EP2089419A2

EP2089419A2 - Inhibition of beta-amyloid aggregation

Info

Publication number: EP2089419A2
Application number: EP07824323A
Authority: EP
Inventors: Jesus Zurdo; Susan Fowler; Ernest Giralt; Meritxell Teixido; Natalia Carulla
Original assignee: Zapaloid Ltd
Current assignee: Zapaloid Ltd
Priority date: 2006-10-27
Filing date: 2007-10-26
Publication date: 2009-08-19
Also published as: WO2008050133A3; WO2008050133A2

Abstract

This invention relates to the peptidyl inhibitors of ABeta1-42 aggregation. These inhibitors may be useful, for example, in the treatment of Alzheimer's disease

Description

Inhibition of Beta-Amyloid Aggregation

This invention relates to the inhibition of protein aggregation, in particular the inhibition of Aβ_1-42 aggregation. This may be useful, for example, in the treatment of Alzheimer's disease

Alzheimer's disease (AD) is characterised by the aggregation of the beta amyloid peptide (AP_1-42) in the brain of patients. The beta amyloid peptide (Aβ_1-42) consists of residues 673 to 713 of the amyloid precursor protein (APP: NCBI Gene ID 351; P05067; NP_000475.l Gl: 4502167) . Methods of reducing the amount or toxicity of these aggregates would be useful in the treatment of AD and other diseases related to the deposition of Aβ_1-42 such as cerebral amyloid angiopathy (CAA) and inclusion-body myositis (IBM) .

The present inventors have identified peptides which inhibit the aggregation of the Aβ_1-42 peptide and may therefore be useful in the treatment of Alzheimer's disease

One aspect of the invention provides a peptide consisting of four to ten D-amino acids having the reverse sequence of a contiguous amino acid sequence within the region between residues 16 and 42 of Aβ_1-42.

A peptide described herein may inhibit the aggregation of Aβ_1-42 and may, for example, consist of 4, 5, 6, I₁ 8, 9 or 10 D-amino acids, preferably 6, 7 or 8 D-amino acids.

In some preferred embodiments, a peptide may consist of four to ten D-amino acids having the reverse sequence of a contiguous amino acid sequence in the region between residues 16-42 of Aβ_i-42. In other words, the D-amino acid sequence in the N terminal to C terminal direction corresponds to the contiguous amino acid sequence of Aβ_1-42 in the C terminal to N terminal direction. A D-amino acid sequence which is the reverse of an L-amino acid sequence is commonly known as a 'retroenantiomer' of that sequence.

For example, a peptide may consist of the reverse sequence of a contiguous amino acid sequence which comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, or all the residues from a region selected from the group consisting of residues 16-22, residues 30-36, residues 32-38, residues 34-40 and residues 36-42 of Aβi_-42. In some preferred embodiments, a peptide may consist of a sequence of D-amino acids selected from the group consisting of: eaffvlk, vmlgiia, wggvml, ggvmlgi and aiwggv.

Peptides of the invention also encompass sequences which consist of an amino acid sequence set out herein with 1, 2, 3 or 4 D-amino acids added, deleted or substituted.

For example, 1, 2, 3 or 4 D-amino acids may be added or deleted from the N-terminal or C-terminal of a peptide sequence set out herein.

The 1, 2, 3 or 4 additional D-amino acids which are added to a peptide set out herein may be the reverse sequence of amino acids which adjoin the N-terminal or C-terminal of the contiguous sequence in Aβχ.₄₂. Alternatively, the 1, 2, 3 or 4 additional D-amino acids may be residues which are not the reverse sequence of amino acids which adjoin the N-terminal or C-terminal of the contiguous sequence in Ap_1-42 (i.e. they may be heterologous amino acids) . In some embodiments, one or more N-methyl-phenylalanine residues may be added to the N terminal, as described below, to facilitate transport across the blood brain barrier.

A substitution may be a conservative or non-conservative substitution. For example, a peptide may consist of sequences having one, two, three or more conservative or non-conservative substitutions relative to a sequence set out herein. A conservative substitution is a replacement of a D-amino acid residue with another of similar properties, such as charge, polarity and/or hydrophobicity. For example, conservative substitutes for an amino acid within the native polypeptide sequence can be selected from other members of the class to which the amino acid belongs . Amino acids can be divided into the following four groups: (1) acidic amino acids, (2) basic amino acids, (3) neutral polar amino acids, and (4) neutral, nonpolar amino acids. Representative amino acids within these various groups include, but are not limited to, (1) acidic (negatively charged) amino acids such as aspartic acid and glutamic acid; (2) basic (positively charged) amino acids such as arginine, histidine, and lysine; (3) neutral polar amino acids such as glycine, serine, threonine, cysteine, cystine, tyrosine, asparagine, and glutamine; and (4) neutral nonpolar (hydrophobic) amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Conservative substitution tables listing functionally similar amino acids are known in the art (Altschul, S. F. 1991 Journal of Molecular Biology 219: 555-665, Crighton (1984) Proteins, W. H. Freeman and Company) . For example, a peptide consisting of a sequence having one, two, three or more conservative substitutions may show 95%, 99% or 100% sequence similarity to a sequence set out herein. Amino acid similarity may be defined with reference to the algorithm GAP (Accelerys) , or the TBLASTN program, of Altschul et al . (1990) J. MoI. Biol. 215: 403- 10.

In embodiments, a peptide may consist of a sequence of D-amino acids in the reverse sequence of a contiguous amino acid sequence in the region between residues 16-42 of Aβ_!_₄₂, with one, two, three or more substitutions which reduce or prevent beta strand association. For example, one or more, 2 or more, 3 or more or 4 D-amino acids in a sequence set out herein may be replaced by D-proline. In some embodiments, the residue at position 2 and/or position 3 may be replaced by D-proline. Examples of such peptides include peptides consisting of a D-amino acid sequence selected from the group consisting of: epffvlk, eapfvlk, vplgiia, vmpgiia, vpggvml, wpgvml , gpvmlgi, ggpmlgi, apwggv, and aipvggv (where lower case letters denote D amino acids) .

Examples of suitable peptides include ZP-0281 to ZP-0307, as set out in table 1.

Another aspect of the invention provides a peptide consisting of three to six D-amino acids, wherein said peptide comprises one more charged residues at both the N and C termini, and one or more central residues between said charged residues, wherein the one or more central residues comprise a hydrophobic residue.

Charged residues may be selected from the group consisting of lysine (K) , histidine (H) , arginine (R) , aspartic acid (D) , glutamic acid (E) and any non naturally occurring amino acids that contain a charge .

Preferably, all the charged residues at one terminal of the peptide have the same charge. For example, the N terminal residues may be positively charged residues, such as R or K, and the C terminal residues may be negatively charged residues such as D or E.

In some preferred embodiments, the one or more charged residues at the N terminal have a different charge from the one or more charged residues at the C terminal. For example, the charged residue at the N terminus of the peptide may be R or K and the charged residue at the C terminus may be D or E or vice versa.

The central region preferably comprises one to three residues. Hydrophobic residues may be selected from the group consisting of tryptophan (W) , phenylalanine (F) , leucine (L) , isoleucine (I) , valine (V) and methionine (M) or any non-naturally occurring amino acids that are hydrophobic in nature.

The central region may further comprise one or more residues selected from the group consisting of glycine (G) , alanine (A) , serine (S) and isoleucine (I) which act as spacers between the charged and hydrophobic amino acids.

In some preferred embodiments, the central region consists of a sequence selected from the group consisting of GWG, GFG, SWS, SFS, AWA, GWI, SWI, two amino acid fragments of any of these, W and F.

Examples of suitable peptides include peptides selected from the group consisting of kgwge rgwge, kgwgd, rgwgd, kgwe, kwge, kwe, kgfge, rgfge, kgfgd, rgfgd, kgfe, kfge, kfe, kswse, rswse, kswsd, rswsd, kswe, kwse, ksfse, rsfse, ksfsd, rsfsd, ksfe, kfse, kawae, rawad, kgwge, rgwge, kgwgd, rgwgd, kgfge, rgfge, kgfgd, rgfgd, kswse, rswse, kswsd, rswsd, ksfse, rsfse, ksfsd, rsfsd, rgwid, rwid, rswid and rwsd (ZP-0326 to ZP- 0375 in Table 1) (where lower case letters denote D amino acids) .

In some embodiments, 1, 2, 3 or 4 D-amino acids may be added to the N-terminal and/or C-terminal of a peptide sequence set out herein. For example, in some embodiments, one or more N-methyl-phenylalanine residues may be added to the N terminal, as described below, to facilitate transport across the blood brain barrier.

Peptides of the invention also encompass sequences which consist of a sequence set out herein with 1, 2, 3 or 4 modified D-amino acids. D-amino acids in peptides described herein may be modified, for example by the introduction of a substituent chemical group, for example at the N position. Suitable substituent groups include halogens such as F, nitrate, and alkyl groups, such as methyl or acetyl. Peptides of the invention may contain one or more substitutions at any position in the sequence with a non natural D or L amino acid. For example β-cyclohexane D-alanine (D-Cha) , D-homophenylalanine (D- HomoPhe) , D-Napthylalanine (D-NaIl), D-2-Napthylalanine (D-Nal2) , D- 2-pyridyalanine (D-PyrAla) , 4-fluoro-D phenylalanine (D-pFPhe) , 1, 2 , 3 , 4-D-tetrahydroisoquinoline-3-carboxylic acid (D-Tic) , 5-phenyl- pyrolidine-2-carboxylic acid(D-2R, 5S) , 4-nitro-D-phenylalanine (D- pNO2Phe)and 3(3,4 dihydroxyphenyl) D-alanine (D-DOPA) have been used here as substitutions in the third amino acid position of the inhibitor series ZP-0387 to ZP-0397 (See tables 1 and 2) .

An amino acid modification may reduce or prevent beta strand association. For example, one or more, for example 2, 3 or 4 D-amino acids in the peptide sequence may be N- substituted, preferably N- alkylated, for example N-methylated or N acetylated.

Examples of peptides comprising modified or non-naturally occurring amino acids include compounds ZP-0387 to ZP-0398 and ZP-0602 to ZP- 0607, as shown in Table 1. These are modified retroenantiomers .

Anther aspect of the invention provides a peptide consisting of four to ten D-amino acids, which forms a beta sheet interaction with at least part of an aggregation region in Aβ_1-42.

The peptide preferably consists of a sequence which is neither the forward or reverse sequence of Ap_1-42 (i.e. a heterologous sequence) which has been designed to specifically interact with at least part of an aggregation region in Aβ_1-42.

Suitable peptides may be designed using the in silico techniques described in US601821553. The sequence of the peptide may be further optimised to maximise interaction with Aβ_1-42 using energy minimisation routines.

Peptides designed using this strategy include ZP-308 to ZP-0316, ZP- 407 and ZP-0671 to ZP-0716 (Table 1) .

Peptides may be generated wholly or partly by chemical synthesis. D-amino acid peptides, such as retroenantiomers, may be produced by employing D- form derivatized amino acid residues in the chemical synthesis. Suitable D-amino acids for solid phase peptide synthesis are commercially available (e.g., Advanced Chem Tech, Louisville; Nova Biochem, San Diego; Sigma, St Louis,- Bachem California Inc., Torrance, etc.) . The peptides can be readily prepared, for example, according to well-established, standard liquid or, preferably, solid-phase peptide synthesis methods, general descriptions of which are broadly available (see, for example, in J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Illinois (1984) , in M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag, New York (1984); in J. H. Jones, The Chemical Synthesis of Peptides. Oxford University Press, Oxford 1991; in Applied Biosystems 430A Users Manual, ABI Inc., Foster City, California , in G. A. Grant, (Ed.) Synthetic Peptides, A User's Guide. W. H. Freeman & Co., New York 1992, E. Atherton and R. C. Sheppard, Solid Phase Peptide Synthesis, A Practical Approach. IRL Press 1989 and in G. B. Fields,

(Ed.) Solid-Phase Peptide Synthesis (Methods in Enzymology Vol.

289) . Academic Press, New York and London 1997) , or they may be prepared in solution, by the liquid phase method or by any combination of solid-phase, liquid phase and solution chemistry, e.g. by first completing the respective peptide portion and then, if desired and appropriate, after removal of any protecting groups being present, by introduction of the residue X by reaction of the respective carbonic or sulfonic acid or a reactive derivative thereof . Peptides as described above may be fused to one or more additional sequences, for example sequences which are not retroenantiomers of Aβ_1-42 sequences. Peptides and oligopeptides comprising peptides as described above are also provided as aspects of the present invention, particularly wherein the peptide is fused to one or more sequences which do not correspond to the forward or reverse sequence of Aβ_1-42, such as retro-enantiomeric sequences (i.e. heterologous sequences) .

By "heterologous" is meant from a different source to the sequence in question, for example, when the peptide is a retro-enantiomer of a natural Aβi_₄₂ sequence, a heterologous sequence is a sequence other than a retro-enantiomer of the natural Aβi_-42 sequence joined by a peptide bond without intervening amino acids to the contiguous A^₁._i2 sequence described herein. Usually, where heterologous amino acids are fused to the peptide, the whole contiguous sequence of amino acids does not occur within beta- Aβ_1-42, and may be 10 or more, preferably 15 or more, more preferably 20 or more, 25 or more or 30 or more amino acids. Heterologous sequences of amino acids which may be fused to a peptide described herein may include antibodies or antibody fragments, such as Fabs, F(ab')₂ ^s _/ dAbs, Fvs, and scFvs, neurotrophins such as NGF BDNF, NT3 , and GDNF, Insulin-like Growth Factors, such as IGFl and IGF2 , transferrin and other peptides that bind to the transferrin receptor, and other coupling partners involved in BBB transport, as described herein.

Peptides and oligopeptides as described herein may be N-terminal and/or C-terminal modified, for example by addition of a coupling partner or moiety. Coupling partners which may be linked to a peptide may include protecting groups or pegylation for example to help to increase the half-life of the peptide in vivo, and targeting groups . Suitable protecting groups are well-known in the art (e.g., Greene e al . , (1991) Protective Groups in Organic Synthesis, 2nd ed. , John Wiley & Sons, Inc. Somerset, N.J.) and include acetyl, amide, and 3 to 20 carbon alkyl groups, Fmoc, t-boc, 9-fluoreneacetyl group, 1- fluorenecarboxylic group, 9-florenecarboxylic group, 9-fluorenone-1- carboxylic group, benzyloxycarbonyl , Xanthyl (Xan) , Trityl (Trt) , 4- methyltrityl (Mtt) , 4-methoxytrityl (Mmt) , 4-methoxy-2 , 3 , 6- trimethylbenzenesulphonyl (Mtr) , Mesitylene-2-sulphonyl (Mts) , 4,4- dimethoxybenzhydryl (Mbh),Tosyl (Tos) , 2 , 2 , 5, 7, 8-pentamethyl chroman-6-sulphonyl (Pmc) , 4-methylbenzyl (MeBzI) , 4-methoxybenzyl (MeOBzI) , Benzyloxy (BzIO) , Benzyl (BzI) , Benzoyl (Bz) , 3-nitro-2- pyridinesulphenyl (Npys) , 1- (4, 4-dimentyl-2 , 6- diaxocyclohexylidene) ethyl (Dde) , 2 , 6-dichlorobenzyl (2 , 6-DiCl-Bzl) , 2-chlorobenzyloxycarbonyl (2-Cl-Z) , 2-bromobenzyloxycarbonyl (2-Br- Z) , Benzyloxymethyl (Bom) , t-butoxycarbonyl (Boc) , cyclohexyloxy (cHxO) ,t-butoxymethyl (Bum), t-butoxy (tBuO) , t-Butyl (tBu) , Trifluoroacetyl (TFA), Caffeic acid, formyl-, biotin and carboxyfluorescein.

In some embodiments, an acetyl group may be used to protect the amino terminus and/or an amide group may be used to protect the carboxyl terminus. Acetylation may, for example, be accomplished during the synthesis when the peptide is on the resin using acetic anhydride. Amide protection may, for example, be achieved by the selection of a proper resin for the synthesis.

Other coupling partners include Blood Brain Barrier (BBB) transport moieties, which may, for example be attached to the N or C terminal of the peptide sequence. A Blood Brain Barrier (BBB) transport moiety may include moieties which facilitate passive diffusion across the BBB and moieties that interact with a receptor or carrier and cross the BBB by receptor or carrier mediated endocytosis, such as Sweet Arrow Peptide (SAP) (Fernandez-Carneado et al Angew Chem Int Ed Engl 2004 43 14 1811-1814), retroviral TAT protein (C. Foerg et al Biochemistry 2005 44 72) , transferrin and transferrin receptor binding peptides. Suitable Blood Brain Barrier (BBB) transport moieties include L or D N-methyl phenylalanine (NMePhe) which has been shown to enhance transport across the BBB (Conradi, R. A. et al Pharm. Res. (1992) 9, 435-439; Chikhale, E. G. et al Pharm. Res. 1994, 11, 412-419; Chikhale, E. G. et al J. Pharmacol. Exp. Ther. (1995) 273, 298-303), transferrin, IGFl, IGF2 and leptin. Peptides may be synthesised with one or more, for example 1, 2, 3 or 4 N-methyl phenylalanine residues using standard synthesis techniques. See example ZP-0387 to ZP-0398, ZP-0602, ZP-0603, ZP- 0606 and ZP-0607 in table 1

Techniques for coupling peptides to both peptidyl and non-peptidyl coupling partners are well-known in the art.

A compound comprising a peptide as described herein linked to one or more coupling partners is provided by another aspect of the invention.

A peptide or compound as described herein, for example a compound described above or shown in Table 1, may be used in a method of treatment of the human or animal body, for example for use in the treatment of Alzheimer's Disease, or in the manufacture of a medicament for the treatment of Alzheimer's Disease and other diseases related to the deposition of Ap_1-42 such as cerebral amyloid angiopathy

A method of treatment of Alzheimer's disease or other disease characterised by the deposition of Ap_1-42 may comprise; administering a peptide as described herein as described herein to an individual in need thereof . Administration of a peptide or compound described herein is preferably in a "prophylactically effective amount" or a "therapeutically effective amount" (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors.

A peptide or compound as described herein described herein may be administered as a pharmaceutical composition. A pharmaceutical composition may include, in addition to the peptide or compound, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, nasal or by injection, e.g. cutaneous, subcutaneous or intravenous.

Another aspect of the invention provides a method of producing a pharmaceutical composition, for example for use in treating Alzheimer's disease or other disease characterised by the deposition of APi_-42, comprising; admixing a peptide or compound described herein or exemplified in Table 1 with a pharmaceutically acceptable excipient, carrier, buffer or stabiliser.

Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

Pharmaceutical compositions suitable for nasal administration, wherein the carrier is a solid, include a coarse powder having a particle size, for example, in the range of about 20 to about 500 microns which is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a liquid for administration as, for example, nasal spray, nasal drops, or by aerosol administration by nebuliser, include aqueous or oily solutions of the active compound.

For intravenous, intramuscular, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen- free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, or Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

A composition comprising a peptide or compound described herein may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

Controls are employed as appropriate within the routine knowledge and expectation of those skilled in the art.

Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure. All documents mentioned in this specification are incorporated herein by reference in their entirety.

The invention encompasses each and every combination and sub- combination of the features that are described above.

Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures described above and tables described below.

Figure 1 shows the inhibition of Aβ_1-42 aggregation by a selection of inhibitors from the group ZP-0326-ZP-0375.

Figure 2 shows the inhibition of Aβ_1-42 aggregation by inhibitors designed to interact with the C-terminal region of Aβl-42.

Figure 3 shows the inhibition of Aβi_-42 aggregation by variants of ZP-0312 with BBB translocation and cell internalisation moieties at the N-terminus.

Figure 4 shows the inhibition of Aβi_-42 aggregation by inhibitors with one NMePhe substitution at position 2 in the sequence.

Figure 5 shows the inhibition of Aβl-42 mediated toxicity for compounds ZP-0336, ZP-0305 and ZP-0312.

Figure 6 shows a reduction in Aβl-42 aggregation related toxicity for compounds ZP-0387 and ZP-0391.

Table 1 shows examples of Aβ_1-42 aggregation inhibitors in accordance with the invention. L and D amino acids are denoted by their prefix. (Ac) denotes acetylation and NMePhe denotes N-methyl phenylalanine. Table 2 shows the chemical structures of the non-naturally occurring amino acids used in inhibitor synthesis

SEQ ID NO: 1 shows the amino acid sequence of Aβ_1-42.

Materials and Methods Peptide Synthesis.

All peptides were synthesized by standard F-moc synthesis. Peptides containing non-natural amino acids were synthesised using standard techniques. Cruz M. et al J. Pept . Res. (2004) 63, 324- 328) .

AB42 aggregation assays.

All AB42 aggregation assays were performed in 25 mM Tris-Cl pH 7.4 with 0.01% sodium azide and 20 μM Thioflavin T. Reactions were set up in a 96 well plate with a final Aβl-42 concentration of 10 μM. Reactions with various concentrations of inhibitor were monitored and compared to an Aβl-42 alone control. Reactions were incubated at 37 °C and Thioflavin T fluorescence was measured every 10 minutes for 24 or 48 hours.

Following acquisition of the aggregation data, kinetic traces were fit using Aggrefit software which fits the data to a Sigmoidal function f (x) =k+A/ (1+exp ( -b (t-tθ) ) ) from which the lag time, rate of aggregation and total change in ThT fluorescence may be calculated.

In-vitro toxicity assay

SHSY5Y cells were seeded overnight on a 96 well plate at 2 x 10⁴ cells per well. Cells were then put in 200 μl serum free media and Aβ_1-42 was added to a final concentration of 10 μM. Inhibitor was added at various concentrations. Plates were incubated at 37⁰C with 5% C02 and toxicity levels due to Aβl-42 aggregation were assessed after 24 and 48 hours using the LDH (CytoTox-One Homogeneous Membrane Integrity Assay - Promega) and MTS (CellTiter 96 Aqueous One Solution Cell Proliferation Assay - Promega) assays.

Results Inhibition of Aβl-42 aggregation

All experiments were performed as described in the methods described with an Aβl-42 concentration of 10 μM. Results are presented in Figures 1 to 4. Figure 1 shows inhibition of Aβl-42 aggregation by a selection of inhibitors from the group ZP-0326-ZP-0375. In this case the inhibitor was added at a 20 fold molar excess. Inhibition was indicated by the resulting reduction in rate of aggregation and reduction in endpoint Thioflavin T (ThT) fluorescence. Figure 2 shows inhibition of Aβl-42 aggregation mediated by inhibitors ZP- 0305 and ZP-0312. Aβl-42. ZP-0305 is an example of a 7 residue peptide designed to inhibit aggregation of Aβl-42 and ZP-0312 is an example of a 5 residue peptide designed using Zyentia's technology to optimise the interaction of the inhibitor with Aβl-42. In both cases inhibition is apparent as an increase in lag phase and a reduction in rate of aggregation and final ThT fluorescence. The level of inhibition is dose dependant and these inhibitors are active at concentrations lower than that of Aβl-42 in the reaction.

Figure 3 shows inhibition of Aβl-42 by variants of ZP-0312 with BBB translocation and cell internalisation moieties at the N-terminus. ZP-0402 has 4 NMePhe residues and ZP-0609 has L-DOPA at the N- terminus . This shows that even with these moieties present the inhibitor is still active. ZP-0402 shows dose dependant inhibition as demonstrated by the increase in lag phase with increasing concentration of compound. In the presence of ZP-0609 aggregation is entirely inhibited in the timeframe of the observation. Figure 4 shows inhibition of Aβl-42 aggregation in the presence of inhibitors from the series with one NMePhe substitution at position 2 in the sequence. ZP- 0387 and ZP- 0391 are shown representative of the series ZP-0387-ZP-0398 which contain a non natural amino acid substitution at position 3 in the inhibitor sequence. The data show that for ZP- 0387 and ZP-0391 inhibition is observed at sub equimolar ratios and at in some cases abolished completely in the timeframe of the observation.

Inhibition of Aβl-42 mediated toxicity

Figure 5 shows inhibition of Aβl-42 mediated toxicity for compounds

ZP-0336, ZP-0305 and ZP-0312. In all reactions, the final concentration of Aβl-42 was 10 μM and ZP-0305 and ZP-0312 are also at a final concentration of 10 μM. ZP-0336 was added at a 5 fold molar excess. In the presence of all inhibitors, cell viability is increased by >30%. Figure 6 shows cell viability for compounds ZP- 0387 and ZP-0391. In all cases, inhibitor was added at a final concentration of 10 μM. A dramatic increase in viability after 48h is seen with all inhibitors compared to Ap_1-42 alone indicating that they are very effective in preventing aggregation mediated toxicity. In all cases, the inhibitor alone was added to the cells as a control . None of the compounds showed any toxicity at the concentrations tested.

Sequences

DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGW IA

SEQ ID NO: 1

Table 1

Table 2

Claims

Claims :

1. A peptide consisting of four to ten D-amino acids having the reverse sequence of a contiguous amino acid sequence within the region between residues 16 and 42 of Aβl-42.

2. A peptide according to claim 1 wherein the peptide inhibits the aggregation of Aβl-42.

3. A peptide according to claim 1 or 2 having the reverse sequence of a contiguous amino acid sequence which comprises one or more residues from a region of Aβ_1-42 selected from the group consisting of residues 16-22, residues 30-36, residues 32-38, residues 34-40 and residues 36-42.

4. A peptide according to claim 3 consisting of a sequence of D- amino acids selected from the group consisting of: eaffvlk, vmlgiia, wggvml , ggvmlgi and aiwggv.

5. A peptide according to claim 3 or claim 4 wherein one or more D-amino acid residues of said peptide is replaced by proline.

6. A peptide according to claim 5 wherein the D-amino acid residue at position 2 or position 3 of said peptide sequence is replaced by proline.

7. A peptide according to claim 6 consisting of the sequence epffvlk, eapfvlk, vplgiia, vmpgiia, vpggvml , wpgvml , gpvmlgi, ggpmlgi, apwggv, and aipvggv, where lower case letters denote D amino acids .

8. A peptide consisting of the d-amino acid sequence of a peptide according to any one of claims 3 to 7 with the addition, deletion or substitution of 1 to 3 D-amino acid residues.

9. A peptide consisting of the d-amino acid sequence of a peptide according to any one of claims 3 to 7 with 1 to 3 additional N terminal residues .

10. A peptide consisting of a sequence of three to six D-amino acid residues, wherein said sequence comprises one more charged residues at both the N and C termini of said sequence and one or more central residues between said charged residues, wherein the one or more central residues comprise a hydrophobic residue .

11. A peptide according to claim 10 wherein the charged residues are independently selected from the group consisting of K, H, R, D and E.

12. A peptide according to claim 11 wherein the charged residue at the N terminus is R or K.

13. A peptide according to claim 11 or claim 12 wherein the charged residue at the C terminus is D or E.

14. A peptide according to any one of claims 10 to 13 wherein the hydrophobic residue is W, F, Y, L, I, V or M.

15. A peptide according to claim 14 wherein the one or more central residues further comprise one or more residues independently selected from the group consisting of G, A, S, I.

16. A peptide according to claim 15 wherein the one or more central residues consist of a sequence selected from the group consisting of GWG, GFG, SWS, SFS, AWA, GWI, SWI, two amino acid fragments of any of these, W and F.

17. A peptide according to claim 16 consisting of a sequence selected from the group consisting of kgwge rgwge, kgwgd, rgwgd, kgwe, kwge, kwe, kgfge, rgfge, kgfgd, rgfgd, kgfe, kfge, kfe, kswse, rswse, kswsd, rswsd, kswe, kwse, ksfse, rsfse, ksfsd, rsfsd, ksfe, kfse, kawae, rawad, kgwge, rgwge, kgwgd, rgwgd, kgfge, rgfge, kgfgd, rgfgd, kswse, rswse, kswsd, rswsd, ksfse, rsfse, ksfsd and rsfsd, where lower case letters denote D amino acids.

18. A peptide which consists of the D-amino acid sequence of a peptide according to any one of claims 10 to 17 with 1 to 3 additional N terminal residues.

19. A peptide consisting of four to ten D-amino acids which forms a beta sheet interaction with at least part of Aβ_1-42.

20. A peptide according to claim 19 consisting of a sequence selected from the group consisting of D-Phe-D-Lys-D-Val-D-Gly-D-Leu, Trp-D-Lys-D-Val-D-Gly-D-Leu, D-Phe-D-Lys-D-VaI-D-Val-D-Leu, D-Phe-D-Lys-D- VaI-D-Lys-D-Leu, D-Phe-D-VaI-D-VaI-D-Lys-D-Leu, D-Phe-D-Val-D-Lys-D-Lys-D- Leu, D-Phe-D-Gly-D-Lys-D-Val-D-Val-D-Leu, D-Tyr-D-Gly-D-Lys-D-Val-D-Val-D- Leu, D-Trp-D-Gly-D-Lys-D-VaI-D-Val-D-Leu, D-Val-D-Lys-D-Leu-D-Gly-D-Ile-D- Ile-D-Ala, D-Val-D-Lys-D-Leu-D-Gln-D-Ile-D-Ile-D-Ala, D-Val-D-Lys-D-Met-D- GIy-D- Ile-D-Ile-D-Ala, D-VaI-D-Lys-D-Met-D-Gln-D-Ile-D-Ile-D-Ala, D-VaI-D- Lys-D-Met-D-Gln-D-Tyr-D-Ile-D-Ala, D-VaI-D-Lys-D-Leu-D-Gly-D-lie-D-Ile-D- Asn, D-Val-D-Lys-D-Leu-D-Gln-D-Ile-D-Ile-D-Asn, D-Val-D-Lys-D-Met-D-Gly-D- Ile-D-Ile-D-Asn, D-Val-D-Lys-D-Met-D-Gln-D-Ile-D-Ile-D-Asn, D-Val-D-Lys-D- Met-D-Gln-D-Tyr-D-Ile-D-Asn, D-AIa-D-Ile-D-Ile-D-Gly-D-Leu-D-Lys-D-VaI, D- AIa-D- lie-D-Ile-D-Gln-D-Leu-D-Lys-D-VaI, D-Ala-D-Ile-D-Ile-D-Gly-D-Met-D- Lys-D-Val, D-AIa-D-Ile-D-Ile-D-Gln-D-Met-D-Lys-D-VaI, D-AIa-D- Ile-D-Tyr-D- Gln-D-Met-D-Lys-D-Val, D-Asn-D-Ile-D-Ile-D-Gly-D-Leu-D-Lys-D-Val , D-Asn-D- Ile-D-Ile-D-Gln-D-Leu-D-Lys-D-Val, D-Asn-D-Ile-D-Ile-D-Gly-D-Met-D-Lys-D- VaI, D-Asn-D- He-D- lie-D-Gln-D-Met-D-Lys-D-VaI, D-Asn-D-Ile-D-Tyr-D-Gln-D- Met-D-Lys-D-Val, D-Phe-D-Phe-D-Val-D-Val-D-Lys-D-Leu, D-Phe-D-Trp-D-VaI-D- Val-D-Lys-D-Leu, D-Trp-D-Phe-D-VaI-D-Val-D-Lys-D-Leu, D-Trp-D-His-D-VaI-D- Val-D-Lys-D-Leu, D-AIa-D-Phe-D-Phe-D-VaI-D-Val-D-Lys-D-Leu, D-Ala-D-Trp-D- Phe-D-Val-D-Val-D-Lys-D-Leu, D-Ala-D-Phe-D-Trp-D-Val-D-Val-D-Lys-D-Leu, D- AIa-D-Trp-D-His-D-VaI-D-VaI-D-Lys-D-Leu, D-His-D- Phe-D-Phe-D-VaI-D-Val-D- Lys-D-Leu, D-His-D-Phe-D-Trp-D-VaI-D-Val-D-Lys-D-Leu, D-Tyr-D-Phe-D-Phe-D- VaI-D-VaI-D-Lys-D-Leu, D-Tyr-D-Trp-D-Phe-D-VaI-D-VaI-D-Lys-D-Leu, D-Phe-D- Phe-D-Phe-D-VaI-D-Val-D-Lys-D-Leu, D-Phe-D-Phe-D-Trp-D-VaI-D-Val-D-Lys-D- Leu, D-Trp-D-Phe-D-Phe-D-Val-D-Val-D-Lys-D-Leu, D-Trp-D-Phe-D-Trp-D-Val-D- VaI-D-Lys-D-Leu, D-Trp-D-Phe-D-Tyr-D-Val-D-VaI-D-Lys-D-Leu, D-Trp-D-Phe-D- Cha-D-Val-D-VaI-D-Lys-D-Leu, D-Trp-D-Phe-D-PyrAla-D-Val-D-VaI-D-Lys-D-Leu, D-Trp-D-Phe-D-pFPhe-D-Val-D-VaI-D-Lys-D-Leu, D-Trp-D-Phe-D-NaIl-D-VaI-D- Val-D-Lys-D-Leu, D-Trp-D-Phe-D-Nal2-D-VaI-D-VaI-D-Lys-D-Leu, D-Trp-D-Phe-D- Homophe-D-VaI-D-Val-D-Lys-D-Leu, D-Trp-D-Phe-D-pNO2Phe-D-VaI-D-Val-D-Lys-D- Leu, D-Trp-D- Phe-D-Dopa-D-Val-D-VaI-D-Lys-D-Leu, D-Trp-D- Phe-D-Tic-D-VaI-D- Val-D-Lys-D-Leu, and D-Trp-D-Phe-D-2R, 5S-D-Val-D-Val-D-Lys-D-Leu.

21. A peptide according to any one of the preceding claims which is linked to one or more coupling partners.

22. A peptide according to claim 21 wherein the coupling partner is a heterologous amino acid sequence.

23. A peptide according to claim 21 wherein the coupling partner is a protecting group.

24. A peptide according to claim 23 wherein the protecting group is an acetyl or 3 to 20 carbon alkyl group.

25. A peptide according to claim 23 wherein the protecting group is an amide group.

26. A peptide according to claim 21 or 22 wherein the coupling partner is a dopamine analogue or DOPA agonist.

27. A peptide according to claim 26 wherein the coupling partner is L-DOPA or pyroglutamic acid.

28. A peptide according to claim 21 or claim 22 wherein the coupling partner is a Blood Brain Barrier (BBB) transport moiety.

29. A peptide according to claim 28 wherein the Blood Brain Barrier (BBB) transport moiety is N-methyl phenylalanine (NMePhe) .

30. A peptide according to claim 28 comprising 1 to 5 N-methyl phenylalanine (NMePhe) moieties.

31. A peptide according to claim 28 wherein the Blood Brain Barrier (BBB) transport moiety is selected from the group consisting of transferri, a transferrin receptor binding peptide, sweet arrow peptide (SAP) , and TAT.

32. A polypeptide consisting of a peptide according to any one of claims 1 to 31 fused to a heterologous amino acid sequence.

33. A compound comprising one or more peptides according to any^¬ one of claims 1 to 31 or a polypeptide according to claim 32.

34. A compound according to claim 33 which further comprises one or more polyethylene glycol moieties linked to said peptide or polypeptide .

35. A pharmaceutical composition comprising a peptide according to any one of claims 1 to 31, a polypeptide according to claim 32 or a compound according to claim 33 or claim 34 and pharmaceutically acceptable excipient .

36. A method of formulating a pharmaceutical composition comprising admixing a peptide according to any one of claims 1 to 31, a polypeptide according to claim 32 or a compound according to claim 33 or claim 34 with a pharmaceutically acceptable excipient.

37. A peptide according to any one of claims 1 to 31, a polypeptide according to claim 32, a compound according to claim 33 or claim 34 or a composition according to claim 35 for use in a method of treatment of the human or animal body.

38. A peptide, polypeptide, compound or composition according to claim 37 for use in the treatment of disease characterised by the deposition of Aβi_-42.

39. A peptide, polypeptide, compound or composition according to claim 38 wherein the disease is Alzheimer's Disease cerebral amyloid angiopathy (CAA) or inclusion-body myositis.

40. Use of a peptide according to any one of claims 1 to 31, a polypeptide according to claim 32, a compound according to claim 33 or claim 34 or a composition according to claim 35 in the manufacture of a medicament for use in the treatment of a disease related to the deposition of Aβ_1-42

41. Use according to claim 40 wherein the disease is Alzheimer's disease cerebral amyloid angiopathy (CAA) or inclusion-body myositis.

42. A method of treatment of a disease related to the deposition of Aβ_1-42 comprising; administering peptide according to any one of claims 1 to 31, a polypeptide according to claim 32, a compound according to claim 33 or claim 34 or a composition according to claim 35 to an individual in need thereof .

43. A method according to claim 42 wherein the disease is Alzheimer's disease, cerebral amyloid angiopathy (CAA) or inclusion- body myositis.