Neurodegenerative disorders

Abstract

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

Claims

1. A method of at least one of reversing T30 or amyloid beta induced toxicity to cell viability, decreasing calcium influx, and increasing acetylcholinesterase activity, the method comprising contacting the cell with an effective amount of one or more peptides consisting of an amino acid sequence as set out in SEQ ID No: 30, 32, 40, 42, 48, 51 or 53.

2. The method according to claim 1, comprising contacting the cell with an effective amount of the peptide consisting of the amino acid sequence as set out in SEQ ID No: 30.

3. The method according to claim 1, comprising contacting the cell with an effective amount of the peptide consisting of the amino acid sequence as set out in SEQ ID No: 32.

4. The method according to claim 1, comprising contacting the cell with an effective amount of the peptide consisting of the amino acid sequence as set out in SEQ ID No: 40.

5. The method according to claim 1, comprising contacting the cell with an effective amount of the peptide consisting of the amino acid sequence as set out in SEQ ID No: 42.

6. The method according to claim 1, comprising contacting the cell with an effective amount of the peptide consisting of the amino acid sequence as set out in SEQ ID No: 48.

7. The method according to claim 1, comprising contacting the cell with an effective amount of the peptide consisting of the amino acid sequence as set out in SEQ ID No: 51.

8. The method according to claim 1, comprising contacting the cell with an effective amount of the peptide consisting of the amino acid sequence as set out in SEQ ID No: 53.

9. The method according to claim 1, comprising contacting the cell with an effective amount of the more than one peptide.

10. The method according to claim 9, wherein the one or more peptide which is protective against T30 toxicity is used in combination with the one or more peptide which is protective against A toxicity.

Description

(1) 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:

(2) FIG. 1a shows the binding of cyclic polypeptide NBP-14 (referred to herein as SEQ ID No:1) binding to the 7 nicotinic-receptor, and FIG. 1b shows an enlarged view of the 3D structure of cyclic NBP-14;

(3) FIG. 2 shows the sequence of NBP-14 with the terminal Alanine (A) and Lysine (K) residues forming the cyclisation sites;

(4) FIG. 3 shows the cyclic NBP-14 peptide in which the terminal Alanine and Lysine residues are linked together;

(5) FIG. 4a-4k are tables showing various embodiments of the linear peptide according to the invention. The peptides are divided into respective groups of peptides which have four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen or fourteen amino acids;

(6) FIG. 5 is a graphical representation of the theoretical affinity of the peptides making up various embodiments of the linear peptides of the invention with the target site of the 7 nicotinic-receptor;

(7) FIG. 6 shows the data and corresponding graphs showing the relationship between acetyl cholinesterase release, calcium ion influx and cell viability. FIG. 6a shows the relationship between acetyl cholinesterase release and calcium ion influx (correlation is 0.210), FIG. 6b shows the relationship between calcium ion influx and cell viability (correlation is 0.026), and FIG. 6c shows the relationship between acetyl cholinesterase release and cell viability (correlation is 0.550);

(8) FIG. 7 is a flow chart showing the four filters or criteria used in the determination of active peptides in accordance with the invention. Criterion 1 involves the correlation between AChE release from PC12 cells and PC12 cell viability; criterion 2 involves the theoretical binding affinity of the peptides remaining following application of criterion 1; criterion 3 involves the calcium ion influx in PC12 cells caused by the peptides remaining following application of criterion 2; and criterion 4 involves peptide size, i.e. excluding peptides remaining following application of criterion 3 with more than 8 amino acids;

(9) FIG. 8 are graphs showing the dose-dependent effects on calcium potentiation of linear variant NBP-601 or NBP-603 against T30;

(10) FIG. 9 are graphs showing the dose-dependent effect on calcium potentiation of linear variant NBP-613 against T30

(11) FIG. 10 is a picture (top panel) and matching schematic representation (bottom panel) of coronal rat brain slices containing diagonal band complex (medial sepctal nucleus (MS), ventral limb diagonal band (VDB), horizontal limb diagonal band (HDB); brain sub-regions which constitute part of the basal forebrain. Red stars indicate the approximate location of stimulation for voltage-sensitive dye imaging (VSDI) and electrophysiology experiments;

(12) FIG. 11 shows VSDI averaged time-series showing the amount of fluorescence (dF/F.sub.0) emitted within the region of interest (ROI) during baseline (blue trace), T30 (2 M; green trace) and T30 (2 M) & NBP14 (4 M; red trace) perfusion conditions. The response profile in basal forebrain shows a tri-phasic response, with phase 1 being the initial peak response (0-15 ms after initial stimulation), phase 2 being the short period of quiescence seen directly after the peak response (15-25 ms after initial stimulation), and phase 3 seen as rebound, long-latency (recurrent) activity directly following phase 2 (25 ms+ after stimulation). Data were acquired by summing (E) the emitted fluorescence between 0 and 280 ms following initial stimulation;

(13) FIG. 12 shows compiled raw data graphs (n=14) showing individual data points of maximum peak amplitude (phase 1) for baseline (blue), T30 (green) and T30 & NBP14 (red)top left panel; individual data points of summed fluorescence (dF/F.sub.0) responsesbottom left panel. Same data showing experiment-specific trends in peak response amplitude (top right panel) and summed fluorescence (bottom right panel). Y-axis units for top panels: recorded fluorescence, dF/F.sub.0; bottom panels: sum of recorded fluorescence (0.fwdarw.280 ms post-stimulus), dF/F.sub.0;

(14) FIG. 13 shows individual values of summed long-latency activity taken from the raw data pool (shown in FIG. 12, bottom panels) showing example experiments where T30 induced a decrease in overall basal forebrain neuronal network activity (top panel) as well as where T30 induced an increase in network activity (bottom panel). In both cases, the change in network activity shows at least a 50% change in response (increase/decrease) from baseline level. In both cases, co-perfusion of T30 with NBP14 (third condition) always reverted the change induced by T30 perfusion back towards baseline activity level;

(15) FIG. 14 is a graph showing individual data points of summed emitted fluorescence during baseline response (x-axis) plotted against the change induced by T30 perfusion (T30 responsebaseline response; y-axis). A correlation can be seen where the higher the level of baseline response, the greater the change induced by T30 is; p=0.007;

(16) FIG. 15 is a graph showing individual data points of change induced in summed emitted fluorescence during T30 perfusion (T30 responsebaseline response; x-axis) plotted against the change induced by addition of NBP14 to the perfusate (T30 & NBP14 responseT30 response; y-axis). A correlation can be seen where the greater the change induced by T30, the greater the reversal of that effect is upon addition of NBP14; p=0.015indicating NBP14 is an efficient blocker of T30 action;

(17) FIG. 16 shows VSDI averaged time-series showing the amount of fluorescence (dF/F.sub.0) emitted within the region of interest (ROI) during baseline (blue trace), T30 (2 M; green trace) and T30 (2 M) & NBP-603 (4 M; red trace) perfusion conditions. Data were acquired by summing () the emitted fluorescence between 0 and 280 ms following initial stimulation;

(18) FIG. 17 shows compiled raw data graphs (n=5) showing individual data points of maximum peak amplitude (phase 1) for baseline (blue), T30 (green) and T30 & NBP-603 (red)top left panel; individual data points of summed fluorescence (dF/F.sub.0) responsesbottom left panel. Same data showing experiment-specific trends in peak response amplitude (top right panel) and summed fluorescence (bottom right panel). Y-axis units for top panels: recorded fluorescence, dF/F.sub.0; bottom panels: sum of recorded fluorescence (0.fwdarw.280 ms post-stimulus), dF/F.sub.0;

(19) FIG. 18 is a graph showing individual data points of summed emitted fluorescence during baseline response (x-axis) plotted against the change induced by T30 perfusion (T30 responsebaseline response; y-axis). Due to the low number of data points and the high variability between them, a downward trend is found (just as seen above); p>0.05;

(20) FIG. 19 is a graph showing individual data points of change induced in summed emitted fluorescence during T30 perfusion (T30 responsebaseline response; x-axis) plotted against the change induced by addition of NBP-603 to the perfusate (T30 & NBP-603 responseT30 response; y-axis). A correlation can be seen where the more positive the change induced by T30 perfusion is, the greater the reversal of the effects once NBP-603 is added to the perfusate; p=0.024;

(21) FIG. 20 shows the binding of linear peptide NBP-601 (referred to herein as SEQ ID No:30) binding to the 7 nicotinic-receptor (7-nAChR); and

(22) FIG. 21 shows the chemical structure of cyclic NBP-14.

EXAMPLES

(23) Materials and Methods

(24) PC12 Cell Cultures

(25) 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).

(26) Wild-type PC12 cells were provided by Sigma-Aldrich (St. Louis, Mo.). The PC12 cell culture or preparation 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.

(27) -Amyloid Preparation

(28) -Amyloid (1-42) fibrils were prepared as described by provider (Abcam, Cambridge UK)). 1 mg of -Amyloid (1-42) was dissolved in 1 ml of 100% 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP). This solution was incubated at room temperature for 1 h. Next, the silution was sonicated for 10 min and then dried in a speed vacuum drier (Thermo Fisher Scientific, Loughborough, UK) and stored at 80 C. For experiments, samples were diluted in 100% DMSO and incubated for 2 h at room temperature to ensure fibril formation.

(29) Cell Viability Assay

(30) 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 or A alone or in conjunction with NBP-14 or the smaller peptides are added and incubated for 1 hour in the incubator. 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.

(31) Acetylcholinesterase Activity Assay

(32) 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 T30 or A (5 M) alone or combined with NBP-14 or the small peptides (0.5 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.4 139 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.

(33) Calcium Fluorometry

(34) 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 per provider protocol). Subsequently, 100 l of growth medium is removed and 100 l of Fluo-8 solution is added. Treatments with T30 or A in conjunction with NBP-14 or small peptides are added and incubated for 30 minutes in the incubator and 30 minutes room temperature.

(35) 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 the peptides are then evaluated.

(36) VSDI Methodology:

(37) Preparation of Brain Slices and Ex-Vivo Recordings

(38) Coronal rat brain slices were prepared according to the procedure described in (Badin et al., 2013) but this time containing basal forebrain (+0.70 and 0.26 millimetres (mm) from bregma (Paxinos and Watson, 1998). Optical imaging using voltage-sensitive dyes was then performed as previously described (Badin et al., 2013, Neuropharmacology. 73C:10-18).

(39) Data Analysis and Statistics (VSDI)

(40) VSDI data were recorded in 44 mm 2-dimensional images, equivalent to 100100 pixelseach pixel being 4040 micrometres (m), from which critical data were extracted. VSDI data was not gathered throughout the experimental run, but in fact was recorded in discrete periods of time 15 minutes in length. The inter-stimulus interval (ISI) between stimulations was 28 seconds and therefore, every recording epoch consisted of 32 successive stimulations. The data from all 32 stimulations were then averaged into a single file for each experimental condition and analysed using a VSDI data analysis toolbox specially made for MatLab (Bourgeois et al., 2014, PLoS One. 9:e108686). In short, this toolbox allowed for the selection of a fixed region of interest (ROI) geometry which could be applied to every slice, in order to extract and compound the data from an identical ROI across all slices and experimental conditions. The ROI was selected along the pial surface of the septal region of slices to encompass the medial septal nucleus (MS), the ventral limb of the diagonal band (VDB) and the horizontal limb of the diagonal band (HDB). VSDI data, taken from the ROI, was then plotted as a single averaged time series (FIG. 11). In order to quantify VSDI data however, the area under the curve was calculated (summed fluorescence fractional change, FIG. 11-22) between the moment of stimulation (t=0) and 280 ms after that; this method of quantification takes into account all the components of the immediate and longer-latency response. All statistical tests (one-way Analysis of VarianceANOVAunless stated otherwise) were performed using GraphPad Prism 6 (v6.05; GraphPad Software Inc., CA, USA), the data (were all found to be normally distributed (data not shown). For all statistical tests, p<0.05 was considered significant; data are expressed as meanS.E.M.

(41) Drugs and Reagents

(42) MEM, culture serums, antibiotics, collagen, Cell Counting Kit-8 and buffers reagents were provided by Sigma-Aldrich (St. Louis, Mo.). T30, AChE peptide and Cyclic T14 were synthesized by Genosphere Biotechnologies (France). Amyloid Beta and Fluo-8 were provided by Abcam (Cambridge, UK). The small peptides were synthesized using routine peptide synthetic techniques. Stocks of peptides were diluted in distilled water.

(43) Data Analysis

(44) 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.).

Example 1Cyclic T14 (i.e. NBP14)

(45) The tailed acetylcholinesterase (T-AChE) is expressed at synapses and the inventors have previously identified two peptides that could be cleaved from its C-terminus, one referred to as T14 (14 amino acids long), within the other which is known as T30 (30 amino acids long), and which both have strong sequence homology to the comparable region of -amyloid.

(46) The amino acid sequence of the linear peptide, T14, is AEFHRWSSYMVHWK [SEQ ID No:1].

(47) The amino acid sequence of the linear peptide, T30, is

(48) TABLE-US-00002 [SEQIDNo:156] KAEFHRWSSYMVHWKNQFDHYSKQDRCSDL.

(49) The AChE C-terminal peptide T14 has been identified as being the salient part of the AChE molecule responsible for its range of non-hydrolytic actions. The synthetic 14 amino acids peptide analogue (i.e. T14), and subsequently the larger, more stable, and more potent amino acid sequence in which it is embedded (i.e. T30) display actions comparable to those reported for non-cholinergic AChE.

(50) Referring first to FIGS. 1a and 20, there is shown the binding of a 14 amino acid long cyclic T14 peptide (i.e. NBP-14) to the allosteric site on the 7 nicotinic-receptor to compete for binding with linear peptides T14 and T30 and also to antagonise -amyloid. The cyclic peptide, NBP-14, is based on the amino acid sequence of T14, i.e. AEFHRWSSYMVHWK [SEQ ID No:1], but has been cyclated via the terminal Alanine (A) and Lysine (K) residues. FIG. 1b shows an enlarged view of the 3D structure of cyclic NBP-14 sitting in the binding pocket of the 7 nicotinic-receptor.

(51) Referring now to FIG. 2, there is shown the sequence of NBP-14 with the terminal Alanine (A) and Lysine (K) residues forming the cyclisation sites, and FIG. 3 shows the cyclic NBP-14 peptide in which the terminal Alanine and Lysine residues are linked together. Cyclisation can be achieved by several different means. For example, Genosphere Biotechnologies (France) performed the cyclisation of T14 by transforming the linear peptide into an N-terminal to C-terminal lactam. Cyclisation of T14 to create cyclic NBP14 brings together both ends, i.e. HWK-AEF.

(52) The inventors have previously shown that cyclic NBP-14 selectively inhibits the non-classical effects of AChE (i.e. the effects of AChE that are independent of its enzymatic activity) and/or its terminal peptide in vitro, and can be used to treat neurodegenerative disorders. NBP14 acts as a true antagonist of the 7 nicotinic-receptor, and has been shown to protect cells from linear T14, T30 and -amyloid toxicity. It also blocks compensatory AChE release induced by the toxicity of linear T14 and T30. In addition, when given alone, cyclic NBP14 has no significant effects on Ca.sup.2+ concentrations in rat brain slices, but blocks the effects of -amyloid.

Example 2Production of an Array of Linear Peptides Derived from Cyclic NBP14

(53) Based on their previous surprising observations with NBP-14 discussed in Example 1, the inventors prepared an array of linear peptides (4-14 amino acids in length) based on the sequence of NBP14 in which each linear peptide sequence starts or ends at any amino acid position along the sequence of NBP14. The array of linear peptides is shown in the large table spanning FIGS. 4a-4k (identified as SEQ ID No's:2-155).

(54) The table of FIG. 4a lists 14 linear peptides (SEQ ID No's:2-15) each of which are four amino acids in length. Each linear peptide is given a name, for example NBP-401, which signifies that it is the first peptide with four amino acids, and NBP-402, which is the second peptide with four amino acids, and so on. The table of FIG. 4b lists 14 linear peptides (SEQ ID No's:16-29) each of which are five amino acids in length. Each linear peptide in this table is given a name, for example NBP-501, which signifies that it is the first peptide with five amino acids, and NBP-502, which is the second peptide with five amino acids, and so on. FIG. 4c lists the 14, six amino acid long peptides (SEQ ID No's:30-43), FIG. 4d lists the 14, seven amino acid long peptides (SEQ ID No's:44-57), FIG. 4e lists the 14, eight amino acid long peptides (SEQ ID No's:58-71), FIG. 4f lists the 14, nine amino acid long peptides (SEQ ID No's:72-85), FIG. 4g lists the 14, ten amino acid long peptides (SEQ ID No's:86-99), FIG. 4h lists the 14, eleven amino acid long peptides (SEQ ID No's:100-113), FIG. 4i lists the 14, twelve amino acid long peptides (SEQ ID No's:114-127), FIG. 4j lists the 14, thirteen amino acid long peptides (SEQ ID No's:128-141), and FIG. 4k lists the 14, fourteen amino acid long peptides (SEQ ID No's:142-155).

(55) Each of the linear peptides were synthesized, and then analysis for their activities as below.

Example 3Effect of Small Linear Peptides Derived from NBP-14 on to or A Toxicity and Cell Viability

(56) Each of the small linear peptides derived from NBP-14 (SEQ ID No's:2-155) were analysed with three in vitro systems against the toxic T30 linear peptide [SEQ ID No:156] and wild-type Amyloid Beta (1-42) (A) (SEQ ID No:158), i.e. (i) AChE release from PC12 cells, (iii) PC12 cell viability; and (iii) calcium influx into PC12 cells.

(57) The amino acid sequence of part of 1-amyloid (A) is provided herein as SEQ ID No:158, as follows: DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA [SEQ ID No:158].

(58) The protective or toxic effect of each linear peptide against T30 and/or A was then determined based on a series of four filters or criteria, as summarised in FIG. 7: 1. AChE release vs PC12 cell viability (for which there is a significant negative correlation); 2. In silico binding to the allosteric site of the 7 nicotinic-receptor; 3. Calcium influx into PC12 cells; and 4. Peptide size.
Filter 1AChE Release Vs PC12 Cell Viability

(59) Initially, due to its correlation, the inventors combined assays (i) and (ii) using the equation below in order to obtain a value coefficient (X) that indicates the protective or toxic effect of each linear peptide against T30 and/or A. The inventors determined the VALUE coefficient (X), which is calculated as:
VALUE COEFFICIENT(X)=(% AChE release)/(% Cell viability)
, wherein % AChE release is the percentage of acetylcholinesterase released from a PC12 cell preparation cultured in the presence of a toxic peptide selected from T30 (SEQ ID No: 156) or A (SEQ ID No:158), and the peptide, derivative or analogue thereof, compared to that of the PC12 cell preparation cultured in the absence of any peptide, derivative or analogue thereof (i.e. control), and % PC12 cell viability is the percentage viability of the PC12 cell preparation cultured in the presence of the toxic peptide selected from T30 (SEQ ID No: 156) or A (SEQ ID No:158), and the peptide, derivative or analogue thereof, compared to that of a PC12 cell preparation cultured in the absence of any peptide, derivative or analogue thereof (i.e. control).

(60) As described below, the inventors have surprisingly shown that it is possible to separate active from inactive peptides based on their value coefficients (X) which represent their protective efficacy against T30 and A toxicity.

(61) The control (i.e. absence of any peptide) has a value of 1. As described in the methods section, the control value is obtained from cells non-treated, the toxic value from cells treated with T30 or A, and the protective value from cells treated with NBP-14 in conjunction with T30 or A. The value for T30 is x=169.45/74.309=2.28, and for Amyloid Beta (A) x=124.19/87.42=1.42.

(62) Hence, a peptide with a value coefficient (X) of under 1.0 and over 1.1 is toxic, whereas any peptide with a value coefficient of 1.0 to 1.1 was consider active, and therefore protective. Tables 1 and 2 show the effect of the linear peptides (at a concentration of 0.5 M) on AChE activity and PC12 cell viability against T30 and A toxicity (at a concentration of 5 M). The Value column shows the protective effect of each peptide.

(63) The inventors have shown that the two parameters, AChE activity in perfusate and PC12 cell viability, are very closely inter-related with a correlation coefficient of 0.55 (n=10), which is significant at the P<0.05 level (see FIG. 6c). This initial metric of AChE release against cell viability yielded 55 successful peptides that were protective either against T30 (n=23 peptides as shown in Table 1) or A (n=32 peptides as shown in Table 2).

(64) TABLE-US-00003 TABLE 1 Effects of linear peptides derived from NBP-14 on T30 toxicity T30 + Value Theo- small AChE/ retical Selected peptides AChE Viability Viability Affinity Calcium peptides 601 100.99 93.21 1.08 7.7 118.37 601 602 112.32 107.76 1.04 8 169.86 603 96.06 94.75 1.01 7.1 119.86 603 606 107.88 102.57 1.05 8.4 131.85 613 103.45 103.45; 1 7.1 102.29 613 102.87 705 94.07 90.95 1.03 7.7 111.98 705 707 98.35 98.53 1 8.1 120.17 708 97.79 97.79; 1 7.8 97.04 708 97.41 804 107.69 107.69; 1 7.5 106.7 804 107.20 1013 91.79 85.31 1.08 6.8 181.12 1101 91.9 91.90; 1 7 86.58 89.24 1102 104.31 104.31; 1 7.2 89.28 1102 96.79 1104 102.36 111.09 1 7.1 119.82 1104 1107 93.13 93.13; 1 7.5 95.43 1107 94.28 1205 95.79 89.43 1.07 7.8 132.71 1212 88.29 88.29; 1 6.9 78 83.14 1313 93.64 89 1.05 6.9 118.94 1405 91.57 83.94 1.09 6.6 100.59 1406 92.78 86.6 1.07 6.1 101.06 1407 94.49 90.51 1.04 6.7 111.71 1408 93.64 93.64; 1 6.8 80.25 86.94 1409 94.85 94.85; 1 6.2 94.59 94.72 1410 93.51 93.51; 1 6.7 120.09 106.80

(65) TABLE-US-00004 TABLE 2 Effects of linear peptides derived from NBP-14 on A toxicity AB + Value Theo- small AChE/ retical Selected peptides AChE Viability Viability Affinity Calcium peptides 503 117.63 109.89 1.07 7.2 195.67 508 106.06 97.62 1.09 8 120.72 509 106.20 102.36 1.04 8 363.48 510 104.25 100.92 1.03 6.6 167.56 512 104.81 103.71 1.01 7.2 163.09 607 97.53 94.93 1.03 7.7 168.97 611 88.60 82.20 1.08 7.5 114.39 611 614 85.05 77.72 1.09 7.5 124.34 709 108.99 101.56 1.07 7.6 288.61 710 108.85 108.73 1 7.3 114.33 710 711 108.57 106.28 1.02 7 148.17 801 108.63 102.35 1.06 7.7 168.66 810 96.22 95.33 1.01 7.3 156.05 905 106.42 97.24 1.09 7.1 194.22 1009 101.23 97.49 1.04 7 255.80 1011 84.13 81.66 1.03 7.1 161.87 1108 82.74 77.72 1.06 7 392.11 1112 98.77 93.37 1.06 7.2 127.62 1201 94.84 93.09 1.02 7.7 114.35 1201 1203 99.63 95.50 1.04 6.5 192.38 1206 96.92 93.63 1.04 7.2 127.27 1207 94.30 87.36 1.08 6.9 261.22 1210 98.18 97.55 1.01 6.8 127.83 1213 93.73 90.77 1.03 6.6 146.97 1214 89.28 85.51 1.04 7.1 217.64 1303 96.73 91.22 1.06 6.5 106.33 1307 94.29 92.47 1.02 6.9 218.25 1311 93.51 86.13 1.09 6.6 137.63 1314 104.08 97.90 1.06 7.2 130.85 1402 99.85 91.59 1.09 7 127.81 1410 93.51 93.51 1 6.7 160.54 1413 99.85 95.17 1.05 6.8 125.28
Filter 2in Silico Binding

(66) The inventors then examined the theoretical in silico binding affinity of the active peptides to the allosteric site of 7 nicotinic-receptor, and the values are also shown in Tables 1 and 2. The in silico analysis was performed using the Autodock Vina software published in Journal of Computational Chemistry (O. Trott, A. J. Olson AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading, Journal of Computational Chemistry 31 (2010) 455-461). The analysis was performed as recommended by the authors. The results analysis and the comparison were performed manually comparing the amino acids bound to the receptor and the distances between amino acids.

(67) They found that a theoretical binding affinity inferior to 7.0 corresponded to higher affinity between the peptide and the allosteric site of 7 nicotinic-receptor. Hence, the total number of active peptides was reduced from 55 based on filter 1, down to 28 peptides following filter 2.

(68) Filter 3Calcium Influx

(69) The inventors also investigated the relationship between cell viability and the third parameter (iii) discussed above, i.e. calcium influx into PC12 cells. However, they found that these parameters, which have very different time scales, are not linked, having a correlation coefficient of only 0.026 (n=10), as shown in FIG. 6b. The inventors also investigated the relationship between AChE activity and calcium influx into PC12 cells. However, they found that these parameters, again over different time scales, are also not linked, having a correlation coefficient of only 0.21 (n=10), as shown in FIG. 6a.

(70) Accordingly, the inventors hypothesise that the release of AChE is most likely a direct compensation due to eventual cell death, and not caused by non-specific, immediate entry of calcium, but instead to a slower intracellular cascade.

(71) The values of calcium influx for the 28 peptides produced by filter 2 are also shown in Tables 1 and 2. The inventors found that a calcium influx value of 97 to 120 corresponded to active peptides. Hence, the total number of active peptides was reduced from 28 based on filter 2, down to 12 peptides following filter 3.

(72) Filter 4Peptide Size

(73) Finally, the inventors believe that any peptide that is larger than 8 amino acids (theoretical molecular weight superior to 900 Da) does not present a realistic lead compound candidate for treating neurodegenerative disorders. Accordingly, these larger peptides were discounted. Hence, the total number of active peptides was reduced from 12 based on filter 3, down to 8 peptides following filter 4.

CONCLUSIONS

(74) As such, when all four filters are taken into consideration, the number of possible neuroprotective agents against T30 is six peptides (i.e. NBP-601, 603, 613, 705, 708, and 804), and the number of agents against A is only two peptides (i.e. NBP-611 and 710). Tables 1 and 2 show the green values corresponding to the protective or active peptides, whereas the red values are the toxic or inactive peptides. Accordingly, use of the combined filters enables the isolation of neuroprotective linear peptides by structure as well as by mechanism or function. As such, the inventors are confident of their efficacy in vivo.

Example 4Combination Therapy Using Linear Peptides

(75) The inventors have clearly demonstrated that a small subset of the linear peptides derived from cyclic NBP-14 show surprising protective activity against T30 toxicity, and that another subset of peptides are protective against A. It will be appreciated that A is currently the more commonly accepted mechanism of toxicity for causing neurodegenerative disorders, such as Alzheimer's disease. Accordingly, one or more of the peptides NBP-611 and 710 are especially useful for treating neurodegenerative disorders.

(76) However, the inventor's previous work would suggest that T30 toxicity is in fact the more likely cause for such diseases, and not A toxicity. Accordingly, one or more of the peptides NBP-601, 603, 613, 705, 708, and 804 are especially useful for treating neurodegenerative disorders.

(77) In some embodiments, the inventors believe that it would be beneficial to administer two peptides, one from the T30 protective group shown in Table 1 and one from the A protective group shown in Table 2. For example, NBP-601 could be co-administered with NBP-611, or NBP-705 can be co-administered with NBP-710, and so on.

Example 5Binding Affinity Analyses

(78) Referring to FIGS. 5 and 20, there is shown a graphical representation of the theoretical binding affinity of the peptides making up various embodiments of the linear peptides of the invention as shown in the box with the target site of the 7 nicotinic-receptor.

Example 6Analysis of Preferred Peptides in Dose Dependent Response in PC12 Cells Against T30 and Amyloid

(79) Dose-dependent experiments were conducted with the selected small linear variants of cyclic NBP-14 against T30, and the results are shown in FIGS. 8 and 9. The results obtained show that NBP601, NBP603 and NBP613 protect against T30 effects. These experiments corroborate the results obtained on brain slices discussed in Example 7.

Example 7Analysis of Preferred Peptides (and NBP-14 Control) in Brain Slice Experiments Against T30

(80) FIG. 10 illustrates the position of the basal forebrain comprising the medial septum, vertical diagonal band and horizontal diagonal band. The aim of VSDI experiments was to characterise the responses evoked in basal forebrain with 30V electrical stimulation, and to see how these responses were first modulated by addition of high concentrations of T30 (2 M; FIG. 11).

(81) Evoked responses were indeed found to be modulated by T30 where a majority showed inhibition while some responses showed increased activity (FIG. 12). In all cases, NBP14 addition to the perfusate was found to significantly reverse any kind of change induced by T30 (FIGS. 13, 14 & 15).

(82) Additionally, the NBP14 variant NBP-603 (FIG. 16) was tested in the same exact experimental paradigm as for NBP14. It was found that variant NBP-603 had some significant effect in reversing the changes induced by T30 (FIGS. 17 & 19).