WO1995006477A1 - Inhibition de la liaison du beta amyloïde aux glycosaminoglycannes pour le traitement de la maladie d'alzheimer - Google Patents

Inhibition de la liaison du beta amyloïde aux glycosaminoglycannes pour le traitement de la maladie d'alzheimer Download PDF

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WO1995006477A1
WO1995006477A1 PCT/US1994/009853 US9409853W WO9506477A1 WO 1995006477 A1 WO1995006477 A1 WO 1995006477A1 US 9409853 W US9409853 W US 9409853W WO 9506477 A1 WO9506477 A1 WO 9506477A1
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amino acid
peptide
alanine
histidine
seq
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PCT/US1994/009853
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English (en)
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Kurt R. Brunden
Rekha Gupta-Bansal
Nancy J. Richter-Cook
Robert C. A. Frederickson
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Gliatech, Inc.
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Priority to AU76424/94A priority Critical patent/AU7642494A/en
Publication of WO1995006477A1 publication Critical patent/WO1995006477A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/07—Tetrapeptides
    • 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
    • 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
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • 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
    • C07K5/10—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
    • 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
    • C07K5/10—Tetrapeptides
    • C07K5/1019—Tetrapeptides with the first amino acid being basic
    • 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
    • C07K5/10—Tetrapeptides
    • C07K5/1021—Tetrapeptides with the first amino acid being acidic

Definitions

  • the present invention relates to compounds that inhibit the binding of glycosaminoglycans and/or proteoglycans to beta amyloid peptide (A(beta) ; A/3) , and to compounds that inhibit A(beta) activation of complement.
  • the invention further relates to treatment of Alzheimer's disease by administration of the inhibitory compounds.
  • AD Alzheimer's disease
  • senile neuritic plaques within the cortex, hippocampus and certain subcortical nuclei of the brain
  • the most well-studied component of these plaques is /3A4 or beta amyloid peptide (A/3) , a 40-43 residue peptide that is derived from the amyloid precursor protein (APP) (Glenner, 1984, Biochem. Biophys. Res. Comm.
  • A/3 is a major component of AD plaques
  • many other macromolecules have been identified within these structures, including dermatan sulfate proteoglycan (Snow et al., 1992, J. Histochem. Cytochem. 40:105- 113) and heparan sulfate proteoglycan (HSPG) (Snow et al., 1988, Am. J. Pathol. 133:456-463).
  • the latter appears to be found throughout the plaque core, and recent studies indicate that HSPG binds both APP (Narindrasorasak et al., 1991, J. Biol. Chem. 266:12878-12883) and A/3 (Snow et al., 1991, Soc. Neurosci.
  • the classical complement pathway is an immune- defense mechanism that results in activated molecules that can initiate inflammation, phagocytosis and cell lysis.
  • An ultimate result of complement activation is the formation of "membrane attack complexes" (MACs) composed of C5-C9 that insert into cellular membranes and form large transme brane channels that can lyse or damage foreign cells.
  • MACs membrane attack complexes
  • a side-effect of complement activation can be "bystander" damage due to MAC attachment to nearby cells that are not specifically targeted for destruction.
  • the usual biological signal that causes activation of the classical complement pathway is the binding of specific immunoglobulins to antigens on foreign cells. If two immunoglobulin (Ig) G molecules are within close proximity on a cellular membrane, complement component Clq binds the Fc portions of the IgG proteins, with resulting activation of Cl and subsequent induction of the complement cascade.
  • Ig immunoglobulin
  • Clq binds the Fc portions of the IgG proteins, with resulting activation of Cl and subsequent induction of the complement cascade.
  • Prior research has shown that the binding of a single IgG to Clq is insufficient to cause activation, and it appears that two or more IgG molecules must bind to the multivalent Clq protein.
  • AD Alzheimer's disease
  • Rogers and co-workers have shown directly in vitro and indirectly in situ that /3-amyloid deposited in amyloid plaques activates complement (Rogers et al., 1992, Proc. Natl. Acad. Sci. USA 89:10016-10020).
  • Rogers et al. showed that Clq immunoreactivity colocalizes with A/3 containing AD pathological structures and not immunoglobulins in the brain of AD patients but not of nondemented elderly control patients;
  • A/3 activates the classical complement pathway in a standard complement activation assay and an ELISA complement activation assay, and
  • a ⁇ activates the full classical pathway in vivo (Rogers et al., 1992, Proc. Natl. Acad. Sci.
  • Rogers et al. propose, based on several lines of direct and indirect evidence, that A/8-mediated complement activation may be a pathogenic mechanism in AD (Rogers et al., 1992, Res. Immunol. 143(6) :624-630) .
  • the present invention relates to a compounds that inhibit the binding of glycosaminoglycans and 5 proteoglycans to beta amyloid peptide (A(beta) ; A/3) , and to compounds that inhibit A/3 activation of complement.
  • the compound is a peptide having an amino acid sequence X-X-N-X, in which X is a amino acid with a cationic side chain and N is a 0 neutral amino acid.
  • the compound is a peptide having an amino acid sequence X-X-N-X-Z, in which X is a amino acid with a cationic side chain, and N and Z are each independently a neutral amino acid.
  • the compound is peptide 5 having an amino acid sequence X j -N-X 2 -X 3 , in which at least two of X l t X 2 , and X 3 are independently an amino acid with an anionic side chain and the third X is an amino acid with an anionic side chain or a neutral amino acid, and N is independently a neutral amino o acid.
  • the compound is an anionic disaccharide.
  • the present invention is based, in part, on the surprising discovery that relatively small molecules, such as peptides of 4 or 5 amino acids, and 5 disaccharides, can inhibit the binding of A ⁇ with glycosaminoglycans and proteoglycans.
  • relatively small molecules such as peptides of 4 or 5 amino acids, and 5 disaccharides
  • the compounds of the invention are believed to reverse the glycosaminoglycan-mediated resistance of A / 3 peptide to 0 proteolysis, thus allowing natural degradation of the A ⁇ peptide and elimination of the A ⁇ plaques. Deposition of A ⁇ results in the formation of amyloid plaques in Alzheimer's disease.
  • the invention is also based, in part, on the 5 surprising discovery that relatively small molecules, i.e., the compounds of the invention, inhibit complement activation associated with beta-amyloid. Although not intending to be limited by any particular theory, it is believed that the molecules inhibit the interaction of Clq with A/3.
  • the invention further relates to a pharmaceutical composition
  • a pharmaceutical composition comprising one or more of the inhibitory compounds and a pharmaceutically acceptable carrier.
  • the invention also relates to a method for treating Alzheimer's disease comprising administering a therapeutically effective amount of a compound as described above to a subject suffering from Alzheimer's disease.
  • Figure 1 The amino acid sequence (SEQ ID NO:l) of the beta amyloid peptide (A/3) .
  • FIG. 1 Heparin-induced aggregation of A ⁇ (1-28) examined as a function of pH. A ⁇ (1-28) at a concentration of 0.25 mM was incubated with 20 ⁇ m heparin in saline solutions at a variety of pH values. The percentage of soluble amyloid remaining in solution after incubation and centrifugation was determined for single samples at each pH value.
  • Figure 3 Heparin affinity chromatography of A/8(l-28) at pH 4.0 (A) and 8.0 (B) .
  • A/3(l-28) was injected onto a heparin affinity column as described in Section 6.1, infra , and non-bound peptide was eluted with either 20 mM sodium phosphate pH 8.0 or 20 mM sodium acetate pH 4.0. Bound peptide was eluted with a linear gradient of 0-0.5 M NaCl. Peptide elution was monitored by determining the absorbance at 280 nm.
  • Figure 4 The ability of heparin and heparan sulfate to cause aggregation of A
  • A/3(1-28) at a concentration of 0.5 mM was incubated with various concentrations of either heparin (open circles) or heparan sulfate (closed circles) at pH 4.0, and the 5 amount of soluble peptide remaining after incubation and centrifugation was measured for single samples as described in Section 6.1, infra .
  • FIG. 5 Comparison of the effects of various glycosaminoglycans on the solubility of A/3(1-28) at pH 0 4.0 and 8.0.
  • A/5(1-28) at a concentration of 0.25 mM was incubated either in the absence (-GAG) or presence of 20 ⁇ M dermatan sulfate (light diagonal hatch) , chondroitin sulfate (cross-hatch) or heparan sulfate (heavy diagonal hatch) at pH values of 4.0 and 8.0.
  • 5 Following centrifugation, the amount of peptide remaining soluble was determined at both pH's and expressed as a percentage of peptide found in non- centrifuged samples. All samples were assayed in triplicate, with the standard error of the mean shown o f°r each condition.
  • FIG. 6 Glycosaminoglycan-induced aggregation of A / S(l-40) at pH 3.5 and 8.0.
  • a ⁇ (1-40) at a concentration of 0.25 mM was incubated either in the absence (-GAG) (heavy diagonal hatch) or presence of 5 heparan sulfate (HS) (cross hatch) or chondroitin sulfate (CS) (light diagonal hatch) at pH values of 3.5 and 8.0. After centrifugation, the amount of peptide remaining in solution was determined for each sample and expressed as a percentage of peptide found 0 in non-centrifuged samples (solid bar) . All samples were assayed in triplicate, with the standard error of the mean shown for each condition.
  • FIG. 7 The binding of A ⁇ (13-17) to a heparin affinity column.
  • A/3(13-17) was injected onto a 5 heparin affinity column as described in Section 6, infra , and non-bound peptide was eluted (solid line) with either 20 mM sodium phosphate pH 4.0 (A) (open circles) or 20 mM sodium acetate pH 8.0 (B) , (closed circles) .
  • Bound peptide was eluted with a linear gradient of 0-0.5 M NaCl (dotted line).
  • -Peptide elution was monitored by determining the absorbance at 230 nm of collected fractions.
  • Figure 8 Competition of A ⁇ (13-17) with A ⁇ (1-28) for heparin binding.
  • Reaction solutions (20 ⁇ l, pH 4.0) contained 0.25 mM A/3(l-28), 5.0 mM A/8(13-17) and heparin, or 0.25 mM A/3(l-28) either alone or with heparin as a control. The reactions were incubated 45 in prior to centrifugation. Each bar represents one of the duplicate assay samples.
  • Samples 1 and 2 contained A/5(1-28) alone; 3 and 4 contained A/3(1-28) with heparin; and 5 and 6 contained Aj3(13-17), Aj8(l-28) and heparin.
  • Figure 10
  • FIG. 11 Competition of poly-L-lysine with A/3(1-40) for heparin binding at pH 3.5.
  • Each bar represents a single sample.
  • the assays were performed in duplicate. The specific concentrations of reagents and reaction conditions are described in the notes to Table 5, infra.
  • FIG. 12 Competition of poly-L-lysine with A/5(1-40) for CSPG binding at pH 3.5.
  • Samples 1 and 2 are A ⁇ (l-AO) ; samples 3 and 4 are A/5(l-40) and CSPG; and samples 5 and 6 are A ⁇ (1-40), CSPG, and poly-L- lysine.
  • Each bar represents a single sample.
  • the assays were performed in duplicate. The specific concentrations of reagents and reaction conditions are described in the notes to Table 5, infra .
  • FIG. 13 Effect of CSPG on the proteolysis of A/5(1-40).
  • Sample A contained 19.35 ⁇ g of A/3(1-40);
  • sample B contained A ⁇ (1-40) and papain (2 ⁇ g) ;
  • sample C contained A/3(l-40) and CSPG (30 ⁇ g) , which were incubated together for 1 hour prior to the addition of papain.
  • the amount of A/3(1-40) was determined by densitometry after gel electrophoresis as described in Section 7.1.2, i-nfra.
  • Figure 14 The effect of increasing concentration of CSPG on papain proteolysis of A/3(l- 40). Each sample contained 21.5 ⁇ g of A / S(l-40) at pH 4.0. The amounts of CSPG added were 0, 24 ⁇ g, 48 ⁇ g, 72 ⁇ g and 96 ⁇ g, followed by a 1 hour incubation. The reaction mixtures were then incubated with 2 ⁇ g of papain for 12 hours, electrophoresed, and the amount of A/5(1-40) quantified using a densitometer.
  • Figure 15 The effect of A ⁇ (13-16) and A/3(13-17) on CSPG-mediated protection of A ⁇ (1-40) from papain proteolysis.
  • Figure 16. The effect of A ⁇ (20-24: Phe 20 ⁇ Glu 20 ) on CSPG-mediated protection of A/3(1-40) from papain proteolysis.
  • A/3(1-40) (4.3 ⁇ g) was treated with papain after a 45 minute incubation with CSPG (30 ⁇ g) and a subsequent incubation with the A ⁇ (20-24: Phe 20 ⁇ Glu 20 ) peptide Glu-Ala-Glu-Asp-Val (150 ⁇ g) , all at pH 6.5.
  • Control samples were incubated without the peptide, without the peptide or CSPG, and without the peptide, CSPG, and papain. Papain digestion ran for 18 hours, followed by gel electrophoresis. A/3(1-40) bands were quantitated using a densitometer.
  • Figure 17 The effect of heparin disaccharide on CSPG-mediated protection of A ⁇ (1-40) from papain proteolysis.
  • the reaction conditions were the same as described for Figure 17, with the exception that the heparin disaccharide (9UA-2s-[l->4]-GlcNS-6S) was used at 120 ⁇ g in place of the A/3(20-24: Phe 20 ⁇ Glu 20 ) peptide.
  • Figure 18 A variation of a complement fixation assay, described in Section 8.1.1 infra , was utilized to assay for amyloid-induced complement activation. Complement activity is expressed as a percent of control samples which did not receive A ⁇ during the initial incubation. Two forms of peptides were used; fresh peptide (Fl-40) was used within 3 days of solubilization and there was no evidence of appreciable aggregate formation, while aged peptide (Al-4) was solubilized and stored for 27 days before use. The aged peptide solutions were Congo Red birefringent, indicating that the peptide exists as fibrils.
  • FIG 19. A solid-phase binding assay for the evaluation of Clq binding to A/3(1-28). This assay is described in Section 8.1.2, infra. The amount of Clq binding to the dotted Aj3(l-28) was detected by im unoenzyme staining and quantified through densitometric analysis of the stained membranes.
  • Figure 20 The effect of A ⁇ (13-16) on A/3(1-40)- induced complement activation was investigated in the complement fixation assay described in Section 8.1.1, infra . Heat-aggregated human gamma globulin (AHGG) was included in the experiment since immunoglobulins are the normal activators of complement.
  • AHGG Heat-aggregated human gamma globulin
  • a ⁇ (13-16) was added at 50-fold molar excess relative to A/3(l- 40) .
  • Figure 21 The effect of A ⁇ (20-24: Phe 20 -Glu 20 ) on AjS(1-40)-induced complement activation was investigated using the complement fixation assay described in Section 8.1.1 infra . The pentapeptide was added at 12.5 to 50-fold molar excess relative to A/3(1-40).
  • the present invention relates to a compounds that inhibit the binding of glycosaminoglycans and/or proteoglycans to beta amyloid peptide (A(beta) ; A/3;
  • SEQ ID NO:l SEQ ID NO:l
  • compounds that inhibit A / 3 activation of complement and therapeutic methods and compositions based thereon.
  • the inhibitory compounds of the invention are relatively small molecules, such as tetra- to hexapeptides or sulfated disaccharides that have the ability to inhibit the aggregation of A/3 with proteoglycans or glycosaminoglycans.
  • the molecules of the invention consist of a sequence of not greater than 6 amino acid residues, and generally not greater than 8 amino acid residues, and, in a particular embodiment, comprise the sequences of peptides described in the subsections below.
  • hydrophobic residues are added to the peptides of the invention, to enhance the ability of the peptides to cross the blood-brain barrier.
  • the compounds of the invention have the ability to inhibit A/3-mediated activation of complement.
  • Specific tests for the ability to inhibit the aggregation of A/S with glycosaminoglycans; to reverse the glycosaminoglycan-mediated protection of A/3 from proteolysis; and to inhibit A ⁇ activation of complement are described in the Examples in Sections 6, 7 and 8, respectively.
  • the compounds of the invention can inhibit or reverse the neuropathology associated with Alzheimer's disease. Although not intending to be limited to any particular theory, it is believed that the neuropathology can result from beta amyloid deposition, the inhibitory effects of proteoglycans and glycosaminoglycans in amyloid on nerve growth, and/or inappropriate complement activation that can destroy nerve cells.
  • the present invention provides three classes of compounds that can inhibit aggregation of A/3 and glycosaminoglycans, or inhibit A/3-mediated complement activation, or both, described in detail below.
  • the peptide inhibitory compounds of the invention are preferably prepared using standard synthetic chemistry from, preferably, naturally occurring amino acids, or obtained commercially.
  • the peptides of the invention can also contain non-natural amino acids or cyclic peptides.
  • Non-classical amino acids include but are not limited to the D-isomers of the common amino acids, ⁇ -amino isobutyric acid, 4-aminobutyric acid, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, ⁇ - alanine, designer amino acids such as /5-methyl amino acids, C ⁇ -methyl amino acids, N ⁇ -methyl amino acids, and amino acid analogs in general.
  • the amino acid can be the D (dextrorotary) or L (levorotary) amino acid.
  • the peptide may be prepared by methods that are known in the art.
  • solid phase peptide synthesis consists of coupling the carboxyl group of the C-terminal amino acid to a resin and successively adding N-alpha protected amino acids.
  • the protecting groups may be any known in the art. Before each new amino acid is added to the growing chain, the protecting group of the previous amino acid added to the chain is removed.
  • the coupling of amino acids to appropriate resins is described by Rivier et al., U.S. Patent No. 4,244,946. Such solid phase syntheses have been described, for example, by
  • the inhibitory compound is a peptide that competes with A/S for binding to a glycosaminoglycan or proteoglycan, and therefore inhibits A ⁇ binding thereto.
  • the inhibitory compound of the invention is a peptide of four or five amino acids based on the putative heparin binding domain of A ⁇ .
  • the sequence of the heparin binding domain of A/5 is histidine 13 -histidine 14 -glutamine 15 - lysine 16 -leucine I7 .
  • peptides corresponding to A/5(13-17) and A/S(13-16) were able to inhibit the binding of A/S to heparin, chondroitin sulfate proteoglycan, and dextran sulfate, as shown in aggregation assays and by the ability of the peptides to reverse proteoglycan/glycosaminoglycan-mediated protection of A/5 from papain proteolysis.
  • the compound is a peptide having an amino acid sequence X-X-N-X, in which X is a amino acid with a cationic side chain and N is a neutral amino acid.
  • X is selected from the group consisting of histidine, lysine and arginine
  • N is selected from the group consisting of glycine, alanine, valine, serine, threonine, asparagine, glutamine, methionine, and cysteine, and hydrophobic amino acids such as isoleucine, phenylalanine, and leucine.
  • the peptide has the amino acid sequence histidine-histidine-glutamine- lysine, which corresponds to A ⁇ (13-16).
  • the peptide has the sequence X-X-X-X, with X as defined above; in a specific embodiment, such a peptide has the sequence Lys-Lys-Lys-Lys (SEQ ID NO:2) or His-His-His-His (SEQ ID NO:3).
  • the compound is a peptide having an amino acid sequence X-X-N-X-Z, in which X is a amino acid with a cationic side chain, N and Z are each independently a neutral amino acid.
  • X is selected from the group consisting of histidine, lysine and arginine
  • N is selected from the group consisting of glycine, alanine, valine, serine, threonine, asparagine, glutamine, methionine, cysteine, and hydrophobic amino acids such as isoleucine, phenylalanine, and leucine
  • Z is selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, serine, threonine, asparagine, glutamine, tryptophane, tyrosine, phenylalanine, methionine and cysteine.
  • the peptide has the amino acid sequence histidine-histidine-glutamine-lysine-leucine, which corresponds to A/3(13-17) .
  • Any basic amino acid including but not limited to histidine, lysine, asparagine, di-aminobutaric acid, and amino acid analogs having amines, guanidines, or other basic side chains and D-amino acids, can be substituted for any of the basic amino acids corresponding to positions 13, 14 and 16 of the A ⁇ heparin binding domain.
  • Any neutral (i.e., uncharged) amino acid can be substituted for the amino acid (N) corresponding to the 15 position of the A/3 peptide.
  • the amino acids glycine, alanine, valine, serine, threonine, asparagine, glutamine, methionine, cysteine, isoleucine, phenylalanine, and leucine, as 5 well as analogs and optical isomers thereof, can be used at that position.
  • the compound of the 0 invention is a peptide that competes with glycosaminoglycans for binding to the heparin binding domain of A/5.
  • the 20-24 region of A/S binds to the heparin 5 binding domain of A/3 in an antiparallel fashion.
  • a pentapeptide having the sequence Phe-Ala-Glu-Asp-Val or an analog thereof and thus corresponding to the amino acids 20-24 region of A/S is specifically preferred since such a peptide will target A/3, whereas o a peptide corresponding to A ⁇ (13-16) or (13-17) (see Section 5.1.1) will target all glycosaminoglycans or proteoglycans.
  • the peptide corresponding to the 20-24 region of A ⁇ and having the 5 structure phenylalanine-alanine-glutamic acid-aspartic acid reversed the glycosaminoglycan-mediated protection of A/3 from proteolysis.
  • an analog of the 20-24 region, in which phenylalanine was substituted with glutamic acid 0 (SEQ ID NO:6) or aspartic acid (SEQ ID NO:7) had the same activity.
  • the compound is a peptide having an amino acid sequence X,-N-X 2 -X 3 , in which at least two of Xj, X 2 , and X 3 are independently an amino acid with 5 an anionic side chain, and the third X is an amino acid with an anionic side chain or a neutral amino acid, and N is independently a neutral amino acid.
  • the amino acid with the anionic side chain is selected from the group consisting of aspartic acid and glutamic acid or other anionic amino acid analog known to one skilled in the art
  • the non-anionic X and N are independently selected from the group consisting of any neutral amino acid, but preferably glycine, alanine, valine, serine, threonine, asparagine, glutamine, methionine, cysteine, and the hydrophobic amino acids isoleucine, phenylalanine, and leucine.
  • the peptide has the amino acid sequence phenylalanine-alanine-glutamic acid-aspartic acid, which corresponds to A ⁇ (20-23).
  • the peptide has the amino acid sequence aspartic acid-alanine-glutamic acid- aspartic acid (SEQ ID NO:4). In yet another embodiment, the peptide has the amino acid sequence glutamic acid-alanine-glutamic acid-aspartic acid (SEQ ID NO:5) .
  • a pentapeptide having the sequence X,-N-X 2 -X 3 -B, with X,, N, X 2/ and X 3 as defined above, and B being any hydrophobic amino acid, including but not limited to leucine, valine, isoleucine, and phenylalanine.
  • B being any hydrophobic amino acid, including but not limited to leucine, valine, isoleucine, and phenylalanine.
  • this peptide corresponding to the 20-24 region of A/3, only two acidic amino acids (with an anionic side chain) need to be found in the pentapeptide; preferably, however, there are three acidic amino acids, to act as complements to the three basic amino acids in the heparin binding domain of Aj8.
  • the non-anionic amino acid in the structure can be any neutral amino acid, but preferably glycine, alanine, valine, serine, threonine, asparagine, glutamine, methionine, cysteine, isoleucine, phenylalanine, and leucine. 5.1.3 THE 25-35 REGION OF AiS
  • the compound of the invention is a peptide that inhibits A3-induced 5 activation of the complement cascade.
  • such a peptide is A/5(13-17) or an analog thereof.
  • such a peptide is A ⁇ (25-35) or an analog thereof.
  • the compound of the invention is a sulfated disaccharide that competes with glycosaminoglycans for binding to the heparin binding domain of A/5.
  • the sulfated disaccharide binds to the heparin binding domain of A/S.
  • Such a disaccharide is preferred since it will target A/3 specifically instead of glycosaminoglycans and proteoglycans generally.
  • the compound is an anionic disaccharide.
  • any sulfated disaccharide is envisioned, in a specific embodiment the disaccharide is derived from heparin.
  • sulfated disaccharides of the invention 5 contain either uronic or glucuronic acid. More particularly, the disaccharide is ⁇ -4-deoxy-L-threo- hex-4-enopyranosyluronic acid-[l->4] D-glucosamine-N- sulfate-6-sulfate.
  • the compounds of the invention are modified so as to permit or enhance their ability to cross the blood brain barrier. Such compounds would be preferred for oral or parenteral 5 administration other than intraventricularly.
  • Suitable modifications of the compounds to enhance their ability to cross the blood-brain barrier include, but are not limited to, adding hydrophobic amino acids, coupling the compound to a lipid, coupling to transferrin, coupling to an antibody which recognizes the transferrin receptor, coupling to avidin, etc.
  • the chemical linkage effectuating the coupling is labile, e . g. , a disulfide bond.
  • Other modifications of the peptides of the invention such as chemical modifications known in the art can be carried out, e .g. , acetylation, amidation, phosphorylation, etc. In a specific embodiment, acetylation of the amino terminus and/or amidation of the carboxy-terminus are carried out.
  • any in vitro assay known in the art can be used to detect inhibition of proteoglycan/glycosaminoglycan bind to A/3, or inhibition of complement activation by a peptide disaccharide of the invention.
  • the assays described in the examples sections infra are employed.
  • Peptides or disaccharides demonstrated to have the desired activity in vitro can be tested in vivo for the desired inhibitory activity.
  • such compounds can be tested in suitable animal model systems prior to testing in humans, including but not limited to rats, mice, chicken, cows, monkeys, rabbits, etc.
  • suitable model systems are also used to demonstrate therapeutic utility.
  • any animal model system known in the art may be used.
  • the peptide or disaccharide inhibitory compounds of the invention can be preferably prepared as a pharmaceutical 0 composition with a pharmaceutically acceptable carrier for administration to a subject.
  • a pharmaceutically acceptable carrier for administration to a subject.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally 5 recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and o oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and 5 aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, 0 magnesium carbonate, magnesium stearate, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • These compositions can take the form of solutions, 5 suspensions, tablets, pills, capsules, powders, sustained-release formulations and the like.
  • Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions will contain a therapeutically effective amount of the active compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active, agent.
  • the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • compositions are administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • the peptides or disaccharides of the invention can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxy1 groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • the compounds of the present invention can be used for the treatment of symptoms of amyloidosis associated with Alzheimer's disease or other diseases including but not limited to AA (inflammation- associated)-amyloid, AL-amyloid (amyloid with deposition of immunoglobulin light chains), Down's syndrome, and prion diseases such as Creutzfeldt-Jakob disease, Gerstmann-Straussler syndrome, kuru, and scrapie.
  • AA inflammation- associated
  • AL-amyloid amyloid with deposition of immunoglobulin light chains
  • prion diseases such as Creutzfeldt-Jakob disease, Gerstmann-Straussler syndrome, kuru, and scrapie.
  • AD Alzheimer's disease
  • peptide or disaccharide compounds can be administered to a subject in need of prophylactic or therapeutic treatment.
  • the term "subject" refers to an animal, more preferably a mammal, and most preferably a human.
  • the therapeutic treatment can commence with diagnosis of AD, or the onset of AD, according to the appropriate criteria.
  • a therapeutically effective dose of a compound of the invention can be administered to the subject. What constitutes a therapeutically effective amount in a particular case will depend on a variety of factors within the knowledge of the skilled practitioner. Such factors include the physical condition of the subject being treated, the severity of the condition being treated, the disorder or disease being treated, and so forth.
  • the peptide or disaccharide compounds can be administered systemically, and more preferably parenterally, i.e., via an intraperitoneal, intravenous, perioral, subcutaneous, intramuscular, intraarterial, etc. route, in order to treat Alzheimer's disease.
  • Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • the peptides or disaccharides may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e .g.
  • oral mucosa may be administered together with other biologically active agents.
  • Administration can be systemic or local.
  • the compounds are directly administered to the cerebrospinal fluid by intraventricular injection. Pulmonary administration can also be employed.
  • the therapeutic compound can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid. , pp. 317-327; see generally ibid. )
  • the therapeutic compound can be delivered in a controlled release system.
  • a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)).
  • polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974) ; Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J.
  • a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e . g. , Goodson, in Medical Applications of Controlled Release, supra , vol. 2, pp. 115-138 (1984)).
  • Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)). The invention can be better understood by reference to the following examples, which are provided merely by way of exemplification and are not intended to limit the invention.
  • Val-His-His-Gln-Lys-Leu interacts with glycosaminoglycans in a pH-dependent manner; in particular, whether there is increased association of the peptide with the sulfated polysaccharides below pH
  • A/S(1-28) peptide for binding to heparin and chondroitin sulfate proteoglycan
  • Amyloid peptides (stored as 2 mM stock solutions in water) were diluted to concentrations of 0.25 or 0.5 mM and mixed with glycosaminoglycans (concentrations ranging from 0.1- 100 ⁇ M) for approximately 1 h at room temperature (21- 24° C) in either 20 mM sodium acetate pH 3.5, 4.0 or 5.0 with 150 mM NaCl, or 20 mM sodium phosphate pH 6.0, 7.0 or 8.0 containing 150 mM NaCl.
  • concentrations of amyloid and glycosaminoglycans used for individual experiments are noted in the figure legends and Tables. Typically, the final volume of the mixtures was 10 or 20 ⁇ l.
  • the peptides used were A/3(l-28) ([gln n ]-/S-amyloid; Bachem, Inc.), A/5(l-40) (synthesized by Biosynthesis, Inc.), A/S(25-35) (Bachem, Inc.), and A/3(13-17) (synthesized as described infra) .
  • glycosaminoglycans employed were heparin (porcine intestinal mucosa; CalBiochem, Inc.), heparan sulfate (bovine kidney; Seikagaku Corp.), chondroitin sulfate (bovine trachea chondroitin sulfate A; Sigma Chemical Co.), and dermatan sulfate (bovine mucosa chondroitin sulfate B; Sigma Chemical Co.).
  • A/S(l-28), A/3(25-35) and A/3(13-17) were injected onto a heparin affinity column (Bio-Rad Econo-Pac cartridge; 5 ml volume) that was equilibrated with either 20 mM sodium acetate, pH 4.0 or 20 mM sodium phosphate, pH 8.0.
  • the peptides were at a concentration of 0.25 mM in water, with 0.2 ml injected.
  • Non-bound peptides were eluted from the column with approximately 15 ml of the equilibration 5 buffer (flow rate of 1 ml/min) , and bound peptides were then eluted with a linear gradient of 0 to 0.5 M NaCl in equilibration buffer over 25 min. The change in NaCl concentration was monitored with an in-line conductivity meter. The elution of A ⁇ (1-28) was 0 monitored at 280 nm. For A/3(25-35), and A/S(13-17), 2 ml fractions were collected from the column and assayed for absorbance at 230 nm using a spectrophotometer.
  • Aggregation assays with A ⁇ (1-28) or A/3(1-40) were performed as described in Section 6.1.1, supra , in the presence of excess amounts of test peptides.
  • the test peptides for inhibition of aggregation were A ⁇ (13-17), o A ⁇ (lO-n) , A/8(l-28) (tested for its ability to inhibit aggregation of A/3(l-40), and poly-L-lysine, typically at 5.0 mM (about a 20-fold molar excess) concentration.
  • Tris-Tricine sample buffer 8% SDS, 24% glycerol, 0.1 M Tris base, 0.1 M Tricine, 0.05% 0 Bromophenol Blue.
  • Samples were analyzed on 16.5% Tris-Tricine gel (16.5% acrylamide, 1 M Tris base, 0.1% SDS, 13.3% glycerol, pH 8.45) with a 4% stacking gel (4% acrylamide, 1 M Tris base, 0.1% SDS, pH 8.45).
  • the anode buffer used was 0.2 M Tris base, pH 8.9, and 5 the cathode buffer was 0.1 M Tris base, 0.1 M Tricine, 0.1% SDS.
  • the gels were stained with 0.2% Coomassie brilliant blue in 45% methanol and 10% acetic acid, then destained in 45% methanol and 10% acetic acid. The gels were scanned with densitometer and A ⁇ (1-28) or A/8(l-40) protein bands were quantified.
  • the side chains of lysines were protected with butoxycarbonyl and those of histidines with fluorenylmethoxycarbonyl. All amino acid residues except glutamine were introduced using diisopropylcarbodiimide hydroxybenzotriazole; for glutamine, pentafluorophenyl ester was employed. Fluorenylmethoxycarbonyl groups were removed with piperidine-dimethylformamide and the desired peptide sequences were cleaved from the solid support with trifluoroacetic acid. The resulting peptides were purified by Sephadex G10 chromatography followed by Cjg-reversed phase HPLC.
  • heparin resembles the heparan sulfate glycosaminoglycan chains of HSPG in that both contain predominantly N-acetylglucosamine and uronic acids as constituent monosaccharides, the former has a higher degree of sulfation than the HSPG polysaccharides.
  • amyloid peptide of the AD plaques is 40-43 amino acids in length (SEQ ID NO:l) (Masters et al. , 1985, EMBO J.
  • Peptides corresponding to the heparin binding domain of A ⁇ were tested for the ability to inhibit 5 binding of A ⁇ (1-28) or A/3(1-40) to the glycosamino ⁇ glycans heparin and chondroitin sulfate proteoglycan.
  • the aggregation assay was used to demonstrate binding • of the A ⁇ peptide to the glycosaminoglycan.
  • a ⁇ (13-17) was tested at pH 4.0 (Fig. 10; Table 4).
  • A/3(l-28) aggregated with CSPG.
  • the aggregation was inhibited by A/3(13-17), thus indicating that A/3(13-17) competes with A ⁇ (1-28) for CSPG binding.
  • poly-L-lysine (MW 30,000- 70,000) was tested for the ability to compete with A ⁇ (1-40) for binding to heparin and to CSPG.
  • Poly-L- lysine which had no effect upon or slightly enhanced A ⁇ (1-40) solubility, did compete with A ⁇ (1-40) for binding heparin (Fig. 11) and CSPG (Fig. 12) .
  • Poly-L-lysine was present at 6 ⁇ g per sample.
  • the concentration of heparin was 15 ⁇ M.
  • A/3(1-28) increases as the pH value is lowered below 6- 7, and there is essentially no association of the peptide with the glycosaminoglycans at pH 8.
  • a ⁇ (1-28) In addition to the ability of A ⁇ (1-28) to bind various sulfated polysaccharides, we have shown that A/3(1-40) also associates with glycosaminoglycans. In contrast, a peptide containing residues 25-35 of the amyloid peptide does not show appreciable association with heparin. We propose that the glycosaminoglycan binding site is found at residues 12-17 of the amyloid peptide (also see Fraser et al., 1992, J. Neurochem. 59:1531-1540; and Kisilevsky, 1989, Neurobiol. Aging 10:499-500).
  • This region of the peptide contains the consensus heparin binding sequence (Cardin and Weintraub, 1989, Arteriosclerosis 9:21-32) Val-His- His-Gln-Lys-Leu, and the ionization state of one or both of the tandem histidines could modulate glycosaminoglycan binding. Supporting this conclusion is our observation that a pentapeptide consisting of the A/3(13-17) sequence binds to a heparin affinity column at pH 4.0, but does not show appreciable binding at pH 8.0.
  • the present invention is not limited by any particular theory or hypothesis, one possibility is that the core protein of HSPG (and perhaps other proteoglycans) initially associates with A/3, bringing a glycosaminoglycan chain in close apposition to residues 12-17 of the peptide. This could then alter the microenvironment around the histidine residues at positions 13 and 14, causing an elevation of the apparent pK's.
  • Yates et al. (1990, J. Neurochem. 55:1624-1630) have reported decrease of pH in the AD brain, which may be sufficient in magnitude to cause an increased association of amyloid with glycosaminoglycan chains.
  • proteoglycan would bind APP at the cell surface through its core protein, and some fraction of the APP with associated proteoglycan would escape secretase cleavage and be internalized into the endosomal/lysosomal pathway.
  • a glycosaminoglycan chain of the proteoglycan would bind tightly to the His-His-Gln-Lys domain of APP, thereby shielding the nonamyloidogenic "secretase" cleavage site at lys 16 .
  • glycosaminoglycans bind to A ⁇ (13-17) at pH values below neutrality.
  • the sulfated polysaccharides appear to bind to a consensus glycosaminoglycan-binding domain at residues 12-17 of the peptide, and one or both of two tandem histidines are likely to confer the pH dependence of this interaction.
  • amyloidogenesis may be prevented or even reversed.
  • PENTAPEPTIDE AND A HEPARIN DISACCHARIDE REVERSE THE GLYCOSAMINOGLYCAN MEDIATED RESISTANCE OF A ⁇ PEPTIDE TO DEGRADATION The following Example provides direct evidence that AjS peptide resists proteolytic degradation when aggregated with a proteoglycan, i . e . , that proteoglycans protect aged amyloid from degeneration. Furthermore, molecules that competitively inhibit binding of A ⁇ peptide to glycosaminoglycans were able to reverse this resistance of A ⁇ to proteolytic degradation.
  • CSPG Chondroitin sulfate proteoglycan
  • a ⁇ The ability of A ⁇ to resist proteolysis was evaluated by testing the amount remaining after incubation with polysaccharide followed by treatment with papain.
  • Reaction mixtures consisting of combinations of AjS(l-40), CSPG, dextran, and papain were prepared in 100 mM Tris, pH 6.0-7.4. The reaction mixture was incubated 45 min. Samples were incubated an additional 45 min with addition of the inhibitory molecule. Samples were then treated with papain for 8 - 18 hours.
  • 2X Tris-Tricine sample buffer (8% SDS, 24% glycerol, 0.1 M Tris base, 0.1 M Tricine, 0.05% Bromophenol Blue). Aliquots were loaded on 16.5% Tris-Tricine gel.
  • the separating gel consisted of 16.5% acrylamide, 1 M Tris base, 0.1% SDS, 13.3% glycerol, pH 8.45.
  • the stacking gel consisted of 4% acrylamide, 1 M Tris base, 0.1% SDS, pH 8.45. After electrophoresis; gels were stained with 0.2% Coomassie brilliant blue in 45% methanol, 10% acetic acid. Gels were scanned and A ⁇ bands were quantified using densitometry.
  • AjS from proteolysis potential inhibitors were added after incubation of the A/3 with CSPG, and prior to the addition of papain.
  • the three potential inhibitors of 19 week aged A/3(1-40)- proteoglycan/glycosaminoglycan binding that were tested were the peptide representing the heparin sulfate proteoglycan binding site of A/3 (two peptides, A/3(13-17) and Aj8(13-16), were tested), the peptide Glu-Ala-Glu-Asp-Val, which corresponds to AjS(20-24:Phe 20 ⁇ Glu 20 ) , and a heparin disaccharide - dUA-2s-[l->4]-GlcNS-6S) .
  • a ⁇ (l-40) MW 4329, LOT #ZJ209 from Bachem, Inc.
  • CSPG High molecular weight CSPG was sonicated for 14 hours to generate low molecular weight material.
  • AjS and CSPG were incubated together for 1 hour, following which 120 ⁇ g of AjS(13-16) and (13-17) were added and the incubation repeated for 1 hour at room temperature. 3. Papain (3 ⁇ g) was added and the samples were incubated for 18 hours at 37° C.
  • Tris- Tricine sample buffer (8% SDS, 24% glycerol, 0.1 M Tris base, 0.1 M Tricine, 0.05% Bromophenol blue) and samples were subjected to 16.5% Tris-Tricine SDS- polyacrylamide gel electrophoresis:
  • a ⁇ (13-16) and A ⁇ (13-17) disrupt A/3-CSPG association and cause 70% increase in A ⁇ proteolysis. These peptides bind proteoglycans.
  • the A/3(20-24: Phe 20 ⁇ Glu 20 ) pentapeptide was synthesized based on the sequence of amyloid (residues 20-24) that are believed to bond naturally in an anti- parallel jS-strand to the glycosaminoglycan-binding domain of A ⁇ (residues 13-17) .
  • the naturally occurring 20-24 sequence is Phe-Ala-Glu-Asp-Val, and Glu 22 and Asp 23 are likely to form stabilizing ion-pairs with His 13 and His 14 of the adjacent anti-parallel strand.
  • the peptide A/3 (20-24:Phe 20 ⁇ Glu 20 ) can reverse the CSPG-mediated protection of A ⁇ from papain proteolysis.
  • A/S(20- 24 Phe 20 ⁇ Gl ⁇ 20 ) appears to be effective at reducing proteoglycan-amyloid interaction.
  • This compound may be of greater therapeutic value than the A/3(13-17) or A ⁇ (13-16) compounds, since it should bind / SAP and not the glycosaminoglycan chains of proteoglycans. This could make the pentapeptide a more specific drug.
  • This molecule is similar to the A/3(20-24) peptide in that this disaccharide is derived from the glycosaminoglycan chains that bind residues 13-17 of amyloid.
  • the present example confirms the observation by Rogers et al. (1992, Proc. Natl. Acad. Sci. USA 89:10016-10020; 1992, Res. Immunol. 143(6) :624-630) that the /3-amyloid peptide (A/3) is capable of specifically activating the complement cascade.
  • This example also shows that a tetrapeptide corresponding to A/3(13-16) and a pentapeptide derived from A / S(20-24) are effective inhibitors of AjS-mediated complement activation.
  • s-SRBC sensitized-sheep red blood cells
  • 0.1 ml of buffer were added to each tube and the suspension was incubated for 1 h in a 37°C water bath. Any complement remaining from the initial overnight incubation with peptide causes lysis of the s-SRBCs, releasing hemoglobin into the medium.
  • the suspension was centrifuged for 5 minutes at 300 x g, and the extent of complement-mediated lysis of the s-SRBCs was determined by reading the optical density of the supernatant at 410 nM. Complement activity was expressed as a percent of control samples which did not receive peptide during the initial incubation.
  • a solid-phase binding assay was utilized for the evaluation of Clq binding to amyloid peptide.
  • This assay which is similar to that recently used by Rogers et al. (1992, Proc. Natl. Acad. Sci. USA 89:10016-10020), employs A ⁇ which is immobilized on membranes, followed by incubation with human Clq. Briefly, A/3 is dotted onto pre-soaked PVDF membranes. The membranes are then rinsed in Tris-buffered saline pH 7.4 (TBS), followed by blocking of the membrane with 5% dry milk in TBS.
  • TBS Tris-buffered saline pH 7.4
  • the membrane is incubated in a solution of Clq (Quidel; 10 ⁇ g/ml in TBS + 5% dry milk) for 2 h.
  • Non- bound Clq is removed by rinsing in TBS, and bound Clq is determined by overnight incubation with rabbit anti-human Clq antibody (Quidel; 1:1000 dilution).
  • the Clq antibody is visualized by a 2 h incubation with biotinylated goat anti-rabbit antibody (Quidel; 1:50 dilution) followed by color development with a Vectastain ABC Elite kit (peroxidase type) .
  • the amount of bound Clq was quantified through densitometric analysis of the stained membranes (PDI model DNA35 densitometer) .
  • the peptides used in these studies were A/3(1-40) (Bachem, Inc.), A/3(l-28) (Bachem, Inc.), or scrambled AjS(l-40) (custom synthesized by Biosynthesis, Inc.).
  • the scrambled peptide contains the same amino acid composition as A ⁇ (1-40), with its sequence randomly assigned.
  • Amyloid peptides were examined for activity as a function of their age in solution. In such experiments, peptides are referred to as "fresh” if they have been in aqueous solution for 1-3 days, and as “aged” if they have been in solution >25 days at 4°C.
  • the "aged" A ⁇ is aggregated and of high molecular weight as judged by the sedimentation of the peptide after centrifugation at 100,000 x g for 30 min. Moreover, the aged amyloid appears to exist as jS-fibrils as evidenced by its Congo Red birefringence. Ultracentrifugation reveals that the freshly prepared A/S(1-40) solutions are not appreciably aggregated. The actual ages of the "aged" peptide solutions used in each experiment are indicated in the results. The A/3(13-16) and A/5(20-24) peptides were prepared by solid phase synthesis using Fmoc chemistry.
  • AD is likely to be due in part to the formation of MAC in response to /3-amyloid peptide.
  • Fig. 20 The ability of the tetrapeptide comprising residues 13-16 of AjS to effectively block AjS activation of the entire complement cascade is seen in Fig. 20.
  • AjS(13-16) does not inhibit the activation of complement by aggregated human immunoglobulin, indicating that amyloid must bind to a different region of Clq than immunoglobulin. This is an important observation, since immunoglobulin is the normal activator of complement and compounds based on A/S(13-16) should not interfere with immunoglobulin-mediated complement activation throughout the body.
  • Residues 22 and 23 have negatively charged amino acids that neutralize the positive charge density around residues 13 and 14.
  • the pentapeptide Glu-Ala-Glu-Asp-Val was prepared. This peptide corresponds to AjS(20-24), with the naturally occurring Phe of residue 20 substituted with a negatively charged glutamic acid. This substitution was designed to allow ion-pairing between the glutamic acid and Lys 16 of the full-length A ⁇ :
  • peptides and their derivatives, are attractive therapeutic agents to reverse the formation of amyloid plaques and reduce the activation of complement within the brains of AD patients.
  • disaccharides such as heparin disaccharide are expected to inhibit A/3-mediated complement activation as well.
  • MAC attachment to membranes such as vitronectin, clusterin, or 5 protectin or peptides based on sequences from these proteins, could be administered to AD patients. These agents would reduce C5-C9 (MAC) attachment to neuronal membranes, and hence would reduce the "bystander” damage that leads to dystrophic neurites.

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Abstract

Composés qui inhibent la liaison de glycosaminoglycannes et de protéoglycannes au peptide bêta amyloïde (A(bêta); Aβ), et composés qui inhibent l'activation Aβ du complément. Selon un aspect, ledit composé est un peptide ayant une séquence d'acides aminés X-X-N-X, dans laquelle X est un acide aminé doté d'une chaîne latérale cationique et N est un acide aminé neutre. Selon un autre aspect, ledit composé est un peptide ayant une séquence d'acides aminés X-X-N-X-Z, dans laquelle X est un acide aminé doté d'une chaîne latérale cationique, N est un acide aminé neutre et Z est un acide aminé neutre. Selon un autre aspect encore, ledit composé est un peptide ayant une séquence d'acides aminés X1-N-X2-X3, dans laquelle au moins deux des éléments X1, X2 et X3 sont indépendamment un acide aminé doté d'une chaîne latérale anionique et le troisième X est un acide aminé doté d'une chaîne latérale anionique ou un acide aminé neutre et N est indépendamment un acide aminé neutre. Selon un autre aspect encore, ledit composé est un disaccharide anionique. La présente invention concerne également un procédé de traitement de la maladie d'Alzheimer, qui consiste à administrer une quantitié thérapeutiquement efficace d'un composé décrit ci-dessus à un sujet souffrant de la maladie d'Alzheimer. Des compositions pharmaceutiques sont également décrites.
PCT/US1994/009853 1993-08-31 1994-08-29 Inhibition de la liaison du beta amyloïde aux glycosaminoglycannes pour le traitement de la maladie d'alzheimer WO1995006477A1 (fr)

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997026913A1 (fr) * 1996-01-26 1997-07-31 The Trustees Of Columbia University In The City Of New York POLYPEPTIDE PROVENANT D'UN EXTRAIT DE POUMON ET SE FIXANT A UN PEPTIDE AMYLOÏDE-$g(b)
US6310073B1 (en) * 1998-07-28 2001-10-30 Queen's University At Kingston Methods and compositions to treat glycosaminoglycan-associated molecular interactions
US6562836B1 (en) 1999-05-24 2003-05-13 Queen's University Of Kingston Methods and compounds for inhibiting amyloid deposits
US7371731B2 (en) 1998-08-28 2008-05-13 Gerardo Castillo Methods for using glucose pentasulfate for treating amyloid associated with type II diabetes
US8835654B2 (en) 2004-12-22 2014-09-16 Bhi Limited Partnership Method and compositions for treating amyloid-related diseases
US9499480B2 (en) 2006-10-12 2016-11-22 Bhi Limited Partnership Methods, compounds, compositions and vehicles for delivering 3-amino-1-propanesulfonic acid
US10751382B2 (en) 2016-11-09 2020-08-25 The University Of British Columbia Anti-amyloid beta antibodies binding to a cyclic amyloid beta peptide
US10759837B2 (en) 2015-11-09 2020-09-01 The University Of British Columbia Anti-amyloid beta antibodies binding to a cyclic amyloid beta peptide
US10774120B2 (en) 2015-11-09 2020-09-15 The University Of British Columbia Anti-amyloid beta antibodies binding to a cyclic amyloid beta peptide
US10772969B2 (en) 2015-11-09 2020-09-15 The University Of British Columbia N-terminal epitopes in amyloid beta and conformationally-selective antibodies thereto
US12071472B2 (en) 2016-07-18 2024-08-27 The University Of British Columbia Methods of reducing toxicity induced by Amyloid beta (A-beta) oligomers using antibodies specific to A-beta oligomers

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5164295A (en) * 1991-03-06 1992-11-17 The Upjohn Company Method for identifying amyloid protein-extracellular matrix protein affinity altering compounds

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5164295A (en) * 1991-03-06 1992-11-17 The Upjohn Company Method for identifying amyloid protein-extracellular matrix protein affinity altering compounds

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BRAIN RESEARCH, Vol. 601, issued 1993, L. BUEE et al., "Binding of Secreted Neuroblastoma Proteoglycans to the Alzheimer's Amyloid A4 Peptide", pages 154-163. *
PROC. NATL. ACAD. SCI. U.S.A., Vol. 85, issued April 1988, D. ALLSOP et al., "Immunohistochemical Evidence for the Derivation of a Peptide Ligand From the Amyloid beta-Protein Precursor of Alzheimer Disease", pages 2790-2794. *

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