acidic environment of intact endosomes or lysosomes to destroy the PrPSc. The activity-structure profile of the tested polymers reveals that most of the active compounds have densely packed and regularly separated amino groups, suggesting that these compounds can be bound to a ligand having periodically separated negative charges. Several scenarios are still possible. (1) The branched polyamines can be fixed directly to the PrPSc arranged as an amyloid with negatively charged fractions exposed, and induce a conformational change under acidic conditions. (2) The treatment of PrP 27-30 with acid reduces turbidity and increases the helical-a-content, suggesting that these conditions could dissociate the PrPSc in monomers (Safar, J., Roller, PP, Gajdusek, DC and Gibbs , CJ, Jr. Scrapie amyloid (prion) protein has the conformational characteristics of an aggregate molten globu-le folding intermediate). Accordingly, it is possible for the polyamines to bind to an equilibrium display intermediate of PrPSc present under acidic conditions. (3) Alternatively, the polyamines could sequester a negatively charged cryptic component bound to the PrPSc that is essential for the protease resistance, but that is only released when the PrPSc undergoes an acid-induced conformational change. This component could act as a chaperone for the internal endosomes or lysosomes of PrP? C. (4) Finally, another possibility is that the polyamines activate an endosomic or lysozomic factor that can induce a conformational change in the PrPSc. Clearly, more work will be required to determine the precise mechanism by which the branched polyamines destroy the PrPSc.

GENERAL APPLICABILITY OF THE TEST The in vitro assay described here is generally applicable in the search for compounds that effectively release the conformationally altered proteins present in the food, thereby preventing a number of degenerative diseases, where the accumulation of proteins appears mediate the pathogenesis of these diseases. By simulating lysosomes, where proteases hydrolyze proteins under acidic conditions, the brain in vitro homogenate assay is able to quickly assess the efficacy of a variety of polyamines to induce the degradation of PrPSc. The in vi tro assay that used the scrapie-infected brain homogenate to test the compounds that release the PrPSc could be modified to test compound to release any conformationally altered protein. The assay is carried out by homogenizing the organ or tissue where the conformationally altered protein is present at the highest concentration. The pH of the homogenate is then reduced to less than 5.0, and preferably to 4.0 or less. For example, the pancreatic tissue can be homogenized to produce an assay to test compounds that release amylin, which is associated with type II diabetes. A homogenized kidney could be used to test compounds that release ß2 microglobulin and ho-mogeneized heart or vascular tissue used to test compounds that release atrial natriuretic factor. Those skilled in the art will recognize that other organs and tissue types can be homogenized to test other compounds that release other conformationally altered proteins. In addition to using the in vi tro assay to screen for potential drugs, compounds found by the assay, such as branched polyamines, provide a new tool for exploring the conversion of a protein to a conformationally altered protein, for example PrPc in PrPSc. The mechanism by which the branched polyamines make the PrPSc susceptible to proteolysis still needs to be established. It is unknown whether the interaction of the branched polyamines with PrPSc is reversible. In addition, we do not know if the branched polyamines are capable of solubilizing the PrPSc without irreversibly denaturing the protein. Whatever the mechanism by which branched polyamines interact with PrPSc, it may be different from that found with chaotropes, as well as with detergents and denaturing solvents (Prusiner SB, Groth, D., Serban, A., Stahl , N. and Gabizon, R. Attempts to restore scrapie prion infectivity after exposure to protein denaturants, Proc Na ti. Acad. Sci. USA 90, 2793-2797 (1993)). Using the assays described and disclosed herein, certain specific branched polyamines that mediate the release of PrPSc from cells grown under non-cytotoxic conditions have been found. These compounds offer the intriguing possibility of adding to a wide range of low pH food products to neutralize the conformationally altered proteins present. Because the compounds found act by stimulating the normal cellular trajectories of protein degradation to destroy PrPSc, this class of compounds would also possibly be valuable in the treatment of other degenerative and inherited disorders where abnormally folded proteins are accumulated, of wild type, or mutants. This approach may find merit in the development of effective therapy for one or more of the common degenerative diseases, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, frontotemporal dementia, adult diabetes mellitus, and amyloidosis (Beyreuther, K and Masters, CL Serpents on the road to dementia and death Accumulating evi-dence from several studies points to the normal function of presenilin 1 and suggests how the mutant protein contributes to deposition of amyloid plaques in Alzheimer's disease Na ture Medicine 3, 723 -725 (1997); Masters, C.L. and Beyreuther, K. Alzheimer's desire. BMJ 316, 446-448 (1998); Selkoe, D.J. The cell biology of beta-amyloid precursor protein and prese-nilin in Alzheimer's disease. Trends in Cell Biol. 8, 447-453 (1998); Selkoe, D.J. Translating cell biology into therapeutic advances in Alzheimer's disease. Na ture 339, A23-31 (1999); Wong P.C. and collaborators. An adverse property of a familial AlS-linked SODl mutation causes motor neuron disease characterized by vacuolar degeneration of mitochondria. Neuron 14, 1105-1116 (1995); Spillantini, M.G., Growther, R.A., Jakes, R., Hasegawa, M. and Goedert, M. a-Synuclein in filamen-tous inclusions of Lewy bodies from Parkinson's disease and dementia with Lewy bodies. Proc. Na ti. Acad. Sci. USA 95, 6469-6473 (1998); Hutton, M. et al. Association of Missense and 5 '-splice-site mutations in tau with the inheritance-dementia FTDP-17. Na ture 393, 702-705 (1998); Stone, M.J. Amyloidosis: a final common pathway for protein deposition in tissues. Blood 75, 531-545 (1990)). It remains to be established whether branched polyamines could also be effective in a variety of inherited disorders, where the accumulation of abnormal proteins is a hallmark of the disease; These genetic diseases include the inherited forms of prion disease, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, frontotemporal dementia, Pick's disease, and amyloidosis, as well as triple-recurrent diseases, including Huntington's disease, cerebellar spinal ataxias. , and myotonic dystrophy (Fu, Y.-H., and collaborators.An unstable triplet repeat in a gene related to myotonic muscular dystrophy.Science 225, 1256-1259 (1992); Group, THsDCR A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes, Cell 72, 971-983 (1993)). The compounds identified by the assays of the invention, such as the branched polyamines, will find utility in preventing or delaying the establishment of these genetic diseases, where carriers with decades of anticipation to detectable neurological or systemic dysfunction can often be identified. The invention is based on the discovery that surprisingly it was found that several dendritic polycations, including the narrow dendrimers Superfect ™ (QIAGEN®, Valencia, CA), polyamidoamide (PAMAM), and the hyper-branched polycation-polyethyleneimine (PEI) , eliminate PrPSc from cultured scrapie-infected neuroblastoma cells. These highly branched polycationic compounds provide a novel class of therapeutic agents to combat prion diseases and other degenerative diseases, including amyloidosis. The removal of PrPSc depends on both the concentration of the dendritic polymer and the duration of the exposure. The dendritic polymers were able to release the PrPSc at concentrations that were not cytotoxic. Repeated exposures to PAMAM dendrimer of heat degraded star or PEI caused a dramatic reduction in PrPSc levels, which persisted for a month even after removing the compound. The dendritic polycations did not appear to destroy the purified PrPSc in vitro, and therefore, can act through a generalized mechanism. Dendritic polycations represent a class of compounds that can be used as therapeutic agents in prion diseases and other disorders involving insoluble protein deposits, such as amyloidosis.

EXAMPLES The following examples are provided to provide ordinary experts in the art with a disclosure and complete description of the manner of making and using the present invention, and are not intended to limit the scope of what the inventors consider to be their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to the numbers used (eg, quantities, temperature, etc.), but some experimental errors and deviations must be counted. Unless indicated otherwise, the parts are parts by weight, the molecular weight is the weight average molecular weight, the temperature is in degrees centigrade, and the pressure is at or near atmospheric.

METHODS AND MATERIALS Chemical products. The high molecular weight PEI was purchased from Fluka. The DOTAP cationic lipid was purchased from Boehringer Mannheim, and the SuperFect transfection reagent was purchased from QIAGEN®. All other compounds were purchased from Sigma-Aldrich. All the test compounds were dissolved in water at a supply concentration of 3 milligrams / milliliter, and filtered through a Millipore filter of 0.22 millimeters.

Cultured cells. The cultures of ScN2a cells were maintained in MEM with 10% FBS, 10% Glutamax (Gibco BRL), 100 units penicillin, and 100 milligrams / milliliter streptomycin (DME complemented). Immediately before the addition of the test compounds, the dishes were washed twice with the fresh supplemented DME medium. After exposure to the test compounds, the dishes were drained from the medium, and the cells were harvested by lysis at 0.25-1 ml-liter of 20 mM Tris, pH 8.0, containing 100 mM NaCl, 0.5 N-40 percent, and 0.5 percent sodium deoxycholate, to obtain a total protein concentration of 1 milligram / milliliter, as measured by the BCA assay. The nuclei were removed from the lysate by centrifugation at 2,000 rpm for 5 minutes. For the samples not treated with proteinase K, 40 microliters of the whole lysate (representing 40 micrograms of total protein) were mixed with an equal volume of 2x SDS reducing sample regulator. For digestion with proteinase K, 20 micrograms / milliliter of proteinase K (Boehringer Mannheim) (total protein: enzyme = 50: 1) was added, and the sample was incubated for 1 hour at 37 ° C. The proteolytic digestion was terminated by the addition of Pefabloc to a final concentration of 5 mM. Samples of 1 milliliter were centrifuged at 100,000 x g for 1 hour at 4 ° C, the supernatants were discarded, and the pellets were resuspended in 80 micro-liters of SDS-reducing sample regulator for SDS-PAGE. Brain homogenates Brain homogenates were prepared from CD-1 mice affected with scrapie RML (10 percent (w / v) in sterile water), by repeated extrusion through syringe needles of a successively smaller size, from an 18 gauge. to a 22 gauge. Kernels and debris were removed by centrifugation at 1,000 xg for 5 minutes. The bicinchoninic acid protein assay (BCA) (Pierce) was used to determine the protein concentration. The homogenates were adjusted to 1 milligram / milliliter of protein in 1 percent NP-40. For the reactions, 0.5 milliliters of homogenate were incubated with 25 milliliters of 1.0 M regulator (sodium acetate for a pH of 3 to 6, and Tris acetate for a pH of 7 to 10) plus or minus 10 milliliters. of polyamine supply solution (3 milligrams / milliliter) for 2 hours at 37 ° C with constant agitation. The final pH value of each sample was measured directly with a calibrated pH electrode (Radiometer Copenhagen). Following the incubation, each sample was neutralized with an equal volume of 0.2 M HEPES, pH 7.5, containing 0.3 M NaCl and 4 percent Sarkosyl. Proteinase K was added to reach a final concentration of 20 micrograms / milliliter, and the samples were incubated for 1 hour at 37 ° C. The proteolytic digestion was terminated by the addition of Pefabloc to a final concentration of 5 μM. 10 microliters of digested brain homogenate were mixed with an equal volume of SDS 2x sample buffer, and analyzed by SDS-PAGE, followed by Western blot. Western blotch Following the electrophoresis, the Western blot was performed as described previously (Scott M. et al, Transgenic mice expressing hamster prion pro-tein produces species-specific scrapie infectivity and amyloid plaques Cell 59, 847-857 (1989)). Samples were boiled for 5 minutes, and cleaned by centrifugation for 1 minute at 14,000 rpm in a Beckman ultracentrifuge. SDS-PAGE was performed on 12-percent polyacrylamide gels, 1.5 millimeters (Laemmli, UK Cleavage of structu-ral proteins during the assembly of the head of bacteriophage T-4, Na ture 227, 680-685 (1970) ). The membranes were blocked with 5 percent defatted milk protein in PBST (PBS free of calcium and magnesium plus 0.1 percent Tween 20) for 1 hour at room temperature. Blocked membranes were incubated with primary R073 polyclonal antibody (to detect MoPrP) (Serban, D., Taraboulos, A., DeArmond, SJ and Prusiner, SB Rapid detection of Creutzfeldt-Jakob disease and scrapie prion proteins.) Neurology 40, 110 -117 (1990)) or monoclonal antibody 3F4 (to detect MHM2 PrP) (Kascsak, RJ et al., Mouse polyclonal and monoclonal antibody to scrapie-associated fibril proteins, J. Virol. 61, 3688-3693 (1987)) in a dilution of 1: 5000 in PBST overnight at 4 ° C. Following incubation with the primary antibody, the membranes were washed three times for 10 minutes in PBST, incubated with horseradish peroxidase-labeled secondary antibody (Amersham Life Sciences) diluted 1: 5000 in PBST for 30 to 60 minutes. at 4 ° C, and washed again three times for 10 minutes in PBST. After the chemiluminescent development with the ECL reagent (Amersham) for 1 minute, the spots were sealed in plastic covers, and exposed to the ECL Hypermax film (Amersham). The movies were automatically processed in a Konica movie processor.

EXAMPLE The branched polyamines inhibit the formation of the nascent PrPSc, and induce the release of the previously existing PrPSc. Western blots were probed with monoclonal antibody 3F4, which recognizes the newly expressed MHP2 PrP. The ScN2a cells were exposed to SuperFect for 3 hours, and were retained for three days after the SuperFect was removed. The gels were tested on both the undigested control sample and on a sample subjected to limited proteolysis. Samples were tested on separate lanes 1-6 with a control sample and a limited proteolysis sample for each of the six lanes as follows: Lane 1: DOTAP-mediated transfection. Lane 2: 30 microgra-mos / milliliter of SuperFect, 5 micrograms of pSPOX MHM2. Lane 3: 75 micrograms / milliliter of SuperFect, 5 micrograms of pSPOX MHM2. Lane 4: 150 micrograms / milliliter of SuperFect, 5 micrograms of pSPOX MHM2. Lane 5: 150 micrograms / milliliter of SuperFect, 10 micrograms of pSPOX MHM2. Lane 6: No addition of transfection reagent or DNA. 40 microliters of undigested brain homogenate were used in these studies, while the samples were subjected to limited digestion with proteinase K, and concentrated 25-fold before SDS-PAGE. One milliliter of the digester was centrifuged at 100,000 x g for 1 hour at 4 ° C, and the pellets were suspended in 80 microliters of SDS sample buffer before SDS-PAGE, followed by Western blot. The apparent molecular weights, based on the migration of protein standards, are 34.2, 28.3, and 19.9 kDa. All control tracks 1 to 6 show multiple bands, as expected. However, of the samples subjected to limited proteolysis, only track 1 shows bands. Unexpectedly, all the tracks of partially digested samples 2 to 5, do not show bands, and as expected, there are no bands in the partially digested track 6. These results show the effect of using SuperFect to clean the PrPSc.

EXAMPLE IB The spot described above was separated from the antibody, exposed to labeled R073, and re-developed. The 3F4 antibody used in Example 1 binds to PrPc, but not to PrPSc. However, R073 is set to PrPSc and PrPc. Lanes 1, 2, and 3 show decreasing amounts of PrPSc, and lanes 4 and 5 show no detectable PrPSc.

EJEEMPLE 2A The gels were tested on the undigested controls 1 to 4, and as before, the samples were subjected to limited pro-theolysis. The tracks were as follows: Track 1: without SuperFect. Track 2: 30 micrograms / milliliter of SuperFect. Lane 3: 75 micrograms / milliliter of SuperFect. Track 4: 150 micrograms / milliliter of SuperFect. ScN2a cells were exposed to SuperFect for 3 hours, and harvested 3 days after the SuperFect was removed. The apparent molecular weights, based on the migration of protein standards, are 33.9, 28.8, and 20.5 kDa. Because each sample was tested after the same time period, the results show the dose-dependent effect of SuperFect on the removal of PrPSc. Tracks 1, 2, and 3 show decreasing amounts of PrPSc, and track 4 does not show detectable PrPSc.

EXAMPLE 2B To determine the time-dependent effect of the SuperFect, 3 different panels were prepared with 4 tracks each, and were tested as follows: ScN2a cells were exposed to 7.5 micrograms / milliliter of: SuperFect (lanes 1-4), PEI (average molecular weight of approximately 60,000) (Pistes 5-8), or PAMAM, generation 4.0 (lanes 9-12). Exposure time for each polyamine: 0 hours (tracks 1, 5, and 9), 4 hours (tracks 2, 6, and 10), 8 hours (tracks 3, 7, and 11), 16 hours (tracks 4, 8 , and 12). All samples underwent limited proteolysis to measure PrP? C. The apparent molecular weights, based on the migration of protein standards, are 38, 26, and 15 kDa. The tracks of each of the three panels show decreasing amounts of PrPSc.

EXAMPLE 3 In this example, 4 panels A, B, C, and D were created, the panels having three double tracks each (control and test). ScN2a cells were exposed to 1.5 micrograms / milliliter of: (A) SuperFect. (B) PEI (average molecular weight of approximately 60,000), (C) PAMAM, generation 4.0, or (D) no addition. The cells were harvested: Pisto 1, before the addition; step 2, immediately following a week of continuous exposure to the test compounds; and Track 3, 3 weeks after removing the test compounds. The minus (-) symbol denotes the undigested control sample, and the plus (+) symbol designates the sample subjected to limited proteolysis. The apparent molecular weights, based on the migration of protein standards, are 33.9, 28.8, and 20.5 kDa. Test tracks 3 on panel A showed a slight PrPSc after 3 weeks, and test tracks 3 on panels B and C did not show detectable PrPSc, while PrPSc was present on all tracks on panel D.

EXAMPLE 4A Four separate gels were tested to demonstrate the effect that chloroquine would have on the levels of PrPSc. Control lanes 1 and 3 where chloroquine was added, show clear bands for PrPSc, while lanes 2 and 4 without chloroquine show barely detectable amounts of PrPSc. The four tracks were prepared as follows: The ScN2a cells were treated with Track 1: control medium. Lane 2: 7.5 micrograms / milliliter of PEI (average molecular weight of approximately 60,000). Track 3: PEI plus chlorine-quina 100 μM. Lane 4: PEI plus NH4C1 30 μM. Chloroquine and NH4C1 were added 1 hour before the addition of PEI. The cells were harvested 16 hours after the addition of PEI. All submitted samples underwent limited proteolysis to measure PrPSc. The apparent molecular weights, based on the migration of protein standards, are 38, 26, and 15 kDa.

EXAMPLE 4B 8 tracks were prepared with SuperFect (+ SF) and 8 tracks without SuperFect (-SF). Tracks 1 to 8 of each group had an adjusted pH of 3.6, 4, 5, 6, 7, 8, 9, and 9.6. Mixing of crude mouse brain homogenates with SuperFect under a range of pH conditions was performed as described in the methods (measured final pH of each sample denoted above the tracks). The addition of 60 microliters / milliliter of SuperFect denoted as "+ SF", and the control without addition as "-SF". All submitted samples underwent limited proteolysis to measure PrPSc. The apparent molecular weights, based on the migration of protein standards, are 30 and 27 kDa. All the tracks of the group -SF showed PrPSc present. Clues 3 to 8 of the group + SF showed PrPSc. However, lanes 1 and 2 with the respective pH levels of 3.6 and 4.0 showed very slight detectable PrPSc. The results show that the ability of a branched polycation, such as SuperFect, to clean the PrPSc depends on the pH.

EXAMPLE 5 16 different tracks were prepared as described. Lanes 1 and 2 were the control lanes, and each of lanes 3 to 16 contained a different compound, as tested in Table 1. The test compounds were all polyamines. Accordingly, the results show the re-motion of PrPSc of the brain homogenate in vi tro by different polyamines. The samples were incubated with polyamines at a pH of 3.6, and processed as described in Methods. Each polyamines was tested at a concentration of 60 micrograms / milliliter. Tracks 1 and 2: control. Lane 3: poly- (L) lysine. Track 4: PAMAM, generation 0.0. Track 5: PAMAM, generation 1.0. Track 6: PAMAM, generation 2.0. Track 7: PAMAM, generation 3.0. Track 8: PAMAM, generation 4.0. Track 9: PAMAM-OH, generation 4.0. Track 10: PPI, generation 2.0. Track 11: PPI, generation 4.0. Track 12: Lineal PEI. Clue 13: PEI of high molecular weight. Lane 14: PEI of low molecular weight. Lane 15: PEI of average molecular weight. Track 16: SuperFect. All submitted samples underwent limited proteolysis to measure PrPSc. The apparent molecular weights, based on the migration of protein standards, are 30 and 27 kDa. Table 1. Removal of PrPSc by the polymeric compounds. IC50 = approximate concentration of polymer required to reduce PrPSc to 50 percent of control levels in ScN2a cells after exposure for 16 hours. All the compounds were tested in 5 different concentrations. The levels of PrP? C were measured by densitometry of Western blot signals.

TABLE 1 (Note that Table 1 includes information on the characteristics of the compounds used, but that the list does not correspond directly to lanes 1 to 16) Compound molecular weight Group NH2 primary molecular IC50 (ng / ml) PAMAM generation 0.0 517 4 > 10,000 PAMAM generation 1.0 1,430 8 > 10,000 PAMAM generation 2.0 3,256 16 2,000 PAMAM generation 3.0 6,909 32 400 PAMAM generation 4.0 14,215 64 80 PAMAM-OH generation 4.0 14,279 0 > 10,000 PPI generation 2.0 773 8 2,000 PPI generation 4.0 3,514 32 80 PEI low molecular weight ~ 25,000 2,000 PEI average molecular weight ~ 60,000 400 PEI high molecular weight -800,000 80 PEI linear ~ 60,000 2,000 poly- (L) lysine ~ 60,000 > 500 10,000 SuperFect 400 Tracks 7, 8, 11, and 13 showed the best results, that is, the best ability to clean the PrPSc under these conditions. Specifically, PAMAM generation 4.0 on lane 8, showed the best ability to clean the PrPSc under these conditions, while PAMAM-OH generation 4.0 showed an almost undetectable ability to clean the PrPSc, and was comparable with the control.

EXAMPLE 6 Transfection of PrPSc Expressing Cells with Dendrimer Compounds Cells of neuronal origin expressing PrPSc were examined to determine the ability of the compounds to suppress PrPSc formation.

Transfection Studies The supply cultures of N2a and ScN2a cells were maintained in MEM with 10 percent FBS, 10 percent Glutamax (Gibco BRL), 100 units of penicillin, and 100 micrograms / milliliter streptomycin. Cells from a single 100-millimeter confluent plate were trypsinized and divided into 10 separate 60-millimeter plates containing DME plus 10 percent FBS, 10 percent glutamax, 100 units of penicillin, and 100 micrograms / milliliter of Streptomycin (DME supplemented) one day before transfection. Immediately prior to transfection, dishes were washed twice with 4 milliliters of the supplemented DME medium, and then drained.

For DOTAP mediated transfection, 15 micrograms of pSPOX MHM2 were resuspended in 150 milliliters of sterile Hepes Regulated Serum (HBS) on the day of transfection. The DNA solution was then mixed with an equal volume of 333 micrograms / milliliter of DOTAP (Boehringer Man-nheim) in HBS in Falcon 2059 tubes, and incubated at room temperature for 10 minutes to allow the formation of DNA complexes. lipid DME supplemented (2.5 milliliters) was added to the mixture, and then pipetted onto monolayers of drained cells. The next day, the medium containing DNA / lipid was removed and replaced with fresh supplemented DME. The cells were harvested three days later. For transfections / exposures mediated by SuperFect MR, * SuperFect ™ with or without DNA was added to a milli-liter of DME supplemented in a Falcon 2059 tube, to reach the specific concentrations required for each experiment. This mixture was pipetted up and down twice, and then over drained cell monolayers. After exposure during the indicated times, the medium containing SuperFect ™ was removed and replaced with fresh supplemented DME. Cells were harvested at the specified times after removing the SuperFect ™ "Exposures were made to PPI (DAB-Am-8, polypropyleneimine octa-amine dendrimer, Generation 2.0, Aldrich 46,072-9), intact PAMAM (Star Dendrimer (PAMAM), Generation 4. Aldrich 41.244-9), PEI (Sigma), poly- (L) lysine (Sigma), and poly- (D) lysine (Sigma), as described above for SuperFect MR, Isolation of the Protein from the Treated Cells The cells were harvested by lysis in 1.2 milliliters of 20 mM Tris pH 8.0, containing 100 mM NaCl, 0.5 percent NP-40, and 0.5 percent sodium deoxycholate. The nuclei were removed from the lysate by centrifugation at 2, 000 rpm for 5 minutes. This lysate normally had a protein concentration of 0.5 milligrams / milliliter, as measured by the BCA assay. For the samples not treated with proteinase K, 40 micro-liters of the whole lysate (representing 20 micrograms of total protein) were mixed with 40 microliters of SDS 2 x sample regulator. Digestion with proteinase K, 1 milliliter of the lysate was incubated with 20 micrograms / milliliter of proteinase K (total protein: enzyme = 25: 1) for 1 hour at 37 ° C. The proteolytic digestion was terminated by the addition of 8 microliters of 0.5 M PMSF in absolute ethanol. The samples were then centrifuged for 75 minutes in a Beckman TLA-45 rotor at 100,000 x g at 4 ° C. The pellet was re-suspended by repeated pipetting in 80 ml-croliters of SDS IX sample regulator. The entire sample (representing 0.5 milligrams of total protein before digestion) was loaded for SDS-PAGE.

Western blot analysis Immunoreactive PrP bands were detected from DOTAP-mediated transfection before and after proteinase K digestion with the monoclonal antibody 3F4. The construct used to express PrPSc in ScN2a cells is MHM2, a chimeric construct that differs from wild-type MoPrP (wt) at positions 108 and 111 (Scott et al., (1992) Protein Sci. 1: 986-997 ). Substitution at these positions with the corresponding residues (109 and 112 respectively) from the Syrian hamster PrP sequence (SHa) creates an epitope for 3F4 (Kascsak et al., (1987), J. Virol. 61: 3688- 3693), which does not recognize the endogenous wild-type MoPrP in ScN2a cells, and therefore, facilitates specific detection of the transgene by Western blot. Following the electrophoresis, the Western hand was performed as described previously (Scott and co-workers, 1989) Cell 59: 847-857). Samples were boiled for 5 minutes, and cleaned by centrifugation for 1 minute at 14,000 rpm in a Beckman ultracentrifuge. The SDS-PAGE was performed on 12 percent polyacrylamide gels, 1.5 millimeters (Laemmli (1970) Na ture 227: 661-665). The membranes were blocked with 5 percent defatted milk protein in PBST (PBS free of calcium and magnesium + 0.1 percent Tween 20) for 1 hour at room temperature. The blocked membranes were incubated with the primary polyclonal antibody R073, or with the monoclonal antibody 3F4, at a dilution of 1: 5000 in PBST overnight at 4 ° C. Following incubation with the primary antibody, the membranes were washed three times for 10 minutes in PBST, incubated with horseradish peroxidase-labeled secondary antibody (Amersham Life Sciences) diluted 1: 5000 in PBST for 25 minutes at room temperature, and washed again three times for 10 minutes in PBST. After the chemiluminescent development with the ECL reagent (Amersham) for 1 minute, the spots were sealed in plastic covers and exposed to the ECL Hyper-max film (Amersham). The movies were automatically processed in a Konica movie processor. In contrast to the cells transfected with DOTAP, the ScN2a cells transfected with different concentrations of SuperFect ™ and DNA, did not appear to contain MHM2 resistant to the protease. Close scrutiny revealed that, prior to protease digestion, samples transfected with Super-FectMR express the MHM2 bands that are not seen in the background pattern of the control sample. These observations indicate that the MHM2 PrP was expressed successfully using the SuperFect ™ transfection reagent, but that the conversion of the MHP2 PrPc to the protease-resistant MHP2 PrPSc was inhibited by the SuperFect ™. To examine whether the SuperFect ™ had affected the levels of the previously existing PrPSc in the ScN2a cells, the Western blot probed with the 3F4 antibody was re-probed with the polyclonal antibody R073, which is capable of recognizing the endogenous MoPrP. Notably, the Super-FectMR caused the disappearance of the previously existing MoPrPSc of ScN2a cells in a dose-dependent manner. After treatment with SuperFect ™, PrPSc could not be detected in the nuclear fraction, in the granule, in the supernatant, or in the medium. The concentration of SuperFect ™ required to completely remove the previously existing PrPSc at a 3-hour exposure was 300 micrograms / milliliter, while 30 micrograms / milliliter was sufficient to interfere with the formation of new MHM2 PrPSc within the same time frame. The duration of the exposure had a dramatic influence on the ability of the SuperFect to remove PrPSc from ScN2a cells. Whereas a 3-hour exposure to 150 micrograms / milliliter of SuperFect ™ significantly reduced the levels of PrPSc in ScN2a cells, exposure for 10 minutes at the same dose of SuperFect ™ did not affect PrPSc levels. When the ScN2a cells were exposed to 2 micrograms / milliliter of Super-Fect ™ continuously for 1 week, the PrPSc disappeared completely. The tested conditions did not appear to be toxic to the cells. Neither 150 micrograms / milliliter of SuperFect ™ for 3 hours, nor 2 micrograms / milliliter of SuperFect ™ continuously for 1 week, caused obvious changes in morphology, viability, or cell growth, as judged by the phase contrast microscope.

EXAMPLE 7 Elimination of PrPSc by repeated exposures to SuperFect "1 The duration of reduction of PrPSc levels after exposure to SuperFect ™ was examined, and it was shown that this reduction could persist for prolonged periods after removal of SuperFect ™. Exposure of ScN2a cells to a single dose of 150 micrograms / milliliter of SuperFect ™ for 3 hours, the levels of PrPSc remained low for 1 week, but returned to almost baseline levels after 3 weeks in culture In contrast, when ScN2a cells were exposed to four separate doses of SuperFect ™ over the course of 16 days, very little PrPSc could be detected four weeks after the final exposure to SuperFect ™, which gives the hope that an exposure Prolonged to Su-perFect ™ can lead to a long-term cure for scrapie infection in cells ltivadas.

EXAMPLE 8 SuperFect .MR does not deduce the TPWrPI-.SC directly. The SuperFectMR dendrimer was used to determine if it could exert a similar inhibitory effect on the PrPSc either in crude brain homogenates, or in PrP rods 27-30 purified. Brain homogenates of normal and scrapie-treated Syrian hamsters were prepared (10 percent (w / v) in sterile PBS), by repeated extrusion through syringe needles of a successively smaller size, from an 18 gauge to a gauge 22. Nuclei and debris were removed by centrifugation at 1,000 xg for 10 minutes. The bicin-ninic acid protein (BCA) assay (Pierce) was used to determine the protein concentration. The homogenates were adjusted to 10 milligrams / milliliter of protein with PBS, and 50 microliters were added to 450 microliters of lysis buffer containing 100 mM NaCl, 1 mM EDTA, 0.55 percent sodium deoxycholate, Triton X-100 at 0.55 percent. one hundred, and 50 mM Tris-HCl, pH 7.5. This mixture was then incubated with 0-300 micrograms / milliliter of SuperFect ™ for 3 hours at 37 ° C, and then centrifuged for 10 minutes at 14,000 rpm in a Beckman ultracentrifuge. The granule was resuspended in 450 microliters of lysis buffer without SuperFect ™. Proteinase K (Boehringer Mannheim) was added to reach a final concentration of 20 micrograms / milliliter, and thus, the total protein / enzyme ratio of 50: 1. The samples were incubated for 1 hour at 37 ° C. The proteolytic digestion was terminated by the addition of 8 microliters of 0.5 M PMSF in ethanol. The samples were then centrifuged for 75 minutes in a Beckman TLA-45 rotor at 100,000 x g at 4 ° C. Undigested samples (10 microliters) were mixed with an equal volume of 2x SDS sample buffer. For the digested samples, the granule was resuspended by repeated pipetting in 100 microliters of the SDS lx sample regulator. 20 microliters (equivalent to 100 micrograms of total protein before digestion with proteinase K) of each sample was loaded for SDS-PAGE. The PrP 27-30 canes were purified from Syrian hamster brains affected with scrapie, and previously described (Prusiner et al., (1983) Cell 35: 349-358). The purified sticks (3.5 micrograms / milliliter) were incubated with or without 900 micrograms / milliliter of SuperFect ™ in 100 microliters of DME supplemented. After 16 hours at 37 ° C, the suspension was centrifuged at 100,000 x g at 4 ° C. The granule was resuspended in 500 microliters of buffer containing 1 milligram / milliliter of BSA, 100 mM NaCl, 1 mM EDTA, 0.55 percent sodium deoxycholate, 0.55 percent Triton X-100, and 50 mM Tris-HCl. , pH of 7.5. Proteinase K was added to reach a final concentration of 20 micrograms / milliliter. The samples were incubated for 1 hour at 37 ° C. The proteolytic digestion was terminated by the addition of 8 micro-liters of Pefabloc 0.5 M. (Boehringer Mannheim). Then the samples were centrifuged for 75 minutes at 100, 000 x g at 4 ° C. Undigested samples (50 microliters) were mixed with an equal volume of SDS 2x sample buffer. For digested samples, the granule was resuspended by repeated pipetting in 100 microliters of SDS lx sample buffer. 40 microliters of each sample was loaded for SDS-PAGE. When the SuperFect ™ was mixed with the crude homogenates of Syrian hamsters affected with scrapie, or with PrP 27-30 purified Syrian hamster, there was no significant change in the level of PrPSc resistant to proteinase K. These results suggest that the Removal of PrPSc from ScN2a cells by SuperFect ™ depends on the presence of intact cellular machinery.Was.

EXAMPLE 9 Cleaning of PrPSc levels by other dendritic polycations The SuperFect ™ compound is a high molecular weight component of heat degraded PAMAM star dendrimers, which is a highly branched cationic monodisperse polymer (Tang et al., (1996) Bioconjugal Chem 7: 703-714). To identify other potentially useful an-ti-prion theutics, we tracked three other dendritic poly-cations and two linear cationic polymers to determine their ability to clear the PrPSc from ScN2a cells. Among the dendritic macromolecules tested, polyethyleneimine (PEI) was the most potent, removing most of the PrPSc from ScN2a cells after 3 hours, when used at a concentration of 10 micrograms / milliliter. The intact PAMAM exhibited a power comparable to SuperFect ™, removing approximately half of the detectable PrPSc when used at a concentration of 50 micrograms / milliliter. In contrast, the polypropylene imine dendrimer (PPI), the poly- (L) lysine, and the linear poly- (D) lysine polycation failed to reduce PrPSc levels in concentrations between 10 and 50 micrograms / milliliter. These results demonstrate that a branched polymeric architecture is required to clean the PrPSc. In addition, exposing the ScN2a cells to the PEI or PAMAM intact for one week at a concentration of 1.5 micrograms / milliliter completely removes the PrPSc, effectively curing the scrapie infection cells. Although the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that different changes can be made and equivalents can be used without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, step or process steps, to the purpose, spirit and scope of the present invention. It is intended that all such modifications be within the scope of the claims appended hereto.

Claims (6)

1. A method for sterilizing an object, which comprises the steps of: contacting the object with a composition of a branched polycation at a pH of 5.0 or less; and allowing the composition to remain in contact with the object for a period of time sufficient to render a conformationally altered protein non-infectious.

2. The method of claim 1, which further comprises: removing the composition of the object. The method of claim 1, wherein the branched polycation is a polycationic dendrimer selected from the group consisting of polypropylene imine, polyethylenimine (PEI) poly (4 '-aza4' -methylheptamethylene D-glucaramide), polyamidoamines, and variants or fragments of them. 4. The method of claim 1, wherein the object is a cell culture. 5. The method of claim 1, wherein the object is a bovine product. 6. A composition, which includes: water in an amount of 1 percent to 99.99