MODIFIED PEPTIDES FOR USE IN TREATING NEURODEGENERATIVE DISORDERS

Abstract

The invention relates to neurodegenerative disorders, and in particular to novel peptides, peptidomimetics, compositions, therapies and methods for treating such conditions, for example Alzheimer's disease.

Claims

1. A method of treating, ameliorating or preventing a neurodegenerative disorder, the method comprising administering, to a subject in need of such treatment, a therapeutically effective amount of a compound of Formula (I), (II), (III), (IV), (V) or (VI): ##STR00121## ##STR00122## wherein: R.sub.1 is —NR.sub.9R.sub.10 or —OH; R.sub.2 is ##STR00123## R.sub.3 is —H or a C.sub.1-5 straight or branched alkyl or alkenyl; R.sub.4 is ##STR00124## R.sub.5 is —H or a C.sub.1-5 straight or branched alkyl or alkenyl; or R.sub.4 and R.sub.5 together with the nitrogen and carbon to which they are bonded form a five membered ring substituted by —OH or —NH.sub.2; R.sub.6 is ##STR00125## R.sub.7 is —H or a C.sub.1-5 straight or branched alkyl or alkenyl; R.sub.8 is —H; a C.sub.1-5 straight or branched alkyl or alkenyl or ##STR00126## X.sub.1 is —NR.sub.9R.sub.10, —OH or ##STR00127## the or each R.sub.9 and R.sub.10 are independently —H or a C.sub.1-5 straight or branched alkyl or alkenyl; R.sub.11 is —NH.sub.2, —OH or an aryl group; the or each m is independently between 0 and 5; and each n is independently between 0 and 10; or a pharmaceutically acceptable salt, solvate, tautomeric form, or polymorphic form thereof.

2. The method according to claim 1, wherein the compound has Formula (Ia), (IIa), (IIIa), (IVa), (Va) or (VIa): ##STR00128## ##STR00129##

3. The method according to claim 1, wherein the compound has Formula (Ib), (IIb), (IIIb), (IVb), (Vb) or (VIb): ##STR00130##

4. The method according to claim 1, wherein R.sub.1 is —NR.sub.9R.sub.10, and is optionally —NH.sub.2.

5. The method according to claim 1, wherein R.sub.2 is ##STR00131##

6. The method according to claim 5, wherein R.sub.2 is ##STR00132## optionally wherein R.sub.2 is ##STR00133##

7. (canceled)

8. The method according to claim 1, wherein R.sub.3 is methyl or H.

9. The method according to claim 1, wherein R.sub.4 is ##STR00134##

10. The method according to claim 9, wherein R.sub.4 is ##STR00135## optionally wherein R.sub.4 is ##STR00136##

11. (canceled)

12. The method according to claim 1, wherein R.sub.5 is methyl or —H.

13. The method according to claim 1, wherein R.sub.4 and R.sub.5 together with the nitrogen and carbon to which they are bonded define the following structure: ##STR00137## wherein X.sub.2 is —OH or —NH.sub.2, optionally wherein R.sub.4 and R.sub.5 together with the nitrogen and carbon to which they are bonded define the following structure: ##STR00138##

14. (canceled)

15. The method according to claim 1, wherein R.sub.6 is ##STR00139## optionally wherein R.sub.6 is ##STR00140##

16. (canceled)

17. (canceled)

18. The method according to claim 1, wherein R.sub.7 is methyl or H.

19. The method according to claim 1, wherein R.sub.8 is ##STR00141##

20. The method according to claim 1, wherein the compound is a compound of Formula (101), (102), (103), (104) or (105): ##STR00142## ##STR00143##

21. The method according to claim 20, wherein the compound is a compound of Formula (101a), (102a), (103a), (104b) or (105a): ##STR00144## ##STR00145##

22. (canceled)

23. The method according to claim 1, wherein the neurodegenerative disorder which is treated is one which is characterised by the damage or death of ‘Global’ neurons.

24. The method according to claim 1, wherein the neurodegenerative disorder is selected from a group consisting of Alzheimer's disease; Parkinson's disease; Huntington's disease; Motor Neurone disease; Spinocerebellar type 1, type 2, and type 3; Amyotrophic Lateral Sclerosis (ALS); schizophrenia; Lewy-body dementia; and Frontotemporal Dementia.

25. A compound of Formula (I), (II), (III), (IV), (V) or (VI): ##STR00146## ##STR00147## wherein: R.sub.1 is —NR.sub.9R.sub.10 or —OH; R.sub.2 is ##STR00148## R.sub.3 is H or a C.sub.1-5 straight or branched alkyl or alkenyl; R.sub.4 is ##STR00149## R.sub.5 is H or a C.sub.1-5 straight or branched alkyl or alkenyl; R.sub.6 is ##STR00150## R.sub.7 is H or a C.sub.1-5 straight or branched alkyl or alkenyl; R.sub.8 is —H, a C.sub.1-5 straight or branched alkyl or alkenyl or ##STR00151## R.sub.9 and R.sub.10 are independently —H or a C.sub.1-5 straight or branched alkyl or alkenyl; and each n is independently between 0 and 10; or R.sub.1 is —NR.sub.9R.sub.10 or —OH; R.sub.2 is ##STR00152## R.sub.3 is H or a C.sub.1-5 straight or branched alkyl or alkenyl; R.sub.4 and R.sub.5 together with the nitrogen and carbon to which they are bonded define the following structure: ##STR00153## R.sub.6 is ##STR00154## and R.sub.7 is H or a C.sub.1-5 straight or branched alkyl or alkenyl; R.sub.8 is —H, a C.sub.1-5 straight or branched alkyl or alkenyl or ##STR00155## R.sub.9 and R.sub.10 are independently —H or a C.sub.1-5 straight or branched alkyl or alkenyl; and each n is independently between 0 and 10; or R.sub.1 is —NR.sub.9R.sub.10 or —OH; R.sub.2 is ##STR00156## R.sub.3 is H or a C.sub.1-5 straight or branched alkyl or alkenyl; R.sub.4 is ##STR00157## R.sub.5 is H or a C.sub.1-5 straight or branched alkyl or alkenyl; R.sub.6 is ##STR00158## and R.sub.7 is H or a C.sub.1-5 straight or branched alkyl or alkenyl; R.sub.8 is —H, a C.sub.1-5 straight or branched alkyl or alkenyl or ##STR00159## R.sub.9 and R.sub.10 are independently —H or a C.sub.1-5 straight or branched alkyl or alkenyl; and each n is independently between 0 and 10; or R.sub.1 is —NR.sub.9R.sub.10 or —OH; R.sub.2 is ##STR00160## R.sub.3 is H or a C.sub.1-5 straight or branched alkyl or alkenyl; R.sub.4 is ##STR00161## R.sub.5 is H or a C.sub.1-5 straight or branched alkyl or alkenyl; R.sub.6 is ##STR00162## and R.sub.7 is H or a C.sub.1-5 straight or branched alkyl or alkenyl; R.sub.8 is —H, a C.sub.1-5 straight or branched alkyl or alkenyl or ##STR00163## R.sub.9 and R.sub.10 are independently —H or a C.sub.1-5 straight or branched alkyl or alkenyl; and each n is independently between 0 and 10; or R.sub.1 is —NR.sub.9R.sub.10 or —OH; R.sub.2 is ##STR00164## R.sub.3 is H or a C.sub.1-5 straight or branched alkyl or alkenyl; R.sub.4 is ##STR00165## R.sub.5 is H or a C.sub.1-5 straight or branched alkyl or alkenyl; R.sub.6 is ##STR00166## and R.sub.7 is H or a C.sub.1-5 straight or branched alkyl or alkenyl; R.sub.8 is —H, a C.sub.1-5 straight or branched alkyl or alkenyl or ##STR00167## R.sub.9 and R.sub.10 are independently —H or a C.sub.1-5 straight or branched alkyl or alkenyl; and each n is independently between 0 and 10; or a pharmaceutically acceptable salt, solvate, tautomeric form, or polymorphic form thereof.

26-29. (canceled)

30. A pharmaceutical composition comprising a compound of Formula (I), (II), (III), (IV), (V) or (VI): ##STR00168## ##STR00169## wherein: R.sub.1 is —NR.sub.9R.sub.10 or —OH; R.sub.2 is ##STR00170## R.sub.3 is —H or a C.sub.1-5 straight or branched alkyl or alkenyl; R.sub.4 is ##STR00171## R.sub.5 is —H or a C.sub.1-5 straight or branched alkyl or alkenyl; or R.sub.4 and R.sub.5 together with the nitrogen and carbon to which they are bonded form a five membered ring substituted by —OH or —NH.sub.2; R.sub.6 is ##STR00172## R.sub.7 is —H or a C.sub.1-5 straight or branched alkyl or alkenyl; R.sub.8 is —H; a C.sub.1-5 straight or branched alkyl or alkenyl or ##STR00173## X.sub.1 is —NR.sub.9R.sub.10, —OH or ##STR00174## the or each R.sub.9 and R.sub.10 are independently —H or a C.sub.1-5 straight or branched alkyl or alkenyl; R.sub.11 is —NH.sub.2, —OH or an aryl group; the or each m is independently between 0 and 5; and each n is independently between 0 and 10; or a pharmaceutically acceptable salt, solvate, tautomeric form or polymorphic form thereof, and a pharmaceutically acceptable vehicle.

31. (canceled)

Description

[0204] For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figures, in which:—

[0205] FIGS. 1a and 1b show different views of a representation of the four key areas (Areas 1, 2, 3 and 4) in the allosteric binding pocket (i.e. the active site) of the α7 nicotinic-receptor which bind to the cyclic peptide NBP-14, and the respective distances between these four areas depending on whether NBP-14 competes with T30 (in FIG. 1a) or amyloid (in FIG. 1b);

[0206] FIG. 2 shows the 3D structure of the α7 nicotinic-receptor binding pocket with a colour coding based on the polarity;

[0207] FIG. 3 shows a stick representation of the α7 nicotinic-receptor binding pocket showing Areas 1-4;

[0208] FIG. 4 merges the two views shown in FIGS. 2 and 3;

[0209] FIG. 5 shows pie charts of the amino acids or the chemical functions involved in the binding at each binding area (Area 1, 2, 3 or 4) in the α7 nicotinic-receptor, and shows if the residue results in an inert peptide, one which is active against toxic T30 or active against A-beta;

[0210] FIG. 6A-6F compares the distance between the amino acids binding in the different areas (Areas 1-4);

[0211] FIG. 7 is a list of chemical functionalities for binding to each of Areas 1, 2, 3 and 4 that are of specific relevance in providing protection against T30 toxicity;

[0212] FIG. 8 is a list of chemical functionalities for binding to each of Areas 1, 2, 3 and 4 that are of specific relevance in providing protection against beta amyloid production;

[0213] FIG. 9 shows cell culture data (i.e. acetylcholinesterase activity) for peptidomimetic compound 1 (i.e. Tri02);

[0214] FIG. 10 shows cell culture data (i.e. calcium ion influx) for peptidomimetic compound 1 (i.e. Tri02);

[0215] FIG. 11 shows the results of voltage-sensitive dye imaging (VSDI) on brain slices for control cyclic peptide NBP-14;

[0216] FIG. 12 shows the results of voltage-sensitive dye imaging (VSDI) on brain slices for peptidomimetic compound 1 (i.e. Tri02);

[0217] FIG. 13 shows correlation analysis of changes induced by addition of peptides against respective baseline response amplitude using voltage-sensitive dye imaging (VSDI) on brain slices. Changes in response amplitude induced by T30 were found to be negatively correlated with the amplitude of their respective baselines (A). Therefore, subsequent correlation analyses were carried out for each experiment in which exogenous peptides were perfused: B) T15, C) NBP14, D) Tri02, E) T30 in the presence of NBP14 and F) T30 in the presence of Tri02. Units on y-axis=ΔF/Fo; x-axis=ΣδF/Fo;

[0218] FIG. 14 shows quantification of effects mediated by the addition of Tri02 and T30;

[0219] FIG. 15 shows a graph comparing the co-application of T30 and NBP14 against that of Tri02 and T30 at blocking the effects of T30 on activity within the basal forebrain. NBP14 co-application was able to totally block the T30-induced effects, whereas T30 w/Tri02 caused a similar but muted modulatory response;

[0220] FIG. 16 shows pharmacokinetic data for cyclic NBP-14 in rat blood;

[0221] FIG. 17 shows pharmacokinetic data for cyclic NBP-14 in human blood;

[0222] FIG. 18 shows pharmacokinetic data for peptidomimetic compound 1 (i.e. Tri02) in rat blood;

[0223] FIG. 19 shows pharmacokinetic data for peptidomimetic compound 1 (i.e. Tri02) in human blood;

[0224] FIG. 20 shows pharmacokinetic data for peptidomimetic compound 3 (i.e. Tri04) in rat blood;

[0225] FIG. 21 shows pharmacokinetic data for peptidomimetic compound 3 (i.e. Tri04) in human blood;

[0226] FIG. 22 shows pharmacokinetic data for procaine in rat blood;

[0227] FIG. 23 shows pharmacokinetic data for procaine in human blood;

[0228] FIG. 24 shows the blood breakdown products from peptidomimetic compound 1 (i.e. Tri02);

[0229] FIG. 25 shows the blood breakdown products from peptidomimetic compound 3 (i.e. Tri04);

[0230] FIG. 26 shows cell culture data (i.e. calcium ion influx) for peptidomimetic compound 3 (i.e. Tri04);

[0231] FIG. 27 shows (A) space-time maps of basal forebrain activity changes induced by addition of peptides (T30 and Tri04) against the baseline response amplitude using voltage-sensitive dye imaging (VSDI) on brain slices. In (B) is shown a graph comparing basal forebrain evoked activity for recordings with T30 and Tri04 (2 uM) with that of T30 alone in the basal forebrain;

[0232] FIG. 28 shows the fluorescence fractional change (response time-series, n=29) for recordings made in the presence of T30 alone or after co-application of T30 and its blocker Tri04 in comparison to the baseline condition; and

[0233] FIG. 29 shows a bar graph of basal forebrain activity using the blocker Tri04 at 4 μM concentration. Tri04 co-application was able to totally block the T30-induced effects in the rat basal forebrain.

EXAMPLES

[0234] The inventors conducted an in silico study in order to design novel peptides and peptidomimetics, which would exhibit affinity for the α-7nChR receptor, and which would therefore block binding to its active site by the endogenous toxic T30 peptide (KAEFHRWSSYMVHWKNQFDHYSKQDRCSDL—SEQ ID No: 1). The in silico study helped to determine the chemical functionalities relevant for the protection against the T30 toxic action and beta-amyloid production by looking at the interaction between the receptor, and cyclic NBP-14 (i.e. AEFHRWSSYMVHWK—SEQ ID No:2), which is known to provide this protection, as demonstrated in previous work (see WO 2005/004430). The following examples describe the in silico study as well as the structures of the various peptides and peptide mimetics that have been identified and tested in vitro.

Example 1—In-Silico Study to Design Novel Peptides which Inhibit α-7nChR Receptor

[0235] By using computational analysis of the affinity of NBP-14 for the drug target receptor, and by structure-based studies, the inventors identified a range of smaller linear peptides with similar in-vitro properties to NBP-14 (SEQ ID No:2). The theoretical interaction between 598 of these smaller linear peptides and the target α-7nChR receptor has been investigated. NBP-14 and the 168 linear peptides derived from the aforementioned computational analysis were chemically synthesised. NBP-14 and all of the 168 peptides were screened in vitro in PC12 cells, which are routinely used as a model system for neuronal differentiation and neurosecretion studies. Screening has been conducted in vitro for toxicity and neurodegenerative bioactivity, the latter via monitoring acetylcholinesterase activity and intracellular calcium levels. From this, a second generation of a range of new molecules with neurodegenerative protective properties against T30 have been identified using in silico analysis of the peptides that have been in vitro tested on PC12 cells to determine the main chemical functionality involved in binding to the receptor.

[0236] The docking of these compounds has been performed on the allosteric site of the α-7nChR receptor. The binding pocket in the receptor contains four areas (denoted Areas 1, 2, 3 and 4) that could be represented as shown in the FIGS. 1-4.

[0237] The in silico analysis comprised a comparison between the peptides to determine and differentiate the chemical features/functionalities that are specific to the protection against T30 toxicity and beta-amyloid production from the chemical features that are inert. FIGS. 2-4 summarizes the 3D structure of the binding pocket of α-7nChR with a colour coding based on the polarity.

[0238] The steps that were followed, as well as their outcomes, are summarised below:

Step 1: Comparison of the Amino Acids Binding in Each Specific Area of the Receptor

[0239] In this analysis, each area was considered separately, only the amino acids that were binding to the area were considered. As shown in FIG. 5, presenting the pie charts of the amino acids or the chemical functions involved in the binding, this step did not reveal amino acids specifically binding in the different areas.

Step 2: Comparison of the Distance Between the Amino Acids Binding in the Different Areas

[0240] In this analysis, the distance between the amino acids is measured taking into consideration the chemical functionality involved in the binding. These data, presented in FIG. 6 reveal no significant changes of distances between the inert variants and the variants with an activity against T30 toxicity and beta amyloid activity.

Step 3: Comparison of the Combination of Amino Acids Involved in the Binding

[0241] This step requires the analysis of the amino acids involved in the binding as a combination of amino acids necessary for the protection against T30 toxicity and beta amyloid production.

[0242] The results shown in the Tables 1 and 2 below indicate that 18 amino acid combinations appear to be necessary for the protection against T30 toxicity and 31 amino acid combinations appear to be necessary for the protection against beta amyloid production.

TABLE-US-00001 TABLE 1 Amino acid combinations that are protection against T30 toxicity Combination Area 1 Area 2 Area 3 Area 4 1 His Lys Phe Trp 2 Amide Phe His — 3 His Met Trp — 4 Amide Arg Glu — 5 Trp His Tyr Met 6 Arg Trp Tyr Met 7 Phe N-ter Arg His 8 Trp Met Phe Lys 9 Arg Amide Tyr — 10 Arg Met Tyr — 11 Lys Trp Tyr — 12 Trp Lys Met His 13 Lys His Tyr — 14 Trp Lys N-ter — 15 Trp Serer Phe Arg 16 N-ter Tyr His Yrp 17 Amide — Glu — 18 His — Ser —

TABLE-US-00002 TABLE 2 Amino acid combinations that are protection against beta amyloid production Combination Area 1 Area 2 Area 3 Area 4 1 Lys Glu C-ter Phe 2 Arg — Tyr Trp 3 Trp — Tyr Met 4 — — Tyr Met 5 Tyr — Hi N-ter 6 Trp His Tyr Arg 7 Trp — Tyr Met 8 Glu — Trp Lys 9 His Trp Tyr Met 10 Trp — Tyr Met 11 N-ter Met — — 12 His Lys Phe Trp 13 His — Tyr Met 14 N-ter His Tyr — 15 Trp Lys Met His 16 — N-ter C-ter — 17 Tyr — N-ter Lys 18 Lys Trp Phe — 19 N-ter Lys Phe Trp 20 His Phe Amide Glu 21 Trp Arg Phe His 22 Tyr Lys His — 23 Trp His Tyr — 24 N-ter Trp His Val 25 Trp Ser His Arg 26 Ala His Tyr — 27 His Phe Trp Arg 28 Amide Trp Val His 29 N-ter — Arg His 30 His — Ser — 31 His Amide — —

[0243] In view of these results, the inventors were then able to determine the ranking of the amino acid residues involved in the binding within each area (Areas 1-4) of the receptor, and thus the chemical functionalities that are of specific relevance in providing protection against both T30 toxicity and beta amyloid production, see FIGS. 7 and 8, respectively.

[0244] The inventors were able to conclude that each area requires specific chemical functionalities that are summarised in the Table 3.

TABLE-US-00003 TABLE 3 The type of interaction occurring in each area of the allosteric site of the α-7nChR receptor Type of Area interaction 1 Hydrogen bond 2 proximal Hydrogen bond 2 distal (optional) Hydrophobic 3 proximal Hydrophobic 3 distal Hydrogen bond 4 distal (optional) Hydrogen bond

[0245] Accordingly, in view of these findings, the inventors have demonstrated that a suitable peptide which would block the toxic effects of endogenous T30 by preferentially blocking the active site of the nicotinic receptor.

Example 2—Design and Production of Peptidomimetics

[0246] An alternative approach was then used to design and isolate novel peptidomimetic compounds which could (as with the peptides described in Example 2) outcompete T30 for the allosteric active site of the nicotinic receptor. Thus, a further in silico study was carried out in which computation solvent mapping was conducted over the starting initial X-ray structure of the allosteric site of the α-7nChR receptor. This analysis was aimed to elucidate the preferential solvent interaction at the binding site as well as to locate the presence of hot spots (hydrophobic, aromatic, polar or charged). This method identified the expected chemical features required by the ligand in order to become active.

[0247] The IPRO solvent analysis unraveled the high hydrophobic nature of the binding site. Perfect overlapping between IPRO solvent mapping prediction and T14 peptide docking was observed.

[0248] Based on the solvent mapping analysis, the T14 structure was used to generate linear libraries of tripeptides and tetrapeptides. In this step more than 500,000 peptidomimetics were generated for a further evaluation. The peptidomimetics were then evaluated by AutoDock Vina docking engine. The theoretical affinities as well as ligand promiscuity (i.e, tendency to bind in multiple binding sites or different binding modes, denoted by a low intra-RMSD) were taken into account for the analysis. This analysis resulted in five candidate peptidomimetic compounds which are shown below, in which the higher the score is (an absolute value), the better the affinity and the higher the probability the compound is active.

Compound 1—Tri02 (Score: −10.2)

[0249] ##STR00116##

4-((S)-2((S)-2-acetamido-3-(naphthalene-2-yl)propanamido)-3-(((S)-1-amino-3-(1H-indol-3-yl)-1-oxopropan-2-yl)amino)-3-oxopropyl)benzenaminium

Compound 2—Tri03 (Score: −9.8)

[0250] ##STR00117##

(3S,5S)-1_((S)-2-acetamido-3-(naphthalene-2-yl)propanoyl)-5-(((S)-1-amino-6-((amino(iminio)methyl)amino)-1-oxohexan-2-yl)carbamoyl)pyrrolidin-3-aminium

Compound 3—Tri04 (Score: −9.4)

[0251] ##STR00118##

4-((S)-2-((S)-2-((S)-2-acetamido-3-(4-benzoylphenyl)propanamido)-6-((amino(iminio)methyl)amino)hexanamido)-3-amino-3-oxopropyl)benzenaminium

Compound 4—Tri05 (Score: −9.6)

[0252] ##STR00119##

(((R)-4-acetamido-5-(((S)-5-((amino(iminio)methyl)amino)-1-(((S)-1-amino-3-(4-benzoylphenyl)-1-oxopropan-2-yl)amino)-1-oxopentan-2-yl)amino)-5-oxopentyl)amino)(amino)methaniminium

Compound 5—Tri06 (Score: −8.9)

[0253] ##STR00120##

(S)-5-(((S)-2-acetamido-5-((amino(iminio)methyl)amino)pentanamido)-6-(((S)-1-amino-3-(4-benzoylphenyl)-1-oxopropan-2-yl)amino)-6-oxohexanoate

Example 3—Synthesis of Identified Compounds

Materials and Methods

[0254] Compounds 1 and 3 from Example 2 were synthesised by Genosphere Biotechnologies and analysed for purity using RP-HPLC (>.sub.99% pure), and mass by mass spectroscopy (average MS 604.79 for Triol and 628.83 for Tri04).

Brief Stepwise Description of Synthesis of TRI02—Sequence: [acetyl]-[2Nal][4nh2-F]-Trp-[amide]

[0255] 1) Boc-Trp-OH+ClooEt+NH.sub.3.H2O-Boc-Trp-NH2, reaction in THF, extracted by acetic ether.

[0256] 2) Boc-Trp-NH2,4NHcl, removed Boc−, obtained H-Trp-NH2.Hcl, precipitation reaction by diethyl ether.

[0257] 3) (2-Naphtyl)-Ala+Acetic Anhydride-Ac-(2-Naphtyl)-Ala-OH, reaction THF/H2O, extracted by acetic ether.

[0258] 4) Boc-(4-NH2)-Phe-OH+H-Trp-NH2.Hcl-Boc-(4-NH2)-Phe-Trp-NH2, reaction in DMF, extracted by acetic ether.

[0259] 5) Boc-(4-NH2)-Phe-Trp-NH2,4NHcl, removed Boc-, obtained H-(4-NH2)-Phe-Trp-NH2.Hcl, precipitation reaction by diethyl ether.

[0260] 6) Ac-(2-Naphtyl)-Ala-OH+H-(4-NH2)-Phe-Trp-NH2.Hcl-Ac-(2-Naphtyl)-Ala-(4-NH2)-Phe-Trp-NH2 reaction in DMF, extracted by acetic ether.

[0261] 7) Purification

Brief Stepwise Description of Synthesis of TRI04—Sequence: [acetyl]-[bpa]R[4NH2-F]-[amide]

[0262] 1) Rink Amide MBHA.Resin Soak in DCM for 30 mins, pumped dry, washed by DMF for 3 times, pumped dry.

[0263] 2) Add Fmoc-(4-NH2)Phe-OH,DIEA,HBTU,DMF,N2, reaction for 30 mins, pumped dry, washed by DMF for 6 times, pumped dry.

[0264] 3) Add piperidine/DMF to remove Fmoc-, reaction for 20 mins, pumped dry, washed by DMF for 3 times, pumped dry.

[0265] 4) Add Fmoc-Arg(Pbf)-OH,DIEA,HBTU,DMF,N2, reaction for 30 mins, pumped dry, washed by DMF for 6 times, pumped dry.

[0266] 5) Repeat step 3.

[0267] 6) Add Fmoc-Bpa-OH,DIEA,HBTU,DMF,N2, reaction for 30 mins, pumped dry, washed by DMF for 6 times, pumped dry.

[0268] 7) Repeat step 3.

[0269] 8) Add Acetic Anhydride/DMF,N2, reaction for 30 mins, pumped dry, washed by DMF for 3 times, pumped dry, washed by DCM for 3 times, pumped dry, washed by MeOH for 3 times, pumped dry.

[0270] 9) Peptide cleaved from resin, pumped dry, precipitation reaction by diethyl ether, obtain the crude peptide, centrifugal drying.

[0271] 10) Purification

Example 4—Evaluation of Compound 1 (Tri02) and Compound 3 (Tri04) in Cell Cultures

[0272] The inventors tested T30, NBP-14, and Tri02 in cell culture studies to determine their effects on acetylcholinesterase activity and calcium influx, and the effects of Tri04 on calcium influx.

Materials and Methods

1. AChE Activity Assay

[0273] AChE activity was measured using the Ellman reagent that measures the presence of thiol groups as a result of AChE activity. In the case of the G4 experiment, AChE (G4) activity was tested alone and also together with either NBP14 or Tri peptides. PC12 cells were plated the day before the experiment as for the cell viability assay. Cells were treated with T30 (1 μM) alone or combined with NBP14 or Tri peptide (0.5 μM). After treatment, supernatant (perfusate) of each treatment was collected and 25 μL from each condition were added to a new flat bottomed 96 well plate followed by the addition of 175 μl of Ellman reagent (Solution A: KH2PO4 139 mM and K2HPO4 79.66 mM, pH 7.0; solution B (substrate): Acetylthiocholine Iodide 11.5 mM; Solution C (Reagent): 5,5′-dithiobis(2-nitrobenzoic acid) 8 mM and NaHCO.sub.3 15 mM). The Ellman reagent was prepared as a mixture of the 3 solutions in a ratio 33(A):3(B):4(C). Absorbance measurements were taken for an interval of 60 minutes across experiments at 405 nm in a Vmax plate reader (Molecular devices, Wokingham, UK).

2. Calcium Fluorometry

[0274] PC12 cells were plated in 200 μl of Dulbecco's Modified Eagle's medium (DMEM) plus 2 mM of L-glutamine medium the day before the experiment in 96 well plates. On the day of the experiment, the Fluo-8 solution (Abcam) was prepared as described by the provider by adding 20 μl of Fluo-8 in the assay buffer that contains 9 ml of Hank's Balanced Salt Solution (HBSS) and 1 ml of pluronic F127 Plus. Subsequently, 100 μl of growth medium was removed and 100 μl of Fluo-8 solution were added. Treatments with T30 in conjunction with NBP14 or Tri peptides were added and incubated for 30 minutes in the incubator and 30 minutes room temperature. After 1 h, the plate was placed in the fluorescence plate reader (Fluostar, Optima, BMG Labtech, Ortenberg, Germany). Before reading the fluorescence, PNU282987 1 μM, an alpha7 specific agonist of the nicotinic receptors, was prepared and placed in the Fluostar injector. For each well, the reading was formed by a basal fluorescence reading followed by PNU282987 injection that induced an increase of calcium via nicotinic receptors.

3. Data Analysis

[0275] In each of the different cell techniques, the statistics analysis was performed with the average of the percentage values of 3 or more experiments. Comparisons between multiple treatment groups and the same control were performed by one-way analysis of variance (ANOVA) and Tukey's post-hoc tests using GraphPAD Instat (GraphPAD software, San Diego, Calif.). Statistical significance was taken at a p value<0.05.

Results

[0276] The results for Triol are shown in FIGS. 9 and 10, in which n values shown on the subsequent graphs refer to number of repeated experiments. As can be seen, 1 μM T30 increases calcium influx and AChE activity, and, as shown in previous work (see WO 2005/004430), 1 μM NBP14 protects against these toxic effects.

[0277] In addition, as can be seen in the Figures, Triol also clearly protects against the toxic effects of T30 by reducing both calcium influx and AChE activity. As such, the inventors are convinced that Triol is neuroprotective, and, due to its smaller size than NBP-14, will have a much greater chance of passing through the blood-brain barrier.

[0278] The results for Tri04 are shown in FIG. 26. As can be seen, Tri04 also protects the toxic effects of T30 by reducing calcium influx.

Example 5—Evaluation of Compound 1 in Brain Slices

[0279] The inventors tested NBP-14 and Triol in brain slice studies using voltage-sensitive dye imaging (VSDI).

Materials and Methods

1. Brain Slice Preparation

[0280] Male Wistar rats (14 days old) were anaesthetised using isoflurane (˜15 ml, 100% w/w). Isoflurane was applied to the cotton bed at the bottom of an anaesthetic chamber (glass box 20×15×15 cm) where rats were then placed for approximately 45 s until complete anaesthesia was reached. The hind paw of each anaesthetised rat was pinched to check for the appropriate depth of anaesthesia. Upon confirmation of anaesthesia, rats were quickly decapitated, with the brain being quickly removed and immersed in ice cold oxygenated ‘slicing’ artificial cerebrospinal fluid (aCSF in mmol: 120 NaCl, 5 KCL, 20 NaHCO.sub.3, 2.4 CaCl2 2 MgSO4, 1.2 KH2PO4, 10 glucose, 6.7 HEPES salt and 3.3 HEPES acid; pH=7.1). Coronal slices (400 μm thick) were then taken from a block of brain containing the basal forebrain, namely the MS-dBB complex (between +9.20 and +9.48 mm Interaural and +0.48 and +0.2 mm Bregma, FIG. 4A) and the somatosensory barrel field cortex (SiBF, between +8.08 and +7.20 mm Interaural and −0.92 mm and −1.80 mm Bregma) (Paxinos and Watson, 1998) using a Vibratome (Leica VTi000S).

[0281] Slices were then transferred to a bubbler pot containing oxygenated aCSF at room temperature (recording aCSF in mmol: 124 NaCl, 5 KCL, 20 NaHCO3, 2.4 CaCl2 2 MgSO4, 1.3 KH2PO4, 10 glucose; pH=7.4) which was identical to that used in VSDI (voltage sensitive dye imaging) recording. Slices were then left for approximately 1-1.5 hours before preparing them for VSD staining.

2. VSD Setup

[0282] Slices were placed in a dark, high humidity chamber filled with aCSF bubbled with 95% O2, 5% CO2. Once there, the dye solution (4% 0.2 mM styryl dye pyridinium 4-[2-[6-(dibutylamino)-2-napthalenyl]-ethenyl]-1-(3-sulfopropyl)hydroxide (Di-4-NEPPS), Invitrogen, Paisley, UK in 48% aCSF, 48% foetal bovine serum, 3.5% DMSO and 0.4% cremophore EL) (Tominaga et al., 2000) was applied to the slices for 20-25 minutes before being transferred back to a bubbler pot containing oxygenated aCSF kept at room temperature for 30 minutes.

[0283] When starting the VSDI recordings, slices were placed in the recording bath on a small piece of filter paper to allow the flow of oxygenated aCSF on the underside of the slice and in order to keep it alive. The slice was then weighed down by a home-made plastic grid that was placed on top of the slice. The perfusing bath solution was heated to 30±1° C. by a stage heater. A concentric bipolar stimulating electrode (FHC, Maine, USA) was placed in the ventral diagonal band of the basal forebrain with stimulation being set to 30V. For the acquisition of VSD data, 2 dimensional images, equivalent to 88×60 pixels, were recorded using the MiCamo2 High Resolution camera (Brain Vision, Japan) with BV_Analyze imaging software. Acquisition of images was coupled to Spike2 V4.23 software (CED Ltd, Cambridge, UK) in order to align the image capture with the stimulation protocol (every 28 s with 30 repeats) via the Micro 1401 MkII. (CED Ltd, Cambridge, UK). Light was generated using an Osram halogen xenophot 64634 HLX EFR Display/Optic lamp and was filtered to emit green (530±10 nm) light using a MHF-G150LR (Moritex Corporation) coupled to the MiCamo2 High resolution imaging system and filtered the emitted fluorescence through a >590 nm high pass filter.

3. Drug Preparation and Application

[0284] Acetylcholinesterase (AChE)C-terminus 30 amino acid peptide (T30; sequence: ‘N’—KAEFHRWSSYMVHWKNQFDHYSKQDRCSDL—SEQ ID No:1), the cyclic version of the active 14 amino acid region of T30 (NBP14; sequence: c[AEFHRWSSYMVHWK]—SEQ ID No:2; c[ ]=cyclic, N-terminal to C-terminal) and the inert 15 amino acid peptide contained within the T30 sequence (T15; sequence: ‘N’—NQFDHYSKQDRCSDL—SEQ ID No:3) were custom synthesised and purchased from Genosphere Biotechnologies (Paris, France) at >99% purity. The linear peptidomimetic, Triol was designed in silico by Iproteos (Barcelona, Spain) and synthesised and purchased from Genosphere Biotechnologies at >99% purity. All drug and peptide stocks were prepared in frozen aliquots prior to experiments. For the production of perfusion solutions, stock solutions were thawed and added to recording aCSF as appropriate and bath applied at a constant rate of 1.5 ml/min perfusion using the Minipulse 3 peristaltic pump (Gilson Scientific Ltd., Bedfordshire, UK). Each experimental trial lasted 52 minutes, with 20 minutes to establish a baseline recording (perfusion with recording aCSF only), 12 minutes to allow the drug solution to perfuse into the bath as well as to let the dye molecules reseat themselves in the cell membranes and finally, a 20 minute recording period measuring the response in the presence of the drug solution.

4. Data Analysis and Statistics

[0285] From the 2 dimensional images generated with each drug condition, the critical data such as the time-course of activation, intensity and spread of the overall fluorescent signal were extracted. These data were processed using a custom script to convert them into usable MatLab (Mathworks Inc. Massachusetts, US) files and then analysed using a Matlab toolbox specifically made for VSDI data analysis (Bourgeois et al., 2014). This toolbox allows for the selection of a fixed region of interest (ROI) geometry that can be applied to every slice, in order to extract and collate the data from an identical ROI across all slices used in each experiment. For the basal forebrain slices, the ROI that will be used is the MSdBB complex, chosen as it encompasses the MS (medial septal nuclei), VDB (ventral diagonal band) and HDB (horizontal diagonal band). More crucially, this ROI was chosen in order to include the entirety of the evoked response. This response can be plotted as a single averaged time series or over space and time in a ‘space-time map’ so as to provide a qualitative description of the data. However, in order to produce quantifiable values, the area underneath the time series was calculated (summed fluorescence fractional change) between the moment of stimulation (t=0) and 156 ms after that. Due to the variability of responses seen between each individual slice, the raw data generated from each experiment was normalised with respect its own baseline to give normalised fluorescence values. This method of quantification was chosen in order to account for the multiple components of the signal generated by VSDI (Chemla and Chavane, 2010) namely the immediate peak and the long latency response (Badin et al., 2016). Statistics were carried out in Prism Graphpad 6.

5. Analysis of Modulatory Peptides

[0286] Throughout the experiments in which T30 was used, an increase or a decrease in signal was observed. Thus upon averaging these results together, no change was detected. However, given the past observed modulatory effects of this peptide in various preparations (Bon and Greenfield, 2003, Day and Greenfield, 2004, Greenfield et al., 2004, Badin et al., 2013) and the fact that the changes induced by application of T30 in this type of preparation are moderately negatively correlated (r=−0.4286, p=0.0257, Spearman's rank correlation, n=27, FIG. 13A) with baseline response amplitude, it was decided that these results should be dichotomised by whether an increase or a decrease was seen.

[0287] Subsequently, a similar correlation analysis was performed for each experiment in which an exogenous compound was added (FIG. 13). Upon determination of a significant correlation, data was then categorised based on whether and increase or a decrease was seen.

Results

[0288] Referring to FIGS. 2, 11 and 12, addition of 4 μM Tri02 recapitulates results seen with application of 4 μM NBP14.

[0289] Referring to FIG. 11, addition of NBP14 (4 uM) to the perfusate induced small, non-significant alterations to the magnitude (summed fluorescence) of evoked responses. Though insignificant, these small induced changes were found to be inversely correlated with magnitude of baseline response; as a result, data were split into trials which caused slight decreases (left histograms) and those which caused increase (right histograms), both in real (top) and normalised (bottom) data format. If considered together, the dataset would show no change from baseline (as increases and decreases would cancel each other out), yet it was crucial to check that no significant effects were induced by NBP14 even when the fluorescence changes were considered separately.

[0290] As shown in FIG. 12, addition of Tri02 (4 uM) to the perfusate induced small alterations to the magnitude (summed fluorescence) of evoked responses, with induced decreases (n=8 of ii total) showing a significant deviation from normalised baseline level (bottom left histogram, p<0.05). These changes were also found to be inversely correlated with magnitude of baseline response; as a result, data were split into trials which caused decreases (left histograms) and those which caused increases (right histograms), both in real (top) and normalised (bottom) data format. If considered together, the dataset would show no change from baseline (as increases and decreases would cancel each other out), yet it was crucial to check that no significant effects were induced by NBP14 even when the fluorescence changes were considered separately.

Analysis of Modulatory Peptidomimetics

[0291] Referring to FIG. 13, there is shown correlation analysis for Tri02 (4 uM) and T30 (2 uM) data (n=15) showing that their co-perfusion induces some changes to the magnitude of evoked responses, with some slices featuring slight increases in activity (n=6) whilst most showed slight decreases (n=9). This correlation was found to be significant (p=0.0405; r.sup.2=−0.534), providing justification to split the data into those that showed increases and decreases in evoked activity as a result of Tri02 and T30 application, just as was done for the addition of NBP14 and Tri02 (FIGS. 11 & 12, respectively).

[0292] Referring to FIG. 14, there is shown quantification of effects mediated by the addition of Tri02 and T30: Both in the case of induced increases and decreases, Tri02 was not found to protect against T30-induced deviations from baseline, with significant decreases (left panel, p<0.01, n=9) and increases (right panel, p<0.05, n=6) reported in overall effects.

[0293] As shown in FIG. 15, overall line graph of normalised effects respective to baseline for experiments testing the effects of normal aCSF (black line), 2 uM T30 (red lines), T30 (2 uM) and 4 uM NBP14 (blue lines), T30 (2 uM) and 4 uM Tri02, control NBP14 (4 uM) experiments (FIG. 11, orange lines), control Tri02 (4 uM) experiments (FIG. 12, purple lines). This graph shows the normalised decreases relative to baseline and each other, with T30 alone inducing the greatest deviation, and Tri02 showing some efficacy in blocking those T30-induced deviation, yet with significant changes still taking place in their co-perfusion (green lines).

Example 6—Pharmacokinetics

[0294] The inventors investigated the degradation products of NBP-14, Tri-02 and Tri-04 in rat and human blood.

Procedure

[0295] Test compounds were spiked at 10 μg/ml into either PBS or blood (Male Wistar rat or Human) diluted with PBS, and a series of samples taken according to the following scheme:

TABLE-US-00004 Time (min) Matrix 0 5 15 30 60 PBS control A1 A2 A3 A4 A5 Blood dil 5-fold B1 B2 B3 B4 B5 Blood dil 20-fold C1 C2 C3 C4 C5 Blood dil 50-fold D1 D2 D3 D4 D5

[0296] Procaine, a compound known to be unstable in blood, was included as a positive control (ran with 5-fold diluted blood only). The sampling procedure was to add an aliquot to ice-cold acetonitrile, centrifuge, and store the supernatant on dry ice until analysis. Analysis by UHPLC-TOF mass spectrometry using electrospray ionisation was performed on the same day as the incubations were performed.

Results

[0297] Procaine showed 60% and 100% turnover in 5-fold diluted rat and human blood, respectively, indicating acceptable metabolic competence for the blood used, as shown in FIGS. 22 and 23.

[0298] Stability data for NBP-14, Tri-02 and Tri-04 in rat and human blood is plotted in FIGS. 16-21. NBP-14 exhibited good stability, and no degradants were detected. Tri-02 exhibited some instability in both 5-fold and 20-fold diluted rat and human blood, and a variety of degradants were detected as indicated in FIG. 24. Tri-04 exhibited better stability than TRI-02, but nevertheless some degradants were still detected in 5-fold diluted rat blood, as indicated in FIG. 25. Accordingly, Tri-02 and Tri-04 are stable and so are good drug candidates.

Example 7—Evaluation of Compound 3 in Brain Slices

[0299] The inventors tested Tri04 in brain slice studies using voltage-sensitive dye imaging (VSDI).

Materials and Methods

1. Brain Slice Preparation

[0300] Brain slices were prepared as in Example 5.

2. VSD Setup

[0301] Slices were placed in a dark, high humidity chamber filled with aCSF bubbling with 95% O2e5% CO2. Once there, the dye solution (4% 0.2 mM styryl dye pyridinium 4-[2-[6-(dibutylamino)-2-aphthalenyl]-ethenyl]-1-(3-sulfopropyl)hydroxide (Di-4-ANEPPS, Invitrogen, Paisley, UK) (Tominaga et al., 2000) in aCSF 48%, fetal bovine serum 48%, DMSO 3.5% and cremophore EL 0.4%) was applied to the slices for 20-25 min before being transferred to an aCSF bubbler pot (room temperature, 22 C+/−1.5 C) for 1 h to wash off excess dye and recover.

[0302] When starting VSD recordings, slices were placed in the recording bath on a small piece of filter paper to keep slice alive and was weighed down appropriately using a home-made plastic grid placed atop the slice. The perfusing bath solution was heated to 30+/−1 C by a stage heater. A concentric bipolar stimulating electrode (FHC, Maine, US) was placed in the ventral diagonal band of the basal forebrain with stimulation being set at 30 V. For acquiring of VSD data, 16-bit images were recorded with 1 ms resolution with a digital camera (Brain Vision MiCAM Ultima R3-V20 Master) with Ultima 2004/08 imaging software (Brain Vision) coupled to Spike 2 V6.0 (CED Ltd, Cambridge, UK) which was used to trigger stimulations with respect to appropriate ISI. Light was generated using an Osram halogen xenophot 64634 HLX EFR Display/Optic lamp and was filtered to emit green (530+/−10 nm) light using a MHF-G150LR (Moritex Corporation) coupled to MiCAM Ultima ultra-fast imaging system and filtered the emitted fluorescence through a >590 nm high-pass filter.

3. Drug Preparation and Application

[0303] The linear peptidomimetic, Tri04, was designed in silico by Iproteos (Barcelona, Spain) and synthesised and purchased from Genosphere Biotechnologies at >99% purity. All drug and peptide stocks were prepared in frozen aliquots prior to experiments. For the production of perfusion solutions, stock solutions were thawed and added to recording aCSF as appropriate and bath applied at a constant rate of 1.5 ml/min perfusion using the Minipulse 3 peristaltic pump (Gilson Scientific Ltd., Bedfordshire, UK). Each experimental trial lasted 52 minutes, with 20 minutes to establish a baseline recording (perfusion with recording aCSF only), 12 minutes to allow the drug solution to perfuse into the bath as well as to let the dye molecules reseat themselves in the cell membranes and finally, a 15 minute recording period measuring the response in the presence of the drug solution.

5. Analysis of Modulatory Peptides

[0304] Throughout the majority of experiments in which T30 was used, a decrease in signal was observed. T30 induced a net inhibition (n=21) in recorded VSDI signal in the basal forebrain of p14 rats, this value actually includes a minority of instances where negligible or marginally positive effects were seen during T30 perfusion (Badin et al., 2016).

Results and Discussion

[0305] Referring to FIGS. 27, 28 and 29, addition of 4 μM Tri04 recapitulates results previously seen with the application of 4 μM NBP14, while 2 μM Tri04 in the perfusion solution determines a significant effect on basal forebrain population activity.

Analysis of Modulatory Peptidomimetics

[0306] Referring to FIG. 27A, there is shown that space-time maps exhibit a recovery in basal forebrain activity due to the presence of 2 μM Tri04 in the perfusate containing 2 μM of T30 (n=29). More specifically, 2 μM Tri04 determines a reversal of the inhibitory effect of T30 over activity measured by direct stimulation of the rat basal forebrain.

[0307] Referring to FIG. 27B, bar graphs relative to the 3 recording epochs show changes in the evoked response after Tri04 application, confirming that 2 μM Tri04 co-perfusion induces an increase in network activity (n=29, p=0.06, two-tailed paired t-test) caused by a inhibition of T30-induces effects.

[0308] Referring to FIG. 28, there is shown that response time-series across the three recording conditions (baseline, T30 application to the artificial cerebro-spinal fluid (aCSF) and co-application of T30 and Tri04 to the aCSF show a similar activation profile for T30 recordings and T30+Tri04 for the first 100 msec, while a higher activity in recordings made in presence of Tri04 is detectable afterwards, confirming a protective role of Tri04 over T30.

[0309] Referring to FIG. 29, there is shown bar graphs relative to three recording conditions. The co-perfusion of 4 μM Tri04 in the artificial cerebro-spinal fluid (aCSF) containing 2 UM T30 determines a significant effect reversing T30 activity. In particular, Tri04 has been found to be protective against T30-induced deviations from the baseline with a significant increase (n=20, p<0.05, two-tailed paired t-test) in basal forebrain activity in comparison to recordings in the presence of T30 alone. Therefore, Tri04 shows some efficacy blocking T30 toxic effects on meso-scale network activity.