Neurodegenerative disorders

Abstract

A cyclic polypeptide, derivative or analogue thereof, comprising an amino acid sequence derived from the C-terminus of acetylcholinesterase (ACh E), or a truncation thereof.

Claims

1-3. (canceled)

4. A cyclic polypeptide of SEQ ID NO: 4, or a derivative or analogue thereof having at least 65% sequence identity to SEQ ID NO: 4.

5-8. (canceled)

9. A cyclic polypeptide, derivative or analogue thereof according to claim 1, wherein the cyclic polypeptide, derivative or analogue thereof comprises an amino acid sequence derived from the C-terminus of tailed acetylcholinesterase (T-AChE), or a truncation thereof.

10-29. (canceled)

30. A method of using the cyclic polypeptide, derivative or analogue thereof according to claim 1, in an in vitro or ex vivo analytical method for investigating the allosteric site of 7 nicotinic-receptor comprising the step of inhibiting additional influx of calcium through the 7 nicotinic-receptor.

31. (canceled)

32. The cyclic polypeptide, derivative or analogue thereof according to claim 1, having at least 75%, 85%, 95% or 99% sequence identity to SEQ ID No: 4.

33. The cyclic polypeptide, derivative or analogue thereof according to claim 1, wherein the polypeptide, derivative or analogue thereof is the cyclic polypeptide of SEQ ID No: 4.

34. The cyclic polypeptide, derivative or analogue thereof according to claim 1, wherein the cyclic polypeptide, derivative or analogue thereof is a selective allosteric modulator of the 7 nicotinic-receptor.

35. The cyclic polypeptide, derivative or analogue thereof according to claim 1, wherein the cyclic polypeptide, derivative or analogue thereof is a selective allosteric modulator of the 7 nicotinic-receptor, which prevents the additional influx of calcium through an allosteric site of the 7 nicotinic-receptor, and outcompetes binding for -amyloid.

36. A method of treating, ameliorating or preventing a neurodegenerative disorder in a subject, the method comprising, administering to a subject in need of such treatment, a therapeutically effective amount of the cyclic polypeptide, derivative or analogue thereof according to claim 1.

37. The method according to claim 36, wherein the polypeptide, derivative or analogue thereof is the cyclic polypeptide of SEQ ID No: 4.

38. The method according to claim 36, 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); Frontotemporal Dementia; and Schizophrenia.

39. The method according to claim 36, wherein the neurodegenerative disorder is Alzheimer's disease, Motor Neurone disease or Parkinson's disease.

40. The method according to claim 36, wherein the neurodegenerative disorder is Motor Neurone disease.

41. The method according to claim 36, wherein the neurodegenerative disorder is Alzheimer's disease.

42. The method according to claim 36, wherein the neurodegenerative disorder is Parkinson's disease.

43. The method according to claim 36, wherein the cyclic polypeptide, derivative or analogue thereof is administered intranasally.

44. A pharmaceutical composition comprising a therapeutically effective amount of a cyclic polypeptide, derivative or analogue thereof according to claim 1, and a pharmaceutically acceptable vehicle.

45. The pharmaceutical composition according to claim 44, wherein the polypeptide, derivative or analogue thereof is the cyclic polypeptide of SEQ ID No: 4.

46. A process for making the pharmaceutical composition, comprising combining a therapeutically effective amount of a cyclic polypeptide, derivative or analogue thereof according to claim 1 with a pharmaceutically acceptable vehicle.

47. A receptor allosteric modulator comprising a cyclic polypeptide, derivative or analogue thereof according to claim 1.

Description

[0093] All of the features described herein (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.

[0094] 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:FIG. 1 is a bar chart showing no effect of Cyclic T14 alone on PC12 cell viability after 1 hour treatment. Data represent meanSEM, N=6;

[0095] FIG. 2 is a graph showing no change in enzymatic activity of AChE (determined by its ability to cleave increasing concentrations of a substrate) in the presence or absence of Cyclic T14. In contrast, Galanthamine displays highly significant competitive inhibition of enzyme activity. Data represent meanSD, N=4;

[0096] FIG. 3 is a bar chart showing the non-specific toxic effect of H.sub.2O.sub.2 (100 M) alone, and its persistence when combined with Cyclic T14 (100 nM) on cell viability. Data represent meanSEM, N=6. *vs Control; * P<0.05, **P<0.01;

[0097] FIG. 4 (A-C) shows the effect after 1 hour treatment of (A) -Amyloid (10 M), (B) T14 (10 M) and (C) T30 (10 M) alone, and combined at time zero with Cyclic T14 (100 nM). Data represent meanSEM, N=12. *vs Control; * P<0.05, ***P<0.001;

[0098] FIG. 5 is a bar chart showing AChE activity after treatment of PC12 cells with T30 (10 M) alone, T14 (10 M) alone or combined with Cyclic T14 (100 nM). Data represent meanSEM, N=6. *vs Control, ***P<0.001; # within groups, ### P<0.001;

[0099] FIG. 6 shows competition curves of the inhibition of [3H] Ivermectin binding by -Amyloid, T30 and T14 in membranes of PC12 cells, as also shown in rat brain membranes. Data represent the meansSEM, N=3;

[0100] FIG. 7 shows competition curves of the inhibition of [3H] Ivermectin binding by Cyclic T14 and Galanthamine in membranes of PC12 cells, as also shown in rat brain membranes. Data represent the meansSEM, N=6;

[0101] FIG. 8 is a bar chart showing the minimum effective concentration of Galanthamine (100 nM) and Cyclic T14 (1 nM) against -Amyloid (10 M) on cell viability. Data represents meanSEM, N=6. *vs Control; * P<0.05;

[0102] FIG. 9 is a graph showing the dose response effect of Cyclic T14 (1 nM) against -Amyloid (10 M) on cell viability. Data represents meanSEM, N=6. *vs Control; * P<0.05;

[0103] FIG. 10 is a bar graph showing the intracellular levels of Ca.sup.2+ in rat brain slices after 2 hours of different treatments. Data represents meanSEM, N=4;

[0104] FIG. 11 shows the amino acid sequence alignment of the peptides, T30, T15, T14 and -amyloid (A);

[0105] FIG. 12 is a diagram showing the binding sites of -amyloid and T30 on the 7-nAChR;

[0106] FIG. 13 is a graph showing the protective effect of different concentrations of NBP-14 (5, 7, 9, 10, 20, 50, 70, 1000, 5000 nM) on Calcium influx induced by T30. The values were fitted to a non linear curve with the logarithm of the inhibitor concentrations, NBP-14, versus the response of the T30, by using GraphPad Prism;

[0107] FIG. 14 is a graph showing the protective effect of different concentrations of NBP-14 (5, 7, 9, 10, 20, 50, 70, 1000, 5000 nM) on AChE release induced by T30. The values were fitted to a non-linear curve with the logarithm of the inhibitor concentrations, NBP-14, versus the response of the T30, by using GraphPad Prism;

[0108] FIG. 15 is a graph showing the protective effect of different concentrations of NBP-14 (5, 7, 9, 10, 20, 50, 70, 1000, 5000 nM) on cell viability induced by T30. The values were fitted to a non linear curve with the logarithm of the inhibitor concentrations, NBP-14, versus the response of the T30, by using GraphPad Prism;

[0109] FIG. 16 (A, B) include graphs showing overall cortical response to thalamic electrical stimulation under different T30 concentrations. (A) Synchrony of neuronal population activity, measured as fractional change in fluorescence intensity. (B) Spread of population activity, measured as the number of active pixelsdefined as pixels showing more than 20% of the max intensity given off by any single pixel within the Region of Interest;

[0110] FIG. 17 shows isolation and linear analysis of the rise phase of the spread of assemblies under different T30 concentrations, as seen from FIG. 16. Increasing concentrations of T30 show a dose-dependent decrease in the linear slope, equivalent to the velocity of propagation of the assembly;

[0111] FIG. 18 shows analysis of spread dynamics of thalamo-cortically-evoked neuronal assemblies under T30 and NBP-14 treatment, n=3. Analysis of the rise phase (left panel) shows a latent effect of T30 in increasing the slope (velocity) of propagation. The slope shows significant increases only about 45-60 minutes after initial T30 perfusion (yellow bar, effects become apparent during orange perfusionwith perfusion of 5 nM NBP-14). This sharp increase trend is slowed down during the 100 nM NBP-14 perfusion (red bar), and then reversed back to baseline levels during the final 300 nM NBP-14 perfusion (black bar). Plateau phase analysis (right panel) shows a similar profile of effects. The top panel shows a box and whisker plot averaging the behaviour of the assemblies' spread under the different drug treatments: a similar trend as in the rise-phase slope analysis can be seen, and though non-significant, the trend remains that T30 gradually increases the spread of evoked assemblies until a sufficiently high NBP-14 concentration reaches the recording bath (100 nM) where this excitatory trend is reversed back towards control levels (blue) at 300 nM NBP-14 perfusion (black);

[0112] FIG. 19 shows qualitative results from the three experiments (ie left, centre and right-hand columns) where T30 effects were tested against increasing concentrations of NBP-14 (0.1, 5, 100 & 300 nM). Top panel shows the two main averaged-data graphs: leftIntensity of fluorescence signal, and rightspread of evoked assemblies. Bottom panel shows space-time maps mapping the activity of a row of pixels lying over the area of interest (y-axis) over time (310 ms total, x-axis) for each perfusion conditions. The drastic reduction in fluorescence intensity as a result of T30 and NBP-14 co-perfusion is clearly evident, as it is on the Intensity graph. Note: the space-time maps are labelled with their respective perfusion (left), and are colour-coded to their corresponding traces both in the intensity (right) and spread (left) graphs;

[0113] FIG. 20 shows a schematic of the procedure followed during the in vivo testing of NBP-14. The day before the surgery all animals are tested in order to reveal any impairment. The day of the surgery is considered as day 0 of the study. On day one a paw placement test allowed the selection of the 16 out of 24 best subjects in order to inject with the vehicle or NPB-14. On day 2 a paw placement test was performed;

[0114] FIG. 21 shows the effect of NBP-14 against 6-OHDA in comparison with Baseline and 6OHDA alone in the Paw placement test. 6-OHDA **P<0.01 and 6OHDA+NBP14 ***P<0.001 vs Baseline. (N=8);

[0115] FIG. 22 shows the cascade of events resulting from the effect of T30 in a cell;

[0116] FIG. 23 shows full-length APP is reduced by NBP-14. Data represent expression of APP in solubilized PC12 cells after 3 different 1 hour treatments. Data are represented as meanSEM, n=2; and

[0117] FIG. 24 shows immunodetection by western blot represented in the graph. The 3 different treatments show different levels of expression of APP (values represented in FIG. 24. For each condition protein is corrected by levels of GAPDH.

[0118] FIG. 25 shows that T30 induces release of A42, an effect reversed by NBP14. Graph showing the release of A42 in control conditions, in presence of T30 and in presence of T30 and NBP-14. The results are represented as meanSEM (n=4).

EXAMPLES

[0119] It should be noted that SEQ ID No:4 is referred to herein as cyclated T14, CT14 or NBP14.

Materials and Methods

Cyclisation of Peptides

[0120] Three techniques were used to achieve cyclization of linear peptides described herein, i.e. sidechain-to-sidechain, sidechain-to-backbone, and head-to-tail (C-terminus to N-terminus) cyclization. Head-to-tail cyclization has been investigated extensively, and can involve directed Cys-Cys disulphide cyclization (up to two per molecule). Careful monitoring of the reaction ensures 100% cyclization. Two general approaches are used for synthesis: (1) classical solution-phase linear peptide cyclization under high dilution conditions; and (2) resin-based cyclization. Two distinct protocols were employed in the solid phase synthesis (1):

(a) The on-resin cyclization of a peptide anchored via a sidechain functional group, such as imidazole, 3 acid,4 amine or alcohol, was carried out. The peptide was orthogonally protected as an ester at the C-terminus, and the peptide was then assembled through regular Boc or Fmoc synthesis followed by saponification, cyclization and cleavage.
(b) Another protocol that was used was the cyclization cleavage approach, in which the cyclic peptide was synthesized by cyclization after step-wise linear peptide synthesis. One advantage of this method is that the sidechain does not need to be anchored, making the approach more general than (a). (Christopher J. White and Andrei K. Yudin (2011) Nature Chemistry 3; Valero et al (1999) J Peptide Res. 53, 76-67; Lihu Yang and Greg Morriello (1999) Tetrahedron Letters 40, 8197-8200; Parvesh Wadhwani et al (2006) J. Org. Chem. 71, 55-61).

[0121] Resultant samples of cyclic peptides were analysed by MALDI-TOF MS.

PC12 Cell Culture

[0122] PC12 cells are a cloned, pheochromocytoma cell line derived from the adrenal medulla (Greene and Tischler, 1976, Proc Natl Acad Sci USA 73: 2424-2428; Mizrachi et al., 1990, Proc Natl Acad Sci USA 87: 6161-6165). They are easily cultured and readily accessible to experimental manipulations. Since chromaffin cells are derived from the neural crest but are located in the centre of an accessible peripheral organ (the adrenal medulla) they have been described as offering a window into the brain (Bornstein et al., 2012, Mol Psychiatry 17: 354-358). These cells serve as a powerful, albeit novel, in vitro model for studying the still unknown primary process of neurodegeneration and the reasons why they are useful for this project are the following: the adrenal medulla in Alzheimer's patients shows various pathological features reminiscent of those seen in the CNS, e.g. numerous Lewy-body like inclusions, neurofibrillary tangles and paired helical filaments, as well as expression of amyloid precursor protein (APP) (Takeda et al., 1994, Neurosci Lett 168: 57-60). Moreover Appleyard and Macdonald (1991, Lancet 338: 1085-1086) demonstrated a selective reduction only in the soluble i.e. releasable form of AChE from the adrenal gland in AD, perhaps due to its enhanced secretion into the plasma, where it is elevated in AD patients (Atack et al., 1985, J Neurol Sci 70: 1-12; Berson et al., 2008, Brain 131: 109-119).

[0123] Wild-type PC12 cell were provided by Sigma-Aldrich (St. Louis, Mo.). The culture was routinely plated in 100 mm dishes (Corning) coated with collagen (2 g/cm.sup.2) and maintained in growth medium with Minimum Essential Medium Eagle (MEM) supplemented with heat-inactivated 10% horse serum (HS) and 5% foetal bovine serum (FBS), 10 mM HEPES, 2 mM L-Glutamine and 1:400 Penicillin/streptomycin solution. Cells were maintained at 37 C. in a humidified atmosphere 5% CO.sub.2 and the medium was replaced every 2 days. For splitting, cells were dislodged from the dish using a pipette with medium, with a portion of these replated onto new cultured dishes. Cells were used between passages 12 and 25.

Cell Membrane Preparation

[0124] PC12 membranes where obtained to perform binding assays. PC12 cells were grown until confluence on 100 mm plates. Growth medium was removed and ice-cold 50 mM Tris-HCl buffer (pH 7.4) containing 4.5 g/1 aprotinin and 0.1 mM phenylmethylsulphonylfluoride (PMSF) were added. Cells were mechanically detached and pelleted by centrifugation (1040g) for 4 minutes at 4 C. Pellets were homogenized with a Polytron and centrifuged (13000g) for 20 minutes at 4 C. The pellets were resuspended in fresh buffer and incubated at 37 C. for 10 minutes to remove endogenous neurotransmitters. The samples were subsequently re-centrifuged. The final pellet was resuspended in buffer and the protein concentration determined using the Bradford Reagent (Sigma-Aldrich, St. Louis, Mo.). The cell membrane preparation was stored at 80 C.

-Amyloid Preparation

[0125] -Amyloid (1-42) fibrils were prepared as described by provider (Abcam, Cambridge UK)). 1 mg of -Amyloid (1-42) was dissolved in 212 l of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and 10 l of NH.sub.4OH. After sonication and distribution of 10 l of sample per tube, samples were dried in a speed vacuum drier (Thermo Fisher Scientific, Loughborough, UK) and stored at 20 C. For experiments, samples were diluted in 2 of DMSO (5 mM) and 98 l of HCl (0.01 N) to ensure fibril formation and incubated over night at 37 C.

[.SUP.3.H] Ivermectin Binding Assay

[0126] For the binding with PC12 membranes, each incubation was performed in polystyrene tube (VWR International Ltd; Leicestershire, UK) containing 0.25 ml of membranes diluted in Tris-HCl 50 mM buffer (containing 50 g of PC12 membranes) with 5 nM [3H] Ivermectin (American Radiolabeled Chemicals, USA) in the absence or presence of different concentrations of AChE peptide T30, -Amyloid or Cyclic T14 (0.1, 0.5, 0.7, 1, 2, 10 M) diluted in Tris-HCl 50 mM, in a final volume of 0.5 ml for 2 h at 4 C. Thereafter, samples were filtered through Brandel GB glass fibre filters (MD, USA); pre-soaked in 0.5% polyethylenemine by a Harvester (Brandel; MD, USA). Tubes were washed 3 times with ice cold 50 mM Tris-HCl buffer. Radioactivity in the tubes was counted by scintillation spectrometry using a 300SL Liquid scintillation counter (Lablogic Systems Limited, UK). Specific binding was determined by subtracting the non-specific (cells treated with Ivermectin 30 M) value to all the tubes.

Cell Viability Assay

[0127] The cell viability assay used was the sulforhodamine B (SRB) colorimetric assay for toxicity screening. The day before of the experiment cells were seeded onto collagen-coated 96-well plates in a concentration of 40,000 cells/well. Cell concentration was determined by the Fuchs-Rosenthal chamber. Drugs were prepared in MEM containing L-Glutamine and cells were treated with different concentrations of Cyclic T14 (0.1-100 M) and T30, T14 and A (10 M) alone or combined with Cyclic T14 (0.1 and 0.7 M). After treatment, medium was replaced and cells were fixed by adding 100 l of 10% Trichloroacetic Acid (TCA) for 1 h at 4 C. Thereafter, cells were washed with H.sub.2O and stained with 100 l of a 0.057% SRB solution in 1% Acetic acid (HAc) for 30 minutes at room temperature. After staining cells were washed with 1% HAc for removing the excess of SRB and then incubated with 200 l of 10 mM Tris base (pH 10.5) and shake it for 5 minutes to solubilise the protein-bound dye. Measurement of the absorbance took place in a VMax Kinetic Microplate Reader (Molecular Devices) at 490 nm.

Acetylcholinesterase Activity Assay

[0128] AChE activity was measured using the Ellman reagent that measures the presence of thiol groups as a result of AChE activity. Cells were plated the day before the experiment as for the cell viability assay. Cells were treated with different concentrations of Cyclic T14 (0.1-100 M) and T30, T14 and A 10 M alone or combined with Cyclic T14 (0.1 and 0.7 M). After treatment, supernatant (perfusate) of each treatment was collected and 25 L of each condition were added to a new flat bottomed 96 well plate followed by the addition of 175 l of Ellman reagent (Solution A: KH.sub.2PO.sub.4139 mM and K.sub.2HPO.sub.4 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 at regular intervals (3, 10, 30 and 60 mins) across experiments at 405 nm.

Calcium Fluorometry

[0129] Increases in intracellular Ca.sup.2+ were monitored by measuring changes in fluorescence in cells loaded with Fluo-4 (Life Technologies Corporation, UK). The brain slices were incubated for 2 hours in 124 mM NaCl, 3.7 mM KCl, 26 mM NaHCO.sub.3, 2 mM CaCl.sub.2, 1.3 mM MgSO.sub.4, 1.3 mM KH.sub.2PO.sub.4 and 10 mM glucose; pH: 7.1 containing -Amyloid, CyclicT14 or -Amyloid+Cyclic T14. After the 2 hours, slices were incubated in the dark for 40 minutes at room temperature with 1.2 ml/well of loading medium that contained: Tyrode's salt solution (TSS; 137 mM NaCl, 2.7 mM KCl, 1.0 mM MgCl.sub.2, 2.5 mM CaCl.sub.2), 0.2 mM NaH.sub.2PO.sub.4, 12.0 NaHCO.sub.3, 5.5 glucose, pH 7.4), Fluo-4 (2 M), Pluronic F127 (0.02%) and probenecid (2 mM). Probenecid is a blocker of the multidrug resistant protein, an ion transporter, and avoids the excretion of the fluorescent molecule from the cell. After incubation, slices were washed with TSS and 1200 l/well of de-esterification medium, containing TSS and probenecid, were added. Slices were incubated in the dark for 20 minutes at 22 C. Fluorescence measurements (excitation 485 nm, emission 538 nm) were recorded in a Fluostar Optima (BMG, UK) plate reader.

Drugs and Reagents

[0130] MEM, culture serums, antibiotics, collagen, sulforhodamine B, Ivermectin and buffers reagents were provided by Sigma-Aldrich (St. Louis, Mo.). T30, T14 AChE peptides and Cyclic T14 were synthesized by Genosphere Biotechnologies (France). Stocks of peptides were diluted in distilled water.

Data Analysis

[0131] In each of the different techniques, the statistics analysis was performed with the average of the percentage values of 12 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.). These tests compare the means of every treatment to the means of every other treatment; that is, apply simultaneously to the set of all pairwise comparisons and identify where the difference between two means is greater than the standard error would be expected to allow. Statistical significance was taken at a P value <0.05. Graphs were plotted using GraphPAD Prism 6 (GraphPAD software, San Diego, Calif.). In the case of the binding experiment, results were obtained as counts per minute (cpm) and transformed to percentages related to control. Results were fitted to a model of one site competition binding using GraphPad Prism. In the case of the calcium results, the EC.sub.50 values were calculated by fitting the logarithm of the experimental data points to a single site Hill equation using a non-linear regression curve using GraphPad Prism.

Example 1Cyclisation of T14

[0132] The inventor synthesised an agent that selectively targets the allosteric site on the 7 nicotinic acetylcholine receptor, to compete for binding with T14/T30 and also to antagonise -amyloid. The agent is a cyclic form of T14 having the amino acid sequence: AEFHRWSSYMVHWK [SEQ ID No:4], with the N-terminal alanine residue being connected to the C-terminal lysine residue. Genosphere Biotechnologies (France) performed the cyclisation of T14 by transforming the linear peptide into an N-terminal to C-terminal lactam. The following examples demonstrate for the first time how the Cyclic T14 peptide blocks the established toxic effects of the T30 peptide and amyloid in vitro.

Example 2Cyclic T14 is not Toxic when Applied Alone

[0133] Using sulforhodamine B (SRB) as a cell viability detection method, PC12 cells were treated for 1 hour with Cyclic T14 produced in example 1. As a result, no changes in cell viability were observed suggesting no toxicity at concentrations as high as 100 M (100 nM: 98.7615.15; 700 nM: 106.9419.92; 1 M: 104.8210.9; 100 M: 93.5811.62) (see FIG. 1).

Example 3Cyclic T14 does not Affect AChE Enzymatic Activity

[0134] The inventor next decided to confirm whether or not Cyclic T14 affects the enzymatic activity of acetylcholinesterase (AChE). AChE enzymatic activity was measured using the acetylcholinesterase activity assay. The inventor found that the presence of Cyclic T14 (2 M) did not affect enzyme activity of acetylcholinesterase: in contrast Galanthamine (2 M) was strongly inhibitory (see FIG. 2).

Example 4Cyclic T14 does not Protect Against Non-Specific Toxicity of Hydrogen Peroxide

[0135] The inventors then determined whether or not Cyclic T14 protects PC 12 cells against the non-specific cytotoxic effects of the hydrogen peroxide. As can be seen in FIG. 3, there is no significant difference when H.sub.2O.sub.2 is given alone or in combination with Cyclic T14.

[0136] Example 5Cyclic T14 protects cells from T14, T30 and -Amyloid toxicity Using SRB as a cell viability detection method, PC12 cells were treated for 1 hour with (4A) -amyloid, (4B) linear T14, or (4C) T30, either alone or combined with Cyclic T14 (100 nM). As shown in FIG. 4, the three peptides alone induce a decrease in cell viability (A: 69.8754.38; T14: 83.025.385 and T30: 68.3953.095), but when combined with Cyclic T14 cells were surprisingly protected from death (A+C14: 94.4757.4; T14+C14: 99.4120.475; T30+C14: 88.598.785).

Example 6Cyclic T14 Blocks AChE Release Induced by T14 and T30

[0137] The colourmetric Ellman assay was used to assess AChE activity as a compensatory response after a toxic stimulus. Cells were treated for 1 hour with linear T14 and T30 (10 M) alone and combined with Cyclic T14 (100 nM) (see FIG. 5). All the peptides induce an increase of AChE activity (T30: 129.101.18; T14: 123.00.62) that was partially blocked when combined with the Cyclic T14 (T30+C14: 110.580.80; T14+C14: 112.301.39).

Example 7-Amyloid, T30 and T14 Displace [3H] Ivermectin Binding

[0138] In order to demonstrate that the 7nAChR (the 7 nicotinic acetylcholine receptor) is a target for the 3-Amyloid, T30 and T14 in the preparations used here. [.sup.3H] Ivermectin binding assays were performed on PC12 cell membrane and demonstrate in a log dose-response manner a decrease of the affinity of the allosteric site of the receptor where the ligand [.sup.3H] Ivermectin binds (see Table 1, FIG. 6).

TABLE-US-00007 TABLE 1 Data showing the percentage of [.sup.3H] Ivermectin binding on PC12 cells in the presence of different concentrations of -Amyloid, T30 and T14, N = 3. % [.sup.3H] Ivermectin (Mean SEM) -Amyloid T30 T14 1 nM 100.00 9.70 109.16 11.9 .sup.100 9.91 10 nM 73.61 11.12 116.63 13.25 90.92 2.38 100 nM 41.17 8.90 106.15 8.04 88.92 3.82 1 M 29.98 12.20 97.41 7.9 85.17 3.03 5 M 34.49 17.29 80.22 3.81 85.36 3.96

Example 8Cyclic T14 Displaces [3H] Ivermectin Binding with Greater Efficacy than Galanthamine

[0139] Low micromolar concentrations of cyclic T14 displaced[.sup.3H] Ivermectin with similar affinity but with significantly greater efficacy than Galanthamine.

TABLE-US-00008 TABLE 2 Data showing the percentage of [.sup.3H] Ivermectin binding on PC12 cells in the presence of different concentrations of Cyclic T14 and Galantamine, N = 6 % [.sup.3H] Ivermectin (Mean SEM) Cyclic T14 Galanthamine 100 nM 98.10 3.28 100.00 11.48 200 nM 80.81 4.37 97.86 1.40 500 nM 79.72 6.76 90.96 1.87 700 nM 62.26 17.63 69.68 9.87 1 M 29.006 8.23 67.17 6.64 2 M 13.46 10.40 66.32 4.29

Example 9Cyclic T14 Protects Cells from -Amyloid Toxicity with Greater Efficacy than Galanthamine

[0140] Using SRB as a cell viability detection method, PC12 cells were treated for 1 hour with -amyloid either alone or combined with Cyclic T14 (1 nM) or Galanthamine (moo nM). As shown in FIG. 8, Cyclic T14 protected against A toxicity (97.349.57) in a dose two orders of magnitude lower than Galanthamine (98.7914.21).

Example 10Minimum Concentration of Cyclic T14 Required for 100% Protection Against -Amyloid

[0141] Using SRB as a cell viability detection method, PC12 cells were treated for 1 hour with -amyloid combined with Cyclic T14 in concentrations increasing from 0.5 nM to moo nM (0.5: 88.4910; 1:97.349.57; 10:102.288.53; 50:101.7913.99; moo: 103.686.34). The threshold dose for full protection was 1 nM (FIG. 9).

Example 11Cyclic T14 Reduces Ca.SUP.2+ Levels in Rat Brain Slices

[0142] Fluorometry was used to detect variations in calcium levels after treatment for two hours with Cyclic T14 1 M, -Amyloid 10 M and both combined. Cyclic T14 does not change the basal level of intracellular calcium whilst -Amyloid induces to increase the intracellular calcium level, which is returned to baseline by Cyclic T14 (see FIG. 10).

Example 12T30 Exhibits a High Binding Affinity for the Allosteric Site of the 7 Nicotinic-Receptor

[0143] Using tests for viability, the inventor has shown that T30 has a binding affinity approximately three orders of magnitude higher (5 nM) for the allosteric site on the 7 nicotinic-receptor, than drugs currently in clinical use, e.g. galanthamine (10 M).

General Discussion

[0144] Cyclic T14 is a novel 7 nicotinic-receptor inert allosteric modulator of the 7 nicotinic-receptor which antagonises the action of T30 and amyloid beta peptides Cyclic T14 is a novel 7 nicotinic-receptor antagonist. The inefficacy of protection against the non-specific agent hydrogen peroxide suggests that the blocking action of Cyclic T14 is selective and receptor mediated. Cyclic T14 antagonises the toxic effects of T30 in a variety of tests indicating that it prevents the additional influx of calcium through an allosteric site on the 7 receptor by competing for binding with T30 as well as with amyloid. The enhanced stability of cyclic peptides would account for this effective displacement.

Why would a Cyclic T14-Based Drug be More Effective than Currently Available Treatments?

[0145] The inventor has recently shown that T30 has a binding affinity approximately three orders of magnitude higher (5 nM) for the allosteric site on the 7 receptor, than drugs currently in clinical use, e.g. galanthamine (10 M). Indeed, this observation would suggest the reason why such drugs currently being prescribed have proved relatively disappointing (See Table 1; Kramp & Herrling, 2011, Neurodegenerative Dis 8, 44-94): if endogenous T30, in excess in the Alzheimer patient's brain, is already occupying the key site, it will not be displaced by low-affinity competition. However, it would be blocked by an agent with very similar or indeed superior binding affinities, as suggested here (see FIG. 7). Such an agent has therefore the potential for being a highly effective drug.

[0146] A further advantage of the Cyclic T14 is that, unlike galanthamine, which is additionally an AChE inhibitor, it would have no other biological actions, other than to bind to the receptor. If, as the inventor's previous work suggests (Greenfield, 2013, Chem Biol Interact. 203(3)543-6), T30 is indeed the pivotal signalling molecule in neurodegenerative diseases, then its antagonism would be combatting these diseases at the most fundamental and specific level. In any event, the observation that this novel agent also antagonises amyloid would be of great clinical interest, where amyloid is implicated in the degenerative process, irrespective of its precise role. It should be noted that whilst other therapeutic candidates targeting the availability of -Amyloid (e.g. gamma secretase inhibitors) have been ineffective, this is the first instance, of the effective blockade of amyloid toxicity.

TABLE-US-00009 TABLE 3 Comparison of features of Galanthamine vs Cyclic T14-based drug Cyclic Galanthamine Dream Drug T14 Inhibits AChE (side Does not affect AChE activity effects) Known action at various Specific action at 7 receptor receptors Micromolar affinity Nanomolar affinity Blocks -Amyloid at Blocks -Amyloid at low doses1 nM) high doses (0.1 M) Low permeability CNS Should have high permeability CNS High bioavailability Should have low peripheral periphery (Side bioavailability effects as diarrhoea) Post-symptomatic Pre-symptomatic

[0147] The inventor believes that the current results suggest that the conformation of Cyclic T14 allows it to bind to its specific target, 7 nicotinic-receptor. Referring to FIG. 12, there is shown a schematic diagram of the 7 nicotinic-receptor. The homomeric receptor contains five identical 7 subunits, which are each symmetrically arranged around a central pore through which ions, such as Na.sup.+ and Ca.sup.2+, pass when the receptor is activated. Each 7 subunit contains an orthosteric binding site (i.e. the active site) and an allosteric binding site. Normal physiological activation of the receptor is achieved by the binding of a single acetylcholine molecule to the interface of two 7 subunits via each of their orthosteric sites. Other known ligands of the orthosteric site include (but are not limited to) choline and Methyllycaconitine (MLA). Ligands of the allosteric site include (but are not limited to) linear and cyclical T14, cyclical and linear T30, galantamine, ivermectin and PNU12.

[0148] As shown in FIG. 10, although not wishing to be bound by this theory, the inventor believes that -amyloid (A) is capable of either (i) simultaneously binding to both the orthosteric and the allosteric binding sites of the 7 nicotinic-receptor, or (ii) non-specifically binding to one either of these sites. The inventor has found that cyclical T14 acts as an antagonist at the allosteric site.

Drug Design

[0149] The inventor believes that it will be possible to use the particular conformation of Cyclic T14 to design a much smaller chemical compound which nonetheless still mimics the three-dimensional form of Cyclic T14 and is able to cross the blood-brain barrier more readily.

Example 13Physico-Chemical Characterisation of Cyclic T-14 (i.e. Referred to as NBP14)

Background

[0150] The solubility of a compound in aqueous and organic solutions strongly affects its ability to cross physiological barriers in the body, such as gastric or enteral. In the case of drugs targeting brain diseases, e.g. dementia, an additional barrier has to be crossed, the Blood-Brain Barrier. The partition coefficient, also known as Log P, evaluates the ability of a compound to solubilize in water and organic solvent, which correlates with the capacity of a compound to cross the different biological barriers.

Detailed Methods

Solvent Preparation

[0151] Saturation of the solvents was performed as follows. 1-octanol was agitated in the presence of water for 24 h at room temperature. mQ water was agitated in presence of 1-octanol for 24 h at room temperature. Then the solutions were left to equilibrate overnight at room temperature. Saturated solvents were collected, using syringes and needles, and stored at room temperature until further use.

Shake-Tube Method

[0152] Saturated water and saturated i-octanol were placed in a glass tube in the following ratios: Each tube contained the equivalent of 0.25 mg of cyclic T14. All tubes were then mixed for 4 h at room temperature. After agitation the tubes were left at room temperature to equilibrate.

Standard Curve

[0153] The concentrations of cyclic T14 used for the standard curve were: 0.5 mg.Math.ml.sup.1, 0.25 mg.Math.ml.sup.1, 0.13 mg.Math.ml.sup.1, 0.066 mg.Math.ml.sup.1, 0.033 mg.Math.ml.sup.1 and 0.016 mg/ml.sup.1. The absorbance of the standard curve was measured at 280 nm.

Sample Analysis

[0154] Both fractions of each sample were collected separately using a syringe with needle. The absorbance of all fractions was measured at 280 nm and the concentration of all the fraction was estimated based on the standard curve. The partition coefficient of cyclic T14 was calculated using the following equation:

Log P=Log(Concentration in Octanol/Concentration in Water)

[0155] The results from each condition were averaged in order to obtain the Log P of cyclic T14.

Results and their Implications

[0156] The average Log P of cyclic T14 is 0.5899. A negative value for Log P means that the compound is more likely to be hydrophilic. However, a Log P close to 0 corresponds to a compound with the ability to be soluble in a lipophilic environment as well. Hence, NBP14 can be formulated to cross the BBB.

Example 14Effects of Tao and Cyclic T-14 (i.e. NBP-14) in PC12 Cells

[0157] To characterize further the protective effects of NBP-14 against T30 toxicity, the inventors have determined the concentration-effect on three in vitro systems ((A) Calcium influx; (B) AChE release; (C) Cell viability), as detailed in the Methods section below.

Methods

(A) Calcium Influx

[0158] PC12 cells are plated in 200 l of complete growth medium the day before the experiment in 96 well plates. On the day of the experiment, the Fluo-8 solution (Abcam) is prepared (as provider protocol). Subsequently, 100 l of growth medium is removed and 100 l of Fluo-8 solution is added. Treatments with T30 and NBP-14 are added and incubated for 30 minutes in the incubator and 30 minutes room temperature.

[0159] After 1 hour, the plate is placed in the fluorescence plate reader (Fluostar). Before reading the fluorescence, acetylcholine (ACh) 100 M is prepared and placed in the Fluostar injector. For each well, the reading will be formed by a basal fluorescence followed by acetylcholine injection that will induce an increase of calcium via nicotinic receptors. The effects of T30 and NBP-14 are then evaluated.

(B) AChE Release

[0160] The protocol used to detect changes in AChE activity is the same as described previously.

(C) Cell Viability

[0161] A Cell Counting Kit-8 (CCK-8) was used as an improvement of the SRB technique used before. By utilizing the highly water-soluble tetrazolium salt WST-8, CCK-8 produces a water-soluble formazan dye upon reduction in the presence of an electron carrier. WST-8 is reduced by dehydrogenases in cells to give a yellow colored product (formazan), which is soluble in the tissue culture medium. The amount of the formazan dye generated by the activity of dehydrogenases in cells is directly proportional to the number of living cells. PC12 cells are plated in 200 l of complete growth medium the day before the experiment in 96 well plates. Treatments with T30 and NBP-14 are added and incubated for 1 hour in the incubator.

[0162] Subsequently, 100 l of growth medium is removed and 10 l of CCK-8 (Cell Counting Kit-8) solution is added. The plate is incubated for 2 hours in the incubator and then placed in the absorbance plate reader. The absorbance must be measured at 450 nm.

(A) Calcium Influx

[0163] As stated previously, T30 is a positive allosteric modulator of the 7 nicotinic receptor. Hence the primary agonist acetylcholine was used to benchmark the control calcium influx as 100%). T30 (5 uM) enhanced this effect until 171.05%6.21%; N=3. Increasing concentrations of NBP-14 (5, 7, 9, 10, 20, 50, 70, 1000, 5000 nM) were subsequently added to determine the antagonism of these T30-induced increases. The values are (respectively) (%): 134.24976.85, 120.86128.65, 113.91628.82, 140.77612.16, 115.837.67, 110.321313.21, 125.95960.1, 99.850.32, 115.19429.84, 79.9914.04. FIG. 13 shows that NBP-14 blocks T30 effects in a concentration manner, being protective at low nanomolar range.

(B) AChE Release

[0164] As described above, PC12 cells respond to the toxic effect of T30 with a compensatory response, i.e. an increase in released AChE activity: 169.45%2.11%; N=3. The inventors determined the dose-dependent effect of NBP-14 against 5 uM T30. The results show (FIG. 14) that NBP-14 protects from AChE compensatory effects at high nanomolar concentrations. The values are (respectively) (%): 130.731.84, 111.682.26, 92.780.99, 82.562.38, 68.900.92, 65.121.32, 61.040.97, 79.431.691.24, 83.911.24, 89.551.25.

(C) Cell Viability

[0165] T30 (5 uM) induces a 25% (74.309%2.87%; N=3) decrease of cell viability that is progressively blocked by NBP-14 in a concentration-effect manner (FIG. 15). The values are (respectively) (%): 76.257.51, 67.044.35, 76.044.22, 71.361.64, 79.0210.22, 75.193.9, 62.433.01, 78.102.16, 116.653.62, 107.795.10. NBP-14 protects from T30 induced cell death in the high nanomolar range.

Example 15Effects of T30 and Cyclic T-14 (i.e. NBP14) on In Vitro Cortical Networks in Rat Brain Slices

Background

[0166] The T30 peptide is a 30-amino acid segment of acetylcholinesterase (AChE), from which the T14 is also cleaved. Both induce the same effect suggesting their active sequence is present on the T14, and in turn present on the T30. The research has already shown the bioactivity of T14 on mammalian brain slices as a highly modulatory agent. The effects of the T14 have been reported to modulate cortical networks, inducing both excitation and inhibition at different concentrations: at low concentrations the peptide triggers enhanced calcium influx via the alpha-7 receptor, but high concentrations induce such excessive amounts of calcium that the channel inactivates (Badin et al., 2013; Bon and Greenfield, 2003), as well as triggering neuronal plasticity (Greenfield et al., 2004).

[0167] In order to gain further understanding of the actions of T14/30 on whole cortical networks, the relatively recent technique of voltage-sensitive dye imaging (VSDI) was used in order to monitor the dynamics of collective neuronal population activity, neuronal assemblies, in brain slices on a temporal scale of milliseconds (ms, commensurate with physiological events) and micrometres (m). Such a technique exploits the sensitivity of specific lipophilic molecules containing a fluorescent core to changes in electrical potentials (Tominaga et al., 2000). Due to their lipophilic nature, these dye molecules embed themselves in cell membranes, and alter their fluorescence reading with regards to the voltage potential across that specific membrane, which are captured with a millisecond-resolution high-speed camera. As a result, imaging using voltage-sensitive dyes provides a direct and on-line readout of electrical potential changes across neuronal cell membranes with an unparalleled spatio-temporal resolution.

[0168] Using this technique the inventors can obtain comprehensive sets of data on neuronal population activity such as (a) the intensity of the response in any given area, from which (b) the spread of elicited neuronal assemblies can be measured, and from this parameter (c) the velocity of propagation of the activity wave-front from the point of initiation (d) measured as the slope of the spread. Each of these parameters have been measured independently for two experiments carried out so far: i) an investigation of the effects induced by increasing concentrations (0.5, 0.75, 1 & 5 M) of T30 on cortical population activity and responsiveness, and 2) assessing the antagonistic effects of NBP-14 on a single, relatively high (1 uM), T30 concentration. [0169] Technique used: voltage-sensitive dye imaging (Di-4-ANEPPS) of thalamocortical (TC) p14 rat (Wistar) brain slices. [0170] Stimulation paradigm: 40 Hz (consistent with thalamo-cortical recurrent stimulation) paired pulse stimulation. [0171] Perfusion paradigm: epochs carried out in two phasesfor every drug perfusion (say: control, 0.1 uM T30 etc.), the new perfusion was applied and left to perfuse for 15 minutes before starting the recording period (15 minutes also), such that the drug had time to reach its actual concentration and induce its concentration-dependent effects once recording. Meaning one perfusion epoch lasted 30 minutes, with the recording only taking in account the last 15 minutes of its respective perfusion epoch.

Detailed Methods

Brain Slice Preparation

[0172] Male Wistar rats (14-17 day old; 15 individual animals in total) were anaesthetised using isoflurane: 10 mL 100% w/w isoflurane was applied to the cotton bed at the bottom of an anaesthesia chamber (glass box 201515 cm) where rats were then placed for 45 seconds until onset of anaesthesia. The hind paw of each anaesthetised rat was pinched to check for appropriate depth of anaesthesia. Once anaesthesia was confirmed, rats were quickly decapitated before immersing the brain in oxygenated ice-cold artificial cerebrospinal fluid (slicing aCSF in mmol: 120 NaCl, 5 KCl, 20 NaHCO.sub.3, 2.4 CaCl.sub.2), 2 MgSO.sub.4, 1.2 KH.sub.2PO.sub.4, 10 glucose, 6.7 HEPES salt and 3.3 HEPES acid; pH: 7.1) for 7-8 minutes, the time taken to cut the brain into slices. Para-saggital sections (400 m thick) were cut from a block of brain containing both Thalamus (VPN) and primary somato-sensory cortex (barrel field) using a Vibratome (Leica VT1000S) and transferred to a bubbler pot containing aCSF at room temperature (recording aCSF in mmol: 124 NaCl, 3.7 KCl, 26 NaHCO.sub.3, 2 CaCl.sub.2, 1.3 MgSO.sub.4, 1.3 KH.sub.2PO.sub.4 and 10 glucose; pH: 70.1), which was identical to that which was used during electrophysiological recordings and VSDI. Slices were left in oxygenated (95% O.sub.2-5% CO.sub.2) recording aCSF to recuperate for at least 1 hour before VSD staining.

VSD Setup

[0173] Slices were placed in a dark, high humidity chamber filled with aCSF bubbling with 95% O.sub.2-5% CO.sub.2. The dye solution (4% 0.2 mM styryl dye pyridinium 4-[2-[6-(dibutylamino)-2-naphthalenyl]-ethenyl]-1-(3-sulfopropyl)hydroxide (Di-4-ANEPPS, Invitrogen, Paisley, UK) (Tominaga et al., 2000) in aCSF 46%, fetal bovine serum 46%, DMSO 3.5% and cremophore EL 0.4%) was then applied to the slices as previously described (Badin et al., 2013). 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. Because of the fluorescent VSD, all of the handling of slices during and after staining with Di-4-ANEPPS was carried out in almost complete darkness in order to keep the detrimental effects of photo-toxicity and bleaching to a minimum. VPN (where stimulating electrodes were placed) was identified with respect to distance from the tip of the hippocampus and to the side of the internal capsule.

[0174] Stimulating electrodes, with impedance (measured at 1000 Hz): 500 k, were placed in VPN, where paired-pulse stimulations (2100 s in duration; 25 milliseconds inter-stimulus intervalISIpaired-pulse at 40 Hz) were triggered to evoke fast-paced propagating waves of activity in the innervated barrels using Spike 2 V6.0 (CED Ltd, Cambridge, UK) with respect to appropriate ISI. Such transient neuronal assemblies were recorded by acquiring 16-bit images with a 1 ms resolution using MiCAM Ultima ultra-fast imaging system coupled to a digital camera (Brain Vision MiCAM Ultima R3-V20 Master) with Ultima 2004/08 imaging software (Brain Vision). Light was generated using an Osram halogen xenophot 64634 HLX EFR Display/Optic lamp and was filtered to emit green light (53010 nm) using a MHF-G150LR (Moritex Corporation). The emitted fluorescence was passed through a dichroic mirror and a >590 nm high-pass filter as described previously (Collins et al., 2007; Devonshire et al., 2010a; Devonshire et al., 2010b; Grandy et al., 2012; Mann et al., 2005).

Drug Preparation & Application

[0175] T30 and NBP-14 solutions were prepared fresh at the start of each experiment, stock solution aliquots were added to recording aCSF as appropriate and bath applied at a constant rate of 1.5 mL per min perfusion using a Minipulse 3 pump (Gilson Scientific Ltd, Bedfordshire, UK). Perfusion conditions were split in 2: the first part consisted of a 15 minute perfusion with no recording taking place, such that the appropriate concentration could be achieved in the recording bath before starting the second part of the perfusion conditionwhere the recording took place for the next 15 minutes of perfusion (30 averaged snapshots)giving a total of 30 minutes per perfusion condition.

Data Analysis and Statistics

[0176] VSDI produced 44 mm (100100 pixels) 2-Dimensional images from which critical data were extracted such as the time-course of activation, spread and intensity of the overall elicited signal. For each VSDI experiment, each snapshot's data between 0 and 200 ms after stimulation, encapsulating the peak response, had their parameters measured and averaged for each condition (total of 30 snapshots per condition for both T30 and T30 v NBP-14 experiments). In order to achieve this, a region of interest (ROI) was selected over the active area, which encompassed the width of the maximum response after it had been filtered with a threshold that isolated active pixels as those showing activity greater than 20% of the maximum activity recorded within that region of interest. Such data were then compiled to produce detailed quantitative graphs of the extent of activation intensity (FIG. 16, 17, 18) as well as qualitative space-time maps (FIG. 19) to measure the effects recorded as well as to produce accurate visual representations of the spatio-temporal data acquired. All statistical tests (Analysis of VarianceANOVA) were performed using non-linear mixed effects models fitted to the data using R Studio while all the data handling and analysis was performed using Mathematica 8 (Wolfram Research, USA). For all statistical tests P<0.05 was considered significant. Data are expressed as meanS.E.M.

Results and Discussion

[0177] FIG. 16 shows the effect of increasing dose of T30 on (A) intensity of emitted fluorescence, as well as (B) spread of active pixels. Each pixel has dimensions 4040 m meaning its specific fluorescence arises from the activity of less than 10 independent neurons. As the T30 concentration increases within the recording bath, the intensity of fluorescence given off by active pixels diminishes. This indicates a de-synchronisation of triggered neuronal population activity, as sizeable and simultaneous neuronal membrane depolarisation from a single pixel amounts to a higher fluorescence reading, the opposite effect is seen here (less synchrony). Additionally, here a 40 Hz paired-pulse stimulation paradigm is used, as can be seen from the Intensity graph (FIG. 16A). The results show that not only is the overall fluorescence reduced as a result of T30 treatment, but that the second pulse (which is triggered at 75 ms, 25 ms after the first one40 Hz paired pulse) shows a much reduced facilitation compared to the magnitude of the original pulse.

[0178] Furthermore, as can be seen from the spread graph (FIG. 16B), the spread of cortical assemblies is not significantly affected by T30 treatment until very high levels (5 uM) are achieved, this corroborates the theory that T30 acts as a modulating agent on whole cortical networks. It is also important to keep in mind that the different parameters highlighted by VSDI, such as the velocity of propagation, the spread and the intensity of fluorescence, often rely on underlying principles of cortical dynamics which are not necessarily related, meaning each of these parameters must be analysed separately and that their results must at first be interpreted independently from each other.

[0179] FIG. 17 shows the effect of increasing T30 application upon the dynamics of assembly initiation and propagation were investigated in greater detail. FIG. 17 shows that T30 induces a reduction in neuronal activity propagation speed (here acquired as the slope of the rise phase), consistent with a decrease in the synchrony of cortical networks, as shown in FIG. 16, with a maximum of a 3.5-fold decrease in propagation speed under 5 M T30 treatment. Resulting from the above, and previous experiments, it was concluded that the 5 M T30 concentration was too high as it was probably inducing sufficient calcium influx to be at the threshold for inactivation of the ion channel, thus leading to a mixture of excitatory/inhibitory effects depending on the sensitivity of the particular preparation, as reported previously (Bon and Greenfield, 2003). The inventors therefore used a lower, yet sufficiently potent, concentration, 1 M T30, to carry out the subsequent experiments exploring the possible antagonistic effects of NBP14. This T30 concentration, although still quite high, was chosen because of the nature of the present study, where an inevitable dilution effect is to be expected as the peptide penetrates the brain slice. Meanwhile, for NPB14, they used increasing concentrations of: 0.1, 5, 100 & 300 nM, i.e. 2 to 4 orders of magnitude lower than the concentrations of T30, since previous in vitro studies in PC 12 cells had indicated a far higher affinity for alpha-7 nicotinic acetylcholine receptors compared to that of T30.

[0180] FIG. 18 shows antagonism of T30 effects by increasing concentrations of NBP-14. The Figure shows the antagonistic nature of NBP-14 on the modulatory effects induced by T30 perfusion on neuronal populations. An important consideration is the dilution factor as T30 penetrates slices, including all physiological processes potentially still present and active within brain slices, such as proteases, neurotransmitter uptake and the density of the extracellular matrix; it therefore seems highly probable that a concentration of 1 M T30 would take time to induce its full effects (45-60 minutes, as suggested by the data presented above). Bearing this in mind, the effects of T30 become apparent during the 5 nM NBP-14 perfusion (orange line/bar), after which the trend induced by T30 is reversed back towards baseline (blue bar/line).

[0181] It is important to also note that NBP-14 has been shown to be inert, never inducing any modulatory effects on its own, implying that the effects seen here are at first attributable to T30, and their reversal to antagonism by increasing concentrations of NBP-14. Importantly, the vast majority of T14 effects are reduced back to control levels under the 300 nM NBP-14 perfusion, while T30 is perfused at a concentration of moo nM. This suggests a significantly higher affinity of NBP-14 for its target compared to T30.

[0182] FIG. 19 shows qualitative results from the three experiments (ie left, centre and right-hand columns) where T30 (1 M) effects were tested against increasing concentrations of NBP-14 (0.1, 5, 100 & 300 nM). Top panel shows the two main averaged-data graphs: leftIntensity of fluorescence signal, and rightspread of evoked assemblies. Bottom panel shows space-time maps mapping the activity of a row of pixels lying over the area of interest (y-axis) over time (310 ms total, x-axis) for each perfusion conditions. The drastic reduction in fluorescence intensity as a result of T30 and NBP-14 co-perfusion is clearly evident, as it is on the Intensity graph. Note: the space-time maps are labelled with their respective perfusion (left), and are colour-coded to their corresponding traces both in the intensity (right) and spread (left) graphs.

REFERENCES

[0183] Badin, A. S., J. Eraifej, and S. Greenfield. 2013. High-resolution spatio-temporal bioactivity of a novel peptide revealed by optical imaging in rat orbitofrontal cortex in vitro: Possible implications for neurodegenerative diseases. Neuropharmacology. 73C:10-18. [0184] Bon, C. L., and S. A. Greenfield. 2003. Bioactivity of a peptide derived from acetylcholinesterase: electrophysiological characterization in guinea-pig hippocampus. Eur J Neurosci. 17:1991-1995. [0185] Collins, T. F., E. O. Mann, M. R. Hill, E. J. Dommett, and S. A. Greenfield. 2007. Dynamics of neuronal assemblies are modulated by anaesthetics but not analgesics. Eur J Anaesthesiol. 24:609-614. [0186] Devonshire, I. M., E. J. Dommett, T. H. Grandy, A. C. Halliday, and S. A. Greenfield. 2010a. Environmental enrichment differentially modifies specific components of sensory-evoked activity in rat barrel cortex as revealed by simultaneous electrophysiological recordings and optical imaging in vivo. Neuroscience. 170:662-669. [0187] Devonshire, I. M., T. H. Grandy, E. J. Dommett, and S. A. Greenfield. 2010b. Effects of urethane anaesthesia on sensory processing in the rat barrel cortex revealed by combined optical imaging and electrophysiology. Eur J Neurosci. 32:786-797. [0188] Grandy, T. H., S. A. Greenfield, and I. M. Devonshire. 2012. An evaluation of in vivo voltage-sensitive dyes: pharmacological side effects and signal-to-noise ratios after effective removal of brain-pulsation artifacts. Journal of neurophysiology. 108:2931-2945. [0189] Greenfield, S. A., T. Day, E. O. Mann, and I. Bermudez. 2004. A novel peptide modulates alpha7 nicotinic receptor responses: implications for a possible trophic-toxic mechanism within the brain. J Neurochem. 90:325-331. [0190] Mann, E. O., T. Tominaga, M. Ichikawa, and S. A. Greenfield. 2005. Cholinergic modulation of the spatiotemporal pattern of hippocampal activity in vitro. Neuropharmacology. 48:118-133. [0191] Tominaga, T., Y. Tominaga, H. Yamada, G. Matsumoto, and M. Ichikawa. 2000. Quantification of optical signals with electrophysiological signals in neural activities of Di-4-ANEPPS stained rat hippocampal slices. Journal of neuroscience methods. 102:11-23.

Example 16Effects of NBP 14 in the Freely Moving Rat Background

[0192] Unlike animal models for Alzheimer's disease, the rat model for hemi-Parkinsonism is very well established and readily quantifiable. Accordingly, a unilateral intra striatum injection of the T30 was administered to observe any behavioural effects of the toxin. In a subsequent experiment, the potential protective effects of NBP14 were observed against the well-known neurotoxin 6-hydroxydopamine (6-OHDA), which led to DA neuron loss on the injected side whilst sparing the contralateral DA neurons. NBP-14 was administered via implanted cannula into the medial forebrain bundle (MFB). 6-hydroxydopamine was injected at 10 mg/kg.

Detailed Methods

[0193] Animals are anaesthetized using Ketamine (10%; 0.1 ml/kg body weight) and Xylazine (2%; 0.01 ml/kg). The animals are then stereotactically injected into the MFB with 2 L 6-OHDA at a concentration of 20 mg/ml in 0.02% ascorbic acid. Lesion coordinates are set according to bregma and dura in cm: L-1.7 mm; AP-3.6 mm; DV-8.0 mm. Following the injection (injection rate 2 l/5 min), the injecting needle is left for another 1 minute to avoid back flow and then slowly retracted.

[0194] Paw Placement Test (Cylinder Test): This test assesses a rat's independent forelimb use to support the body against the walls of a cylindrical enclosure. The test takes advantage of the animals' innate drive to explore a novel environment by standing on the hindlimbs and leaning towards the enclosing walls. To perform this test, rats are placed individually in a glass cylinder (21 cm diameter, 34 cm height) and wall exploration is recorded for 3 minutes. No habituation to the cylinder prior to recording is allowed. Wall exploration is expressed in terms of the ratio between the intact (R) and impaired legs (L) and calculated as the values of intact right+both forelimbs divided into the values of impaired left+both forelimbs (R/L). The paw placement test is conducted on Day 1 to obtain baseline data, on Day 1 for selection and on days 2.

[0195] Selection criteria (Day 1): According to Paw placement test all animals with statistically significant difference between paws will be included in the study (ratio between the intact (R) and impaired legs (L) is expressed as the values of intact right+both forelimbs divided into the values of impaired left+both forelimbs).

[0196] The results from all tests will be presented as MEAN group valueSEM. Analysis of the data by one-way ANOVA following by Tukey test will be applied to determine significance of treatment effects. This study was performed following approval of an application form submitted to the Committee for Ethical Conduct in the Care and Use of Laboratory Animals that states that the present study complied with the rules and regulations set forth.

[0197] FIG. 20 summarises the procedure followed during the in vivo testing of NBP-14.

Results

Effects NBP14

[0198] Analysis of the paw placement R/L ratio reflects unilateral injury of motor function. On day 2, i.e. two days post 6-OHDA injection and one day after injection of NBP-14, there was a significant difference in the R/L ratio of paw placement between 6-OHDA vehicle and treated: 7.541.63 vs. 3.620.55, respectively (p<0.05). Treatment with NBP-14 improved mobility of impaired forelimb after one dosing as was shown in the paw placement test (FIG. 21).

Example 17Effects of T30 and NBP14 on APP and Amyloid Background

[0199] It is has already been established that an excess of calcium can trigger abnormal cleavage of Amyloid Precursor Protein (APP) and hence Amyloid beta (A) release (Hartigan & Johnson,1999; Cai et al., 2012). Since the inventors have shown that T30 increases calcium influx by about 70% in PC12 cells, it is possible that such a calcium increase will trigger the production of amyloid and a consequent decrease in the full length APP molecule.

Detailed Methodology: Detection of APP

Protocol for Solubilizing Protein

[0200] PC12 cells are plated with growth medium in Petri Dishes for a week in order to have enough protein to detect APP in PC12 membranes and treated for 1 hour with T30 and NBP-14 before solubilizing the protein. Once the cells have grown until 90% confluence, the growth medium is removed and cells are re-suspended in 2 ml of HBSS. The cells suspension is transferred to a 15 ml tube and centrifuge 5 minutes at 1000 rpm. Then the supernatant is discarded and the pellet is re-suspended in Lysis buffer (20 mM Tris, 137 mM NaCl, 1% Triton X-100, 2 mM EDTA; pH 8) plus protease inhibitors (1 l:1 ml PMSF and 3 l:1 ml Aprotinin) and triturated by using a Polytron for to seconds. Subsequently, the triturated pellet is distributed in 1.5 ml eppendorfs and rotated or shaken for 2 h at 4 C. After 2 h, the eppendorfs are centrifuged at 15000 rpm for 20 minutes and the supernatant is kept. The Bradford reagent is used to quantify the protein contained in each eppendorf.

Protocol for Electrophoresis

[0201] For APP detection, an aliquot of 25 g of protein is used. Before starting the protocol the reagents are prepared as follows:

Lower Gel (10%) (20 Min to Polymerize)

[0202] For to ml (2 gels):3.6 ml H2O MQ, 2.42 ml Acrilamide and 1.3 ml Bis-Acrilamide, 2.5 ml Tris-HCl 1.5 M pH 8.8, 0.11 ml SDS 10%, 0.06 ml Ammonium persulfate 10%, 6.67 l TEMED (last ingredient).

Upper Gel (5%) (20 Min to Polymerize)

[0203] For 5 ml (2 gels): 3.67 ml H2O MQ, 0.48 ml Acrilamide and 0.26 ml Bis-Acrilamide, 0.625 ml Tris-hCl 1 M pH 6.8, 0.05 ml SDS 10%, 25 l Amonium persulfate 10%, 5 l TEMED.

Tris-HCl 1.5 M pH 8.8

[0204] For 100 ml: 18.16 gr Tris Base, qsp 100 ml H2O MQ, pH 8.8.

Tris-HCl 1 M pH 6.8

[0205] For 100 ml: 12.1 gr Tris Base, qsp too ml H2O MQ, pH 6.8.

Sample buffer (4)

[0206] For 8 ml: 3.2 ml SDS 10%, 1.6 ml Glicerol, 2 ml Tris-HCl 1 M pH 6.8, 0.8 ml B-Mercaptoethanol, 0.4 ml Bromophenol Blue 0.1% or Red. (Use 1 for experiment)

Running Buffer (10)

[0207] For 1 L: 30.3 g Tris base, 144 gr Glycine, 10 gr SDS, qsp 1 L H2O MQ. (Use 1 for Experiment)

[0208] The steps for electrophoresis are the following: [0209] a) Prepare the lower and the upper acrilamide gels. The % for APP gel is 10% lower gel and 5% stacking gel. [0210] b) Prepare 24 l of sample at a concentration of 25 g (determined by the Bradford Assay) (6 l SB 4 cpontaining -mercaptoethanol+protein+lisis) and boil them at 100 C. for 5 minutes to denaturalize them. [0211] c) Put the protein marker and the samples in the wells of the gel (20-30 L). [0212] d) Proceed to Migration: 35 mA (nearly 1 hour).

Protocol for Western Blot

[0213] Before starting the protocol the reagents are prepared as follows:

Transfer buffer (1)

[0214] For 1 L: 3.03 g Tris base, 14.4 gr Glycine, 200 ml Methanol, qsp 1 L H2O MQ.

TBS buffer (4)

[0215] For 1 L: 24.25 gr Tris base, 60 gr NaCl, qsp 1 L H2O MQ, pH 7.5.

TBS-Tween Buffer

[0216] For 1 L: 250 ml TBS 4, 0.5 ml Tween 20, qsp 1 L H2O MQ.

[0217] There are 2 steps to follow, the electrotransfer and the immunodetection of protein, see the steps below:

1) Electrotransfer

[0218] a) Activate the PVDF membrane: 1 minute in MeOH and 2 minutes in MQ H2O. [0219] b) Put the PVDF membranes, papers and sponges in Transfer buffer during 10 minutes. [0220] c) Prepare the sandwich and proceed with the transfer of the proteins from the gel to the PDVF membrane: 0.2 A during 2 hours.

2) Immunodetection of Proteins

[0221] a) Block the inespecific sites of the membrane with milk 5% (dissolve it in TBS-T). [0222] b) Incubation with the primary antibody (dissolved in TBST/milk 5%): Anti-Amyloid Precursor Protein (ab2072, rabbit) at a dilution 1:500 (20 l in 10 ml), over-night at 4 C. [0223] c) Wash the membrane with TBS-T (5 min.sub.2). [0224] d) Incubation with the secondary antibody dissolved in TBS-T: Anti-rabbit-HRP (goat) dilution 1:5000 (20 l in 10 ml) for 45 minutes at room temperature. [0225] e) Wash the membrane with TBS-T (5 min2+10 min1). [0226] f) Take a picture with Chemibox option (white light) to see the position of marker bands. [0227] g) Add ECL reagent (HRP) for antibody detection (1 ml of each component) and take several pictures with Chemibox option (no light).

Detailed Methodology: Detection of A42 in Cell Culture Media

[0228] After 1 hour of treatment, the culture media was collected and diluted to 1:100, using culture media as diluent. Four repeats of each diluted sample were then placed in the plate provided by the ELISA detection kit, from AnaSpec (Fremont, Calif., USA). The detection was then carried out following the manufacturer's protocol. Briefly, the sample was incubated for 4 h in presence of 50 l of detection antibody. The plate was then washed seven times with the washing solution, the samples were then incubated with the 3,3,5,5-Tetramethylbenzidine (TMB) provided with the kit for 15 min. after the sample revelation the reaction was stopped with the stop solution and the optical density was read at 450 nm.

[0229] FIG. 22 is a diagram showing the cascade of events resulting from the effect of T30 in a cell: (1) T30 binds to the allosteric site of the receptor to enhance the opening of the channel for Ca.sup.2+ influx into the cell (Greenfield et al. 2004). (2) Calcium entry induces depolarization and opening of the voltage-dependent (L-VOCC) channel allowing still more Ca.sup.2+ into the cell (Dickinson et al., 2007). (3) This raised intracellular calcium induces an increase in AChE G4 release that includes T30 (Greenfield, 2013). (4) Calcium also induces upregulation of the 7 nicotinic receptor that will allow more Ca.sup.2+ get in the cell by providing still more targets for T30 (Bond et al., 2009). (5) Calcium activates enzymes (ie GSK-3) that will (a) increase Tau, (b) activate -secretase/-secretase that will trigger cleavage of extracellular toxic Amyloid that (c) together with T30 will promote a still further toxic amount of Ca.sup.2+ into the cell. (Hartigan & Johnson (1999), Cai et al. (2012), Garcia-Rats et al (2013)).

[0230] Accordingly, using immunodetection, the inventors have determined (i) APP levels and (ii) release of amyloid following administration of T30 (5 uM) and NBP14 (0.5 M).

Results

[0231] (i) As shown in FIG. 23, T30 reduces levels of full length APP in PC12 cell membranes, an effect reversed by NBP14. The values are: Control (100%10.98); T30 5 M (67.82%6.23%) and T30+NBP-14 0.5 M (126%1.12%).

[0232] FIG. 24 shows immunodetection by western blot represented in the graph. The 3 different treatments show different levels of expression of APP (values represented in FIG. 24). For each condition protein is corrected by levels of GAPDH.

[0233] The production of APP is reduced by T30 peptide, an effect which is reversed by NBP-14. This suggests that APP could be cleaved, releasing Amyloid- 1-42 peptide (A42). [0234] (ii) In order to determine the release of A42 we used an ELISA kit, commercially available, measuring A42 present in solution. This test showed that T30 increases the release of A42 up to approximately 175% compared to control and NBP-14 brings the release of A42 to a value close to control (see FIG. 25).