US20070021345A1 - Peptides antibodies directed thereagainst and methods using same for diagnosing and treating amyloid-associated diseases - Google Patents

Peptides antibodies directed thereagainst and methods using same for diagnosing and treating amyloid-associated diseases Download PDF

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US20070021345A1
US20070021345A1 US10/562,852 US56285204A US2007021345A1 US 20070021345 A1 US20070021345 A1 US 20070021345A1 US 56285204 A US56285204 A US 56285204A US 2007021345 A1 US2007021345 A1 US 2007021345A1
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peptide
amino acid
amyloid
pharmaceutical composition
peptides
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Ehud Gazit
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Tel Aviv University Future Technology Development LP
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Assigned to TEL AVIV UNIVERSITY FUTURE TECHNOLOGY DEVELOPMENT L.P. reassignment TEL AVIV UNIVERSITY FUTURE TECHNOLOGY DEVELOPMENT L.P. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: GAZIT, EHUD
Priority to US11/471,657 priority patent/US7781396B2/en
Publication of US20070021345A1 publication Critical patent/US20070021345A1/en
Priority to US12/458,163 priority patent/US8012929B2/en
Priority to US12/654,461 priority patent/US8697634B2/en
Priority to US13/975,414 priority patent/US8993510B2/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06139—Dipeptides with the first amino acid being heterocyclic
    • C07K5/06156—Dipeptides with the first amino acid being heterocyclic and Trp-amino acid; Derivatives thereof
    • A—HUMAN NECESSITIES
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    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
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    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
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    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/20—Hypnotics; Sedatives
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    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
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    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06078—Dipeptides with the first amino acid being neutral and aromatic or cycloaliphatic
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06139—Dipeptides with the first amino acid being heterocyclic
    • C07K5/06165—Dipeptides with the first amino acid being heterocyclic and Pro-amino acid; Derivatives thereof
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    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
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    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0812—Tripeptides with the first amino acid being neutral and aromatic or cycloaliphatic
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    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
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    • C07K5/0819—Tripeptides with the first amino acid being acidic
    • C—CHEMISTRY; METALLURGY
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    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0821—Tripeptides with the first amino acid being heterocyclic, e.g. His, Pro, Trp
    • C07K5/0823—Tripeptides with the first amino acid being heterocyclic, e.g. His, Pro, Trp and Pro-amino acid; Derivatives thereof
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    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
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    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/1008—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
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    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/101—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
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    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
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    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1021—Tetrapeptides with the first amino acid being acidic
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1024—Tetrapeptides with the first amino acid being heterocyclic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to peptides and antibodies directed thereagainst which can be used to diagnose, prevent, and treat amyloid-associated diseases, such as Type II diabetes mellitus and Alzheimer's disease.
  • Amyloid material deposition (also referred to as amyloid plaque formation) is a central feature of a variety of unrelated pathological conditions including Alzheimer's disease, prion-related encephalopathies, type II diabetes mellitus, familial amyloidosis and light-chain amyloidosis.
  • Amyloid material is composed of a dense network of rigid, nonbranching proteinaceous fibrils of indefinite length that are about 80 to 100 A in diameter.
  • Amyloid fibrils contain a core structure of polypeptide chains arranged in antiparallel or parallel ⁇ -pleated sheets lying with their long axes perpendicular to the long axis of the fibril [Both et al. (1997) Nature 385:787-93; Glenner (1980) N. Eng. J. Med. 302:1283-92; Balbach et al. (2002) Biophys J. 83:1205-16].
  • amyloid fibril proteins Approximately twenty amyloid fibril proteins have been identified in-vivo and correlated with specific diseases. These proteins share little or no amino acid sequence homology, however the core structure of the amyloid fibrils is essentially the same. This common core structure of amyloid fibrils and the presence of common substances in amyloid deposits suggest that data characterizing a particular form of amyloid material may also be relevant to other forms of amyloid material and thus can be implemented in template design for the development of drugs against amyloid-associated diseases such as type II diabetes mellitus, Alzheimer's dementia or diseases and prion-related encephalopathies.
  • amyloid deposits do not appear to be inert in vivo, but rather are in a dynamic state of turnover and can even regress if the formation of fibrils is halted [Gillmore et al. (1997) Br. J. Haematol. 99:245-56].
  • therapies designed to inhibiting the production of amyloid polypeptides or inhibiting amyloidosis may be useful for treating amyloid associated diseases.
  • Inhibition of the production of amyloid polypeptides may be accomplished, for example, through the use of antisense oligonucleotides such as against human islet amyloid polypeptide messenger RNA (mRNA).
  • mRNA messenger RNA
  • the addition of antisense oligonucleotides or the expression of antisense complementary DNA against islet amyloid polypeptide mRNA increased the insulin mRNA and protein content of cells, demonstrating the potential effectiveness of this approach [Kulkarni et al. (1996) J. Endocrinol. 151:341-8; Novials et al. (1998) Pancreas 17:182-6].
  • no experimental results demonstrating the in vivo effectiveness of such antisense molecules have been demonstrated.
  • amyloid fibrils including islet amyloid, contains potential stabilizing or protective substances, such as serum amyloid P component, apolipoprotein E, and perlecan. Blocking their binding to developing amyloid fibrils could inhibit amyloidogenesis [Kahn et al. (1999) Diabetes 48:241-53], as could treatment with antibodies specific for certain parts of an amyloidogenic protein [Solomon et al. (1997) Proc. Natl. Acad. Sci. USA 94:4109-12].
  • Heparin sulfate has been identified as a component of all amyloids and has also been implicated in the earliest stages of inflammation-associated amyloid induction. Kisilevsky and co-workers (Nature Med. 1:143-148, 1995) described the use of low molecular weight anionic sulfonate or sulfate compounds that interfere with the interaction of heparin sulfate with the inflammation-associated amyloid precursor and the ⁇ peptide of Alzheimer's disease (AD). Heparin sulfate specifically influences the soluble amyloid precursor (SAA2) to adopt an increased ⁇ -sheet structure characteristic of the protein-folding pattern of amyloids.
  • SAA2 soluble amyloid precursor
  • anionic sulfonate or sulfate compounds were shown to inhibit heparin accelerated A ⁇ fibril formation and were able to disassemble preformed fibrils in vitro, as monitored by electron micrography. Moreover, these compounds substantially arrested murine splenic inflammation-associated amyloid progression in vivo in acute and chronic models. However, the most potent compound [i.e., poly-(vinylsulfonate)]showed acute toxicity.
  • IDOX Anthracycline 4′-iodo-4′-deoxy-doxorubicin
  • Anti- ⁇ -amyloid monoclonal antibodies have been shown to be effective in disaggregating ⁇ -amyloid plaques and preventing ⁇ -amyloid plaque formation in vitro (U.S. Pat. No. 5,688,561). However, no experimental results demonstrating the in vivo effectiveness of such antibodies have been demonstrated.
  • ⁇ -sheet breakers synthetic peptides that disrupt the ⁇ -pleated sheets
  • amyloidosis a penta-residue peptide inhibited amyloid beta-protein fibrillogenesis, disassembled preformed fibrils in vitro and prevents neuronal death induced by fibrils in cell culture.
  • the beta-sheet breaker peptide significantly reduced amyloid beta-protein deposition in vivo and completely blocked the formation of amyloid fibrils in a rat brain model of amyloidosis.
  • Antioxidants Another proposed therapy has been the intake of antioxidants in order to avoid oxidative stress and maintain amyloid proteins in their reduced state (i.e., monomers and dimers). The use of sulfite was shown to lead to more stable monomers of the TTR both in vitro and in vivo [Altland (1999) Neurogenetics 2:183-188]. However, a complete characterization of the antioxidant effect is still not available and the interpretation of results concerning possible therapeutic strategies remains difficult.
  • a peptide comprising amino acid sequence X-Y or Y-X, wherein X is an aromatic amino acid and Y is any amino acid other than glycine, the peptide being at least 2 and no more than 15 amino acids in length .
  • a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID) NOs. 4, 12-19, 2745, 112-123, 125 and 127, the peptide being at least 2 and no more than 15 amino acids in length.
  • a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 4, 12-19, 27-45, 112-123, 125 and 127, the peptide being at least 2 and no more than 15 amino acids in length.
  • a peptide selected from the group consisting of SEQ ID NOs. 4, 12-19, 27-45, 112-123, 125 and 127.
  • a method of treating or preventing an amyloid-associated disease in an individual comprising providing to the individual a therapeutically effective amount of a peptide including the amino acid sequence X-Y or Y-X, wherein X is an aromatic amino acid and Y is any amino acid other than glycine, the peptide being at least 2 and no more than 15 amino acids in length.
  • the peptide is an active ingredient of a pharmaceutical composition which also includes a physiologically acceptable carrier.
  • the peptide is expressed from a nucleic acid construct.
  • a pharmaceutical composition for treating or preventing an amyloid-associated disease comprising as an active ingredient a peptide including the amino acid sequence X-Y or Y-X, wherein X is an aromatic amino acid and Y is any amino acid other than glycine, the peptide being at least 2 and no more than 15 amino acids in length and a pharmaceutically acceptable carrier or diluent.
  • At least one amino acid of the at least 2 and no more than 15 amino acids of the peptide is a D stereoisomer.
  • At least one amino acid of the at least 2 and no more than 15 amino acids of the peptide is an L stereoisomer.
  • the peptide is two amino acids in length and Y is a ⁇ -sheet breaker amino acid.
  • the peptide is as set forth in SEQ ID NO: 145.
  • the peptide is 3 amino acids in length, whereas Y is an aromatic amino acid and an amino acid residue attached to the amino acid sequence X-Y or Y-X is a ⁇ -sheet breaker amino acid.
  • the ⁇ -sheet breaker amino acid is at a C-terminus of the peptide.
  • the peptide is at least 3 amino acids in length and includes a thiolated amino acid at an N-terminus thereof.
  • nucleic acid construct comprising a polynucleotide segment encoding a peptide including the amino acid sequence X-Y or Y-X, wherein X is an aromatic amino acid and Y is any amino acid other than glycine, the peptide being at least 2 and no more than 15 amino acids in length.
  • nucleic acid construct further comprises a promoter
  • an antibody or an antibody fragment comprising an antigen recognition region capable of binding a peptide including the amino acid sequence X-Y or Y-X, wherein X is an aromatic amino acid and Y is any amino acid other than glycine, the peptide being at least 2 and no more than 15 amino acids in length.
  • Y is a polar uncharged amino acid selected from the group consisting of serine, threonine, asparagine, glutamine and natural derivatives thereof.
  • Y is a ⁇ -sheet breaker amino acid.
  • the ⁇ -sheet breaker amino acid is a naturally occurring amino acid.
  • the naturally occurring amino acid is selected from the group consisting of proline, aspartic acid, glutamic acid, glycine, lysine and serine.
  • the ⁇ -sheet breaker amino acid is a synthetic amino acid.
  • the synthetic amino acid is a C ⁇ -methylated amino acid.
  • the C ⁇ -ethylated amino acid is ⁇ -aminoisobutyric acid.
  • the peptide is a linear or cyclic peptide.
  • the peptide is selected from the group consisting of SEQ ID NOs. 4, 12-19, 27-45, 112-123, 125 and 127.
  • the peptide is at least 4 amino acids in length and includes at least two serine residues at a C-terminus thereof.
  • the peptide is at least 3 amino acids in length and whereas at least one of the amino acids of the peptide other than X-Y is a polar uncharged amino acid selected from the group consisting of serine, threonine, asparagine, glutamine and natural derivatives thereof.
  • the peptide is at least 3 amino acids in length and whereas at least one of the amino acids of the peptide other than X-Y is a is a ⁇ -sheet breaker amino acid.
  • the ⁇ -sheet breaker amino acid is a naturally occurring amino acid.
  • the naturally occurring amino acid is selected from the group consisting of proline, aspartic acid, glutamic acid, glycine, lysine and serine.
  • the ⁇ -sheet breaker amino acid is a synthetic amino acid.
  • the synthetic amino acid is a Ca-methylated amino acid.
  • the C ⁇ -methylated amino acid is ⁇ -saminoisobutyric acid.
  • the ⁇ -sheet breaker amino acid is located downstream to the X-Y in the peptide.
  • the ⁇ -sheet breaker amino acid is located upstream to the X-Y in the peptide.
  • the peptide is at least 3 amino acids in length and whereas at least one of the amino acids of the peptide is a positively charged amino acid and at least one of the amino acid residues of the peptide is a negatively charged amino acid.
  • the positively charged amino acid is selected from the group consisting of lysine, arginine, and natural and synthetic derivatives thereof.
  • the negatively charged amino acid is selected from the group consisting of aspartic acid, glutamic acid and natural and synthetic derivatives thereof.
  • a pharmaceutical composition for treating or preventing an amyloid-associated disease comprising as an active ingredient an antibody or an antibody fragment having an antigen recognition region capable of binding a peptide including the amino acid sequence X-Y or Y-X, wherein X is an aromatic amino acid and Y is any amino acid other than glycine, the peptide being at least 2 and no more than 15 amino acids in length and a pharmaceutical acceptable carrier or diluent.
  • a method of treating or preventing an amyloid-associated disease in an individual comprising providing to the individual therapeutically effective amount of an antibody or an antibody fragment having an antigen recognition region capable of binding a peptide including the amino acid sequence X-Y or Y-X, wherein X is an aromatic amino acid and Y is any amino acid other than glycine, the peptide being at least 2 and no more than 15 amino acids in length.
  • C* is a chiral carbon having a D configuration.
  • R 1 and R 2 are each independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, carboxy, C-thiocarb;
  • R 3 is selected from the group consisting of hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, halo and amine;
  • R 4 is alkyl
  • a method of treating or preventing an amyloid-associated disease in an individual comprising providing to the individual a therapeutically effective amount of a peptide having the general Formula:
  • C* is a chiral carbon having a D configuration.
  • R 1 and R 2 are each independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, carboxy, C-thiocarb;
  • R 3 is selected from the group consisting of hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, halo and amine;
  • R 4 is alkyl
  • R 4 is methyl
  • R 1 and R 2 are each hydrogen and R 3 is hydroxy.
  • the peptide is a cyclic peptide.
  • the present invention successfully addresses the shortcomings of the presently known configurations by providing novel peptides, compositions and methods, which can be used to diagnose and treat amyloid associated diseases such as type II Diabetes mellitus.
  • FIG. 1 is a schematic illustration depicting the self-assembly ability and hydrophobicity of a group of peptides from a number of amyloid proteins as deduced using Kyte and Dolittle scale. Note, that no correlation is observed between hydrophobicity and the amyloidogenic potential of the analyzed peptides. The only apparent indication for potential amyloid fibril formation in this group of peptide is a combination of aromatic nature and minimal length.
  • FIGS. 3 a - c are schematic illustrations of a primary sequence comparison between human and rodent IAPP and the synthetic peptides of the present invention.
  • FIG. 3 a is a sequence alignment of human and rodent IAPP.
  • a block indicates a seven amino acid sub-sequence illustrating the major inconsistencies between the sequences. The “basic amyloidogenic unit” is presented by bold letters and underlined.
  • FIG. 3 b illustrates the chemical structure of the wild type IAPP peptide (SEQ ID NO: 1).
  • FIG. 3 c illustrates the primary sequences and SEQ ID NOs. of the peptides derived from the basic amyloidogenic unit.
  • FIGS. 4 a - b are graphs illustrating light absorbance at 405 nm as a function of time during fibril formation thus reflecting the aggregation kinetics of IAPP-derived peptides.
  • the following symbols are used: closed squares—N1A, opened circles—G3A, closed circles—wild type, opened triangles—L6A, opened squares—I5A and closed triangles—F2A.
  • FIG. 5 is a histogram depicting mean particle size of assembled IAPP peptide and derivatives as measured by light scattering. Each column represents the results of 3-5 independent measurements.
  • FIGS. 6 a - n are photomicrographs illustrating Congo Red binding to pre-assembled IAPP peptides. Normal field and polarized field micrographs are shown respectively for each of the following aged peptide suspensions: N1A peptide ( FIGS. 6 a - b ), F2A peptide ( FIGS. 6 c - d ), G3A peptide ( FIGS. 6 e - f ), wild type peptide ( FIGS. 6 g - h ), I5A peptide ( FIGS. 6 i - j ) and L6A ( FIGS. 6 k - 1 ). Buffer with Congo red reagent was used as a negative control visualized with and without polarized light as shown in FIGS. 6 m and 6 n , respectively.
  • FIGS. 7 a - f are electron micrographs of “aged” IAPP peptide and derivatives.
  • NIA peptide FIG. 7 a
  • F2A peptide FIG. 7 b
  • G3A peptide FIG. 7 c
  • wild type peptide FIG. 7 d
  • I5A peptide FIG. 7 e
  • L6A FIG. 7 f
  • the indicated scale bar represents 100 nm.
  • FIG. 8 a is a nucleic acid sequence alignment of wild type hIAPP and a corresponding sequence modified according to a bacterial codon usage. Modified bases are underlined.
  • FIG. 8 b is a schematic illustration of the pMALc2x-NN vector which is used for cytoplasmic expression of the 48 kDa MBP-IAPP protein.
  • the V8 Ek cleavage site and the (His) 6 tag are fused C-terminally to the malE tag vector sequence.
  • a factor Xa cleavage site for removal of the MBP tag is indicated.
  • FIG. 9 is a protein gel GelCode Blue staining depicting bacterial expression and purification of MBP and MBP-IAPP fusion protein.
  • Bacterial cell extracts were generated and proteins were purified on an amylose resin column. Samples including 25 ⁇ g protein were loaded in each of Lanes 1-3 whereas 5 ⁇ g protein were loaded on each of lanes 4-5. Proteins were resolved on a 12% SDS-PAGE and visualized with GelCode Blue staining. A molecular weight marker is indicated on the left.
  • Lane 4 purified MBP.
  • Lane 5 purified MBP-IAPP.
  • An arrow marks the MBP-IAPP.
  • FIGS. 10 a - b are a dot-blot image ( FIG. 10 a ) and densitometric quantitation thereof ( FIG. 10 b ) depicting putative amyloidogenic sequences in hIAPP.
  • FIG. 11 is a graphic illustration depicting light absorbance at 405 nm as a fuiction of time during fibril formation thus reflecting the aggregation kinetics of IAPP-derived peptides (SEQ ID NOs. 14-19).
  • FIGS. 12 a - f are photomicrographs illustrating Congo Red binding to pre-assembled IAPP peptides. Polarized field micrographs are shown for each of the following one day aged peptide suspensions: NFLVHSSNN peptide ( FIGS. 12 a ), NFLVHSS ( FIG. 12 b ), FLVHSS ( FIG. 12 c ), NFLVH ( FIG. 12 d ), FLVHS ( FIG. 12 e ) and FLVH ( FIG. 12 f ).
  • FIGS. 13 a - f are electron micrographs of “aged” IAPP peptides.
  • NFLVHSSNN peptide FIGS. 13 a
  • NFLVHSS FIG. 13 b
  • FLVHSS FIG. 13 c
  • NFLVH FIG. 13 d
  • FLVHS FIG. 13 e
  • FLVH FIG. 13 f
  • the indicated scale bar represents 100 nm.
  • FIGS. 14 a - f are graphs showing secondary structures in the insoluble IAPP aggregates as determined by Fourier transformed infrared spectroscopy.
  • NFLVHSSNN peptide FIGS. 14 a
  • NFLVHSS FIG. 14 b
  • FLVHSS FIG. 14 c
  • NFLVH FIG. 14 d
  • FLVHS FIG. 14 e
  • FLVH FIG. 14 f
  • FIG. 15 is a chemical structure of a previously reported amyloidogenic peptide fragment of Medin [Haggqvist (1999) Proc. Natl. Acad. Sci. U S A 96:8669-8674).
  • FIGS. 16 a - b are graphs illustrating light absorbance at 405 nm as a function of time during fibril formation thus reflecting the aggregation kinetics of Medin-derived peptides.
  • FIG. 16 a illustrates a short-term kinetic assay.
  • FIG. 16 b illustrates a long-term kinetic assay.
  • FIGS. 17 a - f are electron micrographs of “aged” Medin-derived peptides.
  • NFGSVQFA FIGS. 17 a
  • NFGSVQ— FIG. 17 b NFGSV— FIG. 17 c
  • FGSVQ— FIG. 17 d FGSVQ— FIG. 17 d
  • GSVQ— FIG. 17 e and FGSV— FIG. 17 f The indicated scale bar represents 100 nm.
  • FIGS. 18 a - f are photomicrographs illustrating Congo Red binding to pre-assembled Medin-derived peptides. Polarized field micrographs are shown for each of the following aged peptide suspensions: NFGSVQFA— FIGS. 18 a , NFGSVQ— FIG. 18 b , NFGSV— FIG. 18 c , FGSVQ— FIG. 18 d , GSVQ— FIG. 18 e and FGSV— FIG. 18 f.
  • FIGS. 19 a - c depict the effect of an alanine mutation on the amyloidogenic features of the hexapeptide amyloidogenic fragment of Medin.
  • FIG. 19 a is a graph illustrating light absorbance at 405 nm as a function of time during fibril formation thus reflecting the aggregation kinetics of Medin-derived alanine mutant;
  • FIG. 19 b is an electron micrograph of “aged” Medin—derived alanine mutant, The scale bar represents 100 nm;
  • FIG. 19 c is a photomicrograph illustrating Congo Red binding to pre-assembled Medin-derived peptide mutant.
  • FIGS. 20 a - b are the amino acid sequence of human Calcitonin ( FIG. 20 a ) and chemical structure of an amyloidogenic peptide fragment of human Calcitonin ( FIG. 20 b ). Underlined are residues 17 and 18 which are important to the oligomerization state and hormonal activity of Calcitonin [Kazantzis (2001) Eur. J. Biochem. 269:780-791].
  • FIGS. 21 a - d are electron micrographs of “aged” Calcitonin-derived peptides.
  • DFNKF FIG. 21 a
  • DFNK FIG. 21 b
  • FNKF FIG. 21 c
  • DFN FIG. 21 d
  • the indicated scale bar represents 100 nm.
  • FIGS. 22 a - d are photomicrographs illustrating Congo Red binding to pre-assembled Calcitonin-derived peptides. Polarized field micrographs are shown for each of the following aged peptide suspensions: DFNKF— FIG. 22 a , DFNK— FIG. 22 b , FNKF— FIG. 22 c and DFN— FIG. 22 d.
  • FIG. 23 is a graphic illustration showing secondary structures in the insoluble Calcitonin aggregates as determined by Fourier transformed infrared spectroscopy.
  • FIGS. 24 a - c depict the effect an alanine mutation on the amyloidogenic features of the pentapeptide amyloidogenic fragment of Calcitonin.
  • FIG. 24 a is an electron micrograph of “aged” Calcitonin-derived alanine mutant. The scale bar represents 100 nm;
  • FIG. 24 b is a photomicrograph illustrating Congo Red binding to pre-assembled Calcitonin-derived peptide mutant;
  • FIG. 24 c is a graph showing secondary structures in the mutant peptide as determined by Fourier transformed infrared spectroscopy.
  • FIG. 25 is an electron micrograph depicting self-assembly of “aged” Lactotransferrin-derived peptide.
  • the scale bar represents 100 nm.
  • FIG. 26 is an electron micrograph depicting self-assembly of “aged” Serum amyloid A protein-derived peptide.
  • the scale bar represents 100 nm.
  • FIG. 27 is an electron micrograph depicting self-assembly of “aged” BriL-derived peptide.
  • the scale bar represents 100 nm.
  • FIG. 28 is an electron micrograph depicting self-assembly of “aged” Gelsolin-derived peptide.
  • the scale bar represents 100 nm.
  • FIG. 29 is an electron micrograph depicting self-assembly of “aged” Serum amyloid P-derived peptide.
  • the scale bar represents 100 nm.
  • FIG. 30 is an electron micrograph depicting self-assembly of “aged” Immunoglobulin light chain-derived peptide.
  • the scale bar represents 100 nm.
  • FIG. 31 is an electron micrograph depicting self-assembly of “aged” Cystatin C-derived peptide.
  • the scale bar represents 100 nm.
  • FIG. 32 is an electron micrograph depicting self-assembly of “aged” Transthyretin-derived peptide.
  • the scale bar represents 100 nm.
  • FIG. 33 is an electron micrograph depicting self-assembly of “aged” Lysozyme-derived peptide.
  • the scale bar represents 100 nm.
  • FIG. 34 is an electron micrograph depicting self-assembly of “aged” Fibrinogen-derived peptide.
  • the scale bar represents 100 nm.
  • FIG. 35 is an electron micrograph depicting self-assembly of “aged” Insulin-derived peptide.
  • the scale bar represents 100 nm.
  • FIG. 36 is an electron micrograph depicting self-assembly of “aged” Prolactin-derived peptide.
  • the scale bar represents 100 nm.
  • FIG. 37 is an electron micrograph depicting self-assembly of “aged” Beta 2 microglobulin-derived peptide.
  • the scale bar represents 100 nm.
  • FIG. 38 is a graphic representation of the effect of an inhibitory peptide on IAPP self-assembly. Squares—wild type (wt) IAPP peptide; triangles—wt-IAPP+inhibitory peptide; circles—no peptides.
  • FIG. 39 is a graphic illustration depicting light absorbance at 405 nm as a function of time during fibril formation thus reflecting the aggregation kinetics of IAPP-derived peptides (SEQ ID NOs. 46-49).
  • FIG. 40 is a histogram representation illustrating turbidity of IAPP analogues following seven day aging.
  • FIG. 41 a - f are electron rnicrographs of “aged” IAPP analogues.
  • NFGAILSS FIG. 41 a
  • NFGAILSS FIG. 41 b
  • NIGAILSS FIG. 41 c
  • NLGAILSS FIG. 41 d
  • NVGAILSS FIG. 41 e
  • NAGAILSS FIG. 41 f
  • the indicated scale bar represents 100 nm.
  • FIG. 42 a shows short exposure of the bound peptide-array.
  • FIG. 42 b shows long exposure of the bound peptide-array.
  • FIG. 42 c shows quantitation of the short exposure ( FIG. 42 a ) using densitometry and arbitrary units.
  • FIG. 43 a is a Ramachandran plot showing the sterically allowed regions for all residues (yellow for fully allowed, orange for partially allowed), for L-Proline (blue) and for the achiral Aib residue (magenta).
  • FIGS. 43 b - c are schematic illustrations showing the chemical structure of the longer wild-type IAPP peptide (ANFLVH, SEQ ID NO: 124, FIG. 43 b ) and the Aib modified structure thereof peptide (Aib-NF-Aib-VH, SEQ ID NO: 125, FIG. 43 c ).
  • Functional groups suitable for modicifcation are marked in blue ( FIG. 43 b ) while modified groups are marked in red ( FIG. 43 c ).
  • FIGS. 44 a - d are electron micrographs of “aged” IAPP analogues.
  • FIG. 44 a ANFLVH
  • FIG. 44 b ANFLV
  • FIG. 44 c Aib-NF-Aib-VH
  • FIG. 44 d Aib-NF-Aib-V.
  • the indicated scale bar represents 100 nm.
  • FIGS. 45 a - d are photomicrographs illustrating Congo Red binding to pre-assembled wild type and Aib modified IAPP peptides. Polarized field micrographs are shown for each of the following aged (i.e., 11 days) peptide suspensions.
  • FIG. 45 a ANFLVH
  • FIG. 45 b ANFLV
  • FIG. 45 c Aib-NF-Aib-VH
  • FIG. 45 d —Aib-NF-Aib-V.
  • FIGS. 46 a - b are graphs showing secondary structures in the insoluble wild type and Aib modified hIAPP aggregates as determined by Fourier transformed infrared spectroscopy (FT-IR).
  • FIG. 46 a wild-type peptide ANFLVH and the corresponding Aib modified peptide as designated by arrows.
  • FIG. 46 b wild-type ANFLVH and the corresponding Aib modified peptide as designated by errows.
  • FIG. 47 is a graph showing the inhibitory effect of Aib modified peptides on amyloid fibril formation. Wild type IAPP was incubated alone or with the various peptides of the present invention. Fibril formation as a function of time was determined using ThT fluorescence.
  • FIG. 48 is a histogram showing the inhibitory effect of short aromatic sequences (SEQ ID NOs. 112-123) on IAPP self-assembly.
  • FIGS. 49 a - d are graphs depicting iterative cycles of selection of IAPP fibrilization inhibitors. Fibrilization was monitored by ThT fluorescence assay. Fluorescence values of IAPP alone (4 ⁇ M) or in the presence of assayed compounds (40 AM) were tested. Measurements were taken once IAPP fluorescence reached a plateau. IAPP fluorescence was arbitrary set as 100. FIG. 49 a shows the results of the first round of selection of IAPP fibrilization inhibitors.
  • FIG. 49 b shows the results of the second round of selection of IAPP fibrilization inhibitors.
  • FIG. 49 c shows the results of the third round of selection of IAPP fibrilization inhibitors.
  • FIG. 49 d shows the results of the forth round of selection of IAPP fibrilization inhibitors.
  • FIG. 50 is a graph depicting Inhibition of A ⁇ (140) fibril formation by D-Trp-Aib.
  • a ⁇ 1-40 stock solution was diluted to a final concentration of 5 ⁇ M in 100 mM NaCI, 10 mM sodium phosphate buffer (pH 7.4) with 10 ⁇ M D-Trp-Aib (triangles) or without any addition (squares). Fluorescence values were measured after addition of 0.3 ⁇ M Tht to each sample. The results represent the mean of two independent measurements.
  • FIGS. 51 a - c are photomicrographs depicting the inhibitory effect of D-Trp-Aib on the fibrilization of A ⁇ as visualized by TEM.
  • FIG. 51 a shows A ⁇ alone.
  • FIGS. 51 b - c shows two different field of A ⁇ incubated in the presence of the inhibitor.
  • the present invention is of novel peptides, antibodies directed thereagainst, compositions including same and methods of utilizing each for diagnosing or treating amyloid associated diseases such as type II Diabetes mellitus.
  • amyloid associated diseases such as Type diabetes mellitus
  • the present inventor has identified a sequence characteristic of amyloid forming peptides, which directs fibril formation. This finding suggests that ordered amyloidogenesis involves a specific pattern of molecular interactions rather than the previously described mechanism involving non-specific hydrophobic interactions [Petkova (2002) Proc. Natl. Acad. Sci. U S A 99:16742-16747].
  • ⁇ -stacking interactions are non-bonded interactions which are formed between planar aromatic rings.
  • the steric constrains associated with the formation of those ordered stacking structures have a fundamental role in self-assembly processes that lead to the formation of supramolecular structures.
  • ⁇ -stacking interactions which are probably entropy driven, play a central role in many biological processes such as stabilization of the double-helix structure of DNA, core-packing and stabilization of the tertiary structure of proteins, host-guest interactions, and porphyrin aggregation in solution [for further review on the possible role of ⁇ -stacking interaction in the self-assembly of amyloid fibrils see Gazit (2002) FASEB J. 16:77-83].
  • the present inventor demonstrated the ability of short aromatic peptide sequences, as short as di-peptides (see Example 45-47), to mediate molecular recognition, enabling for the first time, to generate highly efficient diagnostic, prophylactic and therapeutic peptides which can be utilized to treat or diagnose diseases characterized by amyloid plaque formation.
  • a peptide which includes the amino acid sequence X-Y or Y-X, wherein X is an aromatic amino acid and Y is any amino acid other than glycine.
  • Examples of peptides that include this sequence are set forth in SEQ ID Nos. 4, 12-19, 2745, 112-123, 125 and 127.
  • the present inventor have uncovered that contrary to the teachings of the prior art, it is aromaticity rather than hydrophobicity which dictates amyloid self-assembly.
  • the aromatic amino acid of the peptides of the present invention is pivotal to the formation of amyloid fibrils.
  • the aromatic amino acid can be any naturally occurring or synthetic aromatic residue including, but not limited to, phenylalanine, tyrosine, tryptophan, phenylglycine, or modificants, precursors or functional aromatic portions thereof. Examples of aromatic residues which can form a part of the peptides of present invention are provided in Table 2 below.
  • the present invention facilitates the design of peptides exhibiting varying degrees of self-aggregation kinetics and aggregate structure.
  • self-aggregation refers to the capability of a peptide to form aggregates (e.g. fibrils) in an aqueous solution.
  • aggregates e.g. fibrils
  • the ability of a peptide to self-aggregate and the kinetics and type of such self-aggregation determines a use for the peptide in treating or diagnosing amyloid diseases.
  • the present invention encompasses both longer peptides (e.g., 10-50 amino acids), which include the sequences set forth in SEQ ID NOs: 4.12-19, 27-45, 112-123, 125, 127, 128-147 or 148, or preferably shorter peptides (e.g., 2-15 amino acids, preferably at least 2, at least 3, at least 4, at least 5, at least 6, at least 8, at least-10, say 12 amino acids, preferably no more than 15 amino acids) including any of these sequences.
  • longer peptides e.g., 10-50 amino acids
  • SEQ ID NOs: 4.12-19, 27-45, 112-123, 125, 127, 128-147 or 148 or preferably shorter peptides (e.g., 2-15 amino acids, preferably at least 2, at least 3, at least 4, at least 5, at least 6, at least 8, at least-10, say 12 amino acids, preferably no more than 15 amino acids) including any of these sequences.
  • the peptides of the present invention preferably include at least one polar and uncharged amino acid including but not limited to serine, threonine, asparagine, glutamine or natural or synthetic derivatives thereof (see Table 2).
  • amino acid residue Y is the polar and uncharged amino acid.
  • the peptide includes at least 3 amino acids, the X-Y/Y-X amino acid sequence described hereinabove and an additional polar and uncharged amino acid positioned either upstream (N-Terminal end) or downstream (C-Terminal end) of the X-Y/Y-X sequence.
  • the peptides of the present invention can be at least 3 amino acid in length and may include at least one pair of positively charged (e.g., lysine and arginine) and negatively charged (e.g., aspartic acid and glutamic acid) amino acids (e.g., SEQ ID NOs. 27-29).
  • Such amino acid composition may be preferable, since as shown in Examples 21 of the Examples section, it is likely that electrostatic interactions between opposing charges may direct the formation of ordered antiparallel structure.
  • the peptide of the present invention can be 4 amino acids in length and include two serine residues at the C-terminal end of the X-Y/Y-X sequence.
  • the peptide of the present invention can be at least 3 amino acids in length and include a thiolated amino acid residue (i.e., including a sulfur ion), preferably at an N-terminal end thereof (e.g., SEQ ID NOs: 149 and 150, D-Cys-D-Trp-Aib and L-Cys-D-Trp-Aib, respectively as well as their acytelated and amidated forms).
  • a thiolated amino acids include, but are not limited to, the naturally occurring amino acids cysteine and methionine and synthetic amino acids such as Tyr (SO 3 H).
  • the teachings of the present invention also enable design of peptides which would not aggregate into fibrils and be capable of either preventing or reducing fibril formation or disrupting preformed fibrils and thus can be used as a therapeutic agents.
  • a peptide encompassed by SEQ ID NO: 9, 10, 11, 17, 19, 25 or 30 can be utilized for therapy since as is shown in the Examples section which follows, such a peptide displays no aggregation (SEQ ID NO: 9) or slow aggregation kinetics as compared to the wild type peptide (SEQ ID NOs: 9 and 10). It is conceivable that since amyloid formation is a very slow process, these peptide sequences will completely inhibit or significantly delay amyloidosis under physiological conditions.
  • peptide encompasses native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and peptidomimetics (typically, synthetically synthesized peptides), as well as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells.
  • Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, C.A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if fully set forth herein. Further details in this respect are provided hereinunder.
  • Natural aromatic amino acids, Trp, Tyr and Phe may be substituted for synthetic non-natural acid such as Phenylglycine, Tic, naphtylalanine (Nal), phenylisoserine, threoninol, ring-methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr.
  • synthetic non-natural acid such as Phenylglycine, Tic, naphtylalanine (Nal), phenylisoserine, threoninol, ring-methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr.
  • the peptides of the present invention may also include one or more modified amino acids or one or more non-amino acid monomers (e.g. fatty acids, complex carbohydrates etc).
  • amino acid or “amino acids” is understood to include the 20 naturally occurring amino acids; those amino acids often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine.
  • amino acid includes both D- and L-amino acids.
  • Tables 1 and 2 below list naturally occurring amino acids (Table 1) and non-conventional or modified amino acids (e.g., synthetic, Table 2) which can be used with the present invention.
  • Table 1 Three-Letter Amino Acid Abbreviation One-letter Symbol alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamine Gln Q Glutamic Acid Glu E glycine Gly G Histidine His H isoleucine Iie I leucine Leu L Lysine Lys K Methionine Met M phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T tryptophan Trp W tyrosine Tyr Y Valine Val V Any amino acid as above Xaa X
  • the present peptides are preferably utilized in therapeutics or diagnostics which require the peptides to be in soluble form
  • the peptides of the present invention preferably include one or more non-natural or natural polar amino acids, including but not limited to serine and threonine which are capable of increasing peptide solubility due to their hydroxyl-containing side chain.
  • the peptides of the present invention preferably include at least one ⁇ -sheet breaker amino acid residue, which is positioned in the peptide sequence as described below.
  • Peptides which include such ⁇ -sheet breaker amino acids retain recognition of amyloid polypeptides but prevent aggregation thereof (see Examples 40-45 of the Examples section which follows).
  • the ⁇ -sheet breaker amino acid is a naturally occurring amino acid such as proline (e.g., SEQ ID NOs.
  • ⁇ -sheet breaker amino acid residues include, but are not limited to aspartic acid, glutamic acid, glycine, lysine and serine (according to Chou and Fasman (1978) Annu. Rev. Biochem. 47, 258).
  • the ⁇ -sheet breaker amino acid residue is a synthetic amino acid such as a C ⁇ -amethylated amino acid, which conformational constrains are restricted [Balaram, (1999) J. Pept. Res. 54, 195-199].
  • C ⁇ -methylated amino acids have a hydrogen atom attached to the C ⁇ , which affects widely their sterical properties regarding the ⁇ and ⁇ angels of the amide bond.
  • ⁇ -aminoisobutyric acid (Aib, see Table 2, above) has limited ⁇ and ⁇ conformations.
  • peptides of the present invention which are substituted with at least one Aib residue are capable of binding amyloid polypeptides but prevent aggregation thereof (see Examples 40-44).
  • Such peptides are set forth in SEQ ID NOs: 113, 114, 117, 118, 121, 135, 136, 137,143, 145, 149, 129 and 131.
  • the ⁇ -sheet breaker amino acid of this aspect of the present invention can be located at position Y of the X-Y/Y-X amino acid sequence of the peptide (see for Example SEQ ID NOs: 123, 143, 144, 145, 146, 147, 148).
  • the peptides of this aspect of the present invention can be at least 3 amino acids and include the breaker amino acid in any position other than the X-Y/Y-X amino acid sequence (see for example SEQ ID NO: 117).
  • the ⁇ -sheet breaker amino acid may be positioned upstream of the aromatic residue (see SEQ ID NO: 122) or downstream thereto (see SEQ ID NO: 123).
  • the peptide is three amino acids in length, wherein Y is an aromatic amino acid and an amino acid residure attached to the amino acid sequence X-Y or Y-X is a ⁇ -sheet breaker amino acid, which is preferably attached at the C-terminus of the peptide (e.g., SEQ ID NOs: 135 and 140).
  • the peptide is two amino acids in length and Y is a ⁇ -sheet breaker amino acid (e.g., SEQ ID NOs: 121, 143-148).
  • C* is a chiral carbon having a D configuration (also referred to in the art as R-configuration).
  • R 1 and R 2 are each independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, carboxy, thiocarboxy, C-carboxylate and C-thiocarboxylate;
  • R 3 is selected from the group consisting of hydroxy, alkoxy, aryloxy, thiobydroxy, thioalkoxy, thioaryloxy, halo and amine;
  • R 4 is alkyl
  • alkyl refers to a saturated aliphatic hydrocarbon including straight chain and branched chain groups.
  • the alkyl group has 1 to 20 carbon atoms. Whenever a numerical range; e.g., “1-20”, is stated herein, it implies that the group, in this case the alkyl group, may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms. More preferably, the alkyl is a medium size alkyl having 1 to 10 carbon atoms. Most preferably, unless otherwise indicated, the alkyl is a lower alkyl having 1 to 4 carbon atoms.
  • the alkyl group may be substituted or unsubstituted. When substituted, the substituent group can be, for example, halo, hydroxy, cyano, nitro and amino.
  • a “cycloalkyl” group refers to an all-carbon monocyclic or fused ring (i.e., rings which share an adjacent pair of carbon atoms) group wherein one of more of the rings does not have a completely conjugated pi-electron system.
  • examples, without limitation, of cycloalkyl groups are cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexadiene, cycloheptane, cycloheptatriene, and adamantane.
  • a cycloalkyl group may be substituted or unsubstituted. When substituted, the substituent group can be, for example, alkyl, halo, hydroxy, cyano, nitro and amino.
  • aryl group refers to an all-carbon monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups having a completely conjugated pi-electron system. Examples, without limitation, of aryl groups are phenyl, naphthalenyl and anthracenyl.
  • the aryl group may be substituted or unsubstituted. When substituted, the substituent group can be, for example, alkyl, cycloalkyl, halo, hydroxy, alkoxy, thiohydroxy, thioalkoxy, cyano, nitro and amino.
  • a “hydroxy” group refers to an—OH group.
  • alkoxy refers to both an—O-alkyl and an—O-cycloalkyl group, as defined herein.
  • aryloxy refers to an —O-aryl group, as defined herein.
  • a “thiohydroxy” group refers to a —SH group.
  • a “thioalkoxy” group refers to both an —S-alkyl group, and an —S-cycloalkyl group, as defined herein.
  • a “thioaryloxy” group refers to an —S-aryl group, as defined herein.
  • a “carboxy” group refers to a —C( ⁇ O)—R′ group, where R′ is hydrogen, halo, alkyl, cycloalkyl or aryl, as defined herein.
  • a “thiocarboxy” group refers to a —C( ⁇ S)—R′ group, where R′ is as defined herein for R′.
  • C-carboxylate refers to a —C( ⁇ O)—O—R′ groups, where R′ is as defined herein.
  • C-thiocarboxylate refers to a —C( ⁇ S)—O—R′ groups, where R′ is as defined herein.
  • halo refers to fluorine, chlorine, bromie or iodine.
  • amine refers to an —NR′R′′ group where R′ is as defined herein and R′′ is as defined for R′.
  • a “nitro” group refers to an —NO 2 group.
  • a “cyano” group refers to a —C ⁇ N group.
  • R 4 is methyl, such that the compound above is D-tryptophane-alpha-aminobutyric acid (also referred to herein as D-Trp-aib or D-tryptophane-alpha-methyl-alanine), or a derivative thereof.
  • D-tryptophane-alpha-aminobutyric acid also referred to herein as D-Trp-aib or D-tryptophane-alpha-methyl-alanine
  • unmodified di-peptides, peptides of L-configuration, peptides which are of a reversed configuration (i.e., C-to-N sequence of tryptophane (D/L) and alpha-methyl alanine), or alternatively, macromolecules (e.g., peptides, immobilized peptides) which encompass the above-described peptide sequence, are known (see e.g., WO 02/094857, WO 02/094857, EP Pat. No. 966,975, U.S. Pat. Nos. 6,255,286, 6,251,625, 6,162,828 and 5,304,470).
  • such molecules are chemically and biologically different than the above described peptide, which unique activity is strictly dependent on its structure.
  • the peptides of the present invention are preferably utilized in a linear form, although it will be appreciated that in cases where cyclization does not severely interfere with peptide characteristics, cyclic forms of the peptide can also be utilized.
  • Cyclic peptides can either be synthesized in a cyclic form or configured so as to assume a cyclic form under desired conditions (e.g., physiological conditions).
  • a peptide according to the teachings of the present invention can include at least two cysteine residues flanking the core peptide sequence.
  • cyclization can be generated via formation of S—S bonds between the two Cys residues.
  • cyclization can be obtained, for example, through amide bond formation, e.g., by incorporating Glu, Asp, Lys, Orn, di-amino butyric (Dab) acid, di-aminopropionic (Dap) acid at various positions in the chain (—CO—NH or —NH—CO bonds).
  • the present invention provides conclusive data as to the identity of the structural determinant of amyloid peptides, which directs fibril assembly.
  • the present invention enables design of a range of peptide sequences, which can be utilized for prevention/treatment or diagnosis of amyloidosis.
  • the present inventor identified the consensus aromatic sequence of the present invention (SEQ ID NO: 7) in numerous amyloid related proteins , thereby conclusively showing that the present invention enables accurate identification of amyloidogenic fragments in essentially all amyloidogenic proteins.
  • peptides of the present invention allows for the generation of antibodies directed thereagainst, which may be used to dissociate or prevent the formation of amyloid plaques (U.S. Pat. No. 5,688,561).
  • antibody refers to intact antibody molecules as well as functional fragments thereof, such as Fab, F(ab′) 2 , and Fv that are capable of binding to macrophages.
  • functional antibody fragments are defined as follows: (i) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (ii) Fab′, the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab′ fragments are obtained per antibody molecule; (iii) (Fab′) 2 , the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab′) 2 is a dimer of two Fab′ fragments held together by two disulfide bonds; (iv) Fv, defined as a genetically engineered fragment
  • Antibodies may be generated via any one of several methods known in the art, which methods can employ induction of in vivo production of antibody molecules, screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed [Orlandi D.R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837, Winter G. et al. (1991) Nature 349:293-299] or generation of monoclonal antibody molecules by continuous cell lines in culture.
  • Antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment.
  • E. coli or mammalian cells e.g. Chinese hamster ovary cell culture or other protein expression systems
  • Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
  • antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab′) 2 .
  • This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab′ monovalent fragments.
  • an enzymatic cleavage using pepsin produces two monovalent Fab′ fragments and an Fc fragment directly.
  • Fv fragments comprise an association of V H and V L chains. This association may be noncovalent, as described in Inbar et al., Proc. Nat'l Acad. Sci. USA 69:2659-62, 1972.
  • the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde.
  • the Fv fragments comprise V H and V L chains connected by a peptide linker.
  • sFv single-chain antigen binding proteins
  • the structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli .
  • the recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V Wdomains.
  • Methods for producing sFvs are described, for example, by Whitlow and Filpula, Methods, 2: 97-105, 1991; Bird et al., Science 242:423-426, 1988; Pack et al., Bio/Technology 11:1271-77, 1993; and Ladner et al., U.S. Pat. No. 4,946,778, which is hereby incorporated by reference in its entirety.
  • CDR peptides (“minimal recognition units”) can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry, Methods, 2: 106-10, 1991.
  • the antibodies of the present invention are preferably humanized.
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′).sub.2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will include at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].
  • Fc immunoglobulin constant region
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)].
  • the techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boemer et al., J. immunol., 147(1):86-95 (1991)].
  • human can be made by introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos.
  • one specific use for the peptides of the present invention is prevention or treatment of diseases associated with amyloid plaque formation.
  • a method of treating an amyloid-associated disease in an individual Preferred individual subjects according to the present invention are mammals such as canines, felines, ovines, porcines, equines, bovines, humans and the like.
  • the term “treating” refers to reducing or preventing amyloid plaque formation, or substantially decreasing plaque occurrence in the affected tissue.
  • amyloid plaque refers to fibrillar amyloid as well as aggregated but not fibrillar amyloid, hereinafter “protofibrillar amyloid”, which may be pathogenic as well.
  • protofibrillar amyloid an aggregated but not necessarily fibrillar form of IAPP was found to be toxic in culture.
  • protofibrillar IAPP like protofibrillar ⁇ -synucelin, which is implicated in Parkinson's disease pathogenesis, permeabilized synthetic vesicles by a pore-like mechanism.
  • IAPP amyloid pore was temporally correlated to the formation of early IAPP oligomers and disappearance thereof to the appearance of amyloid fibrils.
  • Amyloid-associated diseases treated according to the present invention include, but are not limited to, type II diabetes mellitus, Alzheimer's disease (AD), early onset Alzheimer's disease, late onset Alzheimer's disease, presymptomatic Alzheimer's disease, Perkinson's disease, SAA amyloidosis, hereditary Icelandic syndrome, multiple myeloma, medullary carcinoma, aortic medical amyloid, Insulin injection amyloidosis, prion-systematic arnyloidosis, choronic inflammation amyloidosis, Huntington's disease, senile systemic amyloidosis, pituitary gland amyloidosis, Hereditary renal amyloidosis, familial British dementia, Finnish hereditary amyloidosis, familial non-neuropathic amyloidosis [Gazit (2002) Curr.
  • prion diseases including scrapie of sheep and goats and bovine spongiform encephalopathy (BSE) of cattle [Wilesmith and Wells (1991) Curr Top Microbiol Immunol 172: 21-38] and human prion diseases including (i) kuru, (ii) Creutzfeldt-Jakob Disease (CJD), (iii) Gerstmann-Streussler-Sheinker Disease (GSS), and (iv) fatal familial insomnia (FFI) [Gajdusek (1977) Science 197: 943-960; Medori, Tritschler et al. (1992) N Engl J Med 326: 444-449].
  • CJD Creutzfeldt-Jakob Disease
  • GSS Gerstmann-Streussler-Sheinker Disease
  • FFI fatal familial insomnia
  • the method includes providing to the individual a therapeutically effective amount of the peptide of the present invention.
  • the peptide can be provided using any one of a variety of delivery methods. Delivery methods and suitable formulations are described hereinbelow with respect to pharmaceutical compositions.
  • the peptide of the present invention when utilized for treatment of amyloid diseases, includes an amino acid sequence suitable for preventing fibril formation, reducing fibril formation, or disaggregating formed aggregates by competitive destabilization of the preformed aggregate.
  • SEQ ID NOs: 45, 112-123, 125, 127, 128-149 and 150 can be utilized for treatment of amyloid diseases, particularly type II diabetes mellitus since as shown in Example 35 and in Example 45 of the Examples section which follows, such sequences interfere with IAPP self-assembly as demonstrated by the decreased ability of the amyloidogenic peptide to bind thioflavin T in the presence of inhibitory peptides.
  • the peptides set forth in SEQ ID NOs: 10 or 11 can be used as potent inhibitors of type II diabetes since as shown in the Examples section which follows, substitution of either leucine or isoleucine in the peptide elicits very slow kinetics of aggregation. Since amyloid formation in vivo is a very slow process, it is conceivable that under physiological conditions no fibrilization will occur upon the substitution of isoleucine or leucine to alanine in the context of the full length IAPP.
  • self-aggregating peptides such as those set forth in SEQ ID NOs. 17, 19 and 28-30, can be used as potent inhibitors of amyloid fibrilization, since such peptides can form heteromolecular complexes which are not as ordered as the homomolecular assemblies formed by amyloid fragments.
  • the peptides of the present invention are preferably synthesized from D-isomers of natural amino acids [i.e., inverso peptide analogues, Tjernberg (1997) J. Biol. Chem. 272:12601-5, Gazit (2002) Curr. Med. Chem. 9:1667-1675].
  • the peptides of the present invention include retro, inverso and retro-inverso analogues thereof. It will be appreciated that complete or extended partial retro-inverso analogues of hormones have generally been found to retain or enhance biological activity. Retro-inversion has also found application in the area of rational design of enzyme inhibitors (see U.S. Pat. No. 6,261,569).
  • a “retro peptide” refers to peptides which are made up of L-amino acid residues which are assembled in opposite direction to the native peptide sequence.
  • Retro-inverso modification of naturally occurring polypeptides involves the synthetic assembly of amino acids with ⁇ -carbon stereochemistry opposite to that of the corresponding L-amino acids, i.e., D- or D-allo-amino acids in inverse order to the native peptide sequence.
  • a rerto inverso analogue thus, has reversed termini and reversed direction of peptide bonds, while essentially maintaining the topology of the side chains as in the native peptide sequence.
  • inhibitory peptides preferably include N-methylated amino acids which constrain peptide-backbone due to steric effects [Kapurniotu (2002) 315:339-350].
  • aminoisobutyric acid (Aib or methyl alanine) is known to stabilize an ⁇ helical structure in short natural peptides.
  • the N-methylation also affects the intermolecular NH to CO H-bonding ability, thus suppressing the formation of multiplayer ⁇ -strands, which are stabilized by H-bonding interactions.
  • the antibodies of the present invention may also be used to treat amyloid-associated diseases.
  • the peptides and/or antibodies of the present invention can be provided to an individual per se, or as part of a pharmaceutical composition where it is mixed with a pharmaceutically acceptable carrier.
  • a “pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
  • active ingredient refers to the peptide or antibody preparation, which is accountable for the biological effect.
  • physiologically acceptable carrier and “pharmaceutically acceptable carrier” which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
  • An adjuvant is included under these phrases.
  • One of the ingredients included in the pharmaceutically acceptable carrier can be for example polyethylene glycol (PEG), a biocompatible polymer with a wide range of solubility in both organic and aqueous media (Mutter et al. (1979).
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
  • excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
  • Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, inrtaperitoneal, intranasal, or intraocular injections.
  • compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the active ingredients of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
  • Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • compositions described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.
  • the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
  • a suitable vehicle e.g., sterile, pyrogen-free water based solution
  • the preparation of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
  • compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated.
  • the therapeutically effective amount or dose can be estimated initially from in vitro assays.
  • a dose can be formulated in animal models and such information can be used to more accurately determine useful doses in humans.
  • Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals.
  • the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage may vary depending upon the dosage form employed and the route of administration utilized.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. [See e.g., Fingl, et al., (1975) “The Pharmacological Basis of Therapeutics”, Ch. 1 p.1].
  • dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
  • compositions to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
  • compositions including the preparation of the present invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
  • compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may, for example, comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration.
  • Such notice for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
  • the peptides or antibodies of the present invention can also be expressed from a nucleic acid construct administered to the individual employing any suitable mode of administration, described hereinabove (i.e., in-vivo gene therapy).
  • the nucleic acid construct is introduced into a suitable cell via an appropriate gene delivery vehicle/method (transfection, transduction, homologous recombination, etc.) and an expression system as needed and then the modified cells are expanded in culture and returned to the individual (i.e., ex-vivo gene therapy).
  • the nucleic acid construct of the present invention further includes at least one cis acting regulatory element.
  • cis acting regulatory element refers to a polynucleotide sequence, preferably a promoter, which binds a trans acting regulator and regulates the transcription of a coding sequence located downstream thereto.
  • the promoter utilized by the nucleic acid construct of the present invention is active in the specific cell population transformed.
  • cell type-specific and/or tissue-specific promoters include promoters such as albumin that is liver specific [Pinkert et al., (1987) Genes Dev. 1:268-277], lymphoid specific promoters [Calame et al., (1988) Adv. Immunol. 43:235-275]; in particular promoters of T-cell receptors [Winoto et al., (1989) EMBO J. 8:729-733] and immunoglobulins; [Banerji et al.
  • the nucleic acid construct of the present invention can further include an enhancer, which can be adjacent or distant to the promoter sequence and can function in up regulating the transcription therefrom.
  • the constructs of the present methodology preferably further include an appropriate selectable marker and/or an origin of replication.
  • the construct utilized is a shuttle vector, which can propagate both in E. coli (wherein the construct comprises an appropriate selectable marker and origin of replication) and be compatible for propagation in cells, or integration in a gene and a tissue of choice.
  • the construct according to the present invention can be, for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus or an artificial chromosome.
  • nucleic acid transfer techniques include transfection with viral or non-viral constructs, such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems.
  • viral or non-viral constructs such as adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated virus (AAV) and lipid-based systems.
  • Useful lipids for lipid-mediated transfer of the gene are, for example, DOTMA, DOPE, and DC-Chol [Tonkinson et al., Cancer Investigation, 14(1): 54-65 (1996)].
  • the most preferred constructs for use in gene therapy are viruses, most preferably adenoviruses, AAV, lentiviruses, or retroviruses.
  • a viral construct such as a retroviral construct includes at least one transcriptional promoter/enhancer or locus-defining element(s), or other elements that control gene expression by other means such as alternate splicing, nuclear RNA export, or post-translational modification of messenger.
  • Such vector constructs also include a packaging signal, long terminal repeats (LTRs) or portions thereof, and positive and negative strand primer binding sites appropriate to the virus used, unless it is already present in the viral construct.
  • LTRs long terminal repeats
  • such a construct typically includes a signal sequence for secretion of the peptide or antibody from a host cell in which it is placed.
  • the signal sequence for this purpose is a mammalian signal sequence.
  • the construct may also include a signal that directs polyadenylation, as well as one or more restriction sites and a translation termination sequence.
  • a signal that directs polyadenylation will typically include a 5′ LTR, a tRNA binding site, a packaging signal, an origin of second-strand DNA synthesis, and a 3′ LTR or a portion thereof.
  • Other vectors can be used that are non-viral, such as cationic lipids, polylysine, and dendrimers.
  • peptides of the present invention can also be used as potent detectors of amyloid fibrils/plaques in biological samples. This is of a special significance to amyloid-associated diseases such as Alzheimer's disease wherein unequivocal diagnosis can only be made after postmortem examination of brain tissues for the hallmark neurofibrillary tangles (NFT) and neuritic plaques.
  • NFT neurofibrillary tangles
  • the method is effected by incubating the biological sample with a peptide of the present invention capable of co-aggregating with the amyloid fibril and detecting the peptide, to thereby detect the presence or the absence of amyloid fibril in the biological sample.
  • a peptide of the present invention capable of co-aggregating with the amyloid fibril and detecting the peptide, to thereby detect the presence or the absence of amyloid fibril in the biological sample.
  • the biological sample utilized for detection can be any body sample such as blood (serum or plasma), sputum, ascites fluids, pleural effusions, urine, biopsy specimens, isolated cells and/or cell membrane preparation.
  • body sample such as blood (serum or plasma), sputum, ascites fluids, pleural effusions, urine, biopsy specimens, isolated cells and/or cell membrane preparation.
  • the peptide of the present invention is contacted with the biological sample under conditions suitable for aggregate formation (i.e., buffer, temperature, incubation time etc.); suitable conditions are described in Example 2 of the Examples section. Measures are taken not to allow pre-aggregation of peptides prior to incubation with the biological sample. To this end freshly prepared peptide stocks are preferably used.
  • Protein complexes within a biological sample can be detected via any one of several methods known in the art, which methods can employ biochemical and/or optical detection schemes.
  • the peptides of the present invention are highlighted preferably by a tag or an antibody. It will be appreciated that highlighting can be effected prior to, concomitant with or following aggregate formation, depending on the highlighting method.
  • the term “tag” refers to a molecule, which exhibits a quantifiable activity or characteristic.
  • a tag can be a fluorescent molecule including chemical fluorescers such as fluorescein or polypeptide fluorescers such as the green fluorescent protein (GFP) or related proteins (www.clontech.com). In such case, the tag can be quantified via its fluorescence, which is generated upon the application of a suitable excitatory light.
  • a tag can be an epitope tag, a fairly unique polypeptide sequence to which a specific antibody can bind without substantially cross reacting with other cellular epitopes.
  • epitope tags include a Myc tag, a Flag tag, a His tag, a leucine tag, an IgG tag, a streptavidin tag and the like.
  • aggregate detection can be effected by the antibodies of the present invention.
  • this aspect of the present invention provides a method of assaying or screening biological samples, such as body tissue or fluid suspected of including an amyloid fibril.
  • the present invention may be used for high throughput screening of test compounds.
  • the co-aggregating peptides of the present invention are radiolabeled, to reduce assay volume.
  • a competition assay is then effected by monitoring displacement of the label by a test compound [Han (1996) J. Am. Chem. Soc. 118:4506-7 and Esler (1996) Chem. 271:8545-8].
  • the peptides of the present invention may also be used as potent detectors of amyloid deposits in-vivo.
  • a designed peptide capable of binding amyloid deposits, labeled non-radioactively or with a radio-isotope, as is well known in the art can be administered to an individual to diagnose the onset or presence of amyloid-related disease, discussed hereinabove.
  • the binding of such a labeled peptide after administration to amyloid or amyloid-ike deposits can be detected by in vivo imaging techniques known in the art.
  • peptides of the present invention can be included in a diagnostic or therapeutic kit.
  • peptide sets of specific disease related proteins or antibodies directed thereagainst can be packaged in a one or more containers with appropriate buffers and preservatives and used for diagnosis or for directing therapeutic treatment.
  • the peptides can be each mixed in a single container or placed in individual containers.
  • the containers include a label.
  • Suitable containers include, for example, bottles, vials, syringes, and test tubes.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • additives such as stabilizers, buffers, blockers and the like may also be added.
  • kits can also be attached to a solid support, such as beads, array substrate (e.g., chips) and the like and used for diagnostic purposes.
  • a solid support such as beads, array substrate (e.g., chips) and the like and used for diagnostic purposes.
  • Peptides included in kits or immobilized to substrates may be conjugated to a detectable label such as described hereinabove.
  • the kit can also include instructions for determining if the tested subject is suffering from, or is at risk of developing, a condition, disorder, or disease associated with amyloid polypeptide of interest.
  • Pancreatic amyloid is found in more than 95% of type II diabetes patients.
  • Pancreatic amyloid is formed by the aggregation of a 37 amino acid long islet amyloid polypeptide (IAPP, GenBank Accession No. gi:4557655), the cytotoxicity thereof being directly associated with the development of the disease.
  • IAPP amyloid formation follows a nucleation-dependent polymerization process, which proceeds through conformational transition of soluble IAPP into aggregated ⁇ -sheets.
  • NFGAIL hexapeptide (NFGAIL, SEQ ID NO: 1) of IAPP, also termed as the “basic amyloidogenic unit” is sufficient for the formation of ⁇ -sheet-containing amyloid fibrils [Konstantinos et al. (2000) J. Mol. Biol. 295:1055-1071].
  • FIG. 3 a shows a schematic representation of the chemical structure of the wild-type peptide while FIG. 3 c indicates the amino-acid substitutions in the different mutant peptides that were generated.
  • Kinetic aggregation assay Frsh peptide stock solutions were prepared by dissolving lyophilized form of the peptides in DMSO, a disaggregating solvent, at a concentration of 100 mM. To avoid any pre-aggregation, fresh stock solutions were prepared prior to each and every experiment. Peptide stock solutions were diluted into assay buffer and plated in 96-well plates as follows: 2 ⁇ l of peptides stock solutions were added to 98 ⁇ l of 10 mM Tris pH 7.2, resulting in a 2 mM final concentration of the peptide in the presence of 2% DMSO. Turbidity data was measured at 405 nm. A buffer solution including 2% DMSO was used as a blank. Turbidity was measured at room temperature over several time points.
  • wild-type peptide fragment (SEQ ID NO: 1) showed an aggregation kinetic profile that was very similar to those previously reported for non-seeded hIAPP hexapeptide [Tenidis et al. (2000) J. Mol. Biol 295:1055-71]. Such a profile is strongly indicative of a nucleation-dependent polymerization mechanism [Jarrett and Lansbury (1992) Biochemistry 31:6865-70]. Following a lag-time of 20 minutes, wild type peptide self-assembled into insoluble fibrils. Peptide G3A (SEQ ID NO: 4) showed essentially the same profile as that of wild type peptide.
  • N1A peptide (SEQ ID NO: 2) mediated higher kinetics of aggregation, albeit with different kinetic profile as compared to that of wild-type peptide. Interestingly, the aggregation of N1A seemed to be less nucleation-dependent. Substitution of the isoleucine or leucine to alanine (peptides I5A, SEQ ID NO: 5 and L6A, SEQ ID NO: 6 respectively) reduced the kinetics of aggregation but did not abolish it completely. Substitution of the phenylalanine residue to alanine (peptide F2A, SEQ ID NO:3) led to a total loss of peptide ability to aggregate. The F2A peptide was completely soluble in the aqueous assay buffer.
  • the average size of the aggregates, formed by the various peptides was determined using dynamic light scattering (DLS) experiments.
  • Results The average apparent hydrodynamic diameters of the structures that were formed by the various peptides are presented in FIG. 5 .
  • the apparent hydrodynamic diameter of the structures formed by the various peptides seemed to be consistent with the results obtained by the turbidity assay.
  • the wild-type peptide and G3A peptide formed particles of very similar hydrodynamic diameters. Smaller structures were observed with the derivative peptides: N1A, I5A and L6A.
  • the DLS experiments clearly illustrate that no large particles were formed by the F2A peptide under the indicated experimental conditions.
  • Congo red (CR) staining combined with polarization microscopy was utilized to test amyloidogenicity of the peptides of the present invention.
  • the fibrillogenic potential of the various peptides was assessed by electron microscopy analysis.
  • hIAPP GenBank Accession No. gi:4557655
  • hIAPP 1-10 hIAPP 1-10
  • hIAPP 2-11 . . .
  • hIAPP 28-37 a sequence of HIAPP
  • Bacterial strains E. coli strain TG-1 (Amersham Pharmacia, Sweden) was used for molecular cloning and plasmid propagation.
  • fusion sequence was then constructed using the IAPP synthetic template, which was amplified using primer YAR2 (SEQ ID NO: 60) and primer YAR1 (SEQ ID NO. 59), thereby introducing a V8 Ek cleavage site and a (His) 6 tag at the N-terminus of IAPP.
  • the two primers included a Not I and an Nco I cloning sites, respectively.
  • the resultant PCR product was digested with Nco I and Not I and ligated into the pMALc2x-NN expression vector.
  • the pMALc2x-NN expression vector was constructed by cloning the polylinker site of pMALc-NN 9 into pMALc2x (New England Biolabs, USA) [BACH (2001) J. Mol. Biol. 312:79-93].
  • Cell extracts were prepared in 20 mM Tric-HCI (pH 7.4), 1 mM EDTA, 200 mM NaCi and a protease inhibitors cocktail (Sigma) using a freeze-thaw followed by a brief sonication as previously described [Gazit (1999) J. Biol. Chem. 274:2652-2657]. Protein extracts were clarified by centrifugation at 20,000 g and stored at 4° C. MBP-IAPP fusion protein was purified by passing the extract over an amylose resin column (New England Biolabs, USA) and recovered by elution with 20 mM maltose in the same buffer. Purified MBP-IAPP was stored at 4 20 C.
  • Protein concentration was determined using the Pierce Coomassie plus reagent (Pierce, USA) with BSA as a standard. MBP and MBP-IAPP protein fractions were analyzed on SDS12% polyacrylamide gels, which were stained with GelCode Blue (Pierce, USA).
  • MBP and MBP-IAPP proteins were reacted with 5 equivalents of N-iodoacetyl-N′-(8-sulfo-1-naphthyl) ethylenediamine (IAEDANS) (Sigma, Rehovot, Israel) for overnight at room temperature in the dark. Free dye was separated from labeled protein by gel filtration chromatography on a QuickSpin G-25 Sephadex column. MBP and MBP-IAPP fluorescence was then determined. Only small fluorescence labeling was detected (on average less than 0.1 probe molecules per protein molecules) and there was no significant difference between the labeling of MBP and MBP-IAPP, which suggested that the disulfide bridge in the expressed IAPP molecules was predominantly oxidized.
  • IAEDANS N-iodoacetyl-N′-(8-sulfo-1-naphthyl) ethylenediamine
  • IPTG induction resulted in the accumulation of high levels of MBP-IAPP in the soluble fraction with less then 5% of the MBP-IAPP fusion protein was found in the insoluble fraction of the cell extract (data not shown).
  • Aliquots from typical purification steps of MBP and MBP-IAPP are shown in FIG. 9 .
  • the 48 kDa MBP-IAPP accumulated to 25% of the total soluble protein as calculated by densitometric scanning of GelCode Blue-stained SDS/Polyacrylamide gels.
  • MBP-IAPP was purified to near-homogeneity at a yield of 80 mg/l of cells.
  • an additional His-Tag was also included ( FIG. 8 b ). The His-Tag could be removed by Ek V8 cleavage at the N-terminal Lys residue of the IAPP sequence, resulting in the release of wild type IAPP.
  • IAPP Peptide Array Construction Decamers Corresponding to Consecutive overlapping sequences of hIAPP 1-37 SEQ ID NOs. 61-88) were synthesizes on a cellulose membrane matrix using the SPOT technique (Jerini AG, Berlin, Germany). The peptides were covalently bound to a Whatman 50 cellulose support (Whatman, Maidstone, England) via the C-terminal amino-acids. N-terminal acetylation was used for peptide scanning because of higher stability to peptide degradation, and better representation of the native recognition motif.
  • Peptides Synthesis Peptide synthesis was effected using solid-phase synthesis methods performed by Peptron, Inc. (Taejeon, Korea). Correct identity of the peptides was confirmed by ion spray mass-spectrometry using a HP 1100 series LC/MSD [Hewlett-Packard Company, Palo Alto, CA]. The purity of the peptides was confirmed by reverse phase high-pressure liquid chromatography (RP-HPLC) on a C 18 column, using a 30 minute linear gradient of 0 to 100% acetonitrile in water and 0.1% trifluoroacetic acid (TFA) at flow rate of 1 ml/min.
  • RP-HPLC reverse phase high-pressure liquid chromatography
  • Binding studies The cellulose peptide array was initially blocked with 5% (V/V) non fat milk in Tris buffered saline (TBS, 20 mM Tris pH 7.5, 150 mM NaCI). Thereafter, cellulose membrane was incubated in the presence of 10 ⁇ g/ml MBP-IAPP 1-37 at 4° C. for 12 h in the same blocking buffer. The cellulose membrane was then washed repeatedly with 0.05% Tween 20 in TBS. MBP-IAPP 1-37 bound to the cellulose membrane was detected with an anti MBP monoclonal antibody (Sigma, Israel). HRP-conjugated goat anti mouse antibodies (Jackson Laboratories, USA) were used as a secondary antibody.
  • Immunoblots were developed using the Renaissance western blot Chemiluminescence Reagent (NEN, USA) according to Manufacturer's instructions and signal was quantified using densitometry. Regeneration of the cellulose membrane for reuse was carried out by sequential washing with Regeneration buffer I including 62.5 mM Tris, 2% SDS, 100 Mm 2-mercaptoethanol, pH 6.7, and Regeneration buffer II including 8 M urea, 1% SDS, 0.1% 2-mercaptoethanol. Efficiency of the washing steps was monitored by contacting the membrane with the chemiluminescence reagent, as described.
  • FIGS. 10 a - b a number of peptide segments exhibited binding to MBP-IAPP;
  • An amino acid sequence localized to the center of the IAPP polypeptide i.e., hIAPP 7-16 to hIAPP 12-21 ) displayed the most prominent binding to MBP-hIAPP 1-37 .
  • Another binding region was identified at the C-terminal part of IAPP (hIAPP 19-28 to hIAPP 21-30 ), although binding in this case was considerably less prominent;
  • a third binding spot was located to the N-terminal part of IAPP (hIAPP 2-11 ), however, no typical distribution around a central motif was evident in this case, suggesting that this result may be false.
  • the peptide coupled cellulose membrane was incubated with MBP alone and analyzed by immunobloting. No binding was identified after development of the membrane (not shown).
  • Binding analysis of the recombinant MBP-hIAPP fusion protein to the hIAPP peptide array identified a putative self-assembly domain within the central part of the hIAPP protein.
  • peptide stock solutions were generated by dissolving the lyophilized form of the peptides in dimethyl sulfoxide (DMSO) at a concentration of 100 mg/ml. To avoid any pre-aggregation, fresh stock solutions were prepared for each experiment. Peptide stock solutions were diluted into the assay buffer in enzyme-linked immunosorbent assay (ELISA) plate wells as follows: 8 ⁇ L of peptide stock solutions were added to 92 ⁇ L of 10 mM Tris, pH 7.2 (hence the final concentration of the peptide was 8 mg/ml in the presence of 8% DMSO). Turbidity data were collected at 405 nm. Buffer solution containing the same amount of DMSO as the tested samples was used as blank, which was subtracted from the results. Turbidity was measured continuously at room temperature using THERMOmax ELISA plate reader (Molecular Devices, Sunnyvale Calif.).
  • Turbidity assay was performed in-order to determine the ability of the various peptides (Table 3) to aggregate in an aqueous medium. Fresh stock solutions of the different peptide fragments were made in DMSO, and then diluted into a Tris buffer 25 solution and turbidity, as a hallmark of protein aggregation, was monitored for two hours. As shown in FIG. 11 , the peptides NFLVHSS, FLVHSS and FLVHS exhibited high turbidity.
  • Congo red (CR) staining combined with polarization microscopy was utilized to test amyloidogenicity of the peptides of the present invention.
  • Amyloid fibrils bind CR and exhibit gold/green birefringence under polarized light [Puchtler (1966) J. Histochem. Cytochem. 10:355-364].
  • Congo Red Staining and Birefringence A 10 ⁇ L suspension of 8 mg/ml peptide solution in 10 mM Tris buffer, pH 7.2 aged for at least one day was allowed to dry overnight on a glass microscope slide. Staining was performed by the addition of a 10 ⁇ L suspension of saturated Congo Red (CR) and NaCl in 80% ethanol (v/v) solution as previously described [Puchtler (1966) Supra]. The solution was filtered via 0.45 ⁇ m filter. The slide was then dried for few hours. Birefringence was determined with a SZX-12 Stereoscope (Olympus, Hamburg, Germany) equipped with cross polarizers.
  • peptides NFLVHSSNN, NFLVH and FLVH exhibited very weak birefringence or no birefringence at all ( FIGS. 12 a , 12d and 12f).
  • Peptide NFLVHSSNN exhibited a weaker characteristic birefringence ( FIG. 12 a ).
  • T he peptide NFLVH exhibited a powerful smear of birefringence at the edges of the sample ( FIG. 12 d ).
  • the peptide FLVH exhibited no birefringence ( FIG. 12 f ).
  • a sample of five days aged peptide solution was examined. The same peptide was also tested in aqueous solution and at very high concentrations (10 mg/ml), however no Birefringence was detected in all cases indicating the peptide did not form amyloid (data not shown).
  • the fibrillogenic potential of the various peptides was assessed by electron microscopy analysis.
  • the fibrils formed by NFLVH peptide were thin and short and could be considered as protofilaments rather than filaments. Their appearance was at much lower frequency, and the EM picture does not represent the general fields but rather rare events ( FIG. 13 d ). As shown in FIG. 13 f , the FLVH peptide mediated the formation of amorphous aggregates.
  • FT-IR Fourier transform infrared spectroscopy
  • the NFLVHSS peptide spectrum exhibited major minimum band at 1929 cm ⁇ 1675 cm ⁇ 1 . This spectrum is classical for an anti-parallel ⁇ -sheet structure ( FIG. 14 b ).
  • a similar spectrum was observed for the peptide FLVHS with a major minimum at 1625 cm ⁇ 1 . and a minor mininum at 1676 cm ⁇ 1 ( FIG. 14 e ).
  • the spectrum of FLVHSS peptide showed also a major minimum at 1626 cm ⁇ 1 .
  • the spectrum had also some minor minima around 1637-1676 cm ⁇ 1 but those were shaped more like noise than signal ( FIG. 14 c ).
  • Medin (GenBank Accession No. gi:5174557) is the main constitute of aortic medial amyloid deposits [Häggqvist (1999) Proc. Natl. Acad. Sci. USA. 96:8674-8669].
  • Previous studies found aortic medial amyloid in 97% of the subjects above the age of 50 [Mucchiano (1992) Am. J. Pathol. 140:811-877].
  • the pathological role of those amyloid deposits is still unknown. It was suggested that these amyloid play a role in the diminished elasticity of aortic vessels that is related to old age [Mucchiano (1992) Supra; Häggqvist (1999) Supra].
  • the minimal active fragment of Medin was determined using functional and structural analyses of truncated analogues derived from the published octapeptide [Häggqvist (1999) Supra].
  • FIG. 15 a shows a schematic representation of the chemical structure of the largest peptide fragment studied.
  • Turbidity assay was effected as described in Example 8.
  • turbidity assay was performed. Freshly made stocks of the amyloidogenic octapeptide and truncated analogues thereof were prepared in DMSO. The peptides were than diluted to aqueous solution and the turbidity was monitored by following the absorbance at 405 nm as a function of time. As shown in FIG. 16 a , the NFGSV pentapeptide exhibited the highest degree of aggregation within minutes of incubation. Physical examination of the solution indicated that the peptide formed a gel structure.
  • Electron microscopy analysis was effected as described in Example 10.
  • the fibrillization potential of Medin-derived peptide fragments was effected by electron microscopy (EM) using negative staining.
  • Stock solutions of the peptide fragments were suspended and aged for 4 days.
  • Fibrillar structures were clearly seen in solutions that contained both the NFGSVQFA octapeptide ( FIG. 17 a ) and the truncated NFGSVQ ( FIG. 17 b ).
  • the structures were similar to those observed with much longer polypeptides, such the IAPP and the ⁇ -amyloid (A ⁇ ) polypeptides.
  • the shorter gel-forming NFGSV pentapeptide did not form a typical amyloid structure but a network of fibrous structures ( FIG. 17 c ).
  • a CR staining was effected to determine whether the structures formed by the various Medin-derived peptides show a typical birefringence.
  • the NFGSVQ hexapeptide bound CR and exhibited a characteristic bright and strong green-gold birefringence.
  • the NFGSVQFV octapeptide also exhibited significant birefringence ( FIG. 18 a ), although less typical than that observed with the hexapeptide.
  • the gel-forming NFGSV peptide deposits exhibited very low degree of birefringence ( FIG. 18 c ).
  • the FGSVQ and FGSV peptide showed no birefringence upon staining with CR ( FIGS. 18 d and 18f, respectively).
  • FIG. 17 b This is in complete contrast to the high abundance fibrillar structures seen with the wild-type peptide ( FIG. 17 b ). Furthermore, the structures that were visualized did not show any degree of order as observed with the NFGSV and FGSVQ peptides as described above, FIGS. 17 c - d , but were very similar to the completely non-fibrillar structures as were observed with the FGSV tetrapeptide ( FIG. 17 e ). Interestingly, some degree of birefringence could still be detected ( FIG. 19 c ) with the alanine-substituted peptide (as was observed with the GSVQ peptide, FIG. 18 e ). These results raise further doubts regarding the use of CR staining as a sole indicator of amyloid formation [Khurana (2001) J. Biol. Chem. 276:22715-22721].
  • Human Calcitonin (hCT, GenBank Accession No. gi:179880) is a 32 amino acid long polypeptide hormone that is being produced by the C-cells of the thyroid and is involve in calcium homeostasis [Austin and Health (1981) N. Engl. J. Med. 304:269-278; Copp (1970) Annu. Rev. Physiol. 32:61-86; Zaidi (2002) Bone 30:655-6631. Amyloid fibrils composed of hCT were found to be associated with medullary carcinoma of the thyroid [Kedar (1976) Isr. J. Sci. 12:1137; Berger (1988) Arch. A. Pathol. Anat. Histopathol.
  • Electron microscopy experiments have revealed that the fibrils formed by hCT are approximately 80 ⁇ in diameter and up to several micrometers in length. The fibrils are often associated with one another and in vitro amyloid formation is affected by the pH of the medium [Kamihara (2000) Supra.].
  • Calcitonin has been used as a drug for various diseases including Paget's disease and osteoporosis.
  • hCT the tendency of hCT to associate and form amyloid fibrils in aqueous solutions at physiological pH is a significant limit for its efficient use as a drug [Austin (1981) Supra; Copp (1970) Supra; Zaidi (2002) Supra].
  • Salmon CT [Zaidi (2002) Supra], the clinically used alternative to hCT, causes immunogenic reaction in treated patients due to low sequence homology. Therefore, understanding the mechanism of amyloid formation by hCT and controlling this process is highly important not only in the context of amyloid formation mechanism but also as a step toward improved therapeutic use of Calcitonin.
  • Circular dichroism (CD) studies have shown that in water monomeric hCT has little ordered secondary structure at room temperature [Arvinte (1993) Supra]. However, studies of hCT fibrils using circular dichroism, fluorescence, and infrared spectroscopy revealed that fibrillated hCT molecules have both a-helical and ⁇ -sheet secondary structure components [Bauer (1994) Supra]. NMR spectroscopy studies have shown that in various structure promoting solvents like TFE/H 2 O, hCT adopts an amphiphilic ⁇ -helical conformation, predominantly in the residue range 8-22 [Meadows (1991) Biochemistry 30:1247-1254; Motta (1991) Biochemistry 30:10444-10450]. In DMSO/H 2 O, a short double-stranded antiparallel ⁇ -sheet form in the central region made by residues 16-21 [Motta (1991) Biochemistry 30:2364-71].
  • FIG. 20 b shows a schematic representation of the chemical structure of the longest peptide and Table 5 below, indicates the various peptide fragments that were used in the study.
  • TABLE 5 Amino acid coordinates on hCT Peptide sequence SEQ ID NO: 15-19 NH 2 -DFNKF —COOH 27 16-19 NH 2 - FNKF —COOH 28 15-18 NH 2 -DFNK —COOH 29 15-17 NH 2 -DFN —COOH 30 F > A 15-19 NH 2 -DANKF —COOH 31
  • Electron microscopy analysis was effected as described in Example 10.
  • the fibrillization potential of Calcitonin-derived peptide fragments was effected by electron microscopy (EM) using negative staining.
  • Stock solutions of the peptide fragments were suspended in 0.02M NaCl, 0.01M Tris pH 7.2, aged for 2 days and negatively stained.
  • Fibrillar structures, similar to those formed by the full-length polypeptide [Arvinte (1993) Supra; Benvenga (1994) Supra; Bauer (1994) Supra; Kanaori (1995) Supra; Kamihara (2000) Supra], were clearly seen with high frequency in solutions that contained the DFNKF pentapeptide ( FIG. 21 a ).
  • the shorter DFNK tetrapeptide also formed fibrillar structures ( FIG. 21 b ).
  • FIGS. 22 a - d A CR staining was effected to determine whether the structures formed by the various hCT-derived peptides show a typical birefringence. As shown in FIGS. 22 a - d , all the studies peptides showed some degree of birefringence. However, the green birefringence, which was observed with the DFNKF-pentapeptide was clear and strong ( FIG. 22 a ). The level of birefringence that was observed with the other peptides was lower but significant since no birefringence could be detected using control solutions which did not contain the peptides. The lower level of birefringence of the DFNK tetrapeptide ( FIG.
  • Amyloid deposits are characteristic of fibrils rich with ⁇ -pleated sheet structures.
  • FT-IR spectroscopy was used. Aged peptide solutions were dried on CaF2 plates forming thin films as described in Example 11. As shown in FIG. 23 , the DFNKF pentapeptide exhibited a double minima (at 1639 cm ⁇ 1 and 1669 cm ⁇ ) an amide I FT-IR spectrum that is consistent with anti-parallel ⁇ -sheet structure and is remarkably similar to the spectrum of the amyloid-forming hexapeptide fragment of the islet amyloid polypeptide [Tenidis (2000) Supra].
  • the amide I spectrum observed with the DFNK tetrapeptide was less typical of a ⁇ -sheet structure. While it exhibited a minimum at 1666 cm-1 that may reflect an anti-parallel ⁇ -sheet it lacked the typical minimum around 1620-1640 cm ⁇ 1 that is typically observed with ⁇ -sheet structures.
  • the FNKF tetrapeptide exhibited a FT-IR spectrum that is typical of a non-ordered structure ( FIG. 23 ) and is similar to spectra of the short non-amyloidogenic fragments of the islet amyloid polypeptide [Tenidis (2000) Supra].
  • the DFN tripeptide exhibited a double minima (at 1642 cm ⁇ 1 and 1673 cm ⁇ 1 , FIG. 23 ) amide I FT-IR spectrum that is consistent with a mixture of ⁇ -sheet and random structures. This may further indicate that the structures observed by EM visualization may represent some degree of ordered structure composed of predominantly ⁇ -sheet structural elements.
  • the FT-IR spectrum of the DANKA pentapeptide was similar to that of the FNKF tetrapeptide and other short non-amyloidogenic peptide, typical of non-ordered structures [Tenidis (2000) Supra].
  • the effect of the phenylalanine to alanine substitution is very similar to the effect of such a change in the context of a short amyloid-forming fragment of the islet amyloid polypeptide [Azriel (2001) Supra].
  • the hCT fragment seems to be the pentapeptide with the highest amyloidogenic potential similar to the potent amyloidogenic fragment of the ⁇ -amyloid (A ⁇ ) polypeptide, KLVFFAE [Balbach (2000) Biochemistry 39:13748-59]. It is possible that electrostatic interactions between the opposing charges on the lysine and aspartic acids direct the formation of ordered antiparallel structure. Interestingly, the DFNK polypeptide exhibited a significantly lower amyloidogenic potential as compared to the DFNKF peptide. It is possible that a pentapeptide is a lower limit for potent amyloid former.
  • Amyloid fibril formation by lactotransferrin (GenBank Accession No. gi:24895280) is associated familial subepithelial corneal amyloid formation [Sacchettini and Kelly (2002) Nat Rev Drug Discov 1:267-75]. Based on the proposed role of aromatic residues in amyloid self-assembly, the amyloidogenic features of a Lactotransfenin-derived peptide, LFNQTG (SEQ ID NO: 32) were studied.
  • Serum amyloid A proteins (GenBank Accession No. gi:134167) were found in amyloid-state in cases of Chronic inflammation amyloidosis (Westermark et al. (1992) Biochem. Biophys. Res. Commun. 182: 27-33). Based on the proposed role of aromatic residues in amyloid self-assembly, the amyloidogenic features of a Serum amyloid A protein-derived peptide, SFFSFL (SEQ ID NO: 33) were studied.
  • the human BRI gene is located on chromosome 13.
  • the amyloid fibrils of the BriL gene product (GenBank Accession No. gi:12643343) are associated with neuronal dysfunction and dementia (Vidal et al (1999) Nature 399, 776-781).
  • FENKF (SEQ ID NO: 34) were studied.
  • Amyloid fibril formation by beta-amyloid is promoted by interaction with serum amyloid-P (GenBank Accession No. gi:2144884). Based on the proposed role of aromatic residues in amyloid self-assembly, the amyloidogenic features of a Serum amyloid P-derived peptide, LQNFTL (SEQ ID NO: 36) were studied.
  • Amyloid fibrils formation by Immunoglobulin light chain (GenBank Accession No. gi:625508) is associated with primary systemic amyloidosis [Sacchettini and Kelly (2002) Nat Rev Drug Discov 1:267-75. Based on the proposed role of aromatic residues in amyloid self-assembly, the amyloidogenic features of an Immunoglobulin light chain-derived peptide, TLIFGG (SEQ ID NO: 37) were studied.
  • Amyloid fibril formation by Cystatin C (GenBank Accession No. gi:4490944) is associated with hereditary cerebral amyloid angiopathy [Sacchettini and Kelly (2002) Nat Rev Drug Discov 1:267-75]. Based on the proposed role of aromatic residues in amyloid self-assembly, the amyloidogenic features of a Cystatin C-derived peptide, RALDFA (SEQ ID NO: 38) were studied.
  • Amyloid fibril formation by Transthyretin (GenBank Accession No. gi:72095) is associated with familial amyloid polyneuropathy (Sacchettini and Kelly (2002) Nat Rev Drug Discov 1:267-75). Based on the proposed role of aromatic residues in amyloid self-assembly, the amyloidogenic features of an Transthyretin-derived peptide, GLVFVS (SEQ ID NO: 39) were studied.
  • Amyloid fibril formation by Lysozyme (GenBank Accession No. gi:299033) is associated with familial non-neuropathic amyloidosis [Sacchettini and Kelly (2002) Nat Rev Drug Discov 1:267-75]. Based on the proposed role of aromatic residues in amyloid self-assembly, the amyloidogenic features of a Lysozyme-derived peptide, GTFQIN (SEQ ID NO: 40) were studied.
  • Amyloid fibril formation by Fibrinogen (GenBank Accession No. gi: 1 1761629) is associated with hereditary renal amyloidosis (Sacchettini and Kelly (2002) Nat Rev Drug Discov 1:267-75). Based on the proposed role of aromatic residues in amyloid self-assembly, the amyloidogenic features of a Fibrinogen-derived peptide, SGIFTN (SEQ ID NO: 41) were studied.
  • Amyloid fibril formation by Insulin (GenBank Accession No. gi:229122) is associated with injection-localized amyloidosis [Sacchettini and Kelly (2002) Nat Rev Drug Discov 1:267-75]. Based on the proposed role of aromatic residues in amyloid self-assembly, the amytoidogenic features of an insulin-derived peptide, ERGFF (SEQ ID NO: 42) were studied.
  • Amyloid fibrils formation by prolactin (GenBank Accession No. gi:4506105) is associated with pituitary-gland amyloidosis (Sacchettini and Kelly (2002) Nat Rev Drug Discov 1:267-75). Based on the proposed role of aromatic residues in amyloid self-assembly, the amyloidogenic features of a prolactin-derived peptide, RDFLDR (SEQ ID NO: 43) were studied.
  • Amyloid fibrils formation by beta-2-microtublin (GenBank Accession No. gi:70065) is associated haemodialysis-related amyloidosis (Sacchettini and Kelly (2002) Nat Rev Drug Discov 1:267-75). Based on the proposed role of aromatic residues in amyloid self-assembly, the amyloidogenic features of a beta-2-microtublin-derived peptide, SNFLN (SEQ ID NO: 44) were studied.
  • results To characterize the ability of the beta-2-microtublin-derived peptide to form fibrilar supramolecular ultrastructures, negative staining electron microscopy analysis was effected. As shown in FIG. 37 , under mild conditions, filamentous structures were observed for the selected peptide, suggesting that SNFLN of beta-2-microtublin is important for the polypeptide self-assernbly. These results further substantiate the ability of the present invention to predict amyloidogenic peptide sequences.
  • amyloidogenic peptides of IAPP identified according to the teachings of the present invention to inhibit amyloid formation by the full-length polypeptide was tested by the addition of beta-breaker proline residues to the recognition sequence as set forth in the peptide sequence NFLVHPP (SEQ ID NO: 45).
  • the degree of amyloid fibrils formation with and without the inhibitor was assessed using thioflavin T (ThT) as molecular indicator.
  • ThT thioflavin T
  • the degree of fluorescence of the ThT dye is directly correlated with the amount of amyloid fibrils in the solution [LeVine H 3rd. (1993) Protein Sci. 2:404-410.
  • IAPP solutions (4 ⁇ M hIAPPin 10 mM Tris buffer pH 7.2), were incubated in the presence or absence of 40 ⁇ M of the modified peptide (i.e., NFLVHPP) at room temperature.
  • Fibril formation was determined by a ten fold dilution of the solutions into a solution that contained 3 ⁇ M thioflavin T (ThT) in 50 mM sodium phosphate pH 6.0 and determination of fluorescence at 480 nm with excitation at 450 nm using a LS50B spectroflurimeter (Perkin Elmar, Wellesley, Mass.).
  • ThT thioflavin T
  • As a control 10 mM Tris buffer pH 7.2 were diluted into the ThT solution and fluorescence was determined as described.
  • hydrophobic amino acids are similar or even slightly more hydrophobic than phenylalanine [Wolfenden (1981) Biochemistry 20:849-855; Kyte (1982) J. Mol. Biol. 157:105-132; Radzicka (1988) ], they are not aromatic. Furthermore, valine and isoleucine, are considered to be very strong ⁇ -sheet formers [Chou (1974) Biochemistry 13:211-222; Chou (1978) Annu. Rev. Biochem. 47:251-276], which is assumed to be important to the formation of ⁇ -sheet rich amyloid fibrils.
  • turbidity assay was performed. Freshly made stock solutions of the wild-type peptide and the various peptide mutants were made in DMSO. The peptides were then diluted to a buffer solution and the turbidity was monitored by following the absorbance at 405 nm as a function of time. As shown in FIG. 39 , significant increase in turbidity was observed for the wild-type NFGAILSS octapeptide within minutes following dilution thereof into the aqueous solution.
  • the shape of the aggregation curve resembled that of a saturation curve, with a rapid increase in turbidity in the first hour, followed by a much slower increase in turbidity over the entire incubation time monitored. This probably reflects a rapid aggregation process, with the number of free building blocks as the rate limiting factor.
  • none of the analogue peptides revealed any significant aggregative behavior and the turbidity of all the hydrophobic analogues as well as the alanine-substituted analogues remained very low for at least 24 hours ( FIG. 39 ).
  • peptide analogue solutions were incubated for 1 week in the same experimental conditions and endpoint turbidity values were determined. As shown in FIG. 30 , some low degree of turbidity was observed with the NIGAILSS, and lower extent for the NLGAILSS, NAGAILSS, and NVGAILSS peptides in decreasing order of turbidity. However, even for the NIGAILSS, the degree of turbidity was significantly lower as compared to the wild-type NFGAILSS protein ( FIG. 40 ).
  • Electron microscopy analysis was effected as described in Example 10.
  • the short exposure binding was assessed using densitometry ( FIG. 42 c ). It will be appreciated though, that the measured binding should be interpreted as semiquantitative since the coupling efficiency during synthesis and therefore the amount of peptide per spot may vary. In this case, however, the marked difference in binding between the various peptide variants was very clear.
  • the minimal amyloid forming region of IAPP polypeptide (i.e., IAPP 14-20, see Table 3 above) was selected as the target sequence for designing inhibitors which are able to bind thereto, block it and prevent aggregation thereof.
  • ⁇ -sheet breakers are incorporated into the target sequence, such that the peptides cannot display a ⁇ -sheet conformation by which the monomers are stacked together to form fibrils.
  • ⁇ -aminoisobutyric acid (Aib) is an unnatural amino acid which contains two methyl residues attached to C ⁇ of the carboxylic group. Unlike natural amino acids, this molecule does not have a hydrogen atom attached to the C ⁇ . This affects widely the sterical properties of the amino acid especially with respect to the ⁇ and ⁇ angels of the amide bond.
  • FIG. 43 a shows the conformational map of Aib derived from the superposition of the Ramachandran plots of L-alanine and D-alanine. As is evident for FIG. 43 a , the allowed angels are limited to small regions and the overall structure is much more suitable for an ⁇ -helix conformation rather than a ⁇ -strand conformation.
  • Aib can be used to prevent ⁇ -sheet conformation which is central to amyloid aggregation.
  • a comparison between the Ramachandran plots of Aib and proline shows that Aib is a more potent ⁇ -sheet breaker than proline ( FIG. 43 a ).
  • ANFLVH and ANFLV, SEQ ID NOs: 124 and 126, respectively Two peptides including IAPP amyloid forming regions (i.e., ANFLVH and ANFLV, SEQ ID NOs: 124 and 126, respectively) were synthesized to include Aib substituting the alanine and the leucine residues.
  • the newly synthesized peptides including the following amino acid sequences Aib-NF-Aib-VH (SEQ ID NO: 125) and Aib-NF-Aib-V (SEQ ID NO: 127), are illustrated in FIGS. 43 b - c , respectively.
  • Peptides Synthesis Peptides were synthesized by Peptron, Inc. (Taejeon, Korea) using solid-phase techniques. The correct identity of the peptides was confirmed by ion spray mass-spectrometry using a HP 1100 series LC/MSD. The purity of the peptides was confirmed by reverse phase high-pressure liquid chromatography (RP-HPLC) on a C 18 column, using a 30 minutes linear gradient of 0 to 100% acetonitrile in water and 0.1% trifluoroacetic acid (TFA) at flow rate of 1 ml/min.
  • RP-HPLC reverse phase high-pressure liquid chromatography
  • Peptide solutions freshly prepared stock solutions were prepared by dissolving the lyophilized form of the peptides in dimethyl sulfoxide (DMSO) at a concentration of 100 mM. To avoid any pre-aggregation, fresh stock solutions were prepared for each experiment. Peptide stock solutions were diluted into microtubes as follows: 5 ⁇ L of peptides stock solutions added to 95 ⁇ L of 10 mM Tris, pH 7.2 (hence the final concentration of the peptide was 5 mM in the presence of 5% DMSO).
  • DMSO dimethyl sulfoxide
  • Aib containing peptides was examined in comparison to native IAPP peptides.
  • Aged solutions of the Aib-modified and wild-type peptides were examined under electronic microscope (EM) using negative staining.
  • EM electronic microscope
  • no fibrillar structures were evident for the Aib containing peptides, Aib-NF-Aib-VH ( FIG.
  • Congo Red Staining and Birefringence A 10 ⁇ L suspension of 5 mM peptide solution in 10 mM Tris buffer, pH 7.2 aged for 10 days was allowed to dry overnight on a glass microscope slide. Staining was performed by the addition of a 10 ⁇ L suspension of saturated Congo Red (CR) and NaCl in 80% ethanol (v/v) solution. The solution was filtered via 0.2 ⁇ m filter. The slide was then dried for a few hours. Birefringence was determined with a SZX-12 Stereoscope (Olympus, Hamburg, Germany) equipped with cross polarizers. 100 ⁇ magnification is shown.
  • FIGS. 45 a - b show a typical yellow-green birefringence for both ANFLVH and ANFLV peptides ( FIG. 45 a and b, respectively).
  • FIGS. 45 c - d show a typical yellow-green birefringence for both ANFLVH and ANFLV peptides.
  • the Aib-modified peptides ,Aib-NF-Aib-VH and Aib-NF-Aib-V exhibited no birefringence suggesting that Aib modified peptides can not form amyloid fibrils ( FIGS. 45 c - d ).
  • FT-IR Fourier transform infrared spectroscopy
  • FT-IR spectroscopy was used to elucidate the internal conformation of the observed structures (see Examples 42 and 43, above).
  • IAPP peptides displayed a sharp change in the IR spectra.
  • the ANFLVH and ANFLV peptides spectra were typical of ⁇ -sheet spectra, with minima at 1630 cm ⁇ 1 and 1632 cm ⁇ 1 respectively
  • the Aib-NF-Aib-VH and Aib-NF-Aib-V peptides displayed minima at 1670 cm ⁇ 1 and 1666 cm 31 1 , respectively, which are characteristic to a random coil conformation.
  • the degree of amyloid fibril formation with and without the Aib inhibitor was assessed using thioflavin T (ThT) as molecular indicator.
  • ThT thioflavin T
  • the degree of fluorescence of the ThT dye is directly correlated with the amount of amyloid fibrils in the solution [LeVine H 3rd. (1993) Protein Sci. 2:404-410].
  • IAPP solutions (4 ⁇ M peptide in 10 mM Tris buffer pH 7.2), were incubated with or without 40 ⁇ M of the various peptide solutions at room temperature. Fibril formation was assessed by a ten fold diluation of the solutions into a solution which contained 3 ⁇ M thioflavin T (ThT) in 50 mM sodium phosphate pH 6.0 and determination of fluorescence at 480 nm with excitation at 450 nm using a Perkin Elmar LS50B spectroflurimeter. As a control a 10 mM Tris buffer pH 7.2 was diluted into the ThT solution and fluorescence was determined as described.
  • Thioflavin T Thioflavin T
  • Di- and Tri-aromatic Peptides can Inhibit Aggregation of IAPP Polypeptide
  • FIG. 48 shows endpoint values after IAPP aggregation reaches a plateau, following 142 hours of incubation.
  • the aggregation assay was preformed in the presence of IAPP polypeptide (4 ⁇ M) and the inhibitory peptides (40 ⁇ M).
  • Peptides Synthesis Peptide synthesis (excluding EG5, EG6, and EG7, D-Phe-Pro, and D-Pro-Phe), was effected by using solid-phase synthesis (Peptron, Inc., Taejeon, Korea). Peptide identity was confirmed by ion spray mass-spectrometry. Peptide purity was confirmed by reverse phase high-pressure liquid chromatography (RP-HPLC). EG5, EG6, and EG7, D-Phe-Pro, and D-Pro-Phe were purchased from Bachem (Bubendorf, Switzerland). Islet amyloid polypeptide (IAPP) was purchase from CalBiochem (La Jolla Calif., USA).
  • hIAPP fibrillization was monitored by Thioflavin T dye binding assay.
  • hIAPPI 1-37 stock solution was diluted to a final concentration of 4 ⁇ M in 10 ⁇ M sodium acetate buffer (pH 6.5) with or without inhibitors (40 ⁇ M), and a fmal concentration of HFIP of 1% (vol). Immediately after dilution, sample was centrifuged for 20 minutes in 20,000 ⁇ g at 4° C. and the supernatant fraction was used for fluorescence measurements.
  • Tht was added to a 1 ml sample at a final concentration of 3 ⁇ M and measurements were effected by using Perkin-Elmer (excitation 450 nm, 2.5 nm slit; emission 480 nm 10 nm slit). Background was subtracted from all samples.
  • the first round of selection shown in FIG. 49 a demonstrated that peptides as short as tripeptides can efficiently inhibit the formation of amyloid by IAPP.
  • Comparison of EG01 and EG03 suggests that presence of Asn residue within the short peptide does not contribute to the inhibition of amyloid formation and further supports the use of aromatic moieties along with beta-breakers for optimal inhibition.
  • the effective inhibition of IAPP fibrillization by EG05 a known inhibitor of amyloid formation [Tjemberg et al. (1996) J. Biol. Chem. 271: 8545-854], suggests a generic inhibition of amyloid formation by aromatic residues. Indeed, the similar activity of the EG05 and the much shorter EG08 (Aib conjugated to the Phe-Phe) element clearly suggest that the generic inhibition of EG05 stems from its aromatic nature.
  • FIG. 49 c shows the results of a third round of selection.
  • the forth round also demonstrated the inhibition potential of four putuavely metabolically stable dipeptides EG28, EG29, EG30, EG31. Again, stressing the value of beta-breakers and aromatic amino acids for the inhibitory sequences.
  • Thioflavin T fluorescence assay Fibrillization of A ⁇ 1-40 was monitored by Thioflavin T dye binding assay.
  • a ⁇ 1-40 stock solution was diluted to a final concentration of 5 ⁇ M in 100 mM NaCl, 10 mM sodium phosphate buffer (pH 7.4) with or without inhibitors.
  • Tht was added to 0.1 ml sample to a final concentration of 0.3 ⁇ M ThT and 0.4 ⁇ M polypeptide. Measurements were effected using Jobin Yvon FluroMax-3 (excitation 450 nm, 2.5 mn slit; emission 480 nm 5 nm slit, integration time 1 second). Background was subtracted from all samples.
  • a ⁇ Alzheimer's ⁇ -amyloid 1-40
  • aromatic inhibitors may serve as generic amyloid inhibitors and a common mechanism of assembly [Gazit (2002) FASEB J. 16, 77-83].
  • the D-Trp-Aib compound offers a unique combination of pharmaceutical properties in an extremely small molecule:
  • Hetero-aromatic interactions The interaction between the tryptophan indole and the aromatic recognition interfaces of the growing amyloid fibrils (Gazit, 2002) allows specific and oriented binding that directly and precisely blocks further homo-molecular self-assembly of the growing chain.
  • Aib conformational restriction The conjugation of the ⁇ -aminoisobutyric acid (Aib), an amino acid with exceptional geometrical constrain, induce a very strong ⁇ -sheet breakage effect, a key measure to halt the growing of amyloid fibrils.
  • This ⁇ -breakage strategy shows clear a advantage as compared to prior art (proline introduction) in terms of size and complexity of the molecule.
  • a stable D-isomer conformation The inhibitor is built of a D-amino acid and a non-chiral Aib moiety. This results in the formation of a non-cleavable peptide bond and thus with a presumably high physiological stability.
  • Peptide bond stacking In spite of its small size, a typical peptide bond (although isomerically stable one) is retained within the molecule. The unique planar characteristic of such a peptide bond allows a specific and geometrically-constrained stacking of the molecule on the growing amyloid chain, due to their partially planar resonating structures with the exact gemetry that is consistant with ⁇ -strand interaction.
  • Electrostatic repulsion The existence of charged terimi results in electrostatic repulsion of further binding monomers. Such repulsion is achieved by introduction of a charge aspartic acid in other peptide inhibitors [Soto et al. (2003) J. Biol. Chem 278:13905] as there is a need to block the termini of these peptides to decrease proteolyic degradation.
  • the non-native stable isomeric configuration of the D-Trp-Aib allows the retention of charged termini within the minimal framework resulting in a significantly small molecule.
  • the indole group is also known to act as antioxidant by the scavenging of free-radicals. Indeed some of the drug candidates for treating AD are based on this property (e.g. indole-3-propionic acid of MindSet). However, while such molecules are effective in the protection of neural cells from AD related oxidation stress, they lack the unique fibrillization inhibitory properties of the D-Trp-Aib molecular frame.
  • D-Trp-Aib is a remarkably small active molecule. All the unique properties that were described in the previous paragraphs (1-7) are maintained within a molecule of less than 300 Da. The small size of this non-cleavable molecule suggests an oral bioavailability, long half-life, and transfer of the BBB, while maintaining low immunogenic potential.

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US20060079454A1 (en) * 2002-12-09 2006-04-13 Ramot At Tel Aviv University Ltd. Peptide nanostructures and methods of generating and using the same
US20060079455A1 (en) * 2003-01-07 2006-04-13 Ramot At Tel Aviv University Ltd. Peptide nanostructures encapsulating a foreign material and method of manufacturing same
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US20070298043A1 (en) * 2003-10-02 2007-12-27 Ehud Gazit Novel Antibacterial Agents and Methods of Identifying and Utilizing Same
US20080009434A1 (en) * 2004-09-08 2008-01-10 Meital Reches Peptide Nanostructures Containing End-Capping Modified Peptides And Methods Of Generating And Using The Same
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US20090013087A1 (en) * 2005-01-18 2009-01-08 Zlango Ltd. Communications Network System and Methods For Using Same
US20090023627A1 (en) * 2004-07-20 2009-01-22 Regents Of The University Of Colorado Compositions and methods for alzheimer's disease
US20090035307A1 (en) * 2006-11-30 2009-02-05 Stefan Barghorn Abeta CONFORMER SELECTIVE ANTI-Abeta GLOBULOMER MONOCLONAL ANTIBODIES
US20090061190A1 (en) * 2004-08-02 2009-03-05 Ramot At Tel Aviv University Ltd. Articles of peptide nanostructures and method of forming the same
US20090156471A1 (en) * 2004-07-15 2009-06-18 Ramot At Tel Aviv University Ltd. Use of anti-amyloid agents for treating and typing pathogen infections
US20090175785A1 (en) * 2005-10-11 2009-07-09 Ehud Gazit Self-Assembled Fmoc-Ff Hydrogels
US20090191190A1 (en) * 2005-11-30 2009-07-30 Stefan Barghorn Anti-ABeta Globulomer Antibodies, Antigen-Binding Moieties Thereof, Corresponding Hybridomas, Nucleic Acids, Vectors, Host Cells, Methods of Producing Said Antibodies, Compositions Comprising Said Antibodies, Uses Of Said Antibodies And Methods Of Using Said Antibodies
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US9176150B2 (en) 2003-01-31 2015-11-03 AbbVie Deutschland GmbH & Co. KG Amyloid beta(1-42) oligomers, derivatives thereof and antibodies thereto, methods of preparation thereof and use thereof
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US20180118682A1 (en) * 2010-07-22 2018-05-03 Ramot At Tel-Aviv University Ltd. Pharmaceutical composition containing a tryptophan derivative

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Citations (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4299917A (en) * 1979-02-14 1981-11-10 Boehringer Manneheim Gmbh Diagnostic agents for the detection of leukocytes in body fluids
US5210215A (en) * 1989-10-04 1993-05-11 Polifarma S.P.A. Tryptophane and 3-indolepyruvic acid, methods of production therefor
US5304470A (en) * 1991-01-23 1994-04-19 Forschungszentrum Juelich Gmbh Process for the enzymatic preparation of protected and unprotected di- and oligopeptides in aqueous solutions
US5332648A (en) * 1990-12-27 1994-07-26 Kabushiki Kaisha Toshiba Potosensitive composition and method of forming a pattern using the same
US5556744A (en) * 1992-05-29 1996-09-17 The Trustees Of The University Of Pennsylvania Methods and compositions for diagnosing and treating certain HIV infected patients
US5916642A (en) * 1995-11-22 1999-06-29 Northwestern University Method of encapsulating a material in a carbon nanotube
US6162828A (en) * 1995-03-31 2000-12-19 Takeda Chemical Industries, Ltd. Cysteine protease inhibitor
US6261569B1 (en) * 1992-08-27 2001-07-17 Deakin Research Limited Retro-, inverso- and retro-inverso synthetic peptide analogues
US20020006954A1 (en) * 2000-03-02 2002-01-17 Oklahoma Medical Research Foundation Desmethyl tocopherols for preventing or slowing degenerative neurological diseases
US6361861B2 (en) * 1999-06-14 2002-03-26 Battelle Memorial Institute Carbon nanotubes on a substrate
US20020130484A1 (en) * 2001-03-13 2002-09-19 Liang Fa-Kuang Standing-pedaling bike
US6472436B1 (en) * 2000-07-17 2002-10-29 The Salk Institute For Biological Studies Methods for protecting cells from amyloid toxicity and for inhibiting amyloid protein production
US20030158237A1 (en) * 2001-09-04 2003-08-21 Colba R & D Inc. Combination of antioxidant substances for the treatment of alzheimer's disease
US6613875B1 (en) * 1993-10-14 2003-09-02 The Scripps Research Institute Cyclic peptide tube
US6689753B1 (en) * 1999-11-05 2004-02-10 Axonyx, Inc. β sheet breaker peptide analogs that inhibit β pleated sheet formation in amyloid β-peptide
US6858318B2 (en) * 2001-03-08 2005-02-22 Japan Science And Technology Corporation Metalic nanowire and process for producing the same
US20050069950A1 (en) * 2003-08-29 2005-03-31 Haynie Donald T. Method for designing polypeptides for the nanofabrication of thin films, coatings, and microcapsules by electrostatic layer-by-layer self assembly
US6976639B2 (en) * 2001-10-29 2005-12-20 Edc Biosystems, Inc. Apparatus and method for droplet steering
US20060234947A1 (en) * 2002-01-31 2006-10-19 Tel Aviv University Future Technology Development L.P Peptides antibodies directed thereagainst and methods using same for diagnosing and treating amyloid-associated diseases
US20070135334A1 (en) * 2002-09-06 2007-06-14 Tel Aviv University Future Technology Development L.P. Peptides and methods using same for diagnosing and treating amyloid-associated diseases
US20070298043A1 (en) * 2003-10-02 2007-12-27 Ehud Gazit Novel Antibacterial Agents and Methods of Identifying and Utilizing Same
US20080305040A1 (en) * 2005-09-16 2008-12-11 University Of Pittsburgh In Vivo or in Vitro Method For Detecting Amyloid Deposits Having at Least One Amyloidogenic Protein

Family Cites Families (90)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BE638366A (de) 1963-10-09
NL154600B (nl) 1971-02-10 1977-09-15 Organon Nv Werkwijze voor het aantonen en bepalen van specifiek bindende eiwitten en hun corresponderende bindbare stoffen.
NL154598B (nl) 1970-11-10 1977-09-15 Organon Nv Werkwijze voor het aantonen en bepalen van laagmoleculire verbindingen en van eiwitten die deze verbindingen specifiek kunnen binden, alsmede testverpakking.
NL154599B (nl) 1970-12-28 1977-09-15 Organon Nv Werkwijze voor het aantonen en bepalen van specifiek bindende eiwitten en hun corresponderende bindbare stoffen, alsmede testverpakking.
US3901654A (en) 1971-06-21 1975-08-26 Biological Developments Receptor assays of biologically active compounds employing biologically specific receptors
US3853987A (en) 1971-09-01 1974-12-10 W Dreyer Immunological reagent and radioimmuno assay
US3867517A (en) 1971-12-21 1975-02-18 Abbott Lab Direct radioimmunoassay for antigens and their antibodies
NL171930C (nl) 1972-05-11 1983-06-01 Akzo Nv Werkwijze voor het aantonen en bepalen van haptenen, alsmede testverpakkingen.
US3850578A (en) 1973-03-12 1974-11-26 H Mcconnell Process for assaying for biologically active molecules
US3935074A (en) 1973-12-17 1976-01-27 Syva Company Antibody steric hindrance immunoassay with two antibodies
US3996345A (en) 1974-08-12 1976-12-07 Syva Company Fluorescence quenching with immunological pairs in immunoassays
US4034074A (en) 1974-09-19 1977-07-05 The Board Of Trustees Of Leland Stanford Junior University Universal reagent 2-site immunoradiometric assay using labelled anti (IgG)
US3984533A (en) 1975-11-13 1976-10-05 General Electric Company Electrophoretic method of detecting antigen-antibody reaction
US4036945A (en) 1976-05-03 1977-07-19 The Massachusetts General Hospital Composition and method for determining the size and location of myocardial infarcts
US4098876A (en) 1976-10-26 1978-07-04 Corning Glass Works Reverse sandwich immunoassay
PH14917A (en) 1978-10-23 1982-01-29 Abbott Lab Anti-bacterial peptide
US4331647A (en) 1980-03-03 1982-05-25 Goldenberg Milton David Tumor localization and therapy with labeled antibody fragments specific to tumor-associated markers
US4879219A (en) 1980-09-19 1989-11-07 General Hospital Corporation Immunoassay utilizing monoclonal high affinity IgM antibodies
DE3233679A1 (de) 1981-12-08 1984-03-08 Wilhelm Dr.med. 8900 Augsburg Meyer-Glauner Carrier-tripeptid mit antifungischer wirksamkeit
JPS5944313A (ja) 1982-09-07 1984-03-12 Yakult Honsha Co Ltd 抗菌性組成物
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
JPS6040061A (ja) 1983-08-12 1985-03-02 ユニチカ株式会社 抗菌剤徐放性導尿カテ−テル
US4626540A (en) * 1983-11-08 1986-12-02 Warner-Lambert Company Substituted 1-amino-4-nitro-acridinones and methods of treating bacterial infections and leukemia with them
DE3412445C2 (de) 1984-03-31 1986-10-30 Wilhelm Dr. 7400 Tübingen Meyer-Glauner Tripeptid und dieses enthaltende antifungische Mittel
US5011771A (en) 1984-04-12 1991-04-30 The General Hospital Corporation Multiepitopic immunometric assay
US4666828A (en) 1984-08-15 1987-05-19 The General Hospital Corporation Test for Huntington's disease
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4801531A (en) 1985-04-17 1989-01-31 Biotechnology Research Partners, Ltd. Apo AI/CIII genomic polymorphisms predictive of atherosclerosis
JP2874751B2 (ja) 1986-04-09 1999-03-24 ジェンザイム・コーポレーション 希望する蛋白質をミルク中へ分泌する遺伝子移植動物
JPS6344895A (ja) 1986-08-13 1988-02-25 Kyowa Hakko Kogyo Co Ltd 抗アミロイドａ蛋白質単クロ−ン性抗体
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
US4873316A (en) 1987-06-23 1989-10-10 Biogen, Inc. Isolation of exogenous recombinant proteins from the milk of transgenic mammals
US4970233A (en) * 1987-08-04 1990-11-13 Mchugh John E Treatment of Acquired Immunodeficiency Syndrome (AIDS) HTLV-111/LAV infections and Acquired Immunodeficiency Syndroms (AIDS) Related Complex (ARC)
GB8823869D0 (en) 1988-10-12 1988-11-16 Medical Res Council Production of antibodies
US5272057A (en) 1988-10-14 1993-12-21 Georgetown University Method of detecting a predisposition to cancer by the use of restriction fragment length polymorphism of the gene for human poly (ADP-ribose) polymerase
JPH02295923A (ja) 1989-05-10 1990-12-06 Taiyo Kagaku Co Ltd 腸内クロストリジウム属細菌の増殖阻害剤
US5192659A (en) 1989-08-25 1993-03-09 Genetype Ag Intron sequence analysis method for detection of adjacent and remote locus alleles as haplotypes
US5545806A (en) 1990-08-29 1996-08-13 Genpharm International, Inc. Ransgenic non-human animals for producing heterologous antibodies
US5625126A (en) 1990-08-29 1997-04-29 Genpharm International, Inc. Transgenic non-human animals for producing heterologous antibodies
CA2089661C (en) 1990-08-29 2007-04-03 Nils Lonberg Transgenic non-human animals capable of producing heterologous antibodies
US5633425A (en) 1990-08-29 1997-05-27 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5661016A (en) 1990-08-29 1997-08-26 Genpharm International Inc. Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
US5593967A (en) * 1990-08-31 1997-01-14 Warner-Lambert Company Cholecystokinin antagonists, their preparation and therapeutic use
WO1992019253A1 (en) 1991-04-24 1992-11-12 Warner-Lambert Company CCK ANALOGS CONTAINING α-SUBSTITUTED AMINO ACIDS
US5281521A (en) 1992-07-20 1994-01-25 The Trustees Of The University Of Pennsylvania Modified avidin-biotin technique
CA2167167A1 (en) * 1993-07-19 1995-02-02 Alfred Pollak Hydrazino-type radionuclide chelators having an n3s configuration
DE69403867T2 (de) 1993-07-22 1997-11-13 Ricoh Kk Bildtragendes Element und Verfahren zu seiner Wiederverwertung
US5470951A (en) 1993-09-29 1995-11-28 City Of Hope Peptides for antagonizing the effects of amyloid βprotein
US6303567B1 (en) * 1995-03-14 2001-10-16 Praecis Pharmaceuticals, Inc . Modulators of β-amyloid peptide aggregation comprising D-amino acids
US6319498B1 (en) * 1995-03-14 2001-11-20 Praecis Pharmaceuticals Incorporated Modulators of amyloid aggregation
US5948763A (en) * 1995-06-07 1999-09-07 New York University Peptides and pharmaceutical compositions thereof for treatment of disorders or diseases associated with abnormal protein folding into amyloid or amyloid-like deposits
ATE204291T1 (de) 1995-07-11 2001-09-15 Degussa Verfahren zur herstellung von peptiden und n- carbamoyl-geschützten peptiden
US5972956A (en) 1995-11-02 1999-10-26 Warner-Lambert Company Inhibition of amyloidosis by 9-acridinones
US5688561A (en) 1996-04-16 1997-11-18 Kabushiki Kaisha Nippankenkyusho Coating method
CA2259159A1 (en) 1996-06-25 1997-12-31 Nisshin Flour Milling Co. Ltd. Depsipeptides and drugs containing the same as the active ingredient
US6610478B1 (en) * 1996-08-16 2003-08-26 Yale University Phenotypic conversion of cells mediated by external guide sequences
US6503881B2 (en) * 1996-08-21 2003-01-07 Micrologix Biotech Inc. Compositions and methods for treating infections using cationic peptides alone or in combination with antibiotics
JP2001500852A (ja) * 1996-08-27 2001-01-23 プレーシス ファーマスーティカルズ インコーポレイテッド Ｄ―アミノ酸を含むβ―アミロイドペプチド凝集のモジュレーター
US6617114B1 (en) * 1996-10-31 2003-09-09 Karo Bio Ab Identification of drug complementary combinatorial libraries
EP0941334B1 (de) 1996-11-06 2004-06-02 THE UNITED STATES OF AMERICA, as represented by the Secretary of the Department of Health and Human Services Protease-aktivierbare pseudomonas exotoxin-a-ähnliche proproteine
DE19725619A1 (de) * 1997-06-17 1998-12-24 Fraunhofer Ges Forschung Peptide als Agonisten und/oder Inhibitoren der Amyloidbildung und Zytotoxizität sowie der Verwendung bei Alzheimer'schen Krankheit, beim Typ II Diabetes mellitus und bei spongiformen Encephalopathien
TWI239847B (en) * 1997-12-02 2005-09-21 Elan Pharm Inc N-terminal fragment of Abeta peptide and an adjuvant for preventing and treating amyloidogenic disease
CA2317702A1 (en) * 1998-01-07 1999-07-15 Human Genome Sciences, Inc. 36 human secreted proteins
JP3340699B2 (ja) 1998-06-05 2002-11-05 東京瓦斯株式会社 膵外分泌機能診断剤
JP2000193661A (ja) 1998-12-25 2000-07-14 Tokyo Rika Kikai Kk 痴呆症の検査方法
TWI243672B (en) * 1999-06-01 2005-11-21 Astrazeneca Ab New use of compounds as antibacterial agents
MXPA02001349A (es) 1999-08-09 2002-07-22 Tripep Ab Inhibidores de la polimerizacion de proteinas y metodos de uso.
CA2383172A1 (en) * 1999-09-22 2001-03-29 The Government Of The United States Of America Utilization of fprl1 as a functional receptor by serum amyloid a (saa)
KR20020079731A (ko) * 1999-11-05 2002-10-19 액소닉스, 인코포레이티드. 아밀로이드, 아밀로이드-유사 침착물, 또는 β-쉬이트가풍부한 그의 병리학적 전구체로의 비정상적 단백질 폴딩과관련된 질환의 치료에 있어서 생체내에서 사용하기에적합한 펩티드 동족체 및 유사체
JP2003516151A (ja) * 1999-11-29 2003-05-13 エイブイアイ バイオファーマ， インコーポレイテッド 細菌１６Ｓおよび２３ＳｒＲＮＡに標的化された、荷電していないアンチセンスオリゴヌクレオチド、ならびにその使用
WO2001045726A2 (en) 1999-12-21 2001-06-28 Mars, Incorporated The use of procyanidins in the modulation of cytokine gene expression and protein secretion
CN1981873A (zh) 2000-06-09 2007-06-20 莱古伦公司 制备微胶粒的方法和所述微胶粒的用途
DE10043282A1 (de) 2000-09-02 2002-03-28 Kurt Heininger Verwendung von Inhibitoren der Amyloid-beta-Protein-Bildung
WO2002074931A2 (en) * 2001-03-20 2002-09-26 University Of Chicago Inhibitors and disassemblers of fibrillogenesis
US7214769B2 (en) 2001-05-23 2007-05-08 The Curators Of The University Of Missouri Method for inverse solid phase synthesis of peptides
AU2004203461B2 (en) 2002-01-31 2009-09-03 Tel Aviv University Future Technology Development L.P. Peptides Antibodies Directed Thereagainst and Methods Using Same for Diagnosing and Treating Amyloid-Associated Diseases
KR20040081165A (ko) * 2002-01-31 2004-09-20 텔 아비브 유니버시티 퓨쳐 테크놀로지 디벨롭먼트 엘.피. 펩타이드 항체 및 이를 이용한 아밀로이드-관련 질환의진단 및 치료 방법
US20050020809A1 (en) * 2002-01-31 2005-01-27 Ehud Gazit Peptides antibodies directed thereagainst and methods using same for diagnosing and treating amyloid-associated diseases
US20070021345A1 (en) 2003-06-30 2007-01-25 Ehud Gazit Peptides antibodies directed thereagainst and methods using same for diagnosing and treating amyloid-associated diseases
EP1490140B1 (de) 2002-03-14 2008-06-25 Ash Access Technology, Inc. Medizinische vorrichtungen mit antibakteriellen eigenschaften
CA2479249A1 (en) 2002-03-15 2003-09-25 Samaritan Pharmaceuticals, Inc. Neuroprotective spirostenol pharmaceutical compositions
US6803379B2 (en) * 2002-06-04 2004-10-12 Jose A. Fernandez-Pol Pharmacological agents and methods of treatment that inactivate pathogenic prokaryotic and eukaryotic cells and viruses by attacking highly conserved domains in structural metalloprotein and metalloenzyme targets
IL210418A (en) 2003-06-30 2012-10-31 Ehud Gazit Peptides for treating amyloid-associated diseases
WO2006006172A2 (en) 2004-07-15 2006-01-19 Ramot At Tel Aviv University Ltd. Use of anti-amyloid agents for treating and typing pathogen infections
WO2006018850A2 (en) * 2004-08-19 2006-02-23 Tel Aviv University Future Technology Development L.P. Compositions for treating amyloid associated diseases
CA2625433A1 (en) 2005-10-11 2007-04-19 Lycored Ltd. Carotenoid oxidation products as chemopreventive and chemotherapeutic agents
US7879212B2 (en) * 2005-11-03 2011-02-01 Ramot At Tel-Aviv University Ltd. Peptide nanostructure-coated electrodes
EP2008989A1 (de) 2007-06-26 2008-12-31 Basf Se Kontinuierliches Verfahren zur Herstellung von Neral in reiner oder angereicherter Form
EP2085120A1 (de) 2008-02-01 2009-08-05 Merz Pharma GmbH & Co. KGaA Verwendung von Substanzen zur Behandlung zentraler und peripherer Insulinrezeptorbeeinträchtigung oder Insulinresistenz
CA2817830A1 (en) 2010-11-15 2012-05-24 Ramot At Tel Aviv University Ltd. Dipeptide analogs for treating conditions associated with amyloid fibril formation

Patent Citations (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4299917A (en) * 1979-02-14 1981-11-10 Boehringer Manneheim Gmbh Diagnostic agents for the detection of leukocytes in body fluids
US5210215A (en) * 1989-10-04 1993-05-11 Polifarma S.P.A. Tryptophane and 3-indolepyruvic acid, methods of production therefor
US5332648A (en) * 1990-12-27 1994-07-26 Kabushiki Kaisha Toshiba Potosensitive composition and method of forming a pattern using the same
US5304470A (en) * 1991-01-23 1994-04-19 Forschungszentrum Juelich Gmbh Process for the enzymatic preparation of protected and unprotected di- and oligopeptides in aqueous solutions
US5556744A (en) * 1992-05-29 1996-09-17 The Trustees Of The University Of Pennsylvania Methods and compositions for diagnosing and treating certain HIV infected patients
US6261569B1 (en) * 1992-08-27 2001-07-17 Deakin Research Limited Retro-, inverso- and retro-inverso synthetic peptide analogues
US6613875B1 (en) * 1993-10-14 2003-09-02 The Scripps Research Institute Cyclic peptide tube
US6162828A (en) * 1995-03-31 2000-12-19 Takeda Chemical Industries, Ltd. Cysteine protease inhibitor
US5916642A (en) * 1995-11-22 1999-06-29 Northwestern University Method of encapsulating a material in a carbon nanotube
US6361861B2 (en) * 1999-06-14 2002-03-26 Battelle Memorial Institute Carbon nanotubes on a substrate
US6689753B1 (en) * 1999-11-05 2004-02-10 Axonyx, Inc. β sheet breaker peptide analogs that inhibit β pleated sheet formation in amyloid β-peptide
US20020006954A1 (en) * 2000-03-02 2002-01-17 Oklahoma Medical Research Foundation Desmethyl tocopherols for preventing or slowing degenerative neurological diseases
US6472436B1 (en) * 2000-07-17 2002-10-29 The Salk Institute For Biological Studies Methods for protecting cells from amyloid toxicity and for inhibiting amyloid protein production
US6858318B2 (en) * 2001-03-08 2005-02-22 Japan Science And Technology Corporation Metalic nanowire and process for producing the same
US20020130484A1 (en) * 2001-03-13 2002-09-19 Liang Fa-Kuang Standing-pedaling bike
US20030158237A1 (en) * 2001-09-04 2003-08-21 Colba R & D Inc. Combination of antioxidant substances for the treatment of alzheimer's disease
US6976639B2 (en) * 2001-10-29 2005-12-20 Edc Biosystems, Inc. Apparatus and method for droplet steering
US20060234947A1 (en) * 2002-01-31 2006-10-19 Tel Aviv University Future Technology Development L.P Peptides antibodies directed thereagainst and methods using same for diagnosing and treating amyloid-associated diseases
US20070135334A1 (en) * 2002-09-06 2007-06-14 Tel Aviv University Future Technology Development L.P. Peptides and methods using same for diagnosing and treating amyloid-associated diseases
US20050069950A1 (en) * 2003-08-29 2005-03-31 Haynie Donald T. Method for designing polypeptides for the nanofabrication of thin films, coatings, and microcapsules by electrostatic layer-by-layer self assembly
US20070298043A1 (en) * 2003-10-02 2007-12-27 Ehud Gazit Novel Antibacterial Agents and Methods of Identifying and Utilizing Same
US20080305040A1 (en) * 2005-09-16 2008-12-11 University Of Pittsburgh In Vivo or in Vitro Method For Detecting Amyloid Deposits Having at Least One Amyloidogenic Protein

Cited By (88)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8012929B2 (en) 2002-01-31 2011-09-06 Tel Aviv University Future Technology Development L.P. Peptides directed for diagnosis and treatment of amyloid-associated diseases
US8993510B2 (en) 2002-01-31 2015-03-31 Tel Aviv University Future Technology Development L.P. Peptides and methods using same for diagnosis and treatment of amyloid-associated disease
US20100022459A1 (en) * 2002-01-31 2010-01-28 Tel Aviv University Future Technology Development L.P Peptides directed for diagnosis and treatment of amyloid-associated diseases
US20060234947A1 (en) * 2002-01-31 2006-10-19 Tel Aviv University Future Technology Development L.P Peptides antibodies directed thereagainst and methods using same for diagnosing and treating amyloid-associated diseases
US7781396B2 (en) 2002-01-31 2010-08-24 Tel Aviv University Future Technology Development L.P. Peptides directed for diagnosis and treatment of amyloid-associated disease
US20070135334A1 (en) * 2002-09-06 2007-06-14 Tel Aviv University Future Technology Development L.P. Peptides and methods using same for diagnosing and treating amyloid-associated diseases
US8563273B2 (en) 2002-09-06 2013-10-22 Tel Aviv University Future Technology Development L.P. Method of screening for compounds that disaggregate amyloid aggregates
US20090209041A1 (en) * 2002-09-06 2009-08-20 Tel Aviv University Future Technology Development L.P. Peptides and methods for inhibiting amyloid formation
US20060079454A1 (en) * 2002-12-09 2006-04-13 Ramot At Tel Aviv University Ltd. Peptide nanostructures and methods of generating and using the same
US8927689B2 (en) 2002-12-09 2015-01-06 Ramot At Tel-Aviv University Ltd. Peptide nanostructures and methods of generating and using the same
US8053554B2 (en) 2002-12-09 2011-11-08 Ramot At Tel-Aviv University Ltd. Peptide nanostructures and methods of generating and using the same
US20090123553A1 (en) * 2002-12-09 2009-05-14 Ramot At Tel Aviv University Ltd. Peptide nanostructures and methods of generating and using the same
US8350004B2 (en) 2002-12-09 2013-01-08 Ramot At Tel-Aviv University Ltd. Peptide nanostructures and methods of generating and using the same
US7491699B2 (en) 2002-12-09 2009-02-17 Ramot At Tel Aviv University Ltd. Peptide nanostructures and methods of generating and using the same
US20060079455A1 (en) * 2003-01-07 2006-04-13 Ramot At Tel Aviv University Ltd. Peptide nanostructures encapsulating a foreign material and method of manufacturing same
US7504383B2 (en) 2003-01-07 2009-03-17 Ramot At Tel Aviv University Ltd. Peptide nanostructures encapsulating a foreign material and method of manufacturing same
US8017586B2 (en) 2003-01-07 2011-09-13 Ramot At Tel-Aviv University Ltd. Peptide nanostructures encapsulating a foreign material and method of manufacturing same
US20090121709A1 (en) * 2003-01-07 2009-05-14 Ramot At Tel Aviv University Ltd. Peptide nanostructures encapsulating A foreign material and method of manufacturing same
US8501697B2 (en) 2003-01-07 2013-08-06 Ramot At Tel-Aviv University Ltd. Peptide nanostructures encapsulating a foreign material and method of manufacturing same
US8314069B2 (en) 2003-01-07 2012-11-20 Ramot At Tel-Aviv University Ltd. Peptide nanostructures encapsulating a foreign material and method of manufacturing same
US10464976B2 (en) 2003-01-31 2019-11-05 AbbVie Deutschland GmbH & Co. KG Amyloid β(1-42) oligomers, derivatives thereof and antibodies thereto, methods of preparation thereof and use thereof
US9176150B2 (en) 2003-01-31 2015-11-03 AbbVie Deutschland GmbH & Co. KG Amyloid beta(1-42) oligomers, derivatives thereof and antibodies thereto, methods of preparation thereof and use thereof
US20060194777A1 (en) * 2003-09-25 2006-08-31 Ehud Gazit Compositions and methods using same for treating amyloid-associated diseases
US8372880B2 (en) 2003-09-25 2013-02-12 Tel Aviv University Future Technology Development L.P. Compositions and methods using same for treating amyloid-associated diseases
US7625707B2 (en) 2003-10-02 2009-12-01 Ramot At Tel Aviv University Ltd. Antibacterial agents and methods of identifying and utilizing same
US20070298043A1 (en) * 2003-10-02 2007-12-27 Ehud Gazit Novel Antibacterial Agents and Methods of Identifying and Utilizing Same
US20060278093A1 (en) * 2004-01-13 2006-12-14 Eytan Biderman Feeding formula appliance
US8007847B2 (en) 2004-01-13 2011-08-30 Eytan Biderman Feeding formula appliance
US20090156471A1 (en) * 2004-07-15 2009-06-18 Ramot At Tel Aviv University Ltd. Use of anti-amyloid agents for treating and typing pathogen infections
US7846892B2 (en) * 2004-07-20 2010-12-07 Primex Clinical Laboratories, Inc. Compositions and methods for Alzheimer's disease
US20090023627A1 (en) * 2004-07-20 2009-01-22 Regents Of The University Of Colorado Compositions and methods for alzheimer's disease
US20090061190A1 (en) * 2004-08-02 2009-03-05 Ramot At Tel Aviv University Ltd. Articles of peptide nanostructures and method of forming the same
US8568637B2 (en) 2004-08-02 2013-10-29 Ramot At Tel-Aviv University Ltd. Method of forming a fiber made of peptide nanostructures
US9394628B2 (en) 2004-08-02 2016-07-19 Ramot At Tel-Aviv University Ltd. Method of forming a fiber made of peptide nanostructures
US7732479B2 (en) 2004-08-19 2010-06-08 Tel Aviv University Future Technology Development L.P. Compositions for treating amyloid associated diseases
US7786086B2 (en) 2004-09-08 2010-08-31 Ramot At Tel-Aviv University Ltd. Peptide nanostructures containing end-capping modified peptides and methods of generating and using the same
US20080009434A1 (en) * 2004-09-08 2008-01-10 Meital Reches Peptide Nanostructures Containing End-Capping Modified Peptides And Methods Of Generating And Using The Same
US8796023B2 (en) 2004-09-08 2014-08-05 Ramot At Tel-Aviv University Ltd. Peptide nanostructures containing end-capping modified peptides and methods of generating and using the same
US8375327B2 (en) 2005-01-16 2013-02-12 Zlango Ltd. Iconic communication
US20080082678A1 (en) * 2005-01-16 2008-04-03 Zlango Ltd. Communications Network System and Methods for Using Same
US20080216022A1 (en) * 2005-01-16 2008-09-04 Zlango Ltd. Iconic Communication
US20090013087A1 (en) * 2005-01-18 2009-01-08 Zlango Ltd. Communications Network System and Methods For Using Same
US8019818B2 (en) 2005-01-18 2011-09-13 Zlango Ltd. Communications network system and methods for using same
US20090175785A1 (en) * 2005-10-11 2009-07-09 Ehud Gazit Self-Assembled Fmoc-Ff Hydrogels
US10004828B2 (en) 2005-10-11 2018-06-26 Romat at Tel-Aviv University Ltd. Self-assembled Fmoc-ff hydrogels
US7879212B2 (en) 2005-11-03 2011-02-01 Ramot At Tel-Aviv University Ltd. Peptide nanostructure-coated electrodes
US20070138007A1 (en) * 2005-11-03 2007-06-21 Ramot At Tel Aviv University Ltd. Peptide nanostructure-coated electrodes
US20090191190A1 (en) * 2005-11-30 2009-07-30 Stefan Barghorn Anti-ABeta Globulomer Antibodies, Antigen-Binding Moieties Thereof, Corresponding Hybridomas, Nucleic Acids, Vectors, Host Cells, Methods of Producing Said Antibodies, Compositions Comprising Said Antibodies, Uses Of Said Antibodies And Methods Of Using Said Antibodies
US8691224B2 (en) 2005-11-30 2014-04-08 Abbvie Inc. Anti-Aβ globulomer 5F7 antibodies
US9540432B2 (en) 2005-11-30 2017-01-10 AbbVie Deutschland GmbH & Co. KG Anti-Aβ globulomer 7C6 antibodies
US8497072B2 (en) 2005-11-30 2013-07-30 Abbott Laboratories Amyloid-beta globulomer antibodies
US10538581B2 (en) 2005-11-30 2020-01-21 Abbvie Inc. Anti-Aβ globulomer 4D10 antibodies
US20090238831A1 (en) * 2005-11-30 2009-09-24 Hinz Hillen Monoclonal antibodies and uses thereof
US10323084B2 (en) 2005-11-30 2019-06-18 Abbvie Inc. Monoclonal antibodies against amyloid beta protein and uses thereof
US10208109B2 (en) 2005-11-30 2019-02-19 Abbvie Inc. Monoclonal antibodies against amyloid beta protein and uses thereof
US8775526B2 (en) 2006-01-16 2014-07-08 Zlango Ltd. Iconic communication
US9359430B2 (en) 2006-11-30 2016-06-07 Abbvie Inc. Abeta conformer selective anti-Abeta globulomer monoclonal antibodies
US8877190B2 (en) 2006-11-30 2014-11-04 Abbvie Inc. Aβ conformer selective anti-Aβ globulomer monoclonal antibodies
US20090035307A1 (en) * 2006-11-30 2009-02-05 Stefan Barghorn Abeta CONFORMER SELECTIVE ANTI-Abeta GLOBULOMER MONOCLONAL ANTIBODIES
US9951125B2 (en) 2006-11-30 2018-04-24 Abbvie Inc. Aβ conformer selective anti-Aβ globulomer monoclonal antibodies
US8455626B2 (en) 2006-11-30 2013-06-04 Abbott Laboratories Aβ conformer selective anti-aβ globulomer monoclonal antibodies
US9394360B2 (en) 2006-11-30 2016-07-19 Abbvie Inc. Aβ conformer selective anti-Aβ globulomer monoclonal antibodies
US8895004B2 (en) 2007-02-27 2014-11-25 AbbVie Deutschland GmbH & Co. KG Method for the treatment of amyloidoses
US20110130549A1 (en) * 2007-02-27 2011-06-02 Abbott Gmbh & Co. Kg Method for the treatment of amyloidoses
US10525097B2 (en) 2007-08-21 2020-01-07 Ramot At Tel Aviv University Ltd Methods for the treatment of glaucoma and age-related macular degeneratiion by a peptide D-TRP-AIB
US10987400B2 (en) 2007-08-21 2021-04-27 Ramot At Tel-Aviv University Ltd Methods for the treatment of glaucoma and age-related macular degeneration by a peptide D-TRP-AIB
US20100204137A1 (en) * 2007-08-21 2010-08-12 MERZ PHARMAGMBH & CO. KGaA The use of substances for the treatment of loss of eyesight in humans with glaucoma and other degenerative eye diseases
US20090258818A1 (en) * 2008-04-07 2009-10-15 National Institute Of Immunology Compositions For the Treatment of Diabetes
WO2009125423A3 (en) * 2008-04-07 2010-03-04 National Institute Of Immunology Compositions useful for the treatment of diabetes and other chronic disorder
US20110034385A1 (en) * 2008-04-07 2011-02-10 National Institute Of Immunology Compositions Useful for the Treatment of Diabetes and Other Chronic Disorder
US8426362B2 (en) 2008-04-07 2013-04-23 National Institute Of Immunology Compositions comprising SIA for the treatment of diabetes
WO2009128772A1 (en) * 2008-04-14 2009-10-22 Mivac Development Aktiebolag Stable amyloid beta monomers and oligomers
US10023622B2 (en) 2008-04-14 2018-07-17 Alzinova Ab Method of immunization for reducing toxic damaged caused by oligomeric form of the amyloid-beta protein with stable amyloid beta monomers and oligomers
CN102065881A (zh) * 2008-04-14 2011-05-18 Mivac发展股份公司 稳定的淀粉样beta单体和寡聚体
US9688734B2 (en) 2008-04-14 2017-06-27 Alzinova Ab Stable amyloid beta monomers and oligomers
US20110064741A1 (en) * 2008-04-14 2011-03-17 Mivac Development Aktiebolag Stable amyloid beta monomers and oligomers
US10138281B2 (en) 2008-04-14 2018-11-27 Alzinova Ab Stable amyloid beta monomers and oligomers
US8987419B2 (en) 2010-04-15 2015-03-24 AbbVie Deutschland GmbH & Co. KG Amyloid-beta binding proteins
US9822171B2 (en) 2010-04-15 2017-11-21 AbbVie Deutschland GmbH & Co. KG Amyloid-beta binding proteins
US20180118682A1 (en) * 2010-07-22 2018-05-03 Ramot At Tel-Aviv University Ltd. Pharmaceutical composition containing a tryptophan derivative
US10047121B2 (en) 2010-08-14 2018-08-14 AbbVie Deutschland GmbH & Co. KG Amyloid-beta binding proteins
US9062101B2 (en) 2010-08-14 2015-06-23 AbbVie Deutschland GmbH & Co. KG Amyloid-beta binding proteins
US9630989B2 (en) * 2010-11-15 2017-04-25 Ramot At Tel-Aviv University Ltd. Dipeptide analogs for treating conditions associated with amyloid fibril formation
US9096645B2 (en) 2010-11-15 2015-08-04 Ramot At Tel-Aviv University Ltd. Dipeptide analogs for treating conditions associated with amyloid fibril formation
US20150322110A1 (en) * 2010-11-15 2015-11-12 Ramot At Tel-Aviv University Ltd. Dipeptide analogs for treating conditions associated with amyloid fibril formation
CN109073656A (zh) * 2016-04-29 2018-12-21 于利奇研究中心有限公司 用于鉴别β淀粉样蛋白聚集的一次成核的抑制剂的方法
WO2017186203A1 (de) * 2016-04-29 2017-11-02 Forschungszentrum Jülich GmbH Verfahren zur identifikation von inhibitoren der primären nukleation der amyloid-beta-aggregation
US11346847B2 (en) 2016-04-29 2022-05-31 Forschungszentrum Juelich Gmbh Method for identifying inhibitors of primary nucleation of amyloid-beta aggregation

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CN101415724B (zh) 2015-12-02
US20100105608A1 (en) 2010-04-29
JP5137400B2 (ja) 2013-02-06
CA2530927A1 (en) 2005-01-06
JP2012246299A (ja) 2012-12-13
KR20060025584A (ko) 2006-03-21
EP2719394B1 (de) 2015-07-29

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