CA2375237A1 - Method of sterilizing - Google Patents
Method of sterilizing Download PDFInfo
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- CA2375237A1 CA2375237A1 CA002375237A CA2375237A CA2375237A1 CA 2375237 A1 CA2375237 A1 CA 2375237A1 CA 002375237 A CA002375237 A CA 002375237A CA 2375237 A CA2375237 A CA 2375237A CA 2375237 A1 CA2375237 A1 CA 2375237A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/74—Synthetic polymeric materials
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G83/00—Macromolecular compounds not provided for in groups C08G2/00 - C08G81/00
- C08G83/002—Dendritic macromolecules
- C08G83/003—Dendrimers
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N33/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic nitrogen compounds
- A01N33/02—Amines; Quaternary ammonium compounds
- A01N33/04—Nitrogen directly attached to aliphatic or cycloaliphatic carbon atoms
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/3463—Organic compounds; Microorganisms; Enzymes
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/358—Inorganic compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/74—Synthetic polymeric materials
- A61K31/785—Polymers containing nitrogen
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
- A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
- A61L2/0082—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using chemical substances
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
- A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
- A61L2/0082—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using chemical substances
- A61L2/0088—Liquid substances
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
- A61L2/16—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using chemical substances
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
- A61L2/16—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using chemical substances
- A61L2/18—Liquid substances or solutions comprising solids or dissolved gases
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L101/00—Compositions of unspecified macromolecular compounds
- C08L101/005—Dendritic macromolecules
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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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
METHOD OF STERILIZING
GOVERNMENT SUPPORT
This work was supported, in part, by grants from the National Institutes of Health NS 14069, AG08967, AG02132, AG10770 and K08 NS02048-02. The government may have certain rights in this work.
FIELD OF THE INVENTION
The present invention relates generally to methods of sterilizing materials and particularly to a method of inactivating infectious prions.
BACKGROUND OF THE INVENTION
There are large numbers of known methods of 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 which kill or inactivate pathogens which are contacted by 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, disrupt or kill pathogens in the material. These methods are generally directed toward killing bacteria and inactivating viruses present in or on the material. Although sterilization methods may be quite affective in killing bacteria or inactivating viruses, they do not generally inactivate pathogenic proteins such as prions which can 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, A(3 peptide, al-antichymotrypin, tau and non-A~i component. Many of these diseases are neurological diseases.
However, type II
Diabetes is associated with Amylin and Multiple myeloma-plasma cell dyscrasias is associated with IgGL-chain. The relationship between the disease onset and the transition from the normal protein to the conformationally altered protein has been examined very closely in some instances such as with the association between prion diseases and PrPS°.
Prion diseases are a group of fatal neurodegenerative disorders that can occur in hereditary, sporadic, and infectious forms (Prusiner, S.B. Scrapie prions. Annu. Rev.
Microbiol. 43, 345-374 (1989)). These illnesses occur in humans and a variety of other animals (Prusiner, S.B. Prions.
Proc. Natl. Acad. Sci. USA 95, 13363-13383 (1998)). Prions are infectious proteins. The normal, S cellular form of the prion protein (PrP) designated PrP~ contains three a-helices and has little /3-sheet; in contrast, the protein of the prions denoted PrPs° is rich in (i-sheet structure. The accumulation of PrPs~ in the central nervous system (CNS) precedes neurologic dysfunction accompanied by neuronal vacuolation and astrocytic gliosis.
The spectrum of human prion diseases includes kuru (Gajdusek, D.C., Gibbs, C.J., Jr. &
Alpers, M. Experimental transmission of a kuru-like syndrome to chimpanzees.
Nature 209, 794-796 (1966)), Creutzfeldt-Jakob disease (CJD) (Gibbs, C.J., Jr., et al.
Creutzfeldt-Jakob disease (spongiform encephalopathy): transmission to the chimpanzee. Science 161, 388-389 (1968)), Gerstmann-Straussler-Scheinker disease (GSS) and fatal familial insomnia (FFI) (Goldfarb, L.G., et al. Fatal familial insomnia and familial Creutzfeldt-Jakob disease: disease phenotype determined by a DNA polymorphism. Science 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, new variant CJD (nvCJD), which has emerged in Great Britain and France (Will, R.G. , et al. A new variant of Creutzfeldt-Jakob disease in the UK.
Lancet 347, 921-925 (1996); Cousens, S.N., Vynnycky, E., Zeidler, M., Will, R.G. & Smith, P.G.
Predicting the CJD epidemic in humans. Nature 385, 197-198 (1997); Will, R.G., et al. Deaths from variant Creutzfeldt-Jakob disease. Lancet 353, 979 (1999)). Several lines of evidence have suggested a link between the nvCJD outbreak and a preceding epidemic of bovine spongiform encephalopathy (BSE) (Will, R.G. , et al. A new variant of Creutzfeldt-Jakob disease in the UK.
Lancet 347, 921-925 (1996); Bruce, M.E., et al. Transmissions to mice indicate that 'new variant' CJD is caused by the BSE agent. Nature 389, 498-501 (1997); Hill, A.F., et al.
The same prion strain causes vCJD and BSE. Nature 389, 448-450 (1997); Lasmezas, C.L, et al.
BSE
transmission to macaques. Nature 381, 743-744 (1996)). Although it is too early to predict the number of nvCJD cases that might eventually arise in Great Britain and elsewhere (Cousens, S.N., Vynnycky, E., Zeidler, M., Will, R.G. & Smith, P.G. Predicting the CJD
epidemic in humans.
Nature 385, 197-198 (1997)), it is clear that effective therapeutics for prion diseases are urgently needed. Unfortunately, although a number of compounds including amphotericins, sulfated polyanions, Congo red dye, and anthracycline antibiotics have been reported as prospective therapeutic agents (Ingrosso, L., Ladogana, A. & Pocchiari, M. Congo red prolongs the incubation period in scrapie-infected hamsters. J. Virol. 69, 506-508 (1995); Tagliavini, F., et al.
GOVERNMENT SUPPORT
This work was supported, in part, by grants from the National Institutes of Health NS 14069, AG08967, AG02132, AG10770 and K08 NS02048-02. The government may have certain rights in this work.
FIELD OF THE INVENTION
The present invention relates generally to methods of sterilizing materials and particularly to a method of inactivating infectious prions.
BACKGROUND OF THE INVENTION
There are large numbers of known methods of 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 which kill or inactivate pathogens which are contacted by 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, disrupt or kill pathogens in the material. These methods are generally directed toward killing bacteria and inactivating viruses present in or on the material. Although sterilization methods may be quite affective in killing bacteria or inactivating viruses, they do not generally inactivate pathogenic proteins such as prions which can 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, A(3 peptide, al-antichymotrypin, tau and non-A~i component. Many of these diseases are neurological diseases.
However, type II
Diabetes is associated with Amylin and Multiple myeloma-plasma cell dyscrasias is associated with IgGL-chain. The relationship between the disease onset and the transition from the normal protein to the conformationally altered protein has been examined very closely in some instances such as with the association between prion diseases and PrPS°.
Prion diseases are a group of fatal neurodegenerative disorders that can occur in hereditary, sporadic, and infectious forms (Prusiner, S.B. Scrapie prions. Annu. Rev.
Microbiol. 43, 345-374 (1989)). These illnesses occur in humans and a variety of other animals (Prusiner, S.B. Prions.
Proc. Natl. Acad. Sci. USA 95, 13363-13383 (1998)). Prions are infectious proteins. The normal, S cellular form of the prion protein (PrP) designated PrP~ contains three a-helices and has little /3-sheet; in contrast, the protein of the prions denoted PrPs° is rich in (i-sheet structure. The accumulation of PrPs~ in the central nervous system (CNS) precedes neurologic dysfunction accompanied by neuronal vacuolation and astrocytic gliosis.
The spectrum of human prion diseases includes kuru (Gajdusek, D.C., Gibbs, C.J., Jr. &
Alpers, M. Experimental transmission of a kuru-like syndrome to chimpanzees.
Nature 209, 794-796 (1966)), Creutzfeldt-Jakob disease (CJD) (Gibbs, C.J., Jr., et al.
Creutzfeldt-Jakob disease (spongiform encephalopathy): transmission to the chimpanzee. Science 161, 388-389 (1968)), Gerstmann-Straussler-Scheinker disease (GSS) and fatal familial insomnia (FFI) (Goldfarb, L.G., et al. Fatal familial insomnia and familial Creutzfeldt-Jakob disease: disease phenotype determined by a DNA polymorphism. Science 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, new variant CJD (nvCJD), which has emerged in Great Britain and France (Will, R.G. , et al. A new variant of Creutzfeldt-Jakob disease in the UK.
Lancet 347, 921-925 (1996); Cousens, S.N., Vynnycky, E., Zeidler, M., Will, R.G. & Smith, P.G.
Predicting the CJD epidemic in humans. Nature 385, 197-198 (1997); Will, R.G., et al. Deaths from variant Creutzfeldt-Jakob disease. Lancet 353, 979 (1999)). Several lines of evidence have suggested a link between the nvCJD outbreak and a preceding epidemic of bovine spongiform encephalopathy (BSE) (Will, R.G. , et al. A new variant of Creutzfeldt-Jakob disease in the UK.
Lancet 347, 921-925 (1996); Bruce, M.E., et al. Transmissions to mice indicate that 'new variant' CJD is caused by the BSE agent. Nature 389, 498-501 (1997); Hill, A.F., et al.
The same prion strain causes vCJD and BSE. Nature 389, 448-450 (1997); Lasmezas, C.L, et al.
BSE
transmission to macaques. Nature 381, 743-744 (1996)). Although it is too early to predict the number of nvCJD cases that might eventually arise in Great Britain and elsewhere (Cousens, S.N., Vynnycky, E., Zeidler, M., Will, R.G. & Smith, P.G. Predicting the CJD
epidemic in humans.
Nature 385, 197-198 (1997)), it is clear that effective therapeutics for prion diseases are urgently needed. Unfortunately, although a number of compounds including amphotericins, sulfated polyanions, Congo red dye, and anthracycline antibiotics have been reported as prospective therapeutic agents (Ingrosso, L., Ladogana, A. & Pocchiari, M. Congo red prolongs the incubation period in scrapie-infected hamsters. J. Virol. 69, 506-508 (1995); Tagliavini, F., et al.
Effectiveness of anthracycline against experimental prion disease in Syrian hamsters. Science 276, 1119-1122 (1997); Masullo, C., Macchi, G., Xi, Y.G. & Pocchiari, M. Failure to ameliorate Creutzfeldt-Jakob disease with amphotericin B therapy. J. Infect. Dis. 165, 784-785 (1992);
Ladogana, A., et al. Sulphate polyanions prolong the incubation period of scrapie-infected hamsters.
J. Gen. Tirol. 73, 661-665 (1992)), all have demonstrated only modest potential to impede prion propagation, and none have been shown to effect the removal of pre-existing prions from an infected host.
The PrP gene of mammals expresses a protein which can be the soluble, non-disease form PrP~ or be converted to the insoluble, disease form PrPS'. PrP~ is encoded by a single-copy host gene [Basler, Oesch et al. (1986) Cell 46:417-428] and when PrP~ is expressed it is generally found on the outer surface of neurons. Many lines of evidence indicate that prion diseases result from the transformation of the normal form of prion protein (PrP~) into the abnormal form (PrPs°). There is no detectable difference in the amino acid sequence of the two forms. However, PrPs° when compared with PrP~ has a conformation with higher (i-sheet and lower a-helix content (Pan, Baldwin et al. (1993) Proc Natl Acad Sci USA 90:10962-10966; Safar, Roller et al.
(1993) JBiol Chem 268:20276-20284). The presence of the abnormal PrPS° form in the brains of infected humans or animals is the only disease-specific diagnostic marker of prion diseases.
PrPS' plays a key role in both transmission and pathogenesis of prion diseases (spongiform encephalopathies) and it 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 of humans have been identified: (1) kuru, (2) Creutzfeldt-Jakob Disease (CJD), (3) Gerstmann-Straussler-Scheinker Disease (GSS), and (4) fatal familial insomnia (FFI) [Gajdusek (1977) Science 197:943-960; Medori, Tritschler et al. (1992) NEngl JMed 326:444-449]. Initially, the presentation of the inherited human prion diseases posed a conundrum which has since been explained by the cellular genetic origin of PrP.
The assembly and misassembly of normally soluble proteins into conformationally altered proteins is thought to be a causative process in a variety of other diseases.
Structural conformational changes are required for the conversion of a normally soluble and functional protein into a defined, insoluble state. Examples of such insoluble protein include: A(3 peptide in amyloid plaques of Alzheimer's disease and cerebral amyloid angiopathy (CAA); a-synuclein deposits in Lewy bodies of Parkinson's disease, tau in neurofibrillary tangles in frontal temporal dementia and 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.
Neurol. Sci. 94:1-28; Haan et al. (1990) Clin. Neurol. Neurosurg. 92(4):305-310).
Often these highly insoluble proteins form aggregates composed of nonbranching fibrils with the common characteristic of a (3-pleated sheet conformation. In the CNS, amyloid can be present in cerebral and meningeal blood vessels (cerebrovascular deposits) and in brain parenchyma (plaques).
Neuropathological studies in human and animal models indicate that cells proximal to amyloid deposits are disturbed 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. Pathol.
145:1348-1381;
Kalaria et al. (1995) Neuroreport 6:477-80; Masliah et al. (1996) J. Neurosci.
16:5795-5811).
Other studies additionally indicate that amyloid fibrils may actually initiate neurodegeneration (Lendon et al. (1997) J. Am. Med. Assoc. 277:825-31; Yankner (1996) Nat. Med.
2:850-2; Selkoe (1996) J. Biol. Chem. 271:18295-8; Hardy (1997) Trends Neurosci. 20:154-9).
In both AD and CAA, the main amyloid component is the amyloid (3 protein (A(3). The A(3 peptide, which is generated from the amyloid (3 precursor protein (APP) by two putative secretases, is present at low levels in the normal CNS and blood. Two major variants, A~i 1~
and A(31_a2, are produced by alternative carboxy-terminal truncation of APP
(Selkoe et a1.(1988) Proc. Natl. Acad. Sci. USA 85:7341-7345; Selkoe, (1993) Trends Neurosci 16:403-409). A(3,~2 is the more fibrillogenic and more abundant of the two peptides in amyloid deposits of both AD
and CAA. In addition to the amyloid deposits in AD cases described above, most AD cases are also associated with amyloid deposition 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 CAA, which can exist in the absence of AD.
Human transthyretin (TTR) is a normal plasma protein composed of four identical, predominantly (3-sheet structured units, and serves as a transporter of hormone thyroxin. Abnormal self assembly of TTR into amyloid fibrils causes two forms of human diseases, namely senile systemic amyloidosis (SSA) and familial amyloid polyneuropathy (FAP) (Kelly (1996) Curr Opin Strut Biol 6(1):11-7). The cause of amyloid formation in FAP are point mutations in the TTR gene;
the cause of SSA is unknown. The clinical diagnosis is established histologically by detecting deposits of amyloid in situ in bioptic material.
To date, little is known about the mechanism of TTR conversion into amyloid in vivo.
However, several laboratories have demonstrated that amyloid conversion may 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 mechanism of conformational transition involves monomeric conformational intermediate which polymerizes into linear (3-sheet structured amyloid fibrils [Lai, Colon et al.
(1996) Biochemistry 35(20):6470-82]. The process can be mitigated by binding with stabilizing molecules such as thyroxin or triiodophenol (Miroy, Lai et al. (1996) Proc Natl Acad Sci USA
93(26):15051-6).
The precise mechanisms by which neuritic plaques are formed and the relationship of plaque formation to the disease-associated neurodegenerative processes are not well-defined. The amyloid fibrils in the brains of Alzheimer's and prion disease patients are known to result in the inflammatory activation of certain cells. For example, primary microglial cultures and the THP-1 monocytic cell line are stimulated by fibrillar (3-amyloid and prion peptides to activate identical tyrosine kinase-dependent inflammatory signal transduction cascades. The signaling response elicited by (3-amyloid and prion fibrils leads to the production of neurotoxic products, which are in part responsible for the neurodegenerative . C.K. Combs et al, JNeurosci 19:928-39 (1999).
Although research efforts relating to conformationally altered proteins are advancing efforts to sterilize materials to avoid infections with such proteins axe not keeping pace. The present invention offers a means of sterilizing materials which contain 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 of PAMAM, generation 2.0 (ethylene diamine core).
SUMMARY OF THE INVENTION
A method is disclosed whereby any type of object can be sterilized by combining normal sterilization procedures with the use of a polycationic dendrimer which is capable of rendering a conformationally altered protein such as a prion non-infectious. The method is particularly usefi~l in sterilizing medical devices such as surgical instruments and catheters which have been used and brought into contact with blood or brain tissue. Objects sterilized via the method are also part of the invention and include capsules which are made from geletin extracted from cattle which cattle may be infected with prions, i.e. have undiagnosed BSE known as "mad cow disease."
The polycationic dendrimers can be combined with conventional antibacterial and antiviral agents in aqueous or alcohol solutions to produce disinfecting agents or surgical scrubs. Branched polycations for use in the invention include, but are not limited to, polypropylene imine, polyethyleneimine (PEI) poly(4'-aza-4'-methylheptamethylene D-glucaramide), polyamidoamines and suitable fragments and/or variants of these compounds.
Ladogana, A., et al. Sulphate polyanions prolong the incubation period of scrapie-infected hamsters.
J. Gen. Tirol. 73, 661-665 (1992)), all have demonstrated only modest potential to impede prion propagation, and none have been shown to effect the removal of pre-existing prions from an infected host.
The PrP gene of mammals expresses a protein which can be the soluble, non-disease form PrP~ or be converted to the insoluble, disease form PrPS'. PrP~ is encoded by a single-copy host gene [Basler, Oesch et al. (1986) Cell 46:417-428] and when PrP~ is expressed it is generally found on the outer surface of neurons. Many lines of evidence indicate that prion diseases result from the transformation of the normal form of prion protein (PrP~) into the abnormal form (PrPs°). There is no detectable difference in the amino acid sequence of the two forms. However, PrPs° when compared with PrP~ has a conformation with higher (i-sheet and lower a-helix content (Pan, Baldwin et al. (1993) Proc Natl Acad Sci USA 90:10962-10966; Safar, Roller et al.
(1993) JBiol Chem 268:20276-20284). The presence of the abnormal PrPS° form in the brains of infected humans or animals is the only disease-specific diagnostic marker of prion diseases.
PrPS' plays a key role in both transmission and pathogenesis of prion diseases (spongiform encephalopathies) and it 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 of humans have been identified: (1) kuru, (2) Creutzfeldt-Jakob Disease (CJD), (3) Gerstmann-Straussler-Scheinker Disease (GSS), and (4) fatal familial insomnia (FFI) [Gajdusek (1977) Science 197:943-960; Medori, Tritschler et al. (1992) NEngl JMed 326:444-449]. Initially, the presentation of the inherited human prion diseases posed a conundrum which has since been explained by the cellular genetic origin of PrP.
The assembly and misassembly of normally soluble proteins into conformationally altered proteins is thought to be a causative process in a variety of other diseases.
Structural conformational changes are required for the conversion of a normally soluble and functional protein into a defined, insoluble state. Examples of such insoluble protein include: A(3 peptide in amyloid plaques of Alzheimer's disease and cerebral amyloid angiopathy (CAA); a-synuclein deposits in Lewy bodies of Parkinson's disease, tau in neurofibrillary tangles in frontal temporal dementia and 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.
Neurol. Sci. 94:1-28; Haan et al. (1990) Clin. Neurol. Neurosurg. 92(4):305-310).
Often these highly insoluble proteins form aggregates composed of nonbranching fibrils with the common characteristic of a (3-pleated sheet conformation. In the CNS, amyloid can be present in cerebral and meningeal blood vessels (cerebrovascular deposits) and in brain parenchyma (plaques).
Neuropathological studies in human and animal models indicate that cells proximal to amyloid deposits are disturbed 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. Pathol.
145:1348-1381;
Kalaria et al. (1995) Neuroreport 6:477-80; Masliah et al. (1996) J. Neurosci.
16:5795-5811).
Other studies additionally indicate that amyloid fibrils may actually initiate neurodegeneration (Lendon et al. (1997) J. Am. Med. Assoc. 277:825-31; Yankner (1996) Nat. Med.
2:850-2; Selkoe (1996) J. Biol. Chem. 271:18295-8; Hardy (1997) Trends Neurosci. 20:154-9).
In both AD and CAA, the main amyloid component is the amyloid (3 protein (A(3). The A(3 peptide, which is generated from the amyloid (3 precursor protein (APP) by two putative secretases, is present at low levels in the normal CNS and blood. Two major variants, A~i 1~
and A(31_a2, are produced by alternative carboxy-terminal truncation of APP
(Selkoe et a1.(1988) Proc. Natl. Acad. Sci. USA 85:7341-7345; Selkoe, (1993) Trends Neurosci 16:403-409). A(3,~2 is the more fibrillogenic and more abundant of the two peptides in amyloid deposits of both AD
and CAA. In addition to the amyloid deposits in AD cases described above, most AD cases are also associated with amyloid deposition 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 CAA, which can exist in the absence of AD.
Human transthyretin (TTR) is a normal plasma protein composed of four identical, predominantly (3-sheet structured units, and serves as a transporter of hormone thyroxin. Abnormal self assembly of TTR into amyloid fibrils causes two forms of human diseases, namely senile systemic amyloidosis (SSA) and familial amyloid polyneuropathy (FAP) (Kelly (1996) Curr Opin Strut Biol 6(1):11-7). The cause of amyloid formation in FAP are point mutations in the TTR gene;
the cause of SSA is unknown. The clinical diagnosis is established histologically by detecting deposits of amyloid in situ in bioptic material.
To date, little is known about the mechanism of TTR conversion into amyloid in vivo.
However, several laboratories have demonstrated that amyloid conversion may 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 mechanism of conformational transition involves monomeric conformational intermediate which polymerizes into linear (3-sheet structured amyloid fibrils [Lai, Colon et al.
(1996) Biochemistry 35(20):6470-82]. The process can be mitigated by binding with stabilizing molecules such as thyroxin or triiodophenol (Miroy, Lai et al. (1996) Proc Natl Acad Sci USA
93(26):15051-6).
The precise mechanisms by which neuritic plaques are formed and the relationship of plaque formation to the disease-associated neurodegenerative processes are not well-defined. The amyloid fibrils in the brains of Alzheimer's and prion disease patients are known to result in the inflammatory activation of certain cells. For example, primary microglial cultures and the THP-1 monocytic cell line are stimulated by fibrillar (3-amyloid and prion peptides to activate identical tyrosine kinase-dependent inflammatory signal transduction cascades. The signaling response elicited by (3-amyloid and prion fibrils leads to the production of neurotoxic products, which are in part responsible for the neurodegenerative . C.K. Combs et al, JNeurosci 19:928-39 (1999).
Although research efforts relating to conformationally altered proteins are advancing efforts to sterilize materials to avoid infections with such proteins axe not keeping pace. The present invention offers a means of sterilizing materials which contain 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 of PAMAM, generation 2.0 (ethylene diamine core).
SUMMARY OF THE INVENTION
A method is disclosed whereby any type of object can be sterilized by combining normal sterilization procedures with the use of a polycationic dendrimer which is capable of rendering a conformationally altered protein such as a prion non-infectious. The method is particularly usefi~l in sterilizing medical devices such as surgical instruments and catheters which have been used and brought into contact with blood or brain tissue. Objects sterilized via the method are also part of the invention and include capsules which are made from geletin extracted from cattle which cattle may be infected with prions, i.e. have undiagnosed BSE known as "mad cow disease."
The polycationic dendrimers can be combined with conventional antibacterial and antiviral agents in aqueous or alcohol solutions to produce disinfecting agents or surgical scrubs. Branched polycations for use in the invention include, but are not limited to, polypropylene imine, polyethyleneimine (PEI) poly(4'-aza-4'-methylheptamethylene D-glucaramide), polyamidoamines and suitable fragments and/or variants of these compounds.
An aspect of the invention is a method of treating objects with a composition characterized by its ability to render proteins associated with diseases non-infectious.
An advantage of the invention is that proteins such as prions can be rendered non-infectious without the need for extreme conditions such as exposure to heat over long periods of time, e.g. 1-10 hours at 100°-200°C.
A feature of the invention is that compositions can be useful while containing only very low concentrations of polycationic dendrimers, e.g. 1 % to 0.001 %.
Another aspect of the invention is that capsules made with bovine gelatin can be certified prion free.
Another aspect of the invention is that drugs produced from cell cultures treated with polycationic dendrimers can be certified prion free.
Still another aspect of the invention is that medical devices being reused after exposure to blood or brain tissue can be certified prion free.
Still another aspect of the invention is that hospitals, operating rooms and the devices and equipment within them can be certified prion free by contacting them with polycationic dendrimers at standard temperatures and pressures. A pharmaceutical composition for the treatment of insoluble protein deposit formation 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 may have multiple charged branches. The branches may have the same chemical structure, or the branches may vary in structure within a single molecule. Examples of polymers that may be used in such a pharmaceutical composition include polypropylene imine, polyethyleneimine (PEI) poly(4'-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, anti-inflammatory agent, an antioncogenic agent, an antiviral agent, and the like.
The present invention also provides a method of enhancing clearance of a disease related conformation of a protein from cells by contacting cells with a branched polycation for a time sufficient to enhance the rate of clearance of a disease related conformation of a protein from the 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 such disease related proteins include PrPs°, APP, A(3 peptide, a-1-antichymotrypsin. The method is thus useful for subjects suffering from disorders such as bovine spongiform encephalopathy, Creutzfeldt-Jacob Disease, fatal familial insomnia, GSS for Gerstmann-Straussler-Scheinker Disease, kuru, scrapie, Alzheimer's Disease, Frontal temporal dementia, Huntington's disease, ALS, Pick's disease, Parkinson's disease, Diabetes Type II, multiple myeloma, familial amyloidotic polyneuropathy, medullary carcinoma of thyroid, chronic renal failure, congestive heart failure, senile cardiac and systemic amyloidosis, chronic inflammation, atherosclerosis, and familial amyloidosis. The branched polycation can be administered to a subject in an amount non-toxic to the subject, for example a dosage of 0.001 mg to 1 mg/kg body weight per day. The polycation may be administered in a single dosage form, or it may be repeatedly administered to the subject. The branched polycation may also be administered prophylactically to prevent the formation of the disease conformation of these proteins.
The invention also provides a food composition for the preventing insoluble protein deposit formation in an animal, where the food contains a therapeutically effective amount of a branched canon which enhances clearance of conformationally altered protein. The food can be any food product, including solid foods such as meat (e.g., beef or lamb) and liquid foods such as vinegar, oil, and condiments such as steak sauce and ketchup. The branched polycation is allowed to contact the food prior to ingestion for a time sufficient to allow clearance of the conformationally altered proteins.
The present invention also provides a method of preventing a farm animal from acquiring a disease associated with a conformationally altered protein by feeding the animals animal feed containing a branched polycation. The feed containing the branched polycation may be synthetically produced, and fed directly to the animals, or the branched polycation may be introduced to a natural food source, e.g., the animal feed is grass and the branched polycation is sprayed on the grass or introduced to the grass through a plant fertilizer. One aspect of this embodiment of the invention is a method of preventing disease caused by ingestion of contaminated food products by feeding animals foods containing branched polycations.
The present invention also provides a method of enhancing clearance of a disease related conformation of a protein from a meat food product by contacting the meat with a compound which enhances clearance of a conformationally altered protein at a pH of 5 or less for a time sufficient to allow for destruction of conformationally altered protein.
An advantage of the invention is that conformationally altered protein such as prions can be rendered non-infectious with a method which need only consist of applying a polycationic dendrimer preferably held at a pH of 5.0 or less.
Another aspect of the invention is soaps, surgical scrubs, 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 which might be present on surgical instruments, knives and/or other tools or equipment used by butchers, particularly those used in the butchering of cows or other animals which might be infected with prions.
A feature of the invention is that compositions of the invention can be effective in activating prions when the polycationic dendrimers are present in very low concentrations, e.g. 1 % to 0.001 or less.
These and other aspects, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the compounds, and assay method more fully described below.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
Before the present methods, objects and compositions are described, it is to be understood that this invention is not limited to the particular steps, devices or components described and, as such, may of course vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since 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 belongs.
Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the 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 connection with which the publications are cited.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
DEFINITIONS
The term "detergent" is used to mean any substance that reduces the surface tension of water. The detergent may be a surface active agent which concentrates at oil-water interfaces, exerts emulsifying action and thereby aids in removing soils e.g. common sodium soaps of fatty acids. A
detergent may be anionic, cationic, or monionic depending on their mode of chemical action.
Detergents include linear alkyl sulfonates (LAS) often aided by "builders." A
LAS is preferably an _g_ alkyl benzene sulfonate ABS which is readily decomposed by microorganisms (biodegradable). The LAS is generally a straight chain alkyl comprising 10 to 30 carbon atoms. The detergent may be in a liquid or a solid form.
The term "conformationally altered protein" is used here to describe any protein which has a three dimensional conformation associated with a disease. The conformationally altered protein may cause the disease, be a factor in a symptom of the disease or appear as a result of other factors. The conformationally altered protein appears in another conformation which has the same amino acid sequence. In general, the conformationally altered protein formed is "constricted" in conformation as compared to the other "relaxed" conformation which is not associated with disease. The following is a non-limiting list of diseases with associated proteins which assemble two or more different conformations wherein at least one conformation is an example of a conformationally altered protein.
Disease Insoluble Proteins Alzheimer's Disease APP, A~i peptide, a 1-antichymotrypsin, tau, non-A(3 component, presenillin 1, presenillin apoE
Prion diseases, Creutzfeldt Jakob disease, scrapie and bovine spongiform encephalopathy prps ALS SOD and neurofilament Pick's disease Pick body Parkinson's disease a-synuclein in Lewy bodies Frontotemporal dementia tau in fibrils Diabetes Type II Amylin Multiple myeloma--plasma cell dyscrasias IgGL-chain Familial amyloidotic polyneuropathy Transthyretin Medullary carcinoma of thyroid Procalcitonin Chronic renal failure ~i2--microglobulin Congestive heart failure Atrial natriuretic factor Senile cardiac and systemic amyloidosis Transthyretin 4~ Chronic inflammation Serum amyloid A
Atherosclerosis ApoAl Familial amyloidosis Gelsolin Huntington's disease Huntingtin The term"acid" is used to describe any compound or group of compounds which has one or more characteristics of (a) sour taste; (b) turns litmus dye red; (c) reacts with certain metals to form a salt; (d) reacts with certain bases or a.lkalines to form a salt. An acid comprises hydrogen and in water undergoes ionization so that H30+ ions are formed - also written as H+
and referred to as hydronium ions or simply hydrogen ions. Weak acids such as acetic acid or carbonic acid may be used as may strong acids such as hydrochloric acid, nitric acid and sulfixric acid. In compositions of the invention the acid is preferably present in a concentration so as to obtain a pH of 5 or less, more preferably 4 or less and still more preferably 3.5 ~ 1.
The terms "sterilizing", "making sterile" and the like are used here to mean rendering something non-infectious or rendering something incapable of causing a disease. Specifically, it refers to rendering a protein non-infectious or incapable of causing a disease or the symptoms of a disease. Still more specifically, it refers to rendering a conformationally altered protein (e.g. PrPS°
known as prions) incapable of causing a disease or the symptoms of a disease.
By "effective dose" or "amount effective" is meant an amount of a compound sufficient to provide the desired sterilizing 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 which might be present. Polycations of the invention or more specifically polycationic dendrimer compounds of the invention could be mixed with a material in an amount in a range 1 to 500 ~g of dendrimer per ml or mg of material being sterilized. The concentration is sufficient if the resulting composition is effective in decreasing the infectivity of conformationally altered proteins such that the treated material over time would 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 effective dose or concentration range needed to sterilize can vary considerably. It is also pointed out that the dose needed to treat an amount of material may vary somewhat based on the pH the treatment is carried out at and the amount of time the compound is maintained in contact with the material at the desired low pH (e.g., 5.0 or less) level.
The term "LDSO" as used herein is the dose of an active substance that will result in 50 percent lethality in all treated experimental animals. Although this usually refers to invasive administration, such as oral, pa,renteral, and the like, it may also apply to toxicity using less invasive methods of administration, such as topical applications of the active substance.
The term "amine-terminated" includes primary, secondary and tertiary amines.
The terms "PrP protein", "PrP" and like are used interchangeably herein and shall mean both the infectious particle form PrPs° known to cause diseases (spongiform encephalopathies) in humans and animals and the noninfectious form PrP~ which, under appropriate conditions is converted to the infectious PrPs° form.
The terms "prion", "prion protein", "PrPs°protein" and the like are used interchangeably herein to refer to the infectious PrPS° form of a PrP protein, and is a contraction of the words "protein" and "infection." Particles are comprised largely, if not exclusively, of PrPs° molecules encoded by a PrP gene. Prions are distinct 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 known to affect humans are (1) kuru, (2) Creutzfeldt-Jakob Disease (CJD), (3) Gerstmann-Straussler-Scheinker Disease (GSS), and (4) fatal familial insomnia (FFI). As used herein "prion" includes all forms of prions causing all or any of these diseases or others in any animals used - and in particular in humans and domesticated farm animals.
The term "PrP gene" is used herein to describe genetic material which expresses proteins including known polymorphisms and pathogenic mutations. The term "PrP gene"
refers generally to any gene of any species which encodes any form of a prion protein. Some commonly known PrP
sequences are described in Gabriel et al., Proc. Natl. Acad. Sci. USA 89:9097-9101 (1992) and U.S.
Patent No. 5,565,186, incorporated herein by reference to disclose and describe such 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, it being recognized that the terms include other such PrP genes that are yet to be discovered. The protein expressed by such a gene can assume either a PrP~ (non-disease) or PrPs' (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 may (1) include a transgene as described herein; (2) have and ablated endogenous prion protein gene; (3) have a high number of prion protein gene from a genetically diverse species; and/or (4) be a hybrid with an ablated endogenous prion protein gene and a prion protein gene from a genetically diverse species.
Different combinations of 1-4 are possible, e.g., 1 and 2. The mammals from which standardized pr~ion preparations are obtained exhibit clinical signs of CNS dysfunction as a result of inoculation with priors and/or due to developing the disease of their genetically modified make up, e.g., high copy number of prior protein genes. Standardized prior preparations and methods of making such are described and disclosed in U.S. Patent 5,908,969 issued June l, 1999 and application serial no. 09/199,523 filed 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 formation of neuritic plaques comprising amyloid ~i protein, primarily in the hippocampus and cerebral cortex, as well as impairment in both learning and memory.
"AD" as used herein is meant to encompass both AD as well as AD-type pathologies.
The term "AD-type pathology" as used herein refers to a combination of CNS
alterations including, but not limited to, formation of neuritic plaques containing amyloid ~3 protein in the hippocampus and cerebral cortex. Such AD-type pathologies can include, but are not necessarily limited to, disorders associated with aberrant expression and/or deposition of APP, overexpression of APP, expression of aberrant APP gene products, and other phenomena associated with AD. Exemplary AD-type pathologies include, but are not necessarily limited to, AD-type pathologies associated with Down's 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 AD, particularly such an event that is readily assessable in an animal model. Such events include, but are not limited to, amyloid deposition, neuropathological developments, learning and memory deficits, and other AD-associated characteristics.
The term "cerebral amyloid angiopathy" (abbreviated herein as CAA) as used herein refers to a condition associated with formation of amyloid deposition within cerebral vessels which can be complicated by cerebral parenchymal hemorrhage. CAA is also associated with increased risk of stroke as well as development of cerebellar and subarachnoid hemorrhages (Vinters (1987) Stroke 18:311-324; Haan et al. (1994) Dementia 5:210-213; Itoh et al. (1993) J.
Neurol. Sci. 116:135-414). CAA can also be associated with dementia prior to onset of hemorrhages. The vascular amyloid deposits associated with CAA can exist in the absence of AD, but are more frequently associated with AD.
The term "phenomenon associated with cerebral amyloid angiopathy" as used herein refers to a molecular, structural, or functional event associated with CAA, particularly such an event that is readily assessable in an animal model. Such events include, but are not limited to, amyloid deposition, cerebral parenchymal hemorrhage, and other CAA-associated characteristics.
The term "(3-amyloid deposit" as used herein refers to a deposit in the brain composed of A(3 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-Straussler-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 a Syrian hamster;
SHaPrP for a Syrian hamster prion protein;
PAMAM for polyamidoamide dendrimers PEI for polyethyleneimine PPI for polypropyleneimine PrPs~ for the scrapie isoform of the prion protein;
PrP~ for the cellular contained common, normal isoform of the prion protein;
PrP 27-30 or PrPs~ 27-30 for the treatment or protease resistant form of PrPs~;
MoPrPs' for the scrapie isoform of the mouse prion protein;
N2a for an established neuroblastoma cell line used in the present studies;
ScN2a for a chronically scrapie-infected neuroblastoma cell line;
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 rendering conformationally altered proteins non-infective. The compositions are preferably low pH solutions comprised of a non toxic weak acid such as acetic acid having dissolved therein a branched polycation. Preferred compositions of the invention are in the form of aqueous or alcohol solutions which are comprised of a branched polycation, an antibacterial, an antifungal and an antiviral compound. The compositions are coated on, mixed with, injected into or otherwise brought into contact with a material to be sterilized. The composition is applied in a manner so that the branched polycation is maintained at a low pH (e.g. ~ or less and preferably 3.5 t 1) in an amount of 1 ~cg or more polycation per ml or mg of material to be sterilized. The composition is maintained in the desired pH range at normal temperature (e.g., 15 ° C to 30 ° C) for a sufficient period of time (e.g. 1 hour to 1 week) to cause conformationally altered protein present on or in the material to be destroyed (e.g. hydrolyzed) or rendered non-infective. Preferred compositions of the invention are useful in cleaning and sterilizing and may be comprised of a polycationic dendrimers, a detergent, and an acid proving a pH of about 3.5 ~1.
DENDRIMER COMPOUNDS WHICH CLEAR PRIONS
Dendrimers are branched compounds also known as "starburst" or "star" polymers due to a characteristic star-like structure (see Figure 1). Dendrimers of the invention are polymers with structures built from AB" monomers, with n>_2, and preferably n=2 or 3.
Such 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. Dendrimers may be cationic (full generation dendrimers) or anionic (half generation dendrimers). For a review on the general synthesis, physical properties, and applications of dendrimers, see, e. g. , 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, 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. Pat. 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 hereby incorporated by reference to disclose and describe such compounds.
Dendrimers typically have tertiary amines which have a pKa of 5.7. The dendrimers can optionally be chemically or heat treated to remove some of the tertiary amines. Other suitable canons include polypropylene imine, polyethyleneimine (PEI), which has tertiary amines with a pKa of 5.9, and poly(4'-aza-4'-methylheptamethylene D-glucaramide), which has tertiary amines with a pKa of 6Ø The cationic dendrimer is preferably dissolved in the low pH solvent such as vinegar in a concentration of 0.0001 % or more, preferably 0.01 % or more and more preferably about 1 % .
Preferably, the dendrimers for use in the invention are polyamidoamines (hereinafter "PAMAM"). PAMAM dendrimers are particularly biocompatible, since polyamidoamine groups resemble peptide bonds of proteins.
Dendrimers are prepared in tiers called generations (see generations 0, l and 2 in Figure 1) and therefore have specific molecular weights. The full generation PAMAM
dendrimers have amine terminal groups, and are cationic, whereas the half generation dendrimers are carboxyl terminated. Full generation PAMAM dendrimers are thus preferred for use in the present invention. PAMAM dendrimers may be prepared having different molecular weights and have specific values as described in Table 1 below for generations 0 through 10.
TABLE A
LIST OF PAMAM DENDRIMERS AND THEIR
MOLECULAR WEIGHTS (Ethylene Diamine core, amine terminated), GENERATION TERMINAL GROUPS MO L. WT. /mole 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 PAMAM dendrimers generations 0 through 10 range from 4 to 4,096, with molecular weights of from 517 to 934,720.
PAMAM dendrimers are available commercially from Aldrich or Dendritech.
Polyethyleneimine or polypropylene dendrimers or quaternized forms of amine-terminated dendrimers may be prepared as described by Tomalia et. al, Angew, Chem. Int. Ed. Engl., 29:138-175 (1990) incorporated by reference to describe and disclose methods of making dendrimers.
STERILIZING COMPOSITIONS
Examples provided here show that highly-branched polycations, e.g. dendrimer compounds, affect the extent and distribution of PrPs° 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 detectable PrP S~ in cells and brain homogenates. Thus, 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 in a low pH
environment, (e.g. a polycationic dendrimer) in solution, suspension or mixture.
STERILIZING FORMULATIONS
Sterilizing compositions of the invention preferably contain highly branched polycations, e.g. polycationic dendrimer, in a concentration from 0.0001 to 10% of the formulation. The following methods and excipients are merely exemplary and are in no way limiting.
In addition to including the compound such as a highly branched cationic compound in the formulation it is important to maintain that compound in a low pH
environment. Any number of known acids or mixtures of acids could be used with the invention. Non-limiting examples of commercially available products which could be supplemented with the cationic compounds are described below. In these formulations the percentage amount of each ingredient can vary. In general a solvent ingredient (e.g. water or alcohol) is present in amounts of 40% to 100% and the last listed ingredient is present in a range of 0.5 % to 5 % . The other ingredients are present in an amount in a range of 1 % to 60 % and more generally 5 % to 20 % . In each case the polycationic compounds of the invention are added in amounts of about 0.01 %
to 5 % and preferably 0.1 % to 2 % and more preferably about 1 % . The amount added is an amount needed to obtain the desired effect.
Component wt %
acid 90 - 99.99 polycationic dendrimer0.01 - 10 Component wt %
water 10 - 99 acid 1- 20 polycationic dendrimer0.01 - 10 Component wt %
water 10 - 98 acid 1 - 20 detergent 1 - 20 polycationic dendrimer0.01 - 5 Component wt %
water 10 - 98 acetic acid 1 - 20 linear alkyl sulfonate 1 - 20 polycationic dendrimer 0.01 - 5 Component wt %
water 1 - 98 alcohol 0 - 98 acid 1 - 20 detergent 1 - 20 polycationic dendrimer0.1 - 5 Component wt %
water 1 - 99 acid 1 - 20 antibacterial 0.1 - 5 detergent 1 - 20 polycationic dendrimer0.1 - 5 Component wt %
water 3 - 98.889 antimicrobial active 0.001- 5 agent anionic surfactant 1 - 80 protein donating 0.1 - 12 agent polycationic dendrimer 0.01 - 5 Component wt Polycationic Dendrimer0.5 Ethanol 74.0 Benzalkonium chloride0.2 CAE 0.02 Glycerine 1.0 Chain silicone 0.5 Triglyceride 0.5 Lactic acid 10.0 Purified water 13.28 Component wt Polycationic Dendrimer1.0 Ethanol 75.0 Benzalkonium chloride0.2 CAE 0.02 Glycerine 1.0 Cyclic silicone 0.2 Triglyceride 0.3 Acetic Acid 20.0 Purified water 2.28 Component wt Polycationic Dendrimer0.25 Ethanol 74.0 Chlorhexedine gluconate0.75 Benzalkonium chloride0.2 CAE 0.02 Glycerine 2.0 Chain silicone 0.2 Cyclic silicone 0.2 Triglyceride 0.3 Acetic Acid 20.0 Purified water 2.08 Component wt Polycationic Dendrimer0.1 Ethanol 75.0 Chlorhexedine gluconate0.9 Benzalkonium chloride0.2 CAE 0.02 Glycerine 1.0 Chain silicone 0.5 Cyclic silicone 0.5 Triglyceride 0.3 Lactic acid 14.0 Purified water 7.98 Component wt Polycationic Dendrimer0.01 Ethanol 75.0 Benzalkonium chloride0.2 CAE 0.02 Glycerine 2.0 Chain silicone 0.99 Cyclic silicone 2.0 Triglyceride 3.0 Lactic acid 9 Purified water 7.78 Component wt Polycationic Dendrimer1 Ethanol 75.0 Chlorhexedine gluconate0.2 Benzalkonium chloride0.2 CAE 0.02 Glycerine 0.8 Chain silicone 0.2 Cyclic silicone 0.2 Triglyceride .38 Acetic acid 10 Purified water 12 Component wt Polycationic Dendrimer0.001 Ethanol 75.99 Chlorhexedine gluconate0.2 CAE 0.02 Glycerine 1.0 Chain silicone 0.2 Triglyceride 0.3 Lactic acid 14 Purified water 8.28 Component wt Polycationic Dendrimer1 Ethanol 75.0 Benzalkonium chloride0.2 CAE 0.02 l, 3-butylene glycol 1.0 Metylphenyl polysiloxane0.2 Isopropyl myristate 0.3 (IPM) ~ Purified water ~ 22.28 By using the disclosure provided here and other information such as taught in U.S.
Patents 5,767,054; 6,007,831; 5,830,488; 5,968,539; 5,416,075; 5,296,158; and patents and publications cited therein those skilled in the art can produce countless other formulations of the invention. Further, such formulations can be used as described in such publications and can be packaged in any suitable container or dispenser device, e.g. taught in 5,992,698.
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 nontoxic at a dosage of 0.1 mg/mouse, which is an LD
So of less than one at 40 mg/Kg. Various nutrient formulations and/or injectable formulations of the type known to those skilled in the art can be used to prepare formulations for treating 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 accomplished by adding any desired acid. If desired, the pH can be raised to a normal level after treatment is complete, i.e. after a sufficient amount of any conformationally altered protein present are destroyed.
Compounds effective in sterilizing compositions containing conformationally altered proteins are determined via a cell culture assay and an organ homogenate assay each of which is described below in detail.
ScN2a CELL BASED ASSAY
Efforts were made to optimize the transfection of ScN2a cells with pSPOX
expression plasmids (Scott, M.R., Kohler, R., Foster, D. & Prusiner, S.B. Chimeric prion protein expression in cultured cells and transgenic mice. Protein Sci. l, 986-997 (1992)). In connection with those effects an evaluation was made of a transfection protocol that used SuperFect reagent (QIAGEN~). It was found that epitope-tagged (MHM2) PrPs° (Scott, M.R., Kohler, R., Foster, D. & Prusiner, S.B.
Chimeric prion protein expression in cultured cells and transgenic mice.
Protein Sci. 1, 986-997 WU 00/72851 CA 02375237 2001-11-26 pCT/US00/14353 (1992)) could not be detected in ScN2a cells following SuperFect-mediated transfection, whereas MHM2 PrPs' was efficiently formed when a cationic liposome method for DNA
delivery was used.
Close scrutiny revealed that, prior to protease digestion, SuperFect transfected samples expressed MHM2 bands, which are not seen in the background pattern of an untransfected sample. The 3F4 monoclonal antibody does not react with MoPrP but does exhibit high background staining on Western blots of mouse ScN2a cells. Increased immunostaining in the 20-30 kDa region was observed compared to the non-transfected sample. These observations led us to conclude that MHM2 PrP was successfully expressed using SuperFect transfection reagent, but that conversion of MHM2 PrP~ to protease-resistant MHM2 PrPs° was inhibited by SuperFect.
To investigate this apparent inhibition, a Western blot was reprobed with 8073 polyclonal antiserum to detect endogenous MoPrPs°, the presence of which is diagnostic for prion infection in ScN2a cells (Butler, D.A., et al. Scrapie-infected murine neuroblastoma cells produce protease-resistant prion proteins. J. Virol. 62, 1558-1564 (1988)). Surprisingly, it was found that the SuperFect treated ScN2a cells no longer contained detectable quantities of MoPrPs° - also confirmed in Western blots. To investigate the mechanism by which SuperFect reduced the level of pre-existing PrPs° in chronically infected ScN2a cells, measurements were made of endogenous PrPs' in ScN2a cells exposed to various concentrations of SuperFect in the absence of plasmid DNA. The results showed that treatment with SuperFect (a branched polycation) caused the disappearance of PrPs°
from ScN2a cells in a dose-dependent manner. The concentration of SuperFect required to eliminate >95% of pre-existing PrPs° with a three hour exposure was found to be about 150 ~cg/ml. Duration of treatment also influenced the ability of SuperFect to remove PrPs°
from ScN2a cells: exposure to 150 /.cg/ml SuperFect for 10 min did not affect PrPs° levels, whereas 7.5 /,cg/ml SuperFect eliminated all detectable PrPs' with a t'/Z= 8 h.
SuperFect is a mixture of branched polyamines derived from heat-induced degradation of a PAMAM dendrimer (Tang, M.X., Redemann, C.T. & Szoka, F.C.J. In vitro gene delivery by degraded polyamidoamine dendrimers. Bioconjug. Chem. 7, 703-714 (1996)).
Knowing this structure the ability of several other branched and unbranched polymers to eliminate PrPS° from ScN2a cells (Table 1). The branched polymers investigated include various preparations of PEI, as well as intact PAMAM and PPI dendrimers. Dendrimers are manufactured by a repetitive divergent growth technique, allowing the synthesis of successive, well-defined "generations" of homodisperse structures (Figure 1). The potency of both PAMAM and PPI dendrimers in eliminating PrPs~ from ScN2a cells increased as the generation level increased. The most potent compounds with respect to eliminating PrPs° were PAMAM generation 4.0 and PPI generation 4.0, whereas PAMAM
generation 1.0 showed very little ability to eliminate PrPS° (Table 1).
Similarly, a high MW fraction of PEI was more potent than low MW PEI.
From the foregoing data, it is clear that for all three branched polyamines tested, increasing molecular size corresponded to an increased potency for eliminating PrPs° . To determine whether this trend was directly attributable to increased surface density of amino groups on the larger molecules, PAMAM-OH generation 4.0 was tested. This is a dendrimer that resembles PAMAM
generation 4.0 except that hydroxyls replace amino groups on its surface.
Unlike PAMAM
generation 4.0, PAMAM-OH generation 4.0 did not cause a reduction of PrPs° levels even at the highest concentration tested ( 10 mg/ml), establishing that the amino groups are required for the elimination of PrPs' by PAMAM (Table 1).
In an effort to assess the contribution of the branched architecture to the clearing ability of polyamines for PrPs°, the linear molecules poly-(L)lysine and linear PEI were also tested. Both of these linear compounds were less potent than a preparation of branched PEI
with similar average molecular weight (Table 1), establishing that a branched molecular architecture optimizes the ability of polyamines to eliminate PrPs°, presumably because the branched structures achieve a higher density of surface amino groups.
Kinetics of PrPSc elimination by polyamines.
The preceding results demonstrate the potent ability of branched polyamines to clear PrPs°
from ScN2a cells within a few hours of treatment. The utility of these compounds to act as therapeutics for treatment of prion disease was tested by determining whether they were cytotoxic for ScN2a cells, using as criteria cell growth, morphology, and viability as measured by trypan blue staining. None of the compounds was cytotoxic to ScN2a cells after exposure for one week at concentrations up to 7.5 ~g/ml. To determine whether branched polyamines can cure ScN2a cells of 2~ scrapie infection without affecting cell viability, the kinetics of prion clearance was examined in the presence of a non-cytotoxic concentration (7.5 ~g/ml) of three different branched polyamines. ScN2a cells were exposed to SuperFect, PEI, or PAMAM generation 4.0 for varying periods of time. The kinetics of PrPs' elimination were assessed by Western blotting. All three compounds caused a substantial reduction in PrPS' levels after 8-16 h of treatment, and of the three compounds, PEI
appeared to remove PrPs' most quickly, with a t'/2= 4 h.
Curing neuroblastoma cells of scrapie infection.
The above results show that it is possible to reverse the accumulation of PrPS° in ScN2a cells under non-cytotoxic conditions. It was also found that extended exposure to even lower levels WO 00/72851 CA 02375237 2001-11-26 pCT/US00/14353 of the branched polyamines ( 1.5 ~g/ml) was sufficient to eliminate PrPS°. Based on these findings, this protocol was used to determine whether the severe reduction in PrPs~
levels following exposure to branched polyamines would persist after removal of the compounds. Following the exposure of ScN2a cells to a 1.5 ~g/ml SuperFect for 1 week, PrPs~ was reduced to <1 % of the baseline level, but then increased back to ~5% of the baseline level after 3 additional weeks in culture in the absence of polyamine. In contrast, following exposure to 1.5 ~g/ml of either PEI or PAMAM generation 4.0 for 1 week, PrPSc was completely eliminated and did not return even after 3 weeks in culture without polyamines. A more intensive course of treatment with 1.8 ~cg/ml SuperFect for 9 d also cured ScN2a cells of scrapie infection fully, manifested by the absence of PrPs° 1 month after removal of SuperFect.
Evidence for polyamines acting within an acidic compartment.
The above results showed the potent activity of branched polyamines in rapidly clearing scrapie prions from cultured ScN2a cells. Based on these results the mechanism by which these compounds act was investigated. All of the compounds which effect removal of PrPSc from ScN2a cells are known to traffic through endosomes (Boussif, O., et al. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethyleneimine.
Proc. Natl. Acad. Sci.
U.S.A. 92, 7297-7301 (1995); Haensler, J. & Szoka, F.C.J. Polyamidoamine cascade polymers mediate efficient transfection of cells in culture. Bioconjug. Chem. 4, 372-379 (1993)). Since PrP~
is converted into PrPs° in caveolae-like domains (CLDs) or rafts (Gorodinsky, A. & Hams, D.A.
Glycolipid-anchored proteins in neuroblastoma cells form detergent-resistant complexes without caveolin. J. Cell 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 colocalization of the cellular and scrapie prion proteins in caveolae-like membranous domains.
Proc. Natl. Acad. Sci.
USA 93, 14945-14949 (1996); Kaneko, K., et al. COOH-terminal sequence ofthe cellular prion protein directs subcellular trafficking and controls conversion into the scrapie isoform. Proc. Natl.
Acad. Sci. USA 94, 2333-2338 (1997)) and is then internalized through the endocytic pathway (Caughey, B., Raymond, G.J., Ernst, D. & Race, R.E. N terminal truncation 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. I~irol. 65, 6597-6603 (1991); Borchelt, D.R., Taraboulos, A. &
Prusiner, S.B. Evidence for synthesis of scrapie prion proteins in the endocytic pathway. J. Biol.
Chem. 267, 16188-16199 (1992)), it was deduced that polyamines act upon PrPs' in endosomes or lysosomes. This deduction was investigated by determining the effect of pretreatment with the lysosomotropic agents chloroquine and NH4C1 on the ability of polyamines to eliminate PrPs'. These lysosomotropic agents alkalinize endosomes and have no effect on PrP'' levels when administered to ScN2a cells (Taraboulos, A., Raeber, A.J., Borchelt, D.R., Serban, D. &
Prusiner, S.B. Synthesis and trafficking of prion proteins in cultured cells. Mol. Biol. Cell 3, 851-863 (1992)). Experimental results obtained shows that 100 ~cM chloroquine, but not 30 ~cM NH4C1, blocked the ability of PEI to eliminate PrPs'. Similar results were obtained with SuperFect and PAMAM, generation 4Ø
Although the failure of NH4C1 to affect PrPs' levels is not easily explained, the ability of chloroquine to attenuate the ability of branched polyamines to remove PrPs' is consistent with the notion that these agents act in endosomes or lysosomes.
ORGAN HOMOGENATE ASSAY
The above results with cell cultures prompted investigating the possibility that in an acidic environment branched polyamines, either by indirectly interacting with PrPs' or with another cellular component, could cause PrPs' to become susceptible to hydrolases present in the endosome/lysozome. An in vitro degradation assay was developed to evaluate the effect of pH on the ability of polyamines to render PrPs' sensitive to protease. Crude homogenates of scrapie-infected mouse brain were exposed to a broad range of pH values in the presence or absence of SuperFect and then treated with proteinase K prior to Western blotting. Whereas PrPs' remained resistant to protease hydrolysis throughout the pH range (3.6-9.6) in the absence of Superfect, addition of the branched polyamine at pH 4.0 or below caused PrPS' to become almost completely degraded by protease.
Polyamine addition showed a dramatic effect on clearance in vitro which was optimized at pH 4 or less. These results show that polyamines act on PrPs' in an acidic compartment. To establish that the in vitro degradation assay is a valid approximation of the mechanism by which branched polyamines enhance the clearance of PrPs' from cultured cells, a structure activity analysis was performed with several of the compounds tested in culture cells. An excellent correlation was found between the clearance of PrPs' in cultured ScN2a cells (Table 1) and the ability to render PrPS' susceptible to protease at acidic pH in vitro. Notably, PAMAM-OH
generation 4.0 failed to render PrPs' susceptible to protease, whereas PAMAM generation 4.0 and PPI, generation 4.0 exhibited an even stronger activity than Superfect in vitro, as expected from their observed potency in cultured ScN2a cells (Table 1).
MECHANISM OF ACTION
The results discussed here show that certain branched polyamines cause the rapid elimination of PrPS° from ScN2a cells in a dose- and time-dependent manner. These compounds demonstrate a potent ability to remove prions from cultured cells at concentrations that are completely non-cytotoxic. The cells may be maintained indefinitely in culture in the presence of therapeutic levels of branched polyamines. Furthermore, when ScN2a cells were exposed to these compounds for ~ 1 week, PrPs' was reduced to undetectable levels and remained so for at least one month after removal of the polyamine.
Clarification of the exact mechanism of PrPS~ elimination by branched polyamines is an important objective. Although a number of possible scenarios exist, several possibilities may be excluded already. One possibility that was eliminated was that polyamines act by induction of chaperones such as heat shock proteins that mediate prion protein refolding because the above results show that it was possible to reproduce the phenomenon in vitro. Furthermore polyamines seem to offer advantages over other putative therapeutics that would seek to promote refolding: at very high concentrations, dimethyl sulfoxide (DMSO) and glycerol act as direct "chemical chaperones" and inhibit the formation of new PrPs° (Tatzelt, J., Prusiner, S.B. &
Welch, W.J. Chemical chaperones interfere with the formation of scrapie prion protein. EMBO J. 15, 6363-6373 (1996)), but these compounds cannot reduce pre-existing PrPS° levels. Furthermore, polyamines inhibit PrPs° formation at much lower concentrations than these agents. The ability of polyamines to effect the rapid clearance of PrPS' also contrasts with the activity of other potential prion therapeutics. Sulfated polyanions may inhibit PrPs° accumulation in ScN2a cells by directly binding to PrP~ (Gabizon, R., Meiner, Z., Halimi, M. & Ben-Sasson, S.A. Heparin-like molecules bind differentially to prion-proteins and change their intracellular metabolic fate. J. Cell. Physiol. 157, (1993); Caughey, B., Brown, K., Raymond, G.J., Katzenstein, G.E. & Thresher, W. Binding of the protease-sensitive form of PrP (prion protein) to sulfated glycosaminoglycan and Congo red. J.
Ifirol. 68, 2135-2141 (1994)), but because branched polyamines are able to clear pre-existing PrPs°, their mechanism of action cannot simply involve binding to PrP~ and inhibiting de novo synthesis.
Another possible mechanism which can be excluded is endosomal rupture. The branched polyamines which were effective in clearing PrPs° from ScN2a cells in our experiments, PEI, SuperFect and PAMAM, are also potent lysosomotropic, osmotic agents which can swell in acidic environments and rupture endosomes (Boussif, O., et al. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethyleneimine.
Proc. Natl. Acad. Sci.
U.S.A. 92, 7297-7301 (1995); Haensler, J. & Szoka, F.C.J. Polyamidoamine cascade polymers mediate efficient transfection of cells in culture. Bioconjug. Chem. 4, 372-379 (1993)). This might suggest that branched polyamines clear PrPs° from ScN2a cells by rupturing endosomes and exposing PrPs' to cytosolic degradation processes. However, it is known that the lysosomotropic, endosome-rupturing agents NH4C1, chloroquine, and monensin do not interfere with the formation of PrPs° in ScN2a cells (Taraboulos, A., Raeber, A.J., Borchelt, D.R., Serban, D. & Prusiner, S.B.
Synthesis and trafficking of prion proteins in cultured cells. Mol. Biol. Cell 3, 851-863 (1992)).
Furthermore, the results also show that chloroquine interferes with the ability of branched polyamines to clear PrPs° and that polyamines can clear PrPs' in vitro at acidic pH in the absence of cell membranes. Together, these observations rule out endosome rupture as the mechanism by which branched polyamines remove PrPS°.
Without committing to any particular mechanism of action it appears likely that branched polyamines require the acidic environment of intact endosomes or lyzosomes to destroy PrPS~. The structure-activity profile of polymers tested reveals that the most active compounds possess densely packed, regularly-spaced amino groups, suggesting that these compounds may bind to a ligand which has periodically-spaced negative charges. Several scenarios remain possible.
(1) Branched polyamines may bind directly to PrPs° arranged as an amyloid with exposed negatively-charged moieties and induce a conformational change under acidic conditions. (2) Treatment of PrP 27-30 with acid decreases turbidity and increases a-helical content, suggesting that such conditions might dissociate PrPs~ into monomers (Safar, J., Roller, P.P., Gajdusek, D.C. &
Gibbs, C.J., Jr. Scrapie amyloid (prion) protein has the conformational characteristics of an aggregated molten globule folding intermediate). It is therefore possible that polyamines bind to an equilibrium unfolding intermediate of PrPs' present under acidic conditions. (3) Alternatively, polyamines might sequester a cryptic, negatively charged component bound to PrPS' that is essential for protease resistance, but which is only released when PrPS' undergoes an acid-induced conformational change. Such a component might act as a chaperone for PrPs° inside endosomes or lysosomes. (4) Finally, another possibility is that polyamines activate an endosomal or lysosomal factor which can induce a conformational change in PrPs'. Clearly, more work will be required to determine the precise mechanism by which branched polyamines destroy PrPs' GENERAL APPLICABILITY OF ASSAY
The in vitro assay described here is generally applicable in the search for compounds that effectively clear conformationally altered proteins present in food thereby preventing a number of degenerative diseases, where the accumulation of proteins seems to mediate the pathogenesis of these illnesses. By simulating lysosomes, where proteases hydrolyze proteins under acidic conditions, the in vitro brain homogenate assay is able to rapidly evaluate the efficacy of a variety of polyamines to induce degradation of PrPS'.
The in vitro assay which used scrapie infected brain homogenate to test for compounds which clear PrPs' could be modified to assay for compounds which would clear any conformationally altered protein. The assay is carried out by homogenizing the organ or tissue where the conformationally altered protein is present in the highest concentration. The pH of the homogenate is then reduced to less than 5.0 and preferably 4.0 or less. For example pancreatic tissue can be homogenized to produce an assay to test for compounds which clear amylin which is associated with type II Diabetes. Homogenized kidney could be used to test for compounds which clear (3z -microglobulin and homogenized heart or vascular tissue used to test for compounds which clear atrial natriuretic factor. Those skilled in the art will recognize other organs and tissue types which can be homogenized to test for other compounds which clear other conformationally altered proteins.
Besides using the in vitro assay to screen for potential drugs, the compounds found via the assay such as branched polyamines provide a new tool for exploring the conversion of a protein to conformationally altered protein, e.g. PrP~ into PrPS°. The mechanism by which branched polyamines render PrPs° susceptible to proteolysis, remains to be established. Whether the interaction of branched polyamines with PrPS' is reversible is unknown. In addition, we do not know whether branched polyamines are able to solubilize PrPs' without irreversibly denaturing the protein.
Whatever the mechanism by which branched polyamines interact with PrPs~, it is likely to be different from that found with chaotropes as well as denaturing detergents and solvents (Prusiner, S.B., Groth, D., Serban, A., Stahl, N. & Gabizon, R. Attempts to restore scrapie prion infectivity after exposure to protein denaturants. Proc. Natl. Acad. Sci. USA 90, 2793-2797 (1993)).
Using the assays described and disclosed here certain specific branched polyamines have been found which mediate the clearance of PrPS' from cultured cells under non-cytotoxic conditions.
These compounds offer the intriguing possibility of being added to a wide range of low pH food products to neutralize conformational altered proteins present. Since the compounds found act by stimulating normal cellular pathways of protein degradation to destroy PrPs°, this class of compounds would also likely be of value in the treatment of other degenerative and hereditary disorders where abnormally folded, wild-type or mutant proteins accumulate.
Such an approach may find merit in developing an effective therapeutics for one or more of the common, degenerative illnesses including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, frontotemporal dementia, adult onset diabetes mellitus and the amyloidoses (Beyreuther, K. &
Masters, C.L. Serpents on the road to dementia and death. Accumulating evidence from several studies points to the normal function of presenilin l and suggests how the mutant protein contributes to deposition of amyloid plaques in Alzheimer's disease. Nature Medicine 3, 723-725 (1997);
Masters, C.L. & Beyreuther, K. Alzheimer's disease. BMJ316, 446-448 (1998);
Selkoe, D.J. The cell biology of beta-amyloid precursor protein and presenilin 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. Nature 399, A23-31 (1999); Wong, P.C., et al. An adverse property of a familial ALS-linked SOD1 mutation causes motor neuron disease characterized by vacuolar degeneration ofmitochondria. Neuron 14, 1105-1116 (1995); Spillantini, M.G., Crowther, R.A., fakes, R., Hasegawa, M. & Goedert, M. a-Synuclein in filamentous inclusions of Lewy bodies from Parkinson's disease and dementia with Lewy bodies. Proc. Natl. Acad. Sci. USA
95, 6469-6473 (1998); Hutton, M., et al. Association of missense and 5'-splice-site mutations in tau with the inherited dementia FTDP-17. Nature 393, 702-705 (1998); Stone, M.J.
Amyloidosis: a final common pathway for protein deposition in tissues. Blood 75 , 531-545 (1990)).
Whether branched polyamines might also prove efficacious in a variety of inherited disorders where the accumulation of abnormal proteins is a hallmark of the illness remains to be established;
these genetic maladies include heritable forms of prion disease, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, frontotemporal dementia, Pick's disease and amyloidosis, as well as the triplet repeat diseases including Huntington's disease, spinal cerebellar ataxias and myotonic dystrophy (Fu, Y.-H., et al. An unstable triplet repeat in a gene related to myotonic muscular dystrophy. Science 255, 1256-1259 (1992); Group, T.H.s.D.C.R. A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. Cell 72, 971-983 (1993)).
Compounds identified via assays of the invention such as branched polyamines will find utility in preventing or delaying the onset of these genetic diseases where earners can often be identified decades in advance of detectable neurologic or systemic dysfunction.
The invention is based on the discovery that several dendritic polycations, including the starburst dendrimers SuperfectT"' (QIAGEN~, Valencia, CA), polyamidoamide (PAMAM), and the hyperbranched polycation polyethyleneimine (PEI), were surprisingly found to eliminate PrPs° 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 disease including the amyloidoses. The removal of PrPs° is dependent on both the concentration of dendritic polymer and length of exposure. Dendritic polymers were able to cleax PrPS° at concentrations which were not cytotoxic. Repeated exposures to heat-degraded starburst PAMAM
dendrimer or PEI caused a dramatic reduction in PrPs° levels which persisted for a month even after removal of the compound. Dendritic polycations did not appear to destroy purified PrPS' in vitro, and therefore may act through a generalized mechanism. Dendritic polycations represent a class of compounds which can be used as therapeutic agents in prion diseases and other disorders involving insoluble protein deposits, such as the amyloidoses.
EXAMPLES
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
METHODS AND MATERIALS
Chemicals. High molecular weight PEI was purchased from Fluka. DOTAP cationic lipid was purchased from Boehringer Mannheim and SuperFect transfection reagent was purchased from QIAGEN~. All other compounds were purchased from Sigma-Aldrich. All test compounds were dissolved in water at stock concentration of 3 mg/ml and filtered through a Millipore 0.22 m m filter.
Cultured cells. Stock cultures of ScN2a cells were maintained in MEM with 10%
FBS, 10% Glutamax (Gibco BRL), 100 U penicillin, and 100 mg/ml streptomycin (supplemented DME).
hrunediately prior to addition of test compounds, the dishes were washed twice with fresh supplemented DME media. After exposure to test compounds, dishes were drained of media and cells were harvested by lysis in 0.25-1 ml 20 mM Tris pH 8.0 containing 100 mM
NaCl, 0.5%
NP-40, and 0.5% sodium deoxycholate to obtain a total protein concentration of 1 mg/ml measured by the BCA assay. Nuclei were removed from the lysate by centrifugation at 2000 rpm for 5 min.
For samples not treated with proteinase K, 40 ~1 of whole lysate (representing 40 ~g total protein) was mixed with an equal volume of 2x SDS reducing sample buffer. For proteinase K digestion, 20 ~g/ml proteinase K (Boehringer Mannheim) (total protein:enzyme ratio = 50:1) was added, and the sample was incubated for 1 h at 37°C. Proteolytic digestion was terminated by the addition of Pefabloc to a final concentration of 5 mM. One ml samples were centrifuged at 100,000 x g for 1 h at 4°C, the supernatants were discarded, and the pellets were resuspended in 80 /.c1 of reducing SDS
sample buffer for SDS-PAGE.
Brain homogenates. Brain homogenates from RML scrapie-affected CD-1 mice (10%
(w/v) in sterile water) were prepared by repeated extrusion through syringe needles of successively smaller size, from 18 to 22 gauge. Nuclei and debris were removed by centrifugation at 1000 x g for min. The bicinchnoninic acid (BCA) protein assay (Pierce) was used to determine protein 5 concentration. Homogenates were adjusted to 1 mg/ml protein in 1 % NP-40.
For reactions, 0.5 ml homogenate was incubated with 25 ml 1.0 M buffer (sodium acetate for pH 3-6 and Tris acetate for pH 7-10) plus or minus 10 ml of polyamine stock solution (3 mg/ml) for 2 h at 37 ° C with constant shaking. The final pH value of each sample was measured directly with a calibrated pH electrode (Radiometer Copenhagen). Following incubation, each sample was neutralized with an equal volume 0.2 M HEPES pH 7.5 containing 0.3 M NaCI and 4% Sarkosyl. Proteinase K was added to achieve a final concentration of 20 ~cg/ml, and samples were incubated for 1 h at 37°C. Proteolytic digestion was terminated by the addition of Pefabloc to a final concentration of 5 ~M.
Ten ~cl of digested brain homogenate was mixed with equal volume 2x SDS sample buffer and analyzed by SDS-PAGE
followed by Western blotting.
Western blotting. Following electrophoresis, Western blotting was performed as previously described (Scott, M., et al. Transgenic mice expressing hamster prion protein produce species-specific scrapie infectivity and amyloid plaques. Cell 59, 847-857 (1989)).
Samples were boiled for 5 min and cleared by centrifugation for 1 min at 14,000 rpm in aBeckman ultrafuge. SDS-PAGE
was carned out in 1.5 mm, 12% polyacrylamide gels(Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T-4. Nature 227, 680-685 (1970)). Membranes were blocked with 5% non-fat milk protein in PBST (calcium- and magnesium-free PBS plus 0.1%
Tween 20) for 1 h at room temperature. Blocked membranes were incubated with primary 8073 polyclonal antibody (to detect MoPrP) (Serban, D., Taraboulos, A., DeArmond, S.J. & Prusiner, S.B. Rapid detection of Creutzfeldt-Jakob disease and scrapie prion proteins.
Neurology 40, 110-117 (1990)) or 3F4 monoclonal antibody (to detect MHM2 PrP) (Kascsak, R.J., et al. Mouse polyclonal and monoclonal antibody to scrapie-associated fibril proteins. J.
I~irol. 61, 3688-3693 (1987)) at 1:5000 dilution in PBST overnight at 4°C. Following incubation with primary antibody, membranes were washed 3 x 10 min in PBST, incubated with horseradish peroxidase-labeled secondary antibody (Amersham Life Sciences) diluted 1:5000 in PBST for 30 to 60 min at 4°C and washed again for 3 x 10 min in PBST. After chemiluminescent development with ECL reagent (Amersham) for 1 min, blots were sealed in plastic covers and exposed to ECL
Hypermax film (Amersham). Films were processed automatically in a Konica film processor.
Branched polyamines inhibit formation of nascent PrPs' and induce clearance of pre-existing PrPs' Western blots were probed with 3F4 monoclonal antibody which recognizes newly expressed MHM2 PrP. ScN2a cells were exposed to SuperFect for 3 h and harvested 3 d after removal of SuperFect. Gells were run on both undigested, control sample and a sample subjected to limited proteolysis. The samples were run in separate lanes 1-6 with a control and limited proteolysis sample for each of the 6 lanes as follows: Lane 1: DOTAP-mediated transfection. Lane 2: 30 ~cg/ml SuperFect, 5 ~cg pSPOX MHM2. Lane 3: 75 ~cg/ml SuperFect, 5 ~g pSPOX MHM2.
Lane 4: 150 ~cg/ml SuperFect, 5 ~g pSOX MHM2. Lane 5: 150 ~g/ml SuperFect, 10 ~g pSPOX
MHM2.
Lane 6: No addition of either transfection reagent or DNA. Forty ~1 of undigested brain homogenate was used in these studies while those samples subjected to limited digestion with proteinase K were concentrated 25-fold prior to SDS-PAGE. One ml of the digest were centrifuged at 100,000 x g for 1 h at 4 ° C and the pellets suspended in 80 ~l of SDS sample buffer prior to SDS-PAGE followed by Western blotting. Apparent molecular weights based on migration of protein standards are 34.2, 28.3, and 19.9 kDa.
All of the control lanes 1-6 show multiple bands as expected. However, of the samples subjected to limited proteolytic only lane 1 shows bands. Unexpectedly, all of the partially digested sample lanes 2-5 show no bands and as expected no bands in the partially digested lane 6. These results show the effect of using SuperFect in clearing PrPs'.
The blot described above was stripped of antibody, exposed to labeled 8073 and redeveloped. The antibody 3F4 used in Example 1 binds to PrP~ but not to PrPs'. However, 8073 binds to PrPs' and PrP~. Lanes 1, 2 and 3 show decreasing amounts of PrPs' and lanes 4 and 5 show no detectable PrPS'.
Gels were run on undigested controls 1-4 and as above, samples subjected to limited proteolysis. The lanes were as follows: Lane l: No SuperFect. Lane 2: 30 /.cg/ml SuperFect. Lane 3: 75 /.cg/ml SuperFect. Lane 4: 150 /.cg/ml SuperFect. ScN2a cells were exposed to SuperFect for 3 h and harvested 3 d after removal of SuperFect. Apparent molecular weights based on migration of protein standards are 33.9, 28.8, and 20.5 kDa. In that each sample was tested after the same time period the results show the dose-dependent effect of SuperFect on PrPs° removal. Lanes 1, 2 and 3 show decreasing amounts of PrPs° and lane 4 shows no detectable PrPs'.
To determine the time-dependent effect of SuperFect three different panels with four lanes each were prepared and run as follows: ScN2a cells were exposed to 7.5 ~cg/ml:
SuperFect (lanes 1-4), PEI (average molecular weight ~60,000)(lanes 5-8), or PAMAM, generation 4.0 (lanes 9-12).
Time of exposure times for each polyamine: 0 hours (lanes 1, 5, and 9), 4 hours (lanes 2, 6, and 10), 8 hours (lanes 3, 7, and 11), 16 hours (lanes 4, 8, and 12). All samples were subjected to limited proteolysis to measure PrPs~. Apparent molecular weights based on migration of protein standards are 38, 26, and 15 kDa. Lanes of each of the three panels show decreasing amounts of PrPs°.
In this example four panels A,B, C and D were created with panels having three double (control and test) lanes each. ScN2a cells were exposed to 1.5 ~g/ml: (A) SuperFect, (B) PEI
(average molecular weight 60,000), (C) PAMAM, generation 4.0, or (D) no addition. Cells were harvested: Lane 1, before addition; Lane 2, immediately following 1 week continuous exposure to test compounds; and Lane 3, three weeks after removal of test compounds. Minus (-) symbol denotes undigested, control sample and plus (+) symbol designates sample subjected to limited proteolysis. Apparent molecular weights based on migration of protein standards are 33.9, 28.8, and 20.5 kDa. Test lanes 3 in panel A showed slight PrPs' after three weeks and test lanes 3 in panels B
and C showed no detectable PrPs~ whereas PrPs° was present in all lanes in panel D.
Four separate gels were run to demonstrate the effect of adding chloroquine would have on PrPs° levels. The lanes 1 control and 3 where chloroquine was added show clear bands for PrPs°
whereas lanes 2 and 4 with no chloroquine show barely detectable amounts of PrPs°. The four lanes were prepared as follows: ScN2a cells were treated Lane 1: Control media. Lane 2: 7.5 ~g/ml PEI
(average molecular weight 60,000). Lane 3: PEI plus 100 ~cM chloroquine. Lane 4: PEI plus 30 ~cM NH4Cl. Chloroquine and NH4Cl were added 1 h prior to addition of PEI.
Cells were harvested 16 hours after addition of PEI. All samples shown were subjected to limited proteolysis to measure PrPs'. Apparent molecular weights based on migration of protein standards are 3 8, 26, and 15 kDa.
Eight lanes with SuperFect (+SF) and eight lanes without SuperFect (-SF) were prepared.
Lanes 1-8 of each group had an adjusted pH of 3.6" 4, 5, 6, 7, 8, 9 and 9.6.
In vitro mixture of crude mouse brain homogenates with SuperFect under a range of pH conditions was performed as described in methods (measured final pH of each sample denoted above the lanes). Addition of 60 ~g/ml SuperFect denoted as "+SF" and control with no addition as "-SF". All samples shown were subjected to limited proteolysis to measure PrPs'. Apparent molecular weights based on migration of protein standards are 30 and 27 kDa. All lanes ofthe -SF group showed PrPS' present. Lanes 3-8 of the +SF group showed PrPs'. However, lanes l and 2 with respective pH levels of 3.6 and 4.0 showed very slight detectable PrPs'. The results show that the ability of a blanched polycation such as SuperFect to clear PrPs' is pH dependent.
Sixteen different lanes were prepared as described. Lanes 1 and 2 were control lanes and each of lanes 3-16 contained a different compound as tested in Table 1. The test compounds were all polyamines. Thus, the results show removal of PrPs' from brain homogenate in vitro by various polyamines. Samples were incubated with polyamines at pH 3.6 and processed as described in Methods. Each polyamine was tested at 60 ~cg/ml concentration. Lanes 1 and 2:
control. Lane 3:
poly-(L)lysine. Lane 4: PAMAM, generation 0Ø Lane 5: PAMAM, generation 1Ø
Lane 6:
PAMAM, generation 2Ø Lane 7: PAMAM, generation 3Ø Lane 8: PAMAM, generation 4Ø
Lane 9: PAMAM-OH, generation 4Ø Lane 10: PPI, generation 2Ø Lane 11: PPI, generation 4Ø
Lane 12: linear PEI. Lane 13: high MW PEI. Lane 14: low MW PEI. Lane 15:
average MW PEI.
Lane 16: SuperFect. All samples shown were subjected to limited proteolysis to measure PrPs'.
Apparent molecular weights based on migration of protein standards are 30 and 27 kDa.Table 1.
Removal of PrPs~ by polymer compounds. IC50 = approximate concentration of polymer required to reduce PrPs' to 50% of control levels in ScN2a cells after exposure for 16 hours. All compounds were tested at 5 different concentrations. PrPS' levels were measured by densitometry of Western blot signals.
(Note that Table 1 includes information on the characteristics of compounds used but that the list does not correspond directly to lanes 1-16) Molecular Primary NH2 ICSp (ng/xnl) Weight groups 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 Low MW PEI 25,000 2,000 Average MW PEI 60,000 400 High MW PEI 800,000 80 Linear PEI 60,000 2,000 poly-(L)lysine 60,000 >500 10,000 SuperFect 400 Lanes 7, 8, 11 and 13 showed the best results, i.e. best ability to clear PrPs' under these conditions. Specifically, PAMAM generation 4.0 in lane 8 showed the best ability to clear PrPS' under these conditions whereas PAMAM-OH generation 4.0 showed almost no detectable ability to clear PrPs' and was comparable to the control.
Transfection of PrPs' Expressing Cells with Dendrimer Compounds Cells of neuronal origin expressing PrPs' were examined for the ability of compounds to suppress PrPs° formation.
Transfection Studies Stock cultures of N2a and ScN2a cells were maintained in MEM with 10% FBS, 10%
Glutamax (Gibco BRL), 100 U penicillin, and 100 ~cg/ml streptomycin. Cells from a single confluent 100 mm dish were trypsinized and split into 10 separate 60 mm dishes containing DME
plus 10% FBS, 10% Glutamax, 100 U penicillin, and 100 ~cg/ml streptomycin (supplemented DME) one day prior to transfection. Immediately prior to transfection, the dishes were washed twice with 4 ml supplemented DME media and then drained.
For DOTAP-mediated transfection, 15 ~g pSPOX MHM2 was resuspended in 150 ~cl sterile Hepes Buffered Saline (HBS) on the day of transfection. The DNA solution was then mixed with an equal volume of 333 ~cg/ml DOTAP (Boehringer Mannheim) in HBS in Falcon 2059 tubes and incubated at room temperature for 10 minutes to allow formation of DNA/lipid complexes.
Supplemented DME (2.5 ml) was added to the mixture, and this was then pipetted onto drained cell monolayers. The following day, the medium containing DNA/lipid was removed and replaced with fresh supplemented DME. Cells were harvested three days later.
For SuperfectT"'-mediated transfections/exposures, SuperfectT~~ with or without DNA was added to 1 ml supplemented DME in a Falcon 2059 tube to achieve the specific concentrations needed for each experiment. This mixture was pipetted up and down twice and then onto drained cell monolayers. After exposure for the indicated times, the medium containing SuperfectT"~ was removed and replaced with fresh supplemented DME. Cells were harvested at specified times after removal of SuperfectT~~.
Exposures to PPI (DAB-Am-8, Polypropylenimine octaamine Dendrimer, Generation 2.0 Aldrich 46,072-9), Intact PAMAM (Starburst (PAMAM)Dendrimer, Generation 4.
Aldrich 41,244-9), PEI (Sigma), poly-(L)lysine (Sigma), and poly-(D) lysine (Sigma) were performed as described above for SuperfectT"'.
Isolation of Protein from Treated Cells Cells were harvested by lysis in 1.2 ml of 20 mM Tris pH 8.0 containing 100 mM
NaCI, 0.5% NP-40, and 0.5% sodium deoxycholate. Nuclei were removed from the lysate by centrifugation at 2000 rpm for 5 min. This lysate typically had a protein concentration of 0.5 mg/ml measured by the BCA assay. For samples not treated with proteinase K, 40 ~l of whole lysate (representing 20 ~g total protein) was mixed with 40 ~l of 2x SDS sample buffer. For proteinase K
digestion, 1 ml of lysate was incubated with 20 ~g/ml proteinase K (total protein:enzyme ratio =
25:1) for 1 hr at 37°C. Proteolytic digestion was terminated by the addition of 8 ~1 of 0.5M PMSF
in absolute ethanol. Samples were then centrifuged for 75 min in a Beckman TLA-45 rotor at 100,000 x g at 4 ° C. The pellet was resuspended by repeated pipetting in 80 ~1 of 1X SDS sample buffer. The entire sample (representing 0.5 mg total protein before digestion) was loaded for SDS-PAGE.
Western Blot Analysis Immunoreactive PrP bands from the DOTAP-mediated transfection were detected before and after digestion with proteinase K with monoclonal antibody 3F4. The construct used to express PrPs° in the ScN2a cells is MHM2 a chimeric construct that differs from wild-type (wt) MoPrP 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 (SHa) PrP
sequence creates an epitope for 3F4 (Kascsak et al., (1987) J. ljirol. 61:3688-3693), which does not recognize endogenous wt MoPrP in ScN2a cells and hence facilitates specific detection of the transgene by Western blot.
Following electrophoresis, Western blotting was performed as previously described (Scott et al., (1989) Cell 59:847-857). Samples were boiled for 5 minutes and cleared by centrifixgation for 1 minute at 14,000 rpm in a Beckman ultrafuge. SDS-PAGE was carried out in 1.5 mm, 12%
polyacrylamide gels (Laemtnli (1970) Nature 227:661-665). Membranes were blocked with 5%
nonfat milk protein in PBST (calcium- and magnesium-free PBS plus 0.1% Tween 20) for 1 hour at room temperature. Blocked membranes were incubated with primary 8073 polyclonal or 3F4 monoclonal antibody at a 1:5000 dilution in PBST overnight at 4 °C.
Following incubation with primary antibody, membranes were washed 3 x 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 for 3x 10 minutes in PBST. After ehemiluminescent development with ECL reagent (Amersham) for 1 minute, blots were sealed in plastic covers and exposed to ECL Hypermax filin (Amersham). Films were processed automatically in a Konica film processor.
In contrast to DOTAP transfected cells, ScN2a cells transfected with varying concentrations of SuperfectT"~ and DNA did not appear to contain protease-resistant MHM2.
Close scrutiny revealed that, prior to protease digestion, SuperfectTM transfected samples express MHM2 bands which are not seen in the background pattern of the control sample. These observations indicate that MHM2 PrP was successfully expressed using SuperfectT"' transfection reagent, but conversion of MHM2 PrP~ to protease-resistant MHM2 PrPs° was inhibited by SuperfectT"'.
To examine whether SuperfectT"' had affected levels of preexisting PrPs' in ScN2a cells, the Western blot probed with 3F4 antibody was reprobed with polyclonal antibody 8073, which is able to recognize endogenous MoPrP. Remarkably, SuperfectTM caused the disappearance of preexisting MoPrPs° from ScN2a cells in a dose-dependent manner. After treatment with SuperfectT"~, PrPs' could not be detected in the nuclear fraction, pellet, supernatant, or media.
The concentration of SuperfectT"" required to fi>Zly remove preexisting PrPs° with a three hour exposure was 300 pg/ml, whereas 30 ~g/ml was sufficient to interfere with the formation of new MHM2 PrPs' within the same time frame.
Length of exposure dramatically influenced the ability of SuperfectT"' to remove PrPs' from ScN2a cells. Whereas a 3 hour exposure to 150 ~g/ml SuperfectT"" significantly lowered PrPs' levels in ScN2a cells, exposure for 10 min to the same dose of SuperfectT"' did not affect PrPs' levels.
When ScN2a cells were exposed to 2 pg/ml SuperfectT"' continuously for 1 week, PrPs' disappeared completely.
The conditions tested did not appear to be toxic for the cells. Neither 150 ~g/ml SuperfectT""
for 3 hrs nor 2 ~g/ml SuperfectT"' continuously for 1 week caused any obvious changes in cell morphology, viability, or growth as judged by phase contrast microscopy.
Elimination of PrPs' by repeated exposures to SuperfectT'"
The duration in the reduction in PrPs' levels after exposure to SuperfectT"' was examined, and it was shown that this reduction could persist for extended periods after removal of SuperfectT"' Following the exposure of ScN2a cells to a single dose of 150 ~g/ml SuperfectT"" for 3 hrs, PrPs' levels remained low for one week, but returned to near baseline levels after 3 weeks in culture without SuperfectT~A
In contrast, when ScN2a cells were exposed to 4 separate doses of SuperfectT"~
over the course of 16 days, very little PrPs' could be detected 4 weeks after the final exposure to SuperfectT"' This result offers hope that prolonged exposure to SuperfectT"' may lead to long term cure of scrapie infection in cultured cells.
SuperfectT"' does not destroy PrPs' directly The dendrimer SuperfectT"' was used to determine if it could exert a similar inhibitory effect on PrPs' in either crude brain homogenates or purified PrP 27-30 rods.
Brain homogenates from normal and scrapie-affected Syrian hamsters (10% (w/v) in sterile PBS) were prepared by repeated extrusion through syringe needles of successively smaller size, from 18 to 22 gauge. Nuclei and debris were removed by centrifugation at 1000 x g for 10 min. The bicinchnoninic acid (BCA) protein assay (Pierce) was used to determine protein concentration.
Homogenates were adjusted to 10 mg/ml protein with PBS and 50 p1 was added to 450 p1 of lysis buffer containing 100 mM NaCI, 1 mM EDTA, 0.55% sodium deoxycholate, 0.55%
Triton X-100, and 50 mM Tris-HCl pH 7.5. This mixture was then incubated with 0-300 ~g/ml SuperfectT"' for 3 hrs at 37 °C and then centrifuged for 10 min at 14,000 rpm in a Beckman Ultrafuge. The pellet was resuspended in 450 ~l lysis buffer without SuperfectT"'. Proteinase K
(Boehringer Mannheim) was added to achieve a final concentration of 20 ~g/ml, and thus the ratio of total protein/enzyme was 50:1. Samples were incubated for 1 h at 37 °C. Proteolytic digestion was terminated by the addition of 8 ~1 of 0.5 M PMSF in ethanol. Samples were then centrifuged for 75 min in a Beckman TLA-45 rotor at 100,000 x g at 4 °C. Undigested samples (10 1t1) were mixed with an equal volume of 2x SDS sample buffer. For digested samples, the pellet was resuspended by repeated pipetting in 100 ~l lx SDS sample buffer. Twenty ~1 (equivalent to 100 ~g of total protein prior to proteinase K
digestion) of each sample was loaded for SDS-PAGE.
PrP 27-30 rods were purified from scrapie-affected Syrian hamster brains and previously described (Prusiner et al., (1983) Cell 35:349-358). Purified rods (3.5 ~g/ml) were incubated with or without 900 ~g/ml SuperfectrM in 100 ~l supplemented DME. After 16 hrs at 37 °C, the suspension was centrifuged at 100,000 x g at 4 °C. The pellet was resuspended in 500 ~1 of buffer containing 1 mg/ml BSA, 100 mM NaCI, 1 mM EDTA, 0.55% sodium deoxycholate, 0.55% Triton X-100, and 50 mM Tris-HCl pH 7.5. Proteinase K was added to achieve a final concentration of 20 pg/ml. Samples were incubated for 1 h at 37 ° C. Proteolytic digestion was terminated by the addition of 8 p.1 of 0.5 M Pefabloc (Boehringer Mannheim). Samples were then centrifuged for 75 min at 100,000 x g at 4 °C. Undigested samples (50 p1) were mixed with an equal volume of 2x SDS
sample buffer. For digested samples, the pellet was resuspended by repeated pipetting in 100 p1 lx SDS sample buffer. Forty p1 of each sample was loaded for SDS-PAGE.
When SuperfectT~~ was mixed with either crude homogenates of scrapie-affected Syrian hamsters or with purified Syrian hamster PrP 27-30, there was no significant change in the level of proteinase K-resistant PrPS°. These results suggest that the removal of PrPS' from ScN2a cells by SuperfectTM depends on the presence of intact cellular machinery.
Clearance of PrPS' levels by other dendritic polycations The SuperfectT"~ compound is a high molecular weight component of heat-degraded PAMAM Starburst dendrimers, which is a cationic, highly-branched, monodisperse polymers (Tang et al., (1996) Bioconjugate Chem. 7:703-714). To identify other potentially usefi~l anti prion therapeutic agents, we screened three other dendritic polycations and two linear cationic polymers for their ability to clear PrPs' from ScN2a cells. Among the dendritic macromolecules tested, polyetheleneimine (PEI) was the most potent, removing the majority of PrPs' from ScN2a cells after 3 hrs when used at a concentration of 10 ~g/ml. Intact PAMAM displayed a potency comparable to Superfectr"', removing approximately half of the detectable PrPs~ when used at a concentration of 50 pg/ml. In contrast, the dendrimer polypropyleneimine (PPI), poly-(L)lysine, and the linear polycation poly-(D)lysine failed to reduce PrPs° levels at concentrations between 10-50 ~g/ml.
These results demonstrate that a branched polymeric architecture is required to clear PrPs'.
Furthermore, exposure of ScN2a cells to either PEI or intact PAMAM for one week at a concentration of 1.5 ~cg/ml completely removes PrPs~, effectively curing the cells of scrapie infection.
While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted 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, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.
An advantage of the invention is that proteins such as prions can be rendered non-infectious without the need for extreme conditions such as exposure to heat over long periods of time, e.g. 1-10 hours at 100°-200°C.
A feature of the invention is that compositions can be useful while containing only very low concentrations of polycationic dendrimers, e.g. 1 % to 0.001 %.
Another aspect of the invention is that capsules made with bovine gelatin can be certified prion free.
Another aspect of the invention is that drugs produced from cell cultures treated with polycationic dendrimers can be certified prion free.
Still another aspect of the invention is that medical devices being reused after exposure to blood or brain tissue can be certified prion free.
Still another aspect of the invention is that hospitals, operating rooms and the devices and equipment within them can be certified prion free by contacting them with polycationic dendrimers at standard temperatures and pressures. A pharmaceutical composition for the treatment of insoluble protein deposit formation 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 may have multiple charged branches. The branches may have the same chemical structure, or the branches may vary in structure within a single molecule. Examples of polymers that may be used in such a pharmaceutical composition include polypropylene imine, polyethyleneimine (PEI) poly(4'-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, anti-inflammatory agent, an antioncogenic agent, an antiviral agent, and the like.
The present invention also provides a method of enhancing clearance of a disease related conformation of a protein from cells by contacting cells with a branched polycation for a time sufficient to enhance the rate of clearance of a disease related conformation of a protein from the 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 such disease related proteins include PrPs°, APP, A(3 peptide, a-1-antichymotrypsin. The method is thus useful for subjects suffering from disorders such as bovine spongiform encephalopathy, Creutzfeldt-Jacob Disease, fatal familial insomnia, GSS for Gerstmann-Straussler-Scheinker Disease, kuru, scrapie, Alzheimer's Disease, Frontal temporal dementia, Huntington's disease, ALS, Pick's disease, Parkinson's disease, Diabetes Type II, multiple myeloma, familial amyloidotic polyneuropathy, medullary carcinoma of thyroid, chronic renal failure, congestive heart failure, senile cardiac and systemic amyloidosis, chronic inflammation, atherosclerosis, and familial amyloidosis. The branched polycation can be administered to a subject in an amount non-toxic to the subject, for example a dosage of 0.001 mg to 1 mg/kg body weight per day. The polycation may be administered in a single dosage form, or it may be repeatedly administered to the subject. The branched polycation may also be administered prophylactically to prevent the formation of the disease conformation of these proteins.
The invention also provides a food composition for the preventing insoluble protein deposit formation in an animal, where the food contains a therapeutically effective amount of a branched canon which enhances clearance of conformationally altered protein. The food can be any food product, including solid foods such as meat (e.g., beef or lamb) and liquid foods such as vinegar, oil, and condiments such as steak sauce and ketchup. The branched polycation is allowed to contact the food prior to ingestion for a time sufficient to allow clearance of the conformationally altered proteins.
The present invention also provides a method of preventing a farm animal from acquiring a disease associated with a conformationally altered protein by feeding the animals animal feed containing a branched polycation. The feed containing the branched polycation may be synthetically produced, and fed directly to the animals, or the branched polycation may be introduced to a natural food source, e.g., the animal feed is grass and the branched polycation is sprayed on the grass or introduced to the grass through a plant fertilizer. One aspect of this embodiment of the invention is a method of preventing disease caused by ingestion of contaminated food products by feeding animals foods containing branched polycations.
The present invention also provides a method of enhancing clearance of a disease related conformation of a protein from a meat food product by contacting the meat with a compound which enhances clearance of a conformationally altered protein at a pH of 5 or less for a time sufficient to allow for destruction of conformationally altered protein.
An advantage of the invention is that conformationally altered protein such as prions can be rendered non-infectious with a method which need only consist of applying a polycationic dendrimer preferably held at a pH of 5.0 or less.
Another aspect of the invention is soaps, surgical scrubs, 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 which might be present on surgical instruments, knives and/or other tools or equipment used by butchers, particularly those used in the butchering of cows or other animals which might be infected with prions.
A feature of the invention is that compositions of the invention can be effective in activating prions when the polycationic dendrimers are present in very low concentrations, e.g. 1 % to 0.001 or less.
These and other aspects, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the compounds, and assay method more fully described below.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
Before the present methods, objects and compositions are described, it is to be understood that this invention is not limited to the particular steps, devices or components described and, as such, may of course vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since 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 belongs.
Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the 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 connection with which the publications are cited.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
DEFINITIONS
The term "detergent" is used to mean any substance that reduces the surface tension of water. The detergent may be a surface active agent which concentrates at oil-water interfaces, exerts emulsifying action and thereby aids in removing soils e.g. common sodium soaps of fatty acids. A
detergent may be anionic, cationic, or monionic depending on their mode of chemical action.
Detergents include linear alkyl sulfonates (LAS) often aided by "builders." A
LAS is preferably an _g_ alkyl benzene sulfonate ABS which is readily decomposed by microorganisms (biodegradable). The LAS is generally a straight chain alkyl comprising 10 to 30 carbon atoms. The detergent may be in a liquid or a solid form.
The term "conformationally altered protein" is used here to describe any protein which has a three dimensional conformation associated with a disease. The conformationally altered protein may cause the disease, be a factor in a symptom of the disease or appear as a result of other factors. The conformationally altered protein appears in another conformation which has the same amino acid sequence. In general, the conformationally altered protein formed is "constricted" in conformation as compared to the other "relaxed" conformation which is not associated with disease. The following is a non-limiting list of diseases with associated proteins which assemble two or more different conformations wherein at least one conformation is an example of a conformationally altered protein.
Disease Insoluble Proteins Alzheimer's Disease APP, A~i peptide, a 1-antichymotrypsin, tau, non-A(3 component, presenillin 1, presenillin apoE
Prion diseases, Creutzfeldt Jakob disease, scrapie and bovine spongiform encephalopathy prps ALS SOD and neurofilament Pick's disease Pick body Parkinson's disease a-synuclein in Lewy bodies Frontotemporal dementia tau in fibrils Diabetes Type II Amylin Multiple myeloma--plasma cell dyscrasias IgGL-chain Familial amyloidotic polyneuropathy Transthyretin Medullary carcinoma of thyroid Procalcitonin Chronic renal failure ~i2--microglobulin Congestive heart failure Atrial natriuretic factor Senile cardiac and systemic amyloidosis Transthyretin 4~ Chronic inflammation Serum amyloid A
Atherosclerosis ApoAl Familial amyloidosis Gelsolin Huntington's disease Huntingtin The term"acid" is used to describe any compound or group of compounds which has one or more characteristics of (a) sour taste; (b) turns litmus dye red; (c) reacts with certain metals to form a salt; (d) reacts with certain bases or a.lkalines to form a salt. An acid comprises hydrogen and in water undergoes ionization so that H30+ ions are formed - also written as H+
and referred to as hydronium ions or simply hydrogen ions. Weak acids such as acetic acid or carbonic acid may be used as may strong acids such as hydrochloric acid, nitric acid and sulfixric acid. In compositions of the invention the acid is preferably present in a concentration so as to obtain a pH of 5 or less, more preferably 4 or less and still more preferably 3.5 ~ 1.
The terms "sterilizing", "making sterile" and the like are used here to mean rendering something non-infectious or rendering something incapable of causing a disease. Specifically, it refers to rendering a protein non-infectious or incapable of causing a disease or the symptoms of a disease. Still more specifically, it refers to rendering a conformationally altered protein (e.g. PrPS°
known as prions) incapable of causing a disease or the symptoms of a disease.
By "effective dose" or "amount effective" is meant an amount of a compound sufficient to provide the desired sterilizing 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 which might be present. Polycations of the invention or more specifically polycationic dendrimer compounds of the invention could be mixed with a material in an amount in a range 1 to 500 ~g of dendrimer per ml or mg of material being sterilized. The concentration is sufficient if the resulting composition is effective in decreasing the infectivity of conformationally altered proteins such that the treated material over time would 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 effective dose or concentration range needed to sterilize can vary considerably. It is also pointed out that the dose needed to treat an amount of material may vary somewhat based on the pH the treatment is carried out at and the amount of time the compound is maintained in contact with the material at the desired low pH (e.g., 5.0 or less) level.
The term "LDSO" as used herein is the dose of an active substance that will result in 50 percent lethality in all treated experimental animals. Although this usually refers to invasive administration, such as oral, pa,renteral, and the like, it may also apply to toxicity using less invasive methods of administration, such as topical applications of the active substance.
The term "amine-terminated" includes primary, secondary and tertiary amines.
The terms "PrP protein", "PrP" and like are used interchangeably herein and shall mean both the infectious particle form PrPs° known to cause diseases (spongiform encephalopathies) in humans and animals and the noninfectious form PrP~ which, under appropriate conditions is converted to the infectious PrPs° form.
The terms "prion", "prion protein", "PrPs°protein" and the like are used interchangeably herein to refer to the infectious PrPS° form of a PrP protein, and is a contraction of the words "protein" and "infection." Particles are comprised largely, if not exclusively, of PrPs° molecules encoded by a PrP gene. Prions are distinct 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 known to affect humans are (1) kuru, (2) Creutzfeldt-Jakob Disease (CJD), (3) Gerstmann-Straussler-Scheinker Disease (GSS), and (4) fatal familial insomnia (FFI). As used herein "prion" includes all forms of prions causing all or any of these diseases or others in any animals used - and in particular in humans and domesticated farm animals.
The term "PrP gene" is used herein to describe genetic material which expresses proteins including known polymorphisms and pathogenic mutations. The term "PrP gene"
refers generally to any gene of any species which encodes any form of a prion protein. Some commonly known PrP
sequences are described in Gabriel et al., Proc. Natl. Acad. Sci. USA 89:9097-9101 (1992) and U.S.
Patent No. 5,565,186, incorporated herein by reference to disclose and describe such 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, it being recognized that the terms include other such PrP genes that are yet to be discovered. The protein expressed by such a gene can assume either a PrP~ (non-disease) or PrPs' (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 may (1) include a transgene as described herein; (2) have and ablated endogenous prion protein gene; (3) have a high number of prion protein gene from a genetically diverse species; and/or (4) be a hybrid with an ablated endogenous prion protein gene and a prion protein gene from a genetically diverse species.
Different combinations of 1-4 are possible, e.g., 1 and 2. The mammals from which standardized pr~ion preparations are obtained exhibit clinical signs of CNS dysfunction as a result of inoculation with priors and/or due to developing the disease of their genetically modified make up, e.g., high copy number of prior protein genes. Standardized prior preparations and methods of making such are described and disclosed in U.S. Patent 5,908,969 issued June l, 1999 and application serial no. 09/199,523 filed 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 formation of neuritic plaques comprising amyloid ~i protein, primarily in the hippocampus and cerebral cortex, as well as impairment in both learning and memory.
"AD" as used herein is meant to encompass both AD as well as AD-type pathologies.
The term "AD-type pathology" as used herein refers to a combination of CNS
alterations including, but not limited to, formation of neuritic plaques containing amyloid ~3 protein in the hippocampus and cerebral cortex. Such AD-type pathologies can include, but are not necessarily limited to, disorders associated with aberrant expression and/or deposition of APP, overexpression of APP, expression of aberrant APP gene products, and other phenomena associated with AD. Exemplary AD-type pathologies include, but are not necessarily limited to, AD-type pathologies associated with Down's 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 AD, particularly such an event that is readily assessable in an animal model. Such events include, but are not limited to, amyloid deposition, neuropathological developments, learning and memory deficits, and other AD-associated characteristics.
The term "cerebral amyloid angiopathy" (abbreviated herein as CAA) as used herein refers to a condition associated with formation of amyloid deposition within cerebral vessels which can be complicated by cerebral parenchymal hemorrhage. CAA is also associated with increased risk of stroke as well as development of cerebellar and subarachnoid hemorrhages (Vinters (1987) Stroke 18:311-324; Haan et al. (1994) Dementia 5:210-213; Itoh et al. (1993) J.
Neurol. Sci. 116:135-414). CAA can also be associated with dementia prior to onset of hemorrhages. The vascular amyloid deposits associated with CAA can exist in the absence of AD, but are more frequently associated with AD.
The term "phenomenon associated with cerebral amyloid angiopathy" as used herein refers to a molecular, structural, or functional event associated with CAA, particularly such an event that is readily assessable in an animal model. Such events include, but are not limited to, amyloid deposition, cerebral parenchymal hemorrhage, and other CAA-associated characteristics.
The term "(3-amyloid deposit" as used herein refers to a deposit in the brain composed of A(3 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-Straussler-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 a Syrian hamster;
SHaPrP for a Syrian hamster prion protein;
PAMAM for polyamidoamide dendrimers PEI for polyethyleneimine PPI for polypropyleneimine PrPs~ for the scrapie isoform of the prion protein;
PrP~ for the cellular contained common, normal isoform of the prion protein;
PrP 27-30 or PrPs~ 27-30 for the treatment or protease resistant form of PrPs~;
MoPrPs' for the scrapie isoform of the mouse prion protein;
N2a for an established neuroblastoma cell line used in the present studies;
ScN2a for a chronically scrapie-infected neuroblastoma cell line;
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 rendering conformationally altered proteins non-infective. The compositions are preferably low pH solutions comprised of a non toxic weak acid such as acetic acid having dissolved therein a branched polycation. Preferred compositions of the invention are in the form of aqueous or alcohol solutions which are comprised of a branched polycation, an antibacterial, an antifungal and an antiviral compound. The compositions are coated on, mixed with, injected into or otherwise brought into contact with a material to be sterilized. The composition is applied in a manner so that the branched polycation is maintained at a low pH (e.g. ~ or less and preferably 3.5 t 1) in an amount of 1 ~cg or more polycation per ml or mg of material to be sterilized. The composition is maintained in the desired pH range at normal temperature (e.g., 15 ° C to 30 ° C) for a sufficient period of time (e.g. 1 hour to 1 week) to cause conformationally altered protein present on or in the material to be destroyed (e.g. hydrolyzed) or rendered non-infective. Preferred compositions of the invention are useful in cleaning and sterilizing and may be comprised of a polycationic dendrimers, a detergent, and an acid proving a pH of about 3.5 ~1.
DENDRIMER COMPOUNDS WHICH CLEAR PRIONS
Dendrimers are branched compounds also known as "starburst" or "star" polymers due to a characteristic star-like structure (see Figure 1). Dendrimers of the invention are polymers with structures built from AB" monomers, with n>_2, and preferably n=2 or 3.
Such 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. Dendrimers may be cationic (full generation dendrimers) or anionic (half generation dendrimers). For a review on the general synthesis, physical properties, and applications of dendrimers, see, e. g. , 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, 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. Pat. 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 hereby incorporated by reference to disclose and describe such compounds.
Dendrimers typically have tertiary amines which have a pKa of 5.7. The dendrimers can optionally be chemically or heat treated to remove some of the tertiary amines. Other suitable canons include polypropylene imine, polyethyleneimine (PEI), which has tertiary amines with a pKa of 5.9, and poly(4'-aza-4'-methylheptamethylene D-glucaramide), which has tertiary amines with a pKa of 6Ø The cationic dendrimer is preferably dissolved in the low pH solvent such as vinegar in a concentration of 0.0001 % or more, preferably 0.01 % or more and more preferably about 1 % .
Preferably, the dendrimers for use in the invention are polyamidoamines (hereinafter "PAMAM"). PAMAM dendrimers are particularly biocompatible, since polyamidoamine groups resemble peptide bonds of proteins.
Dendrimers are prepared in tiers called generations (see generations 0, l and 2 in Figure 1) and therefore have specific molecular weights. The full generation PAMAM
dendrimers have amine terminal groups, and are cationic, whereas the half generation dendrimers are carboxyl terminated. Full generation PAMAM dendrimers are thus preferred for use in the present invention. PAMAM dendrimers may be prepared having different molecular weights and have specific values as described in Table 1 below for generations 0 through 10.
TABLE A
LIST OF PAMAM DENDRIMERS AND THEIR
MOLECULAR WEIGHTS (Ethylene Diamine core, amine terminated), GENERATION TERMINAL GROUPS MO L. WT. /mole 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 PAMAM dendrimers generations 0 through 10 range from 4 to 4,096, with molecular weights of from 517 to 934,720.
PAMAM dendrimers are available commercially from Aldrich or Dendritech.
Polyethyleneimine or polypropylene dendrimers or quaternized forms of amine-terminated dendrimers may be prepared as described by Tomalia et. al, Angew, Chem. Int. Ed. Engl., 29:138-175 (1990) incorporated by reference to describe and disclose methods of making dendrimers.
STERILIZING COMPOSITIONS
Examples provided here show that highly-branched polycations, e.g. dendrimer compounds, affect the extent and distribution of PrPs° 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 detectable PrP S~ in cells and brain homogenates. Thus, 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 in a low pH
environment, (e.g. a polycationic dendrimer) in solution, suspension or mixture.
STERILIZING FORMULATIONS
Sterilizing compositions of the invention preferably contain highly branched polycations, e.g. polycationic dendrimer, in a concentration from 0.0001 to 10% of the formulation. The following methods and excipients are merely exemplary and are in no way limiting.
In addition to including the compound such as a highly branched cationic compound in the formulation it is important to maintain that compound in a low pH
environment. Any number of known acids or mixtures of acids could be used with the invention. Non-limiting examples of commercially available products which could be supplemented with the cationic compounds are described below. In these formulations the percentage amount of each ingredient can vary. In general a solvent ingredient (e.g. water or alcohol) is present in amounts of 40% to 100% and the last listed ingredient is present in a range of 0.5 % to 5 % . The other ingredients are present in an amount in a range of 1 % to 60 % and more generally 5 % to 20 % . In each case the polycationic compounds of the invention are added in amounts of about 0.01 %
to 5 % and preferably 0.1 % to 2 % and more preferably about 1 % . The amount added is an amount needed to obtain the desired effect.
Component wt %
acid 90 - 99.99 polycationic dendrimer0.01 - 10 Component wt %
water 10 - 99 acid 1- 20 polycationic dendrimer0.01 - 10 Component wt %
water 10 - 98 acid 1 - 20 detergent 1 - 20 polycationic dendrimer0.01 - 5 Component wt %
water 10 - 98 acetic acid 1 - 20 linear alkyl sulfonate 1 - 20 polycationic dendrimer 0.01 - 5 Component wt %
water 1 - 98 alcohol 0 - 98 acid 1 - 20 detergent 1 - 20 polycationic dendrimer0.1 - 5 Component wt %
water 1 - 99 acid 1 - 20 antibacterial 0.1 - 5 detergent 1 - 20 polycationic dendrimer0.1 - 5 Component wt %
water 3 - 98.889 antimicrobial active 0.001- 5 agent anionic surfactant 1 - 80 protein donating 0.1 - 12 agent polycationic dendrimer 0.01 - 5 Component wt Polycationic Dendrimer0.5 Ethanol 74.0 Benzalkonium chloride0.2 CAE 0.02 Glycerine 1.0 Chain silicone 0.5 Triglyceride 0.5 Lactic acid 10.0 Purified water 13.28 Component wt Polycationic Dendrimer1.0 Ethanol 75.0 Benzalkonium chloride0.2 CAE 0.02 Glycerine 1.0 Cyclic silicone 0.2 Triglyceride 0.3 Acetic Acid 20.0 Purified water 2.28 Component wt Polycationic Dendrimer0.25 Ethanol 74.0 Chlorhexedine gluconate0.75 Benzalkonium chloride0.2 CAE 0.02 Glycerine 2.0 Chain silicone 0.2 Cyclic silicone 0.2 Triglyceride 0.3 Acetic Acid 20.0 Purified water 2.08 Component wt Polycationic Dendrimer0.1 Ethanol 75.0 Chlorhexedine gluconate0.9 Benzalkonium chloride0.2 CAE 0.02 Glycerine 1.0 Chain silicone 0.5 Cyclic silicone 0.5 Triglyceride 0.3 Lactic acid 14.0 Purified water 7.98 Component wt Polycationic Dendrimer0.01 Ethanol 75.0 Benzalkonium chloride0.2 CAE 0.02 Glycerine 2.0 Chain silicone 0.99 Cyclic silicone 2.0 Triglyceride 3.0 Lactic acid 9 Purified water 7.78 Component wt Polycationic Dendrimer1 Ethanol 75.0 Chlorhexedine gluconate0.2 Benzalkonium chloride0.2 CAE 0.02 Glycerine 0.8 Chain silicone 0.2 Cyclic silicone 0.2 Triglyceride .38 Acetic acid 10 Purified water 12 Component wt Polycationic Dendrimer0.001 Ethanol 75.99 Chlorhexedine gluconate0.2 CAE 0.02 Glycerine 1.0 Chain silicone 0.2 Triglyceride 0.3 Lactic acid 14 Purified water 8.28 Component wt Polycationic Dendrimer1 Ethanol 75.0 Benzalkonium chloride0.2 CAE 0.02 l, 3-butylene glycol 1.0 Metylphenyl polysiloxane0.2 Isopropyl myristate 0.3 (IPM) ~ Purified water ~ 22.28 By using the disclosure provided here and other information such as taught in U.S.
Patents 5,767,054; 6,007,831; 5,830,488; 5,968,539; 5,416,075; 5,296,158; and patents and publications cited therein those skilled in the art can produce countless other formulations of the invention. Further, such formulations can be used as described in such publications and can be packaged in any suitable container or dispenser device, e.g. taught in 5,992,698.
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 nontoxic at a dosage of 0.1 mg/mouse, which is an LD
So of less than one at 40 mg/Kg. Various nutrient formulations and/or injectable formulations of the type known to those skilled in the art can be used to prepare formulations for treating 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 accomplished by adding any desired acid. If desired, the pH can be raised to a normal level after treatment is complete, i.e. after a sufficient amount of any conformationally altered protein present are destroyed.
Compounds effective in sterilizing compositions containing conformationally altered proteins are determined via a cell culture assay and an organ homogenate assay each of which is described below in detail.
ScN2a CELL BASED ASSAY
Efforts were made to optimize the transfection of ScN2a cells with pSPOX
expression plasmids (Scott, M.R., Kohler, R., Foster, D. & Prusiner, S.B. Chimeric prion protein expression in cultured cells and transgenic mice. Protein Sci. l, 986-997 (1992)). In connection with those effects an evaluation was made of a transfection protocol that used SuperFect reagent (QIAGEN~). It was found that epitope-tagged (MHM2) PrPs° (Scott, M.R., Kohler, R., Foster, D. & Prusiner, S.B.
Chimeric prion protein expression in cultured cells and transgenic mice.
Protein Sci. 1, 986-997 WU 00/72851 CA 02375237 2001-11-26 pCT/US00/14353 (1992)) could not be detected in ScN2a cells following SuperFect-mediated transfection, whereas MHM2 PrPs' was efficiently formed when a cationic liposome method for DNA
delivery was used.
Close scrutiny revealed that, prior to protease digestion, SuperFect transfected samples expressed MHM2 bands, which are not seen in the background pattern of an untransfected sample. The 3F4 monoclonal antibody does not react with MoPrP but does exhibit high background staining on Western blots of mouse ScN2a cells. Increased immunostaining in the 20-30 kDa region was observed compared to the non-transfected sample. These observations led us to conclude that MHM2 PrP was successfully expressed using SuperFect transfection reagent, but that conversion of MHM2 PrP~ to protease-resistant MHM2 PrPs° was inhibited by SuperFect.
To investigate this apparent inhibition, a Western blot was reprobed with 8073 polyclonal antiserum to detect endogenous MoPrPs°, the presence of which is diagnostic for prion infection in ScN2a cells (Butler, D.A., et al. Scrapie-infected murine neuroblastoma cells produce protease-resistant prion proteins. J. Virol. 62, 1558-1564 (1988)). Surprisingly, it was found that the SuperFect treated ScN2a cells no longer contained detectable quantities of MoPrPs° - also confirmed in Western blots. To investigate the mechanism by which SuperFect reduced the level of pre-existing PrPs° in chronically infected ScN2a cells, measurements were made of endogenous PrPs' in ScN2a cells exposed to various concentrations of SuperFect in the absence of plasmid DNA. The results showed that treatment with SuperFect (a branched polycation) caused the disappearance of PrPs°
from ScN2a cells in a dose-dependent manner. The concentration of SuperFect required to eliminate >95% of pre-existing PrPs° with a three hour exposure was found to be about 150 ~cg/ml. Duration of treatment also influenced the ability of SuperFect to remove PrPs°
from ScN2a cells: exposure to 150 /.cg/ml SuperFect for 10 min did not affect PrPs° levels, whereas 7.5 /,cg/ml SuperFect eliminated all detectable PrPs' with a t'/Z= 8 h.
SuperFect is a mixture of branched polyamines derived from heat-induced degradation of a PAMAM dendrimer (Tang, M.X., Redemann, C.T. & Szoka, F.C.J. In vitro gene delivery by degraded polyamidoamine dendrimers. Bioconjug. Chem. 7, 703-714 (1996)).
Knowing this structure the ability of several other branched and unbranched polymers to eliminate PrPS° from ScN2a cells (Table 1). The branched polymers investigated include various preparations of PEI, as well as intact PAMAM and PPI dendrimers. Dendrimers are manufactured by a repetitive divergent growth technique, allowing the synthesis of successive, well-defined "generations" of homodisperse structures (Figure 1). The potency of both PAMAM and PPI dendrimers in eliminating PrPs~ from ScN2a cells increased as the generation level increased. The most potent compounds with respect to eliminating PrPs° were PAMAM generation 4.0 and PPI generation 4.0, whereas PAMAM
generation 1.0 showed very little ability to eliminate PrPS° (Table 1).
Similarly, a high MW fraction of PEI was more potent than low MW PEI.
From the foregoing data, it is clear that for all three branched polyamines tested, increasing molecular size corresponded to an increased potency for eliminating PrPs° . To determine whether this trend was directly attributable to increased surface density of amino groups on the larger molecules, PAMAM-OH generation 4.0 was tested. This is a dendrimer that resembles PAMAM
generation 4.0 except that hydroxyls replace amino groups on its surface.
Unlike PAMAM
generation 4.0, PAMAM-OH generation 4.0 did not cause a reduction of PrPs° levels even at the highest concentration tested ( 10 mg/ml), establishing that the amino groups are required for the elimination of PrPs' by PAMAM (Table 1).
In an effort to assess the contribution of the branched architecture to the clearing ability of polyamines for PrPs°, the linear molecules poly-(L)lysine and linear PEI were also tested. Both of these linear compounds were less potent than a preparation of branched PEI
with similar average molecular weight (Table 1), establishing that a branched molecular architecture optimizes the ability of polyamines to eliminate PrPs°, presumably because the branched structures achieve a higher density of surface amino groups.
Kinetics of PrPSc elimination by polyamines.
The preceding results demonstrate the potent ability of branched polyamines to clear PrPs°
from ScN2a cells within a few hours of treatment. The utility of these compounds to act as therapeutics for treatment of prion disease was tested by determining whether they were cytotoxic for ScN2a cells, using as criteria cell growth, morphology, and viability as measured by trypan blue staining. None of the compounds was cytotoxic to ScN2a cells after exposure for one week at concentrations up to 7.5 ~g/ml. To determine whether branched polyamines can cure ScN2a cells of 2~ scrapie infection without affecting cell viability, the kinetics of prion clearance was examined in the presence of a non-cytotoxic concentration (7.5 ~g/ml) of three different branched polyamines. ScN2a cells were exposed to SuperFect, PEI, or PAMAM generation 4.0 for varying periods of time. The kinetics of PrPs' elimination were assessed by Western blotting. All three compounds caused a substantial reduction in PrPS' levels after 8-16 h of treatment, and of the three compounds, PEI
appeared to remove PrPs' most quickly, with a t'/2= 4 h.
Curing neuroblastoma cells of scrapie infection.
The above results show that it is possible to reverse the accumulation of PrPS° in ScN2a cells under non-cytotoxic conditions. It was also found that extended exposure to even lower levels WO 00/72851 CA 02375237 2001-11-26 pCT/US00/14353 of the branched polyamines ( 1.5 ~g/ml) was sufficient to eliminate PrPS°. Based on these findings, this protocol was used to determine whether the severe reduction in PrPs~
levels following exposure to branched polyamines would persist after removal of the compounds. Following the exposure of ScN2a cells to a 1.5 ~g/ml SuperFect for 1 week, PrPs~ was reduced to <1 % of the baseline level, but then increased back to ~5% of the baseline level after 3 additional weeks in culture in the absence of polyamine. In contrast, following exposure to 1.5 ~g/ml of either PEI or PAMAM generation 4.0 for 1 week, PrPSc was completely eliminated and did not return even after 3 weeks in culture without polyamines. A more intensive course of treatment with 1.8 ~cg/ml SuperFect for 9 d also cured ScN2a cells of scrapie infection fully, manifested by the absence of PrPs° 1 month after removal of SuperFect.
Evidence for polyamines acting within an acidic compartment.
The above results showed the potent activity of branched polyamines in rapidly clearing scrapie prions from cultured ScN2a cells. Based on these results the mechanism by which these compounds act was investigated. All of the compounds which effect removal of PrPSc from ScN2a cells are known to traffic through endosomes (Boussif, O., et al. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethyleneimine.
Proc. Natl. Acad. Sci.
U.S.A. 92, 7297-7301 (1995); Haensler, J. & Szoka, F.C.J. Polyamidoamine cascade polymers mediate efficient transfection of cells in culture. Bioconjug. Chem. 4, 372-379 (1993)). Since PrP~
is converted into PrPs° in caveolae-like domains (CLDs) or rafts (Gorodinsky, A. & Hams, D.A.
Glycolipid-anchored proteins in neuroblastoma cells form detergent-resistant complexes without caveolin. J. Cell 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 colocalization of the cellular and scrapie prion proteins in caveolae-like membranous domains.
Proc. Natl. Acad. Sci.
USA 93, 14945-14949 (1996); Kaneko, K., et al. COOH-terminal sequence ofthe cellular prion protein directs subcellular trafficking and controls conversion into the scrapie isoform. Proc. Natl.
Acad. Sci. USA 94, 2333-2338 (1997)) and is then internalized through the endocytic pathway (Caughey, B., Raymond, G.J., Ernst, D. & Race, R.E. N terminal truncation 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. I~irol. 65, 6597-6603 (1991); Borchelt, D.R., Taraboulos, A. &
Prusiner, S.B. Evidence for synthesis of scrapie prion proteins in the endocytic pathway. J. Biol.
Chem. 267, 16188-16199 (1992)), it was deduced that polyamines act upon PrPs' in endosomes or lysosomes. This deduction was investigated by determining the effect of pretreatment with the lysosomotropic agents chloroquine and NH4C1 on the ability of polyamines to eliminate PrPs'. These lysosomotropic agents alkalinize endosomes and have no effect on PrP'' levels when administered to ScN2a cells (Taraboulos, A., Raeber, A.J., Borchelt, D.R., Serban, D. &
Prusiner, S.B. Synthesis and trafficking of prion proteins in cultured cells. Mol. Biol. Cell 3, 851-863 (1992)). Experimental results obtained shows that 100 ~cM chloroquine, but not 30 ~cM NH4C1, blocked the ability of PEI to eliminate PrPs'. Similar results were obtained with SuperFect and PAMAM, generation 4Ø
Although the failure of NH4C1 to affect PrPs' levels is not easily explained, the ability of chloroquine to attenuate the ability of branched polyamines to remove PrPs' is consistent with the notion that these agents act in endosomes or lysosomes.
ORGAN HOMOGENATE ASSAY
The above results with cell cultures prompted investigating the possibility that in an acidic environment branched polyamines, either by indirectly interacting with PrPs' or with another cellular component, could cause PrPs' to become susceptible to hydrolases present in the endosome/lysozome. An in vitro degradation assay was developed to evaluate the effect of pH on the ability of polyamines to render PrPs' sensitive to protease. Crude homogenates of scrapie-infected mouse brain were exposed to a broad range of pH values in the presence or absence of SuperFect and then treated with proteinase K prior to Western blotting. Whereas PrPs' remained resistant to protease hydrolysis throughout the pH range (3.6-9.6) in the absence of Superfect, addition of the branched polyamine at pH 4.0 or below caused PrPS' to become almost completely degraded by protease.
Polyamine addition showed a dramatic effect on clearance in vitro which was optimized at pH 4 or less. These results show that polyamines act on PrPs' in an acidic compartment. To establish that the in vitro degradation assay is a valid approximation of the mechanism by which branched polyamines enhance the clearance of PrPs' from cultured cells, a structure activity analysis was performed with several of the compounds tested in culture cells. An excellent correlation was found between the clearance of PrPs' in cultured ScN2a cells (Table 1) and the ability to render PrPS' susceptible to protease at acidic pH in vitro. Notably, PAMAM-OH
generation 4.0 failed to render PrPs' susceptible to protease, whereas PAMAM generation 4.0 and PPI, generation 4.0 exhibited an even stronger activity than Superfect in vitro, as expected from their observed potency in cultured ScN2a cells (Table 1).
MECHANISM OF ACTION
The results discussed here show that certain branched polyamines cause the rapid elimination of PrPS° from ScN2a cells in a dose- and time-dependent manner. These compounds demonstrate a potent ability to remove prions from cultured cells at concentrations that are completely non-cytotoxic. The cells may be maintained indefinitely in culture in the presence of therapeutic levels of branched polyamines. Furthermore, when ScN2a cells were exposed to these compounds for ~ 1 week, PrPs' was reduced to undetectable levels and remained so for at least one month after removal of the polyamine.
Clarification of the exact mechanism of PrPS~ elimination by branched polyamines is an important objective. Although a number of possible scenarios exist, several possibilities may be excluded already. One possibility that was eliminated was that polyamines act by induction of chaperones such as heat shock proteins that mediate prion protein refolding because the above results show that it was possible to reproduce the phenomenon in vitro. Furthermore polyamines seem to offer advantages over other putative therapeutics that would seek to promote refolding: at very high concentrations, dimethyl sulfoxide (DMSO) and glycerol act as direct "chemical chaperones" and inhibit the formation of new PrPs° (Tatzelt, J., Prusiner, S.B. &
Welch, W.J. Chemical chaperones interfere with the formation of scrapie prion protein. EMBO J. 15, 6363-6373 (1996)), but these compounds cannot reduce pre-existing PrPS° levels. Furthermore, polyamines inhibit PrPs° formation at much lower concentrations than these agents. The ability of polyamines to effect the rapid clearance of PrPS' also contrasts with the activity of other potential prion therapeutics. Sulfated polyanions may inhibit PrPs° accumulation in ScN2a cells by directly binding to PrP~ (Gabizon, R., Meiner, Z., Halimi, M. & Ben-Sasson, S.A. Heparin-like molecules bind differentially to prion-proteins and change their intracellular metabolic fate. J. Cell. Physiol. 157, (1993); Caughey, B., Brown, K., Raymond, G.J., Katzenstein, G.E. & Thresher, W. Binding of the protease-sensitive form of PrP (prion protein) to sulfated glycosaminoglycan and Congo red. J.
Ifirol. 68, 2135-2141 (1994)), but because branched polyamines are able to clear pre-existing PrPs°, their mechanism of action cannot simply involve binding to PrP~ and inhibiting de novo synthesis.
Another possible mechanism which can be excluded is endosomal rupture. The branched polyamines which were effective in clearing PrPs° from ScN2a cells in our experiments, PEI, SuperFect and PAMAM, are also potent lysosomotropic, osmotic agents which can swell in acidic environments and rupture endosomes (Boussif, O., et al. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethyleneimine.
Proc. Natl. Acad. Sci.
U.S.A. 92, 7297-7301 (1995); Haensler, J. & Szoka, F.C.J. Polyamidoamine cascade polymers mediate efficient transfection of cells in culture. Bioconjug. Chem. 4, 372-379 (1993)). This might suggest that branched polyamines clear PrPs° from ScN2a cells by rupturing endosomes and exposing PrPs' to cytosolic degradation processes. However, it is known that the lysosomotropic, endosome-rupturing agents NH4C1, chloroquine, and monensin do not interfere with the formation of PrPs° in ScN2a cells (Taraboulos, A., Raeber, A.J., Borchelt, D.R., Serban, D. & Prusiner, S.B.
Synthesis and trafficking of prion proteins in cultured cells. Mol. Biol. Cell 3, 851-863 (1992)).
Furthermore, the results also show that chloroquine interferes with the ability of branched polyamines to clear PrPs° and that polyamines can clear PrPs' in vitro at acidic pH in the absence of cell membranes. Together, these observations rule out endosome rupture as the mechanism by which branched polyamines remove PrPS°.
Without committing to any particular mechanism of action it appears likely that branched polyamines require the acidic environment of intact endosomes or lyzosomes to destroy PrPS~. The structure-activity profile of polymers tested reveals that the most active compounds possess densely packed, regularly-spaced amino groups, suggesting that these compounds may bind to a ligand which has periodically-spaced negative charges. Several scenarios remain possible.
(1) Branched polyamines may bind directly to PrPs° arranged as an amyloid with exposed negatively-charged moieties and induce a conformational change under acidic conditions. (2) Treatment of PrP 27-30 with acid decreases turbidity and increases a-helical content, suggesting that such conditions might dissociate PrPs~ into monomers (Safar, J., Roller, P.P., Gajdusek, D.C. &
Gibbs, C.J., Jr. Scrapie amyloid (prion) protein has the conformational characteristics of an aggregated molten globule folding intermediate). It is therefore possible that polyamines bind to an equilibrium unfolding intermediate of PrPs' present under acidic conditions. (3) Alternatively, polyamines might sequester a cryptic, negatively charged component bound to PrPS' that is essential for protease resistance, but which is only released when PrPS' undergoes an acid-induced conformational change. Such a component might act as a chaperone for PrPs° inside endosomes or lysosomes. (4) Finally, another possibility is that polyamines activate an endosomal or lysosomal factor which can induce a conformational change in PrPs'. Clearly, more work will be required to determine the precise mechanism by which branched polyamines destroy PrPs' GENERAL APPLICABILITY OF ASSAY
The in vitro assay described here is generally applicable in the search for compounds that effectively clear conformationally altered proteins present in food thereby preventing a number of degenerative diseases, where the accumulation of proteins seems to mediate the pathogenesis of these illnesses. By simulating lysosomes, where proteases hydrolyze proteins under acidic conditions, the in vitro brain homogenate assay is able to rapidly evaluate the efficacy of a variety of polyamines to induce degradation of PrPS'.
The in vitro assay which used scrapie infected brain homogenate to test for compounds which clear PrPs' could be modified to assay for compounds which would clear any conformationally altered protein. The assay is carried out by homogenizing the organ or tissue where the conformationally altered protein is present in the highest concentration. The pH of the homogenate is then reduced to less than 5.0 and preferably 4.0 or less. For example pancreatic tissue can be homogenized to produce an assay to test for compounds which clear amylin which is associated with type II Diabetes. Homogenized kidney could be used to test for compounds which clear (3z -microglobulin and homogenized heart or vascular tissue used to test for compounds which clear atrial natriuretic factor. Those skilled in the art will recognize other organs and tissue types which can be homogenized to test for other compounds which clear other conformationally altered proteins.
Besides using the in vitro assay to screen for potential drugs, the compounds found via the assay such as branched polyamines provide a new tool for exploring the conversion of a protein to conformationally altered protein, e.g. PrP~ into PrPS°. The mechanism by which branched polyamines render PrPs° susceptible to proteolysis, remains to be established. Whether the interaction of branched polyamines with PrPS' is reversible is unknown. In addition, we do not know whether branched polyamines are able to solubilize PrPs' without irreversibly denaturing the protein.
Whatever the mechanism by which branched polyamines interact with PrPs~, it is likely to be different from that found with chaotropes as well as denaturing detergents and solvents (Prusiner, S.B., Groth, D., Serban, A., Stahl, N. & Gabizon, R. Attempts to restore scrapie prion infectivity after exposure to protein denaturants. Proc. Natl. Acad. Sci. USA 90, 2793-2797 (1993)).
Using the assays described and disclosed here certain specific branched polyamines have been found which mediate the clearance of PrPS' from cultured cells under non-cytotoxic conditions.
These compounds offer the intriguing possibility of being added to a wide range of low pH food products to neutralize conformational altered proteins present. Since the compounds found act by stimulating normal cellular pathways of protein degradation to destroy PrPs°, this class of compounds would also likely be of value in the treatment of other degenerative and hereditary disorders where abnormally folded, wild-type or mutant proteins accumulate.
Such an approach may find merit in developing an effective therapeutics for one or more of the common, degenerative illnesses including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, frontotemporal dementia, adult onset diabetes mellitus and the amyloidoses (Beyreuther, K. &
Masters, C.L. Serpents on the road to dementia and death. Accumulating evidence from several studies points to the normal function of presenilin l and suggests how the mutant protein contributes to deposition of amyloid plaques in Alzheimer's disease. Nature Medicine 3, 723-725 (1997);
Masters, C.L. & Beyreuther, K. Alzheimer's disease. BMJ316, 446-448 (1998);
Selkoe, D.J. The cell biology of beta-amyloid precursor protein and presenilin 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. Nature 399, A23-31 (1999); Wong, P.C., et al. An adverse property of a familial ALS-linked SOD1 mutation causes motor neuron disease characterized by vacuolar degeneration ofmitochondria. Neuron 14, 1105-1116 (1995); Spillantini, M.G., Crowther, R.A., fakes, R., Hasegawa, M. & Goedert, M. a-Synuclein in filamentous inclusions of Lewy bodies from Parkinson's disease and dementia with Lewy bodies. Proc. Natl. Acad. Sci. USA
95, 6469-6473 (1998); Hutton, M., et al. Association of missense and 5'-splice-site mutations in tau with the inherited dementia FTDP-17. Nature 393, 702-705 (1998); Stone, M.J.
Amyloidosis: a final common pathway for protein deposition in tissues. Blood 75 , 531-545 (1990)).
Whether branched polyamines might also prove efficacious in a variety of inherited disorders where the accumulation of abnormal proteins is a hallmark of the illness remains to be established;
these genetic maladies include heritable forms of prion disease, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, frontotemporal dementia, Pick's disease and amyloidosis, as well as the triplet repeat diseases including Huntington's disease, spinal cerebellar ataxias and myotonic dystrophy (Fu, Y.-H., et al. An unstable triplet repeat in a gene related to myotonic muscular dystrophy. Science 255, 1256-1259 (1992); Group, T.H.s.D.C.R. A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. Cell 72, 971-983 (1993)).
Compounds identified via assays of the invention such as branched polyamines will find utility in preventing or delaying the onset of these genetic diseases where earners can often be identified decades in advance of detectable neurologic or systemic dysfunction.
The invention is based on the discovery that several dendritic polycations, including the starburst dendrimers SuperfectT"' (QIAGEN~, Valencia, CA), polyamidoamide (PAMAM), and the hyperbranched polycation polyethyleneimine (PEI), were surprisingly found to eliminate PrPs° 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 disease including the amyloidoses. The removal of PrPs° is dependent on both the concentration of dendritic polymer and length of exposure. Dendritic polymers were able to cleax PrPS° at concentrations which were not cytotoxic. Repeated exposures to heat-degraded starburst PAMAM
dendrimer or PEI caused a dramatic reduction in PrPs° levels which persisted for a month even after removal of the compound. Dendritic polycations did not appear to destroy purified PrPS' in vitro, and therefore may act through a generalized mechanism. Dendritic polycations represent a class of compounds which can be used as therapeutic agents in prion diseases and other disorders involving insoluble protein deposits, such as the amyloidoses.
EXAMPLES
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
METHODS AND MATERIALS
Chemicals. High molecular weight PEI was purchased from Fluka. DOTAP cationic lipid was purchased from Boehringer Mannheim and SuperFect transfection reagent was purchased from QIAGEN~. All other compounds were purchased from Sigma-Aldrich. All test compounds were dissolved in water at stock concentration of 3 mg/ml and filtered through a Millipore 0.22 m m filter.
Cultured cells. Stock cultures of ScN2a cells were maintained in MEM with 10%
FBS, 10% Glutamax (Gibco BRL), 100 U penicillin, and 100 mg/ml streptomycin (supplemented DME).
hrunediately prior to addition of test compounds, the dishes were washed twice with fresh supplemented DME media. After exposure to test compounds, dishes were drained of media and cells were harvested by lysis in 0.25-1 ml 20 mM Tris pH 8.0 containing 100 mM
NaCl, 0.5%
NP-40, and 0.5% sodium deoxycholate to obtain a total protein concentration of 1 mg/ml measured by the BCA assay. Nuclei were removed from the lysate by centrifugation at 2000 rpm for 5 min.
For samples not treated with proteinase K, 40 ~1 of whole lysate (representing 40 ~g total protein) was mixed with an equal volume of 2x SDS reducing sample buffer. For proteinase K digestion, 20 ~g/ml proteinase K (Boehringer Mannheim) (total protein:enzyme ratio = 50:1) was added, and the sample was incubated for 1 h at 37°C. Proteolytic digestion was terminated by the addition of Pefabloc to a final concentration of 5 mM. One ml samples were centrifuged at 100,000 x g for 1 h at 4°C, the supernatants were discarded, and the pellets were resuspended in 80 /.c1 of reducing SDS
sample buffer for SDS-PAGE.
Brain homogenates. Brain homogenates from RML scrapie-affected CD-1 mice (10%
(w/v) in sterile water) were prepared by repeated extrusion through syringe needles of successively smaller size, from 18 to 22 gauge. Nuclei and debris were removed by centrifugation at 1000 x g for min. The bicinchnoninic acid (BCA) protein assay (Pierce) was used to determine protein 5 concentration. Homogenates were adjusted to 1 mg/ml protein in 1 % NP-40.
For reactions, 0.5 ml homogenate was incubated with 25 ml 1.0 M buffer (sodium acetate for pH 3-6 and Tris acetate for pH 7-10) plus or minus 10 ml of polyamine stock solution (3 mg/ml) for 2 h at 37 ° C with constant shaking. The final pH value of each sample was measured directly with a calibrated pH electrode (Radiometer Copenhagen). Following incubation, each sample was neutralized with an equal volume 0.2 M HEPES pH 7.5 containing 0.3 M NaCI and 4% Sarkosyl. Proteinase K was added to achieve a final concentration of 20 ~cg/ml, and samples were incubated for 1 h at 37°C. Proteolytic digestion was terminated by the addition of Pefabloc to a final concentration of 5 ~M.
Ten ~cl of digested brain homogenate was mixed with equal volume 2x SDS sample buffer and analyzed by SDS-PAGE
followed by Western blotting.
Western blotting. Following electrophoresis, Western blotting was performed as previously described (Scott, M., et al. Transgenic mice expressing hamster prion protein produce species-specific scrapie infectivity and amyloid plaques. Cell 59, 847-857 (1989)).
Samples were boiled for 5 min and cleared by centrifugation for 1 min at 14,000 rpm in aBeckman ultrafuge. SDS-PAGE
was carned out in 1.5 mm, 12% polyacrylamide gels(Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T-4. Nature 227, 680-685 (1970)). Membranes were blocked with 5% non-fat milk protein in PBST (calcium- and magnesium-free PBS plus 0.1%
Tween 20) for 1 h at room temperature. Blocked membranes were incubated with primary 8073 polyclonal antibody (to detect MoPrP) (Serban, D., Taraboulos, A., DeArmond, S.J. & Prusiner, S.B. Rapid detection of Creutzfeldt-Jakob disease and scrapie prion proteins.
Neurology 40, 110-117 (1990)) or 3F4 monoclonal antibody (to detect MHM2 PrP) (Kascsak, R.J., et al. Mouse polyclonal and monoclonal antibody to scrapie-associated fibril proteins. J.
I~irol. 61, 3688-3693 (1987)) at 1:5000 dilution in PBST overnight at 4°C. Following incubation with primary antibody, membranes were washed 3 x 10 min in PBST, incubated with horseradish peroxidase-labeled secondary antibody (Amersham Life Sciences) diluted 1:5000 in PBST for 30 to 60 min at 4°C and washed again for 3 x 10 min in PBST. After chemiluminescent development with ECL reagent (Amersham) for 1 min, blots were sealed in plastic covers and exposed to ECL
Hypermax film (Amersham). Films were processed automatically in a Konica film processor.
Branched polyamines inhibit formation of nascent PrPs' and induce clearance of pre-existing PrPs' Western blots were probed with 3F4 monoclonal antibody which recognizes newly expressed MHM2 PrP. ScN2a cells were exposed to SuperFect for 3 h and harvested 3 d after removal of SuperFect. Gells were run on both undigested, control sample and a sample subjected to limited proteolysis. The samples were run in separate lanes 1-6 with a control and limited proteolysis sample for each of the 6 lanes as follows: Lane 1: DOTAP-mediated transfection. Lane 2: 30 ~cg/ml SuperFect, 5 ~cg pSPOX MHM2. Lane 3: 75 ~cg/ml SuperFect, 5 ~g pSPOX MHM2.
Lane 4: 150 ~cg/ml SuperFect, 5 ~g pSOX MHM2. Lane 5: 150 ~g/ml SuperFect, 10 ~g pSPOX
MHM2.
Lane 6: No addition of either transfection reagent or DNA. Forty ~1 of undigested brain homogenate was used in these studies while those samples subjected to limited digestion with proteinase K were concentrated 25-fold prior to SDS-PAGE. One ml of the digest were centrifuged at 100,000 x g for 1 h at 4 ° C and the pellets suspended in 80 ~l of SDS sample buffer prior to SDS-PAGE followed by Western blotting. Apparent molecular weights based on migration of protein standards are 34.2, 28.3, and 19.9 kDa.
All of the control lanes 1-6 show multiple bands as expected. However, of the samples subjected to limited proteolytic only lane 1 shows bands. Unexpectedly, all of the partially digested sample lanes 2-5 show no bands and as expected no bands in the partially digested lane 6. These results show the effect of using SuperFect in clearing PrPs'.
The blot described above was stripped of antibody, exposed to labeled 8073 and redeveloped. The antibody 3F4 used in Example 1 binds to PrP~ but not to PrPs'. However, 8073 binds to PrPs' and PrP~. Lanes 1, 2 and 3 show decreasing amounts of PrPs' and lanes 4 and 5 show no detectable PrPS'.
Gels were run on undigested controls 1-4 and as above, samples subjected to limited proteolysis. The lanes were as follows: Lane l: No SuperFect. Lane 2: 30 /.cg/ml SuperFect. Lane 3: 75 /.cg/ml SuperFect. Lane 4: 150 /.cg/ml SuperFect. ScN2a cells were exposed to SuperFect for 3 h and harvested 3 d after removal of SuperFect. Apparent molecular weights based on migration of protein standards are 33.9, 28.8, and 20.5 kDa. In that each sample was tested after the same time period the results show the dose-dependent effect of SuperFect on PrPs° removal. Lanes 1, 2 and 3 show decreasing amounts of PrPs° and lane 4 shows no detectable PrPs'.
To determine the time-dependent effect of SuperFect three different panels with four lanes each were prepared and run as follows: ScN2a cells were exposed to 7.5 ~cg/ml:
SuperFect (lanes 1-4), PEI (average molecular weight ~60,000)(lanes 5-8), or PAMAM, generation 4.0 (lanes 9-12).
Time of exposure times for each polyamine: 0 hours (lanes 1, 5, and 9), 4 hours (lanes 2, 6, and 10), 8 hours (lanes 3, 7, and 11), 16 hours (lanes 4, 8, and 12). All samples were subjected to limited proteolysis to measure PrPs~. Apparent molecular weights based on migration of protein standards are 38, 26, and 15 kDa. Lanes of each of the three panels show decreasing amounts of PrPs°.
In this example four panels A,B, C and D were created with panels having three double (control and test) lanes each. ScN2a cells were exposed to 1.5 ~g/ml: (A) SuperFect, (B) PEI
(average molecular weight 60,000), (C) PAMAM, generation 4.0, or (D) no addition. Cells were harvested: Lane 1, before addition; Lane 2, immediately following 1 week continuous exposure to test compounds; and Lane 3, three weeks after removal of test compounds. Minus (-) symbol denotes undigested, control sample and plus (+) symbol designates sample subjected to limited proteolysis. Apparent molecular weights based on migration of protein standards are 33.9, 28.8, and 20.5 kDa. Test lanes 3 in panel A showed slight PrPs' after three weeks and test lanes 3 in panels B
and C showed no detectable PrPs~ whereas PrPs° was present in all lanes in panel D.
Four separate gels were run to demonstrate the effect of adding chloroquine would have on PrPs° levels. The lanes 1 control and 3 where chloroquine was added show clear bands for PrPs°
whereas lanes 2 and 4 with no chloroquine show barely detectable amounts of PrPs°. The four lanes were prepared as follows: ScN2a cells were treated Lane 1: Control media. Lane 2: 7.5 ~g/ml PEI
(average molecular weight 60,000). Lane 3: PEI plus 100 ~cM chloroquine. Lane 4: PEI plus 30 ~cM NH4Cl. Chloroquine and NH4Cl were added 1 h prior to addition of PEI.
Cells were harvested 16 hours after addition of PEI. All samples shown were subjected to limited proteolysis to measure PrPs'. Apparent molecular weights based on migration of protein standards are 3 8, 26, and 15 kDa.
Eight lanes with SuperFect (+SF) and eight lanes without SuperFect (-SF) were prepared.
Lanes 1-8 of each group had an adjusted pH of 3.6" 4, 5, 6, 7, 8, 9 and 9.6.
In vitro mixture of crude mouse brain homogenates with SuperFect under a range of pH conditions was performed as described in methods (measured final pH of each sample denoted above the lanes). Addition of 60 ~g/ml SuperFect denoted as "+SF" and control with no addition as "-SF". All samples shown were subjected to limited proteolysis to measure PrPs'. Apparent molecular weights based on migration of protein standards are 30 and 27 kDa. All lanes ofthe -SF group showed PrPS' present. Lanes 3-8 of the +SF group showed PrPs'. However, lanes l and 2 with respective pH levels of 3.6 and 4.0 showed very slight detectable PrPs'. The results show that the ability of a blanched polycation such as SuperFect to clear PrPs' is pH dependent.
Sixteen different lanes were prepared as described. Lanes 1 and 2 were control lanes and each of lanes 3-16 contained a different compound as tested in Table 1. The test compounds were all polyamines. Thus, the results show removal of PrPs' from brain homogenate in vitro by various polyamines. Samples were incubated with polyamines at pH 3.6 and processed as described in Methods. Each polyamine was tested at 60 ~cg/ml concentration. Lanes 1 and 2:
control. Lane 3:
poly-(L)lysine. Lane 4: PAMAM, generation 0Ø Lane 5: PAMAM, generation 1Ø
Lane 6:
PAMAM, generation 2Ø Lane 7: PAMAM, generation 3Ø Lane 8: PAMAM, generation 4Ø
Lane 9: PAMAM-OH, generation 4Ø Lane 10: PPI, generation 2Ø Lane 11: PPI, generation 4Ø
Lane 12: linear PEI. Lane 13: high MW PEI. Lane 14: low MW PEI. Lane 15:
average MW PEI.
Lane 16: SuperFect. All samples shown were subjected to limited proteolysis to measure PrPs'.
Apparent molecular weights based on migration of protein standards are 30 and 27 kDa.Table 1.
Removal of PrPs~ by polymer compounds. IC50 = approximate concentration of polymer required to reduce PrPs' to 50% of control levels in ScN2a cells after exposure for 16 hours. All compounds were tested at 5 different concentrations. PrPS' levels were measured by densitometry of Western blot signals.
(Note that Table 1 includes information on the characteristics of compounds used but that the list does not correspond directly to lanes 1-16) Molecular Primary NH2 ICSp (ng/xnl) Weight groups 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 Low MW PEI 25,000 2,000 Average MW PEI 60,000 400 High MW PEI 800,000 80 Linear PEI 60,000 2,000 poly-(L)lysine 60,000 >500 10,000 SuperFect 400 Lanes 7, 8, 11 and 13 showed the best results, i.e. best ability to clear PrPs' under these conditions. Specifically, PAMAM generation 4.0 in lane 8 showed the best ability to clear PrPS' under these conditions whereas PAMAM-OH generation 4.0 showed almost no detectable ability to clear PrPs' and was comparable to the control.
Transfection of PrPs' Expressing Cells with Dendrimer Compounds Cells of neuronal origin expressing PrPs' were examined for the ability of compounds to suppress PrPs° formation.
Transfection Studies Stock cultures of N2a and ScN2a cells were maintained in MEM with 10% FBS, 10%
Glutamax (Gibco BRL), 100 U penicillin, and 100 ~cg/ml streptomycin. Cells from a single confluent 100 mm dish were trypsinized and split into 10 separate 60 mm dishes containing DME
plus 10% FBS, 10% Glutamax, 100 U penicillin, and 100 ~cg/ml streptomycin (supplemented DME) one day prior to transfection. Immediately prior to transfection, the dishes were washed twice with 4 ml supplemented DME media and then drained.
For DOTAP-mediated transfection, 15 ~g pSPOX MHM2 was resuspended in 150 ~cl sterile Hepes Buffered Saline (HBS) on the day of transfection. The DNA solution was then mixed with an equal volume of 333 ~cg/ml DOTAP (Boehringer Mannheim) in HBS in Falcon 2059 tubes and incubated at room temperature for 10 minutes to allow formation of DNA/lipid complexes.
Supplemented DME (2.5 ml) was added to the mixture, and this was then pipetted onto drained cell monolayers. The following day, the medium containing DNA/lipid was removed and replaced with fresh supplemented DME. Cells were harvested three days later.
For SuperfectT"'-mediated transfections/exposures, SuperfectT~~ with or without DNA was added to 1 ml supplemented DME in a Falcon 2059 tube to achieve the specific concentrations needed for each experiment. This mixture was pipetted up and down twice and then onto drained cell monolayers. After exposure for the indicated times, the medium containing SuperfectT"~ was removed and replaced with fresh supplemented DME. Cells were harvested at specified times after removal of SuperfectT~~.
Exposures to PPI (DAB-Am-8, Polypropylenimine octaamine Dendrimer, Generation 2.0 Aldrich 46,072-9), Intact PAMAM (Starburst (PAMAM)Dendrimer, Generation 4.
Aldrich 41,244-9), PEI (Sigma), poly-(L)lysine (Sigma), and poly-(D) lysine (Sigma) were performed as described above for SuperfectT"'.
Isolation of Protein from Treated Cells Cells were harvested by lysis in 1.2 ml of 20 mM Tris pH 8.0 containing 100 mM
NaCI, 0.5% NP-40, and 0.5% sodium deoxycholate. Nuclei were removed from the lysate by centrifugation at 2000 rpm for 5 min. This lysate typically had a protein concentration of 0.5 mg/ml measured by the BCA assay. For samples not treated with proteinase K, 40 ~l of whole lysate (representing 20 ~g total protein) was mixed with 40 ~l of 2x SDS sample buffer. For proteinase K
digestion, 1 ml of lysate was incubated with 20 ~g/ml proteinase K (total protein:enzyme ratio =
25:1) for 1 hr at 37°C. Proteolytic digestion was terminated by the addition of 8 ~1 of 0.5M PMSF
in absolute ethanol. Samples were then centrifuged for 75 min in a Beckman TLA-45 rotor at 100,000 x g at 4 ° C. The pellet was resuspended by repeated pipetting in 80 ~1 of 1X SDS sample buffer. The entire sample (representing 0.5 mg total protein before digestion) was loaded for SDS-PAGE.
Western Blot Analysis Immunoreactive PrP bands from the DOTAP-mediated transfection were detected before and after digestion with proteinase K with monoclonal antibody 3F4. The construct used to express PrPs° in the ScN2a cells is MHM2 a chimeric construct that differs from wild-type (wt) MoPrP 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 (SHa) PrP
sequence creates an epitope for 3F4 (Kascsak et al., (1987) J. ljirol. 61:3688-3693), which does not recognize endogenous wt MoPrP in ScN2a cells and hence facilitates specific detection of the transgene by Western blot.
Following electrophoresis, Western blotting was performed as previously described (Scott et al., (1989) Cell 59:847-857). Samples were boiled for 5 minutes and cleared by centrifixgation for 1 minute at 14,000 rpm in a Beckman ultrafuge. SDS-PAGE was carried out in 1.5 mm, 12%
polyacrylamide gels (Laemtnli (1970) Nature 227:661-665). Membranes were blocked with 5%
nonfat milk protein in PBST (calcium- and magnesium-free PBS plus 0.1% Tween 20) for 1 hour at room temperature. Blocked membranes were incubated with primary 8073 polyclonal or 3F4 monoclonal antibody at a 1:5000 dilution in PBST overnight at 4 °C.
Following incubation with primary antibody, membranes were washed 3 x 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 for 3x 10 minutes in PBST. After ehemiluminescent development with ECL reagent (Amersham) for 1 minute, blots were sealed in plastic covers and exposed to ECL Hypermax filin (Amersham). Films were processed automatically in a Konica film processor.
In contrast to DOTAP transfected cells, ScN2a cells transfected with varying concentrations of SuperfectT"~ and DNA did not appear to contain protease-resistant MHM2.
Close scrutiny revealed that, prior to protease digestion, SuperfectTM transfected samples express MHM2 bands which are not seen in the background pattern of the control sample. These observations indicate that MHM2 PrP was successfully expressed using SuperfectT"' transfection reagent, but conversion of MHM2 PrP~ to protease-resistant MHM2 PrPs° was inhibited by SuperfectT"'.
To examine whether SuperfectT"' had affected levels of preexisting PrPs' in ScN2a cells, the Western blot probed with 3F4 antibody was reprobed with polyclonal antibody 8073, which is able to recognize endogenous MoPrP. Remarkably, SuperfectTM caused the disappearance of preexisting MoPrPs° from ScN2a cells in a dose-dependent manner. After treatment with SuperfectT"~, PrPs' could not be detected in the nuclear fraction, pellet, supernatant, or media.
The concentration of SuperfectT"" required to fi>Zly remove preexisting PrPs° with a three hour exposure was 300 pg/ml, whereas 30 ~g/ml was sufficient to interfere with the formation of new MHM2 PrPs' within the same time frame.
Length of exposure dramatically influenced the ability of SuperfectT"' to remove PrPs' from ScN2a cells. Whereas a 3 hour exposure to 150 ~g/ml SuperfectT"" significantly lowered PrPs' levels in ScN2a cells, exposure for 10 min to the same dose of SuperfectT"' did not affect PrPs' levels.
When ScN2a cells were exposed to 2 pg/ml SuperfectT"' continuously for 1 week, PrPs' disappeared completely.
The conditions tested did not appear to be toxic for the cells. Neither 150 ~g/ml SuperfectT""
for 3 hrs nor 2 ~g/ml SuperfectT"' continuously for 1 week caused any obvious changes in cell morphology, viability, or growth as judged by phase contrast microscopy.
Elimination of PrPs' by repeated exposures to SuperfectT'"
The duration in the reduction in PrPs' levels after exposure to SuperfectT"' was examined, and it was shown that this reduction could persist for extended periods after removal of SuperfectT"' Following the exposure of ScN2a cells to a single dose of 150 ~g/ml SuperfectT"" for 3 hrs, PrPs' levels remained low for one week, but returned to near baseline levels after 3 weeks in culture without SuperfectT~A
In contrast, when ScN2a cells were exposed to 4 separate doses of SuperfectT"~
over the course of 16 days, very little PrPs' could be detected 4 weeks after the final exposure to SuperfectT"' This result offers hope that prolonged exposure to SuperfectT"' may lead to long term cure of scrapie infection in cultured cells.
SuperfectT"' does not destroy PrPs' directly The dendrimer SuperfectT"' was used to determine if it could exert a similar inhibitory effect on PrPs' in either crude brain homogenates or purified PrP 27-30 rods.
Brain homogenates from normal and scrapie-affected Syrian hamsters (10% (w/v) in sterile PBS) were prepared by repeated extrusion through syringe needles of successively smaller size, from 18 to 22 gauge. Nuclei and debris were removed by centrifugation at 1000 x g for 10 min. The bicinchnoninic acid (BCA) protein assay (Pierce) was used to determine protein concentration.
Homogenates were adjusted to 10 mg/ml protein with PBS and 50 p1 was added to 450 p1 of lysis buffer containing 100 mM NaCI, 1 mM EDTA, 0.55% sodium deoxycholate, 0.55%
Triton X-100, and 50 mM Tris-HCl pH 7.5. This mixture was then incubated with 0-300 ~g/ml SuperfectT"' for 3 hrs at 37 °C and then centrifuged for 10 min at 14,000 rpm in a Beckman Ultrafuge. The pellet was resuspended in 450 ~l lysis buffer without SuperfectT"'. Proteinase K
(Boehringer Mannheim) was added to achieve a final concentration of 20 ~g/ml, and thus the ratio of total protein/enzyme was 50:1. Samples were incubated for 1 h at 37 °C. Proteolytic digestion was terminated by the addition of 8 ~1 of 0.5 M PMSF in ethanol. Samples were then centrifuged for 75 min in a Beckman TLA-45 rotor at 100,000 x g at 4 °C. Undigested samples (10 1t1) were mixed with an equal volume of 2x SDS sample buffer. For digested samples, the pellet was resuspended by repeated pipetting in 100 ~l lx SDS sample buffer. Twenty ~1 (equivalent to 100 ~g of total protein prior to proteinase K
digestion) of each sample was loaded for SDS-PAGE.
PrP 27-30 rods were purified from scrapie-affected Syrian hamster brains and previously described (Prusiner et al., (1983) Cell 35:349-358). Purified rods (3.5 ~g/ml) were incubated with or without 900 ~g/ml SuperfectrM in 100 ~l supplemented DME. After 16 hrs at 37 °C, the suspension was centrifuged at 100,000 x g at 4 °C. The pellet was resuspended in 500 ~1 of buffer containing 1 mg/ml BSA, 100 mM NaCI, 1 mM EDTA, 0.55% sodium deoxycholate, 0.55% Triton X-100, and 50 mM Tris-HCl pH 7.5. Proteinase K was added to achieve a final concentration of 20 pg/ml. Samples were incubated for 1 h at 37 ° C. Proteolytic digestion was terminated by the addition of 8 p.1 of 0.5 M Pefabloc (Boehringer Mannheim). Samples were then centrifuged for 75 min at 100,000 x g at 4 °C. Undigested samples (50 p1) were mixed with an equal volume of 2x SDS
sample buffer. For digested samples, the pellet was resuspended by repeated pipetting in 100 p1 lx SDS sample buffer. Forty p1 of each sample was loaded for SDS-PAGE.
When SuperfectT~~ was mixed with either crude homogenates of scrapie-affected Syrian hamsters or with purified Syrian hamster PrP 27-30, there was no significant change in the level of proteinase K-resistant PrPS°. These results suggest that the removal of PrPS' from ScN2a cells by SuperfectTM depends on the presence of intact cellular machinery.
Clearance of PrPS' levels by other dendritic polycations The SuperfectT"~ compound is a high molecular weight component of heat-degraded PAMAM Starburst dendrimers, which is a cationic, highly-branched, monodisperse polymers (Tang et al., (1996) Bioconjugate Chem. 7:703-714). To identify other potentially usefi~l anti prion therapeutic agents, we screened three other dendritic polycations and two linear cationic polymers for their ability to clear PrPs' from ScN2a cells. Among the dendritic macromolecules tested, polyetheleneimine (PEI) was the most potent, removing the majority of PrPs' from ScN2a cells after 3 hrs when used at a concentration of 10 ~g/ml. Intact PAMAM displayed a potency comparable to Superfectr"', removing approximately half of the detectable PrPs~ when used at a concentration of 50 pg/ml. In contrast, the dendrimer polypropyleneimine (PPI), poly-(L)lysine, and the linear polycation poly-(D)lysine failed to reduce PrPs° levels at concentrations between 10-50 ~g/ml.
These results demonstrate that a branched polymeric architecture is required to clear PrPs'.
Furthermore, exposure of ScN2a cells to either PEI or intact PAMAM for one week at a concentration of 1.5 ~cg/ml completely removes PrPs~, effectively curing the cells of scrapie infection.
While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted 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, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.
Claims (10)
1. A method of sterilizing an object, comprising 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.
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, further comprising:
removing the composition from the object.
removing the composition from the object.
3. The method of claim 1, wherein the branched polycation is a polycationic dendrimer selected from the group consisting of polypropylene imine, polyethyleneimine (PEI) poly(4'-aza-4'-methylheptamethylene D-glucaramide), polyamidoamines and variants or fragments thereof.
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, comprising:
water in an amount of from 1% to 99.99% by weight;
polycationic dendrimer in an amount of 0.001% to 10% by weight.
water in an amount of from 1% to 99.99% by weight;
polycationic dendrimer in an amount of 0.001% to 10% by weight.
7. The composition of claim 6, wherein the composition is a pharmaceutical composition comprising a therapeutically effective amount of a branched polycation and a pharmaceutically acceptable excipient.
8. The composition of claim 6, further comprising a second compound selected from the group consisting of: a detergent, an antibacterial compound, an antiviral compound, and an antifungal compound.
9. A capsule for oral administration of a compound, comprising:
gelatin extracted from a cow; and a branched polycation.
gelatin extracted from a cow; and a branched polycation.
10. A cell culture, comprising:
cells genetically engineering to produce a pharmaceutical;
cell nutrients; and a branched polycation.
cells genetically engineering to produce a pharmaceutical;
cell nutrients; and a branched polycation.
Applications Claiming Priority (9)
Application Number | Priority Date | Filing Date | Title |
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US09/322,903 US6214366B1 (en) | 1999-06-01 | 1999-06-01 | Clearance and inhibition of conformationally altered proteins |
US09/322,903 | 1999-06-01 | ||
US09/406,972 | 1999-09-28 | ||
US09/406,972 US6419916B1 (en) | 1999-06-01 | 1999-09-28 | Assay for compounds which affect conformationally altered proteins |
US09/447,456 | 1999-11-22 | ||
US09/447,456 US6331296B1 (en) | 1999-06-01 | 1999-11-22 | Food additives which affect conformationally altered proteins |
US09/494,814 US6322802B1 (en) | 1999-06-01 | 2000-01-31 | Method of sterilizing |
US09/494,814 | 2000-01-31 | ||
PCT/US2000/014353 WO2000072851A1 (en) | 1999-06-01 | 2000-05-24 | Method of sterilizing |
Publications (1)
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CA2375237A1 true CA2375237A1 (en) | 2000-12-07 |
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CA002375237A Abandoned CA2375237A1 (en) | 1999-06-01 | 2000-05-24 | Method of sterilizing |
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EP (1) | EP1187622A4 (en) |
JP (1) | JP2003500169A (en) |
KR (1) | KR20020006050A (en) |
AU (1) | AU771547B2 (en) |
BR (1) | BR0011055A (en) |
CA (1) | CA2375237A1 (en) |
IL (1) | IL146769A0 (en) |
MX (1) | MXPA01012357A (en) |
NZ (1) | NZ515607A (en) |
WO (1) | WO2000072851A1 (en) |
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US6719988B2 (en) | 1997-02-21 | 2004-04-13 | The Regents Of The University Of California | Antiseptic compositions for inactivating prions |
US6720355B2 (en) | 1997-02-21 | 2004-04-13 | The Regents Of The University Of California | Sodium dodecyl sulfate compositions for inactivating prions |
US6322802B1 (en) | 1999-06-01 | 2001-11-27 | The Regents Of The University Of California | Method of sterilizing |
AU2001232994A1 (en) * | 2000-01-31 | 2001-08-07 | The Regents Of The University Of California | Compositions treated to inactivate infectious proteins |
CN1639575A (en) * | 2001-10-05 | 2005-07-13 | 斯特里斯公司 | In vitro model for priocidal activity |
ES2564293T5 (en) * | 2002-02-28 | 2019-04-04 | Microsens Biophage Ltd | Enlazamiento of pathological forms of prion proteins |
ATE485839T1 (en) * | 2002-11-26 | 2010-11-15 | Upfront Chromatography As | DENDRIMER CONJUGATES FOR SELECTIVE SOLUTION OF PROTEIN AGGREGATES |
US7985424B2 (en) | 2004-04-20 | 2011-07-26 | Dendritic Nanotechnologies Inc. | Dendritic polymers with enhanced amplification and interior functionality |
US7981444B2 (en) | 2004-04-20 | 2011-07-19 | Dendritic Nanotechnologies, Inc. | Dendritic polymers with enhanced amplification and interior functionality |
DE102004040119A1 (en) * | 2004-08-18 | 2006-04-27 | Heinrich-Heine-Universität Düsseldorf | Agent, useful for e.g. inactivating infectious prion and decontaminating device, instrument or appliance infected with prion, comprises a nano-particle |
US11806718B2 (en) | 2006-03-24 | 2023-11-07 | Handylab, Inc. | Fluorescence detector for microfluidic diagnostic system |
ES2692380T3 (en) | 2006-03-24 | 2018-12-03 | Handylab, Inc. | Method to perform PCR with a cartridge with several tracks |
US10900066B2 (en) | 2006-03-24 | 2021-01-26 | Handylab, Inc. | Microfluidic system for amplifying and detecting polynucleotides in parallel |
WO2008061165A2 (en) | 2006-11-14 | 2008-05-22 | Handylab, Inc. | Microfluidic cartridge and method of making same |
US20080274929A1 (en) * | 2007-05-01 | 2008-11-06 | Whitekettle Wilson K | Method for removing microbes from surfaces |
US9186677B2 (en) | 2007-07-13 | 2015-11-17 | Handylab, Inc. | Integrated apparatus for performing nucleic acid extraction and diagnostic testing on multiple biological samples |
WO2009012185A1 (en) * | 2007-07-13 | 2009-01-22 | Handylab, Inc. | Polynucleotide capture materials, and methods of using same |
EP3709025A1 (en) | 2011-04-15 | 2020-09-16 | Becton, Dickinson and Company | Scanning real-time microfluidic thermocycler and methods for synchronized thermocycling and scanning optical detection |
GB201405660D0 (en) * | 2014-03-28 | 2014-05-14 | Gama Healthcare Ltd | A liquid disinfecting composition |
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US4587329A (en) * | 1984-08-17 | 1986-05-06 | The Dow Chemical Company | Dense star polymers having two dimensional molecular diameter |
US5499979A (en) * | 1987-06-25 | 1996-03-19 | Alza Corporation | Delivery system comprising kinetic forces |
AT408191B (en) * | 1991-08-19 | 2001-09-25 | Haemosan Erzeugung Pharmazeuti | METHOD FOR INACTIVATING PRIONS |
GB9407812D0 (en) * | 1994-04-20 | 1994-06-15 | Nycomed Salutar Inc | Compounds |
US5514388A (en) * | 1994-08-31 | 1996-05-07 | Rohwer; Gary L. | Encapsulated lipid-containing feed |
US5919442A (en) * | 1995-08-11 | 1999-07-06 | Dendritech, Inc. | Hyper comb-branched polymer conjugates |
US5658574A (en) * | 1995-10-13 | 1997-08-19 | Chesebrough-Pond's Usa Co., Division Of Conopco, Inc. | Cleansing compositions with dendrimers as mildness agents |
AU3880197A (en) * | 1996-06-27 | 1998-01-14 | American Ink Jet Corporation | Waterfast pigmented ink jet inks |
FR2759582A1 (en) * | 1997-02-14 | 1998-08-21 | Oreal | DEODORANT COMPOSITION |
AUPP584398A0 (en) * | 1998-09-14 | 1998-10-08 | Starpharma Limited | Inhibition of toxic materials or substances |
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- 2000-05-24 MX MXPA01012357A patent/MXPA01012357A/en not_active Application Discontinuation
- 2000-05-24 JP JP2000620963A patent/JP2003500169A/en not_active Withdrawn
- 2000-05-24 AU AU50441/00A patent/AU771547B2/en not_active Ceased
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MXPA01012357A (en) | 2002-09-02 |
KR20020006050A (en) | 2002-01-18 |
EP1187622A4 (en) | 2005-06-22 |
NZ515607A (en) | 2003-07-25 |
BR0011055A (en) | 2002-05-21 |
EP1187622A1 (en) | 2002-03-20 |
AU5044100A (en) | 2000-12-18 |
JP2003500169A (en) | 2003-01-07 |
AU771547B2 (en) | 2004-03-25 |
IL146769A0 (en) | 2002-07-25 |
WO2000072851A1 (en) | 2000-12-07 |
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