WO2014184596A2 - SMALL PEPTIDE INHIBITORS OF β-AMYLOID TOXICITY - Google Patents

Abstract

The invention relates to peptides and peptidomimetics useful for reducing the neurotoxicity of amyloid peptide aggregates or prion-like (prionoid) protein aggregates, the invention further relates to pharmaceutical compositions containing said peptides and/or peptidomimetics, to the use of said peptides and/or peptidomimetics in the manufacture of medicines useful for the prophylaxis or treatment of diseases that can be cured by protection against the detrimental effect of Αβ-peptides /abnormal Αβ aggregation leading to amyloid plaque formation, and it relates to the use of said peptides and/or peptidomimetics in the prophylaxis or treatment of the above mentioned diseases. The invention aimed at developing treatments of neurodegenerative diseases, particularly Alzheimer's disease (AD), during which accumulation of misfolded and/or aggregated proteins occurs.

Description

Small Peptide inhibitors of β-amyloid toxicity

Field of the invention

The invention relates to peptides and peptidomimetics useful for reducing the neurotoxicity of amyloid peptide aggregates or prion-like (prionoid) protein aggregates, the invention further relates to pharmaceutical compositions containing said peptides and/or peptidomimetics, to the use of said peptides and/or peptidomimetics in the manufacture of medicines useful for the prophylaxis or treatment of diseases that can be cured by protection against the detrimental effect of Αβ-peptides /abnormal Αβ aggregation leading to amyloid plaque formation, and it relates to the use of said peptides and/or peptidomimetics in the prophylaxis or treatment of the above mentioned diseases. The invention aimed at developing treatments of neurodegenerative diseases, particularly Alzheimer' s disease (AD) , during which accumulation of misfolded and/or aggregated proteins occurs.

The following abbreviations are used throughout the present description :

Αβ, β-amyloid peptide; AD, Alzheimer's disease; PD, Parkinson's, Disease; Ac, acetyl; ACN, acetonitrile; APP, amyloid precursor protein; APP x PS1, APP/ presenilin 1 (PS1) mouse model of AD; apape, pentapeptide of SEQ ID NO: 51; Boc, terc-butyloxycarbonyl; BSB, β-sheet breaker; DCC, N,N- dicyclohexylcarbodiimide; DCM, dichloromethane; DIPEA, diisopropylethylamine; DMF, N, N-dimethylformamide; Fmoc, flourenylmethoxycarbonyl; GPCR, G protein coupled receptor; HFIP, 1, 1, 1, 3, 3, 3-hexafluoro-2-propanol; HOBt, 1- hydroxybenzotriazole; IDP, intrinsically disordered protein; i.p., intraperitoneal; LTP, long-term potentiation; NT, neurofibrillary tangles; MBHA, 4-methylbenzhydrylamine ; MWM, Morris water maze; NMDA, N-methyl-D-aspartic acid; PAM, 4- hydroxymethylphenylacetamide; PBS, phosphate buffered saline; TFA, triflouroacetic acid; Thioflavin-T (ThT) ; ACN, acetonitrile; Boc, tert-butyloxycarbonyl ; DCC, N, N- dicyclohexylcarbodiimide; DCM, dichloromethane ; DIPEA, diisopropylethylamine; DMF, N, N-dimethylformamide; EDCxHCL 1- Ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride;

NH2, C-terminal amide group; MBHA 4-methylbenzhydrylamine; PAM, 4-hydroxymethylphenylacetamide; TFA, triflouroacetic acid.

Abbreviations of non-standard amino acids and similar moieties (abbreviations of standard amino acids are known by the person skilled in the art) :

Aib, 2-aminoisobutyric acid; ala, D-alanine; pro, D-proline; glu, D-glutamic acid; asp, D-aspartic acid; Glp, L- pyroglutamic acid; Sar, sarcosin; Pip, L-pipecolic acid; sue, succinyl; GABA, gamma-aminobutyric acid; Abu, 2-aminobutyric acid; Tic, Tetrahydro-isoquinoline-3-carboxylic acid.

As obvious for a person skilled in the art chiral amino acids can have either L- or D-configuration . If an amino acid is mentioned in the present description both L- or D- configuration has to be understood, unless the configuration is specified.

Background of the invention According to the 2010 World Alzheimer Report, the number of people with Alzheimer's or other dementia was about 35.6 million, which number is expected to nearly double in every 20 years. Worldwide, the economic cost of dementia has been estimated as USD 315 billion annually. AD, the most common of senile dementias, is a global health crisis with devastating effects on individuals, families and societies. There is an urgent need to develop efficient and relatively cheap treatments for the early stage of the disease and/or prevention strategies to replace currently available medications which only reduce the symptoms and the progression of the disease but does not solve the underlying problem.

Several diseases, including for example AD, Parkinson's disease, type II diabetes, and amyloidosis are associated with the transformation of normally soluble proteins into amyloid fibrils (Dobson, Trends Biochem Sci. 24 ( 9) : 329-32, 1999; Sipe and Cohen, J Struct Biol 130 (2-3 ): 88-98 , 2000). Two distinct types of fibrillar aggregates were found in AD brain samples: amyloid plaques comprising deposits of Αβ and neurofibrillary tangles consisting of tau, a microtubule-associated protein (Selkoe, J Alzheimers Dis 3(l):75-80, 2001). Αβ aggregates are mainly β-sheets with all the hallmark characteristics of amyloid fibrils, including a cross-beta diffraction pattern and characteristic staining by the dyes Congo Red and thioflavin T (Eanes and Glenner, J Histochem Cytochem 16 (11) : 673-7, 1968; LeVine, Protein Sci 2(3):404-10, 1993; Sipe and Cohen, J Struct Biol 130 (2-3) : 88-98, 2000) . Tau filaments adopt various morphologies, including paired helical filaments, which are β- sheet rich fibrils that appear as twisted structures under electron microscopy, and straight filaments, which lack the twisted morphology (Goedert et al., Curr Opin Neurobiol 8(5): 619-32, Review, 1998). Tau filaments bind the dye thioflavine S (ThS) and yield fluorescent signal and have a cross-beta diffraction pattern (Berriman et al., Proc Natl Acad Sci U.S.A. 100 (15) :9034-8, 2003; Friedhoff et al., Biochemistry 37 (28) :10223-30, 1998) .

AD is characterized by the formation of extracellular amyloid plaques, cerebrovascular amyloid deposits, intracellular neurofibrillary tangles (NT) and neuronal loss in the brain. While NTs mainly consist of the microtubule-associated protein tau (Selkoe, Physiol Rev 81: 741-766, 2001), the amyloid plaques contain large amounts of amyloid beta (Αβ) peptides (a length of 39-43 amino acids) , which are produced by the proteolytic cleavage of a much longer amyloid precursor protein (APP) in neurons and other cells throughout life (Haass et al. Nature 359: 322-325, 1992). According to the amyloid hypothesis it was suggested that dysregulation of APP processing is the key initiating event in AD pathogenesis, subsequently leading to increased levels and aggregation of extracellular Αβ (εΑβ) , specifically θΑβ 1-42 (that is, the 42 amino acid length version of Αβ) . Αβ oligomerization and/or fibrillogenesis trigger a cascade of cellular and molecular events, including disturbed axonal transport, development of synaptic failure, neuronal loss, reduction in neurotransmitter levels, all of which result in defects in cognition, synaptic plasticity and in development of tau pathology (Hardy and Higgins, Science 256: 184-185, 1992) . Some G protein coupled receptors (GPCRs) directly influence the amyloid cascade through modulation of a- , β- and γ-secretases that participate in the proteolysis of APP, and regulation of Αβ degradation. Additionally, Αβ has been shown to disturb the functioning of several GPCRs (Verdier and Penke, Curr Prot &. Peptide Science: 19-31, 2004; Verdier et al. J Neurochem 94: 617-628 2005). Extracellular Αβ triggers aberrant signaling and therefore may cause cognitive dysfunction and consequently neurodegeneration in the brain (Patel and Jhamandas Expert Rev Mol Med 14: e2 201; Xu et al. Prog Neurobiol 97: 1-13, 2012). Emerging insights into the link between GPCRs and AD highlight the potential of this class of receptors as a therapeutic target for AD (for a review, see Thathiah and De Strooper, Nat Rev Neurosci 12(2): 73-87, 2011; United States Patent Application 0100137207, 2010) .

The original ^amyloid cascade' hypothesis has been radically changed several times during the last decade. Recently, the interest of AD researchers has turned to the intracellular Αβ peptides (ίΑβ) . It is now well accepted that two pools of Αβ exist in the brain: extra- and intracellular, and a dynamic relationship exists between them. More and more evidences suggest that θΑβ may have reduced impact on AD pathology and εΑβ depositions/plaques alone cannot be responsible for all kinds of AD pathology, e.g. like apoptotic cell death. Hartmann published already in 1999 the formation of ίΑβ (and specially its highly toxic ίΑβ 1-42 species) in different subcellular organelles (Hartmann Eur Arch Psychiatry Clin Neurosxci 249: 291-298, 1999) . The ^λ intracellular Αβ hypothesis' (LaFerla et al. Nat Rev Neurosci 8: 499-509, 2007) assumes that ϊΑβ accumulation is the early, causative event in AD development. ίΑβ is the cytotoxic substance and eAp deposition (in the later stage of AD) is rather the result of cell death and destruction. Mechanism of amyloid plaque formation also suggests an intracellular basis of Αβ pathogenecity (Friedrich et al. Proc Natl Acad Sci USA 107: 1942-1947, 2010). A series of review articles have summarized the mechanism of ίΑβ accumulation and the results of ίΑβ studies performed during the last ten years (LaFerla et al. Nat Rev Neurosci 8: 499-509, 2007; Gimenez-Llort et al. Neurosci Biobehav Rev 31: 125-147, 2007; Li et al. Prog Neurobiol 83: 131-139, 2007; Gouras et al Acta Neuropathol 119: 523-541, 2010; Bayer and irths Nervenartz 79Supp3: 117, 2010; book chapter review on the role of ίΑβ (Zhang in: Jelinek, R. (Ed.), Lipids and cellular membranes in amyloid diseases, Wiley 2011, pp. 143-159) . ίΑβ has been widely detected in neuronal cells and primary human neurons, and shown to have a broad range of interaction with proteins and subcellular organelles. Studies in several mouse models of AD with intraneuronal Αβ-expression and accumulation (Casas et al Am J Pathol 165: 1289-1300, 2004; Billings Neuron 45: 675-688, 2005; Tomiyama J Neurosci 30: 4845-4856, 2010; Abramowski et al. J Neurosci 32: 1273-1283, 2011) have proven that ϊΑβ causes the onset of early AD cognitive deficits in transgenic mice.

Another hypothesis for understanding AD focuses on the microtubular protein tau (Iqbal et al. Acta Neuropathol 109 (1) :25-312, 2009). Tau is an important protein in AD pathology: the Braak staging of the disease (Braak and Braak Neurobiol Aging 16(3):271-8, 1995) is based on the localization of tau deposits in brain structures. Similarly to Αβ, hyperphosphorylated tau can also form toxic intracellular aggregates. As a consequence of formation of tau deposits, the microtubular system collapse and the axonal transport fails (Iqbal et al. Acta Neuropathol 109 (1) : 25-312, 2009; Takashima Curr Alzheimer Res 5(6): 591-8, 2008), which results in neuronal dysfunction and cell death.

Recently, the two hypotheses for AD etiopathology were combined (Small and Duff Neuron 60: 534-542, 2008). AD is regarded as a special form of tauopathies with primacy of Αβ, that is accumulation of εΑβ, and ίΑβ starts the pathological processes of AD. Both species of Αβ trigger tau-hyperphosphorylation, however, tau protein is also required for Αβ-induced neuronal dysfunction (Shipton et al. J Neurosci 31: 1688-1692, 2011).

The newest theory of Ittner and Gotz (Nature Rev Neuroscience 12, 67-72, 2011), the 'tau axis' hypothesis allows the primacy of Αβ, however, it puts tau and its effects in the dendritic compartment of neurons in the center stating that Αβ-toxicity is tau dependent. Αβ causes tau hyperphosphorylation and detaching from microtubules. Simultaneously, the progressively increasing level of dendritic tau makes neurons vulnerable to Αβ and it is linked to increased tau hyperphosphorylation, thereby establishing a vicious circle. As Αβ is the inducer of pathological processes its effects should be neutralized.

For two decades AD drug research has used the amyloid cascade hypothesis. Putative neuroprotective agents were tried in the pipeline that selectively block Αβ aggregation and/or enhance clearance of Αβ peptides. Beside the traditional organic compounds, which were reported to reduce or inhibit the aggregation and toxicity of Αβ (Kisilevsky et al. Nat Med 1(2): 143-148, 1995; Wood et al. J Biol Chem 271(8): 4086-4092, 1996) , several other approaches have been developed to reduce neuronal loss (neuroprotection) caused by Αβ. One of them is targeting the risk factors in an attempt to neutralize or inhibit their action. In the presence of β- and γ-secretase inhibitors, Αβ 1-42 is not formed. However, these enzyme inhibitors show poor specificity combined with undesirable side effects. Other inhibitors block the interaction between Αβ 1-42 and membranes, e.g. amyloid surface binding molecules, competitive inhibitors (RGD - peptides) and Memantine® /NMDA. Αβ immunization was also considered as another promising approach for AD therapy. However, clinical studies revealed a strong brain inflammatory response in immunized patients (Munch and Robinson, J Neural Transm 109(708): 1081-1087, 2002). Dramatic advances in understanding the neurobiology of AD have revealed a new direction for drug development, e.g. the identification of specific aggregation states of Αβ (for a review, see Broersen et al., Alz. Res Ther 2/12: 1-14, 2010). Recently, a self propagating special conformation that could transmit AD from neuron to neuron was identified (Aguzzi and Falig, Nature Neurosci. 15(7) 936-939, 2012). Recent studies have shown that AD severity correlates more closely with soluble oligomeric than the fibrillar forms of Αβ (McLean et al. Ann Neurol 46: 860-866, 1999; Lue et al . Am J Pathol 155: 853-862, 1999) . Αβ monomer is non-toxic and unstructured in solution, but acquires a β-sheet structure on aggregation and amyloid formation. It has been suggested that amyloid formation is a nucleation-polymerization reaction, that is characterized by an initial lag phase dominated by monomeric (M) species, followed by the formation of low-molecular weight (LMW) soluble oligomers (dimer, trimer and < 8-mers) . The β-sheet rich oligomeric intermediates are referred to as prefibrillar aggregates, which disappear upon fibril formation. Some of the key features of protofibrils are: (i) they contain a mixture of different secondary structural elements with a predominance of β-sheets; (ii) bind amyloid-specific dyes, e.g. Congo red and Thioflavin-T (ThT) ; and (iii) are less stable than mature fibrils. Amyloid fibrils are insoluble, β-sheet rich structures that contain 2-6 protofilament subunits. Thus, the key pathogenic event in the onset of AD is believed to be the formation of soluble forms of neurotoxic/synaptotoxic β-amyloid peptides (A β 42 oligomers and protofibrils), which are intrinsically disordered proteins that possess many metastabil conformational states, thereby able to interact with several proteins of the neural cells (Tompa FEBS J 276 (19) : 5406-15, 2009; Broersen et al. Alzheimer's Res & Therapy 2(4): 12-14, 2010) . Understanding the precise chemical nature of the toxic Αβ oligomeric species is an important goal that may ultimately help the design of specific molecules, which will be able to correct a particular cellular dysfunction by restoring normal signal transduction and the ability of nerve cells to communicate . W

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Based on the findings in the field, a strategy was to design inhibitory peptides of various types and conformations, which were directed to block Αβ aggregation or to dissolve amyloid fibrils (for a review, see Fiilop et al. Research Signpost 37/661 82, 161-188, 2009) . However, the results have not confirmed the nature and conformation of the Αβ species that are responsible for transmission of the disease from cell to cell, thus the design of the first series of inhibitory peptides was unsuccessful.

In the past five years strong experimental evidences from AD and PD model systems demonstrated that infectious substance can be generated in vitro using different proteins. This breakthrough has proven that a misfolded protein is the active component of the infectious agent. This finding binds AD to prion diseases. According to the protein only' hypothesis, infectious prions are composed predominantly, if not entirely, of aggregates of misfolded, host-encoded, cellular prion protein (PrPSC) (Griffith Nature 215:1043-44, 1967). PrPSC arises from normal PrPC through conformational conversion and then PrPSC accumulates predominantly in the extracellular space .

Thus Αβ may be directly toxic to neuronal cells and synapses. The intrinsically disordered (ID) structures of Αβ represent transient intermediates in the aggregation cascade in patient brains. The pathology of AD, the Braak stages (Braak H. and Braak E. Neurobiol. Aging 16:271-278, 1995) also support the idea that AD spreads from neuron to neuron. It is almost forgotten that at the beginning of eighties-nineties and AD- research, the disease was suspected of being a prion disease (Prusiner Biochemistry 23(25): 5898-906, 1984; Gajdusek Mol Neurobiol 8(1): 1-13.1994). Recently, it was demonstrated, that extracts containing soluble Αβ aggregates induce amyloidosis in mice that otherwise never develop amyloid plaques (Morales et al. Mol Psychiatry 17, 1347-1353, 2012). Pyroglutamylated Αβ 3- 42 shows prion-like behavior in mice (Nussbaum et al Nature 485: 651-655, 2012). Pathological similarities between AD and prion diseases suggest that the formation and spread of the proteinaceous lesions might involve a common molecular mechanism - corruptive protein templating (Eisele et al. Science 330: 980-982, 2010, Jucker and Walker, Ann Neurol 70: 532-540, 2011) . Experimentally, cerebral β-amyloidosis can be exogenously induced by exposure to dilute brain extracts containing aggregated Αβ seeds (seeding-nucleation model) . It has been shown recently that pure Αβ injections into the brain may induce plaques throughout the whole brain within 5 to 6 months (Stohr et al. Proc Natl Acad Sci U S A. 27, 11025-30, 2012) . If the peptides were injected to one hemisphere, plaque formation started in both halves of the brain. The amyloid- inducing agent probably is Αβ itself (Auer et al. Physa Rev Lett 101: 258101, 2008), in a special toxic conformation (synonyms :' hypertoxic Αβ' , ^primordial cytotoxic Αβ' , ^prionoid Αβ') generated most effectively in the living brain (Polymenidou and Cleveland, J Exp Med 209: 889-893, 2012). The term ^rionoid' was introduced by Aguzzi for self-propagating, transmissible protein aggregates (Aguzzi and Rajendran Neuron 64: 783:790, 2009). Infectious prions and prionoids can be prepared by protein misfolding cyclic amplification (PMCA) (Soto et al, TINS 25: 390394, 2002, Saa et al. J Biol Chem 281:^' 35245-35252, 2006; Murayama et al. Neurosci Lett 413: 270-273, 2007; Soto and Satani Trend Mol Med 17: 14-24, 2011; Gonzalez- Montalban et al. PLoS Pathog 7:el001277, 2011; Moreno-Gonzalez and Soto Semin Cell Dev Biol 22: 482-487, 2011). The precise molecular mechanism of conversion of a non-transmissible protein molecule to the pathogenic form (e.g. PrPc→PrPSc) is not completely understood, but available data support the seeding-nucleation model in which the infectious conformer (e.g. PrPSc) is an oligomer that acts as a seed to bind native protein (PrPc) and catalyze its conversion into the misfolded form by incorporation into the growing polymer (Soto et al. TINS 25: 390-394. 2002; Soto et al. TIBS 31: 150-155, 2006). It has remained unclear whether other compounds, besides the misfolded prion/prionoid protein, for example a co-factor, might be an essential element of the infectious agent. Such a co-factor might act as an essential catalyst for prion/prionoid conversion, help to stabilize the hypertoxic conformation and might increase the biological stability of prions/prionoids (Soto TIBS 36(3):151-8, 2010).

Summary of the invention As outlined above, there were several attempts to inhibit Αβ aggregation by small molecules (i.e. conventional organic compounds) , some of them are undergoing clinical trials (for a review, see Amijee and Scopes, J Alz Dis 17: 33-47, 2009). Taking in account the most recent results of AD research the amyloid peptide is the target of most AD-modifying therapies. A novel compound for the prophylaxis and treatment of AD should prevent the formation of toxic Αβ oligomers and the conformational change into infectious prionoid species. Such compound preferably also prevents any interaction between toxic extra- or intracellular Αβ and neuronal cell membranes, membrane proteins and intraneuronal as well as molecular organelles. If these interactions are hindered, Αβ cannot initiate tau-pathology, the release of lysosomal enzymes and apoptotic cell death.

Thus, the problem according the present invention is that an effective means is missing to inhibit Αβ aggregation to toxic oligomers in order to prevent or treat diseases that are the consequence of Αβ aggregation, that is that can be cured by protection against the detrimental effect of Αβ-peptides.

Therefore the object of the present invention is to provide compounds that can effectively inhibit Αβ aggregation to toxic oligomers and inititate aggregation to big, non-toxic assemblies. These compounds will be useful in the prophylaxis and treatment of diseases associated with Αβ aggregation, wherein such diseases include, without limitation, neurodegenerative diseases, like AD and Parkinson's disease, Down syndrome, type II diabetes, amyloidosis etc.

Surprisingly, we found that members of the novel class of compounds according to the present invention act as β-amyloid structure modifying agents. Therefore, to solve the above outlined problem, the present invention provides a novel group of short peptides and peptidomimetics. These peptides - among other useful effects - inhibit Αβ aggregation. Briefly, the present invention relates to the following

peptides and peptidomimetics of the general formula (I) :

X-A1-A2-A3-A4-A5-Y (I) wherein

X is absent or represents: acetyl, propionyl, succinyl group Al is absent or represents: Ala, Gly, Aib, Sar, N-methyl-Ala, oi-methyl-Ala, Abu, norvaline, Asp, Glu, iminodiacetic acid, GABA, succinyl, Pro, acetyl, 2-amino-adipoic acid, propionyl group;

A2 is absent or represents: Pro, Tic, thiaproline, a-methyl-

Pro, γ-hidroxy-Pro, Sar, 4-fluoro-Pro, Pip, Nipecotic acid, Glu, Asp, 2-carboxy-piperazine, succinyl, Glp, acetyl,

GABA, propionyl group;

A3 is absent or represents: Ala, Gly, Aib, Sar, N-methyl-Ala, α-methyl-Ala, Abu, norvaline, Asp, Glu, Glp, acetyl, succinyl, GABA, propionyl group;

A4 is absent or represents: Pro, Tic, thiaproline, a-methyl-

Pro, γ-hidroxy-Pro, Sar, 4-fluoro-Pro, Pip, Nipecotic acid,

Glu, Asp, 2-carboxy-piperazine, succinyl, Glp group;

A5 represents: Ala, Gly, Aib, Sar, N-methyl-Ala, a-methyl-Ala,

Abu, norvaline, Asp, Glu, iminodiacetic acid, GABA, 2- amino-adipoic acid, Pro;

Y is absent or represents: amide, N-methylamide, or N, N- dimethyl-amide;

provided that if A2 is absent than Al is also absent and if A3 is absent than Al and A2 are also absent, and if A4 is absent than Al, A2 and A3 are also absent;

and to their pharmaceutically acceptable salts and esters.

As can be seen in the above general formula (I), the present invention relates to peptides containing from 1 to 5 amino acid moieties, where - apart from the naturally occuring amino acids - also some artificial amino acids and peptide chain ends are also present.

Al, A2, A3, A4 and A5 are selected in such a way that the resulting peptide or peptidomimetic bind to the natural Αβ, thereby modulating its aggregation properties and inhibits its neurotoxicity via a conformational change.

The present invention relates preferably to peptides and peptidomimetics listed in Table 2. (see below)

The present invention relates more preferably to peptides and peptidomimetics of the following formulas: EPP-amide (SEQ ID NO 13),

ape-amide (SEQ ID NO 14),

GABA-pe-amide (SEQ ID NO 16),

DPA-amide (SEQ ID NO 17),

pppe-amide (SEQ ID NO 21),

EPAPA (SEQ ID NO 28) ,

EPPPA(SEQ ID NO 29) ,

epppa-amide (SEQ ID NO 32),

apapq (SEQ ID NO 46) ,

apapn (SEQ ID NO 48) ,

apape (SEQ ID NO 51)

and pharmaceutically acceptable salts and esters thereof.

Furthermore, the invention relates to pharmaceutical

preparations, which contain at least one peptide or

peptidomimetic of the general formula (I) , its salt and ester and at least one pharmaceutically acceptable additive.

Preferably this additive is a matrix ensuring controlled release .

The invention relates preferably to pharmaceutical

preparations, which contain at least one of the peptides or peptidomimetics of the following formulas: EPP-amide (SEQ ID NO 13),

ape-amide (SEQ ID NO 14),

GABA-pe-amide (SEQ ID NO 16),

DPA-amide (SEQ ID NO 17),

pppe-amide (SEQ ID NO 21) ,

EPAPA (SEQ ID NO 28),

EPPPA (SEQ ID NO 29) ,

epppa-amide (SEQ ID NO 32),

apapq (SEQ ID NO 46) , apapn (SEQ ID NO 48) ,

apape (SEQ ID NO 51)

and pharmaceutically acceptable salts and esters thereof. The invention also relates to the use of peptides and/or peptidomimetics according to general formula (I) and their pharmaceutically acceptable salt and ester in the manufacture of a pharmaceutical preparation suitable for the prophylaxis and/or treatment of diseases selected from the group of neurodegenerative diseases associated with Αβ aggregation, Down syndrome, type II diabetes, and amyloidosis.

Brief description of the drawings

Fig. 1 shows the TEM images of oligomeric and fibrillar structures of Αβ 1-42 prepared from iso-Αβ 1-42 (75 μΜ) at 0 min A) , 4 h B) , and 24 h C) in the absence and presence

of apape (SEQ ID NO: 51) (1:5 molar ratio).

Fig. 2 shows the effect of the apape pentapeptide on the size distribution of the amyloid aggregates (50 μΜ) . dH: hydrodynamical diameter, CG7: control peptide Cys-(Gly)7. The apape pentapeptide (1:5 molar ratios) facilitated the formation of large agregates of beta amyloid, which formed already after 4 h of incubation, whereas the control peptide did not show a similar effect. After 24 h the presence of fibrillar aggregates could be observed in both samples. Fig. 3 displays the ECD spectra recorded over a 168 h period for Αβ 1-42, apape-NH2 and a 1:5 molar ratio mixture of Αβ 1-42 and apape-NH2. Difference spectra were obtained by subtracting the corresponding pure apape-NH2 spectra from those of the Αβ 1-42 and apape-NH2 mixture. Fig. 4 shows the backbone-fitted representatives of the indicated peptides obtained by cluster analysis. The ribbon is always fitted to the representative of the largest cluster.

Fig. 5 indicates the time-dependence of the signal intensity of apape in the presence of Αβ 1-42 in 1H NMR spectra (a). Concentrations were 250 μΜ for iso Αβ 1-42 and 1.9 mM for apape. After 24 hours, the bound fraction of apape was 3.5 % (diamond). In this case 66.5 μΜ apape bound to Αβ 1-42, which assumes 1:4 binding, respectively. The control signal intensity of apape without Αβ 1-42 was marked by square, (b) : After 1 week, the final value of the bound fraction of apape was 9 % (diamond). In this case, one apape can bind to one Αβ 1-42. The control signal intensity of apape without Αβ 1-42 was 100 ± 0.3 % during the examined period (square) . (c) : Scaled 1H NMR methyl signals of the free apape sample (dark grey) and apape + Αβ 1-42 after 24 hours incubation (light grey) . (d) : Signal assignment of apape; (e) : part of 1H NMR spectrum of apape. Shifting of the N-terminal Ala protons was observed upon addition of iso Αβ 1-42. Lower spectrum is the control apape spectrum and upper spectrum is after 24 hours addition of iso Αβ 1-42. Fig. 6 demonstrates that the peptide pape in vitro protects against the LTP impairment caused by Αβ 1-42 in mouse hippocampal slices. LTP was induced by theta burst stimulation (TBS) , and was followed for 75 min (A) . Panel B shows the mean for field excitatory postsynaptic potential (fEPSP) amplitudes 85-90 min after TBS. * p < 0.05 versus control; one-way ANOVA followed by Dunnett's test.

Fig. 7 shows the effect of intraperitonially administered apape and pentaglycine (GGGGG) on the NMDA response-enhancing effect of Αβ 1-42 (the first grey bar) . The peptide apape had a protective effect between «50 and =250 min, and the control pentaglycine did not interfere with the Αβ 1-42-induced excitation. Mean of maximum NMDA-evoked responses, normalized by the control data (the total spike number during each excitation epoch before Αβ 1-42) ± SEM. Asterisks indicate significant differences, p < 0.05, n=4 for all data.

Fig. 8 depicts the neuroprotective effect of apape in the Morris water maze task in rats. Bar graphs show the mean ± SEM. Latencies to reach the platform in second on each of five days. Animals were divided into the following four groups: hydrogen carbonate buffer (HCBS) treated, physiological saline injected control (white); HCBS treated, apape (10 mg/bwkg, i.p.) injected (light grey) ; injected with oligomeric Αβ 1-42 (75 μΜ, aggregated for 136 h, i.e. v.) and apape (10.0 mg/bwkg, i.p.), (black) ; injected with oligomeric Αβ 1-42 (75 μΜ, aggregated for 136 h, i.c.v.j and physiological saline solution (i.p), (dark grey) . According to one-way ANOVA, the significant level between the groups on the third day F₄₈.₃=3.449, p=0.024. The asterisk denotes significant (p < 0.05) difference in the post hoc LSD test between the experimental groups on the third day.

Fig. 9 shows the neuroprotective effect of apape in the Morris water maze task in APP x PS1 mice. Bar graphs show the mean ± SEM. latencies to reach the platform in second on each of five days. Animals were divided into the following four groups: placebo injected, wild group (white) ; apape injected wild group (light grey); apape injected APPxPSl mice (black); placebo injected APPxPSl mice (dark grey) . According to the repeated measure of ANOVA we found significant differences between the groups (F_{30 4}=10.480 p=0.0001), in post hoc LSD test the difference between the wild-placebo injected group, and the APPXPS1 placebo injected group on the fourth day p=0.001, difference between the apape injected wild and APPXPSl group and the transgenic placebo injected group on the fourth day, p^0.0001. Fig. 10 shows the neuroprotective effect of apape during the first swimming in the Morris water maze task in APP x PS1 mice. Bar graphs show the mean ± SEM. latencies to reach the platform in second on each of five days. Animals were divided into the following four groups: placebo injected, wild group (white); apape injected wild group (light grey) ; apape injected APPxPSl mice (black) ; placebo injected APPxPSl mice (dark grey) . According to the repeated measure of ANOVA we found significant difference between the groups

p=0.002), in post hoc LSD test the difference between the wild-placebo injected group, and the APPXPSl placebo injected group on the fourth day p=0.002, difference between the apape injected wild and APPXPSl placebo injected group p<0.0001, furthermore difference between transgenic placebo injected mice and transgenic apape administered group p=0.037.

Fig. 11 displays tau histology in 50 micrometer thick hippocampal sections of female APPxPSl transgenic mouse strain. The vertical axis shows the mean of the number of tau immune positive cells in wild-placebo injected (white) ; APPxPSl transgenic-placebo administered (light grey) ; apape injected wild (black) ; and the apape injected transgenic (dark grey) animals. According to the one way ANOVA, the difference between the experimental groups was significant (F62, 3=15.642, p<0.0001) In the multiple comparisons post hoc LSD test we also found significant differences between the APPxPSl transgenic-placebo injected group and the other three experimental groups. Data are expressed as mean ± SEM. Fig. 12 reports tau histology in 50 micrometer thick hippocampal sections of male APPxPSl transgenic mouse strain. Bar graphs show the mean of the number of tau immune positive cells ± SEM. For details, see Fig. 11. According to the one way ANOVA, the difference between the experimental groups was significant (Fie, 3=12.756, p<0.0001). The multiple comparisons post hoc LSD test shows significant difference between the transgenic-placebo injected and wild placebo injected, furthermore the wild-apape administered groups (p=0.001). Additionally, the number of tau positive cells was significantly higher in the APPxPSl transgenic-placebo injected group than in the APPxPSl-BFR-106 injected group.

Fig. 13 shows cresyl violet staining in 50 micrometer thick cross sections of hippocampus of female APPxPSl transgenic mouse strain. Bar graphs show the number of tau immune positive cells ± SEM. White: wild-placebo injected; light grey: APPxPSl transgenic-placebo administered group; black: wild-apape injected group; and dark grey: APPxPSl transgenic-apape injected animals. According to the one way ANOVA, the difference between the experimental groups was significant (F4i, 3=8.915) . In the multiple comparisons post hoc LSD test the wild-placebo injected group compared to the transgenic and wild-apape administered groups show significant difference (p=0.0001), furthermore the wild-placebo injected group compared to the APPxPSl-apape injected group show significant different (p=0.003). We also found significant difference between the transgenic-placebo administered and the APPxPSl+apape injected animals (p=0.021). Table 1. General structure of the novel peptides. The table lists the possible moieties certain positions of the novel peptides.

Where X= acyl group; Y= organic functional group; Al, A2, A3, A4 and A5 are amino acids (coded and non coded, L or D) or acyl group

Detailed description of the invention

Peptide structures of the present invention were designed on the basis of computer performed analysis of the common binding sequences of Αβ 1-42 binding proteins (Verdier et al. J. Neurochem. 94, 617-28, 2005). (Table 4). The common Αβ-binding sequences of these proteins revealed by proteomic methods are very similar, containing mainly hydrophobic amino acids, proline and airtinodicarboxylic acids. Hence proline residues were incorporated in different part of the molecule since it is known to have greater β-breaking potential than the other proteinogenic amino acids (Chou and Fasman, Annu Rev Biochem 47: 251-276, 1978). Moreover, incorporation of proline into short peptides homologous to Αβ resulted in nonamyloidogenic analogues (Soto et al. Biochem Biophys Res Commun 226(3): 672- 680, 1996) . Peptides replacing L-amino acids by their D counterparts were also designed since the D-amino acids are more resistant to proteases and mostly non-immunogenic (Poduslo et al. J Neurobiol 39(3): 371-382, 2003: Dintzis et al . Proteins 16(3): 306-308, 1993). To improve the properties of the peptides, suitable chemical modifications, i.e. N- and C- terminal modifications and incorporation of peptidomimetics have been performed (Table 1 and 2) . Due to the complexity of the issue, it is clear that multidisciplinary approaches are needed, including methods to detect the early effects (synaptic activity, long-term potentiation (LTP) and late effects (cytotoxic effects) of Αβ on a cellular level combined with analysis for AD-linked pathophysiology (MTT test) and cognition assays to evaluate the progression of the disease and develop effective treatments.

The soluble forms of neurotoxic/synaptotoxic β-amyloid peptides (Αβ 1-42 oligomers and protofibrils) have no definite conformations, only some secondary structural elements (e.g. β- sheet parts) . The Αβ-peptides belong to the family of intrinsically disordered proteins (IDP) . These proteins interact non-specifically with a big number of important cellular proteins causing neuronal dysfunction. The new class of compounds acts as β-amyloid structure modifying agents. They bind to the soluble, toxic Αβ oligomers and protofibrils via a salt bridge and apolar interaction (s) , and convert them to nontoxic conformation. Afterwards, these peptides initiate aggregation to big, non-toxic Αβ-aggregates . Simultaneously, the novel class of compounds stimulates the formation of dendrites and improves communication between the neurons.

The present invention relates to peptides and peptidomimetics of the following formula:

X-A Aa-A _s-Y

Where X=acyl group; Y= organic functional group; A1-A5 are amino acids (coded and non coded, R or S) or acyl group (see Table 1) .

The present invention also relates to amino acid sequences, which are sequentially analogous to the described sequences and the biological activity of which is also analogous when

compared to the described sequences. A person skilled in the art finds it obvious that certain side change modifications or amino acid replacements can be performed without altering the biological function of the peptide in question. Such

modifications may be based on the relative similarity of the amino acid side chains, for example on similarities in size, charge, hydrophobicity, hydrophilicity, etc. The aim of such changes may be to increase the stability of the peptide against enzymatic decomposition or to improve certain pharmacokinetic parameters .

The scope of protection of the present invention also includes peptides, in which elements ensuring detectability (e.g.

fluorescent group, radioactive atom, etc.) are integrated.

Furthermore, the scope of protection of the present invention also includes peptides, which contain a few further amino acids at their N-terminal, C-terminal, or both ends, if these further amino acids do not have a significant influence on the

biological activity of the original sequence. The aim of such further amino acids positioned at the ends may be to facilitate immobilisation, ensure the possibility of linking to other reagents, influence solubility, absorption and other

characteristics .

We used the IUPAC recommendations to mark the amino acid side chains in the given sequences (Nomenclature of -Amino Acids, Recommendations, 1974 - Biochemistry, 14(2), 1975).

The present invention also relates to the pharmaceutically acceptable salts of the peptides with general formula (I) according to the invention. By this we mean salts, which, during contact with human or animal tissues, do not result in an unnecessary degree of toxicity, irritation, allergic symptoms or similar phenomena. As non-restrictive examples of acid addition salts the following are mentioned: acetate, citrate, aspartate, benzoate, benzene sulphonate, butyrate, digluconate, hemisulphate, fumarate, hydrochloride,

hydrobromide, hydroiodide, lactate, maleate, methane

sulphonate, oxalate, propionate, succinate, tartarate,

phosphate, glutamate. As non-restrictive examples of base addition salts, salts based on the following are mentioned: alkali metals and alkaline earth metals (lithium, potassium, sodium, calcium, magnesium, aluminium) , quaternary ammonium salts, amine cations (methylamine, ethylamine, diethylamine, etc. ) .

The peptides according to the invention can be used in

pharmaceutical preparations, where one or more additives are needed to reach the appropriate biological effect. Such preparations may be pharmaceutical preparations combined for example with matrices ensuring controlled active substance release, widely known by a person skilled in the art. Generally matrices ensuring controlled active substance release are polymers, which, when entering the appropriate tissue (e.g. blood plasma) decompose for example in the course of enzymatic or acid-base hydrolysis (e.g. polylactide, polyglycolide) .

In the pharmaceutical preparations according to the invention other state-of-the-art additives can also be used, such as diluents, fillers, pH regulators, substances promoting

dissolution, colour additives, antioxidants, preservatives, isotonic agents, etc. These are state-of-the-art additives.

Preferably, the pharmaceutical preparations according to the invention can be entered in the organism via parenteral

(intravenous, intramuscular, subcutaneous, etc.)

administration. Taking this into consideration, favourable pharmaceutical compositions may be aqueous or non-aqueous solutions, dispersions, suspensions, emulsions, or solid (e.g. powdered) preparations, which can be transformed into one of the above fluids directly before use. In such fluids suitable vehicles, carriers, diluents or solvents may be for example water, ethanol, different polyols (e.g. glycerine, propylene glycol, polyethylene glycols and similar substances) , carboxymethyl cellulose, different (vegetable) oils, organic esters, and mixtures of all these substances.

The favourable forms of packaging of the pharmaceutical

preparations according to the invention include for example tablets, powders, granules, suppositories, injections, syrups, etc .

The administered dose depends on the type of the given disease, the patient's sex, age, weight, and on the severity of the disease. In the case of oral administration the favourable daily dose may vary for example between 0.01 mg and 1 g, in the case of parenteral administration (e.g. a preparation

administered intravenously) the favourable daily dose may vary for example between 0.001 mg and 100 mg in respect of the active agent.

Furthermore, the pharmaceutical preparations can also be used in state-of-the-art liposomes or microcapsules. The peptides according to the invention can also be entered in the target organism by state-of-the-art means of gene therapy.

The peptides according to the invention can be used first of all in the medical treatment of diseases, in the case of which the inhibition of aggregation of β-amyloid to toxic species is beneficial, wherein the target β-amyloid peptide comprises a β- sheet sequence susceptible to further aggregation up to

fibrillation. Consequently the present invention also relates to the use of peptides in the manufacture of drugs for the treatment of such diseases. As it has been explained above in detail, such diseases are first of all certain

neurodegenerative diseases and type 2 diabetes mellitus, especially the following diseases: Alzheimer's and Parkinson's disease . To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention.

As used herein, 'aggregation' means the association of peptide moieties, whether the resulting structure is regular or irregular, stable or unstable or with ordered or disordered native states. Such association can occur through intermolecular interactions, hydrophobic interactions, hydrogen bonds, van der aals forces, ionic bonds or any force or substance that can result in the collection or association together of two or more peptides or peptide regions. As used herein, "aggregation" encompasses, for example, fibrillation, or the formation of fibrils.

'Amyloid' describes various types of protein aggregations that share specific traits when examined microscopically. The name amyloid comes from the early mistaken identification of the substance as starch (amylum in Latin) , based on crude iodine- staining techniques. Amyloid is typically identified by a change in the fluorescence intensity of planar aromatic dyes such as Thioflavin T or Congo Red. This is generally attributed to the environmental change as these dyes intercalate between beta-strands. The deposits are characterized by the abnormal folding of amyloidogenic protein from a normal secondary structure (often an a-helix, but can also be a random coil) into a pathological β-sheet structure, which permits aggregation into insoluble fibrils. Ultrastructurally, amyloid fibrils are made up of a helical configuration of two protofilaments . These deposits are characteristically resistant to protease digestion. Amyloid polymerization is generally sequence-sensitive, that is, causing mutations in the sequence can prevent self-assembly, especially if the mutation is a beta-sheet breaker, such as proline. For example, humans produce an amyloidogenic peptide associated with type II diabetes, but in rodentia, a proline is substituted in a critical location and amyloidogenesis does not occur. Approximately 25 different proteins are known that can form amyloid in humans.

The term 'amyloidosis' refers to a disease or disorder associated with abnormal protein folding into amyloid or amyloid-like fibrillar deposits in which the abnormally folded proteins in the deposits have a characteristic pathological beta-sheet structure.

As used herein, the terms ' β-amyloid peptide', 'amyloid β peptide' or Άβ peptide' refer to a human amyloidogenic peptide that derives from APP by proteolytic cleavage. Various pathogenic forms of Αβ peptides include Αβ 1-42, Αβ 1-40, Αβ 5- 42, etc. (the numbers following Αβ refer to the starting and ending positions in the amino acid sequence) . The amino acid sequence of the human Αβ 1-42 peptide is the following:

DAEFRHDSGYEVHHNKLVFFAEDVGSNKGAIIGLMVGGVVIA. (SEQ ID NO 66) Mutant human Αβ 1-42 peptides are also known in some familial ADs (like e.g. Swedish mutation). Unless otherwise stated, under the term 'β-amyloid peptide' these mutations are also meant.

As used herein, 'protein folding' is the physical process by which a polypeptide folds into its characteristic and functional three-dimensional structure from random coil. The correct three-dimensional structure is essential for biological activity. Failure to fold into native structure produces inactive proteins that are usually toxic. Several neurodegenerative diseases are believed to result from the accumulation of amyloid fibrils formed by misfolded proteins. As used herein, 'intrinsically disordered proteins' (IDPs) are proteins which lack stable tertiary and/or secondary structure in solution yet fulfill key biological functions.

A protein may undergo reversible structural changes in performing its biological function. The alternative structures of the same protein are referred to as different

'conformations' and transitions between them are called

'conformational changes' . As used herein, the term 'peptide' , when used in reference to an agent that binds amyloid or amyloid-like deposits refers to a peptidic compound made up of 2, 3, 4, 5, 6, or more amino acid residues in length, and also includes amino acid residues with D or L stereochemistry. A peptide' as used in this context specifically excludes an antibody or an antigen-binding fragment (e.g. an Fab) of an antibody, and also an amyloidogenic sequence, such as Αβ 1-40, which aggregates with other like molecules and forms a β-sheet structure. As used herein, 'peptidomimetic' , also referred to as 'peptide mimetic' , means any compound containing amino acid residues, whether D- or L-, whether natural or non-naturally occurring, and it can also comprise non-amino acid moieties. A 'peptidomimetic' is designed to mimic the biological action (s) of a natural mimicked peptide, including, for example, those designed to mimic the structure and/or binding activity (such as, for example, hydrogen bonds and hydrophobic packing interactions) of the peptides according to the methods disclosed herein.

As used herein, the term 'pentapeptide' refers to a compound of five amino acids, modified amino acids or amino acid substitutes. For example, the compound apape (SEQ ID NO: 51) is a pentapeptide consisting of D-Ala, D-Pro, D-Ala, D-Pro and D- Ala-amide .

As used herein, the term 'resistant to. rotease digestion' or 'enzymatically resistant' entails that a given folded form of a polypeptide is cleaved at least 25% less than an alternatively folded form of that polypeptide when both forms are contacted with a similar amount of a given protease enzyme under the same conditions. Since the terms is used for peptides and peptidomimetics for reducing the toxicity of amyloid or amyloid-like deposits, a peptidomimetic is 'resistant to protease cleavage' if it is cleaved at least 10% less (and preferably 20, 30, 40, 50, 60, 70, 80, 90 or even 100% less) by a given amount of a given protease, relative to the cleavage of a corresponding peptide under like conditions.

As used herein, the term ^reduce' or ^inhibits' refers to at least a 10% decrease of the parameter being measured in the presence of an agent relative to the absence of that agent. For example, 'reducing neurotoxicity' means inhibiting the toxic effect of a composition or environment against neural cells or tissue by at least 10% relative to cells or tissue not treated in a manner to reduce such toxic effect.

As used herein, the term 'neurotoxicity' describes the ability of a substance, condition or state to impair, or even kill the functioning of a neural tissue or cell.

As used herein, 'binding' encompasses physical association of one molecule with another. Binding can be both covalent and non-covalent interatomic and intermolecular interaction, whether long lasting or transient. Examples include, without limitation, ionic bonds, hydrophobic interactions, hydrogen bonds, Van der Waals forces and dipole-dipole bonds. As used herein, the term 'neuronal cell' refers to a cell of the central nervous system, including, but not limited to a neuron and a glia.

As used herein, 'screening test' encompasses tests conducted to determine if candidate inhibitory peptides are effective in disrupting Αβ toxicity. Examples of such screening tests are described herein. However, a person of ordinary skill would recognize that other screening tests can be employed without departing from the spirit and scope of the invention.

Examples Below the present invention is described in detail on the basis of examples, which, however, should not be regarded as examples to which the invention is restricted.

Until recently, the synthetic commercial Αβ 1-42 samples were not homogenous as for aggregation grade and water solubility. As a consequence, the biological studies performed by these peptides were not always reproducible. We and others have shown that Αβ assemblies with defined aggregation grade (oligomers and fibrils) can be reproducibly prepared from iso-Αβ 1-42 under specific conditions (Bozso et al. Peptides 31: 248-256, 2010) . This will greatly facilitate studies aiming at

understanding the molecular basis of AD.

Example 1: Design of small peptides inhibiting amyloid toxicity

Oligomerization and aggregation of some polypeptides and proteins (β-amyloid, -synuclein, huntingtin, prion protein) appears to be a key factor in the neurodegeneration process in a group of diseases (e.g. AD) . The soluble forms of neurotoxic/synaptotoxic β-amyloid peptides (Αβ-1-42 oligomers and protofibrils) have no definite conformations, only some secondary structural elements (e.g. β-sheet parts) . The Αβ- peptides belong to the family of intrinsically disordered proteins (IDP) . These proteins interact non-specifically with a big number of important cellular proteins causing neuronal dysfunction. Our novel neuroprotective compounds (see Table 2.) bind to the soluble, toxic Αβ oligomers and protofibrils via a salt bridge and apolar interaction (s) . The novel class of compounds acts as β-amyloid structure modifying agents. These novel compounds convert the intrinsically disordered Αβ- peptides to nontoxic conformation, and then initiate their aggregation to big, non-toxic Αβ-aggregates . Simultaneously, the novel class of compounds stimulates the formation of dendrites and improves communication between the neurons, and thereby decreases the detrimental effect of Αβ-peptides in the brain .

1. Peptide synthesis

Materials

All N-terminally protected amino acids were purchased from Orpegen (Heidelberg, Germany) Bachem (Bubendorf, Switzerland) and GL Biochem (Shanghai, China) . DCC, HOBt and MBHAxHCl resin were from GL Biochem (Shanghai, China) . Boc-Ala-PAM resin was from Bachem (Bubendorf, Switzerland) . Boc-D-Gln-Merrifield resin, Boc-D-Asn-Merrifield resin, Boc-D-Glu (Bzl) -Merrifield resin and EDC*HC1 were from IRIS Biotech GmbH (Marktredwitz, Germany) . Solvents and DIPEA were obtained from Sigma-Aldrich. HPLC grade TFA was ordered from Pierce (Rockford, IL, USA).

A) β-amyloid peptides Controlled in situ preparation of Αβ 1-42 oligomers from the isopeptide 'ϊεο-Αβ 1-42' was performed as published (Bozso et al, . Peptides 31: 248-256, 2010). B) Synthesis of the novel inbibitory peptides and

peptidomimetics

Boc-chemistry protocol

a. Protocol for the following peptides: EPAPA-NH2, EPPPA-NH2, APAPE-NH2, APPPE-NH2 , epapa-NH2, epppa-NH2, apape-NH2, apppe- NH2, EPAP-NH2, EPPP-NH2 , RPAPA-NH2, KPAPA-NH2, RPPPA-NH2 , KPPPA-NH2, pape-NH2, pppe-NH2, DPAPA-NH2, Succinyl-PAPA-NH2 , Propionyl-PAPA-NH2, -Ala-PAPA-NH2 , Iminodiacetyl-PAPA-NH2, E- Sar-APA-NH2, EPA-Sar-A-NH2 , E-Pip-APA-NH2 , EPA-Pip-A-NH2 , E- Sar-A-Sar-A-NH2, apapd-NH2, apapq-NH2, apapn-NH2, Propionyl- pape-NH2, Propionyl-papq-NH2 , apGpe-NH2, ap-N-Me-ala-pe-NH2, a- tic-ape-NH2, apa-tic-e-NH2 , ap-tic-pe-NH2 , a-Sar-ape-NH2 , apa- Sar-e-NH2, a-pip-ape-NH2, apa-pip-e-NH2, Gpape-NH2 , (N-Me-ala) - pape-NH2, ap-Aib-pe-NH2 , Aib-pape-NH2 , apap-NH2, APEPA-NH2, ape-NH2, Ac-ape-NH2, GABA-pe-NH2.

Peptides were synthesized on BHAxHCl resin. The resin was swollen in DCM then neutralized with 5% DIPEA/DCM solution (2x1 min) . After neutralization the resin was washed three times with DCM. Four-fold excess of Na-Boc protected amino acids were activated with DCC/HOBt in DCM/DMF (1:1) then the resin was incubated in this mixture for 3 hours. The resin was washed twice with DMF, DCM and methanol, it was partially dried and the coupling was monitored with qualitative ninhydrin test. If the ninhydrin test indicated incomplete acylation the coupling step was repeated. The resin was resuspended in DCM, then the Boc-protecting group was removed with 50% TFA/DCM (5+25 min) . The peptide-resin was washed 3x with DCM then neutralized as detailed above and a new cycle was started. The peptide was cleaved from the resin with HF (cleavage cocktail: 10 mL HF, 0.8 mL dimethyl-sulfide and 0.2 mL anisole for 1 g of peptide-resin, 0 °C, 45 min) . The crude peptide was precipitated with diethyl-ether , dissolved in 50% ACN/water and lyophilized. b. Protocol for the following peptides: EPAPA, EPPPA, apapq, apapn, apape. Peptides were synthesized on the appropriate resin, depending on the C-terminal amino acid (Boc-Ala-PAM resin, Boc-D-Gln- Merrifield resin, Boc-D-Asn-Merrifield resin, Boc-D-Glu (Bzl ) - Merrifield resin) . The resin was swollen in DCM, and then the Boc protecting group was removed as detailed above. Neutralization, coupling and cleavage from the resin was performed following the procedures detailed in section a.

Fmoc-chemistry protocol

PPPA-NH2, PAPA-NH2 , AC-EPAP-NH2, EPA-NH2, AC-EPA-NH2, EPP-NH2, DPA-NH2, DP-NH2, Succinyl-PA-NH2 , Succinyl-pa-NH2 and Glp-Asp- NH2 were synthesized with Fmoc-chemistry.

Peptides were synthesized on Fmoc-Rink amide resin. The resin was swollen in DCM, the washed with DMF and Fmoc protecting group was removed by treating it with 20% piperidine/DMF (5+15 min). The resin was washed with DMF five-times. Three-fold excess of Na-Fmoc protected amino acids were activated with DCC/HOBt in DCM/DMF (1:1) then the resin was incubated in this mixture for 3 hours. The resin was washed twice with DMF, DCM and methanol, it was partially dried and the coupling was monitored with qualitative ninhydrin test. If the ninhydrin test indicated incomplete acylation the coupling step was repeated. The resin was resuspended in DMF, and the Fmoc group was removed as detailed above. Cycles were repeated until the required sequence was obtained. The peptide was cleaved from the resin using a mixture of TFA (95%) , water (2%) , triisopropyl-silane (1.5%) and dithiothreitol (1.5%). The cleavage was performed on 0 °C for 15 min, then at room temperature for 2 hours 45 min. The crude peptide was precipitated with diethyl-ether, dissolved in 50% ACN/water and lyophilized.

Synthesis of Glp-Gly Glp, Gly-OtBu and EDC*HC1 (1 mmol of each) were mixed in 15 mL NaHC03 (100 mM) for 6 h. The mixture was then extracted with ethyl-acetate (3x) . Ethyl-acetate was evaporated and the resulting oil was mixed with 50% TFA/DCM for 30 min. TFA and DCM was evaporated and the oil was purified on HPLC.

Purification

Peptides were analyzed and purified using RP-HPLC. 0.1 % TFA in d.i. water and 80% ACN, 0.1% TFA in d.i. water was used as eluent A and eluent B, respectively. Analytical analysis was done on a Hewlett-Packard Agilent 1100 Series HPLC apparatus using a Luna C18 column (100 A, 5 μιη, 250x4, 60 mm, Phenomenex) , the flow rate was 1.2 mL/min and the gradient was as it is indicated in Table 2. Preparative chromatography was done on a Shimadzu HPLC apparatus equipped with a Luna C18 column (100 A, 10 m, 250x21.2 mm, Phenomenex) with a flow rate of 4 mL/min. The gradient was 0-30% eluent B in eluent A over 60 min.

Mass spectrometry

Mass spectrometry measurements were done on a FinniganMat TSQ 7000 mass spectrometer in ESI-MS mode.

2. Transmission electron microscopy (TEM) Oligomeric and fibrillar structures of Αβ 1-42 were prepared from iso-Αβ 1-42 (75 μΜ) as published (Bozso et al. Peptides 31: 248-256, 2010). Ten ul droplets of Αβ 1-42 solution (75 μΜ) with or without apape (SEQ ID NO: 51) (1:5 molar ratios) were placed on formvar-carbon coated 400 mesh copper grids (Electron Microscopy Sciences, Washington, PA, USA) and stained negatively with uranyl acetate. The aggregates were characterized by transmission electron microscopy with a Philips CM 10 transmission electron microscope (FEI Company, Hillsboro, Oregon, USA) operating at 100 kV. Images were taken by a Megaview II Soft Imaging System at magnifications of x

46,000 and x 64,000, and analyzed by an Analysis® 3.2 software package (Soft Imaging System GmbH, Miinster, Germany) .

3. Dynamic light scattering (DLS)

The following solutions were prepared in PBS, and placed in a low volume sizing cuvette: Αβ 1-42 (50 μΜ) , Αβ 1-42. (50 μΜ) + apape (SEQ ID NO 51) (250 μΜ) , Αβ 1-42 (50 μΜ) + Cys-Gly7-amide (250 μΜ) . Size distribution was measured at 25 °C with a Malvern Zetasizer Nano ZS Instrument (Malvern Instruments Ltd. Worcestershire, UK) equipped with a He-Ne laser (633 nm) applying the Non-Invasive Back Scatter (NIBS®) technology, which means detection of the scattered light at an angle of 173°. The change in size distribution was followed for 22 hrs . For one single measurement, correlation function and distribution of the apparent hydrodynamic diameter (dh) over the scattered intensity of the particles were determined on the basis of 14 scans. The translational diffusion coefficients were obtained from the measured autocorrelation functions using a fitting algorithm built in the software package Dispersion Technology Software 5.1 (Malvern Instruments Ltd. Worcestershire, UK) . 4. Electronic circular dichroism (ECD) spectroscopy

ECD spectra of the Αβ 1-42, apape-NH2 and their mixture were recorded on a Jasco (Tokyo, Japan) J815 spectropolarimeter equipped with a Peltier temperature controller using a 1 or 2 mm path length quartz cell. Peptide samples were dissolved in HCBS buffer. The concentration of Αβ 1-42 was 12.5 μΜ, while the concentration of apape-NH2 was 62.5 μΜ. Spectra of peptide solutions in the 200-250 nm wavelength region were recorded at 37 °C and 100 nm/s scan speed over a one week period at the following time points: 0 min, 10 min, 20 min, 30 min, 1 h, 3 h, 6 h, 24 h, 48 h, 72 h, 168 h. Spectra presented here are accumulations of 10 scans and the corresponding solvent spectra similarly recorded were subtracted.

5. Computational Binding Study

The fibril model is based on the NMR structure published by Liihrs et a. (PNAS 102/48: 17342-17347, 2005) and contains eight Αβ 1-42 monomer units. The missing residues at the N-terminal part were completed assuming a β-structure. The accomplished structure was embedded into explicit water (TIP3P type) environment and 4 ns long molecular dynamics simulation was performed with the help of the AMBER8 program package (Case et al. AMBER 8, University of California, San Francisco, 2004) applying the FF03 forcefield with its own atomic charges. To map the possible binding positions of the small molecules onto the Αβ 1-42 fibril surface, the ^blind docking' procedure (Hetenyi and van der Spoel, Protein Science 11:1729-1737, 2002) was applied using the Autodock program (Morris et al. J. Comput. Chem. 19: 1639-1662, 1998). Ligand set was re-docked onto the possible binding regions with better grid-map, and those molecules were considered as binding-ones which had large binding free energy (BFE) value in all the regions. Two scores were considered for every peptide: the best BFE value of the docked positions on one hand and the average of the best 10 poses according to the BFE value of a given ligand on the other hand. Taking the geometries from the docking calculation, 1 ns explicit water simulations were performed for selected complexes (fibril model + ligand) and BFEs were calculated with the molecular mechanics-generalized Born surface area (MM-GBSA) method (Srinavasan et al. J. Am. Chem. Soc. 120 9401-9409, 1998).

6. Conformational Analyses

Replica-Exchange Molecular Dynamics (REMD) calculations with 24 trajectories were performed for each molecule between 280 - 430 K using explicit water (TIP3P) environment. 50 ns long simulations were accomplished in all cases by the GROMACS package (Hess et al. J. Chem. Theory Comput . 4: 435-447, 2008) applying the amber99sb forcefield (http://ambermd.org). Taking the trajectory at the lowest temperature, cluster analyses were performed at the 10 -50 ns interval by the ptraj program from the AmberTools package (http://ambermd.org). 4000 snapshots were selected from the trajectory in each case, and the ^''average linkage' method was applied with two cut-off (epsilon) values: ε=1.0 for the clusterization on the main-chain atoms, and ε =1.5 values for the clusterization on the heavy atoms.

Conclusions Based on computer simulation to find common sequences in amyloid binding proteins (Table 1), 72 novel peptides were designed of which 65 are discussed within the frame of the present invention. The MS and HPLC analytical parameters of the novel peptides are summarized in Table 2. The physico-chemical parameters of Αβ oligomers and fibrils in the absence and in the presence of the novel peptides were determined. The morphology of the aggregates can be seen on the TEM images

(Fig. 2) . It was observed that the apape peptide (SEQ ID NO: 51) (in 1:5 molar ratios) promoted the formation of the fibrillar aggregates, which formed already after 4 h of incubation, while the control sample contained only protofibrils and high-n oligomers. The presence of fibrillar aggregates could be observed in both samples after 24 h. These observations, together with the DLS data, indicate that apape

(SEQ ID NO: 51) acts as an aggregation promoter instead of an aggregation inhibitor of the beta amyloid.

ECD spectra of the Αβ 1-42 shows a strong minimum at 200 nm and another peak at 215 nm, indicative of a mixture of random and β-sheet structures even right after the dissolution of the peptide sample (Fig. 3A) . apape-NH2 displayed a random structure by itself and spectra recorded in later time points were not different from the one acquired immediately after dissolution of the sample (Fig. 3B) . This suggests that no major structural change of the pure peptide takes place during the one week period. Spectra recorded for the 1:5 molar ratio mixture of Αβ 1-42 and apape-NH2 appears to be a simple combination of the spectra of the pure component peptides. The aggregation iso-amyloid β 1-42 in the presence of apape-NH2 appears to be less gradual as compared to the case of pure iso- amyloid β1-42. A major structural change was observed after 6 hours. However, after the initial lagging period, structural transformation comes to completion similarly to that observed in the absence of apape-NH2. Difference spectra obtained by subtracting the corresponding pure apape-NH2 spectra from those of the Αβ 1-42 and apape-NH2 mixture are markedly different from those recorded for pure Αβ 1-42. The most conspicuous difference is the blueshift of the 215 minima, which indicates a small-scale structural change (Fig. 3C-D) . Such structural change may take place as a result of an intermolecular interaction between the two component peptides. The presence of apape-NH2 may slightly affect the aggregation kinetics of Αβ 1- 42 as indicated by spectra of Fig. 3D. However, this difference in the aggregation behaviour is less apparent in the difference spectra shown on Figs. 3C and D.

It was revealed from the computational binding simulation (docking studies augmented with MM-GBSA method (Fig. 4) that there is an elongated, poly-proline II helix-like backbone structure which can be found in all cases except the ^xpape' peptide which seems to be too short to form this structure (Fig. 4). However, an elongated conformation is also typical even for the ^xpape' peptide.

Example 2. In vitro studies of the neuroprotective effect of the peptides NaCl, KC1, CaC12, MgCl2, HEPES, NaHC03, D-Glucose, MIT and 96- well plates (Costar) were purchased from Sigma-Aldrich (Hungary) . The ExViS mini-chamber system was from our institute. The animal protocols applied in this study had been approved by the National Institute of Health and by the University of Szeged; permission number: 1-02442/001/2006.

Preparation and treatment of acute hippocampal slices

A slightly modified version of the method reported (Datki et al. Brain Res Bull. 74/1-3, 183-7, 2007) was used in our slice viability experiments. After anesthesia with chloral hydrate (0.4g/kg), 10 ± 1 -weeks-old male istar rats were decapitated and the whole heads (without scalp-leather) were put in ice- cold distilled water for 1 min. The brains were quickly removed and immersed in H-ACSF/1 (preparation solution) with very low Ca2+ and with elevated Mg2+ at 4 °C (Ref . 4) . The composition of this preparation solution (in mM) : NaCl 122, KC1 3, CaC12 0.3, MgC12 3.7, NaHC03 25, HEPES 5.0, D-glucose 10.0, pH=7.4. Brain slices (400 μπι thick) were prepared from the hippocampus with a Mcllwain tissue chopper at 4 °C in ice cold H-ACSF/1 solution followed by taking photos of them (for measuring slice area) . The slices (with an area of approximately 9 mm2) were rapidly transferred into the ExViS mini-chamber (maximum 10 slice in 1 ml) for conditioning (30 min) in the carboxygenated (02:C02= 95 : 5%) preparation solution at room temperature (24 °C) . After resting (30 min) in the carboxygenated H-ACSF/1 solution at room temperature, the brain slices were transferred from the mini-chamber into the plastic Petri dish with cut-off pipettes (type 200 μΐ) in glucose- and carboxigenated-free H- ACSF/2 (2 ml/Petri dish) for one hour. The composition of this H-ACSF/2 (in mM) : NaCl 132, KC1 3.0, CaC12 2.0, MgC12 2.0, NaHC03 25, HEPES 5.0, pH=7.4. The Petri dish was stirred continuously at 370 rpm (modified BIOSAN TS-100 thermo shaker) . After resting/treated in glucose- and oxygen-free H-ACSF/2, the Petri dish content (supernatant) was changed to the normal H- ACSF/1 solution (2 ml/Peri dish) . The slices were rapidly transferred into the ExViS mini-chamber (maximum 10 slice in 1 ml) for treating with Αβ 1-42 (50 μΐ stock solution into 950 μΐ per chamber; 20 μΜ Αβ 1-42 in final concentration) . Before each slice viability assay, the stock solution of Αβ peptide (0.4 mM) was freshly prepared (and stored for maximum 10 min) in distilled water (pH=5) . The peptidomimetics were used in 40 μΜ and 100 μΜ final concentration, alone or together with Αβ 1-42 (mixed in DW before slice treatment) . After treating the slices with Αβ 1-42 and/or peptidomimetics for 4 hours, 0.1 ml MTT (stock solution 5 mg/ml H-ACSF/1) was added to the wells in which they were rested for 15 min without carboxigenation. To stop the reduction of the MTT to formazane crystals, the slices were transferred from H-ACSF/1 to DMSO and incubated for 30 min (50 μΐ DMSO/one slice) . The optical density (OD) of the dissolved formazane was measured at 550 and 620 nm. The following formula: (OD550 - OD620)/area of slice (mm2) = 100% in control (Αβ 1-42 untreated slices) was used for data synchronization.

Conclusions Table 3 summarizes the results of the MTT-assay of several novel peptides in two concentrations. It can be seen that among the peptides studied pape-NH2 has the least protective effect against and all the other peptides studied almost completely eliminated the toxic effect of Αβ . On the basis of these results, we have selected apape (SEQ ID NO: 51) as the leading compound for further biological experiments.

Example 3. In vivo studies using the experimental rat model for electrophysiology

Αβ 1-42 preparation

The synthesized Αβ 1-42 was dissolved in 1,1,1,3,3,3- hexafluoro-2-propanol (HFIP) and incubated overnight at ambient temperature. After removal of the HFIP in vacuo, the peptide was dissolved in d.i. water and incubated at 37 °C for 3 days. Fibrils were collected by centrifugation at 10 000 x g for 15 min and the pellet was washed with d.i. water and lyophilized. Prior to use, the peptide was freshly dissolved in pH 6.4 saline to a final concentration of 5x10-5 M, gently sonicated for 15 min in order to facilitate the homogenization of the sample, and used in the biological experiments.

Extracellular recordings and microiontophoresis Extracellular single-unit recordings were performed in the rat hippocampus (Wistar male 250 to 330 g) after chloral hydrate anesthesia (intraperitonial, i.p. 0.4 g/kg initial dose, supplementary doses as required) . The animals were handled and surgery performed in accordance with the European Communities Council Directives (86/609/ECC) and the Hungarian Act for the Protection of Animals in Research (XXVIII. tv. Section 32). Ethical approvement number was 1-74-13/2010. All efforts were made to minimize pain and reduce the number of animals used. The head of each animal was mounted in a stereotaxic frame, the skull was opened above the hippocampus (a-p:-3.8 mm from bregma; lat: ±2 mm from the midline on either side), and the dura mater was carefully removed. Following a 1 hour recovery period, single-unit activity of the CAl hippocampal neurons was recorded extracellularly between depths of 2 and 3 mm by means of a low-impedance (< 1 ΜΩ) 7 μπι carbon fiber-containing multibarrel microelectrode ( ation Scientific, Minneapolis, N, USA) , and drugs were delivered from the surrounding capillary barrels. Action potentials were amplified by an ExAmp-20KB extracellular amplifier (Kation Scientific) and monitored with an oscilloscope. The filter bandpass frequencies were 300 to 8000 Hz. A window discriminator (WD-2, Dagan Corporation, MN, USA) was used for spike discrimination. The amplified signals were sampled and digitalized at 50 kHz. The number of action potentials per second was counted by the computer and peristimulus time histograms were calculated and displayed in line. Iontophoretic drug delivery and experimental data collection were governed by a PCI-1200 multifunction instrument control and data acquisition board (National Instruments, Austin, TX, USA) placed in a computer programmed in LabVIEW 6, and by iontophoretic pumps (Minion-16 and BAB-350, Kation Scientific) . A multibarrel electrode affixed to the recording electrode was used for iontophoretic ejection of the following drugs: (a) 100 mM NMDANa (Sigma-Aldrich, Budapest, Hungary) in 100 mM NaCl (pH 8.0); (b) 5 χ 10-5 M Αβ1-42 and (c) 2.5 χ 10-4 M peptides dissolved in saline (pH 6.4); (d) a mixture containing Αβ1-42 (5 χ 10-5 M) and the peptides to be studied (2.5 x 10-4 M) stored for 1 h, and (e) to mark the position of the electrode, 4% Pontamine Sky Blue (BDH Chemicals, Poole, England) in 100 mM sodium acetate. The Αβ 1-42 was ultrasonicated for 15 min prior to use. Neurons were excited by repetitive iontophoresis of NMDA at 1 min intervals for 5 s by applying negative iontophoretic currents ranging from 5 to 100 nA and the ejection current was selected so that the maximum firing rate fell between 30 and 80 spikes/s. Retaining current of the opposite direction in the range 2-21 nA was used. The peristimulus time histograms of the neurons were recorded. After establishment of a stable control, the peptides to be tested were co-iontophorized for 3 min at +100 nA, followed by Αβ 1-42 for 1 min at -0.5 μΑ. In the second part of the experimental procedure, a mixture of Αβ 1-42 and a pentapeptide was ejected for 1 min at -0.5 μΑ. Recording sites were marked by the iontophoretic ejection of PSB at a negative current of 5 μΑ negative current for 10 min. At the end of each experiment, the animals were euthanized with an overdose of chloral hydrate. The brain was quickly removed and fixed in 4% paraformaldehyde. Brain sections (50 μιη thick) were counterstained with Neutral Red, and the PSB localization was verified according to the stereotaxic atlas of Paxinos and Watson (1986) . Statistical evaluation was performed using the total number of spikes evoked during each epoch of excitation by the iontophoretic application of NMDA. Differences in magnitude between the different response epochs of a single cell were tested by comparing the total numbers of spikes per excitation period by one-way A OVA followed by the Dunett' s post hoc analysis. The mean of pre-Αβ 1-42 NMDA-evoked responses was taken as 100% in every experiment. The maximum response rate of post-Αβ 1-42 was given as percentage of the control. Data were pooled for statistical analysis. Shown are means ± SEM. Statistical significance was set at p <0.05.

Intraperitoneal administration of peptides and in vivo single- unit recordings

Extracellular single-unit recordings were obtained from 72 CA1 neurons from a total of 32 anesthetized rats. Pentapeptides were administered i.p. (0.5 mg/100 g) , and peristimulus histograms were then taken. The data from all 40-min intervals were pooled and means ± SEM of percentage values were calculated. The pre-Αβ 1-42 control firing rate was set between ~30-80 spike/sec. In that way, the rate of amyloid induced excitation was not dependent of the initial firing rate. The pre- Αβ 1-42 firing values were considered as 100% at each recording, and the maximum post Αβ 1-42 firing rates in % value were used for statistical evaluations.

Conclusion These studies indicated that the peptide apape protected against the NMDA response-enhancing effect of Αβ 1-42 between _* 50-250 min after i.p. administration (Fig. 7) suggesting that apape may cross the blood brain barrier. Example . Behavior and learning studies in rat model

Morris water maze (M M) task prot The maze consisted of a circular pool (diameter: 130 cm, height: 60 cm). The pool was filled to a depth of 40 cm with water (23±1 °C) containing milk. The pool was divided into four virtual quadrants. A circular glassy platform (diameter: 10 cm) was placed in the middle of one of the four quadrants of the pool. The location of the platform was the same during five days. The platform was submerged 1.5 cm below the water surface, so that it was invisible at water level. There was a black curtain around the pool. We placed different geometric shapes in all four directions on the curtain. The tank was placed in a dimly lit, soundproof test room.

The experiment lasted for five days. We used two different starting points. One of the two points was nearly the platform, the other one far from the location of the platform. The mice swam every day two times (two trials) and were placed into the water facing the wall of the pool. The time interval between each trial sessions was 30 seconds. The animals had 90 seconds to find the platform, and 15 seconds to stay on it. Those animals, which did not find the platform were gently guided and placed on it until 15 seconds. The animal was taken to its home cage and was allowed to dry up after the end of the two trials.

During each trial session, the time taken to find the hidden platform (latency) was recorded using a video camera-based Ethovision System (Noldus, Wageningen, Netherlands) . The total duration in arena (the time spent in the arena) was investigated on each day. Statistical analysis was performed with repeated measured analysis of variance (ANOVA) , and Fisher's LSD post hoc test. Conclusion

Behavior and learning experiments proved the applicability of the novel peptides as neuroprotective agents in vivo (Fig. 8- 10) . Morris water maze experiments for studying the reference memory showed that the studied novel peptides and peptidomimetics significantly decrease the time for learning, and they also prevent the neurotoxic effect of Αβ 1-42 aggregates .

Example 5. Histological studies in rats Cresyl violet staining

The animals were anaesthetized with 5 % chloral hydrate (400 mg/bwkg) and perfused transcardially with physiological saline followed by 4% paraformaldehyde in phosphate buffer (0.2 M, pH 7.4). The brains were carefully removed from the skull, postfixed for 1 day in 4% paraformaldehyde, and then left for 2 days in phosphate-buffered saline containing 30% sucrose and 0.01% sodium azide at 4°C. Fifty i thick cross sections were cut and mounted onto gelatine-coated microscopy slides for Nissl staining. The slides were rinsed in 0.2 M PBS, and stained in 0.5% cresyl violet acetate for 1.5-2 minutes. After the staining procedure, the slides were rinsed again in PBS for 20-30 seconds. The slides were immersed consecutively in 70%, 80% and 95% ethanol followed by absolute alcohol and 99% butanol for 20-30 seconds. Finally, slides were placed in Hellendal staining jars containing xylene and mounted with DPX. The slides were scanned in a Zeiss MIRAX MIDI Slide Scanner (Carl Zeiss Inc., Germany). The left and right side of the hippocampus were annotated and the HistoQuant software (3D Histech Kft . , Budapest, Hungary) was used to calculate the density of cells.

TAU immunohistochemistry

The animals were anaesthetized and perfused as above. Their brains were dissected and hippocampus isolated. Fifty μιη thick cross sections were cut in a cryostat. The sections were collected into wells containing PBS and processed for immunohistochemistry. Monoclonal mouse anti-human PHF-tau (paired helical filament tau), clone AT100 antibody (1:800 diluted) was used as the primer antibody (Thermo Scientific, Rockford, IL, U.S. A). Biotinylated anti mouse IgG (1:400 diluted) (Vector Laboratories, Inc. Burlingame) was used as a secondary antibody. Vectastain ABC kit (Vector Laboratories Inc.) was used to visualize the secondary antibody. The peroxidase reaction was developed with 3, 3 ' -diaminobenzidine tetrahydrochloride (DAB Sigma Aldrich Co. Ltd.) enhanced with 1% nickel-ammonium-sulphate. Finally, the f ee-floating slices were mounted on slides and digitally scanned in a Zeiss MIRAX MIDI Slide Scanner (Carl Zeiss Inc., Germany). The left and right side of the hippocampus were annotated and the HistoQuant software (3D Histech Kft., Budapest, Hungary) was used to calculate the number of tau immunopositive cells.

Conclusions

These studies showed that the novel peptides and peptidomimetics may stimulate the formation of dendrites and improve the communication between the neurons (Figs. 11-13).

epapa-NH₂ 483.2 482.53 0-25% / 25 min 12.28 epppa-NH₂ 509.2 508.57 0-25% / 25 min 12.35 apape-N¾ 483.1 482.53 5-25% / 20 min 7.24 apppe-N¾ 509.20 508.57 0-25% / 25 min 12.34

RPAPA-NH2 510.13 509.60 0-25% / 25 min 12.08

KPAPA-NH2 482.07 481.59 0-25% / 25 min 10.29

RPPPA-NHa 534.49

KPPPA-NH2 508.15 507.63 0-25% / 25 min 10.88

DPAPA-NH2 468.93 468.5 0-20% / 20 min 11.89 β-Ala-PAPA- NH₂ 425.05 424.49 0-20% / 20 min 11.88

E-Sar-APA-NH₂ 456.94 456.49 0-20% / 20 min 11.36

EPA-Sar-A-NH₂ 456.95 456.49 0-20% / 20 min 10.83

E-Pip-APA-NH₂ 497.0 496.56 0-20% / 20 min 16.81

EPA-Pip-A-NH₂ 496.82 496.56 10-30% / 20 min 11.02

E-Sar-A-Sar-A- NH₂ 431.35 430.45 0-20% / 20 min 9.68 apapq 482.53 0-20% / 20 min 11.74 apapd-N¾ 469.00 468.50 0-20% / 20 min 15.69 apapn 468.85 468.50 0-20% / 20 min 11.05 apapq-NH₂ 482.41 481.55 0-20% / 20 min 10.58 apapn-NH₂ 467.97 467.52 0-20% / 20 min 10.39 apape 484.01 483.51 0-20% / 20 min 13.38 apGpe-NH₂ 469.49 0-20% / 20 min 13.37 ap-N-Me-ala-pe- NH₂ 497.06 497.54 0-20% / 20 min 13.39 a-tic-ape-NH₂ 8.86

545.18 545.57 15-35% / 20 min 9.38 apa-tic-e-NH₂ 11.77

545.25 545.57 15-35% / 20 min 11.26 ap-tic-pe-NH₂ 10.28

571.26 571.61 10-30% / 20 min 11.29 a-Sar-ape-NH₂ 456.95 456.49 0-20% / 20 min 8.49 apa-Sar-e-NH₂ 456.92 456.49 0-20% / 20 min 11.81 a-pip-ape-NH₂ 497.21 496.56 5-25% / 20 min 12.20 apa-pip-e-NH₂ 497.29 496.56 10-30% / 20 min 6.59

Gpape NH₂ 468.94 469.49 0-20% / 20 min 13.30 (N-Me-ala)-

62 pape-NH₂ 496.99 497.54 0-20% / 20 min 10.34

63 ap-Aib-pe-NH₂ 497.24 497.54 10-30% / 20 min 12.39

64 Aib-pape-NH₂ 497.24 497.54 0-20% / 20 min 6.75

65 APEPA-NH₂ 483.01 482.53 0-20% / 20 min 7.50

Table 2. MS and HPLC analytical parameters of the novel peptides

Table 3 . The neuroprotective effect of the novel peptides measured by MTT-assay in hippocampal slices

Table 4. Binding proteins for Αβ 1-42 identified by mass spectrometry in rat brain synaptic plasma membranes (Verdier et al. J Neurochem 94 (3) : 617-28 , 2005)

Claims

Claims

1. Peptides or peptidomimetics according to general formula I: X-A1-A2-A3-A4-A5-Y (I) wherein

X is absent or represents: acetyl, propionyl, succinyl group Al is absent or represents: Ala, Gly, Aib, Sar, N-methyl-Ala, a-methyl-Ala, Abu, norvaline, Asp, Glu, iminodiacetic acid, GABA, succinyl, Pro, acetyl, 2-amino-adipoic acid, propionyl group;

A2 is absent or represents: Pro, Tic, thiaproline, a-methyl-

Pro, γ-hidroxy-Pro, Sar, 4-fluoro-Pro, Pip, Nipecotic acid,

Glu, Asp, 2-carboxy-piperazine, succinyl, Glp, acetyl, GABA, propionyl group;

A3 is absent or represents: Ala, Gly, Aib, Sar, N-methyl-Ala, a-methyl-Ala, Abu, norvaline, Asp, Glu, Glp, acetyl, succinyl, GABA, propionyl group;

A4 is absent or represents: Pro, Tic, thiaproline, a-methyl- Pro, γ-hidroxy-Pro, Sar, 4-fluoro-Pro, Pip, Nipecotic acid,

Glu, Asp, 2-carboxy-piperazine, succinyl, Glp group;

A5 represents: Ala, Gly, Aib, Sar, N-methyl-Ala, a-methyl-Ala,

Abu, norvaline, Asp, Glu, iminodiacetic acid, GABA, 2- amino-adipoic acid, Pro;

Y is absent or represents: amide, N-methylamide, or N, N- dimethyl-amide;

provided that if A2 is absent than Al is also absent and if A3 is absent than Al and A2 are also absent, and if A4 is absent than Al, A2 and A3 are also absent;

and to their pharmaceutically acceptable salts and esters.

2. Peptides or peptidomimetics according to claim 1, where the peptides or peptidomimetics are selected from peptides with the following sequences: DP-amide (SEQ ID NO 1),

EP-amide (SEQ ID NO 2),

Suc-Pro-amide (SEQ ID NO 3),

Suc-PA-amide (SEQ ID NO 4),

Suc-pa-amide (SEQ ID NO 5),

Glp-Gly (SEQ ID NO 6),

Ac-Glp-G (SEQ ID NO 7),

glp-G (SEQ ID NO 8) ,

Glp-Asp-amide (SEQ ID NO 9),

glp-asp-amide (SEQ ID NO 10),

EPA-amide (SEQ ID NO 11),

Ac-EPA-amide (SEQ ID NO 12),

EPP-amide (SEQ ID NO 13),

ape-amide (SEQ ID NO 14),

Ac-ape-amide (SEQ ID NO 15),

GABA-pe-amide (SEQ ID NO 16),

DPA-amide (SEQ ID NO 17),

EPAP -amide (SEQ ID NO 18),

Ac-EPAP-amide (SEQ ID NO 19),

pape-amide (SEQ ID NO 20),

pppe-amide (SEQ ID NO 21),

Suc-PAPA-amide (SEQ ID NO 22),

Propionyl-PAPA-amide (SEQ ID NO 23),

Iminodiacetyl-PAPA-amide (SEQ ID NO 24),

Propionyl-pape-amide (SEQ ID NO 25),

Propionyl-papq-amide (SEQ ID NO 26),

apap-amide (SEQ ID NO 27),

EPAPA (SEQ ID NO 28) ,

EPPPA (SEQ ID NO 29) ,

APAPE-amide (SEQ ID NO 30), epapa-amide (SEQ ID NO 31), epppa -amide (SEQ ID NO 32),

apape -amide (SEQ ID NO 33),

apppe -amide (SEQ ID NO 34),

RPAPA -amide (SEQ ID NO 35),

KPAPA -amide (SEQ ID NO 36),

RPPPA -amide (SEQ ID NO 37),

KPPPA-amide (SEQ ID NO 38),

DPAPA-amide (SEQ ID NO 39),

β-Ala-PAPA-amide (SEQ ID NO 40),

E-Sar-APA-amide (SEQ ID NO 41),

EPA-Sar-A-amide (SEQ ID NO 42),

E-Pip-APA-amide (SEQ ID NO 43),

EPA-Pip-A-amide (SEQ ID NO 44),

E-Sar-A-Sar-A-amide (SEQ ID NO 45), apapq (SEQ ID NO 46) ,

apapd-amide (SEQ ID NO 47),

apapn (SEQ ID NO 48) ,

apapq-amide (SEQ ID NO 49),

apapn-amide (SEQ ID NO 50),

apape (SEQ ID NO 51) ,

apGpe-amide (SEQ ID NO 52),

ap-N-Me-ala-pe-amide (SEQ ID NO 53), a-tic-ape-amide (SEQ ID NO 54), apa-tic-e-amide (SEQ ID NO 55), ap-tic-pe-amide (SEQ ID NO 56), a-Sar-ape-amide (SEQ ID NO 57), apa-Sar-e-amide (SEQ ID NO 58), a-pip-ape-amide (SEQ ID NO 59), apa-pip-e-amide (SEQ ID NO 60),

Gpape amide (SEQ ID NO 61),

(N- e-ala) -pape-amide (SEQ ID NO 62), ap-Aib-pe-amide (SEQ ID NO 63),

Aib-pape-amide (SEQ ID NO 64), APEPA-amide (SEQ ID NO 65),

3. Peptides or peptidomimetics according to claim 2, where the peptides or peptidomimetics are selected from the following sequences :

EPP-amide (SEQ ID NO 13),

ape-amide (SEQ ID NO 14),

GABA-pe-amide (SEQ ID NO 16),

DPA-amide (SEQ ID NO 17),

pppe-amide (SEQ ID NO 21),

EPAPA (SEQ ID NO 28) ,

EPPPA (SEQ ID NO 29) ,

epppa-amide (SEQ ID NO 32),

apapq (SEQ ID NO 46) ,

apapn (SEQ ID NO 48) ,

apape (SEQ ID NO 51) and pharmaceutically acceptable salts and esters thereof.

4. Pharmaceutical preparation, which contains at least one peptide or peptidomimetic of the general formula (I)

wherein

X is absent or represents: acetyl, propionyl, succinyl group Al is absent or represents: Ala, Gly, Aib, Sar, N-methyl-Ala, a-methyl-Ala, Abu, norvaline, Asp, Glu, iminodiacetic acid, GABA, succinyl, Pro, acetyl, 2-amino-adipoic acid, propionyl group;

A2 is absent or represents: Pro, Tic, thiaproline, a-methyl- Pro, γ-hidroxy-Pro, Sar, 4-fluoro-Pro, Pip, Nipecotic acid, Glu, Asp, 2-carboxy-piperazine, succinyl, Glp, acetyl,

GABA, propionyl group;

A3 is absent or represents: Ala, Gly, Aib, Sar, N-methyl-Ala,

Oi-methyl-Ala, Abu, norvaline, Asp, Glu, Glp, acetyl, succinyl, GABA, propionyl group;

A4 is absent or represents: Pro, Tic, thiaproline, a-methyl-

Pro, γ-hidroxy-Pro, Sar, 4-fluoro-Pro, Pip, Nipecotic acid,

Glu, Asp, 2-carboxy-piperazine, succinyl, Glp group;

A5 represents: Ala, Gly, Aib, Sar, N-methyl-Ala, -methyl-Ala, Abu, norvaline, Asp, Glu, iminodiacetic acid, GABA, 2- amino-adipoic acid, Pro;

Y is absent or represents: amide, N-methylamide, or N, N- dimethyl-amide ;

provided that if A2 is absent than Al is also absent and if A3 is absent than Al and A2 are also absent, and if A4 is absent than Al, A2 and A3 are also absent;

and/or contains the pharmaceutically acceptable salt and ester of a peptide according to general formula (I) , and at least one pharmaceutically acceptable additive.

5. Pharmaceutical preparation according to claim 3, characterised by that at least one pharmaceutically acceptable additive is a matrix ensuring controlled active agent release.

6. Pharmaceutical preparation according to claim 3 or 4, characterised by that the peptide according to general formula (I) is selected from peptides or peptidomimetics with the following sequences: EPP-amide (SEQ ID NO 13),

ape-amide (SEQ ID NO 14),

GABA-pe-amide (SEQ ID NO 16),

DPA-amide (SEQ ID NO 17),

pppe-amide (SEQ ID NO 21), EPAPA (SEQ ID NO 28) ,

EPPPA (SEQ ID NO 29) ,

epppa-amide (SEQ ID NO 32),

apapq (SEQ ID NO 46) ,

apapn (SEQ ID NO 48) ,

apape (SEQ ID NO 51)

and pharmaceutically acceptable salts and esters thereof.

7. The use of peptides or peptidomimetics according to general formula (I)

X-A1-A2-A3-A4-A5-Y (I) wherein

X is absent or represents: acetyl, propionyl, succinyl group Al is absent or represents: Ala, Gly, Aib, Sar, N-methyl-Ala, a-methyl-Ala, Abu, norvaline, Asp, Glu, iminodiacetic acid, GABA, succinyl, Pro, acetyl, 2-amino-adipoic acid, propionyl group;

A2 is absent or represents: Pro, Tic, thiaproline, a-methyl- Pro, γ-hidroxy-Pro, Sar, 4-fluoro-Pro, Pip, Nipecotic acid, Glu, Asp, 2-carboxy-piperazine, succinyl, Glp, acetyl, GABA, propionyl group;

A3 is absent or represents: Ala, Gly, Aib, Sar, N-methyl-Ala, a-methyl-Ala, Abu, norvaline, Asp, Glu, Glp, acetyl, succinyl, GABA, propionyl group;

A4 is absent or represents: Pro, Tic, thiaproline, -methyl- Pro, γ-hidroxy-Pro, Sar, 4-fluoro-Pro, Pip, Nipecotic acid, Glu, Asp, 2-carboxy-piperazine, succinyl, Glp group;

A5 represents: Ala, Gly, Aib, Sar, N-methyl-Ala, a-methyl-Ala, Abu, norvaline, Asp, Glu, iminodiacetic acid, GABA, 2- amino-adipoic acid, Pro,

Y is absent or represents: amide, N-methylamide, or N, N- dimethyl-amide; provided that if A2 is absent than Al is also absent and if A3 is absent than Al and A2 are also absent, and if A4 is absent than Al, A2 and A3 are also absent;

and their pharmaceutically acceptable salt and ester in the manufacture of a pharmaceutical preparation useful for the prophylaxis or treatment of diseases that can be cured by protection against the detrimental effect of Αβ-peptides.

8. Use according to claim 7, where the disease that can be cured by protection against the detrimental effect of Αβ- peptides is selected from neurodegenerative diseases and type II diabetes mellitus.

9. Use according to claim 7, where the disease that can be cured by protection against the detrimental effect of Αβ- peptides is selected from the following: both familial and late-onset Alzheimer's disease, Parkinson's disease, Down syndrome, type II diabetes mellitus and amyloidosis.