The present invention relates to a peptide based compound and its use in a method of prophylactic or therapeutic treatment of at least one amyloidogenic disease or use in a method of non-invasive in vivo and/or in vitro diagnosis of at least one amyloidogenic (protein aggregation) disease.

Alzheimer's disease and type 2 Diabetes, also called Diabetes type 2, are two cell degenerative diseases and major representatives of the class of protein aggregation diseases (Westermark P. Aspects on human amyloid forms and their fibril polypeptides. FEBS J. 2005; 272: 5942-9; Dobson CM. Protein aggregation and its consequences for human disease. Protein Pept Lett. 2006; 13: 219-27).

The key amyloid forming polypeptide in Alzheimer's disease is the 40 to 42 residue polypeptide beta-amyloid peptide, also called Αβ or A- beta. This peptide is present in the blood and cerebrospinal fluid at low nanomolar concentrations and aggregates under conditions of Alzheimer's disease into cell toxic oligomers and amyloid fibrils in the brain. Formation of these cell toxic oligomers and of insoluble amyloid plaques is linked with neurodegeneration and Alzheimer's dis- ease pathogenesis.

In the case of type 2 Diabetes, insoluble amyloid plaques are present in the pancreata of more than 95% of the type 2 Diabetes patients. These amyloid deposits consist mainly of aggregates of the 37- residue islet amyloid polypeptide, abbreviated as IAPP. IAPP is syn- thesized and secreted from the beta-cells of pancreas together with insulin. In its soluble form it acts as a neuroendocrine regulator of glucose homeostasis. However, formation of IAPP amyloid aggregates in the pancreas is associated with pancreatic β-cell damage and type 2 Diabetes pathogenesis.

Mounting amounts of recent pathophysiological, clinical, and epide- miological evidence suggest that onset and pathogenesis of Alzheimer's disease and type 2 Diabetes are linked to each other. Epidemiological studies show, for example, that persons suffering from type 2 Diabetes have an increased risk of Alzheimer's disease and vice versa while based on a number of identified biochemical and pathological links, Alzheimer's disease has been suggested to be "type 3 diabetes" (O'Nuallain B, Williams AD, Westermark P, Wetzel R. Seeding specificity in amyloid growth induced by heterologous fibrils. J Biol Chem. 2004; 279: 17490-9; Yan LM, Velkova A, Tata- rek-Nossol M, Andreetto E, Kapurniotu A. IAPP mimic blocks Ab cy- totoxic self-assembly: cross-suppression of amyloid toxicity of Ab and IAPP suggests a molecular link between Alzheimer's disease and type II diabetes. Angew Chem Int Ed Engl. 2007; 46: 1246-52; Miklossy J, Qing H, Radenovic A, et al. Beta amyloid and hyper- phosphorylated tau deposits in the pancreas in type 2 diabetes. Neu- robiol Aging. 2010; 31 : 1503-15).

Furthermore, Alzheimer's disease and type 2 Diabetes appear to be linked also on the molecular level (O'Nuallain B, 2004, above; Yan LM, 2007, above; Miklossy J, above; Andreetto E, Yan LM, Tatarek- Nossol M, et al. Identification of hot regions of the Abeta-IAPP inter- action interface as high-affinity binding sites in both cross- and self- association. Angew Chem Int Ed Engl. 2010; 49: 3081 -5). In fact, Αβ and IAPP which are both confornnationally flexible but these highly amyloidogenic peptides have a sequence similarity of 25% and a sequence identity of 50%.

Alzheimer's disease and type 2 Diabetes are two yet incurable cell degenerative diseases. Protein misfolding and aggregation are be- lieved to be closely linked to their onset and pathogenesis. Although several compounds including low molecular weight (MW) organic compounds, peptides and antibodies have been reported to prevent aggregation and formation of cytotoxic assemblies of Αβ or IAPP in the past 20 years, only few of them have reached the stage of clini- cal studies and none of them is being currently therapeutically applicable (Citron M. Alzheimer's disease: strategies for disease modification. Nat Rev Drug Discov. 2010; 9: 387-98; Mullard A. Sting of Alzheimer's failures offset by upcoming prevention trials. Nat Rev Drug Discov. 2012; 1 1 : 657-60). Few years ago, a synthetic 37 residue polypeptide [(N-Me)G24, (N- Me)l26]-IAPP (IAPP-GI) has been developed, which is a soluble N- methylated IAPP analogue and IAPP receptor agonist and is able to bind both IAPP and Αβ with nanomolar affinity and to completely block cytotoxic self-assembly and fibril formation of both polypep- tides (Yan LM, 2007, above; Andreetto E, 2010, above; Yan LM, Ta- tarek-Nossol M, Velkova A, Kazantzis A, Kapurniotu A. Design of a mimic of nonamyloidogenic and bioactive human islet amyloid polypeptide (IAPP) as nanomolar affinity inhibitor of IAPP cytotoxic fibril- logenesis. Proc Natl Acad Sci USA. 2006; 103: 2046-51 ). IAPP-GI and other N-methylated analogues of full length IAPP (which have been described in Kapurniotu & Bernhagen, Patent application US 2010/0221240; DE 102004051014.8) are thus soluble IAPP receptor agonists and at the same time high affinity inhibitors of cytotoxic self-assembly of both Αβ and IAPP. Such IAPP mimics could thus find application in the treatment of both type 2 Diabetes and Alzheimer's disease. In fact, based on the favourable biological properties of IAPP, a designed soluble analogue of full length IAPP (Pramlintide or Symlin from Amylin Pharmaceuticals) has been approved by the FDA few years ago and is being used for the treatment of diabetes (Lebovitz HE. Adjunct therapy for type 1 diabetes melli- tus. Nat Rev Endocrinol 2010; 6: 326-34; Schmitz O, Brock B, Rungby J. Amylin agonists: a novel approach in the treatment of diabetes. Diabetes. 2004; 53s3: 233-8). However, as pramlintide is an analogue of the natively nonamyloidogenic rat IAPP sequence which does not or only weakly inhibits amyloid formation and cytotoxicity by IAPP or A-beta (Yan LM, PNAS 2006, Angew. Chem. Int. Ed. 2007, see above) it is not expected to be a potent inhibitor of the cytotoxic aggregation process of these two polypeptides.

As stated above, most of the compounds of the prior art have been tested as inappropriate for being used in treating Alzheimer's disease and/or type 2 Diabetes and can solely be produced in a compli- cated and time- and/or cost intensive way. In addition, all analogues of full length IAPP have certain bioactivity properties, for instance IAPP receptor agonists, which may have beneficial or unwanted properties in the context of a therapeutic application in both type 2 Diabetes and Alzheimer's disease or Alzheimer's disease alone. Ac- cordingly, there is still a need in compounds, preferably low molecular weight compounds, which can be used in prophylactic or therapeutic treatment of Alzheimer's disease and/or type 2 Diabetes. Thus, the technical problem underlying the present invention is to solve preferably the above-identified disadvantages and especially to provide sequences of synthetic peptides that are high affinity ligands of Αβ or lAPP or both and inhibit preferably aggregation or misfolding of Αβ and/or lAPP and cell degeneration by either Αβ or lAPP or both of them and which could therefore find application in the treatment and non-invasive in vivo and/or in vitro diagnosis of Alzheimer's disease, type 2 Diabetes or both diseases. Furthermore, the synthetic peptides should be easily producible and saving thereby cost and time.

Said technical problem has been solved by the teaching of the independent claims.

The present invention relates particularly to a peptide based compound for inhibiting aggregation, preferably fibrillogenesis, and/or toxicity of beta-amyloid peptide (Αβ) and/or islet amyloid polypeptide (lAPP), wherein the peptide based compound comprises a C-terminal end CO-R and an N-terminal end NH-R' and is represented by formula (I), which is SEQ ID NO: 2-IC-SEQ ID NO: 3, wherein R is selected from the group consisting of -OH, -NH ₂ , -NHR ¹ , -NR ¹ R ² and a solubilising tag, wherein R ¹ and R ² are the same or different and are selected from the group consisting of alkyl, haloalkyl, heteroalkyl, cycloalkyl, heter- ocycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, arylheteroalkyl, alkoxy, alkoxyalkyl and alkoxyaryl, wherein R' is selected from the group consisting of -H, -CO-R ³ and a solubilising tag, wherein R ³ is selected from the group consisting of alkyl, haloalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, arylheteroalkyl, alkoxy, alkoxyalkyl and alkoxyaryl, wherein IC (inhibitory core) is selected from the group consisting of 8-amino-3,6-dioxaoctanoic acid, aminodecanoic acid (Adc), ami- nomethylbenzoic acid (Amba), aminobenzoic acid (Aba), Amba-Aba and a sequence consisting of three units selected from the group consisting of 2-aminooctanoic acid (2-Aoc), diaminopropionic acid (Dap), alanine, valine, leucine, isoleucine, norleucine, phenylalanine, cyclohexylalanine (ChA), lysine, arginine, ornithine and proline, wherein the three units are the same or different, wherein aminomethylbenzoic acid (Amba) is preferably p- aminomethylbenzoic acid (Amba), wherein aminobenzoic acid (Aba) is preferably p-aminobenzoic acid (Aba), wherein diaminopropionic acid (Dap) is preferably 2,3- diaminopropionic acid (Dap), wherein aminodecanoic acid (Adc) is preferably 10-aminodecanoic acid (Adc), and wherein the SEQ ID NO: 2 and/or SEQ ID NO: 3 are non-modified or modified, preferably modified.

In accordance with the present invention "the sequence consisting of three units" is a sequence, wherein the units are covalently linked with each other, preferably is a tripeptide.

In accordance with the present invention SEQ ID No: 2 is covalently linked with the linker, preferably the inhibitory core, which is covalently linked with SEQ ID No: 3.

In strong contrast thereto, a peptide corresponding to a partial lAPP segment, namely IAPP(8-28), or its double N-methylated version IAPP(8-28)-GI is unable to inhibit Αβ or lAPP fibrillogenesis and cytotoxicity (Andreetto E, Yan LM, Caporale A, Kapurniotu A. Dissecting the role of single regions of an lAPP mimic and lAPP in inhibition of Abeta40 amyloid formation and cytotoxicity. ChemBioChem. 201 1 ; 12: 1313-22).

It has been surprisingly shown that the nature of the linker between the SEQ ID No: 2 and the SEQ ID No 3 determines the inhibitory effect on aggregation and cell toxicity of Αβ and lAPP.

Surprisingly, the modification of the lAPP regions IAPP(8-18) and IAPP(22-28) by replacing specific amino acid residues by N- methylated ones results in peptide based compounds with not only improved biophysical properties with regard to solubility, amyloido- genicity and cytotoxicity, but also to more potent inhibitory effects on Αβ or lAPP cytotoxic self-assembly and amyloidogesis than the non- N-methylated peptides. N-methylation improves preferably the inhibitory effects on lAPP or Αβ aggregation and toxicity. In addition, the N-methylated peptides have most likely an increased proteolytic stability towards serum proteases as compared to non-methylated ones which makes them promising candidates for therapeutics.

In addition, the preferred addition of solubilising tags to the N- and C- terminal sequence parts of the peptide based compounds leads to improved solubility properties and improved inhibitory potentials.

Surprisingly, the peptide based compounds are preferably able to bind with high affinity, preferably with a dissociation constant Kd smaller than 5 μΜ, Αβ or IAPP monomers and/or their fibrillar as- semblies. Their ability to bind Αβ or IAPP with high affinity is preferably independent of their effects on Αβ or IAPP aggregation and toxicity suggesting a conformational specificity in their interaction with Αβ or IAPP underlying their inhibitory effects on their aggregation and toxicity pathways. Therefore, the peptide based compounds are preferably promising compounds for the development of conformation specific ligands for the non-invasive detection of Αβ or IAPP assemblies.

The peptide based compounds are preferably nanomolar affinity ligands of both Αβ and IAPP binding both soluble Αβ and IAPP and amyloid aggregates thereof. Based on these properties they can be used as non-invasive tracers for the detection of various forms of Αβ and IAPP, namely their monomers, oligomers and/or amyloid plaques, in body fluids and/or tissues, after they are labelled.

The peptide based compounds can be easily synthesized, preferably in large scale, preferably via solid phase synthesis. Their preparation and purification is easier and significantly less expensive as compared to IAPP-GI, since their sequences are about half as long as the sequence of IAPP-GI or the usually high anti-amyloid MW (molecular weight) antibodies or affibodies.

The peptide based compounds are preferably soluble, nontoxic, nonamyloidogenic and nanomolar activity inhibitors of aggregation and toxicity of Αβ, IAPP, or both and are able to block aggregation and toxicity of both Αβ and IAPP in the nanomolar range.

In comparison to IAPP-GI (SEQ ID No: 54) the peptide based compounds according to the present invention a) are preferably significantly shorter which results in an easier and less expensive synthe- sis and purification and b) are preferably not bioactive - other than IAPP-GI and related IAPP mimics - as they do not preferably bind the IAPP receptor. The N- and C-termini of the IAPP (amino acids 1 - 7 and 29 to 37) which are crucial for binding to the IAPP receptor are preferably not present in the peptide based compounds. Accordingly, the peptide based compounds according to the present invention can be preferably applied as inhibitors of amyloid formation by Αβ or IAPP or both of them without exhibiting IAPP receptor agonistic activity.

The peptide based compounds are preferably promising candidates for the development of bifunctional peptide-based drugs targeting protein aggregation and related cell degeneration in both type 2 Diabetes and Alzheimer's disease.

In a preferred embodiment of the present invention the SEQ ID NO: 2 and/or the SEQ ID NO: 3 are preferably modified by at least one N- alkylation, preferably at least one N-methylation. Preferably, at least one amino acid of the peptide based compounds according to the present invention comprise at least one artificially introduced moiety selected from the group consisting of alkyl, haloalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylakyl, arylheteroalkyl, alkoxy, alkoxyalkyl and alkoxyaryl, wherein each moiety has 1 to 8 carbon atoms, preferably 3 to 6 carbon atoms. Preferably, the peptide based compounds according to the present invention are modified, wherein at least at one amino acid comprises at least one artificially introduced short moiety consisting of at least one carbon atom, at least one hydrogen atom and at least one het- ero atom, wherein the at least one hetero atom is selected from the group consisting of N, O and S, and the sum of the carbon atom(s) and the hetero atom(s) is between 1 to 10, preferably 2 to 8, preferably 3 to 7.

The term "X3", preferably used in the tables and the examples herein, is understood as sequence of three X, wherein X is an amino ac- id, preferably abbreviated with the one letter code, or another chemical compound used in the inhibitory core of the peptide based compound according to the present invention. For instance, R3 means Arg-Arg-Arg.

"A peptide based compound according to the present invention" is preferably a peptide based compound according to the present invention in combination with one or more preferred embodiments of the present invention.

With respect to the present invention the "peptide based compound" is a molecule comprising amino acids linked together through an am- ide bond, such as an oligopeptide, polypeptide or protein. The peptide based compound can comprise solely amino acids, either in D- configuration or L-configuration or both. The peptide based com- pound can also comprise in addition to naturally occurring amino acids artificially produced amino acids and/or other chemical compounds, especially chains, comprising at least one amino group and at least one carboxylic group. The present invention relates in a preferred embodiment to a peptide based compound according to the present invention, wherein the solubilising tag consists of at least three, preferably exactly three solubilising amino acids.

A "solubilising amino acid" is an amino acid having a charged side chain at neutral pH, preferably pH = 7.

The present invention relates in a preferred embodiment to a peptide based compound according to the present invention, wherein the at least three solubilising amino acids are selected from the group consisting of lysine, ornithine, aspartic acid, arginine and glutamic acid. The present invention relates in a preferred embodiment to a peptide based compound according to the present invention, wherein at least one amino acid of the peptide based compound is N-methylated, that is amide bond N-methylated.

The present invention relates in a preferred embodiment to a peptide based compound according to the present invention, wherein the at least one N-methylated amino acid is selected from the group consisting of Leucine-12, Alanine-13, Leucine-16, Valine-17, Glycine-24 and lsoleucine-26 (numbers corresponding to amino acid positions of the full length IAPP protein represented by SEQ ID NO: 1 ). The present invention relates in a preferred embodiment to a peptide based compound according to the present invention, wherein the two N-methylated amino acids are Glycine-24 and lsoleucine-26 (numbers corresponding to amino acid positions of the full length IAPP protein represented by SEQ ID NO: 1 ).

The present invention relates in a preferred embodiment to a peptide based compound according to the present invention, wherein the four N-methylated amino acids are Leucine-12, Alanine-13, Leucine- 16 and Valine-17 (numbers corresponding to amino acid positions of the full length IAPP protein represented by SEQ ID NO: 1 ).

The present invention relates in a preferred embodiment to a peptide based compound according to the present invention, wherein the four N-methylated amino acids are Leucine-12, Leucine-16, Glycine- 24 and lsoleucine-26 (numbers corresponding to amino acid posi- tions of the full length IAPP protein represented by SEQ ID NO: 1 ).

The present invention relates in a preferred embodiment to a peptide based compound according to the present invention, wherein the six N-methylated amino acids are Leucine-12, Alanine-13, Leucine-16, Valine-17, Glycine-24 and lsoleucine-26 (numbers corresponding to amino acid positions of the full length IAPP protein represented by SEQ ID NO: 1 ).

The present invention relates in a preferred embodiment to a peptide based compound according to the present invention, wherein the inhibitory core is selected from the group consisting of Val-Val-Val, Leu-Leu-Leu, lle-lle-lle, Nle-Nle-Nle, (2-Aoc)-(2-Aoc)-(2-Aoc), Phe- Phe-Phe, ChA-ChA-ChA, Dap-Dap-Dap, Lys-Lys-Lys, Orn-Orn-Orn, Arg-Arg-Arg, Ala-Ala-Ala, Pro-Pro-Pro, Adc and Amba-Aba. In a preferred embodiment the inhibitory core is selected from the group consisting of Val-Val-Val, Leu-Leu-Leu, lle-lle-lle, Nle-Nle-Nle, (2-Aoc)-(2-Aoc)-(2-Aoc), Phe-Phe-Phe, ChA-ChA-ChA, Dap-Dap- Dap, Arg-Arg-Arg and Ala-Ala-Ala. The present invention relates in a preferred embodiment to a peptide based compound according to the present invention, wherein the peptide based compound is selected from the group consisting of the peptides represented by SEQ ID NOs: 4 to 53, preferably by SEQ ID NOs 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 38, 40, 41 , 47 and 48. The present invention relates particularly to a peptide based compound according to the present invention for use in a method of prophylactic or therapeutic treatment of at least one amyloidogenic disease, preferably Alzheimer's diseases and/or Diabetes type 2. The amyloidogenic disease is a protein aggregation disease. The present invention relates in a preferred embodiment to a peptide based compound according to the present invention for use in a method according to the present invention, wherein the at least one amyloidogenic disease is Alzheimer's disease or type 2 Diabetes.

The present invention relates in a preferred embodiment to a peptide based compound according to the present invention for use in a method of prophylactic or therapeutic treatment of both Alzheimer's disease and type 2 Diabetes in a patient in need thereof.

The present invention relates especially to a peptide based compound according to the present invention for use in a method of non- invasive in vivo and/or in vitro diagnosis of at least one amyloidogenic disease, preferably Alzheimer's diseases and/or Diabetes type 2, wherein the peptide based compound comprises a C-terminal end CO-R and an N-terminal end NH-R' and is represented by formula (I), which is

SEQ ID NO: 2-LINK-SEQ ID NO: 3, wherein R is selected from the group consisting of -OH, -NH ₂ , -NHR ¹ , -NR ¹ R ² and a solubilising tag, wherein R ¹ and R ² are the same or different and are selected from the group consisting of alkyl, haloalkyi, heteroalkyl, cycloalkyl, heter- ocycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, arylheteroalkyl, alkoxy, alkoxyalkyl and alkoxyaryl, wherein R' is selected from the group consisting of -H, -CO-R ³ and a solubilising tag, wherein R ³ is selected from the group consisting of alkyl, haloalkyi, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, arylheteroalkyl, alkoxy, alkoxyalkyl and alkoxyaryl, wherein LINK (linker) is selected from the group consisting of 8- amino-3,6-dioxaoctanoic acid, 8-aminooctanoic acid (Aoc), ami- nodecanoic acid (Adc), aminomethylbenzoic acid (Amba), amino- benzoic acid (Aba), Amba-Aba and a sequence consisting of three units selected from the group consisting of 2-aminooctanoic acid (2- Aoc), diaminopropionic acid (Dap), glycine, acetylated lysine, alanine, valine, leucine, isoleucine, norleucine, phenylalanine, cyclohex- ylalanine (ChA), lysine, arginine, ornithine and proline, wherein the three units are the same or different, and wherein the SEQ ID NO: 2 and/or SEQ ID NO: 3 are non-modified or modified, preferably modified.

The present invention relates in a preferred embodiment to a peptide based compound according to the present invention for use in a method of non-invasive in vivo and/or in vitro diagnosis of at least one amyloidogenic disease, preferably Alzheimer's diseases and/or Diabetes type 2, wherein the peptide based compound comprises a C-terminal end CO-R and an N-terminal end NH-R' and is represented by formula (I), which is SEQ ID NO: 2-IC-SEQ ID NO: 3, wherein R is selected from the group consisting of -OH, -NH ₂ , -NHR ¹ , -NR ¹ R ² and a solubilising tag, wherein R ¹ and R ² are the same or different and are selected from the group consisting of alkyl, haloalkyl, heteroalkyl, cycloalkyl, heter- ocycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, arylheteroalkyl, alkoxy, alkoxyalkyl and alkoxyaryl, wherein R' is selected from the group consisting of -H, -CO-R ³ and a solubilising tag, wherein R ³ is selected from the group consisting of alkyl, haloalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, arylheteroalkyl, alkoxy, alkoxyalkyl and alkoxyaryl, wherein IC (inhibitory core) is selected from the group consisting of 8-amino-3,6-dioxaoctanoic acid, aminodecanoic acid (Adc), ami- nomethylbenzoic acid (Amba), aminobenzoic acid (Aba), Amba-Aba and a sequence consisting of three units selected from the group consisting of 2-aminooctanoic acid (2-Aoc), diaminopropionic acid (Dap), alanine, valine, leucine, isoleucine, norleucine, phenylalanine, cyclohexylalanine (ChA), lysine, arginine, ornithine and proline, wherein the three units are the same or different, and wherein the SEQ ID NO: 2 and/or SEQ ID NO: 3 are non-modified or modified, preferably modified.

The present invention relates in a preferred embodiment to a peptide based compound according to the present invention for use in a method of non-invasive in vivo and/or in vitro diagnosis of both Alzheimer's disease and type 2 Diabetes.

The peptide based compound according to the present invention acts preferably as conformation-specific ligand in the method of noninvasive in vivo and/or in vitro diagnosis of at least one amyloidogen- ic disease, preferably Alzheimer's diseases and/or Diabetes type 2.

The present invention relates in a preferred embodiment to a peptide based compound according to the present invention for use in a method according to the present invention, wherein the peptide based compound is labelled or derivatized in a suitable manner for use in an in vitro or non-invasive in vivo diagnosis system for at least one amyloidogenic disease, preferably Alzheimer's disease and/or type 2 Diabetes.

The peptide based compound influences preferably peptides which form amyloid or amyloidogenic structures with respect to their ability to aggregate or to misfold, preferably inhibits, reduces or blocks the aggregation and/or misfolding of said peptides. In a particularly pre- ferred embodiment of the present invention the lag phase of aggregation is extended in comparison with the lag phase of naturally oc- curing amyloidogenic peptides by a factor of at least 1 .1 , preferably of at least 1 .5, preferably of at least 2, preferably of at least 10, pref- erably of at least 20, preferably of at least 100.

In a preferred embodiment the peptide based compound is represented by the formula (I) SEQ ID NO: 2-Val-Val-Val-SEQ ID NO: 3, wherein Gly-15 and lle-17 are preferably N-methylated.

In a preferred embodiment the peptide based compound is repre- sented by the formula (I) SEQ ID NO: 2-Leu-Leu-Leu-SEQ ID NO: 3, wherein Gly-15 and lle-17 are preferably N-methylated.

In a preferred embodiment the peptide based compound is represented by the formula (I) SEQ ID NO: 2-Nle-Nle-Nle-SEQ ID NO: 3, wherein Gly-15 and lle-17 are preferably N-methylated. In a preferred embodiment the peptide based compound is represented by the formula (I) SEQ ID NO: 2-(2-Aoc)-(2-Aoc)-(2-Aoc)- SEQ ID NO: 3, wherein 2-Aoc is 2-aminooctanoic acid and wherein Gly-15 and lle-17 are preferably N-methylated.

In a preferred embodiment the peptide based compound is repre- sented by the formula (I) SEQ ID NO: 2-Phe-Phe-Phe-SEQ ID NO: 3, wherein Gly-15 and lle-17 are preferably N-methylated.

In a preferred embodiment the peptide based compound is represented by the formula (I) SEQ ID NO: 2-ChA-ChA-ChA-SEQ ID NO: 3, wherein Gly-15 and lle-17 are preferably N-methylated. In a preferred embodiment the peptide based compound is represented by the formula (I) SEQ ID NO: 2-Dap-Dap-Dap-SEQ ID NO: 3, wherein Dap is 2,3-diaminopropionic acid and wherein Gly-15 and lle-17 are preferably N-methylated. In a preferred embodiment the peptide based compound is represented by the formula (I) SEQ ID NO: 2-Arg-Arg-Arg-SEQ ID NO: 3, wherein Gly-15 and lle-17 are preferably N-methylated.

In a preferred embodiment the peptide based compound is represented by the formula (I) SEQ ID NO: 2-lle-lle-lle-SEQ ID NO: 3, wherein Gly-15 and lle-17 are preferably N-methylated.

In a preferred embodiment the peptide based compound is represented by the formula (I) Lys-Lys-Lys-SEQ ID NO: 2-Ala-Ala-Ala- SEQ ID NO: 3-Lys-Lys-Lys, wherein Gly-18 and lle-20 are preferably N-methylated. In a preferred embodiment the peptide based compound is represented by the formula (I) Lys-Lys-Lys-SEQ ID NO: 2-Leu-Leu-Leu- SEQ ID NO: 3-Lys-Lys-Lys, wherein Gly-18 and lle-20 are preferably N-methylated.

In a preferred embodiment the peptide based compound is repre- sented by the formula (I) SEQ ID NO: 2-Ala-Ala-Ala-SEQ ID NO: 3, wherein Leu-5, Ala-6, Leu-9 Val-10, Gly-15 and lle-17 are preferably N-methylated.

In a preferred embodiment the peptide based compound, especially the above identified and individualised peptide based compounds, comprises a C-terminal end CO-R and a N-terminal end NH-R', wherein R is selected from the group consisting of -OH, -NH ₂ , -NHR ¹ , -NR ¹ R ² and a solubilising tag, wherein R ¹ and R ² are the same or different and are selected from the group consisting of alkyl, haloalkyi, heteroalkyl, cycloalkyl, heter- ocycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, arylheteroalkyl, alkoxy, alkoxyalkyl and alkoxyaryl, wherein R' is selected from the group consisting of -H, -CO-R ³ and a solubilising tag, wherein R ³ is selected from the group consisting of alkyl, haloalkyi, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, arylheteroalkyl, alkoxy, alkoxyalkyl and alkoxyaryl.

In a preferred embodiment the peptide based compound, especially the above identified and individualised peptide based compounds, comprises a C-terminal end CO-R and a N-terminal end NH-R', wherein R is selected the group consisting of -OH, -NH ₂ , -NHR ¹ , -NR ¹ R ² and a solubilising tag, wherein R ¹ and R ² are the same or different and are selected from the group consisting of alkyl, haloalkyi, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, arylheteroalkyl, alkoxy, alkoxyalkyl and alkoxyaryl, wherein R' is selected from the group consisting of -H, -CO-R ³ and a solubilising tag, wherein R ³ is selected from the group consisting of alkyl, haloalkyi, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, arylheteroalkyl, alkoxy, alkoxyalkyl and alkoxyaryl and wherein the solubilising tag is selected from the group of peptides consisting of two to five amino acids, preferably three amino acids, which can be the same or different, preferably the same and are preferably selected from the group consisting of Lys, Orn, Asp, Arg and Glu.

In a preferred embodiment the peptide based compound, especially the above identified and individualised peptide based compounds, comprises a C-terminal end CO-R and a N-terminal end NH-R', wherein R is selected from the group consisting of -OH, -NH ₂ , -NHR ¹ , -NR ¹ R ² and a solubilising tag, wherein R ¹ and R ² are the same or different and are selected from the group consisting of C1 to C6 alkyl, C1 to C6 haloalkyl, C1 to C6 heteroalkyl, C3 to C6 cycloalkyl, C3 to C6 heterocycloalkyl, C3 to C6 aryl, C3 to C6 heteroaryl, C3 to C6 arylalkyl, C3 to C6 heteroaryl C1 to C6 alkyl, C3 to C6 arylhetero C1 to C6 alkyl, C1 to C6 alkoxy, C1 to C6 alkoxyalkyl and C1 to C6 alkoxy C3 to C6 aryl, wherein R' is selected from the group consisting of -H, -CO-R ³ and a solubilising tag, wherein R ³ is selected from the group consisting of C1 to C6 alkyl, C1 to C6 haloalkyl, C1 to C6 heteroalkyl, C3 to C6 cycloalkyl, C3 to C6 heterocycloalkyl, C3 to C6 aryl, C3 to C6 heteroaryl, C3 to C6 arylalkyl, C3 to C6 heteroaryl C1 to C6 alkyl, C3 to C6 arylhetero C1 to C6 alkyl, C1 to C6 alkoxy, C1 to C6 alkoxyalkyl and C1 to C6 alkoxy C3 to C6 aryl, wherein the at least one, preferably exactly one, heteroatom is selected from O, N and S or a combination thereof, and wherein the solubilising tag is selected from the group of peptides consisting of two to five amino acids, preferably three amino acids, which can be the same or different, preferably the same, and are preferably selected from the group consisting of Lys, Orn, Asp, Arg and Glu, preferably Lys and Glu.

In a preferred embodiment the peptide based compound, especially the above identified and individualised peptide based compounds, comprises a C-terminal end CO-R and a N-terminal end NH-R', wherein R is selected from the consisting of -OH, -NH ₂ , -NHR ¹ , -NR ¹ R ² and a solubilising tag, wherein R ¹ and R ² are the same or different and are selected from the group consisting of C1 to C6 alkyl, preferably methyl, wherein R' is selected from the group consisting of -H, -CO-R ³ and a solubilising tag, wherein R ³ is selected from the group consisting of C1 to C6 alkyl, preferably methyl, and wherein the solubilising tag is selected from the group of peptides consisting of three amino acids, which can be the same or different, preferably the same, and are selected from the group consisting of Lys, Orn, Asp, Arg and Glu, preferably Lys and Glu. In a preferred embodiment the peptide based compound according to the present invention, preferably SEQ ID NO: 2-(2-Aoc)-(2-Aoc)- (2-Aoc)-SEQ ID NO: 3, wherein 2-Aoc is 2-aminooctanoic acid, wherein Gly-15 and lle-17 are preferably N-methylated, and/or SEQ ID NO: 2-Ala-Ala-Ala-SEQ ID NO: 3, wherein Leu-5, Ala-6, Leu-9 Val-10, Gly-15 and lle-17 are preferably N-methylated, inhibits, preferably strongly, the IAPP aggregation and toxicity.

In a preferred embodiment the peptide based compound according to the present invention, preferably selected from SEQ ID NO: 2-Arg- Arg-Arg-SEQ ID NO: 3, SEQ ID NO: 2-lle-lle-lle-SEQ ID NO: 3, SEQ ID NO: 2-Phe-Phe-Phe-SEQ ID NO: 3, SEQ ID NO: 2-Val-Val-Val- SEQ ID NO: 3, SEQ ID NO: 2-Leu-Leu-Leu-SEQ ID NO: 3 and Lys- Lys-Lys-SEQ ID NO: 2-Leu-Leu-Leu-SEQ ID NO: 3-Lys-Lys-Lys, wherein Gly-22 and lle-24 are preferably N-methylated (numbers corresponding to amino acid positions of the full length IAPP protein represented by SEQ ID NO: 1 ), inhibits, preferably strongly, the Αβ aggregation and toxicity.

In a preferred embodiment the peptide based compound according to the present invention, preferably selected from SEQ ID NO: 2-Nle- Nle-Nle-SEQ ID NO: 3, SEQ ID NO: 2-ChA-ChA-ChA-SEQ ID NO: 3, SEQ ID NO: 2-Leu-Leu-Leu-SEQ ID NO: 3 and Lys-Lys-Lys-SEQ ID NO: 2-Ala-Ala-Ala-SEQ ID NO: 3-Lys-Lys-Lys, wherein Gly-22 and lle-24 are preferably N-methylated (numbers corresponding to amino acid positions of the full length IAPP protein represented by SEQ ID NO: 1 ), inhibits, preferably strongly, both the IAPP and Αβ aggregation and toxicity. In a preferred embodiment at least one peptide based compound, preferably a mixture of at least two peptide based compounds, preferably exactly one peptide based compound, according to the present invention, preferably selected from SEQ ID NO: 2-Ala-Ala-Ala- SEQ ID NO: 3, wherein Leu-5, Ala-6, Leu-9 Val-10, Gly-15 and lle-17 are preferably N-methylated, and SEQ ID NO: 2-(2-Aoc)-(2-Aoc)-(2- Aoc)-SEQ ID NO: 3, wherein 2-Aoc is 2-amino-octanoic acid and wherein Gly-15 and lle-17 are preferably N-methylated, is used in a method of prophylactic or therapeutic treatment of Diabetes type 2, especially for inhibiting at least partially, preferably completely, the aggregation and/or misfolding of the islet amyloid polypeptide, preferably of SEQ ID NO: 1 . By inhibiting said aggregation and/or misfolding pancreatic beta-cell damage and/or Diabetes type 2 pathogenesis is preferably prevented at least partially, preferably com- pletely.

In a preferred embodiment at least one peptide based compound, preferably a mixture of at least two peptide based compounds, preferably exactly one peptide based compound, according to the present invention, preferably selected from SEQ ID NO: 2-Arg-Arg-Arg- SEQ ID NO: 3, SEQ ID NO: 2-Phe-Phe-Phe-SEQ ID NO: 3, SEQ ID NO: 2-Val-Val-Val-SEQ ID NO: 3, SEQ ID NO: 2-Leu-Leu-Leu-SEQ ID NO: 3 and Lys-Lys-Lys-SEQ ID NO: 2-Leu-Leu-Leu-SEQ ID NO: 3-Lys-Lys-Lys, wherein Gly-24 and lle-26 are preferably N- methylated (numbers corresponding to amino acid positions of the full length IAPP protein represented by SEQ ID NO: 1 ), is used in a method of prophylactic or therapeutic treatment of Diabetes type 2, especially for inhibiting at least partially, preferably completely, the aggregation and/or misfolding of islet amyloid polypeptide, preferably of SEQ ID NO: 1 . By inhibiting said aggregation and/or misfolding pancreatic beta-cell damage and/or Diabetes type 2 pathogenesis is preferably prevented at least partially, preferably completely.

In a preferred embodiment at least one peptide based compound, preferably a mixture of at least two peptide based compounds, pref- erably exactly one peptide based compound, according to the present invention, preferably selected from SEQ ID NO: 2-Nle-Nle-Nle- SEQ ID NO: 3, SEQ ID NO: 2-ChA-ChA-ChA-SEQ ID NO: 3, SEQ ID NO: 2-Leu-Leu-Leu-SEQ ID NO: 3 or Lys-Lys-Lys-SEQ ID NO: 2- Ala-Ala-Ala-SEQ ID NO: 3-Lys-Lys-Lys, wherein Gly-24 and lle-26 are preferably N-methylated (numbers corresponding to amino acid positions of the full length IAPP protein represented by SEQ ID NO: 1 ), is used in a method of prophylactic or therapeutic treatment of Alzheimer's disease and Diabetes type 2, especially for inhibiting at least partially, preferably completely, the aggregation and/or misfold- ing of beta-amyloid peptide and islet amyloid polypeptide, preferably of SEQ ID NOs: 1 , 55 and 56. By inhibiting said aggregation and/or misfolding forming of cell toxic oligomers and amyloid fibrils, preferably in the brain, as well as pancreatic beta-cell damage and/or Diabetes type 2 pathogenesis are preferably prevented at least partially, preferably completely.

In a preferred embodiment at least one peptide based compound, preferably a mixture of at least two peptide based compounds, preferably exactly one peptide based compound, according to the present invention is used in a method of prophylactic or therapeutic treatment of Alzheimer's disease, especially for inhibiting at least partially, preferably completely, the aggregation and/or misfolding of beta-amyloid peptide, preferably of SEQ ID NOs: 55 and/or 56. By inhibiting said aggregation and/or misfolding forming of cell toxic oli- gomers and amyloid fibrils, preferably in blood and cerebrospinal fluid, is preferably prevented at least partially, preferably completely.

In a preferred embodiment at least one peptide based compound, preferably a mixture of at least two peptide based compounds, pref- erably exactly one peptide based compound, according to the present invention is used in a method of prophylactic or therapeutic treatment of Diabetes type 2, especially for inhibiting at least partially, preferably completely, the aggregation and/or misfolding of islet amyloid polypeptide, preferably of SEQ ID NO: 1 . By inhibiting said aggregation and/or misfolding pancreatic beta-cell damage and/or Diabetes type 2 pathogenesis is preferably prevented at least partially, preferably completely.

In a preferred embodiment at least one peptide based compound, preferably a mixture of at least two peptide based compounds, pref- erably exactly one peptide based compound, according to the present invention is used in a method of prophylactic or therapeutic treatment of Alzheimer's disease and Diabetes type 2, especially for inhibiting at least partially, preferably completely, the aggregation and/or misfolding of beta-amyloid peptide and islet amyloid polypep- tide, preferably of SEQ ID NOs: 1 and 55 and/or 1 and 56. By inhibiting said aggregation and/or misfolding forming of cell toxic oligomers and amyloid fibrils, preferably in the brain, as well as pancreatic beta-cell damage and/or Diabetes type 2 pathogenesis are preferably prevented at least partially, preferably completely. The peptide based compound according to the present invention is preferably a highly potent inhibitor of cytotoxic self-assembly and/or fibrillogenesis of Αβ, lAPP, or both Αβ and lAPP, wherein lAPP is preferably represented by SEQ ID NO: 1 and Αβ by SEQ ID NO: 55 and/or 56. The peptide based compound according to the present invention is preferably a high affinity ligand of Αβ, IAPP, or both Αβ and IAPP, wherein IAPP is preferably represented by SEQ ID NO: 1 and Αβ by SEQ ID NO: 55 and/or 56.

The peptide based compound is preferably produced by using the IAPP "hot spot regions" of the ΙΑΡΡ-Αβ cross-interaction interface SEQ ID NO:2, that is IAPP(8-18), and SEQ ID NO:3, that is IAPP(22- 28), as molecular templates (see Figure 1 ). These hot regions are covalently linked to each other by applying various different inhibitory cores, abbreviated as IC.

In a preferred embodiment at least one, preferably exactly one, preferably a mixture of peptide based compound(s) is used in a method of non-invasive in vivo and/or in vitro diagnosis of an amyloidogenic disease, preferably Alzheimer's disease or Diabetes type 2, by binding with high affinity to beta-amyloid peptide and islet amyloid polypeptide, preferably to at least one polypeptide selected from SEQ ID NOs: 1 , 55 and 56, preferably in their monomer, oligomer or amyloid fibril state. The method of non-invasive in vivo and/or in vitro diagno- sis of an amyloidogenic disease is preferably a non-invasive in vivo diagnosis. Preferably said non-invasive in vivo and/or in vitro diagnosis, construed as assay system, can be used for quantification of beta-amyloid peptide and islet amyloid polypeptide, preferably of at least one polypeptide selected from SEQ ID NO: 1 , 55 and 56, pref- erably in their monomer, oligomer or amyloid fibril state.

In a preferred embodiment of the present invention the peptide based compounds are used in a method of non-invasive in vivo and/or in-vitro diagnosis of at least one amyloidogenic disease, wherein the method of in-vitro diagnosis is preferably ELISA (Enzyme Linked Immunosorbent Assay) or RIA (Radioimmunoassay). In said methods the peptide based compound is used together prefera- bly with a marker, preferably with a spin label or fluorescence or luminescence marker, especially in an N-terminal-label, preferably in an N-terminal biotinylated, form or N-terminal fluorescein-labelled form or labelled with another fluorescent marker. The method of noninvasive in vivo diagnosis is preferably a PET (Positron Emission Tomography) und SPECT (Single Photon Emission Computed Tomography) based detection of aggregate forms of Αβ or IAPP associated with Alzheimer's disease and type 2 Diabetes pathogenesis. In said methods the peptide based compound is preferably labelled, preferably N-terminal, with a marker for PET which can be the mark- er 1 ,4,7,10-tetraazacyclododecane-1 ,4,7,10-tetraacetic acid (DOTA). The DOTA comprises preferably additionally a metal ion, preferably gallium.

In a preferred embodiment of the present invention the peptide based compound comprises at least one amino acid being N- methylated. An N-methylated amino acid is an amino acid in which a hydrogen atom of the a-amino group of the amino acid is replaced by a methyl group.

In a preferred embodiment of the present invention the peptide based compound are labelled in particular at the N-terminal a-amino group, in particular with an acetyl group, a radioactive marker, an enzyme marker, fluorescent marker, luminescent marker or a spin label, a marker for PET or SPECT, preferably in such a way that said marker is joined to the peptide based compound by means of a spacer, which is preferably an amino acid.

In a preferred embodiment of the present invention the peptide based compound is selected from the group consisting of the com- pounds in table 1 and/or 2.

Table 1

24 ATQRLANFLVH-/4c/c-NFGAILS Add

25 ATQRLANFLVH-y¾mba-y½a-NFGAILS Amba-Aba

Table 2

42 Glu-Glu-Glu-ATQRLMiFLV -Ala-Ala-Ala- E3-A3-K3-GI NF(N-Me)GA(N-Me)l LS-Lys-Z-ys-Z-ys

43 Glu-Glu-Glu-ATQRLMiFLV -Ala-Ala-Ala- E3-A3-E3-GI

NF(N-Me)GA(N-Me)ILS-G/tv-G/tv-G/tv

44 ATQRLANFLVH-Pro-Pro-Pro-NF(N-Me)GA(N- P3-GI

Me)ILS

45 ATQRLANFLVH-/\oc-NF(N-Me)GA(N-Me)ILS Aoc-GI

46 ATQRLANFLVH-yWc-NF(N-Me)GA(N-Me)ILS Adc-GI

47 ATQRLANFLVH-/\mba-/\ba-NF(N-Me)GA(N- Amba-Aba- Me)ILS Gl

48 ATQR(N-Me)L(N-Me)ANF(N-Me)L(N-Me)VH- 4-N-Me-A3- Ala-Ala-Ala-NF(N-Me)GA(N-Me)ILS Gl

49 ATQR(N-Me)LANF(N-Me)LVH-Ala-Ala-Ala- 2-N-Me-A3- NF(N-Me)GA(N-Me)ILS Gl

50 ATQR(N-Me)L(N-Me)ANF(N-Me)L(N-Me)VH- 4-N-Me-L3- Leu-Leu-Leu-NF(N-Me)GA(N-Me)ILS Gl

51 ATQR(N-Me)LANF(N-Me)LVH-Leu-Leu-Leu- 2-N-Me-L3- NF(N-Me)GA(N-Me)ILS Gl

52 ATQR(N-Me)L(N-Me)ANF(N-Me)L(N-Me)VH- 4-N-Me-L3

Leu-Leu-Leu-NFGAILS

53 ATQR(N-Me)L(N-Me)ANF(N-Me)L(N-Me)VH- 4-N-Me-A3

Ala-Ala-Ala-N FGAI LS

Further preferred embodiments are the subject-matter of the subclaims.

The present invention is illustrated by the figures and examples, below. Figure 1 shows primary structures of Αβ and lAPP. The regions IAPP(8-18) and IAPP(22-28) are the lAPP regions which have been identified as hot regions of the ΙΑΡΡ-Αβ (Αβ40 or Αβ42) interaction interface.

Figure 2 shows in the upper part sequences of lAPP and IAPP-GI which is the potent inhibitor of lAPP and Αβ40 or Αβ42 aggregation and toxicity and in the lower part an inhibitor design concept, basic structures, and classes of designed ligands and inhibitors of aggregation and toxicity of IAPP and Αβ (see also Tables 1 , 2).

Figure 3 shows the results of assays reporting on kinetics of formation of amyloid fibrils via the amyloid specific ThT binding assay (Figure 3A) and cytotoxic aggregates via the MTT reduction assay in aged (0 h - 7 days) solutions of Αβ alone or mixtures of Αβ with each peptide based compound as indicated. Cytotoxicity of the aged solutions of Αβ alone and the mixtures with each of the peptide based compounds was determined at the incubation time point of 72 h (Figure 3B) or 7 days (Figure 3C). The presented data is means (±SEM (scanning electron microscope)) of 3 assays (performed in triplicates).

Figure 4 shows the results of assays reporting on kinetics of formation of amyloid fibrils via the amyloid specific ThT binding assay (Figure 4A) and cytotoxic aggregates via the MTT reduction assay in aged (0 h - 7 days) solutions of Αβ alone or mixtures of Αβ with each of the peptide based compounds as indicated. Cytotoxicity of the aged solutions of Αβ alone and the mixtures with each of the peptide based compounds was determined at the incubation time point of 72 h (Figure 4B) or 7 days (Figure 4C). The presented data is means (±SEM) of 3 assays (performed in triplicates).

Figure 5 shows the results of assays reporting on kinetics of formation of amyloid fibrils via the amyloid specific ThT binding assay (Figure 5A) and cytotoxic aggregates via the MTT reduction assay in aged (0 h - 7 days) solutions of Αβ alone or mixtures of Αβ with each of the peptide based compounds as indicated. Cytotoxicity of the aged solutions of Αβ alone and the mixtures with each of the peptide based compounds was determined at the incubation time point of 72 h (Figure 5B) or 7 days (Figure 5C). The presented data is means (±SEM) of 3 assays (performed in triplicates).

Figure 6 shows the results of assays reporting on kinetics of for- mation of amyloid fibrils via the amyloid specific ThT binding assay (Figure 6A) and cytotoxic aggregates via the MTT reduction assay in aged (0 h - 7 days) solutions of IAPP alone or mixtures of IAPP with each of the peptide based compounds as indicated. Cytotoxicity of the aged solutions of IAPP alone and the mixtures with each of the peptide based compounds was determined at the incubation time point of 24 h (Figure 6B) or 7 days (Figure 6C). The presented data is means (±SEM) of 3 assays (performed in triplicates).

Figure 7 shows the results of assays reporting on kinetics of formation of amyloid fibrils via the amyloid specific ThT binding assay (Figure 7A) and cytotoxic aggregates via the MTT reduction assay in aged (0 h - 7 days) solutions of IAPP alone or mixtures of IAPP with each of the peptide based compounds as indicated. Cytotoxicity of the aged solutions of IAPP alone and the mixtures with each of the peptide based compounds was determined at the incubation time point of 24 h (Figure 7B) or 7 days (Figure 7C). The presented data is means (±SEM) of 3 assays (performed in triplicates).

Figure 8 shows the results of assays reporting on kinetics of formation of amyloid fibrils via the amyloid specific ThT binding assay (Figure 8A) and cytotoxic aggregates via the MTT reduction assay in aged (0 h - 7 days) solutions of A alone or mixtures of Αβ with peptide based compounds (L3-GI or L3) as indicated. Cytotoxicity of the aged solutions of Αβ alone and the mixtures with each of the peptide based compounds was determined at the incubation time point of 72 h (Figure 8B) or 7 days (Figure 8C). The presented data is means (±SEM) of 3 assays (performed in triplicates).

Table 1 shows sequences of representative compounds of classes (a) inhibitors/amyloid ligands described herein.

Table 2 shows sequences of representative compounds of classes (b)-(d) inhibitors/amyloid ligands described herein.

The following abbreviations are inter alia used herein:

2-Aoc = 2-amino octanoic acid

Αβ = beta-amyloid peptide

Adc = amino decanoic acid, preferably 10-amino deca- noic acid

Amba-Aba = p-aminomethylbenzoic acid covalently linked via amid bond with p-amino benzoic acid

Aoc = 8-amino octanoic acid

Dap = 2,3-diaminopropionic acid ChA = cyclohexylalanin

IAPP = islet amyloid polypeptide

IAPP-GI = [(N-Me)G24, (N-Me)l26]-IAPP

IC = inhibitory core

Examples

Example 1

The effects of peptide based compounds containing the IC sequences GGG, AAA, WV, LLL and N-methylations at G24 and I26 (class b inhibitors) on formation of Αβ fibrils and cytotoxic assemblies are compared to each other and to the effect of IAPP(8-28)-GI (partial IAPP sequence IAPP(8-28) which has been N-methylated at G24 and I26) as shown in Fig. 1 . N-methylated positions correspond to amino acid positions of the full length IAPP protein represented by SEQ ID NO: 1 .

It is well known that Αβ forms fibrils and cytotoxic oligomers in a time- and concentration-dependent manner. Experimental assay systems have been established to study in vitro kinetics of these processes (described in Yan LM, 2007, above). Accordingly, kinetics of fibrillogenesis were determined by incubating Αβ (16.5 μΜ in 50 mM sodium phosphate buffer, pH 7.4, containing 100 mM NaCI and 1 % hexafluoroisopropanol (HFIP)) and 1 :1 mixtures of Αβ40 with analogues (16.5 μΜ each in 50 mM sodium phosphate buffer, pH 7.4, containing 100 mM NaCI and 1 % HFIP) for up to 7 days at room temperature. At the indicated time points aliquots were withdrawn from the incubations, mixed with suitable amounts of Thioflavin T (ThT) as described in Yan LM, 2007, above, and the amount of fibrils was quantified by the ThT binding assay (Fig. 3A) (Yan LM, 2007, above). Aliquots of the above aged solutions of Αβ and its mixtures with peptides (as above) were added (following dilution with medium) to PC12 rat pheochromocytoma cells at the time point of 72 h (Fig. 3B) and 7 days (Fig. 3C). Following 24 h incubation with the cells at 37°C cell viabilities were assessed via the 3-[4,5-dimethylthiazol-2- yl]-2,5-diphenyltetrazolium bromide (MTT) reduction assay which is a commonly applied assay to determine amyloid-associated cell damage as previously described (Yan LM, 2007, above). Peptide based compounds which are able to block fibril formation are in most of the cases also able to suppress formation of cytotoxic aggregates since the two processes are linked to each other. Based on the results of the assay system, the peptide based compounds were classified in "highly potent" inhibitors (++++ in Table 3) when they were found to be able to block Αβ fibril formation and toxicity up to the incubation time point of 7 days and in "medium potency" inhibitors ("++" in Table 3) when they were found to be able to inhibit Αβ fibril formation and toxicity up to the 72 h incubation time point. Finally, peptide based compounds were classified in "no inhibitors" when they had no effect on formation of fibrils and cytotoxic aggregates ("- -" in Table 3).

As shown in Figure 3, peptide based compounds with linker sequences L3 and V3 are highly potent inhibitors. By contrast, the A3- linker containing peptide based compound is a medium potency in- hibitor and the G3-linked one has no effect whatsoever on Αβ aggregation and toxicity. Importantly, IAPP(8-28)-GI has also no effect on aggregation and toxicity of Αβ as previously reported (Andreetto E, 201 1 , above). The segments IAPP(8-18), IAPP(22-28), and IAPP(22- 28)-GI alone are unable to affect fibrillogenesis and cytotoxicity of Αβ as well (Andreetto E, 201 1 , above). Taken together, these results provide strong evidence that the nature of the linker sequence between regions (8-18) and (22-28) of IAPP defines the inhibitory po- tential of the IAPP(8-28) analogue peptide based compounds on Αβ aggregation and toxicity.

Example 2

The effects of the double N-methylated (at G24 and 126; N- methylated positions correspond to amino acid positions of the full length IAPP protein represented by SEQ ID NO: 1 ) peptide based compounds K3-GI, KAc3-GI, R3-GI, and Dap3-GI containing polar charged residues in the linker sequence on Αβ fibrillogenesis and cytotoxicity are studied and compared to each other (Figure 4). Ki- netics of fibrillogenesis (Fig. 4A) and formation of cytotoxic assemblies (Fig. 4B and 4C) were determined as described above under Example 1 . The peptide based compounds are classified (Table 3) with regard to their inhibitory potential according to the same criteria as defined in Example 1 . As shown in Figure 4, only the peptide based compound with linker sequence R3 is a highly potent inhibitor whereas the K3- and Dap- linker (Dap: 2,3-Diaminopropionic acid) containing peptide based compounds are medium potency inhibitors and the KAc3-linked peptide based compound has no effect on Αβ aggregation and toxicity. These results exemplify that differences in the nature of the linker sequence between regions (8-18) and (22-28) of IAPP define the inhibitory potential of the IAPP(8-28)-GI analogue peptide based compounds on Αβ aggregation and toxicity and are in good agreement with the results shown in Example 1 . Example 3

The effects of the double N-methylated (at G24 and 126; N- methylated positions correspond to amino acid positions of the full length IAPP protein represented by SEQ ID NO: 1 ) peptide based compounds L3-GI, Nle3-GI, F3-GI, ChA3-GI (ChA, Cyclohexylala- nine), and 2-Aoc3-GI (Aoc, 2-aminooctanoic acid) containing various different hydrophobic residues in the linker sequence on Αβ fibrillo- genesis and cytotoxicity are studied and compared to each other (Figure 5). Kinetics of fibrillogenesis (Fig. 5A) and formation of cyto- toxic assemblies (Fig. 5B and 5C) were determined as described in Example 1 . The analogues are classified (Table 3) with regard to their inhibitory potentials according to the same criteria as defined in Example 1 .

As shown in Figure 5, the peptide based compounds with L3, Nle3, and F3 linkers are highly potent inhibitors while the 2-Aoc3 linker containing peptide based compound does not significantly affect Αβ aggregation and toxicity and the ChA3 linker containing peptide based compound is medium potency inhibitor. These results also exemplify that differences in nature of the linker sequence between IAPP(8-18) and IAPP(22-28)-GI define the inhibitory potential of the IAPP(8-28)-GI analogue peptide based compound on Αβ aggregation and toxicity and are in good agreement with the results shown in Example 1 . Example 4

The effects of peptide based compounds containing the linker sequences GGG, AAA, VW, and LLL and N-methylations at G24 and 126 (N-methylated positions correspond to amino acid positions of the full length IAPP protein represented by SEQ ID NO: 1 ) on formation of IAPP fibrils and cytotoxic assemblies which were studied in Example 1 with regard to their effect on aggregation and toxicity of Αβ are studied and compared to each other and to the effect of IAPP(8-28)-GI as shown in Fig. 6. Similarly to Αβ, IAPP also forms fibrils and cytotoxic oligomers in a time and concentration-dependent manner and experimental assay systems has been established to study in vitro kinetics of IAPP fibril- logenesis and toxicity (Yan LM, 2006, above; Yan LM, 2007, above). Accordingly, kinetics of IAPP fibrillogenesis were determined by in- cubating IAPP (16.5 μΜ in 50 mM sodium phosphate buffer, pH 7.4, containing 100 mM NaCI and 0.5% HFIP) and 1 :2 mixtures of IAPP with analogues (16.5 μΜ IAPP and 33 μΜ each of the analogues in 50 mM sodium phosphate buffer, pH 7.4, containing 100 mM NaCI and 0.5% HFIP) for up to 7 days at room temperature. At the indicat- ed time points aliquots were withdrawn from the incubations, mixed with suitable amounts of ThT and the amount of fibrils was quantified by the ThT binding assay (Fig. 6A) (Yan LM, 2007, above). Aliquots of the above aged solutions of IAPP and its mixtures with peptides were added (following dilution with medium) to the rat insulinoma cell line RIN5fm at the time point of 24 h (Fig. 6B) and 7 days (Fig. 6C). Following 24 h incubation with the cells at 37°C cell viabilities were assessed via the MTT reduction assay as above (Yan LM, 2007, above). Based on the results our assay system, our peptide based compounds were classified in "highly potent" inhibitors (++++ in Table 3) when they were found to be able to block IAPP fibril formation and toxicity up to the incubation time point of 7 days and in "medium potency" inhibitors ("++" in Table 3) when they were found to inhibit IAPP fibril formation and toxicity up to the 24 h incubation time point. Peptide based compounds were "no inhibitors" when they were found to have no effect on formation of IAPP fibrils and cytotoxic aggregates ("- -" in Table 3).

As shown in Figure 6, only the peptide based compound with the linker sequence L3 was found to be a highly potent peptide based compound. By contrast, all other analogues were unable to inhibit aggregation and toxicity of IAPP. These results provided strong evidence that the nature of the linker sequence between IAPP(8-18) and IAPP(22-28)-GI defines the inhibitory potential of the IAPP(8- 28)-GI analogue peptide based compounds on IAPP aggregation and toxicity.

Example 5

Here the effects of peptide based compounds containing the linker sequences with hydrophobic residues and N-methylations at G24 and I26 (N-methylated positions correspond to amino acid positions of the full length IAPP protein represented by SEQ ID NO: 1 ) on formation of IAPP fibrils and cytotoxic assemblies were studied (Fig. 7). These peptide based compounds were studied with regard to their effect on aggregation and toxicity of Αβ. Kinetics of IAPP fibrillogen- esis (Fig. 7A) and formation of cytotoxic assemblies (Fig. 7B and 7C) alone and in the presence of each of the analogues were determined as described above under Example 4. The analogues are classified (Table 3) with regard to their inhibitory potential according to the same criteria as defined in Example 4.

As shown in Figure 7, peptide based compounds with L3, Nle3, 2- Aoc3, and ChA3 linkers are highly potent inhibitors while the Phe3 linker containing peptide based compound does not significantly affect IAPP aggregation and toxicity (Table 3). These results clearly exemplify that differences in the nature of the linker sequence between regions IAPP(8-18) and IAPP(22-28)-GI define the inhibitory potential of the IAPP(8-28)-GI analogue peptide based compounds on IAPP aggregation and toxicity and are consistent with the results shown in Example 4.

Example 6

An example is presented which demonstrates the favourable effects of the double N-methylation at G24 and I26 (N-methylated positions correspond to amino acid positions of the full length IAPP protein represented by SEQ ID NO: 1 ) which was present in all class (b) and (c) analogues on the inhibitory potential of the peptide based compounds on Αβ aggregation and toxicity.

Kinetics of fibrillogenesis (Fig. 8A) and formation of cytotoxic assem- blies (Fig. 8B and 8C) of 0-7 days aged incubations of Αβ alone versus mixtures of Αβ with L3-GI (as also shown in Figure 3) or L3 (which differs from L3-GI only in the absence of the two N-methyl residues) were determined as described above under Example 1 . The peptide based compounds were classified (Table 3) with regard to their inhibitory potential according to the same criteria as under Example 1 . As shown in Figure 8, L3-GI is a highly potent inhibitor while L3 is a medium potency inhibitor (at the 1 :1 molar ratio to Αβ applied here) and in fact similar results were obtained with regard to the inhibitory effecs of IAPP as well (data not shown). These results are consistent with the presence of the double N-methylation at G24 and I26 resulting in improved inhibitory potentials on Αβ and IAPP fibrillogenesis and cytotoxicity.

Example 7

In Table 3 a summary of the effects of several of the class (b) and (c) peptide based compounds, IAPP(8-28)-GI, and each of the linked sequences alone on formation of cytotoxic aggregates of Αβ or IAPP is presented. The effects are classified in: "++", corresponding to medium inhibitory potency; "++++", corresponding to high inhibitory potency; "+", corresponding to very weak inhibitory potency; and corresponding to no inhibitory potency. High inhibitory potency indicates inhibitory effect of the peptide based compound on formation of cytotoxic aggegates by Αβ or IAPP following incubation for up to 7 days; medium potency indicates inhibitory effect on formation of cytotoxic aggregates following incubation for up to 24 h for IAPP or 72 h for Αβ as determined in vitro using the experimental assay systems described in Examples 1 to 6. This Table demonstrates the different effects of the various different linker sequences on the inhibitory potential of the IAPP(8-28)-GI analogue peptide based compounds on Αβ and IAPP fibril formation and cytotoxicity. Table 3 shows a summary of the effects of several of the class (b) and (c) peptide based compounds, IAPP(8-28)-GI, and each of the linked sequences alone on formation of cytotoxic aggregates of Αβ or IAPP. The effects are classified in: "++", corresponding to medium inhibitory potency; "++++", corresponding to high inhibitory potency; "+", corresponding to very weak inhibitory potency; and corresponding to no inhibitory potency (see text under Example 1 ). Table 3

Example 8

In Table 4 a summary of the IC50 values of the effects of highly potent peptide based compounds on formation of cytotoxic Αβ or IAPP assemblies is presented. The assays for the determination of the IC50 values were performed by using mixtures of Αβ or IAPP (at 100 nM) with various amounts of the analogues under the same experimental set-up as described in Examples 1 to 6 and in references Yan LM, 2006, above; Yan LM, 2007, above; Yan LM, 2013, above). IC50s were determined in 3 days aged incubations of Αβ alone or its mixtures with analogues and in 24 h aged incubations of IAPP alone or its mixtures with the peptide based compound as described (Yan LM, 2013, above). The IC50 values shown in Table 4 are means (± SEM) from 3 assays.

These results showed that a number of the peptide based com- pounds exhibits inhibitory potentials in the low nanomolar range which is consistent with these peptide based compounds being under the most potent inhibitors of Αβ and IAPP cytotoxicity.

Table 4 shows IC50 values of effects of some of the highly potent analogues on formation of cytotoxic Αβ or IAPP assemblies. Table 4

V3-GI 1 1 .7 (±1 .2) nM nd ^*

L3 52.8 (±14.7) nM 48.5 (±5.0) nM

Amba-Aba-GI 158 (±36.0) nM 41 .3 (±4.9) nM

K3-A3-K3-GI 292 (±78.0) nM 43.1 (±21 .6) nM

^* nd, not determined as the peptide based compound was found to not inhibit IAPP cytotoxicity

Example 9 In Table 5 a summary of the apparent (app.) binding affinities of the interactions of the peptide based compounds with IAPP or Αβ as determined by fluorescence spectroscopic titrations is presented. The underlying principle of this kind of assay is that changes in the fluorescence emission of fluorescently labelled proteins are highly sensitive indicators of changes in the environment of the fluorophore that might be caused by ligand binding (Eftink MR. Fluorescence methods for studying equilibrium macromolecule-ligand interactions. In: Brand L, Johnson ML, editors. Methods in Enzymology: Fluorescence Spectroscopy. San Diego: Academic Press; 1997. p. 221 -57). The experimental assay system applied to quantify the app. affinities (app. Kd) of interactions of the analogues with IAPP or Αβ monomers has been earlier developed and is described in references (Yan LM, 2006, above; Yan LM, 2007, above; Yan LM, 2013, above). Briefly, all titrations were performed using synthetic Na-amino termi- nal fluorescein-labelled IAPP (Fluos-IAPP) or 7- diethylaminocoumarin-3-carbonyl- (DAC) labelled Αβ (DAC-Αβ). For the titrations of Fluos-IAPP with the peptide based compounds the excitation was at 492 nm and fluorescence emission spectra be- tween 500 and 600 nm were recorded. For the titrations of DAC-Αβ with the peptide based compounds the excitation was at 430 nm and emission spectra between 440 and 550 nm were recorded. Titrations were performed in freshly made solutions containing Fluos-IAPP (5 nM) or DAC-Αβ (10 nM) and varying amounts of the peptide based compounds in 10 mM sodium phosphate buffer, pH 7.4, containing 1 % HFIP at room temperature as described (Yan LM, 2006, above; Yan LM, 2007, above; Yan LM, 2013, above).

To determine whether the peptide based compounds are able also to bind Αβ or IAPP fibrils, Αβ or IAPP fibrils were prepared from aged Αβ or IAPP solutions (for Αβ fibrils incubation conditions were: 16.5 μΜ in 50 mM sodium phosphate buffer, pH 7.4, containing 100 mM NaCI and 1 % HFIP; for IAPP fibrils incubation conditions were: 16.5 μΜ in 50 mM sodium phosphate buffer, pH 7.4, containing 100 mM NaCI and 0.5% HFIP) as described under references (Yan LM, 2007, above; Yan LM, 2013, above). Titrations of synthetic Na-amino terminal fluorescein-labelled peptide based compounds with various amounts of the fibrillar assemblies containing solutions were then performed under the same experimental conditions as above (Yan LM, 2007, above; Yan LM, 2013, above). Analysis of the binding isotherms yielded the app. affinities (app. Kd) of interactions which are shown in Table 5 (Yan LM, 2007, above; Yan LM, 2013, above).

The above results suggested that most of the peptide based compounds bind IAPP and Αβ in monomeric or fibrillar state with high affinity (concentration range: from low μΜ to low nanomolar) and suggested that a conformation specific interaction with Αβ or IAPP conformers/assemblies might play an important role in their various different effects on Αβ or IAPP aggregation and toxicity. Table 5 shows apparent binding affinities (app. Kd) of interactions of peptide based compounds with Αβ or IAPP (monomeric or fibrillar assemblies) as determined by fluorescence spectroscopic titrations. The app. Kds shown are means from 3 assays in the case of Kds for the interaction with monomers and from one assay in the case of app. Kds for the interaction with fibrillar assemblies.

Table 5

nd = not determined