WO2003105677A2 - Methods for treating viral diseases using modulators of amyloidogenic peptide aggregation - Google Patents

Methods for treating viral diseases using modulators of amyloidogenic peptide aggregation Download PDF

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Publication number
WO2003105677A2
WO2003105677A2 PCT/US2003/019365 US0319365W WO03105677A2 WO 2003105677 A2 WO2003105677 A2 WO 2003105677A2 US 0319365 W US0319365 W US 0319365W WO 03105677 A2 WO03105677 A2 WO 03105677A2
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WIPO (PCT)
Prior art keywords
phe
leu
val
modulator compound
virus
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PCT/US2003/019365
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French (fr)
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WO2003105677A3 (en
Inventor
David I. Israel
Tajib Mirzabekov
Woj M. Wojtowicz
Joseph Sodroski
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Praecis Pharmaceuticals, Inc.
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Priority to AU2003276149A priority Critical patent/AU2003276149A1/en
Publication of WO2003105677A2 publication Critical patent/WO2003105677A2/en
Publication of WO2003105677A3 publication Critical patent/WO2003105677A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • HIV like all other viruses, is incapable of replicating on its own and must use the machinery of the infected host cell.
  • Most current therapies are directed toward targets that present themselves after the cell is infected.
  • Current therapeutic agents used during the treatment of chronic phase of HIV infection have not been particularly efficacious and are, at best, only able to slow the progress of infection.
  • drugs have been identified which inhibit the replication of HIV in vitro (Haseltine, W. A. (1989) J. Acquir. Immune Def. Syndr., 2:311-324) and nucleotide chain elongation inhibitors such as 3-azidothymidine (AZT) have received widespread acceptance for clinical use.
  • AZT 3-azidothymidine
  • the present invention is directed to methods and compositions for modulating infection of a cell, e.g., a brain cell, by a virus and methods and compositions for treating a viral disease, e.g., a viral disease of the brain, in a subject.
  • the present invention is based, at least in part, on the discovery that infectivity of cells by the human immunodeficiency virus (HIV) is enhanced in the presence of aggregated /3-amyloid fibrils in the brain. Without intending to be limited by theory, it is believed that aggregates of amyloidogenic peptide fibrils, e.g., /3-amyloid fibrils, in the brain may facilitate infection of brain cells by HIV and contribute to the development of AJDS- related dementia.
  • HIV human immunodeficiency virus
  • the present invention provides a method for treating a viral disease, e.g., a viral disease of the brain, in a subject by administering to the subject a therapeutically or prophylactically effective amount of an amyloidogenic peptide modulator compound, e.g., a ⁇ amyloid modulator compound, thereby treating a viral disease in the subject.
  • a viral disease e.g., a viral disease of the brain
  • an amyloidogenic peptide modulator compound e.g., a ⁇ amyloid modulator compound
  • the viral disease is caused by infection with a virus selected from the group consisting of human immunodeficiency virus, herpes simplex virus, varicella zoster virus, poliomyelitis virus, cytomegalovirus, influenza virus, respiratory syncytial virus, coxsackie virus, ebola virus, hantavirus, human papilloma virus, rotavirus, west nile virus, Epstein-Barr virus, and hepatitis virus.
  • a virus selected from the group consisting of human immunodeficiency virus, herpes simplex virus, varicella zoster virus, poliomyelitis virus, cytomegalovirus, influenza virus, respiratory syncytial virus, coxsackie virus, ebola virus, hantavirus, human papilloma virus, rotavirus, west nile virus, Epstein-Barr virus, and hepatitis virus.
  • the ⁇ amyloid modulator compound may be a peptide (e.g., a vaccine), a peptidomimetic, an antibody, a nucleic acid molecule, a protease inhibitor, a ⁇ -secretase or ⁇ -secretase inhibitor, or a modulator of blood cholesterol levels.
  • the ⁇ amyloid modulator compound is a peptide comprised entirely of D-amino acids and having at least three amino acid residues independently selected from the group consisting of a D-leucine structure, a D-phenylalanine structure, a D-tyrosine structure, a D-iodotyrosine structure and a D-alanine structure.
  • the ⁇ amyloid modulator compound is a compound comprising a formula: '
  • Xaa ⁇ , Xaa 2 , Xaa ⁇ and Xaa4 are each D-amino acid structures and at least three of Xaa l5 Xaa 2 , Xaa and Xaa4 are, independently, selected from the group consisting of a D-leucine structure, a D-phenylalanine structure, a D-tyrosine structure, a D-iodotyrosine structure and a D-alanine structure;
  • Y which may or may not be present, is a structure having the formula (Xaa) a , wherein Xaa is any D-amino acid structure and a is an integer from 1 to 15;
  • Z which may or may not be present, is a structure having the formula (Xaa)b, wherein Xaa is any D-amino acid structure and b is an integer from 1 to 15;
  • A is a modifying group attached to the compound, with the proviso that A is not an amino acid or is a non-natural amino acid that acts as a ⁇ -turn mimetic; and n is an integer from 1 to 15; wherein Xaaj, Xaa 2 , Xaa3, Xaa,4, Y, Z, A and n are selected such that the compound binds to natural ⁇ -amyloid peptides or modulates the aggregation or inhibits the neurotoxicity of natural ⁇ -amyloid peptides when contacted with the natural ⁇ -amyloid peptides.
  • the ⁇ amyloid modulator compound is a compound having a structure selected from the group consisting of: N,N-dimethyl-(Gly-D-Ala-D-Phe-D-Phe-D-Val-D-Leu)-NH 2 ; N,N-dimethyl-(D-Ala-D- Phe-D-Phe-D-Val-D-Leu)-NH 2 ; N-methyl-(Gly-D-Ala-D-Phe-D-Phe-D-Val-D-Leu)- NH 2 ; N-ethyl-(Gly-D-Ala-D-Phe-D-Phe-D-Val-D-Leu)-NH 2 ; N-isopropyl-(Gly-D-Ala- D-Phe-D-Phe-D-Val-D-Leu)-NH 2 ; H-(D-Leu-D-Val-D-Phe-D-Phe-D-Ala
  • the ⁇ amyloid modulator compound is a compound having the structure: N-methyl-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)- ⁇ 2 or the structure: 4-Hydroxybenzoyl-D-Leu-D-Val-D-Phe-D-Phe-D-Ala-NH 2
  • the present invention provides a method for treating AIDS in a subject by administering to the subject a therapeutically or prophylactically effective amount of an amyloidogenic peptide modulator compound, e.g., a ⁇ amyloid modulator compound, thereby treating ADDS in the subject.
  • the present invention provides a method of modulating infection of a cell, e.g., a brain cell, by a human immunodeficiency virus by contacting the cell with an amyloidogenic peptide modulator compound, e.g., a ⁇ amyloid modulator compound, in an amount effective to modulate infection of the cell by a human immunodeficiency virus.
  • a cell e.g., a brain cell
  • an amyloidogenic peptide modulator compound e.g., a ⁇ amyloid modulator compound
  • the present invention provides a method for identifying an amyloidogenic peptide modulator compound, e.g., a ⁇ amyloid modulator compound, capable of treating a viral disease in a subject.
  • the method includes contacting a cell with a virus in the presence of an amyloidogenic peptide (e.g., ⁇ amyloid peptide) and an amyloidogenic peptide modulator compound, e.g., a ⁇ amyloid modulator compound, and determining the ability of the virus to infect the cell, h one embodiment, the ability of the virus to infect the cell is determined by monitoring the cell for a phenotype associated with viral infection, e.g., virus burden, cell lysis, plaque formation, or low pH induced fusion of infected cells.
  • a phenotype associated with viral infection e.g., virus burden, cell lysis, plaque formation, or low pH induced fusion of infected cells.
  • the method may further include administering the amyloidogenic peptide modulator compound, e.g., the ⁇ amyloid modulator compound, to an animal model for a viral disease and monitoring viral burden in the animal before and after the administration of the amyloidogenic peptide modulator compound, e.g., the ⁇ amyloid modulator compound.
  • the amyloidogenic peptide modulator compound e.g., the ⁇ amyloid modulator compound
  • the present invention provides a method for identifying an amyloidogenic peptide modulator compound, e.g., a ⁇ amyloid modulator compound, capable of modulating infection of a cell by a virus.
  • the method includes contacting a cell with a virus in the presence of an amyloidogenic peptide (e.g., ⁇ amyloid peptide) and an amyloidogenic peptide modulator compound, e.g. , a ⁇ amyloid modulator compound, and determining the ability of the virus to infect the cell, thereby identifying an amyloidogenic peptide modulator compound, e.g., a ⁇ amyloid modulator compound, capable of modulating infection of a cell by a virus.
  • an amyloidogenic peptide e.g., ⁇ amyloid peptide
  • an amyloidogenic peptide modulator compound e.g., a ⁇ amyloid modulator compound
  • Figure 1 is a graph demonstrating that A ⁇ stimulates infection by recombinant HIV-1 viruses.
  • Figure 3 is a graph demonstrating that HIV-1 infection in the presence of A ⁇ is dependent on co-receptors.
  • A Cf2Th cells expressing only CCR5 (D), only CD4 ( ⁇ ), both receptors ( ⁇ ) or neither receptor (O) were used as target cells for infection.
  • Cf2Th-CD4/CCR5 cells were infected with recombinant HIV-1 pseudotyped with ADA (A), YU2 (O) and VSV (D) envelope glycoproteins in the presence of varied concentrations of amyloidogenic peptides PPI- 2480 (AGAKWSWWELTWVGG; SEQ ID NO:l) or PPI-2566 (TRQAMCNISRADWND; SEQ ID NO:2).
  • PPI- 2480 AGAKWSWWELTWVGG; SEQ ID NO:l
  • PPI-2566 TRQAMCNISRADWND; SEQ ID NO:2
  • Infection was also performed in the presence of more than 20 different non-amyloido genie peptides of 8-17 amino acids long. None of them, including the peptide shown in the figure, PPI-1966 (APMGSDPPTA; SEQ ID NO:3), affected viral entry. All peptides were amidated at the C-terminus and incubated under fibril-forming conditions. Results of independent assay
  • Figure 5 depicts the results of experiments demonstrating that peptides that enhance viral infection form fibrils and promote lipid vesicle association with cells.
  • Cf2Th cells were incubated with fluorescent liposomes (final concentration of 1 mg of lipid/ml) in the presence of 10 ⁇ M pre-aggregated amyloidogenic peptides A ⁇ i- 2 (gray), PPI-2566 (diagonal lines), A ⁇ 1 . 40 (black), PPI-2480 (dots) or no peptide (white) and analyzed using fluocytometry.
  • Non-amyloidogenic peptides, including PPI-1966 did not promote lipid vesicle association with cells.
  • B-F Negatively stained electron micrographs of amyloidogenic peptides, A ⁇ 1- 0 , A ⁇ i- 42 , PPI-2480, and PPI-2566 are shown (B-E, respectively). The non-amyloidogenic peptide PPI-1966 used for a control is also depicted (F). The scale bar represents 100 nM (F).
  • Figure 6 is a graph demonstrating that A ⁇ fibrils stimulate infection by viruses other than HIV-1.
  • A Cells were infected with a recombinant A-MuLV vector expressing ⁇ -galactosidase in the absence of additive or in the presence of 8 ⁇ g/ml of polybrene or 10 ⁇ M of pre-aggregated A ⁇ 40-1 reverse fragment or A ⁇ M o. The precipitable fraction of A ⁇ i ⁇ o was compared with any soluble fraction of A ⁇ 1-40 remaining in the supernatant following centrifugation. ⁇ -galactosidase expression 24 hours after incubation of viruses and cells was used to evaluate the efficiency of viral infection.
  • the present invention provides methods and compositions for treating a viral disease, e.g., a viral disease of the brain, in a subject by administering to the subject a therapeutically or prophylactically effective amount of an amyloidogenic peptide modulator compound, e.g., a ⁇ amyloid modulator compound.
  • a viral disease e.g., a viral disease of the brain
  • an amyloidogenic peptide modulator compound e.g., a ⁇ amyloid modulator compound.
  • viral disease is intended to include any disease, disorder or condition associated with or caused by a virus.
  • Viral diseases include, but are not limited to, infection with herpes simplex virus (type 1 and type 2), varicella zoster virus, poliomyelitis virus, cytomegalovirus, influenza virus (A and B), respiratory syncytial virus, coxsackie virus, ebola virus, hantavirus, human papilloma virus, rotavirus, west nile virus, Epstein-Barr virus, human immunodeficiency virus, and hepatitis virus (A, B and C).
  • herpes simplex virus type 1 and type 2
  • varicella zoster virus varicella zoster virus
  • poliomyelitis virus poliomyelitis virus
  • cytomegalovirus influenza virus
  • influenza virus A and B
  • respiratory syncytial virus coxsackie virus
  • ebola virus ebola virus
  • hantavirus
  • amyloidogenic peptide modulator compound is intended to include any compound, e.g., a peptide, a nucleic acid molecule or an antibody, capable of modulating, e.g., inhibiting, the aggregation of an amyloidogenic peptide, and/or modulating, e.g., inhibiting, the neurotoxicity of an amyloidogenic peptide (e.g., transthyretin (TTR), prion protein (PrP), islet amyloid polypeptide (IAPP), atrial natriuretic factor (ANF), kappa light chain, lambda light chain, amyloid A, procalcitonin, cystatin C, ⁇ 2 microglobulin, ApoA-I, gelsolin, procalcitonin, calcitonin, fibrinogen and lysozyme).
  • TTR transthyretin
  • PrP prion protein
  • IAPP islet amyloid polypeptide
  • AMF atrial na
  • the amyloidogenic peptide modulator compound may modulate the aggregation of amyloidogenic peptides by modulating: (a) the processing of amyloidogenic peptides (e.g., either by direct or indirect protease inhibition); (b) the synthesis of amyloidogenic peptides; (c) the release of amyloidogenic peptides; or (d) the processes that produce toxic amyloidogenic peptides in vivo.
  • amyloidogenic peptide modulator compound is also intended to include compounds which have the ability to remove aggregated amyloidogenic peptides, such as monoclonal antibodies against an amyloidogenic peptide; or vaccines which, when administered to a subject, elicit the formation of antibodies against an amyloidogenic peptide.
  • amloidogenic peptide is intended to include any peptide that is capable of forming fibrils. Amyloidogenic peptides are typically hydrophobic and may comprise at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 amino acid residues.
  • amyloidogenic proteins include transthyretin (TTR), prion protein (PrP), islet amyloid polypeptide (IAPP), atrial natriuretic factor (ANF), kappa light chain, lambda light chain, amyloid A, procalcitonin, cystatin C, ⁇ 2 microglobulin, ApoA-I, gelsolin, procalcitonin, calcitonin, fibrinogen and lysozyme.
  • TTR transthyretin
  • PrP prion protein
  • IAPP islet amyloid polypeptide
  • AMF atrial natriuretic factor
  • kappa light chain lambda light chain
  • amyloid A procalcitonin
  • cystatin C ⁇ 2 microglobulin
  • ApoA-I amyloid A
  • gelsolin procalcitonin
  • fibrinogen fibrinogen and lysozyme.
  • ⁇ amyloid modulator compound is intended to include any compound, e.g., a peptide, a nucleic acid molecule or an antibody, capable of modulating, e.g., inhibiting, the aggregation of natural ⁇ amyloid peptides ( ⁇ -AP) and/or modulating, e.g., inhibiting, the neurotoxicity of natural ⁇ amyloid peptides.
  • ⁇ amyloid modulator compound is also intended to include compounds which have the ability to remove aggregated ⁇ amyloid peptides, such as monoclonal antibodies against ⁇ amyloid; or vaccines which, when administered to a subject, elicit the formation of antibodies against ⁇ amyloid.
  • ⁇ amyloid modulator compounds are well known and readily available and include those described in, for example, U.S. Patent Nos.
  • the term "subject" includes warm-blooded animals, preferably mammals, including humans, hi a preferred embodiment, the subject is a primate. In an even more preferred embodiment, the primate is a human.
  • administering includes dispensing, delivering or applying an amyloidogenic peptide modulator compound, e.g., a ⁇ amyloid modulator compound, to a subject by any suitable route for delivery of the amyloidogenic peptide modulator compound, e.g., the ⁇ amyloid modulator compound, to the desired location in the subject, including delivery by either the parenteral or oral route, intracerebral injection, intramuscular injection, subcutaneous/intradermal injection, intravenous injection, buccal administration, transdermal delivery and administration by the rectal, colonic, vaginal, intranasal or respiratory tract route.
  • an amyloidogenic peptide modulator compound e.g., a ⁇ amyloid modulator compound
  • a prophylactically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as preventing or inhibiting a viral disease or infection of a cell by a virus in a subject predisposed to a viral disease or viral infection.
  • a prophylactically effective amount can be determined as described above for the therapeutically effective amount. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of viral disease, the prophylactically effective amount will be less than the therapeutically effective amount.
  • ⁇ Amyloid Modulator Compounds Any compound capable of modulating, e.g., inhibiting, the aggregation of natural ⁇ amyloid peptides ( ⁇ -AP) and/or modulatmg, e.g., inhibiting, the neurotoxicity of natural ⁇ -APs may be used in the methods of the present invention.
  • the ⁇ amyloid modulator compound may modulate the aggregation of natural ⁇ amyloid peptides by modulating: (a) the processing of natural ⁇ amyloid peptide (e.g., either by direct or indirect protease inhibition); (b) the synthesis of natural ⁇ amyloid peptide; (c) the release of natural ⁇ amyloid peptide; or (d) the processes that produce toxic ⁇ amyloid peptide, or other APP fragments, in vivo.
  • natural ⁇ amyloid peptide e.g., either by direct or indirect protease inhibition
  • Compounds which have the ability to remove aggregated ⁇ amyloid peptides may also be used in the methods of the present invention.
  • vaccines may be used which, when administered to a subject, elicit the formation of antibodies against ⁇ amyloid.
  • ⁇ amyloid modulator compounds e.g., peptides, peptidomimetics, nucleic acid molecules or antibodies
  • ⁇ amyloid modulator compounds are well known and readily available to those of skill in the art and include those described in, for example, U.S. Patent Nos. 5,985,242; 5,854,215; 5,948,763; 5,854,204; 6,022,859; 5,817,626; 6,051,684; 6,017,887; 6,015,879; 6,251,928; 5,969,100; 5,962,419; 6,177,246; 6,080,778; 6,211,235; 6,207,710;
  • peptide is intended to encompass peptide analogues, peptide derivatives, and peptidomimetics that mimic the chemical structure of a peptide composed of naturally-occurring amino acids.
  • peptide analogue as used herein is intended to include molecules that mimic the chemical structure of a peptide and retain the functional properties of the peptide.
  • peptide analogues include peptides comprising one or more non-natural amino acids.
  • peptide derivative as used herein is intended to include peptides in which an amino acid side chain, the peptide backbone, or the amino- or carboxyl-terminus has been derivatized (e.g., peptidic compounds with methylated amide linkages).
  • peptidomimetic as used herein is intended to include peptidic compounds in which the peptide backbone is substituted with one or more benzodiazepine molecules (see, e.g., James, G.L. et al. (1993) Science 260:1937-1942), "inverso" peptides in which all L-amino acids are substituted with the corresponding D- amino acids, “retro-inverso” peptides (see U.S. Patent No.
  • peptide back-bone i.e., amide bond
  • modifications of the amide nitrogen, the ⁇ -carbon, amide carbonyl including modifications of the amide nitrogen, the ⁇ -carbon, amide carbonyl, complete replacement of the amide bond, extensions, deletions or backbone crosslinks.
  • ⁇ indicates the absence of an amide bond.
  • the structure that replaces the amide group is specified within the brackets.
  • Other possible modifications include an N-alkyl (or aryl) substitution ( ⁇ [CONR]) 5 backbone crosslinking to construct lactams and other cyclic structures, and other derivatives including C-terminal hydroxymethyl derivatives, O-modified derivatives and N-terminally modified derivatives including substituted amides such as alkylamides and hydrazides.
  • the amyloidogenic peptide modulator compound e.g., the ⁇ amyloid modulator compound
  • the amyloidogenic peptide modulator compound e.g., the ⁇ amyloid modulator compound
  • the amyloidogenic peptide modulator compound may be combined with one or more other active ingredients such as other drugs suitable for treating a viral disease, e.g., an HIV-1 infection.
  • Other pharmaceutically active compounds that may be used can be found in Harrison's Principles of Internal Medicine, Thirteenth Edition, Eds. T.R. Harrison et al.
  • the pharmaceutical composition comprises an amyloidogenic peptide modulator compound, e.g., a ⁇ amyloid modulator compound, and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carrier is suitable for parenteral administration or for administration via inhalation.
  • the carrier is suitable for administration into the central nervous system (e.g., intraspinally or intracerebrally).
  • the carrier can be suitable for intravenous, intraperitoneal or intramuscular administration, hi another embodiment, the carrier is suitable for oral administration.
  • Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, or sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.
  • the compounds of the invention can be administered in a time release formulation, for example in a composition which includes a slow release polymer.
  • the amyloidogenic peptide modulator compound e.g., the ⁇ amyloid modulator compound
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers (PLG). Many methods for the preparation of such formulations are patented or generally known to those skilled in the art.
  • Sterile injectable solutions can be prepared by incorporating the amyloidogenic peptide modulator compound, e.g., the ⁇ amyloid modulator compound, in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
  • the amyloidogenic peptide modulator compound e.g., the ⁇ amyloid modulator compound
  • Preferred compounds to be added to formulations to enhance the solubility of an amyloidogenic peptide modulator compound, e.g., a ⁇ amyloid modulator compound are cyclodextrin derivatives, preferably hydroxypropyl- ⁇ - cyclodextrin.
  • amyloidogenic peptide modulator compound e.g., the ⁇ amyloid modulator compound
  • Another formulation for the amyloidogenic peptide modulator compound, e.g., the ⁇ amyloid modulator compound comprises the detergent Tween-80, polyethylene glycol (PEG) and ethanol in a saline solution.
  • Tween-80 polyethylene glycol
  • PEG polyethylene glycol
  • ethanol in a saline solution.
  • a non-limiting example of such a preferred formulation is 0.16% Tween-80, 1.3% PEG-3000 and 2% ethanol in saline.
  • the amyloidogenic peptide modulator compound e.g., the ⁇ amyloid modulator compound
  • a pharmaceutical composition comprising a solid ionic complex of an amyloidogenic peptide modulator compound, e.g., a ⁇ amyloid modulator compound, and a carrier macromolecule, wherein the amyloidogenic peptide modulator compound, e.g., the ⁇ amyloid modulator compound, content of said complex is at least 40% by weight, preferably at least 45%, 50%, 55%, 57%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or
  • Ranges intermediate to the above recited values e.g., at least about 50% to about 80%, at least about 60% to about 90%, or at least about 57% to about 80%, are also intended to be part of this invention.
  • ranges of values using a combination of any of the above recited values as upper and/or lower limits are intended to be included.
  • carrier macromolecule is intended to refer to a macromolecule that can complex with a peptide to form a water-insoluble complex.
  • the macromolecule has a molecular weight of at least 5 kDa, more preferably at least 10 kDa.
  • anionic carrier macromolecule is intended to include negatively charged high molecular weight molecules, such as anionic polymers.
  • cationic carrier macromolecule is intended to include positively charged high molecular weight molecules, such as cationic polymers.
  • water-insoluble complex is intended to refer to a physically and chemically stable complex that forms upon appropriate combining of a ⁇ amyloid modulator compound and carrier macromolecule according to procedures described herein.
  • a water-insoluble complex of the invention may involve (e.g., be mediated at least in part by) hydrophobic interactions. Still further, formation of a water-insoluble complex of the invention may involve (e.g., be mediated at least in part by) covalent interactions. Description of the complex as being "water-insoluble” is intended to indicate that the complex does not substantially or readily dissolve in water, as indicated by its precipitation from aqueous solution. However, it should be understood that a "water- insoluble" complex of the invention may exhibit limited solubility in water either in vitro or in the aqueous physiological environment in vivo.
  • Sustained delivery of the amyloidogenic peptide modulator compound can be demonstrated by, for example, the continued therapeutic effect of the amyloidogenic peptide modulator compound, e.g., the ⁇ amyloid modulator compound, over time.
  • sustained delivery of the amyloidogenic peptide modulator compound, e.g., the ⁇ amyloid modulator compound may be demonstrated by detecting the presence of the amyloidogenic peptide modulator compound, e.g., the ⁇ amyloid modulator compound, in vivo over time.
  • a complex used in the methods of the invention is prepared by combining the amyloidogenic peptide modulator compound, e.g., the ⁇ amyloid modulator compound, and the carrier macromolecule under conditions such that a water-insoluble complex of the amyloidogenic peptide modulator compound, e.g., the ⁇ amyloid modulator compound, and the carrier macromolecule forms.
  • a pharmaceutical formulation of the invention is a sterile formulation.
  • the complex can be sterilized, preferably by gamma radiation or electron beam sterilization.
  • the water- insoluble complex can be isolated using conventional sterile techniques (e.g., using sterile starting materials and carrying out the production process aseptically).
  • the pharmaceutical formulation can be administered to the subject by any route suitable for achieving the desired therapeutic result(s), although prefened routes of administration are parenteral routes, in particular intramuscular (i.m.) injection and subcutaneous/intradermal (s.c./i.d.) injection. Alternatively, the formulation can be administered to the subject orally.
  • Other suitable parental routes include intravenous injection, buccal administration, transdermal delivery and administration by the rectal, vaginal, intranasal or respiratory tract route. It should be noted that when a formulation that provides sustained delivery for weeks to months by the i.m or s.c./i.d.
  • the dosage of the amyloidogenic peptide modulator compound is about 10-500 mg/month, about 20-300 mg/month, or about 30-200 mg/month.
  • the dosage of the amyloidogenic peptide modulator compound is about 30-120 mg/month. Ranges intermediate to the above recited values, e.g., about 10-200 mg/month, about 30-250 mg/month, or about 100-200 mg/month, are also intended to be part of this invention. For example, ranges of values using a combination of any of the above recited values as upper and or lower limits are intended to be included.
  • the dosage of the amyloidogenic peptide modulator compound e.g., the ⁇ amyloid modulator compound
  • the dosage of the amyloidogenic peptide modulator compound is about 5-500 ⁇ g/kg/day, about 10-400 ⁇ g/kg/day, or about 20-200 ⁇ g/kg/day.
  • the dosage of the amyloidogenic peptide modulator compound e.g., the ⁇ amyloid modulator compound
  • dosage values may vary with the severity of the condition to be alleviated.
  • Another aspect of the invention pertains to methods for identifying an amyloidogenic peptide modulator compound, e.g., a ⁇ amyloid modulator compound, capable of treating a viral disease in a subject or capable of modulating infection of a cell by a virus.
  • an amyloidogenic peptide modulator compound e.g., a ⁇ amyloid modulator compound
  • the methods include contacting a cell, e.g., a brain cell, with a virus in the presence of an amyloidogenic peptide (e.g., ⁇ amyloid peptide) and an amyloidogenic peptide modulator compound, e.g., a ⁇ amyloid modulator compound, and determining the ability of the virus to infect the cell, thereby identifying an amyloidogenic peptide modulator compound, e.g., a ⁇ amyloid modulator compound, capable of treating a viral disease in a subject or capable of modulating infection of a cell by a virus.
  • an amyloidogenic peptide e.g., ⁇ amyloid peptide
  • an amyloidogenic peptide modulator compound e.g., a ⁇ amyloid modulator compound
  • Virally infected cells e.g., virally infected brain cells, treated with an amyloidogenic peptide modulator compound, e.g., a ⁇ amyloid modulator compound, can be examined for one or more phenotype(s) associated with viral infection and/or viral disease.
  • Cellular phenotypes that are associated with viral disease include viral infection (e.g., virus burden), cell lysis, plaque formation, and low pH induced fusion of infected cells (Sung T-C et ⁇ l. (1997) EMBO J. 16:4519-4530; Roper RL and Moss B (1999) J. Virol. 73:1108-1117; Blasco R and Moss B (1991) J. Virol 65:5910-5920).
  • animal-based viral disease systems such as those described herein, maybe used to identify compounds capable of ameliorating viral disease symptoms.
  • Such animal models may be exposed to an amyloidogenic peptide modulator compound, e.g., a ⁇ amyloid modulator compound, at a sufficient concentration and for a time sufficient to elicit an amelioration of a viral disease symptom in the exposed animal.
  • the response of the animals to the amyloidogenic peptide modulator compound, e.g., the ⁇ amyloid modulator compound may be monitored by assessing the reversal of disorders associated with viral disease, for example, by monitoring viral burden before and after treatment.
  • amyloidogenic proteins include transthyretin (TTR), prion protein (PrP), islet amyloid polypeptide (IAPP), atrial natriuretic factor (ANF), kappa light chain, lambda light chain, amyloid A, procalcitonin, cystatin C, ⁇ 2 microglobulin, ApoA-I, gelsolin, procalcitonin, calcitonin, fibrinogen and lysozyme.
  • TTR transthyretin
  • PrP prion protein
  • IAPP islet amyloid polypeptide
  • AMF atrial natriuretic factor
  • kappa light chain lambda light chain
  • amyloid A procalcitonin
  • cystatin C ⁇ 2 microglobulin
  • ApoA-I gelsolin
  • procalcitonin calcitonin
  • fibrinogen fibrinogen and lysozyme.
  • the amyloidogenic peptides used in the methods of promoting infection are amyloidogenic peptides which fonn short fibrils, e.g., or PPI-2480 (AGAKWSWWELTWVGG; SEQ ID NO:l).
  • the methods include contacting a cell with an amyloidogenic peptide or an amyloidogenic peptide modulator compound, e.g., a ⁇ amyloid modulator compound, in an amount effective to promote infection of the cell by a virus (e.g., an Adeno-associated virus (AAV) or a retro virus).
  • a virus e.g., an Adeno-associated virus (AAV) or a retro virus.
  • gene therapy vectors can be delivered to a subject in conjunction with an amyloidogenic peptide modulator compound, e.g., a ⁇ amyloid modulator compound, or a non-toxic amyloidogenic peptide by, for example, intravenous injection, local administration (see U.S. Patent 5,328,470) or by stereotactic injection (see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057).
  • an amyloidogenic peptide modulator compound e.g., a ⁇ amyloid modulator compound, or a non-toxic amyloidogenic peptide by, for example, intravenous injection, local administration (see U.S. Patent 5,328,470) or by stereotactic injection (see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057).
  • the pharmaceutical preparation of the gene therapy vector can include the gene therapy vector as well as an effective amount of an amyloidogenic peptide modulator compound, e.g., a ⁇ amyloid modulator compound, or a non-toxic amyloidogenic peptide in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle and the amyloidogenic peptide modulator compound, e.g., the ⁇ amyloid modulator compound, or the non-toxic amyloidogenic peptide are imbedded.
  • an amyloidogenic peptide modulator compound e.g., a ⁇ amyloid modulator compound, or a non-toxic amyloidogenic peptide in an acceptable diluent
  • an effective amount of an amyloidogenic peptide modulator compound e.g., a ⁇ amyloid modulator compound, or a non-toxic amyloidogenic peptide in an acceptable diluent
  • an amyloidogenic peptide modulator compound e.
  • viruses e.g., recombinant retroviruses
  • infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14.
  • suitable retroviruses include pLJ, pZIP, pWE and pEM which are well known to those skilled in the art.
  • Recombinant HIV-1 reporter viruses were constructed by cotransfection of 293T- HEK cells with vectors expressing the pCMV ⁇ Pl ⁇ envpA HIV-1 Gag-Pol packaging construct (Parolin, C. et al. (1996) Virology 222, 415-422) the envelope glycoproteins of AMLV, VSV, and HIV-1 isolates (ADA, YU2, JR-FL and HXBc2), and a reporter gene at a DNA weight ratio of 1 : 1 :3 using Effectene reagents (Qiagen).
  • Cotransfection produced replication-defective (single-round) virions capable of expressing HIV-1 tat and the firefly luciferase gene under control of the HIV-1 long terminal repeat (LTR), or the green fluorescent protein (GFP) gene under control of the cytomegalovirus immediate-early promoter (CMV).
  • Viruses pseudotyped with VSV-G, A-MuLV, and HIV-1 envelope glycoproteins were produced by cotransfecting the pHCMV-G (Yee, J. et al. (1994) Proc Natl Acad Sci USA 91, 9564-9568), SV-A-MLV-Env ( Landau, N. et al.
  • Target cells for viral entry were seeded in 96-well luminometer-compatible tissue culture plates (Dynex) at a density of 6 x 10 3 cells/well and incubated for 24 hours. The medium was removed from the target cells and replaced with fresh complete DMEM containing RT-normalized units of recombinant virus. The amounts of virus varied depending upon the envelope glycoproteins used for pseudotyping: VSV-G, IK cpm; A- MuLV, 30K cpm; ADA, YU2, JR-FL, 89.6, ADA- ⁇ V1/V2, and HXBc2 HIV-1 envelope glycoproteins, 10K cpm).
  • Varying amounts of A ⁇ 1- 0 , A ⁇ 1- 2 (1.25-20 ⁇ M), PPI-2566 or PPI-2480 (1-100 ⁇ M) were added with the recombinant viruses, to a final infection volume of 50 ⁇ l.
  • Target cells were incubated with the infection medium for 48 hours. Following this incubation, the medium was aspirated from each well and the cells were lysed by addition of 30 ⁇ l passive lysis buffer (Promega Corp.), agitation, and 2 freeze-thaw cycles.
  • the luciferase activity of each well was measured for 10 seconds following addition of 100 ⁇ l luciferase buffer (15 mM MgSO 4 , 15 mM KPO 4 , pH 7.8, 1 mM ATP, 1 mM DTT) and 50 ⁇ l 1 mM D-luciferin potassium salt (BD PharMingen) using an EG&G Berthold Microplate Lu inometer LB 96V.
  • SupTl-CCR5 target cells were seeded in 24- well tissue culture plates (Falcon) at a density of 5 x 10 4 cells/well with medium containing RT-normalized units of GFP- expressing recombinant virus (VSV-G, 3K cpm; ADA, 150K cpm; YU2, 150K cpm) and varying amounts of A ⁇ 1-40 or A ⁇ 1-42 (62.5 nM-1 ⁇ M) in a final volume of 0.4 ml.
  • the infection medium-cell mixture was incubated for 48 hours, 1 ml of fresh complete RPMI was added to each well, and the cells were incubated for an additional 24 hours.
  • the cells were then harvested, washed with PBS, fixed in 10% formalin, and analyzed by florescence-activated cell sorting using a Becton Dickinson FACScan with CellQuest software.
  • Rhodamine- labeled liposomes were incubated with Cf2Th cells in the presence and absence of peptide fibrils in the medium used for the viral entry assay (DMEM + 10% fetal bovine serum) supplemented with 0.02% NaN 3 for 1 hour at 37° C.
  • fluorescent A ⁇ 40 fibrils were incubated with Cf2Th cells either lacking or expressing CD4 and/or CCR5, in the absence and presence of recombinant HIV-1 g ⁇ l20 envelope glycoprotein from the JR-FL isolate.
  • Cf2Th-CD4/CCR5 cells were also incubated without additives or with unlabeled pre- aggregated A ⁇ M o, as above, and the CD4 and CCR5 cell surface expression was detected using the anti-CD4 antibody RPA-T4-PE (BD PharMingen) and the anti-CCR5 antibody 2D7-PE (BD PharMingen) at a final concentration of 10 nM.
  • EXAMPLE 1 A ⁇ ⁇ - 40 AND A ⁇ ⁇ _ 42 ENHANCE SIGNAL OF VIRAL ENTRY To investigate whether the presence of A ⁇ affects HIV-1 infection of target cells, recombinant replication-defective HIV-1 vectors expressing firefly luciferase or GFP were used. These single-round viruses were pseudotyped with the envelope glycoproteins of various HIV-1 isolates, or with those of VSV or A-MuLV. The receptors for VSV and A-MuLV are ubiquitously expressed.
  • viruses pseudotyped with the HIV-1 envelope glycoproteins are dependent on the presence of CD4 or a chemokine receptor, CCR5 or CXCR4; the viruses pseudotyped with the VSV or A-MuLV envelope glycoproteins do not require CD4 or chemokine receptor expression on the target cells.
  • Pre-aggregated A ⁇ o and A ⁇ i- 4 2 fibrils dramatically increased infection of Cf2Th-CD4/CCR5 cells by HIV-1 pseudotyped with the envelope glycoproteins of three CCR5-using primary HIV-1 isolates (ADA, YU2, JR-FL) in a dose-dependent manner (Figure 1).
  • a ⁇ 1-40 similarly increased infection of GHOST(3)- CD4/CXCR4 cells by HIV-1 pseudotyped with the envelope glycoproteins of the CXCR4-using isolate, HXBc2 ( Figure 1). Similar results were obtained by infecting a human T-lymphocyte cell line stably expressing CCR5 (SupTl-CCR5) with GFP- expressing viruses pseudotyped with ADA and YU2 envelope glycoproteins.
  • a ⁇ 1-40 was more potent than A ⁇ 1-42 and increased the entry of viruses by 2-10 times in a concentration range of 1-5 ⁇ M, and by 5-30 times at a concentration of 20 ⁇ M. Infection of cells by viruses pseudotyped with the A-MuLV or VSV envelope glycoproteins was also enhanced. The relatively lower enhancement observed with VSV-G-pseudotyped virus may be due to the substantially greater efficiency with which this virus infects cells in the absence of A ⁇ .
  • a ⁇ can substantially increase the efficiency of infection of cells by HIV- 1 pseudotyped with the envelope glycoproteins of a wide range of HIV- 1 isolates, as well as with those of other enveloped viruses.
  • incubation of the recombinant viruses with the Cf2Th-CD4/CCR5 target cells was carried out for only 4 hours, followed by washing.
  • the target cells were incubated with 20 ⁇ M A ⁇ 1-40 concurrently with virus (+/-), immediately following removal of virus (-/+), or throughout both time periods (+/+) ( Figure 2). After the wash, the cells were incubated for an additional 48 hours, at which time luciferase activity was measured. Enhancement of infection was observed only when A ⁇ ⁇ - 0 was present during the initial 4-hour incubation of virus and cells.
  • a ⁇ has been shown to exert a destabilizing effect on cellular membranes (Yip, C. M. et al. (2001) Biophys. J. 80, 1359-1371; and McLaurin, J. et al. (1997) Eur. J. Biochem. 245, 355-363). Therefore, A ⁇ might facilitate fusion of the target cell and viral membrane in a manner that would circumvent the dependence of the virus on its receptors. To investigate this possibility, infection of Cf2Th, Cf2Th-CD4, Cf2Th- CCR5, and Cf2Th-CD4/CCR5 cells by CCR5-dependent HIV-1 isolates was examined.
  • Figure 4 shows that two such peptides, PPI-2480 (AGAKWSWWELTWVGG; SEQ ID NO:l) and PPI-2566 IRQAMCNISRADWND; SEQ ID NO: 2), which form fibrils similar to A ⁇ o and A ⁇ 1- 2 ( Figures B-F), also enhanced the infection efficiency of recombinant HIV-1 virus pseudotyped with the envelope glycoproteins of the ADA and YU2 HIV-1 isolates by 5-20 fold. The stimulation by these fibrils also required the expression of viral entry coreceptors. These compounds enhanced infection of HIV-1 virus pseudotyped with the VSV-G protein by approximately two-fold. A number of control peptides of varying sequences and lengths that did not form fibrils had no effect on HIV-1 infection.
  • EXAMPLE 5 FIBRIL-FORMING PEPTIDES PROMOTE LIPID VESICLE
  • the peptides did not cause the formation of syncytia, nor did they promote liposome-to-cell fusion, as judged by the failure of the rhodamine dye in the liposomes to distribute into the cell membrane. Consistent with their relative ability to enhance infection, A ⁇ 1- 0 promoted the adherence of liposomes better than A ⁇ 1- 2 . PPI-1966, which Figure 5F shows cannot fonn fibrils, had no effect on the association of liposomes with cells. Utilizing fluorescent A ⁇ 1-40 fibrils (FITC- A ⁇ ), it was demonstrated that FITC-A ⁇ associated with cell surfaces independent of CD4 or CCR5 expression.
  • EXAMPLE 7 A ⁇ WEAKLY STIMULATES INFECTION BY AN ENVELOPED VIRUS OTHER THAN A RETROVIRUS
  • CSF cerebrospinal fluid
  • a CSF solution is prepared containing 75% Rhesus monkey CSF (commercially available from Northern Biomedical Research), 23% sterile phosphate buffered saline and 2% dimethylsulfoxide (v/v) (Aldrich Chemical Co., Catalog No. 27,685-5).
  • ⁇ amyloid modulator compounds are added to the CSF solution to a final concentration of 40 ⁇ M or 15 ⁇ M. All sample handling is carried out in a laminar flow hood and test solutions are maintained at 37 °C during the assay.
  • Chromatographic analysis is performed using a Hewlett Packard 1090 series II HPLC.
  • the column used for separation is a C4, 5 ⁇ m, 1 x 250 mm (Vydac #214TP51).
  • the flow rate is 50 ⁇ L/min and the elution profile of the test compounds is monitored at 214, 230, 260 and 280 nm.
  • ASSAY Brain levels of a ⁇ amyloid modulator compound are determined using a rat following intravenous administration. Under ketamine/xylazine anesthesia male Sprague-Dawley rats (219-302g) receive an intravenous injection via a catheter inserted in the left jugular vein (dose volume of 4 mL/kg administered over 1 minute).
  • the left common carotid artery is cannulated to enable perfusion of the left forebrain to remove cerebral blood.
  • the left forebrain, void of blood is subjected to capillary depletion as described by Triguero et al. (1990) J. Neurochem. 54:1882-1888.
  • This established technique separates brain vasculature from the parenchyma and, thus, allows the accurate determination of the concentration of the compound under investigation that has traversed the blood brain barrier.
  • the amount of parent compound that is present within the brain is detennined by LC/MS/MS.

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Abstract

The present invention provides methods and compositions for modulating infection of a cell by a virus and methods and compositions for treating a viral disease, e.g., a viral disease of the brain, in a subject.

Description

METHODS FOR TREATING VIRAL DISEASES USING MODULATORS OF AMYLOIDOGENIC PEPTIDE AGGREGATION
Related Applications This application claims priority to U.S. Provisional Patent Application Serial
No. 60/390,040 filed June 18, 2002 U.S. Provisional Patent Application Serial No. 60/394, 390 filed July 8, 2002 the entire contents of which are incorporated herein by reference.
Background of the Invention
Viral infections have come to be an accepted part of everyday life. From children with chickenpox to the many people each year who develop the flu, viruses are often encountered. Many other viruses affect individuals, such as those that cause warts, herpes, chlamydia and more seriously hepatitis A, B, and C. Of the many viruses that infect humans, the most devastating is the Human
Immunodeficiency Virus (HIV). Millions of individuals world- wide are infected with HIV. Consequently, HIV infection represents a serious public health concern. HIV has been determined to be the etiologic agent for acquired immunodeficiency syndrome (AIDS). The number of people living world wide with AIDS is currently 36.1 million. The number of new HIV cases each year is increasing and so will the number of individuals with AIDS unless a treatment is discovered.
HIV, like all other viruses, is incapable of replicating on its own and must use the machinery of the infected host cell. Most current therapies are directed toward targets that present themselves after the cell is infected. Current therapeutic agents used during the treatment of chronic phase of HIV infection have not been particularly efficacious and are, at best, only able to slow the progress of infection. For example, several drugs have been identified which inhibit the replication of HIV in vitro (Haseltine, W. A. (1989) J. Acquir. Immune Def. Syndr., 2:311-324) and nucleotide chain elongation inhibitors such as 3-azidothymidine (AZT) have received widespread acceptance for clinical use. Despite the success of these drugs in prolonging the onset of AIDS, there are currently no drugs marketed that would stop the virus from infecting a cell.
Summary of the Invention The present invention is directed to methods and compositions for modulating infection of a cell, e.g., a brain cell, by a virus and methods and compositions for treating a viral disease, e.g., a viral disease of the brain, in a subject. The present invention is based, at least in part, on the discovery that infectivity of cells by the human immunodeficiency virus (HIV) is enhanced in the presence of aggregated /3-amyloid fibrils in the brain. Without intending to be limited by theory, it is believed that aggregates of amyloidogenic peptide fibrils, e.g., /3-amyloid fibrils, in the brain may facilitate infection of brain cells by HIV and contribute to the development of AJDS- related dementia.
Accordingly, the present invention provides a method for treating a viral disease, e.g., a viral disease of the brain, in a subject by administering to the subject a therapeutically or prophylactically effective amount of an amyloidogenic peptide modulator compound, e.g., a β amyloid modulator compound, thereby treating a viral disease in the subject.
In one embodiment, the viral disease is caused by infection with a virus selected from the group consisting of human immunodeficiency virus, herpes simplex virus, varicella zoster virus, poliomyelitis virus, cytomegalovirus, influenza virus, respiratory syncytial virus, coxsackie virus, ebola virus, hantavirus, human papilloma virus, rotavirus, west nile virus, Epstein-Barr virus, and hepatitis virus.
The β amyloid modulator compound may be a peptide (e.g., a vaccine), a peptidomimetic, an antibody, a nucleic acid molecule, a protease inhibitor, a β-secretase or γ-secretase inhibitor, or a modulator of blood cholesterol levels. hi one embodiment, the β amyloid modulator compound is a peptide comprised entirely of D-amino acids and having at least three amino acid residues independently selected from the group consisting of a D-leucine structure, a D-phenylalanine structure, a D-tyrosine structure, a D-iodotyrosine structure and a D-alanine structure.
In another embodiment, the β amyloid modulator compound is a compound comprising a formula: '
Figure imgf000003_0001
wherein Xaa^, Xaa2, Xaaβ and Xaa4 are each D-amino acid structures and at least three of Xaal5 Xaa2, Xaa and Xaa4 are, independently, selected from the group consisting of a D-leucine structure, a D-phenylalanine structure, a D-tyrosine structure, a D-iodotyrosine structure and a D-alanine structure;
Y, which may or may not be present, is a structure having the formula (Xaa)a, wherein Xaa is any D-amino acid structure and a is an integer from 1 to 15;
Z, which may or may not be present, is a structure having the formula (Xaa)b, wherein Xaa is any D-amino acid structure and b is an integer from 1 to 15;
A is a modifying group attached to the compound, with the proviso that A is not an amino acid or is a non-natural amino acid that acts as a β-turn mimetic; and n is an integer from 1 to 15; wherein Xaaj, Xaa2, Xaa3, Xaa,4, Y, Z, A and n are selected such that the compound binds to natural β-amyloid peptides or modulates the aggregation or inhibits the neurotoxicity of natural β-amyloid peptides when contacted with the natural β-amyloid peptides.
In a further embodiment, the β amyloid modulator compound is a compound having a structure selected from the group consisting of: N,N-dimethyl-(Gly-D-Ala-D-Phe-D-Phe-D-Val-D-Leu)-NH2; N,N-dimethyl-(D-Ala-D- Phe-D-Phe-D-Val-D-Leu)-NH2; N-methyl-(Gly-D-Ala-D-Phe-D-Phe-D-Val-D-Leu)- NH2; N-ethyl-(Gly-D-Ala-D-Phe-D-Phe-D-Val-D-Leu)-NH2; N-isopropyl-(Gly-D-Ala- D-Phe-D-Phe-D-Val-D-Leu)-NH2; H-(D-Leu-D-Val-D-Phe-D-Phe-D-Ala)- isopropylamide; H-(D-Leu-D-Val-D-Phe-D-Phe-D-Ala)-dimethylamide; N,N-diethyl- (Gly-D-Ala-D-Phe-D-Phe-D-Val-D-Leu)-NH2; N,N-diethyl-(D-Ala-D-Phe-D-Phe-D- Val-D-Leu)-NH2; N,N-dimethyl-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH2; N,N- dimethyl-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH2; N,N-dimethyl-(D-Leu-D-Phe-D- Phe-D-Val-D-Leu)-NH2; H-(Gly-D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH2; N-ethyl- (Gly-D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH2; N-ethyl-(Gly- D-Leu-D-Phe-D-Phe-D- Val-D-Leu)-NH2; N-methyl-(D-Leu-D-Phe-D-Phe-D-Val-D-Leu)-NH2; N-ethyl-(D-Leu- D-Val-D-Phe-D-Phe-D-Leu)-NH2; N-propyl-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH2; N,N-diethyl-(Gly-D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH2; H-(D-Ile-D-Val-D-Phe-D- Phe-D-Ile)-NH2; H-(D-Ile-D-Val-D-Phe-D-Phe-D-Ala-)-NH2; H-( D-Ile- D-Ile-D-Phe- D-Phe- D-Ile)-NH2; H-(D-Nle-D-Val-D-Phe-D-Phe-D-Ala-)-NH2; H-(D-Nle-D-Val-D- Phe-D-Phe-D-Nle)-NH2; l-piρeridine-acetyl-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH2; 1 -ρiperidine-acetyl-(D-Leu-D-Phe-D-Phe-D-Val-D-Leu)-NH2; H-D-Leu-D-Val-D-Phe- D-Phe-D-Leu-isopropylamide; H-D-Leu-D-Phe-D-Phe-D-Val-D-Leu-isopropylamide; H-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-methylamide; H-(D-Leu-D-Phe-D-Phe-D-Val- D-Leu)-methylamide; H-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-OH; N-methyl-(D-Leu-D- Val-D-Phe-D-Phe-D-Leu)-NH2; H-(D-Leu-D-Val-D-Phe-D-Cha-D-Leu)-NH2; H-(D- Leu-D-Val-D-Phe-D-[p-F]Phe-D-Leu)-NH2; H-(D-Leu-D-Val-D-Phe-D-[F5]Phe-D-
Leu)-NH2; H-(D-Leu-D-Phe-D-Cha-D-Val-D-Leu)-NH2; H-(D-Leu-D-Phe- D-[p-F]Phe- D-Val-D-Leu)-NH2; H-(D-Leu-D-Phe- D-[F5]Phe-D-Val-D-Leu)-NH2; H-(D-Leu-D- Phe-D-Lys-D-Val-D-Leu)-NH2; H-(D-Leu-D-Cha-D-Phe-D-Val-D-Leu)-NH2; H-(D- Leu-D-l -F]Phe-D-Phe-D-Val-D-Leu)-NH2; H-(D-Leu-D-[F5]Phe-D-Phe-D-Val-D- Leu)-NH2; H-(D-Leu- D-Lys-D-Phe-D-Val-D-Leu)-NH2; H-(D-Leu-D-Cha-D-Cha-D- Val-D-Leu)-NH2; H-(D-Leu- D-[p-F]Phe-D-I -F]Phe-D-Val-D-Leu)--NH2; H-(D-Leu-D- [F5]Phe-D-[F5]Phe-D-Val-D-Leu)-NH2; H-(D-Leu- D-Lys- D-Lys-D-Val-D-Leu)-NH2; N-methyl-(D-Leu-D-Val-D-Phe-D-Cha-D-Leu)-NH2; N-methyl-(D-Leu-D-Val-D-Phe- D-| -F]Phe-D-Leu)-NH2; N-methyl-(D-Leu-D-Val-D-Phe-D-[F5]Phe-D-Leu)-NH2; H- D-Leu-D-Val-D-Phe-NH-(H-D-Leu-D-Val-D-Phe-)NH; H-D-Leu-D-Val-D-Phe-NH- NH-COCH3; H- D-Leu-D-Val-D-Phe-NH-NH2 H-(lv-[3-I]y-fa)-NH2; H-(lvffl)-NH-Et; H-lvffl-NH-CH2CH2-NH2; H-(GGClvffl)-NH2; H-(GGClvfyl)-NH2; H-(GGClvf-[3-I]y-l)- NH2; H-LVF-NH-NH-FVL-H; H-LVF-NH-NH-M-H; H-lff-(nvl)-l-NH2; H-lf-[pF]f- (nvl)-l-NH2; H-l-[pF]f-[pF]f-(nvl)-l-NH2; Me-lvyfl-NH2; H-(lvyfl)-NH2; Me-(lv-[p-F]f-fl)- NH2; H-(lv-| -F]f-fl)-NH2; H-(lvf-0-F]f-l)-NH2; lvff-[nvl])-NH2; Me-(lvff-[nle])-NH2; Me-(lvffl)-OH; Me-(lvffl)-NH-OH; H-(lv-[p-F]f-f-(nvl))-NH2; Me-(l-v-[p-F]f-f-(nvl))- NH2; H-((nvl)-v-[p-F]f-f-nvl)-NH2; H-(l-(nvl)-| -F]f-f-(nvl)-NH2; H-((nvl)-(nvl)-[p-F]f- f-(nvl))-NH2; Me-(l-(nvl)-[p-F]f-f-(nvl))-NH2; H-(lvff-(nvl))-NH2; Ac-(lvffl)-NH2; Ac- (lvffl)-OH; and H-(lv-[3-I]y-fl)-NH2.
In a preferred embodiment, the β amyloid modulator compound is a compound having the structure: N-methyl-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)- ϊ2 or the structure: 4-Hydroxybenzoyl-D-Leu-D-Val-D-Phe-D-Phe-D-Ala-NH2 In another aspect, the present invention provides a method for treating AIDS in a subject by administering to the subject a therapeutically or prophylactically effective amount of an amyloidogenic peptide modulator compound, e.g., a β amyloid modulator compound, thereby treating ADDS in the subject. h yet another aspect, the present invention provides a method of modulating infection of a cell, e.g. , a brain cell, by a virus by contacting the cell with an amyloidogenic peptide modulator compound, e.g., a β amyloid modulator compound, in an amount effective to modulate infection of the cell by a virus.
In a further aspect, the present invention provides a method of modulating infection of a cell, e.g., a brain cell, by a human immunodeficiency virus by contacting the cell with an amyloidogenic peptide modulator compound, e.g., a β amyloid modulator compound, in an amount effective to modulate infection of the cell by a human immunodeficiency virus.
In another aspect, the present invention provides a method for identifying an amyloidogenic peptide modulator compound, e.g., a β amyloid modulator compound, capable of treating a viral disease in a subject. The method includes contacting a cell with a virus in the presence of an amyloidogenic peptide (e.g., β amyloid peptide) and an amyloidogenic peptide modulator compound, e.g., a β amyloid modulator compound, and determining the ability of the virus to infect the cell, h one embodiment, the ability of the virus to infect the cell is determined by monitoring the cell for a phenotype associated with viral infection, e.g., virus burden, cell lysis, plaque formation, or low pH induced fusion of infected cells. In one embodiment, the method may further include administering the amyloidogenic peptide modulator compound, e.g., the β amyloid modulator compound, to an animal model for a viral disease and determining whether the amyloidogenic peptide modulator compound, e.g., the β amyloid modulator compound, is capable of ameliorating a viral disease symptom in the animal.
In another embodiment, the method may further include administering the amyloidogenic peptide modulator compound, e.g., the β amyloid modulator compound, to an animal model for a viral disease and monitoring viral burden in the animal before and after the administration of the amyloidogenic peptide modulator compound, e.g., the β amyloid modulator compound.
In yet another aspect, the present invention provides a method for identifying an amyloidogenic peptide modulator compound, e.g., a β amyloid modulator compound, capable of modulating infection of a cell by a virus. The method includes contacting a cell with a virus in the presence of an amyloidogenic peptide (e.g., β amyloid peptide) and an amyloidogenic peptide modulator compound, e.g. , a β amyloid modulator compound, and determining the ability of the virus to infect the cell, thereby identifying an amyloidogenic peptide modulator compound, e.g., a β amyloid modulator compound, capable of modulating infection of a cell by a virus.
In another aspect, the present invention provides kits which include an amyloidogenic peptide modulator compound, e.g., a β amyloid modulator compound, and instructions for use in modulating infection of a cell by a virus or instructions for use in treating a viral disease in a subject.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
Brief Description of the Drawings
Figure 1 is a graph demonstrating that Aβ stimulates infection by recombinant HIV-1 viruses. The efficiency of infection of C£2Th-CD4/CCR5 or GHOST(3> CD4/CXCR4 cells by recombinant luciferase-expressing HIV-1 viruses pseudotyped with CCR5-using HIV-1 envelope glycoproteins (ADA, YU2, JR-FL) or the CXCR4- using HIV-1 envelope glycoprotein (HXBc2), respectively, was determined by measuring luciferase activity in the cells. Viruses with VSV-G and A-MuLV envelope proteins, which utilize ubiquitously-expressed receptors, were included for comparison. Increasing concentrations of aggregated Aβ1-40 (left) and Aβ1-42 (right) peptides were incubated with the virus and target cells. Luciferase activities were normalized relative to those observed for each recombinant virus in the absence of peptide. Results are representative of the median values obtained from independent assays performed in duplicate or triplicate.
Figure 2 is a graph demonstrating that Aβ acts at an early stage of viral infection. Cf2Th-CD4/CCR5 cells were incubated with recombinant HIV-1 pseudotyped with HIV-1 (ADA, YU2, JR-FL) or A-MuLV envelope glycoproteins for 4 hours at 37° C, after which the cells were washed to remove the virus. Aβ1-4o was absent (-/-), present only during virus incubation (+/-), present only after virus was washed away (-/+), or present throughout the assay (+/+). The luciferase activity in the target cells 48 hours after incubation with the virus is indicated. Figure 3 is a graph demonstrating that HIV-1 infection in the presence of Aβ is dependent on co-receptors. (A) Cf2Th cells expressing only CCR5 (D), only CD4 (Δ), both receptors (♦) or neither receptor (O) were used as target cells for infection. Infection by recombinant HIV-1 viruses pseudotyped with the envelope glycoproteins of CCR5-using HIV-1 isolates (ADA and YU2), as well as the envelope glycoproteins of A-MuLV and VS V, was assessed in duplicate or triplicate by measuring the luciferase activity in the target cells. Average values are shown. (B) Infection of Cf2Th- CD4/CCR5 cells by recombinant HIV-1 pseudotyped with the envelope glycoproteins of the CCR5-using HIV-1 isolates ADA and YU2 was carried out in the presence of 10 μM Aβ with increasing concentrations of the 2D7 anti-CCR5 monoclonal antibody. Figure 4 is a graph demonstrating that synthetic fibril-forming peptides enhance infection of recombinant HIV-1. Cf2Th-CD4/CCR5 cells were infected with recombinant HIV-1 pseudotyped with ADA (A), YU2 (O) and VSV (D) envelope glycoproteins in the presence of varied concentrations of amyloidogenic peptides PPI- 2480 (AGAKWSWWELTWVGG; SEQ ID NO:l) or PPI-2566 (TRQAMCNISRADWND; SEQ ID NO:2). Infection was also performed in the presence of more than 20 different non-amyloido genie peptides of 8-17 amino acids long. None of them, including the peptide shown in the figure, PPI-1966 (APMGSDPPTA; SEQ ID NO:3), affected viral entry. All peptides were amidated at the C-terminus and incubated under fibril-forming conditions. Results of independent assays are reported as previously described in the legend to Figure 1.
Figure 5 depicts the results of experiments demonstrating that peptides that enhance viral infection form fibrils and promote lipid vesicle association with cells. (A) Cf2Th cells were incubated with fluorescent liposomes (final concentration of 1 mg of lipid/ml) in the presence of 10 μM pre-aggregated amyloidogenic peptides Aβi- 2 (gray), PPI-2566 (diagonal lines), Aβ1.40 (black), PPI-2480 (dots) or no peptide (white) and analyzed using fluocytometry. Non-amyloidogenic peptides, including PPI-1966, did not promote lipid vesicle association with cells. (B-F) Negatively stained electron micrographs of amyloidogenic peptides, Aβ1- 0, Aβi-42, PPI-2480, and PPI-2566 are shown (B-E, respectively). The non-amyloidogenic peptide PPI-1966 used for a control is also depicted (F). The scale bar represents 100 nM (F).
Figure 6 is a graph demonstrating that Aβ fibrils stimulate infection by viruses other than HIV-1. (A) Cells were infected with a recombinant A-MuLV vector expressing β-galactosidase in the absence of additive or in the presence of 8 μg/ml of polybrene or 10 μM of pre-aggregated Aβ40-1 reverse fragment or AβMo. The precipitable fraction of Aβi^o was compared with any soluble fraction of Aβ1-40 remaining in the supernatant following centrifugation. β-galactosidase expression 24 hours after incubation of viruses and cells was used to evaluate the efficiency of viral infection. (B) Cf2Th cells were infected with a recombinant HSV vector (HD-2) containing the β-galactosidase reporter gene in the presence of 5 or 10 μM of pre- aggregated Aβ1-40. The results shown in (a) and (b) are the mean values obtained from duplicate experiments.
Detailed Description of the Invention
The present invention is directed to methods and compositions for treating a viral disease, e.g., a viral disease of the brain, in a subject. The present invention is based, at least in part, on the discovery that infectivity of cells by the human immunodeficiency virus (HIV) is enhanced in the presence of aggregated amyloidogenic peptide fibrils, e.g., /3-amyloid fibrils, in the brain. Without intending to be limited by theory, it is believed that aggregates of /3-amyloid fibrils in the brain may facilitate infection of brain cells by HIV and contribute to the development of AIDS-related dementia.
Accordingly, the present invention provides methods and compositions for treating a viral disease, e.g., a viral disease of the brain, in a subject by administering to the subject a therapeutically or prophylactically effective amount of an amyloidogenic peptide modulator compound, e.g., a β amyloid modulator compound.
As used herein, the term "viral disease" is intended to include any disease, disorder or condition associated with or caused by a virus. Viral diseases include, but are not limited to, infection with herpes simplex virus (type 1 and type 2), varicella zoster virus, poliomyelitis virus, cytomegalovirus, influenza virus (A and B), respiratory syncytial virus, coxsackie virus, ebola virus, hantavirus, human papilloma virus, rotavirus, west nile virus, Epstein-Barr virus, human immunodeficiency virus, and hepatitis virus (A, B and C). The clinical sequelae of viral infection include herpes, AIDS, lassa fever, kaposi's sarcoma, meningitis, mumps, polio, chicken pox, colds and flu, dengue fever, encephalitis, Fifth disease, shingles, genital warts, rubella, yellow fever, hepatitis A, B and C, measles, rabies, and smallpox. In a preferred embodiment, the term "viral disease" is intended to include a viral disease of the brain. As used herein, the term "amyloidogenic peptide modulator compound" is intended to include any compound, e.g., a peptide, a nucleic acid molecule or an antibody, capable of modulating, e.g., inhibiting, the aggregation of an amyloidogenic peptide, and/or modulating, e.g., inhibiting, the neurotoxicity of an amyloidogenic peptide (e.g., transthyretin (TTR), prion protein (PrP), islet amyloid polypeptide (IAPP), atrial natriuretic factor (ANF), kappa light chain, lambda light chain, amyloid A, procalcitonin, cystatin C, β2 microglobulin, ApoA-I, gelsolin, procalcitonin, calcitonin, fibrinogen and lysozyme). The amyloidogenic peptide modulator compound may modulate the aggregation of amyloidogenic peptides by modulating: (a) the processing of amyloidogenic peptides (e.g., either by direct or indirect protease inhibition); (b) the synthesis of amyloidogenic peptides; (c) the release of amyloidogenic peptides; or (d) the processes that produce toxic amyloidogenic peptides in vivo. As used herein, the term amyloidogenic peptide modulator compound is also intended to include compounds which have the ability to remove aggregated amyloidogenic peptides, such as monoclonal antibodies against an amyloidogenic peptide; or vaccines which, when administered to a subject, elicit the formation of antibodies against an amyloidogenic peptide.
As used herein, the term "amyloidogenic peptide" is intended to include any peptide that is capable of forming fibrils. Amyloidogenic peptides are typically hydrophobic and may comprise at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 amino acid residues. Examples of amyloidogenic proteins include transthyretin (TTR), prion protein (PrP), islet amyloid polypeptide (IAPP), atrial natriuretic factor (ANF), kappa light chain, lambda light chain, amyloid A, procalcitonin, cystatin C, β2 microglobulin, ApoA-I, gelsolin, procalcitonin, calcitonin, fibrinogen and lysozyme.
As used herein, the term "β amyloid modulator compound" is intended to include any compound, e.g., a peptide, a nucleic acid molecule or an antibody, capable of modulating, e.g., inhibiting, the aggregation of natural β amyloid peptides (β-AP) and/or modulating, e.g., inhibiting, the neurotoxicity of natural β amyloid peptides. The β amyloid modulator compound may modulate the aggregation of natural β amyloid peptides by modulating: (a) the processing of natural β amyloid peptides (e.g., either by direct or indirect protease inhibition); (b) the synthesis of natural β amyloid peptides; (c) the release of natural β amyloid peptides; or (d) the processes that produce toxic β amyloid peptides, or other APP fragments, in vivo. As used herein, the term β amyloid modulator compound is also intended to include compounds which have the ability to remove aggregated β amyloid peptides, such as monoclonal antibodies against β amyloid; or vaccines which, when administered to a subject, elicit the formation of antibodies against β amyloid. β amyloid modulator compounds are well known and readily available and include those described in, for example, U.S. Patent Nos. 5,985,242; 5,854,215; 5,948,763; 5,854,204; 6,022,859; 5,817,626; 6,051,684; 6,017,887; 6,015,879; 6,251,928; 5,969,100; 5,962,419; 6,177,246; 6,080,778; 6,211,235; 6,207,710; 6,191,166; and 5,814,646; PCT application Nos. WO 00/68263; WO 00/52048; WO 98/08868; WO 00/100663; WO 00/31,548; and WO 00/77,178; and Gordon et al. (2001) Biochemistry 40, 8237-8245, the contents of all of which are incorporated herein by reference.
As used herein, the term "subject" includes warm-blooded animals, preferably mammals, including humans, hi a preferred embodiment, the subject is a primate. In an even more preferred embodiment, the primate is a human.
As used herein, the term "administering" to a subject includes dispensing, delivering or applying an amyloidogenic peptide modulator compound, e.g., a β amyloid modulator compound, to a subject by any suitable route for delivery of the amyloidogenic peptide modulator compound, e.g., the β amyloid modulator compound, to the desired location in the subject, including delivery by either the parenteral or oral route, intracerebral injection, intramuscular injection, subcutaneous/intradermal injection, intravenous injection, buccal administration, transdermal delivery and administration by the rectal, colonic, vaginal, intranasal or respiratory tract route. As used herein, the term "therapeutically effective amount" includes an amount effective, at dosages and for periods of time necessary, to achieve the desired result, e.g., sufficient to treat a subject suffering from a viral disease or sufficient to inhibit infection of a cell by a virus. A therapeutically effective amount of an amyloidogenic peptide modulator compound, e.g., a β amyloid modulator compound, as defined herein may vary according to factors such as the disease state, age, and weight of the subject, and the ability of the compound to elicit a desired response in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects (e.g., side effects) of the amyloidogenic peptide modulator compound, e.g., the β amyloid modulator compound, are outweighed by the therapeutically beneficial effects.
A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as preventing or inhibiting a viral disease or infection of a cell by a virus in a subject predisposed to a viral disease or viral infection. A prophylactically effective amount can be determined as described above for the therapeutically effective amount. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of viral disease, the prophylactically effective amount will be less than the therapeutically effective amount. Various additional aspects of the methods of the invention are described in further detail in the following subsections.
Amyloidogenic Peptide Modulator Compounds
Any compound capable of modulating, e.g., inhibiting, the aggregation of an amyloidogenic peptide and/or modulating, e.g., inhibiting, the neurotoxicity of an amyloidogenic peptide may be used in the methods of the present invention. The amyloidogenic peptide modulator compound may modulate the aggregation of an amyloidogenic peptide by modulating: (a) the processing of an amyloidogenic peptide (e.g. , either by direct or indirect protease inhibition); (b) the synthesis of an amyloidogenic peptide; (c) the release of an amyloidogenic peptide; or (d) the processes that produce toxic amyloidogemc peptides in vivo.
Compounds which have the ability to remove aggregated amyloidogenic peptides, such as monoclonal antibodies against an amyloidogenic peptide, may also be used in the methods of the present invention, hi another embodiment, vaccines may be used which, when administered to a subject, elicit the formation of antibodies against an amyloidogenic peptide.
Examples of suitable amyloidogenic peptide modulator compounds for use in the methods of the present invention include those described in, for example, U.S. Patent Nos. 5,858,326; 6,211,149; 6,355,610; and 6,319,498, the contents of each of which are incorporated herein by reference.
β Amyloid Modulator Compounds Any compound capable of modulating, e.g., inhibiting, the aggregation of natural β amyloid peptides (β-AP) and/or modulatmg, e.g., inhibiting, the neurotoxicity of natural β-APs may be used in the methods of the present invention. The β amyloid modulator compound may modulate the aggregation of natural β amyloid peptides by modulating: (a) the processing of natural β amyloid peptide (e.g., either by direct or indirect protease inhibition); (b) the synthesis of natural β amyloid peptide; (c) the release of natural β amyloid peptide; or (d) the processes that produce toxic β amyloid peptide, or other APP fragments, in vivo.
Compounds which have the ability to remove aggregated β amyloid peptides, such as monoclonal antibodies against β amyloid, may also be used in the methods of the present invention. In another embodiment, vaccines may be used which, when administered to a subject, elicit the formation of antibodies against β amyloid.
Compounds which have the ability to lower blood cholesterol levels may also be used to modulate the production of β amyloid peptide. β amyloid modulator compounds (e.g., peptides, peptidomimetics, nucleic acid molecules or antibodies) are well known and readily available to those of skill in the art and include those described in, for example, U.S. Patent Nos. 5,985,242; 5,854,215; 5,948,763; 5,854,204; 6,022,859; 5,817,626; 6,051,684; 6,017,887; 6,015,879; 6,251,928; 5,969,100; 5,962,419; 6,177,246; 6,080,778; 6,211,235; 6,207,710;
6,191,166; and 5,814,646; PCT application Nos. WO 00/68263; WO 00/52048; WO 98/08868; WO 00/100663; WO 00/31,548; and WO 00/77,178; and Gordon et al. (2001) Biochemistry 40, 8237-8245, the contents of all of which are incorporated herein by reference. As used herein, the term "peptide" is intended to encompass peptide analogues, peptide derivatives, and peptidomimetics that mimic the chemical structure of a peptide composed of naturally-occurring amino acids. The term "peptide analogue" as used herein is intended to include molecules that mimic the chemical structure of a peptide and retain the functional properties of the peptide. Examples of peptide analogues include peptides comprising one or more non-natural amino acids. The term "peptide derivative" as used herein is intended to include peptides in which an amino acid side chain, the peptide backbone, or the amino- or carboxyl-terminus has been derivatized (e.g., peptidic compounds with methylated amide linkages).
The term "peptidomimetic" as used herein is intended to include peptidic compounds in which the peptide backbone is substituted with one or more benzodiazepine molecules (see, e.g., James, G.L. et al. (1993) Science 260:1937-1942), "inverso" peptides in which all L-amino acids are substituted with the corresponding D- amino acids, "retro-inverso" peptides (see U.S. Patent No. 4,522,752 by Sisto) in which the sequence of amino acids is reversed ("retro") and all L-amino acids are replaced with D-amino acids )"inverso") and other isosteres, such as peptide back-bone (i.e., amide bond) mimetics,, including modifications of the amide nitrogen, the α-carbon, amide carbonyl, complete replacement of the amide bond, extensions, deletions or backbone crosslinks. Several peptide backbone modifications are known, including ψ[CH2S], ψ [CH2NH], ψ[CSNH2], ψ[NHCO], ψ[COCH2], and ψ[(E) or (Z) CH=CH]. In the nomenclature used above, ψ indicates the absence of an amide bond. The structure that replaces the amide group is specified within the brackets. Other possible modifications include an N-alkyl (or aryl) substitution (ψ[CONR])5 backbone crosslinking to construct lactams and other cyclic structures, and other derivatives including C-terminal hydroxymethyl derivatives, O-modified derivatives and N-terminally modified derivatives including substituted amides such as alkylamides and hydrazides. Pharmaceutical Compositions
The amyloidogenic peptide modulator compounds, e.g., the β amyloid modulator compounds, used in the methods of the present invention can be incorporated into pharmaceutical compositions suitable for adminis -ration to a subject, such as those described in U.S. Patent Nos. 5,968,895 and 6,180,608 Bl, the contents of which are incorporated herein by reference, which allow for sustained delivery of the amyloidogenic peptide modulator compound, e.g., the β amyloid modulator compound, for a period of at least several weeks to a month or more. Preferably, the amyloidogenic peptide modulator compound, e.g., the β amyloid modulator compound, is the only active ingredient(s) formulated into the pharmaceutical composition, although in certain embodiments the amyloidogenic peptide modulator compound, e.g., the β amyloid modulator compound, may be combined with one or more other active ingredients such as other drugs suitable for treating a viral disease, e.g., an HIV-1 infection. Other pharmaceutically active compounds that may be used can be found in Harrison's Principles of Internal Medicine, Thirteenth Edition, Eds. T.R. Harrison et al. McGraw- Hill N.Y., NY; and the Physicians Desk Reference 50th Edition 1997, Oradell New Jersey, Medical Economics Co., the complete contents of which are expressly incorporated herein by reference. In a preferred embodiment, the pharmaceutical composition comprises an amyloidogenic peptide modulator compound, e.g., a β amyloid modulator compound, and a pharmaceutically acceptable carrier.
As used herein "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In one embodiment, the carrier is suitable for parenteral administration or for administration via inhalation. Preferably, the carrier is suitable for administration into the central nervous system (e.g., intraspinally or intracerebrally). Alternatively, the carrier can be suitable for intravenous, intraperitoneal or intramuscular administration, hi another embodiment, the carrier is suitable for oral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.
Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin. Moreover, the compounds of the invention can be administered in a time release formulation, for example in a composition which includes a slow release polymer. The amyloidogenic peptide modulator compound, e.g., the β amyloid modulator compound, can be prepared with carriers that will protect the amyloidogenic peptide modulator compound, e.g., the β amyloid modulator compound, against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers (PLG). Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. Sterile injectable solutions can be prepared by incorporating the amyloidogenic peptide modulator compound, e.g., the β amyloid modulator compound, in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof. The amyloidogenic peptide modulator compound, e.g., the β amyloid modulator compound, can be formulated with one or more additional compounds that enhance the • solubility of the amyloidogenic peptide modulator compound, e.g., the β amyloid modulator compound. Preferred compounds to be added to formulations to enhance the solubility of an amyloidogenic peptide modulator compound, e.g., a β amyloid modulator compound, are cyclodextrin derivatives, preferably hydroxypropyl-γ- cyclodextrin. For example, inclusion in the formulation of hydroxypropyl-γ-cyclodextrin at a concentration 50-200 mM may increase the aqueous solubility of the amyloidogenic peptide modulator compound, e.g., the β amyloid modulator compound,. Another formulation for the amyloidogenic peptide modulator compound, e.g., the β amyloid modulator compound, comprises the detergent Tween-80, polyethylene glycol (PEG) and ethanol in a saline solution. A non-limiting example of such a preferred formulation is 0.16% Tween-80, 1.3% PEG-3000 and 2% ethanol in saline. Preferably, an amyloidogenic peptide modulator compound, e.g., a β amyloid modulator compound, is administered to the subject as a sustained-release formulation using a pharmaceutical composition comprising a solid ionic complex of an amyloidogenic peptide modulator compound and a carrier macromolecule, wherein the carrier and the amyloidogenic peptide modulator compound used to form the complex are combined at a weight ratio of carrier: amyloidogenic peptide modulator compound of, for example, 0.5:1 to 0.1:1. In other embodiments, the carrier and amyloidogenic peptide modulator compound used to form the complex are combined at a weight ratio of carrier:amyloidogenic peptide modulator compound of 0.8:1, 0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.25:1, 0.2:1, 0.15:1, or 0.1:1. In apreferred embodiment, the complex is not a microcapsule. Ranges intermediate to the above recited values, e.g., 0.8:1 to 0.4:1, 0.6:1 to 0.2:1, or 0.5:1 to 0.1:1 are also intended to be part of this invention. For example, ranges of values using a combination of any of the above recited values as upper and/or lower limits are intended to be included. hi another embodiment, the amyloidogenic peptide modulator compound, e.g., the β amyloid modulator compound, is administered to the subject using a pharmaceutical composition comprising a solid ionic complex of an amyloidogenic peptide modulator compound, e.g., a β amyloid modulator compound, and a carrier macromolecule, wherein the amyloidogenic peptide modulator compound, e.g., the β amyloid modulator compound, content of said complex is at least 40% by weight, preferably at least 45%, 50%, 55%, 57%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or
95%o by weight. Ranges intermediate to the above recited values, e.g., at least about 50% to about 80%, at least about 60% to about 90%, or at least about 57% to about 80%, are also intended to be part of this invention. For example, ranges of values using a combination of any of the above recited values as upper and/or lower limits are intended to be included.
As used herein, the term "carrier macromolecule" is intended to refer to a macromolecule that can complex with a peptide to form a water-insoluble complex. Preferably, the macromolecule has a molecular weight of at least 5 kDa, more preferably at least 10 kDa. The term "anionic carrier macromolecule" is intended to include negatively charged high molecular weight molecules, such as anionic polymers. The term "cationic carrier macromolecule" is intended to include positively charged high molecular weight molecules, such as cationic polymers. As used herein, the term "water-insoluble complex" is intended to refer to a physically and chemically stable complex that forms upon appropriate combining of a β amyloid modulator compound and carrier macromolecule according to procedures described herein. This complex typically takes the form of a precipitate that is produced upon combining aqueous preparations of the amyloidogenic peptide modulator compound, e.g., the β amyloid modulator compound, and carrier macromolecule. Although not intending to be limited by mechanism, the formation of preferred water- insoluble complexes used in the methods of the invention is thought to involve (e.g., be mediated at least in part by) ionic interactions in situations where the amyloidogenic peptide modulator compound, e.g., the β amyloid modulator compound, is cationic and the carrier molecule is anionic or vice versa. Additionally or alternatively, the formation of a water-insoluble complex of the invention may involve (e.g., be mediated at least in part by) hydrophobic interactions. Still further, formation of a water-insoluble complex of the invention may involve (e.g., be mediated at least in part by) covalent interactions. Description of the complex as being "water-insoluble" is intended to indicate that the complex does not substantially or readily dissolve in water, as indicated by its precipitation from aqueous solution. However, it should be understood that a "water- insoluble" complex of the invention may exhibit limited solubility in water either in vitro or in the aqueous physiological environment in vivo. As used herein, the term "sustained delivery" or "sustained release" is intended to refer to continual delivery of an amyloidogenic peptide modulator compound, e.g., a β amyloid modulator compound, in vivo over a period of time following administration, preferably at least several days, a week or several weeks and up to a month or more. In a preferred embodiment, a formulation of the invention achieves sustained delivery for at least about 28 days, at which point the sustained release formulation can be re- administered to achieve sustained delivery for another 28 day period (which re- administration can be repeated every 28 days to achieve sustained delivery for several months to years). Sustained delivery of the amyloidogenic peptide modulator compound, e.g., the β amyloid modulator compound, can be demonstrated by, for example, the continued therapeutic effect of the amyloidogenic peptide modulator compound, e.g., the β amyloid modulator compound, over time. Alternatively, sustained delivery of the amyloidogenic peptide modulator compound, e.g., the β amyloid modulator compound, may be demonstrated by detecting the presence of the amyloidogenic peptide modulator compound, e.g., the β amyloid modulator compound, in vivo over time.
A complex used in the methods of the invention is prepared by combining the amyloidogenic peptide modulator compound, e.g., the β amyloid modulator compound, and the carrier macromolecule under conditions such that a water-insoluble complex of the amyloidogenic peptide modulator compound, e.g., the β amyloid modulator compound, and the carrier macromolecule forms.
In another embodiment, a pharmaceutical formulation of the invention is a sterile formulation. For example, following formation of the water-insoluble complex, the complex can be sterilized, preferably by gamma radiation or electron beam sterilization. Alternatively, to prepare a sterile pharmaceutical formulation, the water- insoluble complex can be isolated using conventional sterile techniques (e.g., using sterile starting materials and carrying out the production process aseptically).
The pharmaceutical formulation can be administered to the subject by any route suitable for achieving the desired therapeutic result(s), although prefened routes of administration are parenteral routes, in particular intramuscular (i.m.) injection and subcutaneous/intradermal (s.c./i.d.) injection. Alternatively, the formulation can be administered to the subject orally. Other suitable parental routes include intravenous injection, buccal administration, transdermal delivery and administration by the rectal, vaginal, intranasal or respiratory tract route. It should be noted that when a formulation that provides sustained delivery for weeks to months by the i.m or s.c./i.d. route is administered by an alternative route, there may not be sustained delivery of the agent for an equivalent length of time due to clearance of the agent by other physiological mechanisms (i.e., the dosage form maybe cleared from the site of delivery such that prolonged therapeutic effects are not observed for time periods as long as those observed with i.m or s.c./i.d. injection).
The pharmaceutical formulation contains a therapeutically or prophylactically effective amount of the amyloidogenic peptide modulator compound, e.g., the β amyloid modulator compound. A therapeutically or prophylactically effective amount of an amyloidogenic peptide modulator compound, e.g., a β amyloid modulator compound, may vary according to factors such as the disease state, age, and weight of the individual, and the ability of the amyloidogenic peptide modulator compound, e.g., the β amyloid modulator compound (alone or in combination with one or more other drugs) to elicit a desired response in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically or prophylactically effective amount is also one in which any toxic or detrimental effects of the amyloidogenic peptide modulator compound, e.g., the β amyloid modulator compound, are outweighed by the therapeutically or prophylactically beneficial effects.
In one embodiment, the dosage of the amyloidogenic peptide modulator compound, e.g., the β amyloid modulator compound, is about 10-500 mg/month, about 20-300 mg/month, or about 30-200 mg/month. h a preferred embodiment, the dosage of the amyloidogenic peptide modulator compound, e.g., the β amyloid modulator compound, is about 30-120 mg/month. Ranges intermediate to the above recited values, e.g., about 10-200 mg/month, about 30-250 mg/month, or about 100-200 mg/month, are also intended to be part of this invention. For example, ranges of values using a combination of any of the above recited values as upper and or lower limits are intended to be included. The above recited dosages may also be calculated and expressed in mg/kg/day. Accordingly, in another embodiment, the dosage of the amyloidogenic peptide modulator compound, e.g., the β amyloid modulator compound, is about 5-500 μg/kg/day, about 10-400 μg/kg/day, or about 20-200 μg/kg/day. In a preferred embodiment, the dosage of the amyloidogenic peptide modulator compound, e.g., the β amyloid modulator compound, is about 100 μg/kg/day. It is to be noted that dosage values may vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.
In one embodiment, the amyloidogenic peptide modulator compound, e.g., the β amyloid modulator compound, is co-administered to a subject along with another agent which functions to permeabilize the blood brain barrier (BBB). Examples of such BBB "permeabilizers" include bradykinin and bradykinin agonists (see, e.g., U.S. Patent No. 5,112,596, the entire contents of which are incorporated by reference) and peptidic compounds disclosed in U.S. Patent No. 5,268,164, the entire contents of which are incorporated by reference.
Assays that measure the in vitro stability of the amyloidogenic peptide modulator compound, e.g., the β amyloid modulator compound, in cerebrospinal fluid (CSF) and the degree of brain uptake of the modulator compounds in animal models can be used as predictors of in vivo efficacy of the compounds. Suitable assays for measuring CSF stability and brain uptake are described herein in the Examples section.
Screening Assays Another aspect of the invention pertains to methods for identifying an amyloidogenic peptide modulator compound, e.g., a β amyloid modulator compound, capable of treating a viral disease in a subject or capable of modulating infection of a cell by a virus. The methods include contacting a cell, e.g., a brain cell, with a virus in the presence of an amyloidogenic peptide (e.g., β amyloid peptide) and an amyloidogenic peptide modulator compound, e.g., a β amyloid modulator compound, and determining the ability of the virus to infect the cell, thereby identifying an amyloidogenic peptide modulator compound, e.g., a β amyloid modulator compound, capable of treating a viral disease in a subject or capable of modulating infection of a cell by a virus. Virally infected cells, e.g., virally infected brain cells, treated with an amyloidogenic peptide modulator compound, e.g., a β amyloid modulator compound, can be examined for one or more phenotype(s) associated with viral infection and/or viral disease. Cellular phenotypes that are associated with viral disease include viral infection (e.g., virus burden), cell lysis, plaque formation, and low pH induced fusion of infected cells (Sung T-C et αl. (1997) EMBO J. 16:4519-4530; Roper RL and Moss B (1999) J. Virol. 73:1108-1117; Blasco R and Moss B (1991) J. Virol 65:5910-5920).
In addition, animal-based viral disease systems, such as those described herein, maybe used to identify compounds capable of ameliorating viral disease symptoms. Such animal models may be exposed to an amyloidogenic peptide modulator compound, e.g., a β amyloid modulator compound, at a sufficient concentration and for a time sufficient to elicit an amelioration of a viral disease symptom in the exposed animal. The response of the animals to the amyloidogenic peptide modulator compound, e.g., the β amyloid modulator compound, may be monitored by assessing the reversal of disorders associated with viral disease, for example, by monitoring viral burden before and after treatment.
An amyloidogenic peptide modulator compound, e.g., a β amyloid modulator compound, identified using the assays described herein may also be administered to an animal model for a viral disease to determine the efficacy, toxicity, or side effects of treatment with such a compound.
Transgenic mouse models for viral disease that may be used in the methods of the invention include those described in Rail GF et αl. (Virol. (2000) 271 :220-226), Eckert RL et αl. (Int. J. Oncol. (2000) 16:853-70), and Morrey JD et αl. (Antiviral Ther. (1998) 3:59-68). Non-recombinant, non-genetic animal models of viral disease that may also be used include, for example, animal models in which the animal has been exposed to viral infection, as described in, for example, Mosier, D (2000), Virol. 271:215-219; Lavi, E et al. (1999) J. Neuropathol. Exp. Neurol. 58:1197-1206; Briese, T et al. (1999) J. Neurovirol. 5:604-612; Johannessen, I et al. (1999) Rev. Med. Virol. 9:263-277; Hayashi, K et al. (2000) Pathol. Int. 50:85-97; Michalak, TI (2000) Immunol. Rev. 174:98-111; McSharry, JJ (1999) Antiviral Res. 43:1-21; Bernstein, Ωl et al. (2000) Antiviral Res. 47:159-169; Thackray, AM et al. (2000) J. Gen. Virol. 81:2385-2396; Nakazato, I et al. (2000) Pathol. Res. Pract. 196:635-645; and Takasaki, I et al. (2000) Jpn. J. Pharmacol. 83:319-326. Methods for Promoting Infection by a Virus hi another aspect, the present invention provides methods for promoting infection by a virus using amyloidogenic peptides or amyloidogenic peptide modulator compounds, e.g., β amyloid modulator compounds, that are able to promote infection (e.g., with the same or greater efficiency as Aβι- 0). Such methods are useful in applications like gene therapy or cell transfection. Without intending to be limited by theory, it is believed that amyloidogenic peptides enhance viral infection by mediating a physical association of viral envelopes with the cell lipid bilayer.
As used herein, the term "amyloidogenic peptide" is intended to include any peptide that is capable of forming fibrils. Amyloidogenic peptides are typically hydrophobic and may comprise at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 amino acid residues. Examples of amyloidogenic peptides suitable for use in the methods of promoting infection include naturally occurring proteolytic cleavage products of the β amyloid precursor protein (APP) which are involved in β-AP aggregation and β-amyloidosis, e.g. , Aβ 1.39, Aβ ^.40, Aβ ι_^\, Aβ 1- 2 and Aβι.43. (The 43 amino acid long form of natural β-AP has the amino acid sequence DAEFRHDSGYEVHHQKLVFFA EDVGS-NKLGA-EGLMVGG IAT (SEQ ID NO:4)). Other examples of amyloidogenic proteins include transthyretin (TTR), prion protein (PrP), islet amyloid polypeptide (IAPP), atrial natriuretic factor (ANF), kappa light chain, lambda light chain, amyloid A, procalcitonin, cystatin C, β2 microglobulin, ApoA-I, gelsolin, procalcitonin, calcitonin, fibrinogen and lysozyme.
In a prefened embodiment, the amyloidogenic peptides used in the methods of promoting infection, are amyloidogenic peptides which fonn short fibrils, e.g.,
Figure imgf000020_0001
or PPI-2480 (AGAKWSWWELTWVGG; SEQ ID NO:l). The methods include contacting a cell with an amyloidogenic peptide or an amyloidogenic peptide modulator compound, e.g., a β amyloid modulator compound, in an amount effective to promote infection of the cell by a virus (e.g., an Adeno-associated virus (AAV) or a retro virus).
For cell transfection applications, a cell may be contacted in vitro with an amyloidogenic peptide and the virus of interest under conditions suitable for infection of the cell. Such conditions are well known in the art and described in, for example, Curcent Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) Greene Publishing Associates, (1989) and other standard laboratory manuals.
For gene therapy applications, gene therapy vectors can be delivered to a subject in conjunction with an amyloidogenic peptide modulator compound, e.g., a β amyloid modulator compound, or a non-toxic amyloidogenic peptide by, for example, intravenous injection, local administration (see U.S. Patent 5,328,470) or by stereotactic injection (see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector as well as an effective amount of an amyloidogenic peptide modulator compound, e.g., a β amyloid modulator compound, or a non-toxic amyloidogenic peptide in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle and the amyloidogenic peptide modulator compound, e.g., the β amyloid modulator compound, or the non-toxic amyloidogenic peptide are imbedded.
Protocols for producing viruses, e.g., recombinant retroviruses, and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM which are well known to those skilled in the art.
This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application, as well as the Figures, are hereby incorporated by reference.
EXAMPLES
The following experimental procedures were used in the Examples.
Peptides and Fibrils
Lyophilized beta-amyloid (Aβ) 1-40 and 1-42 fragments (California Peptide Research, hie. and New England Peptide, Inc.), Aβ40-1 reverse fragment (Sigma- Aldrich), and small molecular weight peptides, PPI-2480 and PPI-2566, were dissolved in DMSO to 5 mM and subsequently diluted to 200 μM in PBS (10 mM HEPES, pH 7.4 for Aβ1-42). Peptides were used fresh or incubated under conditions that support fibril formation as follows. Aβ^o, PPI-2480, and PPI-2566 peptide solutions were incubated for 8 days at 37° C, sonicated, aliquotted, and stored at -20° C. Fibril formation for the Aβi-42 peptide solution was allowed to proceed for 24 hours at room temperature with stirring. The efficiency of fibril formation was verified by reaction with Congo Red or electron microscopy. Purity of all peptides was >98%. .
Cell lines and culture
All cell lines were grown at 37° C and 5% CO2 in Dulbecco's modified Eagle medium (GibcoBRL) containing 10% fetal bovine serum (Sigma- Aldrich) and 100 μg/ml penicillin-streptomycin (Mediatech, Inc.) (complete DMEM) supplemented with antibiotics as noted. Cf2Th canine thymocytes, 293T human embryonal kidney (HEK), and NTH-3T3 mouse embryonal fibroblast cells were obtained from the American Type Culture Collection (ATCC CRL 1430, 1573 and 1658, respectively). Stable cell lines included Cf2Th expressing human CD4 and CCR5 (Cf2Th-CD4/CCR5) (Farzan, M. et /.(1999) Cell 96, 661-616) grown in medium supplemented with 0.5 mg/ml Geneticin (GibcoBRL) and 0.15 mg/ml Hygromycin B (Roche Diagnostics Corp.), Cf2Th-CD4 (31) with 0.15 mg/ml Hygromycin B, Cf2Th-CCR5 (30) with 0.5 mg/ml Geneticin, and GHOST(3)-CD4/CXCR4 human osteosarcoma cells expressing human CD4 and CXCR4 (32) with 0.5 mg/ml Geneticin, 50 μg/ml Hygromycin B and 1 μg/ml Puromycin (Sigma-Aldrich). SupTl-CCR5 cells (Means, R. E. et al. (2001) J. Virol. 75, 3903- 3915) were cultured in complete RPMI medium (GibcoBRL) supplemented with 0.2 μg/ml Puromycin.
Recombinant Reporter Viruses
Recombinant HIV-1 reporter viruses were constructed by cotransfection of 293T- HEK cells with vectors expressing the pCMVΔPlΔenvpA HIV-1 Gag-Pol packaging construct (Parolin, C. et al. (1996) Virology 222, 415-422) the envelope glycoproteins of AMLV, VSV, and HIV-1 isolates (ADA, YU2, JR-FL and HXBc2), and a reporter gene at a DNA weight ratio of 1 : 1 :3 using Effectene reagents (Qiagen). Cotransfection produced replication-defective (single-round) virions capable of expressing HIV-1 tat and the firefly luciferase gene under control of the HIV-1 long terminal repeat (LTR), or the green fluorescent protein (GFP) gene under control of the cytomegalovirus immediate-early promoter (CMV). Viruses pseudotyped with VSV-G, A-MuLV, and HIV-1 envelope glycoproteins were produced by cotransfecting the pHCMV-G (Yee, J. et al. (1994) Proc Natl Acad Sci USA 91, 9564-9568), SV-A-MLV-Env ( Landau, N. et al. (1991) J Virol 65, 162-169), or pSVIϋenv (Helseth, E. et al. (1990) J Virol 64, 2416- 2420; Sullivan, N. et al. (1995) J Virol 69, 4413-4422; Gao, F. et al. (1996) J Virol 70, 1651-1667; and Kolchinsky, P. et al. (2001) J Virol 75, 3435-3443) plasmids, respectively. Production of the VSV-G and A-MuLV recombinant viruses also required cotransfection of pCMV-Rev, a plasmid expressing the HIV-1 Rev protein ( LaBonte, J. A. et al. (2000) J Virol 1A, 10690-10698). Thirty hours following transfection, the virus- containing cell supernatants were harvested, filtered (0.45 μm), aliquotted and kept frozen until use. The reverse transcriptase (RT) activities of all viruses were quantified and normalized by cpm as described previously ( Rho, H. M. et al. (1981) Virology 112, 355-360). Replication-deficient A-MuLV (Retropack, Clonetech) and HSV (HD-2) ( Morrison, L. A. et al. (1994) J Virol 68, 689-696) vectors containing β-galactosidase reporter genes were produced using NIH-3T3 cells, according to the manufacturer's protocol; infection efficiencies were estimated by reporter gene activity in the target cells. Luciferase and β-galactosidase activity was quantitated as described in Promega protocols using an EG&G Berthold Microplate Luminometer LB 96V. Infection by Single-Round Viruses Expressing Luciferase
Target cells for viral entry were seeded in 96-well luminometer-compatible tissue culture plates (Dynex) at a density of 6 x 103 cells/well and incubated for 24 hours. The medium was removed from the target cells and replaced with fresh complete DMEM containing RT-normalized units of recombinant virus. The amounts of virus varied depending upon the envelope glycoproteins used for pseudotyping: VSV-G, IK cpm; A- MuLV, 30K cpm; ADA, YU2, JR-FL, 89.6, ADA-ΔV1/V2, and HXBc2 HIV-1 envelope glycoproteins, 10K cpm). Varying amounts of Aβ1- 0, Aβ1- 2 (1.25-20 μM), PPI-2566 or PPI-2480 (1-100 μM) were added with the recombinant viruses, to a final infection volume of 50 μl. The anti-CCR5 antibody 2D7 (Wu, L. et al. (1997) JExp Med 186, 1373-1381) (BD PharMingen) or TAK-779, a small molecular weight nonpeptide compound that specifically binds CCR5 (Baba, M. et al. (1999) Proc Natl Acad Sci US A 96, 5698-5703) (Takeda Chemical Industries, Ltd.), was also included in some assays. Target cells were incubated with the infection medium for 48 hours. Following this incubation, the medium was aspirated from each well and the cells were lysed by addition of 30 μl passive lysis buffer (Promega Corp.), agitation, and 2 freeze-thaw cycles. The luciferase activity of each well was measured for 10 seconds following addition of 100 μl luciferase buffer (15 mM MgSO4, 15 mM KPO4, pH 7.8, 1 mM ATP, 1 mM DTT) and 50 μl 1 mM D-luciferin potassium salt (BD PharMingen) using an EG&G Berthold Microplate Lu inometer LB 96V.
Infection by Single-Round Viruses Expressing GFP
SupTl-CCR5 target cells were seeded in 24- well tissue culture plates (Falcon) at a density of 5 x 104 cells/well with medium containing RT-normalized units of GFP- expressing recombinant virus (VSV-G, 3K cpm; ADA, 150K cpm; YU2, 150K cpm) and varying amounts of Aβ1-40 or Aβ1-42 (62.5 nM-1 μM) in a final volume of 0.4 ml. The infection medium-cell mixture was incubated for 48 hours, 1 ml of fresh complete RPMI was added to each well, and the cells were incubated for an additional 24 hours. The cells were then harvested, washed with PBS, fixed in 10% formalin, and analyzed by florescence-activated cell sorting using a Becton Dickinson FACScan with CellQuest software.
Infection by Single-Round Viruses Expressing β-galactosidase Cf2Th cells were infected with an A-MuLV vector expressing β-glactosidase without additives, in the presence of 8 μg/ml of polybrene, or in the presence of 10 μM pre-aggregated Aβ40-ι reverse fragment or Aβ1- 0. The precipitable fraction of Aβ1-40 was recovered by pelleting pre-aggregated Aβ1- 0 at 15,000 x g for 5 minutes at 4° C, after which the supernatant was removed and retained. The precipitated peptide fibrils were resuspended in PBS, washed two more times and resuspended in the starting volume. The β-galactosidase expression in the target cells 24 hours after infection was estimated using a chemiluminescent assay (Galacto-Star, Tropix, Inc.). Cf2Th cells were also infected with a single-round HSV virus vector (HD-2) (Morrison, L. A. et al. (1994) J. Virol. 68, 689-696.) containing the β-galactosidase reporter gene in the presence of 5 or 10 μM pre-aggregated Aβ1-40. Cells were stained according to the Promega protocol and counted under the microscope 24 hours following infection.
FACS analysis of fibril interactions with liposomes and cells
Unilamellar small liposomes (liposomes) similar in size to HIV-1 were prepared from a 2/1 (M7M) mixture of l-palmitoyl-2-oleoyl--?n~glycero-3-phosphocholine and 1- palmitoyl-2-oleoyl--s,w-glycero-3-phosphoethanolamine supplemented with 1% fluorescent rhodamine-lissamine B-phosphatidylethanolamine (Avanti Polar Lipids), as previously reported (Pedersen, S. et al. (1996) Biophys. J. 71, 554-560). Rhodamine- labeled liposomes were incubated with Cf2Th cells in the presence and absence of peptide fibrils in the medium used for the viral entry assay (DMEM + 10% fetal bovine serum) supplemented with 0.02% NaN3 for 1 hour at 37° C. Similarly, fluorescent Aβμ 40 fibrils (FITC-Aβ) were incubated with Cf2Th cells either lacking or expressing CD4 and/or CCR5, in the absence and presence of recombinant HIV-1 gρl20 envelope glycoprotein from the JR-FL isolate. Following incubation, the cells were washed with PBS containing 2% bovine serum albumin and the association of rhodamine-labeled liposomes or FITC-Aβ with cells was analyzed using FACScan, as described above. Cf2Th-CD4/CCR5 cells were also incubated without additives or with unlabeled pre- aggregated AβMo, as above, and the CD4 and CCR5 cell surface expression was detected using the anti-CD4 antibody RPA-T4-PE (BD PharMingen) and the anti-CCR5 antibody 2D7-PE (BD PharMingen) at a final concentration of 10 nM.
EXAMPLE 1 : Aβ ι-40 AND Aβ ι_42 ENHANCE SIGNAL OF VIRAL ENTRY To investigate whether the presence of Aβ affects HIV-1 infection of target cells, recombinant replication-defective HIV-1 vectors expressing firefly luciferase or GFP were used. These single-round viruses were pseudotyped with the envelope glycoproteins of various HIV-1 isolates, or with those of VSV or A-MuLV. The receptors for VSV and A-MuLV are ubiquitously expressed. Entry of viruses pseudotyped with the HIV-1 envelope glycoproteins is dependent on the presence of CD4 or a chemokine receptor, CCR5 or CXCR4; the viruses pseudotyped with the VSV or A-MuLV envelope glycoproteins do not require CD4 or chemokine receptor expression on the target cells. Pre-aggregated Aβ^o and Aβi-42 fibrils dramatically increased infection of Cf2Th-CD4/CCR5 cells by HIV-1 pseudotyped with the envelope glycoproteins of three CCR5-using primary HIV-1 isolates (ADA, YU2, JR-FL) in a dose-dependent manner (Figure 1). Aβ1-40 similarly increased infection of GHOST(3)- CD4/CXCR4 cells by HIV-1 pseudotyped with the envelope glycoproteins of the CXCR4-using isolate, HXBc2 (Figure 1). Similar results were obtained by infecting a human T-lymphocyte cell line stably expressing CCR5 (SupTl-CCR5) with GFP- expressing viruses pseudotyped with ADA and YU2 envelope glycoproteins. Aβ1-40 was more potent than Aβ1-42 and increased the entry of viruses by 2-10 times in a concentration range of 1-5 μM, and by 5-30 times at a concentration of 20 μM. Infection of cells by viruses pseudotyped with the A-MuLV or VSV envelope glycoproteins was also enhanced. The relatively lower enhancement observed with VSV-G-pseudotyped virus may be due to the substantially greater efficiency with which this virus infects cells in the absence of Aβ.
These data show that Aβ can substantially increase the efficiency of infection of cells by HIV- 1 pseudotyped with the envelope glycoproteins of a wide range of HIV- 1 isolates, as well as with those of other enveloped viruses. The fact that enhancement of infection was observed for viruses containing several different envelope glycoproteins that utilize unrelated receptors, indicates that enhancement does not require specific protein-protein interactions. Consistent with this, these peptides substantially enhanced the association of liposomes with cells (see below).
EXAMPLE 2 : Aβ 1 40 ENHANCES AN EARLY STEP IN VIRUS INFECTION
To elucidate whether the enhancement of virus infection by Aβ was mediated by an increased efficiency of early or late events in the virus life cycle, incubation of the recombinant viruses with the Cf2Th-CD4/CCR5 target cells was carried out for only 4 hours, followed by washing. The target cells were incubated with 20 μM Aβ1-40 concurrently with virus (+/-), immediately following removal of virus (-/+), or throughout both time periods (+/+) (Figure 2). After the wash, the cells were incubated for an additional 48 hours, at which time luciferase activity was measured. Enhancement of infection was observed only when Aβ \ - 0 was present during the initial 4-hour incubation of virus and cells. These data indicate that Aβ exerts its effect at an early stage of viral infection. Because the first 4 hours of HIV-1 infection involves virus attachment and entry into the host cell, Aβ likely enhances these processes. EXAMPLE 3 : ENHANCEMENT OF VIRAL INFECTION BY Aβ IS
RECEPTOR-MEDIATED
Aβ has been shown to exert a destabilizing effect on cellular membranes (Yip, C. M. et al. (2001) Biophys. J. 80, 1359-1371; and McLaurin, J. et al. (1997) Eur. J. Biochem. 245, 355-363). Therefore, Aβ might facilitate fusion of the target cell and viral membrane in a manner that would circumvent the dependence of the virus on its receptors. To investigate this possibility, infection of Cf2Th, Cf2Th-CD4, Cf2Th- CCR5, and Cf2Th-CD4/CCR5 cells by CCR5-dependent HIV-1 isolates was examined. No infection by CCR5-dependent HIV-1 isolates was observed in the presence or absence of Aβ with cells lacking CD4 and/or CCR5 (Figure 3 A), whereas infection by viruses pseudotyped by VSV and A-MuLV envelope glycoproteins, which do not require these cellular receptors, was enhanced by Aβ on all cells examined. Aβ-enhanced CCR5-dependent viral entry remained sensitive to inhibition by CCR5 ligands, including the 2D7 antibody (Wu, L. et al. (1997) JExp Med 186, 1373-1381) (Figure 3B) and the small-molecule antagonist TAK-779 (Baba, M. et al. (1999) Proc. Natl. Acad. Sci. USA 96, 5698-5703). These data demonstrate that HIV-1 infection in the presence of Aβ remains dependent on the expression of CD4 and a chemokine co-receptor.
EXAMPLE 4: OTHER FIBRIL-FORMING PEPTIDES ENHANCE VIRAL INFECTION
Aβ aggregates into fibrils ( Selkoe, D. J. (2001) Physiol. Rev. 81, 741-766; Yip, C. M. et al. (2001) Biophys. J. 80, 1359-1371; McLaurin, J. et al. (2000) J. Struct. Biol. 130, 259-270; Serpell, L. C. (2000) Biochim. Biophys. Ada 1502, 16-30; and Kowalewski, T. et al. (1999) Proc. Natl. Acad. Sci. USA 96, 3688-3693). The peurpose of this experiment was to investigate whether other fibril-forming peptides unrelated to Aβ could enhance virus infection. Figure 4 shows that two such peptides, PPI-2480 (AGAKWSWWELTWVGG; SEQ ID NO:l) and PPI-2566 IRQAMCNISRADWND; SEQ ID NO: 2), which form fibrils similar to Aβ^o and Aβ1- 2 (Figures B-F), also enhanced the infection efficiency of recombinant HIV-1 virus pseudotyped with the envelope glycoproteins of the ADA and YU2 HIV-1 isolates by 5-20 fold. The stimulation by these fibrils also required the expression of viral entry coreceptors. These compounds enhanced infection of HIV-1 virus pseudotyped with the VSV-G protein by approximately two-fold. A number of control peptides of varying sequences and lengths that did not form fibrils had no effect on HIV-1 infection. An example is the peptide PPI-1966 shown in Figures 4 and 5F. These data demonstrate that the ability of a peptide to enhance viral infection conelates with its propensity to form fibrils in solution. Interestingly, the peptides that most potently enhance infection (Aβϊ-4o and PPI-2480) formed shorter fibrils (Figures 5B and 5D), while peptides forming longer fibrils (Aβ!-42 and PPI-2566, Figures 5C and 5E) were less efficient.
EXAMPLE 5: FIBRIL-FORMING PEPTIDES PROMOTE LIPID VESICLE
ASSOCIATION WITH CELLS
To investigate further the mechanism by which these fibril-forming peptides stimulate viral infection, the enveloped virus interaction with cells was modeled using liposomes. Rhodamine-labeled liposomes, approximately the size of HIV-1 (ViUar, A. et al. (1998) FEBSLett 432, 150-154), were incubated with cells under the conditions of viral infection in the presence and absence of Aβ1-40, Aβμ42, PPI-2566, PPI-2480 and PPI-1966. The liposome-cell mixtures were then analyzed by FACScan (Figure 5 A). Each of the fibril-forming peptides that enhanced infection also promoted irreversible association of liposomes with cells. The peptides did not cause the formation of syncytia, nor did they promote liposome-to-cell fusion, as judged by the failure of the rhodamine dye in the liposomes to distribute into the cell membrane. Consistent with their relative ability to enhance infection, Aβ1- 0 promoted the adherence of liposomes better than Aβ1- 2. PPI-1966, which Figure 5F shows cannot fonn fibrils, had no effect on the association of liposomes with cells. Utilizing fluorescent Aβ1-40 fibrils (FITC- Aβ), it was demonstrated that FITC-Aβ associated with cell surfaces independent of CD4 or CCR5 expression. The presence of recombinant HIV-1 envelope glycoprotein (JR-FL gpl20) in the medium did not promote the association of FITC-Aβ with cell membranes. Additionally, the presence of Aβ1- 0 did not induce changes in cell surface expression of CD4 or CCR5. These data support a model in which Aβ and other fibril- forming peptides enhance viral infection by mediating a physical association of viral envelopes with the cell lipid bilayer.
EXAMPLE 6: MAGNITUDE OF INFECTION ENHANCEMENT BY THE PRECIPITABLE FRACTION OF Aβ EXCEEDS THE
EFFECT OF POLYBRENE
As shown in Figures 1-3, infection by HIV-1 pseudotyped with the envelope glycoprotein of A-MuLV was enhanced by Aβ. Infection by complete A-MuLV was also strikingly enhanced, from 30-50 fold, in the presence of pre-aggregated Aβi_4o (Figure 6A). This effect was 2-3-fold greater than that observed for polybrene, a cationic polymer commonly used to increase the efficiency of retro viral gene delivery systems (Le Doux, J. M. et αZ. (2001) Hum. Gene Ther. 12, 1611-1621). hi this experiment, Aβ1--4o fibrils were precipitated by multiple centrifugation and washing steps, and compared with the supernatant of the first centrifugation. Figure 6 A demonstrates that the precipitable Aβ^o fraction, but not any residual soluble peptide, enlianced A-MuLV infection comparably to Aβi- 0 that had not been centrifuged. Conversely, the Aβ4o-i reverse fragment did not enhance the infection efficiency of A-MuLV. These data underscore the substantial enhancement of retroviral infectivity by
Figure imgf000028_0001
and demonstrate that the precipitable, and presumably fibril-forming, fraction of Aβ mediates its ability to enhance infection.
EXAMPLE 7: Aβ WEAKLY STIMULATES INFECTION BY AN ENVELOPED VIRUS OTHER THAN A RETROVIRUS
Because HSV has been suggested to play a role in AD and is a major opportunistic infection observed in late-stage HIV-1 infection, the ability of Aβ to enhance HSV infection was investigated in this experiment. A dose-dependent enhancement of the infection mediated by an HSV vector was observed (Figure 6B). However, relative to the enhancement observed with retroviruses, Aβi-40 was substantially less efficient in enhancing HSV infection. This less pronounced ability of Aβ to enhance HSV infection may be a consequence of differences in accessibility or composition of the HSV lipid membrane.
EXAMPLE 8: ASSAY OF β AMYLOID MODULATOR COMPOUND
STABILITY IN CEREBROSPINAL FLUID
The stability of a β amyloid modulator compound in cerebrospinal fluid (CSF) can be assayed in an in vitro assay as follows. A CSF solution is prepared containing 75% Rhesus monkey CSF (commercially available from Northern Biomedical Research), 23% sterile phosphate buffered saline and 2% dimethylsulfoxide (v/v) (Aldrich Chemical Co., Catalog No. 27,685-5). β amyloid modulator compounds are added to the CSF solution to a final concentration of 40 μM or 15 μM. All sample handling is carried out in a laminar flow hood and test solutions are maintained at 37 °C during the assay. After 24 hours, enzymatic activity in the solutions is quenched by adding acetonitrile to produce a final concentration of 25% (v/v). Samples (at the 0 time point and the 24 hour time point) are analyzed at room temperature using reverse-phase HPLC. A microbore column is used to maximize sensitivity. The parameters for analytical HPLC are as follows:
Solvent System
A: 0.1% Trifluoroacetic acid (TFA) in water (v/v) B: 0.085% TFA/Acetonitrile, l% H2O (v/v) Injection and Gradient
Inject: 100-250 μL of test sample
Run: 10% for B for 5 min., then 10-70% B over 60 min.
Chromatographic analysis is performed using a Hewlett Packard 1090 series II HPLC. The column used for separation is a C4, 5 μm, 1 x 250 mm (Vydac #214TP51). The flow rate is 50 μL/min and the elution profile of the test compounds is monitored at 214, 230, 260 and 280 nm.
EXAMPLE 9: β AMYLOID MODULATOR COMPOUND BRAIN UPTAKE
ASSAY Brain levels of a β amyloid modulator compound are determined using a rat following intravenous administration. Under ketamine/xylazine anesthesia male Sprague-Dawley rats (219-302g) receive an intravenous injection via a catheter inserted in the left jugular vein (dose volume of 4 mL/kg administered over 1 minute).
At 60 minutes post administration the left common carotid artery is cannulated to enable perfusion of the left forebrain to remove cerebral blood. The left forebrain, void of blood is subjected to capillary depletion as described by Triguero et al. (1990) J. Neurochem. 54:1882-1888. This established technique separates brain vasculature from the parenchyma and, thus, allows the accurate determination of the concentration of the compound under investigation that has traversed the blood brain barrier. The amount of parent compound that is present within the brain is detennined by LC/MS/MS.
Equivalents
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

We claim;
1. A method for treating a viral disease in a subject comprising administering to said subject a therapeutically or prophylactically effective amount of an amyloidogenic peptide modulator compound, thereby treating a viral disease in said subject.
2. The method of claim 1, wherein said viral disease is a viral disease of the brain.
3. The method of claim 1 , wherein said viral disease is caused by infection with a virus selected from the group consisting of human immunodeficiency virus, herpes simplex virus, varicella zoster virus, poliomyelitis virus, cytomegalovirus, influenza virus, respiratory syncytial virus, coxsackie virus, ebola virus, hantavirus, human papilloma virus, rotavirus, west nile virus, Epstein-Barr virus, and hepatitis virus.
4. The method of claim 1 , wherein said subject is a human subject.
5. The method of claim 1 , wherein said amyloidogenic peptide modulator compound is a β amyloid modulator compound.
6. The method of claim 5, wherein said β amyloid modulator compound is a peptide.
7. The method of claim 5, wherein said β amyloid modulator compound is an antibody.
8. The method of claim 5, wherein said β amyloid modulator compound is a nucleic acid molecule.
9. The method of claim 5, wherein said β amyloid modulator compound is a protease inhibitor.
10. The method of claim 5, wherein said β amyloid modulator compound is a β-secretase or γ-secretase inhibitor.
11. The method of claim 5, wherein said β amyloid modulator compound is a peptide comprised entirely of D-amino acids and having at least three amino acid residues independently selected from the group consisting of a D-leucine structure, a D- phenylalanine structure, a D-tyrosine structure, a D-iodotyrosine structure and a D- alanine structure.
12. The method of claim 5, wherein said β amyloid modulator compound is a compound comprising a formula:
Figure imgf000032_0001
wherein Xaa^, Xaa2, Xaa3 and Xaa4 are each D-amino acid structures and at least three of Xaaj, Xaa2, Xaa3 and Xaa4 are, independently, selected from the group consisting of a D-leucine structure, a D-phenylalanine structure, a D-tyrosine structure, a D-iodotyrosine structure and a D-alanine structure;
Y, which may or may not be present, is a structure having the formula (Xaa)a, wherein Xaa is any D-amino acid structure and a is an integer from 1 to 15;
Z, which may or may not be present, is a structure having the fonnula (Xaa)!-,, wherein Xaa is any D-amino acid structure and b is an integer from 1 to 15; A is a modifying group attached to the compound, with the proviso that A is not an amino acid or is a non-natural amino acid that acts as a β-turn mimetic; and n is an integer from 1 to 15; wherein Xaal5 Xaa2, Xaa3, Xaa4, Y, Z, A and n are selected such that the compound binds to natural β-amyloid peptides or modulates the aggregation or inhibits the neurotoxicity of natural β-amyloid peptides when contacted with the natural β-amyloid peptides.
13. The method of claim 5, wherein said β amyloid modulator compound is a compound having a structure selected from the group consisting of: N,N-dimethyl-(Gly-D-Ala-D-Phe-D-Phe-D-Val-D-Leu)-NH2; N,N-dimethyl-(D-Ala-D- Phe-D-Phe-D-Val-D-Leu)-NH2; N-methyl-(Gly-D-Ala-D-Phe-D-Phe-D-Val-D-Leu)- NH2; N-ethyl-(Gly-D-Ala-D-Phe-D-Phe-D-Val-D-Leu)-NH2; N-isopropyl-(Gly-D-Ala- D-Phe-D-Phe-D-Val-D-Leu)-NH2; H-(D-Leu-D-Val-D-Phe-D-Phe-D-Ala)- isopropylamide; H-(D-Leu-D-Val-D-Phe-D-Phe-D-Ala)-dimethylamide; N,N-diethyl- (Gly-D-Ala-D-Phe-D-Phe-D-Val-D-Leu)-NH2; N,N-diethyl-(D-Ala-D-Phe-D-Phe-D-
Val-D-Leu)-NH2; N,N-dimethyl-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH2; N,N- dimethyl-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH2; N,N-dimethyl-(D-Leu-D-Phe-D- Phe-D-Val-D-Leu)-NH2; H-(Gly-D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH2; N-ethyl- (Gly-D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH2; N-ethyl-(Gly- D-Leu-D-Phe-D-Phe-D- Val-D-Leu)-NH2; N-methyl-(D-Leu-D-Phe-D-Phe-D-Val-D-Leu)-NH2; N-ethyl-(D-Leu- D-Val-D-Phe-D-Phe-D-Leu)-NH2; N-propyl-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH2; N,N-diethyl-(Gly-D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH2; H-(D-Ile-D-Val-D-Phe-D- Phe-D-Ile)-NH2; H-(D-Ile-D-Val-D-Phe-D-Phe-D-Ala-)-NH2; H-( D-Ile- D-Ile-D-Phe- D-Phe- D-Ile)-NH2; H-(D-Nle-D-Val-D-Phe-D-Phe-D-Ala-)-NH2; H-(D-Nle-D-Val-D- Phe-D-Phe-D-Nle)-NH2; l-ρiρeridine-acetyl-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)--NH2; l-piperidine-acetyl-(D-Leu-D-Phe-D-Phe-D-Val-D-Leu)-NH2; H-D-Leu-D-Val-D-Phe- D-Phe-D-Leu-isopropylamide; H-D-Leu-D-Phe-D-Phe-D-Val-D-Leu-isopropylamide; H-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-methylamide; H-(D-Leu-D-Phe-D-Phe-D-Val- D-Leu)-methylamide; H-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-OH; N-methyl-(D-Leu-D- Val-D-Phe-D-Phe-D-Leu)-NH2; H-(D-Leu-D-Val-D-Phe-D-Cha-D-Leu)-NH2; H-(D- Leu-D-Val-D-Phe-D-[p-F]Phe-D-Leu)-NH2; H-(D-Leu-D-Val-D-Phe-D-[F5]Phe-D- Leu)-NH2; H-(D-Leu-D-Phe-D-Cha-D-Val-D-Leu)-NH2; H-(D-Leu-D-Phe- D-[p-F]Phe- D-Val-D-Leu)-NH2; H-(D-Leu-D-Phe- D-[F5]Phe-D-Val-D-Leu)-NH2; H-(D-Leu-D- Phe-D-Lys-D-Val-D-Leu)-NH2; H-(D-Leu-D-Cha-D-Phe-D-Val-D-Leu)-NH2; H-(D- Leu-D-[p-F]Phe-D-Phe-D-Val-D-Leu)-NH2; H-(D-Leu-D-[F5]Phe-D-Phe-D-Val-D- Leu)-NH2; H-(D-Leu- D-Lys-D-Phe-D-Val-D-Leu)-NH2; H-(D-Leu-D-Cha-D-Cha-D- Val-D-Leu)-NH2; H-(D-Leu- D-[p-F]Phe-D-[p-F]Phe-D-Val-D-Leu)-NH2; H-(D-Leu-D- [F5]Phe-D-[F5]Phe-D-Val-D-Leu)-NH2; H-(D-Leu- D-Lys- D-Lys-D-Val-D-Leu)-NH2; N-methyl-(D-Leu-D-Val-D-Phe-D-Cha-D-Leu)-NH2; N-methyl-(D-Leu-D-Val-D-Phe- D-[p-F]Phe-D-Leu)-NH2; N-methyl-(D-Leu-D-Val-D-Phe-D-[F5]Phe-D-Leu)-NH2; H- D-Leu-D-Val-D-Phe-NH-(H-D-Leu-D-Val-D-Phe-)NH; H-D-Leu-D-Val-D-Phe-NH- NH-COCH3; H- D-Leu-D-Val-D-Phe-NH-NH2 H-(lv-[3-I]y-fa)-NH2; H-(lvffl)-NH-Et; H-lvffl-NH-CH2CH2-NH2; H-(GGClvffl)-NH2; H-(GGClvfyl)-NH2; H-(GGClvf-[3-I]y-l)- NH2;H-LVF-NH-NH-FVL-H; H-LVF-NH-NH-M-H; H-lff-(nvl)-l-NH2; H-lf-[pF]f- (nvl)-l-NH2; H-l-[pF]f-[pF]f-(nvl)-l-NH2; Me-lvyfl-NH2; H-(lvyfl)-NH2; Me-(lv-| -F]f-fl)- NH2; H-(lv-[p-F]f-fl)-NH2; H-(lvf-| -F]f-l)-NH2; lvff-[nvl])-NH2; Me-(lvff-[nle])-NH2; Me-(lvffl)-OH; Me-(lvffl)-NH-OH; H-(lv-[p-F]f-f-(nvl))-NH2; Me-(l-v-[p-F]f-f-(nvl))- NH2; H-((nvl)-v-[p-Fjf-f-nvl)-NH2; H-(l-(nvl)-[p-F]f-f-(nvl)-NH2; H-((nvl)-(nvl)-[p-F]f- f-(nvl))-NH2; Me-(l-(nvl)-[ -F]f-f-(nvl))-NH2; H-(lvff-(nvl))-NH2; Ac-(lvffl)-NH2; Ac- (lvffl)-OH; and H-(lv-[3-I]y-fl)-NH2.
14. The method of claim 5, wherein said β amyloid modulator compound is a compound having the structure: N-methyl-(D-Leu-D-Val-D-Phe-D-Phe-D-Leu)-NH2.
15. The method of claim 5, wherein said β amyloid modulator compound is a compound having the structure: 4-Hydroxybenzoyl-D-Leu-D-Val-D-Phe-D-Phe-D-Ala-
NH2.
16. The method of claim 1, wherein said amyloidogenic peptide modulator compound is introduced into the cerebrospinal fluid of the subject.
17. The method of claim 1, wherein said amyloidogenic peptide modulator compound is introduced to the subject intrathecally.
18. The method of claim 1, wherein said amyloidogenic peptide modulator compound is introduced into a region selected from the group consisting of a cerebral ventricle, the lumbar area, and the cisterna magna of the subject.
19. The method of claim 1 , wherein said amyloidogenic peptide modulator compound is administered to the subject in a pharmaceutically acceptable formulation.
20. The method of claim 19, wherein the pharmaceutically acceptable formulation is a dispersion system.
21. The method of claim 19, wherein the pharmaceutically acceptable formulation comprises a lipid-based formulation.
22. The method of claim 21 , wherein the pharmaceutically acceptable formulation comprises a liposome formulation.
23. The method of claim 21 , wherein the pharmaceutically acceptable formulation comprises a multivesicular liposome formulation.
24. The method of claim 19, wherein the pharmaceutically acceptable formulation comprises a polymeric matrix.
25. The method of claim 19, wherein the pharmaceutically acceptable formulation is contained within a minipump.
26. The method of claim 19, wherein the pharmaceutically acceptable formulation provides sustained delivery of said amyloidogenic peptide modulator compound for at least one week after the pharmaceutically acceptable formulation is administered to the subject.
27. The method of claim 19, wherein the pharmaceutically acceptable formulation provides sustained delivery of said amyloidogenic peptide modulator compound for at least one month after the pharmaceutically acceptable formulation is administered to the subject.
28. A method for treating AIDS in a subject comprising administering to said subject a therapeutically or prophylactically effective amount of an amyloidogenic peptide modulator compound, thereby treating AIDS in said subject.
29. The method of claim 28, wherein said amyloidogenic peptide modulator compound is a β amyloid modulator compound.
30. A method of modulating infection of a cell by a virus, comprising contacting said cell with an amyloidogenic peptide modulator compound in an amount effective to modulate infection of said cell by a virus.
31. The method of claim 30, wherein said cell is a brain cell.
32. The method of claim 30, wherein said virus is selected from the group consisting of human immunodeficiency virus, heφes simplex virus, varicella zoster virus, poliomyelitis virus, cytomegalovirus, influenza virus, respiratory syncytial virus, coxsackie virus, ebola virus, hantavirus, human papilloma virus, rotavirus, west nile virus, Epstein-Ban virus, and hepatitis virus.
33. The method of claim 30, wherein said amyloidogenic peptide modulator compound is a β amyloid modulator compound.
34. A method of modulating infection of a cell by a human immunodeficiency virus, comprising contacting said cell with an amyloidogenic peptide modulator compound in an amount effective to modulate infection of said cell by a human immunodeficiency virus.
35. The method of claim 34, wherein said cell is a brain cell.
36. The method of claim 34, wherein said amyloidogenic peptide modulator compound is a β amyloid modulator compound.
37. A method for identifying an amyloidogenic peptide modulator compound capable of treating a viral disease in a subject comprising
(a) contacting a cell with a virus in the presence of an amyloidogenic peptide modulator compound and (b) determining the ability of said virus to infect said cell, thereby identifying an amyloidogenic peptide modulator compound capable of treating a viral disease in a subject.
38. The method of claim 37, wherein the ability of said virus to infect said cell is determined by monitoring said cell for a phenotype associated with viral infection.
39. The method of claim 38, wherein said phenotype is virus burden.
40. The method of claim 37, further comprising administering said amyloidogenic peptide modulator compound to an animal model for a viral disease and determining whether said amyloidogenic peptide modulator compound is capable of ameliorating a viral disease symptom in said animal.
41. The method of claim 37, further comprising administering said amyloidogenic peptide modulator compound to an animal model for a viral disease and monitoring viral burden in said animal before and after the administration of said amyloidogenic peptide modulator compound.
42. The method of claim 37, wherein said amyloidogenic peptide modulator compound is a β amyloid modulator compound.
43. A method for identifying an amyloidogenic peptide modulator compound capable of modulating infection of a cell by a virus, comprising
(a) contacting a cell with a virus in the presence of an amyloidogenic peptide modulator compound; and
(b) determining the ability of said virus to infect said cell, thereby identifying an amyloidogenic peptide modulator compound capable of modulating infection of a cell by a virus.
44. A kit comprising an amyloidogenic peptide modulator compound and instructions for use in modulating infection of a cell by a virus.
45. A kit comprising an amyloidogenic peptide modulator compound and instructions for use in treating a viral disease in a subject.
PCT/US2003/019365 2002-06-01 2003-06-18 Methods for treating viral diseases using modulators of amyloidogenic peptide aggregation WO2003105677A2 (en)

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