ZA200601908B - Compounds comprising LPA - Google Patents

Description

Compounds comprising LPA

Field of invention.

The present invention relates to newv peptide compounds capable of binding to fibroblast growth factor receptor (FGF R), said compounds comprising two individual amino acid sequences, wherein at least one of the two amino acid sequences is capable of binding to FGFR. The invention discloses the amino acid sequences of the compounds and features pharrmaceutical compositions comprising thereof.

Invention also relates to uses of the compounds and pharmaceutical compositions for treatment and/or prevention of different pathological conditions, wherein FGFR plays a role in pathology and/or recovery from the disease. New peptide compounds of the invention are obtainable by the ligand presenting assembly (LPA) method.

Background of invention ne

Brain plasticity and the mechanisms controlling plasticity are central to learning and memory as well as the recovery of function after brain injury. While it is clear that neurotrophic factors are one of the molecular classes that continue to regulate brain plasticity in the adult central nervous system (CNS), less appreciated but equally profound is the role of cell adhesion molecules (CAMs) in plasticity mechanisms such as long term potentiation, preservation of neurons and regeneration. Ironically, however, CAMs can also reorganise the extra-celiular space and cause distur- bances that drive the development of brain pathology in conditions such as Alz- heimer's disease and multiple sclerosis. Candidate molecules include the amyloid precursor protein, which shares many properties of a classical CAM and beta- amyloid, which can masquerade as a pseudo CAM. Beta-Amyloid serves as a nidus for the formation of senile plaques in Alzheimer's disease and like CAMs provides an environment for organising neurotrophic factors and other CAMs. inflammatory responses evolve in this environment and can initiate a vicious cycle of perpetuated neuronal damage that is medicated by microglia, complement and other factors (Cotman et al. (1998) Prog Neurobiol. 55:659-69).

Neural cell adhesion molecules (CA Ms) of the immunoglobulin superfamily nucleate and maintain groups of cells at key sites during early development and in the adult.

In addition to their adhesive properties, CAMs homophylic and heterophyiic interac-

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tions cam affect intracellular signalling. Their ability to influemnce developmental events, including cell migration, proliferation, and differentiation rmay therefore result from both their adhesive and signalling properties.

The neural cell adhesion molecule, NCAM, was the first discovered neural CAM.

Since the discovery NCAM has been intensively studied and now it is well character- ised (Ronn et al. (2000) Int J Dev Neurosci 18:193-9) NCAM beslongs to the immu- noglobultin (Ig) superfamily. Its extracellular part consists of five ig-like and two fi- bronectin type Ill (F3) modules. NCAM assists both the cell-cell and cell-substratum interactions. NCAM binds to various extracellular matrix comporents such as hepa- rin‘hepa ran sulfate, chondroitin sulfate proteoglycans, and different types of colla- gen. Ce ll-cell interactions are mostly assisted by the NCAM hoamophilic interaction.

The diff'erent modules of NCAM have been shown to perforrmn distinct functions.

Thus, NECAM homophilic binding is now believed to depend on the first three Ig moduless. The heparin binding sequence is localised to the Ig2 amodule. NCAM also binds to» the neural cell adhesion molecule L1. This interactiors is believed to take place bextween the fourth Ig module of NCAM and an oligoma._nnosidic moiety ex- pressed on Li. The two membrane-proximal F3 modules of NCAM have been shown involved in fibroblast growth factor receptor (FGFR) bindieng.

A number of research groups has now accumulated a large bo-dy of evidence indi- cating that intracellular signalling cascades underlying the NCAM-mediated axonal outgrowth are similar to signal transduction cascades which aare activated due to stimulation of FGFR (Povisen et al. (2003) Neurochem Res 1:127-41). 2 Fibroblast growth factor receptors (FGFRs) are a family of fouar closely related re- ceptor protein tyrosine kinases consisting extracellularly of three Ig-like modules and intracell ularly of a split tyrosine-kinase module (Powers et al. {2000) Endocr Relat

Cancer 7:165-97).. The receptors are known as key regulatorss of morphogenesis, development, angiogenesis, and wound healing. FGFR activation and signalling are dependent on dimerization of the receptor which is induced by high affinity binding of FGFRR natural ligand, fibroblast growth factor (FGF), and it also requires participa- tion of cell surface heparin or heparan sulphate proteoglycanss. Fibroblast growth factors (FGFs) and their receptors constitute an elaborate signaling system that par- ticipatess in many developmental and repair processes of virtaally all mammalian tissues, in particular, they play a prominent role in functioning of the peripheral and

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central neural system. Thus, among 23 members of the FGF family, ten have been identifiexd in the braln (Jungnickel et al, (2004) Mol Cell Neurosci. 25:21-9; Reuss and vo n Bohlen und Halbach (2003) Ceili Tissue Res. 313:139-57).

NCAM has recently been regarded as a member of a new class of putative alterna- tive ligmands of FGFR, the low affinity binding ligands. There has beeen obtained evi- dence for a direct interaction between NCAM and the receptor (Kiselyov et al. (2003) Structure (Camb) 11:691-701).

The identified NCAM fragment having the sequence EVYVVAEN=QQGKSKA (FGL peptide) involved in the direct interaction between NCAM and FCSFR has recently been suggested as a new candidate drug for the treatment of a variety of pathologic disorders where the regulation of activity of FGFR may play the key role (WO 03/016351). WO 03/016351 describes some biological effects of the FGL peptide due tos binding and activating FGFR. According to WO 03/016351, presentation of a single copy (monomer) of the peptide to neuronal cells in vitro is emough to promote cell survival and differentiation. However, rather a high concentrat¥on of the peptide is nee-ded to achieve the effects.

Multip le presentation of peptide sequences, e.g. antigenic pepotides, has been shown to be a valuable mean to amplify biological responses of p eptide sequences in vitreo (Berezin and Bock (2004) J Mol Neurosci.22:33-39; Ron et al (1999) Nat

Biotechnol 17:1000-5), and it has also been effectively used in anirmal models in vivo (Cambbon et al. (2004) J Neurosci. 17:4197-204).

Synth-etic dendritic polymers of biologically active peptide sequences are now used in reseearch and some clinical applications. A dendritic polymer corsists of a number of copies of a monomeric peptide sequence, which is attached to & core molecule at multiple sites forming thereby a branching polymer. Lysine is mosst commonly used amino acid in the core matrix because it has two reactive amino ge-oups available for branching reactions. The multimerization of peptide sequences orn the lysine core is knowm as the multiple antigen peptide (MAP) presentation. A metimod for the synthe- sis of MAP(s) (dendrimeric peptides) was described in PCT/USS0/«02038. in gereral, two routs can be used for the synthesis of a dendrimer—ic peptide, namely the direct and indirect route. In both cases, the C-terminal core is first assembled on a soli«d support using bi-protected Boc-Lys(Boc) in case of Boc chemistry, or Fmoc-

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Loys(Fmoc) in the case of Fmoc chemistry in order to obtain the desired degree of beranching. im the direct route synthesis is done by stepwise assembling of the particular peptide on the lysinyl core by the well-known Merrifield metiiod. This stepwise method pro- duces peptides with a C to N orientation. The peptide chains may be synthesised in tandem, or, alternatively, each peptide can be synthesised on the different lysine ams of the core using orthogonal deprotection methods.

Although this approach for dendrimeric peptide syn-thesis is convenient, the quality of the final product may be questionable and problems of obtaining a well-defined goroduct have been widely discussed in the literatuare. Problems arise during chain assembly. The MAP type dendrimers are macromolecules, where each single pep- tide chains may uncontrolled interact with each othear and prevent thereby high effi- «ciency coupling to the core leading to production ef non-homogenous compound, =which require effort to purification. Characterisation of these products by, e.g. elec- ~trospray mass spectroscopy (ES-MS) is difficult amd as a result, dendritic peptide products are often used without full characterisation.

In the indirect route the synthesis is done by couplin g of purified unprotected peptide fragments to the core matrix. Two general methodss for this type of ligation may be used, which, respectively, are based on thiol and ca rbonyl chemistries.

The thiol chemistry may be carried out by incomoraation of chloroacetyl group(s) on the lysine matrix and subsequent coupling to a purified, synthetic N-terminal cys- teinyl peptide to yield a dendrimer with unambiguosus structure. The reverse place- ment may be achieved with thiol on the core matrix by using an S-acetyl group on the lysinyl core and the haloacetyl group on the peptide. In the carbonyl chemistry the condensation takes place between a carbonyl group and a weak base. There are two types of weak bases, which may be used fer this reaction. The first includes hydroxylamines and hydrazines, and the second includes 1,2-di-substituted moieties such as 1,2-aminoethanthiol and 1,2-aminoethanol. The last two groups are found in

N-terminal Cys and Thr or Ser respectively, and the condensation products are thia- zolidine and oxazolidine. The carbonyl group on thea core matrix may be obtained by periodate oxidation of N-terminal Ser, Thr or Cys. Both methods are well known in

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the art (see e. g. Lu et al., (1991) Mol Immunol 28:623-630; Defoort et al., (1992) Int

J Pept Prot Res 40:214-221; Drijfhout et al. (1891 ) Int J Pept Prot Res 37.27-32).

Another problem associated with the MAP type compounds is orientation of peptide 5 chains. In the direct route with stepwise assembling of the desired peptide on the lysinyl core only a compound having peptide chains with C to N orientation can only be obtained. The indirect route allows the both owrientations, C to N and N to C, but the process has other major disadvantages, e. ge. undesirable side reactions due to oxidation of disulphides, leading to impure products, interference with cystein resi- dues present in the native peptide chains, and tie procedure demands optimisation for every synthesis.

Finding the optimal number of multiplication of a peptide sequence is another— problem to be solved. Most studies of dendritic p eptide polymers synthesised by the=

MAP method have been carried out using tetra— or octameric polymers. However, application of the compounds comprising 4-8 seequences of 15 or more amino acidss in vivo is challenged by a problem of the capabi lity of a compound to penetrate the= blood-brain barrier, which is crucial if the compound is for the treatment of the brain_

Dimers of the peptide sequences may be a solution to this problem, in case the= dimers possess biological activity of the dendritic tetramer analogues. Productiorm and use of biologically active peptide dimers comprising different linkers is also welll known in the art (see e.g. WO00/18791, WO00/24770).

Summary of invention

Although, a dendrimeric version of the FGL peptide consisting of four sequences of

FGL has been shown to be effective in vivo in raat (Cambon et al. (2004) J Neurosci... 17:4197-204), the athours of the present invertion found that it is preliminary to consider using of the dendrimer as a drug cardidate for the treatment of humar patients. Final purification of the dendritic tetramer of the FGL peptide revealed that the product of the MAP synthesis is highly hetewogenous and its characterisation iss difficult. To solve this problem the authors of the present invention attempted to synthesise different dimers of FGF, using know synthetic procedures. Surprisingly, the only FGL dimer that had biological activity similar to the FGL dendrimer was dimer produced by the ligand presenting assermbly method (LPA), as disclosed im

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WO000/18791. Other dimers of FGL, such as a dime=r wherein the FGL sequences were linked by a Cys residue, as disclosed in Goodwin et al. (1998) Bioorg Med

Chem Lett 8:2231-2234, or by a Lys residue, as disclosed in Rajagopalan et al. (1995) Int J Pept Protein Res 45:173-179, did not hawe same activity.

Accordingly, in the first aspect the present invention relates to a compound compris- ing two individual peptide sequences, wherein at leaast one of the two individual se- quences comprises an amino acid sequence of the feormula

L1-A-L2-B-L3-C-L4-D-L5 wherein one of A, B, C, D is selected from a hydrophobic amino acid residue, one of A, B, C, D is selected from a basic amino acid residue, Asn or Gin, one of A, B, C, D is selected from an acidic amino acid residue, Asn or Gin, one of A, B, C, Dis Gly or Ala, and

L1, L2, L3, L4 and L5 is selected from a chemical bcand or an amino acid sequence having n amino acid residues, wherein n is an integer of from 0 to 5, said two peptide sequences being connected to eac h other through a linker of the general formula

X[(ANCOOH]{(B)ymCOOH] n and m independently are an integer of from 1 to 20,

X is HN, H.N(CR2)pCR, RHN(CR2)pCR, HO(CRX,)pCR, HS(CR:)pCR, halogen- (CR,)pCR, HOOC(CR,)pCR, ROOC(CR:)pCR, HCO(CR:)pCR, RCO(CR:)pCR, [HOOC(ANHOOC(B)M]CR(CR2)PCR, HN(CR2Jp, RHN(CRz)p, HO(CRq)p,

HS(CR2)p, halogen-(CR2)p, HOOC(CRz)p, ROOC(CCR2)p, HCO(CR.)p, RCO(CR.)p, or [HOOC(A)N[HOOC(B)m)(CR.)p , wherein p is 0 or integer of from 1 to 20,

A and B independently are a substituted or unsubstituted C,.,o alkyl, a substituted or unsubstituted C,.4 alkenyl, a substituted or unsubst@tuted cyclic moiety, a substituted or unsubstituted heterocyclic moiety, a substituted or unsubstituted aromatic moiety, or A and B together form a substituted or unsubstituated cyclic moiety, substituted or unsubstituted heterocyclic moiety, substituted or unsubstituted aromatic moiety.

The above compound is according to the invention #is obtainable by the LPA method.

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In another aspect, the invention relates to a pharmaceutical composition comprising a compound as defined above.

In still another aspect, the invention concerns using the above compound for the manufacture of a medicament for a) treatment of conditions of the central and peripheral nervous system associated with postoperative nerve damage, traumatic nerve damage, impaired myelina- tion of nerve fibers, postischaemic damage, e.g. resulting from a stroke, Parkin- 190 son's disease, Alzheimer's disease, Huntington's disease, dementias such as multiinfarct dementia, sclerosis, nerve degeneration associated with diabetes mellitus, disorders affecting the circadian clock or neuro-muscular transmission, and schizophrenia, mood disorders, such &s manic depression; 156 b) treatment of diseases or conditions of the muscles including conditions with im- paired function of neuro-muscular connections, such as after organ transplanta- tion, or such as genetic or traumatic atrophic muscle disorders; or for treatment of diseases or conditions of various organs, such as degenerative conditions of the gonads, of the pancreas such as diabetes mellitus type | and Ii, of the kidney such as nephrosis and of the heart, liver and bowel; c) promotion of wound-healing; d) prevention of death of heart muscle cells, such as after acute myocardial infarc- tion, or after angiogenesis; e) promotion of revascularsation; f) stimulation of the ability to learn and/or the short and/or long-term memory; g) prevention of cell death due to ischemia; h) prevention of body damages due to alcohol consumption; i) treatment of prion diseases;

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Wo’ 0 2005/014623 PCT/DK2004/000527 v 8 j) treatment of cancer.

Description of Drawings

Figure 1 shows the flow chart of the synthesis of the LPA-type FGL dimer (FGL.)

Figure 2 presents the HPLC purification profile off FGL.

Figure 3 presents the HPLC purification profile o=f FGLp

Figure 4 demonstrates the effect of the FGL pegptide on survival of primary neurons treated with various neurotoxic agents.

Dopaminergic neurons (DN) (a and b) from day~ 15 rat embryos grown at a density of 150,000 cells/cm? for six days without or with warious concentrations of peptide on 24-well cell culture plates coated with poly-D-I ysine were exposed to 100 uM 6-

OHDA for two hours. Medium was changed aand various concéntrations of FGLy were added. The neurons were grown for anoth er 24 hours before the cultures they were fixed and immunostained for tyrosine hydroxylase.

Hippocampal neurons (HN) (c and d) from dayy 19 rat embryos were seeded at a density of 40,000 cells/cm? on poly-L-lysine coated 8-well permanox chamber slides and grown for 24 hours in medium containing =20 pM Amyloid-g 25-35 peptide (AB 25-35) at the presence of various concentratioens of FGL,, before they were fixed and stained with Hoechst 33268.

Cerebella granular meurons (CGN) (e and f) freom postnatal day 7 rats were grown at a density of 100,000 cells/cm? for 7 days on poly-L-lysine coated microtiter plates in the medium containing 40 mM KCI, then the- medium was substituted to a 5 mM

KCI containing medium suplemented with vario us concentrations of FGL. After two days of incubation, the cultures were fixed and sstained with Hoechst 33258. (a) - Effect of 10 ng/ml GDNF on survival of d opaminergic neurons treated with 6-

OHDA. (b) Effect of various concentrations of FGLy cn survival of dopaminergic neurons treated with 6-OHDA. (c) Effect of 50 ng/ml BDNF on hippocampal cwultures treated with Ap 25-35.

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(d) Effect of various concentratiornss of FGL4 on survival of hippocampal neurons treated with AR 25-35. (e) Effect of 50 ng/ml IGF-1 on CGN cultures induced to undergo apoptosis by de=- priving the neurons of high potassium. (f) Effect of various concentrations of FGLd on CGN cultures induced to underg © apoptosis.

Results from at least four independent experiments for each type of culture are exx- pressed as percentage + SEM of live neurons as compared to the total number &f neurons. Control cultures induced to undergo cell death without FGL-treatment wewre set at 100 %. + p<0.05, ++ p<0.01 when compared to the untreated controls. * p<0.05, ** p<0.01 and *** p<0.001 when compared to the cultures induced to um- dergo cell death.

Figure 5 demonstrates the effect of the FGL peptide on DNA-fragmentation in CGN cultures induced to undergo apoptosis by changing the medium after the neuroms were grown for six days in high KCl medium (40 mM) to a low KCI medium (5 mM

KCI). The number of neurons with fragmented DNA was measured using the Apo-

Alert apoptosis detection kit and the fraction of TUNEL-positive neurons was est- mated by counting. Results from at feast four independent experiments are shown as percentage of cells showing DNA-fragmentation relative to the total number of cells + SEM with control cultures in low KCl set at 100 %. (a) Effect of depriving CGN cultures of high KCI on number of cells undergoing apoptosis. (b) Effect of FGLA in apoptosis-induced CGN cultures. * p<0.05, ** p<0.01 and *** p<0.001 when compared to low KCl-treated CGN cul- tures. ‘

Figure 6 shows the effect of FGL on phosphorylation of Erk1/2. CGN were grown for three days at a density of 290,000 cells/cm? on poly-L-lysine coated microtiter plates, were subsequently treated with FGL4 for 10 - 90 min, fixed and stained vith antibodies against phospho-p42/44. The total number of neurons was estimaated using crystal violet staining. Results from at least three independent experiments are expressed as percentage + SEM with untreated cultures set at 100 %. * p<0.05 sand ** p<0.01 when compared to the controls.

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I

Figure 7 shows the effect of FGL on phosphorylation oF Akt. CGN were grown for “three days at a density of 290,000 cells/cm’ on poly-L-lysine coated microtiter plates, were subsequently treated with FGL. at various . concentrations for 10 or 30 minutes respectively and subsequently fixed and stained . with antibodies against Akt phosphorylated on Ser473. The total number of neurons was estimated using crystal violet staining. Results from at least three independent experiments are expressed as percentage + SEM with untreated cultures set at 1@00 %. * p<0.05 when com- pared to the controls.

Figure 8 shows the effect of inhibitors of FGFR, MEK. and PI3K on FGL-induced survival of CGN induced to undergo apoptosis. CGN waere grown at a density of 100,000 cells/cm? for seven days on poly-L-lysine coatead 8-well permanox slides in medium containing 40 mM KCl, before the medium was changed to a 5 mM KCI containing medium together with FGLy. Thereafter the MEK inhibitor PDS8059 (e),

PI3K inhibitor LY294002 (A) and FGFR inhibitor SU54(D2 (mu) were added at various concentrations. The addition of the inhibitors followed irn all cases by the addition of 10 pg/ml FGL4. After two days of incubation with the pe=ptide and inhibitors, the cul- tures were fixed and stained with Hoechst 33258. Resumlts from at least four individ- ual experiments are shown as percentage of the numbeer of live neurons relative to the total number of neurons + SEM, with control cultumres treated with FGL, set at 100 %.

Figure 9 shows the effect of the FGL peptide on neurite outgrowth from dopaminer- gic (e), hippocampal (A) and cerebellar granule neuron s (um). Dopaminergic neurons were grown at a density of 100,000 cells/cm? on poly—D-lysine coated 24-well cell culture plates for 72 hours with various concentrations of FGLy. The cultures were subsequently immunostained for tyrosine hydroxylase.. Hippocampal neurons and

CGN were plated at a density of 10,000 cells/cm? ory 8-well permanox chamber slides and incubated for 24 hours in the presence of various concentrations of FGL.

Subsequently the neurons were immunostained for GAAP-43. Results from at {east five independent experiments for each neuronal culture are shown as percentage +

SEM with the untreated controls set at 100 %. * p<0.05,, ** p<0.01, *** p<0.001 when compared to the controls.

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Figure 10 shows the effect of three dimeric versions of the FGL peptide on neurite outgrowth of hippocampal neurons from newborn rats. Primary cultures of hippo- campal neurons were grown for 24 hours at a density of 10,000 cells/cm? with vari- ous concentrations of FOL, (mw), FGLys (A) or FGLcys (e) and then were immu- nostained for GAP-43. Results from at least four independent experiments are shown as percentage + SEM with untreated controls set at 100 %. ** p<0.01 when compared to the controls.

Figure 11 shows the effect of inhibitors of FGFR, MEK and PI3K on FGL-induced neurite outgrowth in CGN cultures. CGN cultures were grown for 24 hours at a den- sity of 10,000 cells/cm? on 8-well permanox slides in the presence of the FGL pep- tide and an inhibitor. PD98059 (#) was added at concentrations of 12.5, 25 and 50 uM simultaneously with 10 pg/ml FGLg; LY294002 (A) was added at concentrations of 3.5, 7 and 10 pM simultaneously with 27 ug/ml FGL,;. SU5402 (m) was added in concentrations of 20, 40 and 80 uM simultaneously with 27 pg/ml FGL,4. After the treatment cells were fixed and immunostained for GAP-43. Results from at least four independent experiments are shown as percentage + SEM, with FGLd-treated con- trol cultures set at 100 %. The dashed line indicates the average neurite length in cultures without FGLtreatment. * p<0.05, ** p<0.01 when compared to the FGL- treated controls.

Figure 12 shows the effect of FGLy, FGLconto and vehicle on the rate of FM 1-43 destaining of primary cultures of hippocampal neurons from E19 rats. Each value represents the mean of 4-8 experiments and error bars indicate S.E.M. Neither treatment with 5 pg/ml FGL4 for any time period, nor the treatment with 20 pg/ml for 24 hours had any effect on the rate of FM 1-43 unioading. in contrast, synapses in cultures treated with 20 pg/ml FGL for 1 hour and 48 hours showed a significantly increased rate of FM 1-43 unloading when compared to control cultures, indicating an enhanced presynaptic response in these cultures. The values were compared using an unpaired t-test. *p= 0.04 (1h) and **p = 0.0032 (48h). FGlconta is @ control peptide consisting the sequence EVYVVAENAAGKSKA (SEQ ID NO: 147). Muta- tion of Gin residues of the FGL sequence to Ala has been shown to abrogate the

FGL activity (Kiselyov et al. 2003 see above. cit).

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Figure 13 dexmonstrates the effect of early sub-occipital administration of FGL.,

FGL; and FG Lowa ON memory (Social Recognition test) of rats in which cognitive impairment w-as induced by intracerebroventricular (i.c.v.) -injection oef the (25-35) #- amyloid fragrcient. The results are from the experiment in which the ssuboccipital ad- ministration o-f 5.0 pg FGL per administration per rat was done at days 7, 10, and 13 after A-B-induaced neurotoxicity. The Sacial Recognition test was pertformed 21 days after i.c.v. administration of the (25-35) B-amyloid fragment and esstimated as the ratio betweerm the times the adult rat spent exploring a juvenile rat dsuring a first (T1) and a second (T,) meeting. The number of animals in the groups waried from 4 to 22. The results were analysed using one-way ANOVA (F(2,27)=15.4,, P<0.0001) and significance eof results was contingent on achieving a p-value of less than 0.05 (**p<0.01), w hen compared to the A-3/V group (Newman-Keuls post-test). A statisti- cally significant effect was also observed between the control and the V/V-treated group compacred to the A-/V group (p<0.001). ‘A-’ and ‘V' are the abbreviations of the (25-35) (3-amyloid fragment and vehicle, respectively. ‘V/V’ stamnds for the rats received vehiicle as replacement of A-p and FGL. FGLconro as above.

Figure 14 sh ows the effect of early suboccipital administration of FGSL, and FGL4 on formation of amyloid burden in the cingulate cortex of rats in which cognitive im- pairment wass induced by i.c.v. -injection of the (25-35) B-amyloid fragment (5.0 ng

FGL per adninistration per rat) on days 7, 10 and 13 after (25-35) (-amyloid (A-8) fragment-induiced neurotoxicity. The number of animals in the grouyos varied from 3 to 13. Resulfts were analysed using one-way ANOVA (F(2,18)=5.2:, p=0.016), and significance -of results was contingent on achieving a p-value of less than 0.05 (*p<0.05 and **p<0.01), when compared with the A-B/V group (Newrman-Keuls post- test). A statistically significant difference was observed between fthe V/V treated group and thme A-B/V treated group (p<0.05). ‘A-B’ and ‘V’ are the abbreviations of the (25-35) B—amyloid fragment and vehicle, respectively.

Figure 15 shows the effect of early sub-occipital administration of FGL, and FGL4 on formation of the amyloid burden in the CA3 area of hippocampuss of rats in which ! cognitive impairment was induced by i.c.v. -injection of the (25-35) p-amyloid frag- ment (6.0 pg FGL per administration per rat) on days 7, 10 and 13 after (25-35) - amyloid (A-p0) fragment-induced neurotoxicity. The number of animals in the groups

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varied from 2 to 12. Results were analysed using one-way ANOVA (F(2,15)==9.54, p=0.002), and significances of results was contingent on achieving a p-value of less than 0.05 (***p<0.001), wken compared with the A-B/V group (Newman-Keuls post- test). A statistically significant difference was observed between the V/V treated group and the A-B/V treated group (p<0.01). ‘A-f’ and *V' are the abbreviations of the (25-35) B-amyloid fraggment and vehicle, respectively.

Figure 16 shows the effexct of early sub-occipital administration of FGLL and FGLq on neuronal death in the hippocampus of rats in which cognitive impairme-nt was induced by i.c.v. -injectiosn of the (25-35) B-amyloid fragment. The effect of 5.0 png

FGL per administration per rat, administered sub-occipitally on days 7, 10 =and 13 after (25-35) B-amyloid (AB) fragment-induced toxicity, on neuronal cell death in the

CA3 zone of the rat hippocampus. The number of animals in the groups varied from 4 to 8. The results were analysed using one-way ANOVA (F(3,17)=13.76, p=<0.001) and significance of results was contingent on achieving a p-value of less than 0.05 (***p<0.001), when com pared with the A-B/V group (Newman-Keuls post-&est). A statistically significant difference was observed between the Control group and the

A-B/V treated group (p<0.001). 'A-f’ and ' are the abbreviations of the (25-35) B- amyloid fragment and ve hicle, respectively.

Figure 17 demonstrates the effects of 5.0 ug FGL, (per administration per rat)ad- ministered sub-occipitally on days 30, 33, and 36 after (25-35) p-amyloid fragment (A-B)-induced neurotoxicity in the Social Recognition test. The Social Rec-ognition test was performed 44 days after i.c.v. administration of the A-p fragment aand esti- mated as the ratio between the times the adult rat spent exploring a juvenile rat dur- ing a first (T4) and a second (T2) meeting. The number of animals per grougp varied from 4 to 5. The results were analysed using unpaired t-test and significance of re- sults was contingent on achieving a p-value of less than 0.05 (*p<0.05), wheen com- pared to the A-B/V group. A significant impairment of short-term memory was in- duced by treatment witth A-B (p<0.05, unpaired t-test between Control andl A-/V). ‘Af’ and ‘V' are the albabreviations of the (25-35) p-amyloid fragment and vehicle, respectively.

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Figure 18 shows the effects of 5.0 ug FGL._ (per administration per rat) administered sub-ocgcipitally on days 30, 33 and 36 after A-B fragment-induced neurotoxicity on the amyloid burden in the cingulate cortex of" rats. The number of animals per group varied from 3 fo 13. The results werse analysed using one-way ANOVA (F(2,12)=18.6, P=0.002) and significance of results was contingent on achieving a p- value of less than. 0.05 (**p<0.001) when compared with the A-B/V group (Bon- ferroni's multiple comparison test). A statistically significant difference was observed between the V/V-treated group and the A-B/"V-treated group (p<0.001). ‘Af’ and V' are the abbreviations of the (25-35) B-amyloi d fragment and vehicle, respectively.

Figure 19 shows the effects of 5.0 pg FGL.. (per administration per rat) administered sub-occipitally on days 30, 33 and 36 after (25-35) B-amyloid (A-B) fragment- induced neurotoxicity on the amyloid burdesn in the CA3 area of hippocampus of rats. The number of animals per group varie-d from 3 to 5. The results were analysed using one-way ANOVA (F(2,9)=10.6, P=0.0 04) and significance of results was con- tingent on achieving a p-value of less than 0.05 (***p<0.001) when compared with the A-B/V group (Newman-Keuls post-test). A statistically significant difference was observed between the Control-treated grou p and the A-B/V-treated group (p<0.01). ‘A-B’ and ‘V' are the abbreviations of the (C25-35) p-amyloid fragment and vehicle, respectively.

Figure 20 shows the effect of 5.0 pg FGL. (per administration per rat) administered sub-occipitally on days 7, 10 and 13 after (225-35) B-amyloid (A-B) fragment-induced toxicity on neuronal cell death in the cingulate cortex. The number of animals in the groups varied from 4 to 8. The results were analysed using one-way ANOVA (F(2,9)=26.9; p<0.001, Newmann-Keuls post-test: A-B/V compared with the control group (***p<0.001) and compared with the A-B/FGL, treated group (**p<0.01). Sig- nificance of results was contingent on achieving a p-value of less than 0.05, when compared with the A-B/V group (Newman-BKeuls post-test). A statistically significant difference was observed between the Coratrol-treated group and the A-B/V-treated group (p<0.001). ‘A-B’ and ‘V’ are the abbwreviations of the (25-35) g-amyloid frag- ment and vehicle, respectively.

SUBSTITUTE SHEET (RWLE 26)

Figure 21 shows the effect of three intranasal administrations of vehicle, 2600 pg

FGLy, and 400 ug FGL, per rat, at days 7, 10 and 13 after i.c.v. administration of the (25-35) B-amyloid (A-B) fragmemt in the Social Recognition test. The social rescogni- tion test was performed 21 days after i.c.v. administration of the (25-35) B-armyloid (B-A) fragment and estimated as the ratio between the times the aduit rat speent on exploration of a juvenile rat during a first (T,) and a second (T, ») meeting. Nurmber of animals in the groups varied from 3 to 8. Results are shown as means + SEM and analysed by unpaired t-test (;2<0.001 of control vs ABV) and one-way A_NOVA (F(2,19)=5.6; p=0.01; Newman-Keuls post-test, p<0.05 of ABV vs A-BIFGL 400; and p>0.05 of ABV vs A-P/FGLL20 whereas p<0.05 of A-B/IFGLL 40 Vs A-

BIFGL_ 200). Significance of results was contingent on achieving a p-value of less than 0.05 (p<0.05) when compared to the A-B/V treated group. ‘A-}’ and 'V' =are the abbreviations of the (25-35) -amyloid fragment and vehicle, respectively.

Figure 22 shows the effect of three intranasal administrations of vehicle, 2200 ug

FGL,, and 400 pg FGL, per rak, at days 7, 10 and 13 after i.c.v. administratior of the (25-35) B-amyloid (B-A) on neuronal cell death in the cingulate cortex. The mumber of animals in the groups varied from 4 to 8B. The results were analysed us@ing un- paired t-test (of the control growup versus the A-B/V-treated group (p<0.001) ard one- way ANOVA (F(2,16)=8.6; p= 0.003), with Newman-Keuls post-test ™p<0.071 of A-

BV vs A-BIFGLL400 and p>0.05 of A-B/V vs. A-B/FGL 200). Significance of results was contingent on achieving a p-value of less than 0.05 (**p<0.01), when cormpared with the A-B/V treated group. ‘A-' and ‘V' are the abbreviations of the (258-35) B- amyloid fragment and vehicle, respectively.

Figure 23 shows the effect of 5.9 ng FGL« administration on rats tested for the

Rearing Activity in the Open Field test. The peptide was applied sub-occipitally at days 7, 10, and 13 after the (25-35) B-amyloid fragment induced neurotoxicity. The numbers ‘1’, ‘2’, and ‘3’ indicante the periods of measurement. ‘1’ denotes 1—3 min,, 2' denotes 8-10 min., and ‘3 denotes 18-20 min. ‘V/V' indicates that the mrats re- ceived vehicle as replacement for the (25-35) B-amyloid fragment and FGHA,, The number of animals per group waried from 8 to 11. ‘Af’ and 'V’ are the abbrewiations of the (25-35) B-amyloid fragrment and vehicle, respectively. The results we re ana- lysed using unpaired t-test and significance of results was contingent on achiieving a

SUBSTITUT E SHEET (RULE 26)

p-vaflue of less than 0.05 (*p<0.05, **p<0.01), when compared with the first meas- urement period (1-3 min.).

Figuare 24 shows the results of performance of the Surface Righting Reflex test by rat pups at PND4 after intranasal administration of 2.6 ng FGL,, FGLg, or FGLcont! per pup at PND 1, 2, and 3. The number of litters of each treatment group was six.

The results were analysed using one-way ANOVA (F=4.05, P=0.039), and the

Newman-Keuls test showed a statistically significant effeect of FGL_ (P<0.05) and

FGL_4 (P<0.05) when compared to the control- and FGL_comrar 9roups. FGleonirat @S above.

Figure 25 shows the results of performance the Negative Geotaxis Reflex test by rat pup-s at PND 6 and PND 9 after intranasal administratiory of 2.6 pg FGL, FGL4, OF

FGL_cmo Per pup at PND 1, 2, and 3. The number of littemrs of each treatment group wass six. The results were analysed using one-way ANOVA (Fenpe=5.14;

Peamne=0.008). At PND 6, statistically significant effects weere observed for the FGL_ (*p<<0.05) and FGL4 (**p<0.01) groups, when compared fo the control- and FGL. (FGSLeont) groups. At PND G, no significant effect of FGI. was observed (One-way

ANCOVA: Fenpe=0.94; Ppnps=0.44). FGLeontral 5 above.

Figsure 26 shows the schematic presentation of the time c-ourse of in vivo studies.

Figure 27 shows the survival effect of the peptide of SEQ ID NO: 2 (EFloop) in cul- turezs of rat cerebellar granule neurons at low KCI concemtration. Control cells were trecated with insulin growth factor 1(IGF-1) at the same cul ture conditions.

Fig ure 28 shows neuritogenic effect of the EFL peptide SEQ ID NO: 2) on rat hip- poczampal neurons in vitro. The length of neurites in tke EFL treated cultures is cormpared to the length of neurites in cultures treated wiiith vehicle (phosphate buf- fred saline). :

Detailed description of the invention 1. Compound

SUBSTITUTE SHEET (RULE 26)

Compaunds capable of modulating the functiorm of fibroblast growth factor receptor (FGFR) are of greatest importance in view of development of effective drugs for therapeutic treatment of a variety of diseases and pathologic conditions. Thus, it is an objection of the present invention to provide rovel compounds capable of binding to FGFR and modulating the FGFR activity. According to the invention the compounds comprise at least two peptide sequences, wherein at least one of the two sequences comprises a structural motif cormmon for the amino acid sequences capable of low affinity binding to FGFR. 1. Peptide sequences

Thus, in one aspect the invention relates to two individual peptide sequences, wherein at least one of said two individual pep tide sequences comprises an -amino acid sequence of the formula

L1-A-L2-B-L3-C-L4-D-L5 wherein one of A, B, C, D is selected from a hydrophaobic amino acid residue, one of A, B, C, D is selected from a basic armnino acid residue, Asn or Gin, one of A, B, C, D is selected from an acidic -amino acid residue, Asn or Gin, one of A, B, C, Dis Gly or Ala, and

L1, L2, L3, L4 and L5 is selected from a cheemical bond or an amino acid se- quence having nh amino acid residues, wherein nis an integer of from 0 to 5. in the present context the standard one-letter «code for amino acid residues as well as the standard three-letter code are applied. Abbreviations for amino acids are in accordance with the recommendations in the I®UPAC-IUB Joint Commission on Bio- chemical Nomenclature Eur. J. Biochem, 198<4, vol. 184, pp 9-37. Throughout the description and claims either the three letter code or the one letter code for natural amino acids are used. Where the L or D form has not been specified it is to be un- derstood that the amino acid in question has the natural L form, cf. Pure & Appl.

Chem. Vol. (56(5) pp 595-624 (1984) or the D form, so that the peptides formed may be constituted of amino acids of L form, D form , or a sequence of mixed L forms and

D forms.

SUBSTITUTE SHEET (RULE 26)

Where nothing is specified it is to be understood that the C-terminal amino acid of a peptide of the invention exists as the free carboxylic acid, this may also be specified as “-OH". However, the C-terminal amino acid of a compound of the invention may be the amidated derivative, which is indicated as “-NH.". Where nothing else is- stated the N-terminal amino acid of a polypeptide comprise a free amino-group, thiss may also be specified as "H-".

Where nothing else is specified amino acid can be selected from any amino acid. whether naturally occurring or net, such as alfa amino acids, beta amino acids , and/or gamma amino acids. Accordingly, the group comprises but are not limited to :

Ala, Val, Leu, Ile, Pro, Phe, Trp, Met, Gly, Ser, Thr, Cys, Tyr, Asn, Gin, Asp, Glu ,

Lys, Arg, His Aib, Nal, Sar, Om, Lysine analogues, DAP, DAPA and 4Hyp.

Also, according to the invention modifications of the compounds/peptides may be performed, such as for example glycosylation and/or acetylation of the amino acids.

Basic amino acid residues are according to invention represented by the residues of amino acids Arg, Lys, and His, acidic amino acid residues — by the residues of amino acids Glu and Asp, and hydrophobic amino acid residues by the residues of amino acids Leu, lle, Val, Phe, Trp, and Tyr. In a preferred embodiment a basic amino acid residue is represented by Arg or Lys. 1.1 Length of the peptide sequences

A compound of the invention may comprise two individual peptide fragments comprising together between 6 —160 amino acid residues, such as 6-150 amino acid residues, for example 6-140 amino acid residues, such as 6-130 amino acid residues, for example 6-120 amino acid residues, such as 6-110 amino acid residues, for example 6-100 amino acid residues, such as 6-90 amino acid residues, for example 6-80 amino acid residues, such as 6-70 amino acid residues, for example 6-60 amino acid residues, such as 6-50 amino acid residues, for examp8e 6-40 amino acid residues, such as 6-30 amino acid residues, for example 6-20 amino acid residues, such as 6-10 amino acid residues. Thus, in one embodimemt the length of the amino acid seqquence of any of the two individual peptide fragmen-ts

SUBSTITUTE SHEET (RULE 26)

of a compound may vary. In another embodiment the length of the amino acid sequence of each peptide fragment of aa compound may be identical.

The wording “individual peptide fragmemnts/sequences” in the present context means that the peptide fragments/sequences (“two or more) are not contiguously connected by a peptide bond in one amino acid sequence, but they may be connected to each other through a linker, such as for exanple a linker discussed below.

Accordingly, either of the individual peptide sequences of a compound may independently consist of 3-80 amino acid residues, such as 3-70, for example 3-60, such as 3-50 amino acid residues, for example 3-30 amino acid residues, such as 3- 20 amino acid residues, for example 3—15 amino acid residues, such as 3-10 amino acid residues

In another embodiment either of the sesquences of the compound may independently have the length of between 4-80 amin acid residues, such as 4-70, for example 4- 60, such as 4-50 amino acid residues, for example 4-40 amino acid residues, such as 4-30 amino acid residues, for exanmple 4-20 amino acid residues, such as 4-15 amino acid residues.

In further embodiment the sequencess may independently have the length of between 5-80 amino acid residues, sLuch as 5-70, for example 5-60, such as 5-50 amino acid residues, for example 5-4 0 amino acid residues, such as 5-30 amino acid residues, for example 5-20 amino acid residues, such as 5-15 amino acid residues, for example 5-10 amino acid residues.

In yet further embodiment the seque nces may independently have the length of between 6-80 amino acid residues, stuch as 6-70, for example 6-60, such as 6-50 amino acid residues, for example 6-4-0 amino acid residues, such as 6-30 amino acid residues, for example 6-20 amimno acid residues, such as 6-15 amino acid residues, for example 6-10 amino acid residues.

The invention also relates to a comppound comprising two individual amino acid sequences, which may independently have the length of between 7-80 amino acid residues, such as 7-70, for example 7-60, such as 7-50 amino acid residues, for

SUBSTITUTE SHEEET (RULE 26)

WO) 2005/014623 PCT/DK2004/000527 example 7-40 amino acid residues, such as “7-30 amino acid residues, for example 7-20 amino acid residues, such as 7-15 amiro acid residues, for example 8, 9, 10, 11, 12, 13, or 14 amino acid residues.

The length of the individual sequences may also be of 8-80 amino acid residues, such as 8-70, for example 8-60, such as 8-50 amino acid residues, for example 8-40 amino acid residues, such as 8-30 amino a_cid residues, for example 8-20 amino acid residues. Or it may be of 9-80 amino ascid residues, such as 9-70 amino acid residues, for example 9-60 amino acid residues, such as 9-50 amino acid residues, for example 9-40 amino acid residues, suech as 9-30 amino acid residues, for example 9-20 amino acid residues.

A compound which comprises two individual amino acid sequences, either of which is having the length of between 10-80 amineo acid residues, such as 10-70 amino acid residues, for example 10-60 amino acid residues, such as 10-50 amino acid residues, for example 10-40 amino acid residues, such as 10-30 amino acid residues, for example 10-20 amino acid ressidues is also within the scope of the invention. in one preferred embodiment a compound comprises two individual amino acid sequences, wherein either of the sequences may independently have the length of between 15-80 amino acid residues, such as 15-70 amino acid residues, for example 15-60 amino acid residues, such as 15-50 amino acid residues, for example 15-40 amino acid residues, such as 15-30 amino acid residues, for example 15-20.

In another preferred embodiment of the invention the minimal length of any of the two of amino acid sequences of the compourmd may independently be of 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acid residues, and the maximal is 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 486, 47, 48, 49 or 50 amino acid residues. It is also a preferred compound, wherein the length of any of the tao individual sequences is of between 25 and 36 amino acid residues.

In still another preferred embodiment the Mength of a peptide fragment of the compound may independently be of 5, 6, 7, 8,. 9, 10, 11, 12, 13, 14 or 15 amino acid

SUBSTITUTE SHEET (RULE 26)

residues. The length of an individual peptide fragment in the r=ange of 9 to 20 amino accid residues is most preferred. i yet another preferred embodiment of the invention at least sone of the two peptide sequences of the compound derives from the sequence of &a polypeptide selected from the group comprising cell adhesion molecules, cell-surfamce receptors, heparan s-ulphate proteoglycans, and metalloproteases, extracellular™ matrix molecules or girowth factors. 2.2 Origin of the peptide sequences

T hus, it is preferred that at least one of the two peptide sequences of the compound iss derived from the sequence of a polypeptide selected fron the group comprising cell adhesion molecules, cell-surface receptors, heparan saulphate proteoglycans, and metalloproteases, extracellular matrix molecules and growth factors.

Whe authors of the present invention have recently identified® a new class of FGFR

I&gands, which differ from the FGFR natural high affinity ligands, FGFs, 1) on their capacity of binding to FGFR and 2) on the structure of their binding site for the re- ceptor. New FGFR ligands are capable of low affinity bindirg to the receptor at a binding site on FGFR, which is also different from the known FGF binding site of the receptor. According to the application, the FGFR binding site2 of the new low affinity

GFR ligands comprises a sequence comprising the above discussed motif. The mew class of FGFR ligands includes according to the application the proteins, which rnay have a biological function independent on the activity of FGFR. Furthermore, a rew low affinity FGFR ligand may have 1) a function that is executed due to its bind- ing and activating FGFR, and 2) a function that is executed tlihrough another mecha- mism. The function (1) and function (2) may have different biological significance.

Due to a relatively low affinity of binding to the receptor the neew ligands according to fnvention are not capable to modulate the receptor activation, which has already een initiated by FGF. According to the invention the new group of low affinity FGFR

Rigands comprises molecules known in the art as cell adEnesion molecules, cell- surface receptors, heparan sulphate proteoglycans, and metalloproteases, extracel

Bular matrix molecules or growth factors.

SUBSTITUTE SHEET (RULE 26)

According tos the invention the cell adhesion molecule may be selected from the group comprising - Neural =Cell Adhesion Molecule (NCAM) (Swiss-Prot Ass. Nos: P13591,

P13595-01, P13595), - Neural ~cell adhesion molecule L1 (Swiss-Prot Ass. Nos: Q8QYQ7, Q9QY38,

P11627, Q05695, P32004), - Neural Cell Adhesion Molecule-2 (NCAM-2) (Swiss—Prot Ass. No: P36335) - Neurona-glia Cell Adhesion Molecule (Ng-CAM) (Sweiss-Prot Ass. No: Q03696;

Q90933), - Neural cell adhesion molecule CALL (Swiss-Prot A=ss. No: 000533), - Neurogglian (Swiss-Prot Ass. No: P91767, P20241).. - Nr-CAM (HBRAVO, NRCAM, NR-CAM 12) (Swiss—Prot Ass. Nos: 092823, 015179, Q9QVN3 - Axonin—1/TAG-1 (Swiss-Prot Ass. Nos: Q02246, P22063, P28685 ), - Axonal—associated Cell Adhesion Molecule (AxCANM) (NCBI Ass. No:

NP_03 1544.1; Swiss-Prot Ass. No: Q8TC35), - Myelin—Assaociated Glycoprotein (MAG) (Swiss-Pro# Ass. No: P20917), - Neural cell adhesion molecule BIG-1 (Swiss-Prot Aass. No: Q62682), - Neural cell adhesion molecule BIG-2 (Swiss-Prot Aass. No: Q62845), - FascicBin (FAS-2) (Swiss-Prot Ass. No: P22648), - Neural cell adhesion molecule HNB-3/NB-3 (Swiss -Prot Ass. Nos: Q9UQ52,

P975283, Q9.JMB8) - Neural cell adhesion molecule HNB-2/NB-2 (Swiss ~Prot Ass. Nos: 094779,

P0740m9, P97527), - Cadhe rin (Swiss-Prot Ass. No: QIVWT71), - Junctional Adhesion Molecule-1 (JAM-1) (Swiss-Pr-ot Ass. Nos: Q9JKDS, 08879_2), - Neural cell adhesion F3/F11(Contactin) (Swiss-Proot Ass. Nos: Q63198, P1260,

Q1286 0, Q28106, P14781, 093250), - Neurof=ascin (Swiss-Prot Ass. Nos: Q90924, Q91Z@50; 042414), - B-lymphocyte. cell adhesion molecule CD22 (Swisss-Prot Ass. Nos: Q9R094,

P202733), - Neoge-nin (NEO1) (Swiss-Prot Ass. Nos: Q92859, P97603, Q90610, P97798), - Interce=liular Cell Adhesion Molecule-5 (ICAM-5/teleencephalin) (Swiss-Prot Ass.

Nos: Ce8TAM9, Q60625) or

SUBSTITUTE SHEET (RULE 26)

- Galactose binding lectin-12 (galectin-1 2) (Swiss-Prot Ass. Nos: Q91VD1,

QOJKX2, QONZ03), - Galactose binding lectin-4 (galectin-4) (Swiss-Prot Ass. No: Q8K419; P38552), or fragments, or variants thereof.

The functional cell-surface receptor may selected from the group comprising - Fibroblast Growth Factor Receptor 1 CFGFR1) (Swiss-Prot Ass. Nos: Q9QZM?7,

QY9AVV7, QOUD50, Q63827), - Fibroblast Growth Factor Receptor 2 (FGFR2) (Swiss-Prot Ass. Nos: Q96KM2,

P21802, Q63241), - Fibroblast Growth Factor Receptor 3 ( FGFR3) (Swiss-Prot Ass. Nos: Q95M13,

AF487554, Q99052), - Fibroblast Growth Factor Receptor 4 CFGFR4) (Swiss-Prot Ass. No: Q91742), - Neurotrophin Tyrosin Kinase Type-2 ( NTRKT-2) (Swiss-Prot Ass. No:

Q8WXUJ5), - Leukocyte Antigen Related Protein-Tyrrosine Phosphatase (LAR-PTPRF) (Swiss-Prot Ass. Nos: Q9EQ17, Q646505, Q64604, Q2QW67, QSVIS8

P10586), - Nephrin (Swiss-Prot Ass. Nos: Q925S85, Q9JIX2, QIETS9, Q9R044, Q9QZS7,

Q06500), - Protein-Tyrosine Phosphatase Receptor type S (PTPRS) (Swiss-Prot Ass.

Nos: Q64699, Q13332, 075870), - Protein-Tyrosine Phosphatase Receptor type kappa (R-PTP-kappa) (Swiss-

Prot Ass. No: Q15262), - Protein-Tyrosine Phosphatase Receptor type D (PTPRD) (Swiss-Prot Ass.

Nos: Q8WX65, Q9IAJ1, P23468, Q64-487), - Ephrin type-A receptor 8 (EPHA8/Tyrosine-Protein Kinase Receptor EEK) (Swiss-Prot Ass. Nos: 009127, P29322), ~ Ephrin type-A receptor 3 (EPHA8/Tyr-osine-Protein Kinase Receptor ETK- 30 . 1/CEK4) (Swiss-Prot Ass. No: P293188), - Ephrin type-A receptor 2 (Swiss-Prot Ass. No: Q8N32Z2) - Insulin Receptor (IR) (Swiss-Prot Ass. No: QOPWNG) - Insulin-like Growth Factor-1 Receptor (IGF-1) (Swiss-Prot Ass. Nos: Q9QVW4,

P08069, P24062, Q60751, P15127, P 15208) - Insulin-related Receptor (IRR) (Swiss- Prot Ass. No: P14616),

SUBSTITUTE SHEEW (RULE 26)

-Tyrosine-Protein Kinase Receptor Tie-1 (Swiss-Prot Ass. Nos: 06805,

P35590, Q06806), - Roundabout receptor-1 (redbo-1) (Swiss-Prot Ass. Nos: 044924, AF041082,

QOY6N7), - Neuronal nicotinic acetylcholine receptor alpha 3 subunit (CHRNAS) (Swiss-

Prot Ass. Nos: Q8VHH6, FP04757, Q8R4G9, P32297) - Neuronal acetylcholine reeceptor alpha 6 subunit (Swiss-Prot Ass, Nos:

Q15825, Q9ROW9) - Platelet-Derived Growth Factor Receptor Beta (PDGFRB) (Swiss-Prot Ass.

Nos: Q8R406, Q05030), - Interleukin-6 Receptor (IL—6R) (Swiss-Prot Ass. No: Q00560), - Interleukin-23 Receptor (I L-23R) (Swiss-Prot Ass. No: AF461422), - Beta-common cytokine re-ceptor of IL-3, IL5 and GmCsf (Swiss-Prot Ass. No:

P32927) - Cytokine Recaptar-Like molecule 3 (CRLF1) (Swiss-Prot Ass. No: Q9JM58), - Class | Cytokine Receptomr (ZCYTORS) (Swiss-Prot Ass. No: Q9UHHS) - Netrin-1 receptor DCC (Swviss-Prot Ass. No: P43146), - Leukocyte Fc Receptor-lit<e Protein (IFGP2) (Swiss-Prot Ass. Nos: QS6PJ6,

QI96KM2), - Macrophage Scavenger Receptor 2 (MSR2) (Swiss-Prot Ass. No: Q91YK7), or - Granulocyte Colony Stimulating Factor Receptor ( G-CSF-R) (Swiss-Prot Ass.

No: Q99062), or fragments, or variants th ereof.

The heparan sulphate proteoglycan according to the invention is perlecan (Swiss—

Prot Ass. No: P98160), or a framgment, or a variant thereof.

The metalloprotease may be selected from the group comprising - ADAM-8 (Swiss-Prot Ass. No: Q05910), - ADAM-19 (Swiss-Prot Asss. Nos: Q9H013, 035674), - ADAM-8 (Swiss-Prot Ass. No: P78325), - ADAM-12 (Swiss-Prot Asss. Nos: 043184, Q61824), - ADAM-28 (Swiss-Prot Ass. Nos: Q9JLN6, Q61824, Q9XSLE, QIUKQ2), - ADAM-33 precursor (Swisss-Prot Ass. Nos: Q8R533, Q923W9), - ADAM-9 (Swiss-Prot Ass. Nos: Q13433, Q61072),

SUBSTITUTE SHEET (RULE 26)

- ADAM-7 (Swviss-Prot Ass. NoS: Q9H2U9, 035227, Q63180), - ADAM-1A F-ertilin alpha (Swiss-Prot Ass. No: Q8R533), - ADAM-15 (Swiss-Prot Ass. Nos: Q9QYV0, 088839, Q13444), - Metalloprotexinase-desintegrin domain containing protein (TECAM) (Swiss-Prot

Ass. No: AF 163291), - Metalloprotezinase 1 (Swiss-Prot Ass. Nos: 095204, Q9BSI6), or fragments, or variants thereof.

The extracellular matrix molecule may selected from the group cosmprising - Collagen tygoe VII (Swiss-Prot Ass. No: Q63870), - Fibronectin (Swiss-Prot Ass. Nos: Q95KV4, QS5KVS5, P0758 9, Q28377,

U42594, 095609, P11276), or - Tenascin-R_ (Swiss-Prot Ass. Nos: Q15568, 000531, Q9099%5, P10039), or fragments _, or variants thereof. “45

The growth factor according to the invention is Cytokine-like factor-1 (CLF-1) (Swiss-Prot Ass. No:075462), or a fragment, or a variant thereof.

The term * frag ment “ in the present context is meant a polygoeptide having the amino acid sequence, which is about 25 - 99 % of the length of the predetermined protein/polypeptiide amino acid sequence, said polypeptide being a fuctional homo- logue of the precletermined polypeptide selected from the above groups.

The term “variarat’ in the present content means a polypeptide having the amino acid sequence, which i) is 50-100 % homologous to the sequence of the predetermined polypeptide, an-d/or ii) comprises other chemical ‘moieties, such various post- translational modifications of amino acid residues, for example phosphorylation, acylation, glycossilation of the amino acids residues, and/or iii} is physically associ- ated with or chemically bound, such as covalently bound, to another biological molecule(s), for example a polypeptide(s), lipid(s) or carbohydra-te(s), said polypep- tide being a fuct ional homologue of the predetermined polypeptide selected from the above groups. Whe term “physically associated “ means that the molecules are as- sociated via hycErophobic or electrostatic interactions.

SUBSTITUTE SHEET (RULE 26)

By the term “funcional homologue” of a predetermined polypeptide is in the present context meant a rnolecule which is capable of one or more biological functions of the predetermined polypeptide, in a preferred embodiment the biological fFunction(s), which is execute through the mechanism of binding and activating FCGFR.

As it has been discussed above, a preferred functional cell-surface resceptor of the invention is a receptor selected from the family of fibroblast growth factor receptors (FGFRs). The ab-ove molecules according to the invention comprise aan alternative low affinity binding site for FGFR, which is different from the known FESFR high affin- ity binding site of FGFs.

A low affinity FGFR binding site of the above molecules is characterissed in that it comprises an amaino acid sequence which has at least 60 %, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%%, more pref- erably 95% homology to the sequence EVYVVAENQQGKSKA (SEQ {D NO 1), The sequence EVYVWAENQQGKSKA is derived from the neural cell adhession molecule (NCAM) and kno=wn in the prior art as the FGL peptide of NCAM. The- homology of one amino acid ssequence with another amino acid sequence is definexd as a per- centage of identiecal amino acids in the two collated sequences. The homology be- tween amino acid sequences may be calculated using well known alggorithms such as BLOSUM 30, BLOSUM 40, BLOSUM 45, BLOSUM 50, BLOSUM 55, BLOSUM 60, BLOSUM 62,. BLOSUM 65, BLOSUM 70, BLOSUM 75, BLOSUM: 80, BLOSUM 85, or BLOSUM 0.

In another embodiment, the FGFR binding site of the above moleculess comprises an amino acid sequence that has an amino acid sequence having at least 60 %, more preferably at leasst 70%, more preferably at least 80%, more preferabtly at least 80%, more preferably #5% positive amino acid matches compared to to thes sequence

EVYVVAENQQGSKSKA (SEQ ID NO 1). Such sequence is defined b-y the applica- tion as a variant =of a predetermined sequence, for example SEQ ID MO: 1. A posi- tive amino acid nnatch is defined as an identity or similarity defined bys physical and/or chemical cproperties of the amino acids having the same positi-on in two com- pared sequencess. Preferred positive amino acid matches of the preseant invention are KioR,EtoD,LtoM,QtoE,ltoV,ItoL, Ato S,YtoW,KtoQ-,StoT,Nto S and QtoR. :

SUBSTITUTE SHEET (RULE 26)

In still another embodiment an FGFR binding site of a new low affinity binding ligand of FGFR may comprise a fragment or a homologue of the sequence, which i) has at least 60 %, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably 95% homology to the sequence EVYVVAENQQGKSKA (SEQ ID NO 1), or ii) has at least 60 %, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably 95% positive amino acid matches compared to to the sequence EVYVVAENQQGKSKA (SEQ ID } NO 1).

In still yet another embodiment the F GFR binding site of a new low affinity FGFR ligand of the invention may be characterised by the specific 3D feature, namely, it may essentially consists of one or m ore “strand-loop-strand” structural motifs. lt is a preferred feature of the sequences discussed above. According to the invention the strand-loop-strand structure is a preferred 3D structure of an amino acid sequence of the formula

L1-A-L2-B-L3-C-L4-D-L5 wherein one of A, B, C, D is selected fron a hydrophobic amino acid residue, one of A, B, C, D Is selected from a basic amino acid residue, Asn or Gin, one of A, B, C, D is selected from an acidic amino acid residue, Asn or Gin, one of A, B, C, Dis Gly or Ala, and

L1, L2, L3, L4 and L5 is selected from a chemical bond or an amino acid se- quence having n amino acid residues, wherein n is an integer of from 0 to 5.

According to the invention at least one of the two individual amino acid sequences preferably comprises at least one of the above identified structural motifs and is preferably derived from any of the above molecules. Such sequence according to the invention may be selected from any of the sequences identified as SEQ ID NOs: 1-146. In some embodiments one of the two individual sequences may be selected from one group of sequences of the groups 1 to 12 as identified below, and another of the two individual sequences may be selected from another group of sequences.

In other embodiments the sequences may be selected from the same group of se- quences.

SUBSTITUTE SHEET (RULE 26)

Thus, in one embodiment one amino acid sequence of the compound or either of the sequences may be independently selected from time sequences of the group 1:

EVYVVAENQQGKSKA (SEQ ID NO 1),

NIEVWVEAENALGKKYV (SEQ ID NO: 2),

ATNRQGKVKAFAHL (SEQ ID NO: 3),

RYVELYVVADSQEFQK (SEQ ID NO: 4)

VAENSRGKNVAKG (SEQ ID NO: 5),

GEYWCVAENQYGQR (SEQ ID NO: 6),

RLAALNGKGLGEIS (SEQ ID NO: 7),

KYIAENMKAQNVAKEI (SEQ ID NO: 8),

TIMGLKPETRYAVR (SEQ ID NO: 9).

In another embodiment at least one of the at least two peptide sequences may be selected from the group 2 consisting of the sequences

NMGIWVQAENALG (SEQ ID NO: 11),

IWVQAENMLG (SEQ ID NO: 12),

EIWVEATNRLG (SEQ ID NO: 13),

VWVQAANALG (SEQ ID NO: 14),

EVWIEKDPAKGRI (SEQ ID NO: 15),

ATNKGGEVKKNGHL (SEQ ID NO: 16). in still another embodiment at least one of the at least: two peptide sequences may be selected from the group 3 consisting of the sequermces

KYVELYLVADYLEFQK (SEQ ID NO: 17),

RYVELYVVVDNAEFQ (SEQ ID NO: 18),

KYVELVIVADNREFQR (SEQ ID NO: 19),

KYIEYYLVLDNGEFKR (SEQ ID NO: 20),

RYLELYIVADHTLF (SEQ ID NO: 21),

KYVELFIVADDTVYRR (SEQ ID NO: 24),

KFIELFVVADEYVYRR (SEQ ID NO: 25),

KIVEKVIVADNSEVRK (SEQ 1D NO: 26),

VELVIVADHSEAQK (SEQ ID NO: 27)

SUBSTITUTE SHEET (RULE 2:6)

Anoth:er embodiment concems the sequence selected from the group 4 consisting of the seaquences

VAENSRGKNIAKG (SEQ ID NO: 28),

IAENSRGKNVARG (SEQ ID NO: 29),

AIEENSRGKNSFRG (SEQ ID NO: 30), 1A SNLRGRNLAKG (SEQ ID NO: 31),

IPENSLGKTYAKG (SEQ ID NO: 32), 1A ENMKAQNEAK (SEQ ID NO: 33)

In stil another embodiment the sequence may be selected from the group 5 consist- ing of” the sequences

G EYWCVAKNRVGQ (SEQ ID NO: 35),

G-SYTCVAENMVGK (SEQ ID NO: 36),

G-KYVCVGTNMVGER (SEQ ID NO: 37), 16 G=NYTCVVENEYG (SEQ ID NO: 38),

GSEYTCLAGNSIG (SEQ ID NO: 39),

Q=YYCVAENGYG (SEQ ID NO: 40),

GEEYYQEAEQNGYG (SEQ iD NO: 41),

GSNYTCLVENEYG (SEQ ID NO: 42),

GsMYQCLAENAYG (SEQ ID NO: 43),

GEMYQCAENTHG (SEQ ID NO: 44),

GSIYYCLASNNYG (SEQ ID NO: 45),

GSGYYCTADNSYG (SEQ ID NO: 46),

GSEYQCFARNDYG (SEQ ID NO: 47),

GSEYFCLASNKMG (SEQ ID NO: 48), :

GSEYQCFARNKFG (SEQ ID NO: 49),

GSEYFCLASNKMG (SEQ ID NO: 50),

GSGYYCTADNNYG (SEQ ID NO: 51),

GENYSCEAENAWGTK (SEQ ID NO: 52),

GEYTCLAENSLG (SEQ ID NO: 53),

GSEYECVAENGRLG (SEQ ID NO: 54),

GENYTCVVENKFGR (SEQ 1D NO: 55),

GSEYTCLAGNSIG (SEQ ID NO: 56),

GEEYFCVASNPIG (SEQ ID NO: 57),

ETYTCIANNQAGE (SEQ ID NO: 58),

SUBSTITUTE SHEET (RULE= 26)

GMYQCVAENIKHLG (SEQ ID NO: 59),

GEYMCTASNTTIGQ (SEQ ID NO: 60),

EYVCIAENKA.GEQ (SEQ ID NO: 61),

GDYTLIAKNE YGK (SEQ ID NO: 62),

GFYQCVAEN EAG (SEQ ID NO: 63),

GKYECVATN=SAGTR (SEQ ID NO: 64),

GEYFCVYNN SLG (SEQ ID NO: 65),

GEYECAATN.AHGR (SEQ ID NO: 66),

GAYWCQGTMNSVGK (SEQ ID NO: 67),

GTYSCVAEN ILG (SEQ ID NO: 68).

In yet another em bodiment one or both sequences are selected from the grosup 6:

RVAAVNGKGQGDYS (SEQ ID NO: 69),

RVAAINGCGHEGPFS (SEQ ID NO: 70),

AVLNGKGLGs (SEQ ID NO: 71),

RLAAKNRAG=LGE (SEQ ID NO: 73),

RLGVVTGKDsLGEI (SEQ ID NO: 74).

Stil, in another embodiment the sequence may be selected from the group 7:

TVTGLKPETSYMVK (SEQ ID NO: 75),

TLTGLQPSTRYRYV (SEQ ID NO: 77),

TLLGLKPDTWYDIK (SEQ ID NO: 78),

TLQGLRPET.AYELR (SEQ ID NO: 79),

TLRGLRPET#AYELR (SEQ ID NO: 80),

TLMNLRPKTeGYSVR (SEQ ID NO: 81),

TISGLKPDTTY (SEQ ID NO: 83),

TLQGLKPDT.AY (SEQ ID NO: 84),

LRGLKPWTQYAV (SEQ ID NO: 85),

IDGLEPDTEWIVR (SEQ ID NO: 86),

LQGLKPWTQYAI (SEQ ID NO: 87),

TITGLEPGTEEYTIQ (SEQ ID NO: 88),

GLKPWTQYA (SEQ ID NO: 89),

TLASLKPWT QYAV (SEQ ID NO: 90),

LMGLQPATEEYIV (SEQ ID NO: 91).

SUBSTITUTE SHEET (RULE 26)

In other em bodiments the invention may concem a compound comprising the se- quences of &he groups 8, 9, 10 «or 11, wherein the grou p 8 is consisting of the sequences

KGMGP-MSEAVQFRT (SEQ ID NO: 92),

TLTGLKIPDTTYDVK (SEQ ID NO: 93),

ISGLQP=ETSYSL (SEQ ID NO: 94),

TLLGLKCPDTTYDIK (SEQ ID NO: 95),

TISGLTEPETTYSI (SEQ ID NO: 96),

GNYSCRLAENRLGR (SEQ ID NO: 97),

GNYTC®WVENRVG (SEQ ID NO: 98),

NGVLTGYVLRY (SEQ ID NO: 101),

NGVLTGSYNLRY (SEQ ID NO: 102),

NGNLTGSYLLQY (SEQ ID NO: 103),

VDENG"VLTGYKIYY (SEQ ID NO: 104),

THNGALLVGYSVRY (SEQ ID NO: 105),

NGILTETYILKY (SEQ ID NO: 106),

NGILIGWTLRY (SEQ ID NO: 107),

THSGQETGYKIRY (SEQ ID NO: 108),

NGKITGSYIIYY (SEQ ID NO: 109),

NGILTETYTLKY (SEQ ID NO: 111),

LDPNGI ITQYEISY (SEQ ID NO: 112),

NGKITGIYIIYY (SEQ ID NO: 113), the group 9 is consisting of the sequences

HLEVQAFNGRGSGPA (SEQ ID NO: 114),

HLTVRAYNGAGYGP (SEQ ID NO: 115),

HLSVKAYNSAGTGPS (SEQ ID NO: 116),

HLAVKAYNSAGTGPS (SEQ ID NO: 117),

NLEVRA\FNSAGDGP (SEQ ID NO: 118),

HLTVLA_YNSKGAGP (SEQ ID NO: 119),

LRVLVF NGRGDGP (SEQ ID NO: 120),

HIDVSA FNSAGYGP (SEQ ID NO: 121),

HLAVEL_FNGR (SEQ ID NO: 122),

LELQSIMNFLGGQPA (SEQ ID NO: 123),

SUBSTITUTE SHEET (RULE 26)

VO 2005/014623 PCT/DK2004/000527

HFTVRAYNGAGYGP (SEQ ID NO: 124),

HLEVQAFNGRGSQPA (SEQ ID NO: 125), the group 10 is consisting of the sequences

VIADQPTFVKYLIK (SEQ ID NO: 128),

TIKGLRPGVVYEGQ (SEQ ID NO: 127),

TLDDLAPDTTYLVQ (SEQ ID NO: 129),

TVSDVTPHAIYTVR (SEQ ID NO: 130),

IIRGLNASTRYLFR (SEQ ID NO:131),

TLMNLRPKTGYSVR (SEQ ID NO:132),

TLTGLKPGTEYEVR (SEQ iD NO: 133),

RVTGLTPKKTYEFR (SEQ ID NO: 135),

LTGLKPGTEYEFR (SEQ ID NO: 136), and the group 11 is consisting of the sequences

EVRVQAVNGGGNGPP (SEQ ID NO: 137),

LIKVVAINDRGE (SEQ ID NO: 138),

VVSIIAVNGREE (SEQ ID NO: 139),

VVSVYAQNQNGE (SEQ ID NO: 140),

TISLVAEKGRHK (SEQ ID NO: 141),

HLEVQAFNGRGSGPA (SEQ ID NO: 142),

HVEVQAFNGRGLGPA (SEQ ID NO: 143),

HVEVQAFNGRGLGPA (SEQ ID NO: 144),

EFRVRAVNGAGEG (SEQ ID NO: 145),

In some embodiments the compound may comprise a sequence selected from the sequences of the group 12:

KYVEMFVVVNHQRFQ (SEQ ID NO: 22),

RYVELFIVVDKERY (SEQ ID NO: 23),

ALNGQGLGATS (SEQ ID NO: 72),

TLTGLKPSTRYRI (SEQ ID NO: 76),

TVSGLKPGTRY (SEQ ID NO: 82),

GTYHCVATNAHG (SEQ ID NO: 99),

LSHNGVLTGYLLSY (SEQ ID NO: 100),

LSHNGIFTLY (SEQ ID NO: 110),

SUBSTITUTE SHEET (RULE 2:6)

TLTELSPSTQYTVK (SEQ ID NO: 128),

GPEHLMPSSTYVAR (SEQ ID NO: 1349),

VARVRTRLAPGSRLS (SEQ ID NO: 1486). or

QFIAENMKSHNETKEV (SEQ ID NO: 34).

The present invention also relates to fragments of the above peptide sequences having at least 40%, more preferably at lseast 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95% of the- length of a predetermined sequence set forth in SEQ ID NOS: 1-146, wherein an a mino acid sequence homology between a fragment and the predetermined sequences is 100%. A variant in the present context is defined as an amino acid sequence hav-ing at least 60 %, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably 95% homology to a sequence selected from SEQ ID NOS: 1-148, or an amino acid sequence having at least 60 %, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably 95% positive amino acid matches compared to a sequence selected from SEQ ID NOS: 1-146. A positive amino acid match is defined as an identity or similarity defined by physical and/or chemical properties of the amino acids having the same position in two compared sequences. Preferred positive amino acid matches of the present invention are Kto

R EtoD,LtoM, QtoE,ltoV,ItoL,AtomS, YtoW,KtoQ,S1oT, NtoSandQto

R. The homology of one amino acid sequesnce with another amino acid is defined as a percentage of identical amino acids in the two collated sequences. A homologue in the present context is defined as an a mino acid sequence which has less then 80% and more then 19 %, such as 50-59%, for example 55%, such as 40-49%, for example 45%, such as 30-39%, for exarmple 35%, such as 20-29%, for example 25% homology to any of the sequences s=et forth in SEQ ID NOS: 1-146 having re- mained some of the physical properties Of the predetermined sequences, such as for example the three-dimensional structure or some of the functional properties, such as for example a capability to interacct with another molecule, in particular with a receptor molecule. A variant of a homodogue in the present context is defined as an amino acid sequence having at least 60 %, more preferably at least 70%, more preferably at least 80%, more preferably amt least 90%, more preferably 95% positive amino acid matches compared to a homologue of any of the sequences selected

SUBSTITUTE SHEECT (RULE 26)

from SEQ ID NOS: 1-846. Preferred embodiments of the positive amino acid matches is as the defineed above. The invention concerns the fragments, variants and homologues, which remain a capability of the predetermined sequences to in- teract with the cell-surface receptor defined below. 6 . in one preferred embodiment of the invention, the selected sequence is

EVYVVAENQQGKSKA (SEQ ID NO: 1). In another embodiment the preferred se- quence is NIEVWVEAENIALGKKYV (SEQ ID NO: 2).

According to the invention, the compound may comprise at least two different indi- vidual peptide sequences selected from any of the above sequences (SE€Q ID NO: 1-148). Yet, the compound may comprise two individual sequences having the iden- tical amino acid sequence selected from any of the above sequences. in one preferred embodiment the compound comprises two identical indivi dual pep- tide sequences, wherein the sequence is EVYVVAENQQGKSKA (SEQ ID INO: 1). In another preferred embodiment the compound comprises two identical individual peptide sequences, whe rein the sequence is NIEVWVEAENALGKKYV (SE Q ID NO: 2). In stilt another preferred embodiment the compound comprises two different indi- vidual peptide sequences, wherein one of the sequences is EVYVVAENQ: QGKSKA (SEQ ID NO: 1), and thee other is NIEVWVEAENALGKKY (SEQ ID NO: 2). Other preferred embodiments encompass compounds comprising two individual amino acid sequences compris ing fragments, variants or homologues of SEQ ID= NO: 1 or 2, wherein said fragments, variants and homologues being as defined aloove. In a preferred embodiment, the fragments, homologues or variants of SEQ ID INO: 1 are selected from the sequences of SEQ ID NOs: 3-9, 69-74, 100, 125 and 1 37-146 of the groups 1, 6, 11 and -12 identified above, and the fragments, homologues or vari- ants of SEQ ID NO: 2 are selected from the sequences of SEQ ID NOs: 75-91, 114- 123, 126-132 of the grousps 7, 9 and 10 identified above.

In one embodiment at least one of the two individual amino sequences of" the com- pound comprise, essentially comprise, or consist of two or more contiguous peptide sequences of SEQ ID NOS: 1-146 co-joined by peptide bonds in one peptide chain.

In other embodiments, the co-joined peptide sequences may be identical, such as for example SEQ ID NO: 1 repeated in one peptide chain 2 to 5 times, or for exam-

SUBSTITUTE SHEET (RULE 26)

ple SEQ ID NO: 2 repeated in a peptide chain 2 to 5 times, or for example any of th-e sequences SEQ ID NO: 3--146 being repeated in a peptide chain 2 to 5 times form- ing thereby an oligomer (nultimer) consisting of identical monomers. In other en- bodiments monomers of thes peptide fragments forming an oligomer may differ in th e amino acid sequence, suche as for example in the oligomer (dimer) consisting of two monomers, one of which iss having the amino acid sequence of SEQ ID NO: 1 an d another of SEQ ID NO: 2, or any of the sequences set forth in SEQ ID NOS: 3-146.

According to the invention, the oligomer (multimer) may consist of 2, 3, 4, 5, 6, 7, 8, 9 or 10 repeated sequencess (monomers).

A compound consisting off two individual peptide sequences connected to eacch other through a linker, whewrein each of said individual peptide sequences is a single copy of any of the sequences identified in SEQ ID NOS:1-146, is a preferred conm- pound of the invention. Even more preferred a compound, wherein each of the twwo 16 individual peptide sequences is the sequence of SEQ ID NO:1, or a compound, wherein each of the two imdividual peptide sequences is the sequence of SEQ I'D

NO: 2.

Orientation of the individuak peptide sequences in a compound may be either N to CC,

C to N or mixed. The weording “N to C orientation* means that two individual : sequences are connected to a linker through their N-terminal amino acid residue=, such as a compound of the structure (COOH)peptide sequence(NHZ2)-linke-r- (NH2)peptide sequence(CCOOH). The wording “C to N orientation“‘means that twwo predetermined sequences are connected to a linker through their C-terminal amirmo acid residue, such as a cormnpound of the structure (NH2)peptide sequence(COOH )}- linker(COOH)peptide sequience(NH2). The term “mixed orientation” is meant that two predetermined sequernces are connected to a linker either through their C- terminal amino acid residue or N-terminal, such as one peptide sequence though iis

N-terminal amino "acid ressidue and another through its C-terminal amino acid residue, for example as a compound of the type:(NH2)peptide sequence(COOH )- linker-(NH2)peptide sequerace(COOH). 1.3 Liker

SUBSTIWUTE SHEET (RULE 26)

According to the invention two individual peeptide sequences of a compound are co- joined by a linker. A linker molecule is according to the invention is selected from achiral di-, tri- or tetracarboxylic acids, saied acids having the general formula

X[(AInCOOH][(B)mCOOH] wherein n and m independently are an integer of from 1 to 20,

X is HN, H.N(CR;)pCR, RHN(CR,)pCR, HO(CR;)pCR, HS(CR;)pCR, halogen— -10 (CR)PCR, HOOC(CR,)PCR, ROOC(CRR pCR, HCO(CR.)pCR, RCO(CR:)pCR , [HOOC(AN][HOOC(B)M]CR(CR.)pCR, H.N(CR2)p, RHN(CR;)p, HO(CR:)p ,

HS(CR;)p, halogen-(CR;)p, HOOC(CR,)p, ROOC(CR,)p, HCO(CR)p, RCO(CR)p , or [HOOC(A)nJ[HOOC(B)M}(CR,)p , wherein p is 0 or integer of from 1 to 20,

A and B independently are a substituted ©r unsubstituted C,.1o alkyl, a substituted om “15 unsubstituted C,.¢ alkenyl, a substituted oer unsubstituted cyclic moiety, a substitute or unsubstituted heterocyclic moiety, a su bstituted or unsubstituted aromatic moiety, or A and B together form a substituted or unsubstituted cyclic moiety, substituted omr unsubstituted heterocyclic moiety, substit_ated or unsubstituted aromatic moiety. 20 Under the term C,.4 alkyl is meant straighmt or branched chain alkyl groups having 1— carbon atoms, e.g. methyl, ethyl, isopropyl, butyl, and tertbutyl.

Under the term Ca. alkenyl is meant stralight or branched chain alkenyl groups hav— ing 2-10 carbon atoms, e.g. ethynyl, propeenyl, isopropenyl, butenyl, and tert-butenyl.-

Under the term cyclic moiety is meant cycllohexan, and cyclopentane.

Under the term aromatic moiety is meant phenyl.

The wording “A and B forms a cyclic, he®erocyclic or aromatic moiety® denotes cy— clohexan, piperidine, benzene, and pyridirme.

According to the invention two individual peeptide sequences comprising two or more= of the defined above amino acid sequences are connected to each other so that one= of these two peptide sequences is covale ntly bound to one of two carboxylic groups

SUBSTITUTE SHEET (FRRULE 26)

of a linker molecule selected from the molecules defired above and another of the peptide sequences is covalently bound to another carboxylic group of said linker molecule. The peptide sequences are covalently boumd to the linker through their amino- or carboxy-groups of the N- or C terminal amimno acid residue, respectively.

Accordingly, a compound of the invention has the formula

COOH/CONH2-peptide sequence-NH-CO-linker-CO-N H-peptide sequence-

COOH/CONH2 or NH2-peptide sequence-CO-NH-linker-NH-CO-peptidie sequence-NH2. 2. Production of the compound 2.1 Production of individual peptide sequences

The peptide sequences of the present invention may be prepared by any conven- tional synthetic methods, recombinant DNA technoloegies, enzymatic cleavage of full-length proteins which the peptide sequences are dserived from, or a combination of said methods.

Recombinant preparation

Thus, in one embodiment the peptides of the inventiomn are produced by use of re- combinant DNA technologies.

The DNA sequence encoding a peptide or the corresponding full-length protein the peptide originates from may be prepared synthetically by established standard methods, e.g. the phosphoamidine method described . by Beaucage and Caruthers, 1981, Tetrahedron Lett. 22:1859-1869, or the method described by Matthes et al., 1984, EMBO J. 3:801-805. According to the phosphoaamidine method, oligonucleo- tides are synthesised, e.g. in an automatic DNA syrithesiser, purified, annealed, ligated and cloned in suitable vectors.

The DNA sequence encoding a peptide may also be prepared by fragmentation of the DNA sequences encoding the corresponding full-length protein of peptide origin, using DNAase | according to a standard protocol (Sambrook et al., Molecular clon- ing: A Laboratory manual. 2 rd ed., CSHL Press, Cold SSpring Harbor, NY, 1989). The

SUBSTITUTE SHEET (RULE 26)

presexnt invention relates to full-length proteins selsected from the groups of proteins identified above. The DNA encoding the full-lengtrm proteins of the invention may al- ternaatively be fragmented using specific restriction endonucleases. The fragments of

DNA. are further purified using standard procedures described in Sambrook et al,

Mole=cular cloning: A Laboratory manual. 2 rd ed., CSHL Press, Cold Spring Harbor,

NY, —1989.

The DNA sequence encoding a full-length protein may also be of genomic or cDNA origian, for instance obtained by preparing a genormic or cDNA library and screening for IDNA sequences coding for all or part of the Full-length protein by hybridisation usingg synthetic oligonucleotide probes in accordemnce with standard techniques (cf.

Sammbrook et al., Molecular Cloning: A Laborato~ry Manual, 2nd Ed., Cold Spring

Harbor, 1989). The DNA sequence may also be gorepared by polymerase chain re- action using specific primers, for instance as described in US 4,683,202 or Saiki et 16 al., W988, Science 239:487-491.

The DNA sequence is then inserted into a recombinant expression vector, which may be any vector, which may conveniently be ssubjected to recombinant DNA pro- cedures. The choice of vector wili often depend omn the host cell into which it is to be introsduced. Thus, the vector may be an autonomaeously replicating vector, i.e. a vec- tor that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alteematively, the vector may be one whicsh, when introduced into a host cell, is integrated into the host cell genome and repli cated together with the chromosome(s) into wehich it has been integrated.

In the vector, the DNA sequence encoding a peptide or a full-length protein should be operably connected to a suitable promoter sequence. The promoter may be any

DNA sequence, which shows transcriptional acti-vity in the host cell of choice and may~ be derived from genes encoding proteins eittier homologous or heterologous to the host cell. Examples of suitable promoters foer directing the transcription of the codi-ng DNA sequence in mammalian cells are the SV 40 promoter (Subramani et al, 1981, Mol. Cell Biol. 1:854-864), the MT-1 (metallothionein gene) promoter (Pal miter et al., 1983, Science 222: 809-814) or the adenovirus 2 major late pro- moter. A suitable promoter for use in insect cells is the polyhedrin promoter (Vasu- vedaan et al., 1992, FEBS Lett. 311:7-11). Suitable promoters for use in yeast host

SUBSTITUTE SHEET (RULE 26)

cells include promoters from yeast g lycolytic genes (Hitzeman et al., 1980, J. Biol.

Chem. 255:12073-12080; Alber and EXawasaki, 1982, J. Mol. Appl. Gen. 1: 419-434) or alcohol dehydrogenase genes (Young et al., 1982, in Genetic Engineering of M i- croorganisms for Chemicals, Hoilaerder et al, eds., Plenum Press, New York), or the TPI1 (US 4,599,311) or ADH2-<4¢ (Russell et al., 1983, Nature 304.652-6543) promoters. Suitable promoters for use in filamentous fungus host cells are, for iM- stance, the ADH3 promoter (McKnight et al., 1985, EMBO J. 4:2093-2099) or thme tpiA promoter.

The coding DNA sequence may also be operably connected to a suitable terminator, such as the human growth hormone terminator (Palmiter et al., op. cit.) or (for fung al hosts) the TPI1 (Alber and Kawasa ki, op. cit.) or ADH3 (McKnight et al., op. cit.) promoters. The vector may further comprise elements such as polyadenylation sieg- nals (e.g. from SV 40 or the adenowirus 5 Elb region), transcriptional enhancer see- quences (e.g. the SV 40 enhancer) and translational enhancer sequences (e.g. the ones encoding adenovirus VA RNAs").

The recombinant expression vector rmay further comprise a DNA sequence enablirg the vector to replicate in the host ceall in question. An example of such a sequence (when the host cell is a mammalian cell) is the SV 40 origin of replication. The vect«or may also comprise a selectable ma rker, e.g. a gene the product of which compl-e- ments a defect in the host cell, suche as the gene coding for dihydrofolate reductase (DHFR) or one which confers resistance to a drug, e.g. neomycin, hydromycin «or methotrexate.

The procedures used to ligate the DENA sequences coding the peptides or full-length proteins, the promoter and the terminator, respectively, and to insert them into sumit- able vectors containing the informat ion necessary for replication, are well known to persons skilled in the art (cf., for instance, Sambrook et al., op.cit.).

To obtain recombinant peptides of tiie invention the coding DNA sequences may ke usefully fused with a second peptide. coding sequence and a protease cleavage site coding sequence, giving a DNA corstruct encoding the fusion protein, wherein tie protease cleavage site coding sequence positioned between the HBP fragment ard second peptide coding DNA, inserted into a recombinant expression vector, ard

SUBSTITUTE SH EET (RULE 26)

expressed in recombinant host cells. In one embodiment, said second peptide se=- lected from, but not limited by the group comprising glutathion-S-reductase, ca If thymosin, bacterial thioredox®in or human ubiquitin natural or synthetic variants, cor peptides thereof. In another embodiment, a peplide sequence comprising a prote- ase cleavage site may be thes Factor Xa, with the amino acid sequence /EGR, em- terokinase, with the amino a«<cid sequence DDDDK, thrombin, with the amino acied sequence LVPR/GS, or Acha rombacter lyticus, with the amino acid sequence XKX, cleavage site.

The host cell into which the expression vector is introduced may be any cell which i-s capable of expression of the gpeptides or full-length proteins, and is preferably a ela- karyotic cell, such as inverte=brate (insect) cells or vertebrate cells, e.g. Xenopus laevis oocytes or mammalian cells, in particular insect and mammalian cells. Exan- ples of suitable mammalian cell lines are the HEK293 (ATCC CRL-1573), COS (ATCC CRL-1650), BHK (AT CC CRL-1632, ATCC CCL-10) or CHO (ATCC CCL—- 61) cell lines. Methods of transfecting mammalian cells and expressing DNA se=- quences introduced in the cealis are described in e.g. Kaufman and Sharp, J. Mo 1.

Biol. 159, 1982, pp. 601-621; Southern and Berg, 1982, J. Mol. Appl. Genet. 1:3277- 341; Loyter et al., 1982, Proc. Natl. Acad. Sci. USA 79: 422-426; Wigler et al., 19783,

Cell 14.725; Corsaro and Pearson, 1981, in Somatic Cell Genetics 7, p. 603; Grem- ham and van der Eb, 1973, Virol. 52:456; and Neumann et al., 1982, EMBO .H. 1:841-845,

Alternatively, fungal cells (including yeast cells) may be used as host cells. Exane- ples of suitable yeast cells include cells of Saccharomyces spp. or Schizosaccharco- myces spp., in particular stramins of Saccharomyces cerevisiae. Examples of othe=r fungal cells are cells of filamentous fungi, e.g. Aspergillus spp. or Neurospora spp—, in particular strains of Aspergillus oryzae or Aspergillus niger. The use of Aspergillus spp. for the expression of proteins is described in, e.g., EP 238 023.

The medium used to culture the cells may be any conventional medium suitable fosr growing mammalian cells, su«ch as a serum-containing or serum-free medium cons- taining appropriate supplemenmts, or a suitable medium for growing insect, yeast oer fungal cells. Suitable media awe available from commercial suppliers or may be pre--

SUBSTITUTE SHEET (RULE 26)

pared accord ing to published recipes (e.g. in catalogues of the American Type Cul- ture Collectio n).

The peptides or full-length proteins recombinantly produced by the cells may then be recovered from the culture medium by conventional proced ures including sepa- rating the host celis from the medium by centrifugation or filtration, precipitating the proteinaceowas components of the supernatant or filtrate by rwieans of a salt, e.g. ammonium ssulphate, purification by a variety of chromatographic procedures, e.g.

HPLC, ion exchange chromatography, affinity chromatography, or the like.

Synthetic preparation

The method s for synthetic production of peptides are well knowvn in the art. Detailed descriptions as well as practical advice for producing synthetic peptides may be found in Synthetic Peptides: A User's Guide (Advances in Molecular Biology), Grant

G. A. ed., O»xford University Press, 2002, or in: Pharmaceutical Formulation: Devel- opment of Peptides and Proteins, Frokjaer and Hovgaard edss., Taylor and Francis, 1990. ‘

Peptides may for example be synthesised by using Fmoc chemistry and with Acm- protected csysteins. After purification by reversed phase HPLC, peptides may be further processed to obtain for example cyclic or C- or N-termi nal modified isoforms. "The methods for cyclization and terminal modification are wel I-kknown in the art and described ic detail in the above-cited manuals.

In a preferr<d embodiment the peptide sequences of the inverytion are produced syntheticalky, in particular, by the Sequence Assisted Peptide Synthesis (SAPS) method.

Sequence Assisted Peptide Synthesis (SAPS)

Peptides rwnay be synthesised either batchwise in a polyethylene vessel equipped with a polypropylene filter for filtration or in the continuous-flow version of the poly- amide solid-phase method (Dryland, A. and Sheppard, R.C., (1986) J.Chem. Soc.

Perkin Trans. I, 125 - 137.) on a fully automated peptide synthesiser using 9-

SUBSTITUTE SHEET (RULE 26)

fluorenylmethsyloxycarbonyl (Fmoc) or tert. -Butyloxycarbonyl, (Boec) as N-a-amino protecting grup and suitable common protection groups for side—chain functional- ity’s.

The following is a list of chemicals and a description of the procesdure that may be helpful when wusing SAPS for the synthesis of the peptide fragmentss of the invention.

Chemicals arsd procedures 1. Solvents

DMF (N,N-dirnethylformamide, Riedelde-Haen, Germany) (may be= purified by pass- ing through a_ column packed with a strong cation exchange resine, for example Le- watit S 100 M/B/H strong acid, Bayer AG Leverkusen, Germany, and analysed for free amines prior to use by additon of 3,4-dihydro-3-hycdroxy-4-oxo-1,2,3- benzotriazine (Dhbt-Oli) giving rise to a yellow colour (Dhbt-O- ani on) if free amines are present.

DCM (dichloromethane, analytical grade, Riedelde-Maen, Germa:ny) may be used directly withowut purification. 2. Amino acidls:

Fmoc-protect ed amino acids and corresponding pentafluorophenyfl (Pfp) esters may be purchasecd from MilliGen, UK, NovaBiochem, Switzerland, and Bachem, Switzer- land, and thee Dhbt-esters from NovaBiochem, Switzerland in swmuitable side-chain protected forrs.

Bocprotected amino acids may be purchased from Bachem, Switzeerland. 3. Coupling re2agents

Diisopropyicaarbodiimide (DIC) may be purchased from Riedelde-+Hllen, Germany and distilled prior to use.

Dicyclohexylcarbodiimide (DCC) may be purchased from Mercik-Schuchardt, Ni- inchen, Germany, and purified by destillation.O-Benzofriazolyl-N, N,N",N"- tetrame- thyluronium tetrafluoroborate (TBTU) (PerSeptive Biosystems Gmbtl Hamburg,

Germany). 4. Linkers

HMPA, Nova®biochem, Switzerland;

SUBSTITUTE SHEET (RULE 26)

4-hydroxymethylbenzoic acid, Nowabiochem; 4-methoxymandelic acid, Aldrich, Germany;

HMPB, Novabiochem; AM, Novabiochem; 3-(4-hydroxymethylphenoxy) propionic acid, Novabiochem, to be coupled to the resin as a preformed I-hydroxybenzotriazole (HObt) ester generated by means of

DIC.

Racemic 4-methoxymandelic acid (98% pure, Aldrich, Germany) may be used di- rectly as linker or resolved by treatment with (+)-cinchonine (85% pure, Aldrich,

Germany) giving the optical active linker (+)-4-methoxymandelic acid, lax20 =+ 146 (water) in 95.8 % optical purity and (-)-4-methoxymandelic acid, [al20 =- 128.6 (wa- ter) in 88.1 % optical purity. Resolution of (+/-)-4-methoxvmandelic acid (A. Mc Ken- zie,D.J.C. Pirie, (1936), Berichte 69, 868; E. Knorr, (1904), Berichte 37, 3172). (+-)— 4-Methoxymandelic acid (10 g, 54.89 mmol; Aldrich, 98%) is dissolved in 500 mi hot water (60-800C) and the solution decanted while still warm in order to remove in— soluble impurities. (+)-Cinchonine (16,16 g, 54.89 mmol, Aldrich, 85%, [alD20 = + 211 (litt.: + 228 0)) is added to the hot solution in small portions. The solution be— came clear after 15 min stirring at 60-800C and is cooled in ice. After 1 h the pre— cipitate is collected by filtration and dried in an exsiccator over night, yielding 9.9 g of the chinconine salt. The salt is recrystallized from boiling water (80 mi); the solu— tion decanted while still warm and then cooled in ice. The precipitate is collected bw filtration after 1h, washed three times with cold water, and dried in an exsiccato-r over night yielding 7.84 g (16.45 mmol). The chinconine salt (2 g, 4,2 mmol) is diss- solved in 40 ml 2NHC1 and immediately extracted with 3 x 30 ml diethylether. Thee ether phase was dried over Na2S04 and evaporated to dryness yielding 0.55 g 4 methoxymandelic acid. The optical purity of the liberated 4methoxymandelic acid was estimated t018.5% ([a]D20 =+ 270) . After a second recrystallisation of th e chinconine salt followed by liberation of the mandelic acid as described above th e optical purity was estimated to 69.0 % ([a]D20 =+ 100.8 0). A third recrystallisatio=n resulted in an optical purity of 95.8% ([a]D20 = 4 140.0 0). 5. Solid supports

Peptides produced according to the Fmoc-strategy are synthesized on three diffe=r- ent types of solid support using 0.05 M or higher concentrations of Fmoc-protected activated amino acid in DMF.

SUBSTITUTE SHEET (RULE 26)

1) PEG-PS (polyethyleneglycol grafted on polystyrene; TentaGel S NH2 resin, 0.27 mmol/g, Rapp Polymere, Germany or NovaSyn TG resin, 0.29 mmol/g, No- vabiochem, Switzerland) ; 2) PepSyn Gel (polydimethylacrylamide resin —functionalized with sarcosine me- thylester, 1.0 mmol/g; MilliGen, UK). 3) PepSyn K (Kieselguhr supported polydimethylacrylamide resin functionalized with sarcosine methylester 0.11mmol/g; MilliGsen, UK).

Peptides synthesised according to the Bac-strate-gy may be synthesised on a Merri- field-resin (polystyrenedivinylbenzene) with the first amino acid attached (Novabio- 140 chem, Switzerland). 6. Catalysts and other reagents

Diisopropylethylamine (DIEA) may be purchased from Aldrich, Germany,

Ethylenediamine from Fluka, Switzerland,

Piperidine from Riedel-deWhen, Frankfurt, Germany. 4-(N,N-dimethylamino)pyridine (DMAP) may be gourchased from Fluka, Switzerland and used as a catalyst in coupling reactions involwing symmetrical anhydrides. 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzo- triazines (Dhbt-OH) may be obtained from

Fluka, Switzerland, 2© I-hydroxybenzotriazole (HObt) from NovaBiochem, Switzerland. 7. Coupling procedures

The first amino acid is coupled as a symmetrical anhydride in DMF generated from the appropriate N-cc-pratected amino acid and DIC. The following amino acids are coupled as Pfp- or Dhbt-esters or as preformed HObt esters made from appropriate

N-protected amino acids and HObt by means of [DIC or TBTU in DMF. In the case of

Fmoc all acylations are checked by the ninhydrin test performed at 800C in order to prevent Fmoc deprotection during the test. 8. Deprotection of the N-terminal amino acid-amiro (N-a-amino) protecting group.

Deprotection of the Fmoc group is performed by treatment with 20% piperidine in

DMF (1x3 and 1x7 min when synthesized batchvwise) or by flowing the deprotection solvent through the resin (10 min, flow rate 1 ml’/min using continuous flow synthe- sis), followed by wash with DMF until no yellow colour (Dhbt-O-) could be detected after addition of Dhbt-OH to the drained DMF. Dezprotection of the Boc group is per-

SUBSTITUTE SHEET (RWLE 26)

formed by treatment with TFA in DCM 10x1.5 min and 10 min followed by wash 6x9 mir each with DCM, neutralisation with 10% triethyl=amine in DCM (v/v) 2x1.5 min each, followed by 6x8 min wash with DCM. 9. Cleawage of peptide from resin with acid.

Peptidess are cleaved from the resins by treatment witty 95% triflouroacetic acid (TFA, Halocarbon Products Corporation, U.S.A.; Biesterfeld & Co. Hamburg, Ger- many)-wvater viv at r.t. for 2 h. The filtered resins are wa=shed with 85% TFA-water and filtarates and washings evaporated under reduced pressure. The residue is washed with ether and freeze dried from acetic acid-watear. The crude freeze dried product is analysed by high-performance liquid chromatosgraphy (HPLC) and identi- fied by matrix assisted laser desorption ionisation time o=f flight mass spectrometry (MALDE TOF MS) or by electrospray ionisation mass spectrometry (ES-MS). 10. Cleavage of peptide from resin with base.

The dried resin (1 g) is treated with 1M sodium hydroxide (10 ml) at 4 °C and left for 15 min at room temperature. The resin is filtered into a flask containing 10% aq. acetic acid. The peptide is isolated by lyophilization and stubmitted to gel filtration. 11. Cleavage of peptide from resin with TFMSA.

The drEed resin (250 mg) is placed in a round-bottomecd flask with a stirring bar.

Thioank sole/ethanedithiol (2:1, 750F1)) is added, the mixtuare chilled in ice, 5 ml TFA is adde=d and the mixture is stirred for 5 - 10 min. TFMSSA (500K]l) is added drop wise amd the reaction continued at room temperature (r.t.0) for 30 - 60 min. The pep- tide is precipitated after addition of ether. 12. Deporotection of side chain protective groups

Preferaably, the side chains are deprotected simultaneous-ly with the cleavage of the peptide= from the resin. 13. Pre=formed HObt-ester

Method a. 3 eq. Na-a-amino protected amino acid is dissolved in DMIF together with 3 eq. HObt and 3 «q DIC. The solution was left at r.t. for 10 minutzes and then added to the

SUBSTITUTE SHEET (RULE 26)

resin, which had been washed with a solution of 0.2 Dhbt-OH in DMF prior to the addition of the preaactivated amino acid.

Method b. 3 eq. N-a-amino p rotected amino acid is dissolved in DMF together with 3 eq. HOD, 3 eq TBTU and 4,55 eq. DIEA. The solution is left at r.t. for 5 minutes and then added to the resin. Prefomrmed symmetrical anhydride 6 eq.N-u-amino protectec] amino acid is dissolved in DC-M and cooled to 0 °C. DCC (3 eq.) is added and the reeaction con- tinued for 10 min. The solvent is removed in vacuum and the remainers dissolved in

DMF. The solutior is filtered and immediately added to the resin followe=d by 0.1 eq. of DMAP. 14. Estimation of ®he coupling yield of the first N-a-amino protected amirmo acid 3 - 5 mg dry Fmosc-protected peptide-resin is treated with 5 mi 20 times piperidine in

DMF for 10 min at r.t. and the UV absorption for the dibenzofulvenep iperidine ad- duct is estimated at 301 nm. The yield is determined using a calculated extension coefficient e 301 based on a Fmoc-Ala-OH standard. In case of Boc-pr-otection, the coupling is estimaated according to the ninhydrin-method after removal of the Boc- group (Sarin, V.K . et al., (1981), Anal. Biochem, 117, 147-157). 15. Peptide synthmesis on PepSyn K resin

Dry PepSyn K (c=a 500 mg) is covered by ethylenediamine and left at rt over night.

The resin is draired and washed with DMF 10 x 15 ml, 5 min each. After draining the resin is washmed with 10% DIEA in DMF viv (2 x 15 ml, 5 min each) and finally washed with DMF until no yellow colour could be detected by addition ©f Dhbt-OH to the drained DMF . 3 eq. HMPA 3 eq. HObt and 3 eq. DIC is dissolved i n 10 ml DMF and left for activation for 10 min, after which the mixture is added to ®he resin and the coupling continued for 24 h. The resin is drained and washed with DMF (10 x 15 ml, 5 min each) , and the acylation was checked by the ninhydrin t-est. The first amino acid is cowupled as the side chain protected preformed symmetri=cal anhydride (see above), andl the coupling yields estimated as described above. It Es in all cases to be better than 70%. The synthesis is then continued either as "contiruous-flow” or as “"batchwise" ass described below. 16. Continued peaptide synthesis on PepSyn K using continuous-flow te-=chnique.

SUBSTITUTE SHEET (RULE 26)

The resin (ca. 500 mg) with the first amino acid attached is p laced in a column con- nected to thes fully automated peptide synthesiser. The Fmo < group is deprotected as describecl above. The remaining amino acids according to the sequence are coupled as Fmocprotected, if necessary side chain protected, Pfp esters (3 eq.) with the addition «of Dhbt-OH (1 eq.). The end-point of each coupling is determined auto- matically by amonitaring the disappearance of the yellow colour of the Dhbt-OH anion spectrophotometrically. After completed synthesis the pepticde-resin is washed with

DMF (10 mir flow rate 1 mUmin), DCM (3x5 ml, 3 min each) and finally diethyl ether (3x5 ml each) removed from the column and dried in vacuum . 17. Continued batchwise peptide synthesis on PepSyn K.

The resin (casa. 500 mg) with the first amino acid attached is placed in a polyethylene vessel equipped with a polypropylene filter for filtration, and the Fmoc-group depro- tected as described above. The remaining amino acids acc-ording to the sequence are coupled as preformed Fmoc-protected, if necessary side chain protected, HObt esters (3 eqg.) in DMF (5 mi) prepared as described above. The couplings are con- tinued for 2 h unless otherwise specified. Excess reagent iss then removed by DMF washing (122 min, flow rate 1 m/min) All acylations are checked by the ninhydrin test performed aat 80 °C. After completed synthesis the peptidle-resin is washed with

DMF (10 miiin, flow rate 1ml/min), DCM (5x5 ml, 1 min each ) and finally with diethyl ether (6x5 nl, 1 min each), and dried in vacuum. 18. Batchwi=se peptide synthesis on PEG-PS.

TentaGel S NH2 or NovaSyn TG resin (250 mg, 0.27-0.29 mmol/g) is placed in a polyethylene= vessel equipped with a polypropylene filter for filtration. The resin is swelled in DMF (5 ml), and treated with 20% piperidine in [DMF to secure the pres- ence of normprotonated amino groups on the resin. The resim is drained and washed with DMF until no yellow color could be detected after addition of Dhbt-OH to the drained DNVIF. HMPA (3 eq.) is coupled as a preformed HObt-ester as described above and tthe coupling is continued for 24 h. The resin is dlirained and washed with

DMF (5 x 5 ml, 5 min each) and the acylation checked by the ninhydrin test. The first amino acid is coupled as a preformed symmetrical anhydriide as described above.

The couplimg yields of the first Fmoc-protected amino acicds are estimated as de- scribed above. It is in all cases to be better than 60%. Thee following amino acids according teo the sequence are coupled as preformed FmocC-protected, if necessary

SUBSTITUTE SHEET (RULE 26)

side chain protected, HODt esters (3 eq.) as described above. The couplings are continued for 2 h. The resin is drained and wasted with DMF (5 x 5 ml, 5 min each) in order to remove excess reagent. All acylatiorss are checked by the ninhydrin test performed at 80 °C. After completed synthesiss the peptide-resin is washed with

DMF (3x5 mi, 5 min each), DCM (3x5 ml, 1 mire each) and finally diethyl ether (3x5 mi, 1 min each) and dried in vacuo. 19. Batchwise peptide synthesis on PepSyn Gel

Dry PepSyn Gel resin (500 mg, 1 mmol/g) i=s placed in a polyethylene vessel “10 equipped with a polypropylene filter for filtration. The resin is swelled in ethyl- enediamine (15 ml) and gently agitated by shakimng for 20 h. The resin is drained and washed with DMF (10 x 15 ml, 5 min each). After draining the resin is washed with 10% DIEA in DMF v/v (2 x 15 ml, 5 min each) and finally washed with DMF (6 x 15 mi, 5 min each) until no yellow colour could be diietected after addition of Dhbt-OH to “15 the drained DMF. HMPA (3eq.) is coupled ass a preactivated HObt-ester as de- scribed above (method a) and the coupling is continued for 24 h. The resin is drained and washed with DMF (5 x 15 ml, § mir each). The acylation is checked by the ninhydrin test. The first amino acid is couple«d as preformed side chain protected symmetrical anhydride as described above. Th e coupling yields of the first Fmoc— protected amino acids are estimated as describead above. It is in all cases to be bet— ter than 70%. The remaining amino acids according to the sequence are coupled as preformed Fmoc-protected, if necessary side chain protected, HObt esters (3 eq.) as described above (method a). The couplings are continued for 2 h and, if necessary, double coupled over night. The resin is drained and washed with DMF (5 x5 ml, § ' 25 min each) in order to remove excess reagent. A_Il acylations are checked by the nin— hydrin test performed at 80 °C. The Fmoc groups is deprotected as described above.

After completed synthesis the peptide-resin is washed with DMF (3x15 ml, 5 min each), DCM (3x15 ml, 2 min each) and finally diethyl ether (3x15 ml, 2 min each) and dried in vacuum. 19 HPLC.

HPLC may be performed on a Waters 600 E instrument equipped with a Waters 996

Photodiode array detector with a Waters Radial Pak 8 x 100 mm C18 reversed— phase column. Buffer A is 0.1 vol % TFA in wateer, and buffer B is 90 vol % acetoni— trile, 9.9 vol % water and 0.1 vol % TFA. The Buffers are pumped through the col—

SUBSTITUTE SHEET (RWILE 26)

umn at a flow rate of 1.5 m/min using the following gradient: linear gradient from 0% - 70% B (20 min), linear gradient frosm 70 - 100% B (1 min), isocratic 100% B (5 min). 2. Isocratic with 0% B (2 min), linear gradient from 0 - 50% B (23 min), linear gradient from 50 - 100% B (5 min), isocratic 100% B (5 min). 2.2 LPA production of the compound

According fo the invention a compound of interest is preferably obtained by the LPA method. :

LPA method

The LPA method is disclosed in WO 0-0/18791. The method essentially comprises the following steps: 16 (a) providing by solid phase synthesiss or fragment coupling peptide fragment(s) comprising the desired sequence(s), the peptide fragment(s) being attached to a solid phase; (b) if necessary, deprotecting any N-terminal amino groups whole the peptide frag- ment(s) are still attached to a solid p=hase, (c) reacting the peptide fragment(s) ha ving unprotected N-terminal groups with an achiral di-, tri-, or tetracarboxylic acid so as to provide a construct having a ring structure, and (d) cleaving the construct from the solid phase so as to provide an LPA comprising the peptide fragment(s) having free €C-terminal groups.

In the above method, prior step (d) the following steps may be performed: (c1) if present, deprotecting any N-protected groups originating from the carboxylic acid used in step (c), (c2) continuing the solid phase synthesi s or fragment coupling so as to provide pep- tide fragment(s) comprising desired secguence(s) having at least one N-protected N- terminal amino acid group and/or attach ing chemical moieties, and (c3) deprotecting, if present, any N-termuinal amino groups (prior or after step (d)).

SUBSTITUTE SHEET (RULE 26)

The method provides i.a. LPAs presenting desired sequences of the invention with

N fo C orientatiora (step (c)). And also simultaneously sequences with C to n orien- tation (step (c2))

Thus, to obtain a compound of the invention two peptide chairs attached to a solid ~ phase are to be assembled by means of achiral di-, tri- or “etracarboxylic acids.

Suitable achiral dli-, tri- or tetracarboxylic acids to be used irm the present method have the general formula

X[(A)nCOOH][(B»mCOOH] wherein n and m independently are an integer of from 1 to 20, X is HN, A and B in- dependently are aa substituted or unsubstituted Cy. alkyl, a sumbstituted or unsubsti- tuted C..1o alkeny~l, a substituted or unsubstituted cyclic moiety», a substituted or un- substituted heterocyclic moiety, a substituted or unsubstituted aromatic moiety, or A and B together form a substituted or unsubstituted cyclic moiety, substituted or un- substituted heterocyclic moiety, substituted or unsubstituted aromatic moiety. in another embodiment suitable achiral di-, tri- or tetracarboxyl ic acids to be used in the present meth«d have the general formula

X[(A)nCOOH]{(BP®mMCOOH] wherein n and rm are 0 or an integer of from 1 to 20, X is H,N(CR2)pCR, or

RHN(CR;)pCR, wwherein p is 0 or integer of from 1 to 20, wherein each Ris H, a substituted or unsubstituted Cio alkyl, a substituted or unsubsstituted C,.1o alkenyl, a substituted or unsubstituted cyclic moiety, a substituted or unsubstituted heterocyclic molety, a substiteuted or unsubstituted aromatic moiety, or A aand B together form a substituted or urn substituted cyclic moiety, substituted or unsubstituted heterocyclic moiety, substituted or unsubstituted aromatic moiety.

In still another embodiment suitable achiral di-, tri- or tetraccarboxylic acids to be used in the presexnt method have the general formula

X[(A)nCOOH]{(BIMCOOH]

SUBSTITUTE SHEET (RULE 26)

wherein n and m are 0 or an integer of from 1 to 20, X is HO&CR)PCR,

HS(CR2)pCR, halogen-(CR2)pCR, HOOC(CR2)pCR, ROOCH&CR)pCR,

HCO(CR2)p»CR, RCO(CR,)pCR, or [HOOC(A)n}[HOOC(B)M|CR(CR2)pCFR, wherein p is 0 or integer of from 1 to 20, each R independently is H or a substitLated or un- substituted C,.40 alkyl, a substituted or unsubstituted C..10 alkenyl, a substituted or unsubstitutead cyclic moiety, a substituted or unsubstituted heterocyclic moiety, a substituted or unsubstituted aromatic moiety, or A and B together form a ssubstituted or unsubstituted cyclic moiety, substituted or unsubstituted heterocyclic mmoiety, sub- stituted or uansubstituted aromatic moiety.

In yet another embodiment suitable achiral di-, tri- or tetracarboxylic a:cids to be used in the present method have the general formula

X[(AICOO=H][(B)ymCOOH]

Wherein n and m are 0 or an integer of from 1 to 20, X is H:N(CR2)p, REHN(CR2)p,

HO(CR,)p, HS(CRy)p, halogen-(CR;)p, HOOC(CR;)p, ROOC(CR2)p, H CO(CR2)p,

RCO(CR2)p, or [HOOC(A)n][HOOC(B)m)(CR2)p, wherein p is 0 or intege=r of from 1 to 20, each R independently is H or a substituted or unsubstituted C;.,, a’lkyl, a sub- stituted or Lunsubstituted C,.1o alkenyl, a substituted or unsubstituted cycliec moiety, a substituted or unsubstituted heterocyclic moiety, a substituted or unsubs-tituted aro- matic moiety, or A and B together form a substituted or unsubstituted cyeclic moiety, substituted or unsubstituted heterocyclic moiety, substituted or unsubstituted aro- matic moiety.

Under the tterm Cy alkyl! is meant straight or branched chain alkyl groupss having 1- 10 carbon &atoms, e.g. methyl, ethyl, isopropyl, butyl, and tertbutyl.

Under the term Cy alkenyl is meant straight or branched chain alkenyl ggroups hav- ing 2-10 ca rbon atoms, e.g. ethynyl, propenyl, isopropenyl, butenyl, and teert-butenyt.

Under the t.erm cyclic moiety is meant cyclohexan, and cyclopentane.

Under the t-erm aromatic moiety is meant phenyl.

SUBSTITUTE SHEET (RULE 26)

The wording “A and B forms a cyclic, heterocyclic or aromatic moiety” denotes cy- clohexan, piperidine, benzene, and pyridine.

By reaction with a carboxylic acid, a construct of the type

X(CO-sequence)2-solid phase, whe rein X as defined above, is obtained.

By the term “sequence” is in the present content meant a peptide comprising n=atu- rally occurring and/or non-naturally €ccurring amino acids, a PNA-sequence, or poep- tidomimetic. By “naturally occurring amino acids” is meant L- and D-forms of thea 20 acids found in nature. Non-naturallzy occurring amino acids are e.g. modified n=atu- rally occurring amino acids. The term sequence is further intended to comprise one or more of such sequences. Examples of suitable peptidomimetics are describe d in

Marshall G.R.,(1993) Tetrahedron, 49:3547-3558. The term "chemical moieties" denotes an entity enhancing the sol ubility or biological activity of the LPA, and ertity. for directing the LPA to its target or a marker. Preferred embodiments for the se- quences are described above.

The group X permits directly or indirectly continued stepwise synthesis or a fragn—ent coupling of the same sequence, or ©f one or more different sequences and/or m-oie- ties. Orientation of peptide fragments (N to C or C to N) in LPA is defined as de- sired. In one embodiment the present invention features LPAs with N to C oriemnta- tion, in another embodiment it concerns the compounds with simultaneous N tec C and C to N presentation of the seq uences, and in yet another embodiment the se- quences have C to N orientation.

In the case where X comprises a temporally protected amino function, synthesiss or coupling can be carried out directly after protection. Suitable activation of all e=ar- boxyl-containing groups providing e#fective formation of the ring system (on step (c), see above) can be ensured using h alf-equivalent carboxy acid. In case of tri- or tet- racarboxylic acids .the activated carboxy group may further be derivatised witth a diamine such as ethylenediamine o r an amine suitably functionalised for further- re- actions such as mercapto-, an oxy—, an oxo or carboxyl group. In the case of edia-

SUBSTITUTE SHEET (RULE 26)

mine, peptide synthesis or fragment coupling can bee continued directly according to the desired sequence or chemical moiety. In a preferred embodiment, the Fmoc- protection strategy is used, but any amino protection group may be used depending on the synthesis or coupling strategy. Examples are the Boc-protection group strat- egy.

Since the continued stepwise synthesis or fragmerat coupling is performed with one or in case of a bifunctional chemical moiety such ly=sine with two amino acid groups, it has surprisingly been found that a much better result can be obtained as com- pared to conventional tetrameric lysine dendimers obtained by the MAP synthesis.

Furthermore, optimal peptide synthesis proceduress or coupling procedures can be used for the single chains attached to the solid phasse, and their homogeneity can be verified prior to forming the LPA. Cleavage from the solid phase and simultaneous side-chain deprotection can be performed by standard peptide synthesis procedures (described above). A final product may thus be oBbtained having optimal and well- defined composition. Purification by standard chwromatography methods such as

HPLC or gel filtration can easily be performed, if dessired or needed.

Favourable di-, tri- and tetracarboxilyc acids for providing the ring structure may be selected from imino diacetic acid, 2-amino maloni~c¢ acid, 3-amino glutaric acid, 3- methyiamino glutaric acid, 3-chloro glutamic acid, 3-methoxy-carbonyl glutaric acid, 3-acetyl glutaruc acid, glutaric acid, tricarballylic &cid,3,4-bis-carboxymethyl adipic acid, 4-(2-carboxyethyl)-pimelic acid, (3,5-bis-camboxymethyl-phenyl)-acetic acid, 3,4-bis-carboxymethyl-adipic acid, benzene-1,2,48 5-tetra carboxylic acid, 4-(3- carboxy-altylamino)-but-2-enoic acid, 4,4-imino-dib=enzoic acid, 1,4-dihydropyridine- 3,5-dicarboxylic acid, 5-amino isophthalic acid, 2:-chloro malonic acid, 3-hydroxy glutaric acid, and benzene-1,3,5-tricarboxylic acid.

Fragment coupling (fragment coupling or fragment econdensation) may be performed according to standard procedures, e.g. as describe d in Peptide Synthesis protocols,

Methods in Molecular Biology Vol. 35, Chapter 15, 303-316, Nyfeler R, Pennington

MW and Dunne BM Eds.,Humana Press, 1994. Accordingly, fragments may be syn- thesised on a solid phase, cleaved from the solid phase with full preservation of pro- tecting groups, purified and characterised as descsribed above. Suitable fragments may also be obtained by other techniques described above.

SUBSTITUTE SHEET (RULE 2:6)

It iss a preferred embodiment of the invention to use the above LPA method for the prosduction of a compound of the invention. 3. Biological activity

According to the invention the compound can oind and modulate activity of a cell surface receptor through a binding site located within the peptide sequence(s) of the cormpound, said binding site comprising at leasst one amino acid sequence of the formula

L1—A-L2-B-L3-C-L4-D-L5 wherein one of A, B, C, D is selected from a hydropshobic amino acid residue, one of A, B, C, D is selected from a basic amino acid residue, Asn or Gin, one of A, B, C, D is selected from an acidic amino acid residue, Asn or Gin, one of A, B, C, D is Gly or Ala, and

L1, L2, L3, L4 and L5 is selected from a chemical bond or an amino acid se- quence having n amino acid residues, whemrein n is an integer of from 0 to 5.

Th erefore, all peptide sequences having the length of 4 to BO amino acid residues comprising the above receptor binding structu ral motif are in the scope of the inven- tiomn. Preferred embodiments of the peptide ssequences are discussed above. The prezsent invention in a preferred embodiment concerns a compound comprising two individual sequences, each of sald sequencess comprising the above motif and being ca pable of binding fo FGFR.

Receptor

Thus, a compound of the invention is the ligand of a receptor, in particular a func- tiosnal cell-surface receptor.

A cell-surface receptor is defined in the comntent of the present invention as any meolecule comprising at least one polypeptide chain, said molecule being associated wi th the plasma membrane of a cell in suche a way that enables said molecule to re ceive a signal at the outer side of the memtorane, transduce the signal through the

SUBSTITUTE SHEET (RUL-E 26)

membrane, and convey said signal further by inducing a certain action on mole=cular level inside the cell, for example by inducing association or dissociation of mole=cular complexes, or initiating a biochemical reaction, for example auto-phosphorylati-on of the receptor, or proteolytic cleavage of the receptor leading to initiation of intraccellu- lar signal transduction.

The invention relates to a fun«ctional cell-surface receptor. By “functional cell-surface receptor” is meant that the cell-surface receptor of the invention has an identi¥iable group of ligands, the binding of these ligands to the receptor induces intracellular signal transduction, which results in a physiological response of the cell. The physio- logical response, such as for example differentiation, proliferation, survival, &pop- tosis or motility of a cell, depe=nds on the ligand that is involved in the interactior with the receptor, and/or characte=ristics of said Interaction, such as the affinity or dura- tion, and/or a species of the cell which expresses the receptor. By the term “ligand” is defined a compound which is capable to bind to the receptor and thereby to acti- vate or inhibit said receptor. ““Activation” of a receptor is meant that after extra celiu- lar binding of a ligand the receptor became capable to transmit the effect of “l&gand binding” into a cascade of bio chemical reactions collectively termed “receptor sEgnal- ling” or “signal transduction” I nside the cell resuiting in one of the above mentioned physiological responses of th e cell. “Inhibition” of a receptor is meant that after ex- tracellular binding of a ligand the receptor became “silent” or “inactive” and Fs not capable anymore initiate a cascade of biochemical reactions which is normally initi- ated in response to receptor ligand binding. According to the invention compounds featured above are capable of activation or inhibition of a functional cell-surface re- ceptor.

The functional cell-surface re«ceptor of the invention, is in a preferred embodiment a receptor of the family of fiberoblast growth factor receptors (FGFRs) comp xrising

FGFR1, FGFR2, FGFR3 and FGFR4. In most preferred embodiment, a receptor of the invention is FGFR1 or a functional homologue thereof.

By the term “functional homologue of the receptor * is meant a molecule which is capable of : i) extracellular binding of a ligand of FGFR and thereby activating the receptor dependent signal transduction cascade in the cell, and/or

SUBSTITUTE SHEET (RULE 26)

ii) intracellular binding of an adapter molecule of FGFR and thereby acti-vating the receptor depe=ndent signal transduction cascade in the cell.

The term “binding” refers to a direct or indirect interaction between FGFR or an

FGFR homologue and = counter molecule having a binding site for FGFR. In the present context the counter molecule is an extracellular ligand of FGFR, or an intracellular adapter molezcule. An “adapter molecule” is defined in the content of the invention as a molecule, which is capable to recognise an “active state’ of the receptor, selectively birmd to such receptor and convey the signal of "activated receptor” in the cell by Sinducing a cascade of reactions of signal transduction. An intracellular adapter moleecule may be represented by for example STN, FRSS, Grb,

SHP2, PLCy, or PIP3.

Activation of a receptor- often depends on the receptor dimerisation induced by 16 ligand binding. Therefore, the capability of a compound to promote dimerisation (or multimerisation) of the receptor molecules is concerned by the invention as an advantageous feature. T hus, in one embodiment the invention features a com. pound that is capable of dimeri-sing of FGFR. Dimerisation of the receptor by a com: pound of the invention may takee place for example In situation when a natural high =affinity ligand of the receptor, e_g. FGF, is absent in receptor environment and the re ceptor is temporary “silent”, ina ctive. However, in the presence of one of the FGFs &he re- ceptor is occupied and activated by this FGF, and the compounds of the invention have therefore a low capacity or are not capable at all to modulate FGFR re-ceptor signalling induced by thee FGF. Nevertheless, compounds of the invention may at- tenuate binding of naturaal high affinity ligands to FGFR by occupying an altermative binding site(s), if the receptor has been exposed to the compound(s) prior tec FGF exposure. In this way tlhe compounds of the invention may modulate the re=ceptor signalling dependent on FGF. It is known in the art that the cellular response £o acti- vation of a receptor depesnds on the strength of receptor stimulation, which m ay, for example, be characteriseed by the value of affinity of interaction of a ligand with the receptor, and/or by the duration of such interaction. Thus, both affinity and duration of interaction of FGF anc the receptor may be affected by a compound of the inven- tion. Accordingly, it is araother embodiment of the invention to provide a compound, which is capable of mowdulating the receptor signalling induced by a high =affinity ligand. Further embodimments concern 1) a compound(s), which is capable of Enhibit-

SUBSTITUTE SHEET (RULE 26)

ing FEGFR activation induced by a low affinity ligand dis cussed above by competing with ssaid ligand for FGFR binding at the binding site ©f the invention , 2) a com- pounci(s), which Is capable of activating FGFR inhibited by a low affinity ligand dis- cussexd above or another molecule, said another molecule being a natural FGFR inhibitor or artificial substance.

Affinity of interaction

By thme term “interaction” is meant that a compound has a transient or permanent, direc or indirect contact with a cell-surface receptor.

Interexction between two molecules may be characterised by affinity of the interac- tion. The affinity of interaction is commonly expressed by the value of dissociation equil3brium constant, Kd, expressed in moles (M). Kd reflects the ratio between the rate -of dissociation of a ligand molecule from a receptor molecule and the rate of binding of said ligand molecule to said receptor molecule, and thus represents a measure of the strength of binding between these two ¥molecules. Stronger the bind- ing, 1-ower is the value of Kd.

The Jnvention relates to compounds having a relatively low affinity binding to FGFR.

The low affinity interaction of the invention is characterised by Kd having a value in the range of 10° to 10® M, such for example from 1072 to 107M, such for example from 10° to 10 M, such for example from 10™ to 10 M, or about 10“M, or about 10° M. :

Modulation of biological function of the receptor

A caapability of a cell-surface receptor to induce and/or maintain a cellular process upor the ligand binding is herein termed "biological fun ction” of the receptor.

The invention features compounds that are capable of modulating the biological functtion of FGFR by modulating the receptor signalling. By “modulating receptor signaling” is meant activation or inhibition of the production of a cascade of messsenger molecules, which normally takes place in the cell in response to activation of the receptor by an extracellular stimulus.

SUBSTITUTE SHEET (RULE 26)

Activation or inhibition of the receptor signalling is normally reflected by a physio- logical response of the cell expressing said receptor. Therefore, by modulating the receptor signalling it is possible to modulate cell response —

Th e invention preferably concems activation or inhibition of FGFR1-associated sig- na lling, which may for example be reflected by a change in i) the degree of tyrosine phosphorylation of FGFR1; ii) the activation status of one= or more intracellular pro- tei ns involved in any of the FGFR1-associated signal transduction pathways, such as for example the STAT1, JNK, PLCy, ERK, STATS, Pi3K, PKC, FRS2 and/or

GFRB2 proteins; and/or iii) cellular response.

W hen concerned FGFR dependent cellular response, the invention relates to a cel- lular response selected from but not limited by induction/"inhibition of cell differentia- tion or dedifferentiation, apoptosis or cell survival, cell mostility.

M edicament

Ceell death plays a key role in normal neuronal development, where 50% of the de- vesloping neurons are eliminated through programmed ceell death, and in the patho- physiology of neurodegenerative conditions, such as A Izheimer's and Parkinson's dEseases, FGFRs and their ligands has been shown to bee important determinants of neauronal survival both during development and during adulthood (Reuss and von

B chlen und Halbach (2003) Cell tissue Res, 313:139-577). Therefore, a compound, which is capable to promote neuronal cell survival by bineding and activation FGFR is hiighly desirable. Thus, in one aspect the invention feamtures compounds that pro- mote survival of neural cells and can be used as medic=aments for the treatment of conditions involving neural cell death. However, a comgoound of the invention may a Iso be used as a medicament for promotion of survival of another type of cells, e.g. d ifferent type of muscle cells, or, alternatively, for promotion of cell death of still an- osther type of cells, e.g. cancer cells, as the FGFR signalBling has been shown to be a s.urvival factor for both muscle and cancer cells (Ozen et al. (2001) J Nat Cancer frst. 93:1783-90; Miyamoto et al. (1998) J Cell Physiosl. 177:58-67; Detilliux et al. (32003) Cardiovasc Res. 57:8-19).

SUBSTITUTE SHEET (RULE 26)

Activity of cell-surface receptors is strictly regulated in a healthy organism. Muta- tions, abnormal expression or processing of a receptor or the receptor ligands lead to abnormalities in activity of the receptor and therefore lead to dysfunction of the receptor. The dysfunction of the receptor is in turn a reason for dysfunction of the cells which use the receptor for induction and/or maintenance of various celiul ar processes. The latter is the manifestation of a disease. It has also been shown trmat attenuation of FGFR signalling leads to development of a number of different patiwo- logic conditions, e.g. diabetes (Hart et al., Nature 2000, 408:864-8). Activation of

FGF receptors is involved in normal, as well as in pathologic angiogenesis (Slavin,

Cell Biol Int 1995, 19:431-44). It is important for development, proliferation, function- ing and survival skeletal muscle cells, cardiomyocytes and neurons (Merle at al. J

Biol Chem 1995, 270:17361-7; Cheng and Mattson, Neuron 1991, 7:1031-41; Zhu et al., Mech Ageing Dev 1999, 108:77-85). It plays a role in maintenance of normal kidney structure (Cancilla et al., Kidney Int 2001, 60:147-55), and it is implicated in mound healing and cancer disease (Powers et al, Endocr Relat Cancer. 20000, 7.165-97).

The present invention provides compounds capable of modulation of the activity of

FGFRs. Consequently, said compounds are concerned by the invention as medica- ment for the treatment of diseases, wherein modulation of the activity of FGFRs nay be considered as an essential condition for the curing.

Thus, the medicament of the invention is in one embodiment for prevention and/or treatment of 1) diseases and conditions of the central and peripheral nervous system, or of &he muscles or of various organs, and/or 2) diseases or conditions of the central and peripheral nervous system, such as postoperative nerve damage, traumatic nerve damage, impaired myelinatiorm of nerve fibers, postischaemic damage, e.g. resulting from a stroke, Parkinsosn's disease, Alzheimer's disease, Huntington's disease, dementias such as multiin- farct dementia, sclerosis, nerve degeneration associated with diabetes mellitus, disorders affecting the circadian clock or neuro-muscular transmission, and schizophrenia, mood disorders, such as manic depression; 3) for treatment of diseases or conditions of the muscles including conditions vavith impaired function of Neuro-muscular connections, such as after organ transpl=an-

SUBSTITUTE SHEET (RULE 26)

WVO 2005/014623 PCT/DK2004/000527 tation, or such as genetic or traumatic atrophic muscle disorders; or for treat- ment of diseases or conditions of various orggans, such as degenerative condi- tions of the gonads, of the pancreas such as diabetes mellitus type | and Il, of the kidney such as nephrosis and of the heart, liver and bowel, and/or 4) cancer disease, and/or 5) prion diseases.

The invention concerns cancer being any type of solid tumors requiring neoangio- genesis. :

The invention concemns prion diseases selected freom the group consisting of scra- pie, Creutzfeldt-Jakob disease. It has been show that FGFRs plays a distinct role in prion diseases (Castelnau et al. (1994) Exp Neurobiol. 130:407-10; Ye and Carp (2002) J Mol Neurosci. 18:179-88).

In another embodiment a compound of the inventlion is for the manufacture of a me- dicament for 1) promotion of wound-healing, and/or 2) prevention of cell death of heart muscle cellss, such as after acute myocardial infarction, or after. angiogenesis, and/or 3) revascularsation.

FGFRs and their ligands play important roles in C=NS, in particular they are involved in the processes associated with memory and leaaring (Reuss and von Bohlen und

Halbach (2003) Cell Tissue Res. 313:139-57). Thhus, in still another embodiment a compound of the invention is for stimulation of the= ability to learn and/or of the short and/or long-term memory. This embodiment is onee of the preferred embodiments of the invention.

In yet another embodiments a compound of the in=vention is for the manufacture of a medicament for the 1) prevention of cell death due to ischemia; 2) prevention of body damages due to alcohol comsumption;

SUBSTITUTE SHEET (RULE. 26)

In yest still another embodiment a compound of the inventior is for manufacture of a medicament for the treatment of normal, degenerated or damaged cells which normally in vivo express one or more of the proteins selected from the group consisting

Neural Cell Adhesion Molecule (NCAM) (Swiss-Prot Ass . Nos: P13591,

P13595-01, P13596), — Neural cell adhesion molecule L1 (Swiss-Prot Ass. Noss: Q9QYQ7, Q9QY38,

P11627, Q05695, P32004), — Neural Cell Adhesion Molecule-2 (NCAM-2) (Swiss-Proat Ass. No: P36335) — Neuron-glia Cell Adhesion Molecule (Ng-CAM) (Swiss-BProt Ass. No: Q03696;

Q90933), — Neural cell adhesion molecule CALL (Swiss-Prot Ass. Mo: 000533), — Neuroglian (Swiss-Prot Ass. No: P91767, P20241), — Nr-CAM (HBRAVO, NRCAM, NR-CAM 12) (Swiss-Prot. Ass. Nos: Q92823, 015179, Q9QVN3 — Axonin-1/TAG-1 (Swiss-Prot Ass. Nos: Q02246, P2206 3, P28685 ) — Axonal-associated Cell Adhesion Molecule (AXCAM) (NJICBI Ass. No:

NP_031544.1; Swiss-Prot Ass. No: Q8TC35), — Myelin-Associated Glycoprotein (MAG) (Swiss-Prot Asss. No: P2091 7, ~ Neural cell adhesion molecule BIG-1 (Swiss-Prot Ass. Mo: Q62682), — Neural cell adhesion molecule BIG-2 (Swiss-Prot Ass. No: Q62845), — Fasciclin (FAS-2) (Swiss-Prot Ass. No: P22648), = Neural cell adhesion molecule HNB-3/NB-3 (Swiss-Proft Ass. Nos: QOUQ52,

P97528, Q3JMBS) - Neural cell adhesion molecule HNB-2/NB-2 (Swiss-Pro& Ass. Nos: 094779,

P0O7409, P97527), ~- Cadherin (Swiss-Prot Ass, No: Q9VW71), - Junctional Adhesion Molecule-1 (JAM-1) (Swiss-Prot Asss. Nos: Q9JKDS, 088792), - Neural cell adhesion F3/F11(Contactin) (Swiss-Prot Asss. Nos: Q63198, P1260,

Q12860, Q28106, P14781, 083250), - Neurofascin (Swiss-Prot Ass. Nos: Q90924, Q91Z60; O»42414), - B-lymphocyte cell adhesion molecule CD22 (Swiss-Pro& Ass. Nos: Q9R094,

P20273), - Neogenin (NEO1) (Swiss-Prot Ass. Nos: Q92859, P976503, Q90610, P97798),

SUBSTITUTE SHEET (RULE 26)

- Intercellular Cell Adhesion Molecuie-5 (ICAM-5/telencephalin) (Swisss-Prot Ass.

Nos: Q8TAMS, Q=60625) or - Galactose bindineg lectin-12 (galectin-12) (Swiss-Prot Ass. Nos: Q91w/D1,

Q9JKX2, QSNZ0=3), - Galactose bindin g lectin-4 (galectin-4) (Swiss-Prot Ass. No: Q8K419 ; P38552), - Fibroblast Growth Factor Receptor 1 (FGFR1) (Swiss-Prot Ass. Nos= Q9QZM7,

Q99AVV7, QIUD50, Q63827), - Fibroblast Growth Factor Receptor 2 (FGFR2) (Swiss-Prot Ass. Nos : Q96KM2,

P21802, Q63241 ), - Fibroblast Growt h Factor Receptor 3 (FGFR3) (Swiss-Prot Ass. Nos : Q95M13,

AF487554, Q99052), - Fibroblast Growt h Factor Receptor 4 (FGFR4) (Swiss-Prot Ass. No: Q91742), - Neurotrophin Tysosin Kinase Type-2 (NTRKT-2) (Swiss-Prot Ass. Noo:

Q8WXJ5), - Leukocyte Antigesn Related Protein-Tyrosine Phosphatase (LAR-PTHEPRF) (Swiss-Prot Ass. Nos: QIEQ17, Q64605, Q64604, QOQWE7, QIVIS8

P10586), - Nephrin (Swiss-Prot Ass. Nos: Q92585, QQJIX2, Q9ETS9, Q9R044 , Q9QZS7,

Q06500), - Protein-Tyrosine= Phosphatase Receptor type S (PTPRS) (Swiss-Preot Ass.

Nos: Q64699, Q13332, 075870), - Protein-Tyrosine= Phosphatase Receptor type kappa (R-PTP-kappa)» (Swiss-

Prot Ass. No: Q #5262), - Protein-Tyrosine= Phosphatase Receptor type D (PTPRD) (Swiss-Pr-ot Ass.

Nos: Q8WX65, QQ9IAJ1, P23468, 064487), - Ephrin type-A rexceptor 8 (EPHA8/Tyrosine-Protein Kinase Receptor EEK) (Swiss-Prot Ass_ Nos: 009127, P29322), - Ephrin type-A receptor 3 (EPHAS8/Tyrosine-Protein Kinase Receptor ETK- 1/CEK4) (Swiss—Prot Ass. No: P29318), - Ephrin type-A resceptor 2 (Swiss-Prot Ass. No: Q8N3Z2) - Insulin Receptor- (IR) (Swiss-Prot Ass. No: QSPWNS) - Insulin-like Grovasth Factor-1 Receptor (IGF-1) (Swiss-Prot Ass. Nos : Q9QVW4,

P08069, P24062, Q60751, P15127, P156208) - Insulin-related Receptor (IRR) (Swiss-Prot Ass. No: P14616), -Tyrosine-Protein Kinase Receptor Tie-1 (Swiss-Prot Ass. Nos: 0680.5,

SUBSTITUTE SHEET (RULE 26)

P35590, Q06806), - Roundabout receptor-1 (robo-1) (Swiss-Prot Ass. Nos: 044924, AF041082,

QOY6N7), - Neuronal nicotinic acetylcholine receptor alpha 3 subunit (CHRNA3) (Swiss-

Prot Ass. Nos: Q8VHH6, P0475, Q8R4G9, P32297) - Neuronal acetylcholine receptor alpha 6 subunit (Swiss-Prot Ass. Nos:

Q15825, Q9ROW9) - Platelet-Derived Growth Factor Receptor Beta (PDGFRB) (Swiss-Prot Ass.

Nos: Q8R406, Q05030), - Interieukin-6 Receptor (IL-6R) (Swiss-Prot Ass. No: Q00560), ~ Interleukin-23 Receptor (IL-23R) (Swiss-Prot Ass. No: AF461422), - Beta-common cytokine receptor of IL-3, ILS and GmCsf (Swiss-Prot Ass. No:

P32927) - Cytokine Receptor-Like molecule 3 (CRLF 1) (Swiss-Prot Ass. No: Q9JM58), - Class | Cytokine Receptor (ZCY TORS) (Swiss-Prot Ass. No: Q9UHHS5) - Netrin-1 receptor DCC (Swiss-Prot Ass. No: P431486), - Leukocyte Fc Receptor-like Protein (IFGP2) (Swiss-Prot Ass. Nos: Q96PJ6,

Q96KM2), - Macrophage Scavenger Receptor 2 (MSR2) (Swiss-Prot Ass. No: Q91YK7), or - Granulocyte Colony Stimulating Factor Receptor ( G-CSF-R) (Swiss-Prot Ass.

No: Q99062), - perlecan (Swiss-Prot Ass. No: P8160), - ADAM-8 (Swiss-Prot Ass. No: Q@Q5310), - ADAM-19 (Swiss-Prot Ass. Nos: Q9H013, 035674), - ADAM-8 (Swiss-Prot Ass. No: P8325), - ADAM-12 (Swiss-Prot Ass. Nos: 043184, Q61824), - ADAM-28 (Swiss-Prot Ass. Nos: Q9JLN6, Q61824, Q9XSL6, QUKQ2), - ADAM-33 precursor (Swiss-Prot Ass. Nos: Q8R533, Q923W9), - ADAM-9 (Swiss-Prot Ass. Nos: Q13433, Q61072), - ADAM-7 (Swiss-Prot Ass. NoS: Q9H2U9, 035227, Q63180), - ADAM-1A Fertilin alpha (Swiss-Prot Ass. No: Q8R533), - ADAM-15 (Swiss-Prot Ass. Nos: QOQYV0, 088839, Q13444), - Metalloproteinase-desintegrin do-main containing protein (TECAM) (Swiss-Prot

Ass. No: AF163291), - Metalloproteinase 1 (Swiss-Prot Ass. Nos: 095204, Q9BSI6),

SUBSTITUTE SHE ET (RULE 26)

- Collagen type Vil (Swiss-Prot Ass. No: Q63870), - Fibronectin (Swiss-Prot Ass. Noes: Q95KV4, QI5KV5, P07589, Q28377,

U42594, 095609, P11276), or - -Tenascin-R (Swiss-Prot Ass. Nos: Q15568, 000531, Q20995, P10039 - = Cytokine-like factor-1 (CLF-1) (Swiss-Prot Ass. No:075462), - or afragment, or a variant thereof.

In particular, the invention concerns normal, degenerated or damaged ENCAM presenting cells.

The medicament of the invention comprises an effective amount of one or- more compounds as defined above, or a pharmaceutical composition comprising «one or more compounds and pharmaceutically acceptable additives.

Thus, the invention in another aspe=ct also concems a pharmaceutical comp osition comprising at least one compound of the invention.

A further aspect of the invention is aa process of producing a pharmaceutical ccompo- sition, comprising mixing an effective amount of one or more of the compousnds of the invention, or a pharmaceutical composition according to the invention with one or more pharmaceutically acceptable additives or carriers. In one embodiment the compounds are used in combinatior with a prosthetic device, wherein the de=vice is a prosthetic nerve guide. Thus, in a further aspect, the present invention relates to a prosthetic nerve guide, characterisexd in that it comprises one or more of the com- pounds or the pharmaceutical connposition as defined above. Nerve guidees are known in the art. : The invention relates to use of a medicament and/or pharmaceutical composition comprising the compound of invention for the treatment or prophylaxis of any» of the diseases and conditions mentioned below.

Such medicament and/or pharmaceutical composition may suitably be formulated for oral, percutaneous, intramuscular, intravenous, intracranial, intrathecal, in- tracerebroventricular, intranasal or pulmonal administration.

SUBSTITUTE SHIEET (RULE 26)

Strategies in formulation development of medicaments and compositions based on the compounds of the present Envention generally correspond to formulation strate- gies for any other protein-based drug product. Potential problems and the guidance required to overcome these problems are dealt with in several textbooks, e.g. “Therapeutic Peptides and Protein Formulation. Processing and Delivery Systems”,

Ed. A.K. Banga, Technomic Publishing AG, Basel, 1995. injectables are usually prepared either as liquid solutions or suspensions, solid forms suitable for solution in, or suspension in, liquid prior to injection. The prepara- tion may also be emulsified. The active ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient.

Suitable excipients are, for exasmple, water, saline, dextrose, glycerol, ethanol or the like, and combinations thereof. In addition, if desired, the preparation may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH- buffering agents, or which enhance the effectiveness or transportation of the prepa- ration.

Formulations of the compounds of the invention can be prepared by techniques known to the person skilled in the art. The formulations may contain pharmaceuti- cally acceptable carriers and excipients including microspheres, liposomes, micro- capsules, nanoparticles or the | ike.

The preparation may suitably be administered by injection, optionally at the site, where the active ingredient is to exert its effect. Additional formulations which are suitable for other modes of administration include suppositories, nasal, pulmonal and, in some cases, oral formulations. For suppositories, traditional binders and carriers include polyalkylene glycols or triglycerides. Such suppositories may be formed from mixtures containing the active ingredient(s) in the range of from 0.5% to 10%, preferably 1-2%. Oral foramulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sus- tained release formulations or powders and generally contain 10-95% of the active ingredient(s), preferably 25-70%4.

SUBSTITUT E SHEET (RULE 26)

Other formulations are such suitable for nasal and pulmonal administrators, e.g. inhalators and aerosols.

The active compound may be formulated as neutral or salt forms. Pharmaceuatically acceptable salts include acid addition salts (formed with the free amino groups of the peptide compound) and which are formed with inorganic acids such as, for ex- ample, hydrochloric or phosphoric acids, or such organic acids as acetic acid, oxalic acid, tartaric acid, mandelic acid, and the like. Salts formed with the free carboxyl group may also be derived from inorganic bases such as, for example, sodium, po- tassium, ammonium, calcium, or ferric hydroxides, and such organic bases as iso- propylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

The preparations are ad ministered in a manner compatible with the dosage fo-rmula- tion, and in such amount as will be therapeutically effective. The quantity to be ad- ministered depends on the subject to be treated, including, e.g. the weight amd age of the subject, the disease to be treated and the stage of disease. Suitable closage ranges are per kilo body weight normally of the order of several hundred pg active ingredient per administration with a preferred range of from about 0.1 ug to 5@00 ug per kilo body weight. U sing monomeric forms of the compounds, the suitable dos- ages are often in the range of from 0.1 ug to 5000 ug per kilo body weight, such as in the range of from about 0.1 pg to 3000 ug per kilo body weight, and especially in the range of from aboust 0.1 pg to 1000 pg per kilo body weight. Using multimeric forms of the compounds, the suitable dosages are often in the range of from 0.1 ug to 1000 pg per kilo body weight, such as in the range of from about 0.1 ug to 750 pg per kilo body weight, and especially in the range of from about 0.1 ug to 500 pg per kilo body weight such as in the range of from about 0.1 ug to 250 pg per kik 0 body weight. In particular, when administering nasally smaller dosages are use=d than when administering by other routes. Administration may be performed once or may be followed by subsequent administrations. The dosage will also depend on the route of administration and will vary with the age and weight of the subject to be treated. A preferred dosage of multimeric forms would be in the interval 1 mg to 70 mg per 70 kg body weight.

For some indications a localised or substantially localised application is prefexred.

SUBSTITUTE SHEET (RULE 26)

For other indications, intranasal application is prefeerred.

Some of the compounds of the present invention sare sufficiently active, but for some of the others, the effect will be enhanced if the preparation further comprises phar- maceutically acceptable additives and/or carriers. Such additives and carriers will be known in the art. In some cases, it will be advantageous fo include a compound, which promotes delivery of the active substance t=o its target.

In many instances, it will be necessary to administrate the formulation multiple times. Administration may be a continuous infusicon, such as intraventricular infusion or administration in more doses such as more times a day, daily, more times a week, weekly, etc. It is preferred that administration of the medicament is initiated before or shortly after the individual has been subjected to the factor(s) that may lead to cell death. Preferably the medicament i=s administered within 8 hours from the factor onset, such as within 5 hours from the factor onset. Many of the com- pounds exhibit a long term effect whereby admiristration of the compounds may be conducted with long intervals, such as 1 week or- 2 weeks.

In connection with the use in nerve guides, the =administration may be continuous or 20m in small portions based upon controlled release of the active compound(s). Further- more, precursors may be used to contro! the ramte of release and/or site of release.

Other kinds of implants and well as oral administration may similarly be based upon controlled release and/or the use of precursors.

Treatment

Treatment by the use of the compounds/compositions according to the invention is in one embodiment useful for inducing differentiation, modulating proliferation, stimulate regeneration, neuronal plasticity and@ survival of cells, for example cells being implanted or transplanted. This is particularly useful when using compounds having a long term effect. in further embodiment the treatment may be fom stimulation of survival of cells which are at risk of dying due to a variety of factors, such as traumas and injuries, acute diseases, chronic diseases and/or disorders, in particular degenerative diseases

SUBSTITUTE SHEET (RULE 26)

normally leading to cell death, other external facteors, such as medical and/or surgi- cal treatments and/or diagnostic methods that maay cause formation of free radicals or otherwise have cytotoxic effects, such as X-rays and chemotherapy. in relation to chemotherapy the FGFR binding compounds according to the invention are useful in cancer treatment.

Thus, the treatment comprises treatment and/or prophylaxis of cell death in relation to diseases or conditions of the central and peripheral nervous system, such as postoperative nerve damage, traumatic nerve damage, e.g. resulting from spinal cord injury, impaired myelination of nerve fibers, postischaemic damage, €.9. result- ing from a stroke, muitiinfarct dementia, multiple sclerosis, nerve degeneration as- sociated with diabetes mellitus, neuro-muscular- degeneration, schizophrenia, Alz- heimer's disease, Parkinson's disease, or Huntington's disease.

Also, in relation to diseases or conditions of the muscles including conditions with impaired function of neuro-muscular connections, such as genetic or traumatic atro- phic muscle disorders; or for the treatment of d iseases or conditions of various or- gans, such as degenerative conditions of the gosnads, of the pancreas, such as dia- betes mellitus type | and lI, of the kidney, such as nephrosis the compounds accord- ing to the invention may be used for inducing differentiation, modulating proliferation, stimulate regeneration, neuronal plasticity and survival , i.e. stimulating survival.

Furthermore, the compound and/or pharmaceutical composition may be for prevent- ing cell death of heart muscle cells, such as after acute myocardial infarction, in or- der to induce angiogenesis. Furthermore, in one embodiment the compound and/or pharmaceutical composition is for the stimulation of the survival of heart muscle cells, such as survival after acute myocardial imfarction. In another aspect the com- pound and/or pharmaceutical composition is for revascularisation, such as after inju- ries.

It is also within the scope of the inventiori a use of the compound and/or pharmaceutical composition for the promotion of wound-healing. The present compounds are capable of stimulating angioge=nesis and thereby they can promote the wound healing process.

SUBSTITUTE SHEET (RULE 26)

Thee invention further discloses a use of the compound and/or pharmaceutical commposition in the treatment of cancer. Regulation of activation of FGFR is imgportant for tumor agiogenesis, proliferation and spreading.

In yet a further embodiment a use of the compound and/eor pharmaceutical composi- tion is for the stimulation of the ability to leam andlor off the short and/or long term meemory, as FGFR activity is important for differentiation «of neural cells.

In still another embodiment a compound and/or pharmaceutical composition of the in~vention is for the treatment of body damages due to alcohol consumption. Devel opmental malformations of foetuses, long-term neurobeavioral alterations, alco- hoolic liver disease are particularly concerned.

Ttherapeutic treatment of prion diseases including usin g a compound and/or phar- 16 rmaceutical composition is still another embodiment of thae invention.

Im particular the compound and/or pharmaceutical composition of the invention may b-e used in the treatment of clinical conditions, such ass Neoplasms such as malig- maant neoplasms, benign neoplasms, carcinoma in situ and neoplasms of uncertain beehavior, diseases of endocrine glands, such as diabetes mellitus, psychoses, such ams senile and presenile organic psychotic conditions, al: coholic psychoses, drug psy- choses, transient organic psychotic conditions, Alzhefimer's disease, cerebral lipi-

Hoses, epilepsy, general paresis [syphilis], hepatolentiicular degeneration, Hunting- teon's chorea, Jakob-Creutzfeldt disease, multiple sclerosis, Pick's disease of the [orain, syphilis, Schizophrenic disorders, affective psychecses, neurotic disorders, per- ssonality disorders, including character neurosis, nonpesychotic personality disorder associated with organic brain syndromes, paranoid personality disorder, fanatic per- ssonality, paranoid personality (disorder), paranoid traifits, sexual deviations and dis- orders, mental retardation, disease in the nervesystenm and sense organs, cognitive sanomalies, inflammatory disease of the central nervou s system, such as meningitis, «encephalitis, Cerebral degenerations such as Alzheimer's disease, Pick's disease, =senile degeneration of brain, communicating hydro-cephalus, obstructive hydro- . cephalus, Parkinson's disease including other extra pyramidal disease and abnor- mal movement - disorders, spinocerebellar di:sease, cerebellar ataxia,

Marie's, Sanger-Brown, Dyssynergia cerebellaris myoclonica, primary cerebellar de-

SUBSTITUTE SHEET (RULE 26)

germeration, such as spinal muscular atrophy, famuilial, juvenile, adult spinal muscular atrophy, motor neuron disease, amyotrophic lateral sclerosis, motor neuron disease, pro-gressive bulbar palsy, pseudobulbar palsy, primary lateral sclerosis, other ante- rior hom cell diseases, anterior horn cell disease, unspecified, other diseases of spimal cord, syringomyelia and syringobuibia, vascular myelopathies, acute infarc- tio of spinal cord (embolic) (nonembolic), arterial thrombosis of spinal cord, edema of -spinal cord, subacute necrotic myelopathy, subacute combined degeneration of spinal cord in diseases classified elsewhere, mayelopathy, drug-induced, radiation- induced myelitis, disorders of the autonomic nenw/ous system, disorders of peripheral autonomic, sympathetic, parasympathetic, or- vegetative system, familial dy- sa=utonomia [Riley-Day syndrome], idiopathic pe=ripheral autonomic neuropathy, ca- roEid sinus syncope or syndrome, cervical symgpathetic dystrophy or paralysis. pe- rip-heral autonomic neuropathy in disorders classsified elsewhere, amyloidosis, dis- eases of the peripheral nerve system, brachial plexus lesions, cervical rib syndrome, cowstociavicular syndrome, scalenus anterior syndrome, thoracic outlet syndrome, brachial neuritis or radiculitis, including in newbom. Inflammatory and toxic neuropa- th=y, including acute infective polyneuritis, Guillain-Barre syndrome, Postinfectious polyneuritis, polyneuropathy in collagen vascular disease, disorders affecting multi- ple structures of eye, purulent endophthalmitis, diseases of the ear and mastoid process, chronic rheumatic heart disease, ischamemic heart disease, arrhythmia, dis- eases in the pulmonary system, abnormality of organs and soft tissues in newbom, including in the nerve system, complications of the administration of anesthetic or - other sedation in labor and delivery, diseases im the skin including infection, insuffi- cisent circulation problem, injuries, including after surgery, crushing injury, burns. irmjuries to nerves and spinal cord, including cGivision of nerve, lesion in continuity (with or without open wound), traumatic neur-oma (with or without open wound), trsaumatic transient paralysis (with or without cepen wound), accidental puncture or lamceration during medical procedure, injury to o ptic nerve and pathways, optic nerve irmjury, second cranial nerve, injury to optic chiassm, injury to optic pathways, injury to visual cortex, unspecified blindness, injury to Other cranial nerve(s), injury to other and unspecified nerves. Poisoning by drugs, rmedicinal and biological substances, g enetic or traumatic atrophic muscle disorderss; or for the treatment of diseases or conditions of various organs, such as degenerative conditions of the gonads, of the pancreas, such as diabetes mellitus type | andi |, of the kidney, such as nephrosis.

SScrapie, Creutzfeldt-Jakob disease, Gerstm ann-Straussier-Sheinker (GSS) dis-

SUBSTITUTE SHEET (RULE 26)

PCT/DK2004/000527 ease.

According to the invention the treatment and/or prevention of the above conditions and symptoms comprises a step of” administering an effective amount of a com- pound and/or pharmaceutical composition to an individual in need.

Examples

Example 1. Production of different formulations of the FGL peptide (SEQ LD

NO: 1) and other peptides of the invention

Solid Phase Synthesis of the indiviadual peptide chain of FGL

The individual peptide chain of FGL and other peptide sequences of the inventiom, such as for example the EFL peptide, is synthesized by the standard Fmoc-sol id phase method as described above . The synthesis of the peptide used in the expenrri- ments is performed on TentaGel S& RAM resin (90 mg, 0.22 mmol/g). The resin was placed in a polyethylene vessel equipped with a polypropylene fitter for filtration. The resin was swelled in DMF, and tresated with 20% piperidine in DMF to secure thie presence of non-protected amino groups on the resin. Afterwards the resin was drained and washed with DMF unti | no yellow colour could be detected after addition of Dhbt-OH to the drained DMF. Thhe amino acids were coupled one at a time alte=r- nating with removal of Fmoc-grou ps. The Fmoc-amino acids were preactivated in

DMF by TBTU /HOBt before the coupling to the growing resin-bound peptide chaim.

For the removal of Fmoc-groups a ssolution of piperidine in DMF was used.

LPA production of the FGL dimer

The LPA-type dimer of FGL (FGLy ) of the application was made by coupling Boec- iminodiacetic acid to the peptide orm the resin, using TBTU/HOBt as described in WO 00/18791. To reduce side reactio-ns multiple coupling were performed with Boc- iminodiacetic acid as the limiting component. The peptide was cleaved from thme resin and simultaneously deprotectted on the side chains in TFA at the presence of

TES and water as scavengers to wield the peptide amide. The amount of TFA waas reduced by evaporation and the pes=ptide precipitated. Final purification of FGL. waas done by reversed phase HPLC. Thez conditions of HPLC were as follows:

SUBSTITUTE SHEET (RULE 26)

Column: YMC ODS-AMQ, 5 pm, 4.6 x 250 mm, 200 A

Flow: 1.0 mm/min

Mobile phases: A: 09.12% TFA, 95% H,0, 5% ACN

B: 08.1% TFA, 95% ACN, 5% H:0

Gradient: 15% B to 35% B in 20 min, 359% B to 85% B in 1 min and hold for 5 min.

Detection: Diode array detector 190-400 nm

Wavelength: 220 nm

Injection volume: 20 pl,

Concentration of FGLL 0.5 mg/ml

The HPLC purified FGL, was= isolated by lyophilisation. The flowchart of FGL, swyn- thesis is shown in Figure1. Figgure 2 depicts the HPLC elution profile of the final pruri- fication of FGL.. :

Abbreviations

Abbreviations for amino acids are in accordance with the recommendations in the

IUPAC-IUB Joint Commissior on Biochemical Nomenclature Eur. J. Biochem, 19984, vol. 184, pp 9-37

Other abbreviations:

AcOH Acetic acid

TBTU O-(Benzoiriazo¥-1-yl}-N,N,N',N"-tetramethyluronium tetrafiuoroborates

Ida Boc iminodiace=tic acid

MTBE t-Butyl methyl esther

DMF Dimethyiformarmide

EtOH Ethanol, 99,9%

DIPEA N-Ethyl-diisopr-opylamine

HOBt 1-Hydroxybenz=otriazol

NMP N-methylpyrrol idone

TFA Trifluoroacetic acid

TES Triethylsilane

Boc N-tertButyl oxywcarbonyl

Fmoc 9-Fluorenylme-thyloxycarbonyl tBu tert-Butyl

SUBSTITUTE SHEET (RULE 26)

HPLC High pressure liquid chromatography

R Amide-TG-resin

AA Amino acid

Physicochemical properties of FGL.

FGL. h as the structural formula: / CHy-CO-Glu-Val- Tyr-Val-Val-Ala-Glu-Asn-Gin-Glin-Gly-Lys-Ser-Lys-Ala-NH2

HN

«CH -CO-Glu-Val-Tyr-Val-Vak-Ala-Glu-Asn-Gin-G i n-Gly-Lys-Ser-Lys-Ala-NH 2 it conssists of two identical peptide sequences of 15 amino acid residues co-joined by the linker group, amidoacetic acid (N-(carboxymethyl)glycine), on their N-terminals.

The molecular weight of FGL, is 3394.8 Dalton. Nonr-covalent aggregation of the peptid e compound in concentrated water solutions (higher then 0,5 mg/ml) is pre- vented by addition of acetic acid.

FGLg,-g and FGL,s dimmers synthesis

The FGLcys dimer consisting of two individual FGL sequences, each containing an additieonal cysteine residue at the N-terminal end bou nd through an S-S bond, was syntheesized using solid-phase synthesis described in in Goodwin et al. (1998) Bio- org Milled Chem Lett 8:2231-2234.

The FGLlys dimer, constructed as two individual FGL. monomer sequences coupled throusgh their C-terminal to lysine, was made using solid-phase synthesis described in Ra_jagopalan et al. (1995) int J Pept Protein Res.45:173-179.

SyntPesis of dendrimeric tetramer of FGL (FGLy)

The MNMAP-type dendrimer of FGL, FGLg, was synthesised according to the standard mettaod as for example described in PCT/US90/02039.

Figuwe 3 demonstrates the HPLC profile of the final purification of FGLo. The hatched area indicates the tevel of heterogeneity of the synthesised product.

SUBSTITUTE SHEET (RULE 26)

Example 2. The effect of different formulations of FGL aen survival of neurons in vitro

Different formulations of the FGL peptide were copared for biological activity in the surviv-al assays well known in the art and described in detalesd below.

Dopaaminergic neurons (DN)

Dopasmminergic neurons were prepared from Wistar rat embryos at embryonic day 15 (Charles River, Sulzfeld, Germany or Mgllegaard, Denmaark). A pregnant rat was sacrificed and the uterus was taken out and kept on ice in Hank's balanced salt solu- tion (BHBSS; Gibco, BRL). The ventral part of the mesencepialon was dissected from the embryonic brains, homogenised on ice in Gey's balaneced salt solution (GBSS;

Gibco, BRL) supplemented with 5 g glucosell (Sigmau-Aldrich) and thereafter trypsilinised. The dissociated cells were washed in the prese nce of DNAse 1 and soy- bean trypsin inhibitor (Sigma-Aldrich).

For the survival assay isolated neurons were seeded zat a density of 150,000 cells®cm? in 24-well cell culture plates coated with poly-D-ly=sine as described above.

The mneurons were left to differentiate for six days without cor with various concentra- tions of the FGL peptide, after which 6-OHDA was added at a concentration of 100 uM feor two hours. A stock solution was prepared by dissolv~ing 6-OHDA to a concen- tration of 10 mM in 0.1 % (w/v) sodium metabisulfite in orrder to prevent oxidation.

After~ two hours with 6-OHDA, the medium was changed to= Neurobasal medium with suppolements and peptide, and the cell cultures were further incubated for 24 hours, fixed and immunostained for tyrosine hydroxylase. 98 imacyes for each experimental condition in each individual experiment were automatically recorded as described for the clopaminergic neurite outgrowth assay below.

Hipoocampal neurons (HN)

For ~the experiments dissociated hippocampal neurons wewre isolated from Wistar rat embwryos at embryonic day 19 or newborn rats as descritoed by Renn et al. (1999)

Nat Biotechnol. 17:1000-5.

Briefly, hippocampus was isolated from the brain in ice ceold modified Krebs Ringer solution, cleared of blood vessels, roughly homogenisesd by chopping and then

SUBSTITUTE SHEET (RULE 26)

trypsinised. The dissociated cells were washed in the presence of DNAse 1 and s©y- bean trypsin inhibitor.

For the assay neurons were plated at a density of 40,000 cells/cm? on 8-well per- manox slides coated with 10 pg/ml poly-L-lysine in Neurobasal medium. 15 minustes after plating, AB 25-35 was added to a final concentration of 20 uM. The Ap 25-35 had prior to plating been dissolved in sterile deionized water to a concentration ©f 3 mM and incubated at 37°C for four days according to Pigino et al. (2001) J Neuro sci. 21:834-42. This treatment induces fibrillation of the Ap-peptide fragment, ther<by increasing its neurctoxic activity. The neurons were incubated with Neurobasal ame- dium containing the FGL peptide for 70 hours, fixed with 4 % (v/v) formaldehyde, and stained with Hoechst 33258 for 25 minutes as described by Kruman et al. (1997).

Images of 1000-1 500 neurons were randomly recorded for each group in each ex- periment using computer assisted fluorescence microscopy as described for the hip- pocampal neurite outgrowth assay. Nuclei from dead and live neurons were courted using the software package Prima developed at the Protein Laborator, Copenhaagen

University, and the fraction of live neurans relative to the total number of neusons was estimated.

Cerebellar granule neurons (CGN)

Cerebellar granule neurons (CGN) are prepared from postnatal day seven W istar rats largely as previously described by Drejer and Schousboe (1989) Neurochem

Res. 14:751-4.

For the experiments cerebellar tissue was dissected in modified Krebs-Ringer solu- tion kept on ice, and treated as described for the hippocampal neurons abovea. All cell cultures were incubated at 37°C in a humidified atmosphere containing § % «CO.

All animals were handled in accordance with the national guidelines for animal wel- fare.

Primary cultures of CGN were plated at a density of 100,000 cells/cm? on pasly-L- lysine coated 8-well permanox slides in Neurobasal-A medium (Gibco, BRL) sup- plemented with 2 % (v/v) B27, 0.5 % (v/v) glutamax, 100 U/ml penicillin, 100 ag/mi streptomycin and KCI, making the final concentration of KCl in the medium 40 mM. 24 hours after plating, cytosine-g-D-arabinofuranoside (Ara-C; Sigma-Aldrich) was added to a final concentration of 10 uM to avoid proliferation of glial cells, after vwhich the neurons were allowed to differentiate for further six days at 37°C. Apoptotic cell

SUBSTITUTE SHEET (RULE 26)

death was induced by washing twice and changing the medium to Basal Medium

Eagle (BME; Gibco BRL) supplemented with 1 %& (v/v) glutamine, 100 U/ml penicillin and 100 pg/ml streptomycin, 3.5 g D-glucose/l ard 1 9% (v/v) sodium pyruvate (Gibco

BRL) together with various concentrations of peptide. Thereby the concentration of potassium in the cultures was reduced to 5 mM KCl. Two days after induction of apoptosis, the cells were fixed with 4 % formaaldehyde and stained with Hoechst 33258 as described for the survival assay emplozying hippocampal neurons.

The assay is performed as described by D’'Mello et al, (1993) Proc Natl Acad Sci u

S A. 90:10989-93.

Other survival assays using the cultures of CGN... 1. PACE assay

Detection of phosphorylated Erk1/2 and Akt relative to the total number of CGN is carried out according to Versteeg et al. (2000) FEBS Lett. 465:69-73. “15 For the experiments CGN were plated at a de mnsity of 290,000 cells/cm? in poly-L- lysine coated 96-well microtiter plates. The nesurons were grown in Neurobasal-A medium supplemented with 0.5 % (v/v) glutamaxax, 100 U/ml penicillin and 100 ug/ml streptomycin. 24 hours after plating, Ara-C wass added to the cultures as described above. After 72 hours of incubation, half of the medium was replaced with-Neuroba- 20 sal-A medium containing the peptide to be tested. For the Erk1/2 phosphorylation assay, the cultures were further incubated for 0» - 90 minutes, centrifuged at 70 g for 8 minutes, fixed with 4 % (v/v) formaldehydee and immunostained using primary phospho-p42-44 rabbit antibodies and peroxidease-conjugated anti-rabbit secondary antibodies. For the detection of phosphorylate Akt, the cultures were further incu- bated for 10 or 30 minutes, centrifuged at 70 -g for 8 minutes, fixed with 4 % (v/v) formaldehyde and immunostained using polysclonal antibodies against Akt phos- phorylated at Ser473 and peroxidase-conjugamted secondary antibodies. The total number of neurons was in both phosphorylation-assays estimated by staining with crystal violet. 2. TUNEL assay

CGN were plated at a density of 60,000 cellss/cm? on poly-L-lysine coated 8-well permanox slides. The cultures were grown in BNeurobasal-A medium supplemented with 2 % (v/v) B27, 0.5 % (v/v) glutamax, 100 LJ/mi penicillin, 100 pg/mi streptomycin

SUBSTITUTE SHEET (RULE 26)

and 40 mM KC! (final concentration) for six days, and apeoptosis was induced as de- scribed for the CGN survival assay above. 24 hours afteer induction of apoptotic cell de=ath, the cultures were fixed and the extent of DNA-fraagmentation was measured ussing the ApoAlert apoptosis detection kit, essentially ass described by the manufac- tuwer. Briefly, blunt ends of double-stranded DNA molecules were enzymatically a- balled with fluorescein-dUTP, and all the neurons were Stained with propidium iodide at a concentration of 750 ng/ml. Images of at least 200 neurons from each group in each experiment were obtained using a BioRad Radiance laser scanning system 2000 coupled to a Nikon Eclipse TE 200 confocal microscope equipped with an oll immersion 60x 1.4 NA objective (Nikon, Tokyo, Japan). The fraction of TUNEL- p-ositive neurons was determined by counting.

RESULTS

GL promotes survival of dopaminergic, hippocampal and cerebellar granule neu- rons

Since FGL is an agoinist of the FGF receptor with a greowth factor-like activity, it was investigated whether FGL could act as a neuroprotectant under various neurotoxic conditions. Cell death in dopaminergic neurons was induced by addition of 6-OHDA.

En Figure 4a it can be seen that the number of livee dopaminergic neurons was strongly decreased after exposure {0 6-OHDA as compared to the untreated control.

Addition of 10 ng/ml glia derived neurotrophic factor (CSDNF) partially prevented this seuronal loss (150 % as compared to the 6-OHDA-treated control cells set to 100 <9). When cultures were grown in the presence of various concentrations of FGLd (Figure 4b), survival of dopaminergic neurons treated “with 6-OHDA increased statis- tically significantly with a maximal rescue of approximaately 135 % at a concentration of 1 pg/ml FGLd and the effect exhibited a bell-shaped dose-response relationship.

Thus, FGLd is able to rescue dopaminergic neurons to approximately the same ex- tent as GDNF in a survival model employing 6-OHDA as the neurotoxic compound. it was also tested whether FGLd is able to protect “hippocampal neurons from the death induced by Af 25.35. Cultures of hippocampal neurons were incubated with pre-aggregated Af 25-35 together with various concentrations of FGLd. In Figure 4c it can be seen that treatment of hippocampal cell cultures with 20 HM AB 25-35 in-

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duced a moderatex-low neuronal cell loss. This loss could in part be mescued by 50 ng/ml brain derivead neurotrophic factor (BDNF). As shown in Figure 4d, treatment with FGLd in concentrations ranging from 0.1 — 50 pg/ml also partly rescued hippo- campal neurons from neurodegeneration induced by 20 pM AB 25-35. The FGLd- induced neuropro-tection reached a maximal level already at a conceantration of 0.3 pg/mi FGLd (110 % as compared to the AB 25-35-treated controls), remaining more or less constant at concentrations of FGLd up to 50 pg/ml.

Thus, FGLd can protect hippocampal neurons against the neurotoxic effect of the AB 25-35 peptide to tthe same extent as that achieved by treatment with EBDNF. it has previously shown that if primary cultures of CGN are grown at high levels of

KCI, and the con centration of KCI subsequently is reduced, apoptosis is induced. A neuroprotective e=ffect of FGLd was investigated in CGN cultures induced to differen- tiate in 40 mM KCl for seven days. Subsequently the KCI concentratieon was reduced to 5 mM, and aftear two days, cell viability was estimated. As seen in BFigure 4e, a low

KCI concentratiomn induced cell death in CGN cultures, and this could be rescued by treatment with in sulin-like growth factor 1 (IGF-1). As shown in Figu re 4f, cell death could also be pamrtially prevented by treatment with FGLd. A maximal neuroprotection of 135 % as cormpared to controls grown at a low KCi concentrati-on was seen at peptide concentrations between 1 - 10 pg/ml.

Thus, the resultss indicate that FGL is capable of promoting survival of dopaminergic, hippocampal ancd cerebellar granule neurons in vitro.

FGL protects cemrebellar granule neurons against apoplosis

In order further to characterise the mechanism of the neuroprotective effects of

FGLd, the externt of DNA-fragmentation (normally associated with agpoptosis) in KCl deprived CGN \was measured (as described in Eidadah et al, (2000) J Neurosci. 20:179-86). CGN maintained in the high KCl medium displayed v-ery few neurons with fragmented DNA. A large proportion of the cells treated with low KCl displayed

DNA-fragmenta®ion. In CGN cultures treated with FGLd in a conce-ntration of 10-20 pg/ml, very few of the neurons displayed fragmented DNA upon KeCl-deprivation. In contrast, the co'ntrol peptide, FGL9, 10diAlad, with a double alanine substitution ex- hibited no neuroprotective effect. The neuroprotective effect of F-GLd was subse-

SUBSTITUTE SHEET (RULE 26)

quently quantified, and in Figure 5 it can be seen that KCl-sdeprivation resulted in a clear increase of the number of CGN containing fragmented DNA as compared to

CGN maintainead in a high KCI medium. Treatment with thes control peptide, FGLS, 10diAlad, did n ot rescue CGN from apoptosis. Addition of ESGLd in various concen- trations reduce=d the number of apoptotic CGN with a mamimal rescue of 40 % at concentrations of 10 — 15 ug/ml FGLd, relative to apoptotics neurons without FGLd- treatment.

FGL-induced signal transduction results in phosphorylation eof Erk and Akt

The FGL peptide has been shown to bind and activate thee FGFR. Signalling path- ways initiated by the FGFR include among others the Ra s-MAPK pathway, as re- flected by acti-vation of MAPK Erk1/2, and the PI3K path=way, which activates the serine/threonirme protein kinase Akt. Both pathways are knoswn to be involved in neu- ronal differentiation and survival. The neurite outgrowth a:nd survival promoting ef- fects of FGL nay therefore depend on activation of the MABPK and PI3K pathways. It was therefore investigated whether Erk1/2 and Akt were phaosphorylated in response to treatment off CGN cultures with the FGL peptide. From F igure 8 it appears that the peptide in a concentration of 10 pg/ml induced a sustained phosphorylation of Erk1/2 in contrast to a concentration of 5 ug/ml, which was unab le to induce phosphoryla- tion of Erk1/2. This activation (115-120 %, as compared to econtrol cultures) was seen 10 minutes after stimulation with FGLd and lasted for at lea st 90 minutes.

The phosphorylation of Akt by FGL was analysed after exposure of CGN cultures to the peptide fowr 10 or 30 minutes (Figure 7). A 10-minute e=xposure period in concen- trations above 0.5 pg/ml led to a significant increase of gohosphorylation of Akt as compared to t_he untreated control. The phosphorylation level reached a maximum at a peptide con centration of 1 ug/ml, and stayed relatively constant at doses increas- ing up to 20 j3g/ml. At exposure times of 30 minutes, no pehosphorylation of Akt was detected indicsating that activation of Akt was transient andl terminated within 30 min- utes in contrast to the phosphorylation response of Erk1/2.

Thus, the results indicate that FGL induces a sustained (eat least 90 minutes) activa- tion of the MAPK pathway and a transient (10 minutes) activation of Akt.

FGL promote.s neuronal cell survival through FGFR, MEK &nd PI3K

SUBSTITUTE SHEET (RULE 26)

in order to investigate whether the FGFRR, MEK and PI3K also were involved in FGL- induced survival, CGN cultures induced to undergo apoptosis were treated with the above-described inhibitors. As shown in Figure 8, survival of cells in the apoptossis- induced CGN cultures treated with FGL«d, but without inhibitors, were set to 100 %, and the apoptosis-induced control neurons not treated with FGLd (marked with a dashed line) had a lower survival rate, as compared to the FGLd-treated neurcans.

The FGER inhibitor, SU5402 (mw), significantly inhibited the survival response to FGLd already at a concentration of 20 uM and a concentration of 40 pM SU5402 reduced the FGLd-induced survival to the survival rate of the untreated apoptosis-indu«ced control neurons. The MEK inhibitor, PL298059 (e), inhibited cell survival induced by

FGLd to the level of the control cultures at a concentration of 12.56 uM. The PI3K inhibitor LY294002 (A) inhibited FGLd-Enduced cell survival significantly at a concen- tration of 3.5 pM. indicating that LY294002 is a very potent and general inhibitor of neuronal survival. 16 Thus, the FGL-induced survival after KKCl-deprivation of CGN cultures is dependent on activation of the FGFR and of the in€racellular kinases MEK and PI3K. The results show that LY294002 is the most potent inhibitor of neuronal survival, followed by the

MEK inhibitor, PD98059, which appeared to be less efficient in the neurite outgrowth assay than In the survival assay (see below), possibly indicating that the main func- tions of the FGL-induced MEK activati on is neuroprotection, rather than differe-ntia- tion.

Example 3. In vitro neurite outgrow th stimulation by different formulatiorss of

FGL

Primary cultures of DN, HN and CGN wvere prepared as described above.

Dopaminergic neurons (DN)

DN were plated at a density of 100,000 cells/cm? in 24-well cell culture plates (fporevi- ously coated with 12.5 pg/ml poly-D-lysine; Sigma-Aldrich) in a medium containing 50 % (viv) Optimem 1 (Gibco, BRL), 25 % (v/v) horse serum (Gibco, BRL), 25 % (v/v) HBSS and § g glucose/l. The cell s were left to adhere for one-two hours, before the medium was changed to Neurobasal medium supplemented with 2 % (VW) B27

Neurobasal Supplement, 0.5 % (v/v) glutamine, 100 U/ml penicillin and 100 gug/ml streptomycin (all from Gibco, BRL) without or with various concentrations of peptide.

After 72 hours of incubation, the neurons were fixed with 4 % (v/v) formaldehyde and

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immunostained using a primary rmouse monoclonal antibody against tyrosine hy- droxylase and secondary biotinylated sheep anti-mouse antibodies followed by incu- bation with peroxidase conjugated streptavidin. 98 images for each experimental condition in each individual experisment were automatically recorded using a comput- erised microscope workstation diescribed by Walmod et al. (2002) Toxicol App!

Pharmacol. 181:1-15. Briefly, the workstation consisted of an Eclipse TE 300 in- verted microscope (Nikon, Tokyo, Japan) equipped with a motorised, movable mi- croscope stage (LUDL Electronic Production Ltd, Hawthorne, NY, USA), a 10x objec- tive and a 1100 analogue b/w CC D video camera (DFA, Denmark). A software pack- age Prima developed at the Protein Laboratory (Copenhagen University, Denmark) was used to make a stereologicaslly based determination of neurite length according to Renn et al., (2000) J Neurosci Methods. 100:25-32).

Hippocampal neurons (HN) and cerebellar granule neurons (CGN)

Postnatal HN or CGN were plated at a density of 10,000 cells/cm? on uncoated 8- well permanox Lab-Tek chamber slides in Neurobasal medium supplemented with 0.4 % (w/v) bovine serum albumin (BSA; Sigma-Aldrich), 2 % (v/v) B27 Neurobasal supplement, 1 % (v/v) glutamax, 100 U/ml penicillin, 100 pg/ml streptomycin and 2 % 1 M HEPES (all from Gibco, BRL). Peptide solutions without or with inhibitors of vari- ous signal transduction pathway s were added to a total volume of 300 pl/fem?, and the slides were incubated at 37°C. After 24 hours, the neurons were fixed with 4 % (v/v) formaldehyde for 20 minute s and thereafter immunostained using primary rabbit antibodies against GAP-43 and Alexa Fluor secondary goat anti-rabbit antibodies.

Images of at least. 200 neurons for each group in each individual experiment were obtained systematically by using computer assisted fluorescence microscopy as pre- viously described (Renn et al., 22000 op. cit.). Briefly, a Nikon Diaphot inverted micro- scope with a Nikon Plan 20x objective (Nikon, Tokyo, Japan) coupled to a video camera (Grundig Electronics, Germany) was used for recordings. The same software package as described above for- the dopaminergic neurite outgrowth assay was used to process the recorded images-

Results

FGL induces neurite outgrowth in primary cultures of dopaminergic, hippocampal and cerebellar granule neurons

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A mononneric form of FGL has been demonstrated to induce neurite outgrowth from primary hippocampal neurons. It is known that multimeric forms of peptide ligands have a higher potency for receptor activation than moneomeric forms (discussed above). “Therefore, various dimeric and tetrameric forms of” FGL were manufactured and tested on their neurite outgrowth stimulatory capability employing primary cul- tures of dopaminergic, hippocampal and cerebellar granule neurons (CGN).

The cultures were grown in the absence or presence of the tetrameric (dendrimeric)

FGL peptide (FGLd). Neurite outgrowth induced by FGLd in all three types of neu- rons was subsequently quantified and shown in Figure ©. Dopaminergic neurons treated with FGLd (e) exhibited a statistically significant inecrease in neurite length in a dose-dependent manner with a maximal average neurite length (125 % as com- pared to the untreated control) obtained at a concentratior of 1 pg/ml FGLd. Hippo- campal neurons treated with FGLd (4) exhibited an increase in neurite length al- ready at a dose of 0.04 pg/ml FGLd, reaching a plateau-li ke level with a stimulation of approximately 150 % as compared to the untreated control cultures employing concentrations of FGLd up to 20 ug/mi. CGN cultures treated with FGLd (m) also exhibiteed a dose-dependent neurite outgrowth response. A maximal stimulation of 255 % as compared to the untreated control was seen at a concentration of 50 pg/ml

FGLd.

Primary cultures of hippocampal neurons were selected to test the effect of three differeret dimeric versions of the FGL peptide, FGL, FGI; and FGLeys, ON neurite outgrowth. As shown in Figure 10, the dimeric lysine deandrimers, FGLys (A) and

FGLee (vo) were both unable to stimulate neurite outgrowth at any of the concentra- tions tested. In contrast, FGL, (m) induced a significant Meurite outgrowth response exhibiting a dose-dependent relation with a bell-shaped curve reaching a maximal stimulation at a concentration of 1 pg/ml. The maximal stimulation was 155 % as compawed to the untreated control (Figure 10), which is reoughly the same as the re- sponse induced by FGLd (see Figure 9).

Neurite outgrowth induced by FGL involves activation of FGFR, MEK and PI3K

In ordesr to elucidate the biological correlates of activation of FGFR, MEK and PI3K, it was te sted the effect of inhibitors of these kinases on neurite outgrowth induced by

FGLd in CGN. The FGFR inhibitor, SU5402, is known to inhibit the tyrosine kinase activity of the FGFR subtype 1 by interacting with the catalytic domain of the recep-

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tor. In Figure 11 the average neurite length in FGLd-treated CGN cultures is set at 100 % =and the average neurite length for control neurons not treatzed with FGLd is marked with a dashed line. It appears that SU5402 (m) significartly inhibited the

FGLd-imduced neurite outgrowth at a concentration of 80 uM (to 3 § % of the initial value). “Treatment with the MEK inhibitor PDO8059 (e) in concentrations above 25 uM statistically significantly decreased neurite outgrowth induced b~y FGLd to 75 %.

Also, the PI3K inhibitor LY294002 (A) significantly inhibited FGLc-induced neurite outgrowth already at a concentration of 3.5 iM to 90 % and at a camncentration of 10 uM LY 294002 the neurite outgrowth response to FGLd was reduced to almost 50 %, which iss lower than for the untreated control neurons. SU5402 and LY294002 were without significant effect on neurite outgrowth in the control culture s not treated with

FGLd ( data not shown). PD98059 has previously been found not to affect the basal neurite outgrowth in CGN. Thus, the FGLd-induced neurite outgr-owth response of

CGN iss dependent on the activation of the FGFR and the subsecguent activation of the MAPK and the PI3K intracellular signalling pathways.

Table 1. Summary of the effects of different formulations of “the FGL peptide on newsronal survival in primary cultures of rat CGN, DN and HN induced to undersgo cell death by withdrawal of high potassium, or additHion of 6-hydroxy dopammine (6-OHDA) or (25-35) B-amyloid peptide fragment . . : . Neuronal

Mechwanism for Tissue source FL . peptide/ Survival inducsing cell (age of rat) effective (% of Coru- death g concentration | 4a

El EY — |FGL, |] (6 mtv) (PND 34) | ng/mL swe

Low KK CGN FGLy4 (5 mim) (PND 3-4) 5 pg/mL 110%* : 20 pg/mL 117%"

Low K' CGN FGL4: (5 mi) (PND 3-4) 1 pg/mL 131%* 10 pg/ml 132%"

Low K CGN FGLy (6 mM) (PND 7-8) 10 pg/mL 110%" 50 pg/mL 119%* 250 pg/mL 142%" (5m NM) (PND 7-8) 20 pg/mL 175%" 6-OHDA DA FGLs (E14) 1 pg/mL 143%*

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p-amyloid Hippocarnpal | FGL4: (E19) 0.01 ng/mL 109%** 3 pg/mL 107%** 9 pg/mL 107%"

B-amyloid Hippocawnpal | FGL.: (E19) 0.3 pg/mL 108%* a gurvival in cultures induced to undergo cell death without FGL treatment is desig- nated as 100% (control) amd compared to cultures where FGL was added.

Statistics (+FGL vs. contro 1 treatment): *p<0.05; **p<0.01, ***n<0.001

Table 2. Summary of the effects of FGL peptides on neurite outgrovwth in pri- mary cultures of rat CGNi, DN and HN.

Statistics (+FGL vs. Contre): *p<0.05; *p<0.01; **p<0.001

PND = Post-natal day; E = Embryonic day

Assay Tissue Peptide Concentra- | Effect on Neurite Length

Sorarce/ tion (pg/mL) (Percent Increase vs. Con-

Age of Rat trol)

Primary culture; | CG N/ —

Incubation: 24 h +/- FGL; | -PIND2-4 | FGL4-9 24%" -PEND 58 | FGL4-3,9,27 30%"*, 93%**, 121%"

Analysis: computer as- * * -* sisted fluorescence mi- FGLy-10, 25, 50, 100 | 37%", 79%", 179%**, E6% croscopy

Primary culture; : DNN/ incubation: 72 h +/- FGL; | E®4 FGLy - 0.04,0.2,1,5 | 12%", 23%***, 24%", 13%*

Analysis: computer as- sisted fluorescence mi- FGL.-0.3,1,3 19%", 34%**, 33%" croscopy FGLn~ 10, 50, 250 15%", 23%", 28%

Primary culture; HM incubation: 24 h +/- FGL; | - EE19 FGLs-3, 9, 27 65%", 51%", 69%" - ewbom | FGLs~0.04,5 38%**, 45%"

Analysis: computer as- " ER0LeY ROLF sisted fluorescence mi- |” Newbom | FGLL-02,1,5 35%", 53%**, 30% croscopy

Conclusions

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1. The FGL peptide is a survival promoting compound for a variety of neuronal cell types in vitro, which is essential in the treatment of neuro-degenerative disor- ders. 2. The FGL peptide is capable of inducing neurite outgrowth in a variety of neu- ronal cell types in vitro and FGL has a potermtial to promote the growth of both axons and dendrites, which is essential in thme treatment of neuro-degenerative disorders. 3. All, monomeric FGLn, dimeric FGL_ and derdrimeric FGLp, formulations of the

FGL peptide are potent in promotion of bot survival and neurite outgrowth. 4. FGL_and FGLp both are more then 200 times more potent then FGLm in promo- tion of neurite outgrowth. 5. FGL, is as potent in promotion of both neur—ite outgrowth and survival as FGLp. 6. Other formulations of the FGL peptide such as dimers FGL,,; and FGLq are inactive in promotion of neurite outgrowth.

Example 4. The effect of FGL on synaptic polasticity of primary cell cultures of rat hippocampal neurons

In the adult nervous system, structures such as the hippocampus continuously un- dergo plastic changes to adjust structure andi function. In particular, the acquisition of memory Is believed to require structural anad functional changes of already estab- lished neuronal connections. NCAM has been shown to play an important role in neuronal plasticity during development and in leaming and memory (Welz! and

Stork, (2003) News Physiol Sci. 18:147-50); however, the mechanism for these ef- fects is unknown. The present study it was irvestigated whether the FGL peptide is able to affect synaptic plasticity.

The effects of FGL4 on vesicle tumover was estimated in primary cultures of hippo- campal neurons from embryonic rats (E19). The cultures were prepared as above.

Following 13-16 days in vitro, FGL, was adcded to the cultures and incubated for 1, 24 or 48 hours. After 48 hours of incubatieon, the FGFR-inhibitor (SU 5402) was added simultaneously with FGL, to some of ithe cultures.

After treatment with FGLy the neurons were labeled with the fluorescent membrane probe FM 1-43 as described by Kiryushko est al (2003) J Biol Chem. 278:12325-34.

The rate of FM 1-43 release (destaining of cells) evoked by depolarisation with 90

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mM potassium was then evaluated by a time-lapse image acquisition using the Ra- diance 2000 scanning system and a Nikeon eclipse TE200 confocal microscopy.

The effect of FGL; on synapse formation was further evaluated using double- & immunostaining of neurites and synaps-es in primary embryonic (E19) hippocampal neurons grown in culture for 13 days. Cells were incubated with FGL4 for 48 or 96 hours, before fixation and immunostainimng. The synapses were detected by antibod- ies against synaptophysin and the neymrites were labelled by antibodies against the growth-associated protein-43 (GAP-43- also called B-50 or F1). The immunostaining was visualised and quantified by confocal fluorescence microscopy as described by

Renn et al. (2000) (op. cit.).

Results

The treatment of hippocampal neurons with 20 ug/ml FGL, for 1 h and 48 h resulted in a statistically significant increase of tine rate of FM 1-43 destaining, compared with the control (from 0.008 sec” to 0.016 sec”, and from 0.013 sec” to 0.028 sec’, re- spectively); whereas 24 h treatment did not result in a significant effect (Figure 12).

This suggests that FGL4 causes both =a short-term facilitation of transmitter release (1 h) and a long-term (48 h) increases of synaptic efficiency. The FGFR inhibitor

SU5402 abolished the effect of FGL, iradicating that the effect of FGLy was mediated through its interactions with FGFR.

The double immunostaining of hippocampal neurons for synaptophysin and GAP-43 showed that FGL4 (20 ug/ml) adminis:tered to hippocampal cultures for 48 and 96 hours significantly increased the numbser of synapses, as reflected by approximately 20% increase in the number of synapstophysin positive spots per 100 pm length of neurite (from 2.40 to 2.96 and from 2.7" 3to 3.24, respectively).

These results suggest that the FGL pe=ptide influences synaptic plasticity by increas- ing the rate of presynaptic vesicle tumeover and the number of the synapses.

Example 5. The effect of FGL on organotypic hippocampal neuronal slice cul— tures and primary cultures of disssoclated hippocampal neurons exposed tos ischemic conditions

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The brain has a haigh consumption of oxygen and glucose and depe- nds almost ex- clusively on oxidative phosphorylation for energy production. Deficiesncy of oxygen and glucose supply results in neuronal cell damage and death. The susceptibility to ischemic injury differs among different parts of the brain and even Ahifferent neuronal populations. The neurons of the hippocampal CA1 area are one of the regions in the brain of high vulmerability.

Methods in the present staudy it was investigated the effect of the FGL pepticle on synaptic plasticity, neuroral function and vulnerability following oxygen and glucose depriva- tion (OGD) in orgyanotypic hippocampal neuronal slice cultures (OHSC) from 7-day- old Wistar rats according to method of Stoppini et al. (1991) Neuromsci Methods. 37:173-82 and primary cultures of dissociated hippocampal neurors from 1-day-old rats according to method of Maar et al.(1997) J Neurosci Res. 47:~163-72. Primary cell cultures were maintained for 11-12 days and OHSC for 12-14 days prior to add- ing FGL4.

The primary cell cultures and OHSC were exposed to OGD for 1.0 and 20 minutes.

Following OGD, the cultures were again supplied with oxygen ancd glucose and ana- lysed at 1, 4 or 24 hour time point after introduction of OGD exposure. FGL4 (or the control peptide FGL) was added 24 hr before, immediately, or 1 hr or 24 hr after

OGD. When adlministered prior to OGD, FGL4 was removed from the medium when introducing OG D. in some cases, the slice cultures were treated “with FGL; only at 4 hr after OGD. lan addition, the FGFR inhibitor, SU5402 (25 uM), vvas added together with FGL4 in so-me of the set-ups.

In hippocampal neuronal cultures, FM 1-43 staining was used to eavaluate pre- synaptic function (described above). Cell viability in both primary cell cultures and

OHSC was me asured using the MTS [3-(4,5-dimethylthiazol-2-yi»-2,5- diphenyltetrazoolium bromide] assay according to Malich et al, (19%97) Toxicology. 124:179-92. In, addition, cell viability in OHSC was measured usi ng propidium io- dide (PI) staining according to Laake et al. (1999) Brain Res Braimn Res Protoc. 4:173-84.

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Resstits

The metabolic activity of dissociated hippocampal cell cultures was significantly re- duesed after 1 hour (31%) and 4 hours (30%) following 20 min of OGD, compared to comtrol cultures, as measured by the MTS assay. When cell cultures were treated with FGL, (24 hours before or immediately after OGD) tine effect of OGD on meta- bo-lic activity was diminished. The metabolic activity in COHSC showed a different ressponse to OGD. One hour after OGD, the metabolic aactivity was significantly in- crezased (19%) compared to control, while a longer pericad of reoxygenation (24 hours) resulted in a decrease (24%) of metabolic activity. At both time-points, treat- meant with FGL4 24 hours prior to OGD caused a normalization of the metabolic ac- tivity in the hippocampal slices, compared to the contro. In both the dissociated cell cultures and OHSC, FGLy treatment alone led to an increase in metabolic activity. In acidition, pretreatment with FGL4 for 24 hours did not dE minish the OGD-induced re=duction in metabolic activity if the FGFR-inhibitor SU 5402 was present together 16 w ith FGLy, indicating that FGLy might influence metabomlic activity through FGFR asctivation.

In dissociated hippocampal cell cultures, the rate of pres-synaptic vesicle release (FM 1—43 destaining) upon high potassium stimulation waas statistically significantly en- h anced (from 0.04 sec” to 0.06 sec™) by FGL, treatmaent and decreased after OGD when analyzed at 1 h and 4 h (both from 0.04 sect to 0.02 sec™). If cell cultures were treated with FGLy either 24 hours before or immediately after OGD, the OGD- irduced decrease in the rate of FM 1-43 release wass diminished. Furthermore, the

F=GFR-inhibitor SU 5402 abrogated the effect of FCSLy on the OGD-induced de- crease in FM 1-43 release. lm OHSC, 10 minutes of OGD induced delayed cell da:mage as estimated by propidium iodide (Pl) staining. There was a significant . increase in the Pl staining of

OHSC (% of total CA1 area) at both 4 hours (0.3% to 5.0%) and 24 hours (0.6% to 223.1%) after OGD. This effect could be almost completely abolished by pre-treating the slices for 24 hours with FGLg. Cell damage was al so avoided by treating slices with FGL, immediately after OGD, but not if FGL, treamtment was delayed until 4

Fnours after OGD. The control peptide FGL. did not affvect cell damage induced by «OGD, and if slices were pre-incubated with the FGFR_-inhibitor SU 5402 together whith FGL, the Pl staining was similar to that of OGD alone.

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Discussion

The FGLy peptide has neuroprotective effects when applied 24 hours before or im- mediately after a transient deprivation of oxygen and glucose in two different in vitro model systems. The OGD caused a rexduction in metabolic activity in dissociated hippocampal cell cultures and in OHSC, and in both cases treatment with FGLy could partially or completely normalize metabolic activity after OGD.

Ischemia has been reported to cause either enhanced synaptic transmission or re- duced synaptic transmission, depending on the mode! used and the severity of the ischemic challenge. in the model used in this study, 20 minutes of OGD caused a reduction in presynaptic vesicle release upon potassium stimulation, and this reduc- tion was attenuated by administering the FGLg peptide either 24 hours before or immediately after OGD.

Thus, for all three parameters investigated (metabolic activity, presynaptic vesicle release and cell viability), the FGL peptide could partially or completely inhibit the effects of OGD. Furthermore, this neuroprotective ability of FGL could in all three cases be inhibited by the FGFR-inhibitor SU 5402, indicating that FGFR activity Is necessary for the action of FGL.

Example 6. Effect of FGL on social behaviour of rats and g-amylold induced neurotoxicity in vivo.

Description of the model

The present study employed a rat model, in which neurotoxicity and cognitive im- pairment were induced by the (25-35) B-amyloid peptide fragment (see Maurice T., et al. (1996) (Brain Res. 706:181-93) and Delobette S., et al. (1997) Eur J Pharma- col.319:1-4). Short-term memory deficit induced by the (25-35) B-amyloid peptide fragment in rat is of special importance to study, as it resembles one of the clinical manifestations of Alzheimer's disease. Short-term memory deficit in rats was evalu- ated using three different behavioural tests: the Social Recognition (Kogan et al, (2000) Hippocampus 10:47-56), Open Fleld (Cerbone and Sadile, (1994) Neurosci

Biobehav Rev. 18:497-518,; Vianna et al, (2000) Learn Mem. 7:333-40) and Y-maze (Clayton & Williams, (2000) Neurobiol Learn Mem. 74:135-45) tests. In addition, neuropathological investigations were performed on brain tissue collected at the

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time of sacrifice. The FGL peptide was administered either sub-occipitally or intra- nasally. }

Methods

The timeline of a representative social-recogniticon study with early treatment is schematically shown in Figure 26 (A).

Following an acclimatisation period of five days, rats were treated with the (25-35) B- amyloid peptide fragment (A-) (25 pg per arnimal), which was injected i.c.v. in the right lateral ventricle of the brain (day 0).

The FGL peptide (either FGL_ or FGL.) was administered sub-occipitally (Sug per animal per administration) or intranasally (400 pag per animal per administration) at days 7, 10 and 13 or at days 30, 33 and 36. Behaviour of animals was evaluated using the Social Recognition test, the Open Fielad test or the Light-dark Discriminat- ing Y-maze. On day 28-30 or day 51 afteri.c.v. administration of the (25-35) §- “15 amyloid fragment, the animals were sacrificed, and in selected studies, the brain tissue was collected, histological sections were immunostained with A-B specific antibodies and the amyloid-burden was calculated as a percentage of the A-B posi- tive area. In addition, the number of neurons in selected areas of brain-sections was calculated using a stereological approach. 20

Effects of (25-35) A-B/Vehicle-administration or social behaviour and memory

In the Social Recognition test, an increase in the time spent exploring a juvenile rat during the second frial was detected In rats that received the (25-35) p-amyloid fragment as compared to the control animals reeceiving vehicle (V). In the Open Field test, (25-35) p-amyloid fragment-treated rats did not decrease exploratory activity during a 20-minute session, in contrast to the edecrease noted in the group of control animals. In the Light-dark Discriminating Y-rmaze with positive food reinforcement, rats treated with the (25-35) B-amyloid fragment demonstrated an increase in the number of errors (entries into the wrong arms) .

Thus, in all three tests, i.c.v. administration woof the (25-35) p-amyloid fragment re- sulted in a cognitive deficit displayed as short-—term memory impairments.

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Effects of FGL administration on behaviour and memory ef rats in which cognitive impairment was induced by Lc..-administration of the (255-36) 8-amyioid fragment

The effects of FGL were estimated in different tests in viwo described below in sev- eral independent studies. ° Social Recognition test and Neuro-histopathology o Study 1: FGL was administered sub-occipitally at days 7, 10 and 13 following i.c.v-. administration of the (25-35) B-amyloid fragmemt. No effect of FGLg was observed in the Social Recognition test at day 14, thee day after the last admini- stration of FGLy and no significant effect was observeed in the neuro- histopathology evaluation of the CA3 area of hippocampus comparing placebo- treated rats with rats receiving either the (25-35) p-ammyloid fragment alone or withs FGLs. 16 o Study 2: FGL was administered sub-occipitally at da ys 7, 10 and 13 following i.c.\v. administration of the (25-35) B-amyloid fragmemt. Administering the (25-35) p-awmyloid fragment resulted in a statistically significant increase in the time spent by the rat exploring a juvenile animal at a second meeting as well as in the amyloid burden and neuronal cell death when comp -ared to control rats. How- evear, treatment with FGL_ and FGLy significantly recluced the increased time spent by the treated animal exploring a juvenile rat curing the second trial, when cormpared to the animals treated only with the p-am=yloid peptide fragment.

Treatment with FGL, and FGL, also significantly recluced the amyloid burden in thes cingulate cortex (from 100% to 39% & 42%, res_pectively) and in the CA3 area of the hippocampus (from 100% to 17% & 44%%, respectively). In addition,

FGsL, and FGL, caused an increase in the density of neurons (from 15 to 21 & 22 neurons/mem?x10%, respectively) in the CA3 zore of the hippocampus, when commpared to the group of rats treated only with the (25-35) p-amyloid fragment.

Th e results are graphically presented in Figure 13-16. o Study 3: FGL, was administered sub-occipitally at c3ays 30, 33 and 36 following i.c_v. administration of the (25-35) -amyloid fragment. A statistically significant reciuction was observed at day 44, one week after &he last administration of

FGSL., in the ratio of the time the rats spent investig ating a novel object (juvenile rat) on the second exposure compared to the (25-385) A-B/Vehicle treated control

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rats (from -0.50 to 0.41), when FGL_ was administered sub-occipitally. tr addition, neuropath-ology investigations showed a statistically significant reduction in amy- loid burdem (from 100% to 24%) in the cingulate cortex and the CA3 area of Hip- pocampuss (from 100% to 29%). In addition, a statistically significant decrease in the percertage of damaged neurons (from 27 to 24 neurons/mem?x10™*) was observed in the cingulate cortex, compared to the group of rats treated only with the (25-355) B-amyloid fragment. The results are graphically presented in Figures 17-20. o Study 4: FGL,_ was administered intranasally at days 7, 10 and 13 following i.c.v. administration of the (25-35) p-amyloid fragment. Treatment with the Amyloid beta peptide induced a short term memory deficit, which was abrogate=d by treatment with FGL-peptides. In the cingulate cortex, a statistically siggnificant in- crease im the neuronal density (from 11 to 14 pm?x 10%) was observe-d in re- sponse teo FGL, administration, compared with the group of rats treated only with the (25-35) p-amyloid fragment. The results are graphically presented in Figures 21-23.

Open Field study

The rats thaat received FGL4 administered sub-occipitally at days 7.10 and 13, fol- lowing i.c.v.—administration of the (25-35) p-amyloid fragment, showed = statistically significant decrease in the time spent exploring holes (from 3.3 s to 0.1 s) and rear- ing (from 7.0 s to 0.7 s) in the last measurement period, compared with the first measureme=nt period at day 14, the day after the last administration eof FGL. The group of rats treated with only the (25-35) B-amyloid fragment showeed no statisti- cally significcant difference between the two measurement periods. The locomotion activity was unaffected at day 14.

Light-dark Discriminating Y-maze study with positive food-reinforcementt

The rats thaat received FGL4 administered sub-occipitally showed a statistically sig- nificant redmuction (from 3.8 to 1.7) in the number of errors at day 25, the= last day of training, while no significant differences were observed at training days 21-24 in comparisor with the (25-35) p-amyloid fragment-treated control rats.

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a3

Discussion

The model usecd in this study, the (25-35) B-amyloid fragment induced neuro-toxicity, reflects many saspects of neuro-pathology and cognitive inmpairment seen in Alz- heimer's patierts. In this model, i.c.v.-administration of the= (25-35) B-amyloid pep- tide fragment r=esults in deposition of the B-amyloid peptide=, starting at day 14 after the (25-35) B-=amyloid peptide fragment administration, wh'ich lead to neuronal cell death and cognitive impairment (short-term memory deficit)~

The experiments of early administration of the FGL pepticte (at days 7, 10 and 13 after the i.c.v.—irjection of the (25-35) p-amyloid fragment) clearly demonstrate that both sub-occipsital and intranasal administration of FGL restlts in prevention of short- term memory adeficit, a decrease of B-amyloid burden in thes various brain areas and a decrease in neuronal cell death. The administration of the FGL peptide at a later stage, at dayss 30, 33 and 36, after i.c.v.-injection of the ( 25-35) p-amyloid peptide 16 fragment ameRiorated both the neuropathological changes in the brain tissue and the memory deficit.

The important: aspect of these studies was to compare thes effectiveness of different routes of FGL administration. The results obtained clearly indicate that the intranasal administrations of the FGL peptide effectively reduced the rmemory deficit induced by treatment with the (25-35) B-amyloid peptide fragment, although the potency of the

FGL peptide administered intranasally was much lower, compared to the potency of the peptide emdministered sub-occipitally. It should be noted that the effect of in- transally adm&nistered FGL on memory was dose-depende=nt. : In conclusion , taken together, these results indicate that the FGL peptide has the potential to readuce neuropathology and to reduce the meemory deficits induced by treatment witkn the (25-35) B-amyloid peptide fragment. These findings also indicate that the FGL peptide has a therapeutic potential for the tareatment of cognitive defi- cits associate=d with B-amyloid deposition in patients with Alzheimer’s Disease.

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Example 7. Sensory Motor Control Developmeznt in Rat Pups

The neonatal CNS of immature rodents is a useeful model for studying drugs influ- encing the maturation of brain functions. The maaturation of postural adjustment (lo- comotion) needs integration of motor and sensory systems. The maturation of the postural and locomotor functions during the firs®t post-natal week is connected with the growth of the descending pathways from borainstem and cortico-spinal tracts, reaching the thoracic levels at post-natal day (P*ND) 3 and the lumbar level at PND 6. Thus, the period between PND 3 and PND 6 canbe considered as a period sen- sitive to the effects of agents able to modify post-natal integration of the sensory- motor system. A number of motor control tests =are available, which can be used for determining the effect of neuro-active drugs such as FGL. In the present study, the

Surface Righting Response performance (Tilney, 1933), the Pivoting Locomotion performance (Altman et al, (1975) Anim Behav. =23:896-920), the Negative Geotaxis performance [Henck et al, (2001) Toxicol Sci. 6s2:80-91), and the Forepaw Suspen- sion performance [Kimier et al, (1998) Brain Rees Dev Brain Res. 107:49-565) of rat pups were chosen to evaluate the effect of FG. on sensory motor control develop- ment. The time-course of the experiments Is s-chematically presented in Figure 26 (8).

Dose-Response study. The effect of intranasal administration of 0.026, 0.26 and 2.6 ug FGLy per day at post-natal day (PND) 1, 2 and 3 on performance of the Surface

Righting Reflex by rat pups was evaluated. A total of five litters, each consisting of 10 pups (five males and five females) were usedd in this study.

Administration of FGLy significantly improved oerformance of the Surface Righting

Reflex at PND 5 (from 3.4 s to 1.3 s) administered at a concentration of 2.6 pg per pup per administration compared to the vehicle—treated control animals.

Sensory Motor Control Development study. T he effect of FGL on sensory motor control development (Surface Righting Reflex. Negative Geotaxis Reflex, Pivoting

Locomotion and Forepaw Suspension performances) of rat pups receiving 2.6 ng peptide intranasally daily at PND 1, 2 and 3 wa:=s evaluated. A total of six litters, each consisting of 10 pups (five males and five fema les), were used in the study.

SUBSTITUTE SHEET «RULE 26)

Adrministration of FGL, statistically significantly improved performance of the Sur- face= Righting reflex at PND 4 (from 21sto 1.6 s) (Figure 24) and of the Negative

Geomtaxis Reflex at PND 6 (from 56.7% to 80.0%) (Figure 25), compared with the vehicle-treated control rats, without affecting thes Pivoting Locomotor activity or the

Foreapaw Suspension performance. This indicate s that FGL promotes the post-natal sen. sory-motor control development, without disturbing other functions. In addition, no -significant effect on Negative Geotaxis performance was observed at PND 9, sugagesting that the motor contro development was already completed by PND 9.

Dis-cussion

The present results demonstrate an effect of thee FGL peptide in modulation of the initial processes of CNS plasticity during post-raatal development, and suggest that the= FGL peptide, by stimulating the FGF receptor, may have a potential to acceler- ate= the development of sensori-motor functions. 16 Excample 8. The Contextual Fear Conditionin g Test and the Morris Water Maze

Te st

Thse Contextual Fear Conditioning test (CFC) teest (Cordero et al, (2003) Horm Be- ha_v. 2003 Nov;44(4):338-45) and the Morris \#/Vater Maze (MWM) test [Morris R., (12984) J Neurosci Methods. 11:47-60) are the &kasks that assess leaming and mem- orvy, and are used to compare the performance of normal control rats and of rats trea ated with drugs.

In the Contextual Fear Conditioning test, rats experienced inescapable shocks when exxposed to a *new environment, which results in the development of a conditioned fesar response, consisting of a characteristic irmamobility or ‘freezing response’, when suabsequently re-exposed to the same environment.

In- the Morris Water Maze test, rats are evaluateed in the spatial training task to find a hi-dden platform in order to escape swimming ir the water.

Thhe present study investigated whether posft-training administration of the FGL4 peeptide is effective in modulating the memory consolidation in aversive and/or new situations.

The Contextual Fear Conditioning test

SUBSTITUTE SHEET ((RULE 26)

The rats were handled for 120 seconds every day for 3 days preceding the first day of the experiment. On day 1 (traini ng), the rats received a 1-second shock (uncondi- tioned stimuli) each minute for two minutes. Animals were injected immediately after training with 5 pl FGLg (5 pg), the control peptide (5 pg) or the vehicle. Each tre-at- ment group consisted of 11-14 rats. In total, 39 animals were evaluated. Animals were tested on days 2, 7 and 30 by placing them in the chamber used for condition- ing, but in the absence of a shock, for 8 minutes. Each rat was rated as either “freezing” or “active”, every 2 seconds, using a time-sampling procedure.

The time-course of the experiments is schematically presented in Figure 26 (C).

A statistically significant increase in percentage of freezing time after FGL4 admini- stration was detected when animals were tested on day 2, (from 50.56% to 73.5%) and day 7 (from 30.0% to 60.3%) after conditioning compared to rats receiving pla- cebo or the control. This indicates that FGL4 is capable of improving the long-term retention of the conditioned fear response when administered post-training in adult male Wistar rats.

The Morris Water Maze test

In the Morris Water Maze study, rats were pre-trained to locate a hidden platform by daily training for 120 seconds for- five days (days —4 to 0) before the experiment. On days 1 and 2, rats were given 3 consecutive trials in which the platform locatior re- mained constant. The time during which the rats were able to locate the platform was recorded within a maximum test-period of 120 seconds. The rats received a 5 ul injection of the FGLy peptide (5 ng), the contro! peptide (5 pg), or the vehicle solu- tion immediately after each traini ng trial on days 1 and 2.

The animals were tested 24 hours (day 3) and 7 days (day 10} after training. Four- teen days after training (day 17), the location of the platform was changed. Rats were trained to find a new locati on of the platform in three consecutive trials (“rever- sal learning”). In total, 48 animals were evaluated. Animals were divided into three groups consisting of 12-18 rats per treatment group.

Administering FGL, improved consolidation of long-term memory in rats as eevalu- ated in the Morris Water Maze test. At day 2, the day after the first administration of

SUBSTI TUTE SHEET (RULE 26)

FGLg, FGL, or vehicle, the FGl_streated rats were able to locate the platform statisti- cally significantly faster (65.8 =) than the untreated rats (93.0 s). The effect was also statistically significant at day 1 7, suggesting an improved learning ability.

The time-course of the experirment is schematically presented in Figure 26 (D).

Additional experiments in thes Open Field test showed that FGL4 did not influence emotional or locomotor behav jour in rats.

Discussion :

The results of the CFC/MWNA studies demonstrated a long-term effect of FGLq on learning and memory of norireal rats. FGL may be acting by triggering signalling cas- cades involved in long-term memory formation. As it is shown above, FGL can modulate synaptic plasticity acting through the FGFR, which results in an increased pre-synaptic vesicle tumover . Together, these results suggest that FGL has a very good therapeutic potential ira the treatment of patients with leaning and memory impairment.

Example 9. In vivo pharmaaco-kinetics of FGL_: Estimation of FGL, in plasma and CSF :

The levels of the administered FGL, in rat and dog plasma and CSF were assessed by the radio-immuno assay.

Assay 8 Rat and dog plasma samples contained lithium heparin as an anti-coagulant. b FGL, was iodinated. lodinzation was done by using the lodogen method. The iodi- nated peptide was purified from unincorporated 1'® on C18 column. 9 The measurements were dione in replicates. 1. Plasma (or CSF) sample (20 pl) was added to EDTA containing tubes (n=2).

Non-specific binding (NSB) tubes containing non-immune rabbit serum (or

CSF from control aniamals) (20 ul) were used as control. 2. Diluted radiolabelled FGL_ (100 pl; approximately 20,000 cpm/tube of tracer) was added. Control tubes containing the tracer only were also prepared.

SUBSTITUTE SHEET (RULE 26)

3. Diluted antisera sample (1: 20,9000 for PAD268: 100 pl) was added to all tubes (except the control tubes. “The contents were vortex mixed. 4. The tubes were incubated overnight at 4°C. 5. Anti-rabbit SAC-Cell solution (O.1 ml) was added to all tubes except the tubes containing the tracer. The» contents were vortex mixed and then incu- bated at room temperature for 3€@ min. 6. Wash buffer (2 ml) was added to all tubes except the tubes containing the tracer and the tubes were centrifuged at 3000 rpm for 5 minutes. 7. The supernatant was decanted and the tubes were left to drain for no more than 1 minute. 8. The wash buffer (1.5 ml) was added to the tubes again and the tubes were centrifuged at 3000 rpm for § minutes. 9. The supematant was decanted and the tubes were left to drain for no more than 1 minute. The tubes were centrifuged at 3000 rpm for 1 minute thereaf- ter. 10. All tubes were counted two times: first for 3 minutes and for 10 min.

Results

A calibration curve precision profile wras prepared in replicate (n=6) in rat plasma over the concentration range 0.080 ng/ml to 20.48 ng/ml of FGL,. Good precision was observed over the whole concentration range with acceptable accuracy (<25%) down to 0.160 ng/ml, which gives an indication of the potential lower limit of quantifi- cation (LLOQ) of the assay in rat plas ma. Increasing the counting time did improve the precision of the assay.

A calibration curve precision profile was prepared in replicate (n=6) in dog plasma over the concentration range 0.080 ng/ml to 20.48 ng/ml.

Pharmacokinetic parameters were calculated using the computer program WinNon- fin Pro version 3.3 (Pharsight Corporation, USA). Values that were below the limit of quantification (BLQ) were entered as zero in the calculation of the mean.

There were also defined some toxicokinetic parameters. The toxicokinetic parame- ters that were measured included: maaximum plasma concentrations of FGLy (Cmax), their times of occurrence (Tne), and plasma concentrations at 24 hours post-dose (C24). Areas under the plasma FGL_, concentration-ime curves within a 24-hour dosing interval (AUC,,) were estimated by the linear trapezoidal rule. In the calcula- tion of AUCs values following intranasal administration, the times of the samples

SUBSTITUTE SHEET (RULE 26)

were relative to the start of administration (assuming a 10-minute break between each of the 5 administration procedures). Terminal rate constants (k) were esti- mated by fitting a linear regression of log concentration against time. Terminal half- lives (2) were calculated as in2/k.

FGL, levels in the CSF from the two rats treated intravenously with 50 mglkg were 321 and 269 ng/ml. FGLy levels in the CSF from other animals treated subcutane- ously with 100 mg/kg were 242, 92 and 81 ng/ml. These data provide evidence that

FGL, enters the CNS when administered parenterally.

Toxicokinetic samples taken after the First dose revealed.

Combined male and female exposures (50 mg/kg/day.i.v.):

AUC = 51,026 h.ng/mL; Crnax = 20,951 ng/mL.

Combined male and female exposures (100 mg/kg/day,s.c.):

AUC = 50,119 h.ng/mL; Ceax = 8,346 ng/mL.

Table 12 shows the toxicokinetic parameters calculated in dogs recelving doses of

FGL, of 15, 30 and 75 mg/kg, s.c. — - — — " i: " —TA0Co] — rey

Dose 9 Day x 124)

No (ng/ml) | (hr) (hr*ng/ml) § (hr*ng/ml) ery tr L| - —— — - SE : 15 7132 20 | 62065 |84820 2 120 0863 81243 [104608 3 mo/k 2709 40 | 38208 |56518 13049 : 75 1 9222 7 161489 mg/kg

Mean levels of FGL, in the CSF from the three dogs (sampled one hour after the last dose at day 8) ranged from 4.1 ng/mi for animal No. 2 to 8.1 ng/ml for animal

No. 3, indicating penetration of FGL._ into the CNS, following subcutaneous dosing.

SUBSTITUTE SHEET (RULE 26)

VO 2005/014623 PCT/DK2004/000527

Example 10. The effects of peptide fragments of SEQ ID NO: 2 and SEQ ID NO: in vitro 5 Individual monomer of the peptide of SEQ ID NO: 2, EFL peptide, is capable to strongly stimulate neurite outgrowth of rat hippocampal neurons and stimulate sur- vival of rat cerebellar neurons in the experimezntal set-ups described above (the ef- fects are shown on Figure 27 and 28 correspondingly). The EFL peptide is derived from the the Fn 3, 2 module of NCAM and represents the E strand-loop-G strand part of the module. For the experiments the peptide was synthesised as described above. The neuritogenic activity of the peptide is FGFR1 dependent, as stimulation by the peptide is specifically blocked by an inhibitor of FGFR1, SU54402. The pep- tide is also capable to bind and stimulate FG FR1 in cells expressing the receptor, wherein the peptide induces the receptor phos phorylation (at concentration 20 pg/ml by more then 300%). Phosphorylation of FG FR1 was studied using the following assay. The cDNA for the rat FGFR1 (WIC isoform) was cloned by RT-PCR using

RNA isolated from the rat PC12 cell line and inserted into a pcONA3.1(+) plasmid (Invitrogen), which allows expression of FGFR1 fused to the N-terminal of hexa- histidine. ~8x10° HEK293 cells were cultured for 24h in 60 mm plates in full medium (DMEM 1965 supplemented with 10% FCS, 100 U/ml penicillin, 100 pg/ml strepto- mycin and 58.4 g/l Giutamax) and then transfected with 0.2 pg plasmid (with FGFR) using the LipofectAMIN PLUS™ reagent kit according to the manufacturer's instruc- tions (Gibco BRL). Cells were grown for another 24 hrs in full medium, and then shifted to starvation media (DMEM 1965) ovemight. FGFR transfected cells were stimulated for 20 min with the EFL monomer, lysed in 8M urea, 1 mM orthovanadate (in PBS) and purified from the lysate via the His-tag as follows: The lysate was loaded on NiZ*/NTA-sepharose (Qiagen), was hed with lysis buffer plus 10 mM imi- dazole, and the FGFR was eluted with lysis buffer plus 250 mM imidazole. The puri- fied FGFR was analysed by immunoblotting using anti-pentahis (Qiagen) or anti- phosphotyrosine (PY20, Transduction Laboratories) antibodies. The bands were visualised by chemilumiscense and the band density was measured using a

GeneGnome apparatus (SynGene).

A peptide derived from the sequence of the aaxonal-associated cell adhesion mole- cule [NCBI: NP_031544.1] was also studied in the assays described above. The

SUBSTITUTE SHEET (RULE 26)

selected pepticie was an 11-amino acids fragment of the sequence set forthin S

ID NO: 5 havirg two amino acids truncated from the C-terminus. Likewise the F GL and EFL peptisdes, the peptide was capable of significant stimulation of neurite Out- growth of rat Fippocampal neurons in vitro. The maximal effect of 500% increase2 In neurite length ~ was observed at 0.3 pg/ml concentration of the peptide.

SUBSTITUTE SHEET (RULE 26)

Claims (11)

PCT/DK2C004/000527 ® 102 Claims

1. A compound comprising two individual peptide sequences, wherein at lea=stone of the two individeial peptide sequences comprises an amino acid sequence of the formula L1-A-12-B-13-C-E 4-D-LS wherein one of A, B, CG, D is selected from a hydrophobic amino acid residue, one of A, B, C, D Is selected from a basic amino acid residue, Asn or Gin, one of A, B, C, D Is selected from an acidic amino acid residue, Ash or Gin, one of A, B, C, D is Gly or Ala, and L1,L2, L3, L4 and LS is selected from a chemical bond or an amino acid se- quence having n amino acid residues, wherein n is an integer of from 0 tos 5, wherein said peptide sequences are connected to each other through a linker of the for- mula ) X{(A)nCOOH][(B YmCOOH] n and m indeperedently are an integer of from 1 to 20, X is HN, HoN(CER)pCR, RHN{CR,)}pCR, HO(CR,)pCR, HS(CR2)pCR, Mmalogen- (CRz)pCR, HOO:C(CR2)pCR, ROOC(CR)pCR, HCO(CR,)pCR, RCO(CR,)pCR, [HOOC(A)N[HOOC(B)MICR(CRPCR, H:N(CR:)p, RHN(CR;)p, HO{(CR.)p, HS(CRz)p, haBogen-(CR)p, HOOC(CRzp, ROOC(CRp, HCCHCR.)p, RCO(CRz)p, or [HOOC(AN[HOOC(BYM{CR)p , wherein-p is 0 or in®eger of from 1 to 20, A and B independently are a substituted or unsubstituted Cup alkyl, am substi- tuted or unsubstituted C,.io alkenyl, a substituted or unsubstituted cyclic moiety, a substituted or unsubstituted heterocyclic moiety, a substituted or unsubostituted aromatic moiety, or A and B together form a substituted or unsubstituteed cyclic moiety, substituted or unsubstituted heterocyclic moiety, or substituted or un- substituted aromatic moiety.

2. The compound according to claim 1, wherein the at least one of the two Peptide sequences is capable of binding to a functional! cell surface receptor. AMENDED SHEET CLEAN COPY

PCT73K2004/000527 ® 103

3. The compound according to claim 2, wherein the functional cell surfamce receptor is a receptor selected from the family of fibroblast growth factor recepstors (FGFRs) comprising FGFR1, FGFR2, FGFR3 and FGFRA.

4. The compound according to claim 2, wherein the at least one of the two peptide sequences is deriveed from the sequence of a polypeptide selected from the group comprising ceall adhesion molecules, cell-surface receptors, tkeparan sul- phate proteoglycanss, and metalloproteases, extracellular matrix mmolecules or growth factors.

5. The compound acc=ording to the claim 4, wherein the cell adhesion molecule is selected from the group comprising - Neural Cell Adhesion Molecule (NCAM) (Swiss-Prot Ass. Nos: P135501, P13595-01, P13535), - Neural cell adhesiosn molecule L1 (Swiss-Prot Ass. Nos: Q9QYQ7, CQ8QY38, P11627, Q05695, F32004), - Neural Cell Adhesieon Molecule-2 (NCAM-2) (Swiss-Prot Ass. No: P236335) - Neuron-glia Cell Acdhesion Molecule (Ng-CAM) (Swiss-Prot Ass. No : Q03696; Qg0933), - Neural cell adhesican molecule CALL (Swiss-Prot Ass. No: 000533) - Neuroglian (Swiss- Prot Ass. No: P81767, P20241), - Nr-CAM (HBRAVO=, NRCAM, NR-CAM 12) (Swiss-Prot Ass. Nos: Q»92823, 015179, QOQVN3 - Axonin-1/TAG-1 (Sswiss-Prot Ass. Nos: Q02246, P22063, P28685 ), - Axonal-associated Cell Adhesion Molecule (AXCAM) (NCBI Ass. No: NP_031544.1; Swisss-Prot Ass, No: Q8TC35), - Myelin-Associated Glycoprotein (MAG) (Swiss-Prot Ass. No: P2031 7), - Neural cell adhesicon molecule BIG-1 (Swiss-Prot Ass. No: Q62682):, - Neural cell adhesion molecule BIG-2 (Swiss-Prot Ass. No: Q62845):, - Fasciclin (FAS-2) (-Swiss-Prot- Ass. No: P22648), - Neural cell adhesicon molecule HNB-3/NB-3 (Swiss-Prot Ass. Nos: CQ9UQ52, P97528, Q9JMBBS) - : - Neural cell adhesicon molecule HNB-2/NB-2 (Swiss-Prot Ass. Nos: £94779, P07408, P97527), AMENDED SHEET :

PCT/DK2004/ 000527 - Cadherin (Swiss-Prot Ass. No: QOVW71), - Junctional Adhesion Mo lecule-1 (JAM-1) (Swiss-Prot Ass. Nos: Q3JKD5, 088792), - Neural cell adhesion F3#/F11(Contactin) {Swiss-Prot Ass. Nos: Q63198, P12630, Q12860, Q28106, P147781, 033250), - Neurofascin (Swiss-Pro& Ass. Nos: 80924, Q91Z60; 042414), - B-lymphocyte cell adhesion molecule CD22 (Swiss-Prot Ass. Nos: QGR094, } P20273), - Neogenin (NEO1) (Swis=s-Prot Ass. Nos: Q92859, P97603, Q90610, P57798-), - Intercellular Cell Adhesion Molecule-5 (ICAM-5/telencephalin} (Swiss-Prot Asss. Nos: Q8TAM9, Q60625® or - Galactose binding lectin ~12 (galectin-12) (Swiss-Prot Ass. Nos: Q91VD1, QoJKX2, QSNZ03) and - Galactose binding lectin:-4 (galectin-4) (Swiss-Prot Ass. No: Q8K4109; P38552).

)

6. The compound accordirg to the claim 4, wherein the cell-surface receptomr is selected from the group comprising - Fibroblast Growth Factor Receptor 1 (FGFR1) (Swiss-Prot Ass. Nos: Q9QZNA7, Q99AVV7, QIUDS0, QES3827), - Fibroblast Growth Factosr Receptor 2 (FGFR2) (Swiss-Prot Ass. Nos: Q96Kiv2, P21802, Q63241), . - Fibroblast Growth Factcer Receptor 3 (FGFR3) (Swiss-Prot Ass. Nos: Q85M1 3, AF487554, Q99052), - Fibroblast Growth Factcer Receptor 4 (FGFR4) (Swiss-Prot Ass. No: Qo1 T4277), - Neurotrophin Tyrosin Kimnase Type-2 (NTRKT-2} (Swiss-Prot Ass. No: QBWXJ5), - Leukocyte Antigen Rela _ted Protein-Tyrosine Phosphatase (LAR-PTPRF) (Swiss-Prot Ass. Nos: CRSEQ17, Q64605, Q64604, QOQWE7, QSVISS P10586), - Nephrin (Swiss-Prot Asss. Nos: Q926S5, Q9JIX2, Q9ET59, Q9R044, Q9QzS-7, Q06500), - Protein-Tyrosine Phosphatase Receptor type S (PTPRS) (Swiss-Prot Ass. Nos: Q64699, Q13332 , 075870), - Protein-Tyrosine Phosplhatase Receptor type kappa (R-PTP-kappa) (Swiss- Prot Ass. No: Q15262), : AM ENDED SHEET C LEAN COPY

PCT/DK2004/000527 ® 105 - Protein-Tyrosine Phosphatase Receptor typ= D (PTPRD) (Swiss-Prot Ass.

Nos: QBWXES, QSIAJ1, P23468, Q64487), - Ephrin type-A receptor 8 (EPHA8/Tyrosine-P rotein Kinase Receptor EEK) (Swiss-Prot Ass.

Nos: 009127, P29322), - Ephrin type-A receptor 3 (EPHA8/Tyrosine-Protein Kinase Receptor ETK- 1/CEK4) (Swiss-Prot Ass.

No: P29318), - Ephrin type-A receptor 2 (Swiss-Prot Ass.

No: Q8N3Z2) - Insulin Receptor (IR) (Swiss-Prot Ass.

No: QEPWNG) - Insulin-like Growth Factor-1 Receptor (IGF-1) (Swiss-Prot Ass.

Nos: Q3Qvw4, P0B069, P24062, QB0751, P15127, P15208) - Insulin-related Receptor (IRR) (Swiss-Prot Asss.

No: P14616), -Tyrosine-Protein Kinase Receptor Tie-1 (Swiss-Prot Ass.

Nos: 06805, P35590, Q06806), - Roundabout receptor-1 (robo-1) {Swiss-Prot Ass.

Nos: 044324, AF041082, QIYBN7), - Neuronal nicotinic acetylcholine receptor alptha 3 subunit (CHRNAS) (Swiss- Prot Ass.

Nos: Q8VHHSE, P04757, Q8R4G9, IPP32297) - Neuronal acetylcholine receplor alpha 6 subunit (Swiss-Prot Ass.

Nos: Q15825, QIROWS) - Platelet-Derived Growth Factor Receptor Beta (PDGFRB) (Swiss-Prot Ass.

Nos: Q8R406, Q05030), - Interleukin-6 Receptor (IL-6R) (Swiss-Prot Ass.

No: Q00560), - Interieukin-23 Receptor (IL-23R) (Swiss-Praot Ass.

No: AF461422), - Beta-common cytokine receptor of IL-3, IL5 and GmCsf (Swiss-Prot Ass.

No: P32927) - - Cytokine Receptor-Like molecule 3 (CRLF1) (Swiss-Prot Ass.

No: Q9JM58), - Class | Cytokine Receptor (ZCYTORS) (Swisss-Prot Ass.

No: QSUHHS) - Netrin-1 receptor DCC (Swiss-Prot Ass.

No: IP43146), - Leukocyte Fc Receptor-like Protein (IFGP2) €Swiss-Prot Ass.

Nos: Q96PJ6, QIBKMR2), - Macrophage Scavenger Receptor 2 (MSR2) (Swiss-Prot Ass.

No: Q91YK7) and - Granulocyte Colony Stimulating Factor Receptor { G-CSF-R) (Swiss-Prot Ass.

No: Q99062).

AMENDED SHEET : CLEAN COPY

PCT/DK2004/000527 ® 106

7. The compound according to the claim 4, wherein the heparan sulphate pro- teogly-can is periecan (Swiss-Prot Ass. No: P98160).

8. The compound according to the claim 4, wherein the metalloprotease is selected from tEhe group comprising - ADAM-8 (Swiss-Prot Ass. No: Q05910), - ADARM-18 (Swiss-Prot Ass. Nos: Q9HO13, 035674), - ADAIM-8 (Swiss-Prot Ass. No: P78325), - ADARMV-12 (Swiss-Prot Ass. Nos: 043184, Q61824), © - ADARM-28 (SwissProt Ass. Nos: QSJLNG, 61824, QIXSLES, QOUKQ2), - ADABM:-33 precursor (Swiss-Prot Ass. Nos: Q8R533, Q923Vw/9), - ADARM-9 (Swiss-Prot Ass. Nos: Q13433, Q61072), - ADABRM-7 (Swiss-Prot Ass. NoS: Q9H2U9, 035227, Q631800), - ADABM-1A Fertilin alpha (Swiss-Prot Ass. No: Q8R533), 16 - ADABM-15 (Swiss-Prot Ass. Nos: Q9QYV0, 088833, Q1 344.4), - Meta_lloproteinase-desintegrin domain containing protein (TEECAM) (Swiss-Prot :

Ass. No: AF163291), and - Meta lloproteinase 1 (Swiss-Prot Ass. Nos: 085204, Q9BSIES).

9. The compound according to the claim 4, wherein the extracellular matrix mole- cule is selected from the group comprising - Colia_gen type VII (Swiss-Prot Ass. No: Q63870), - Fibro nectin (Swiss-Prot Ass. Nos: Q95KV4, QI5KVS5, P075839, 028377, U425=94, 095608, P11276), or - Tena scin-R (Swiss-Prot Ass. Nos: Q15568, 000531, Q909635, P10039).

10. The compound according to the claim 4, wherein the growti— factor is Cytokine- > like factor-1 (CLF-1) (Swiss-Prot Ass. No:075462).

11. The cosmpound according to any of the claims 1 to 10, wherein the at least one of the ®Ewo peptide sequences is a peptide fragment having thee amino acid se- quence= selected from EVYV\WAENQQGKSKA (SEQ ID NO 1), NIEVWJVEAENALGKKYV (SEQ ID NO: 2), ATNRQGKVKAFAHL (SEQ ID NO: 3), AMENDED SHEET CLEAN COPY

PCT/DK2004,/000527

@® RYVELYVVADSQEFQK (SEQ ID NO: &) VAENSRGKNVAKG (SEQ ID NO: 5), GEYWCVAENQYGQR (SEQ ID NO: 6) , RLAALNGKGLGEIS (SEQ ID NO: 7),

KYIAENMKAQNVAKE! (SEQ ID NO: 8), TIMGLKPETRYAVR (SEQ iD NO: 9), KGLGEISAATEFKT (SEQ ID NO: 10), NMGIWVQAENALG (SEQ ID NO: 11), IWVQAENMLG (SEQ ID NO: 12),

EIWVEATNRLG (SEQ ID NO: 13), VWVQAANALG (SEQ ID NO: 14), EVWIEKDPAKGRI (SEQ ID NO: 15), ATNKGGEVKKNGHL (SEQ ID.NO: 16), KYVELYLVADYLEFQK (SEQ ID NO: 17),

RYVELYVVVDNAEFQ (SEQ ID NO: 138), KYVELVIVADNREFQR (SEQ ID NO: 9), KYIEYYLVLDNGEFKR (SEQ ID NO: 20), RYLELYIVADHTLF (SEQ ID NO: 21), KYVEMFVVVNHQRFQ (SEQ ID NO: 222) ,

RYVELFIVVDKERY (SEQ ID NO: 23), KYVELFIVADDTVYRR (SEQ ID NO: 24), KFIELFVVADEYVYRR (SEQ ID NO: 25),

KIVEKVIVADNSEVRK (SEQ ID NO: 226), VELVIVADHSEAQK (SEQ ID NO: 27) , VAENSRGKNIAKG (SEQ ID NO: 28), IAENSRGKNVARG (SEQ ID NO: 29), AENSRGKNSFRG (SEQ ID NO: 30), IASNLRGRNLAKG (SEQ ID NO: 31), IPENSLGKTYAKG (SEQ ID NO: 32), a0 IAENMKAQNEAK (SEQ ID NO: 33), QFIAENMKSHNETKEV (SEQ ID NO: 34), GEYWCVAKNRVGQ (SEQ ID NO: 355), GSYTCVAENMVGK (SEQ ID NO: 36°), GKYVCVGTNMVGER (SEQ ID NO: 537), GNYTCVVENEYG (SEQ ID NO: 38), AMENDED SHIEET CLEAN COPY

PCT/DK2004/000527 ® 108 GEYTCLAGNSIG (SEQ ID NO: 39), QYYCVAENGYG (SEQ ID NO: 40), GEYYQEAEQNGYG (SEQ ID NO: 41), GNYTCLVENEYG (SEQ ID NO: 42),

GMYQCLAENAYG (SEQ ID NO: 43), GMYQCAENTHG (SEQ ID NO: 44), GIYYCLASNNYG (SEQ ID NO: 45), GGYYCTADNSYG (SEQ ID NO: 46), GEYQCFARNDYG (SEQ ID NO: 47),

GEYFCLASNKMG (SEQ ID NO: 48), GEYQCFARNKFG (SEQ ID NO: 49), GEYFCLASNKMG (SEQ ID NO: 50), GGYYCTADNNYG (SEQ ID NO: 51), GNYSCEAENAWGTK (SEQ ID NO: 52),

- GEYTCLAENSLG (SEQ ID NO: 53), GEYECVAENGRLG (SEQ ID NO: 54), GNYTCVVENKFGR (SEQ ID NO: 55), GEYTCLAGNSIG (SEQ ID NO: 56), GEYFCVASNPIG (SEQ ID NO: 57),

EYTCIANNQAGE (SEQ ID NO: 58), GMYQCVAENKHLG (SEQ ID NO: 59), GEYMCTASNTIGQ (SEQ ID NO: 60),

EYVCIAENKAGEQ (SEQ ID NO: 61), GDYTLIAKNEYGK (SEQ ID NO: 62),

GFYQCVAENEAG (SEQ ID NO: 63), GKYECVATNSAGTR (SEQ ID NO: 64), GEYFCVYNNSLG (SEQ ID NO: 65), GEYECAATNAHGR (SEQ ID NO: 66), GAYWCQGTNSVGK (SEQ ID NO: 67),

GTYSCVAENILG (SEQ ID NO: 68), RVAAVNGKGQGDYS (SEQ ID NO: 69), RVAAINGCGIGPFS (SEQ ID NO: 70), AVLNGKGLG (SEQ ID NO: 71), ALNGQGLGATS (SEQ ID NO: 72),

RLAAKNRAGLGE (SEQ ID NO: 73),

AMENDED SHEET CLEAN COPY

PCT/DK2004,/000527 ° 109 . RLGVVTGKDLGEI (SEQ ID NO: 74), TVTGLKPETSYMVK (SEQ ID NO: 75), TLTGLKPSTRYRI (SEQ ID NO: 76), ~ TLTGLQPSTRYRV (SEQ ID NO: 77),

TLLGLKPDTTYDIK (SEQ ID NO: 78), TLQGLRPETAYELR (SEQ ID NO: 79), TLRGLRPETAYELR (SEQ ID NO: 80), TLMNLRPKTGYSVR (SEQ ID NO: 81), TVSGLKPGTRY (SEQ ID NO: 82),

TISGLKPDTTY (SEQ ID NO: 83), TLQGLKPDTAY (SEQ ID NO: 84), LRGLKPWTQYAV (SEQ ID NO: 85), IDGLEPDTEYIVR (SEQ ID NO: 86), LOGLKPWTQYAI (SEQ ID NO: 87),

TITGLEPGTEYTIQ (SEQ ID NO: 88), GLKPWTQYAV (SEQ ID NO: 89), : TLASLKPWTQYAV (SEQ ID NO: 90), LMGLQPATEYIV (SEQ ID NO: 91), KGMGPMSEAVQFRT (SEQ ID NO: 82),

TLTGLKPDTTYDVK (SEQ ID NO: 93), ISGLQPETSYSL (SEQ ID NO: 94), TLLGLKPDTTYDIK (SEQ ID NO: 95), TISGLTPETTYSI (SEQ ID NO: 96), GNYSCLAENRLGR (SEQ ID NO: 97),

GNYTCVVENRVG (SEQ iD NO: 98), GTYHCVATNAHG (SEQ ID NO: 99), LSHNGVLTGYLLSY (SEQ ID NO: 100), NGVLTGYVLRY (SEQ ID NO: 101), NGVLTGYNLRY (SEQ ID NO: 102),

NGNLTGYLLQY (SEQ ID NO: 103), VDENGVLTGYKIYY (SEQ ID NO: 104), THNGALVGYSVRY (SEQ ID NO: 105), NGILTEYILKY (SEQ ID NO: 106), NGILIGYTLRY (SEQ ID NO: 107),

THSGQITGYKIRY (SEQ ID NO: 108),

AMENDED SHEE T CLEAN COPY

PCT/DK2004/000527 C 110 NGKITGYIYY (SEQ ID NO: 108), LSHNGIFTLY (SEQ ID NO: 110), NGILTEYTLKY (SEQ ID NO: 111), LDPNGIITQYEISY (SEQ ID NO: 112), NGKITGYIIYY (SEQ ID NO: 113), HLEVQAFNGRGSGPA (SEQ ID NO: 1 14), HLTVRAYNGAGYGP (SEQ ID NO: 115), HLSVKAYNSAGTGPS (SEQ ID NO: 1-186), HLAVKAYNSAGTGPS (SEQ ID NO: 1 17), NLEVRAFNSAGDGP (SEQ ID NO: 1138), HLTVLAYNSKGAGP (SEQ ID NO: 119), LRVLVFNGRGDGP (SEQ ID NO: 120), HIDVSAFNSAGYGP (SEQ ID NO: 121 ), HLAVELFNGR (SEQ ID NO: 122), LELQSINFLGGQPA (SEQ ID NO: 123), HFTVAAYNGAGYGF (SEQ ID.NO: 1224), HLEVOAFNGRGSQPA (SEQ ID NO: 925), VIADQPTFVKYLIK (SEQ ID NO: 126), TIKGLRPGVVYEGQ (SEQ ID NO: 127), TLTELSPSTQYTVK (SEQ ID NO: 128 ), TLDDLAPDTTYLVQ (SEQ ID NO: 1298), TVSDVTPHAIYTVR (SEQ ID NO: 130), IIRGLNASTRYLFR (SEQ ID NO:131), TLMNLRPKTGYSVR (SEQ ID NO:132), TLTGLKPGTEYEVR (SEQ ID NO: 133), GPEHLMPSSTYVAR (SEQ ID NO: 13:4), RVTGLTPKKTYEFR (SEQ ID NO: 135), LTGLKPGTEYEFR (SEQ ID NO: 136) , : EVRVQAVNGGGNGPP (SEQ ID NO: 137), LIKVVAINDRGE (SEQ ID NO: 138), VVSIIAVNGREE (SEQ ID NO: 139), VVSVYAQNQNGE (SEQ ID NO: 140). TISLVAEKGRHK (SEQ ID NO: 141), HLEVQAFNGRGSGPA (SEQ ID NO: 142), HVEVQAFNGRGLGPA (SEQ ID NO: 143), AMENDED SHEEET CLEAN COPY

PCT/DK2004/0005 27 : pu 111 . HVEVQAFNGRGLGPA (SEQ ID NO: 144), EFRVRAVNGAGEG (SEQ ID NO: $45), or VARVRTRLAPGSRLS (SEQ ID NO»: 146), or or a fragment, or a variant, or homologue thereof, wherein . said fragment is an amino acid sequence which has at least 40% of the length of a sequence selected from SEQ ID [NOs:1-146 and which is capable of binding to fibroblast growth factor receptor, said variant is an amino acid sequence which has at least 60% of homology to a sequence selected from SEQ ID NOs: 1-146 and which is capable of binding to fibroblast growth factor receptor, an d said homologue is an amino acid sequence which has at least 20% homology to a sequence selected from SEQ ID NOs: 1-146 and which is capable of binding to fibroblast growth factor receptor.

12. The compound according to claims 1 to 10, wherein the at least one of the two peptide sequences is SEQ ID NO: 1 (EVYVVAENQQGKSKA), or a fragment, variant, or homologue of said sequence.

18. The compound of claim 12, wherein the variant or homologue of SEQ ID NO: 1 is selected from SEQ ID NOs: 2-9, 100 or 125,

14. The compound according to claims 1 to 10, wherein the at least one of the two peptide sequences is SEQ ID NO: 2 (NIEVWVEAENALGKKV), or a fragment, variant or homologue of said seque nce. -

15. The compound according to any of the preceding claims, wherein the compound comprises two individual peptide fragments comprising different amino acid se- quences, said different amino acid sequences being selected from any of the peptide fragments of claim 11.

16. The compound according to any of the preceding claims, wherein the compound comprises two peptide fragments comprising the identical amino acid sequence, said amino acid sequence being selected from any of the peptide fragments of claim 11. AMENDED SHEET I FAN CNDV ’

PCT/DK2004/000527 ® 112 -

17. The compound according to claim 16, wherein the peptide fragments are inde- pendently having the sequence EVYVVAEENQQGKSKA (SEQ 1D NO: 1).

18. The compound according to claim 16, winerein the peptide fragments are inde- pendently having the sequence NIEVWVEAENALGKKY (SEQ ID NO: 2).

19. The compound according to claim 15, wh-erein one of the two peptide fragments is having the sequence EVYVVAENQQGHECSKA (SEQ ID NO: 1), and the other is having the sequence NIEVWVEAENALGHKY (SEQ ID NO: 2).

20. The compound according to any of the preceding claims, said compound being obtained by a method for preparing zn LPA enabling presentation of se- quence(s) as defined in claim 11 comprising the steps of (a) providing by solid phase synthesis or fragment coupling ligands comprising desired sequence(s), the ligands berg attached 10 a solid phase, : (b) if nessesary, deprotecting any N-terminal amino acid groups while th eli- gands/s) are still attached to the solid phase, (c) reacting the ligand{s) having unprotected N-terminal groups with an achiral di- tri- or tetracarboxylic acid so ass to provide a construct having a ring structure, and (d) cleaving the construct from the solicd phase so as to provide an LPA com- prising ligands having free C-terminami groups.

21. A pharmaceutical composition comprisirg a compound as. defined in claims 1-

20.

22. Use a compound as defined in any of tine claims 1-20 for the manufacture of a medicament for treatment of conditions of the central and peripheral nervous system associated with postoperative n erve damage, traumatic nerve damage, impaired myelination of nerve fibers, po stischaemic damage, e.g. resulting from a stroke, Parkinson's disease, Alzheim=er's disease, Huntington's disease, de- mentias such as muttiinfarct dementia, ssclerosis, nerve degeneration associated with diabetes mellitus, disorders affectirg the circadian clock or neuro-muscular transmission, and schizophrenia, mood -disorders, such as manic depression; for AMENDED SHEET CLEAN COPY

PCT/DK2004/000527 C 113 treatment of diseases or conditions of the muscles including conditions with im- paired function of neuro-muscutar eonnections, such as after organ transplanta- tion, or such as genetic or traumatic atrophic muscle disorders; or for treatment of diseases or conditions of various organs, such as degenerative conditions of the gonads, of the pancreas such &s diabetes mellitus type | and ll, of the kidney such as nephrosis and of the heart, liver and bowel.

23. Use a compound as defined in arwy of the claims 1-20 for the manufacture of a medicament for the treatment of postoperative nerve damage, traumatic nerve damage, impaired myelination of Merve fibers, postischasmic, e.g. resulting from a stroke, Parkinson's disease, AEzheimer's disease, Huntington's disease, de- mentias such as multiinfarct dementia, sclerosis, nerve degeneration associated with diabetes mellitus, disorders affecting the circadian clock or neuro-muscular transmission, and schizophrenia, gnood disorders, such as manic depression.

24. Use a compound as defined in any of the claims 1-20 for the manufacture of a medicament for the promotion of wound-healing.

25. Use a compound as defined in claims 1-20 for the manufacture of a medicament for the treatment of cancer

26. The use according to claim 25, wherein the cancer is any type of solid tumors requiring neoangiogenesis

27. Use a compound as defined in any of the claims 1-20 for the manufacture of a medicament for the prevention of death of heart muscle cells, such as after acute myocardial infarction, or after angiogenesis

28. Use a compound as defined in any of claims 1-20 for the manufacture of a me- dicament for revascularsation.

29. Use a compound as defined in any of the claims 1-20 for the manufacture of a medicament for the stimulatiory of the ability to learn and/or the short and/or long-term memory AMEN DED SHEET CL EAN COPY

® 114 PCT/DK2004/000527

30. Use a compound as defined in any of the claims 1-220 for the manufacture of a medicament for the prevention of cell death duee to ischemia.

31. Use a compound as defined in any of the claims 1-20 for the manufacture of a mnedicament for the prevention of body darmages due to alcohol consurmption.

32. Use a compound as defined in any of the claims 1- 20 for the manufacture of a medicament for the treatment of prion diseases.

33. Use of a medicament of any of the claims 22—31 or pharmaceutical composition according to claim 21 for the treat ment of a disease or condition as defined in claims 22-31.

34. Use of a compound as defined in any of the claims 1 to 20, in the manufacture of a medicament for treatment of an individual in need.

35. A subostance or composition for use in a me thod for treatment of conditions of the central and peripheral nervous system associated with postoperative nerve damage, traumatic nerv € damage, impaired myelimation of nerve fibers, postischaemic damag e, e.g. resulting from a stroke , Parkinson's disease, Alzheimer's disease, Huntington's disease, dementias such as multiinfarct dementia, scleros-is, nerve degeneration "25 associated with diabetes mellitus, disorders affecting the circadian clock or ne uro-muscular transmission, and schizophr enia, mood disorders, such &as manic depression; for treatment of diseases or conditions of the muscl e€s including conditions with impaired func-tion of neuro-muscular conne=ctions, such as after organ transplantation, or such as genetic or traumatic atrophic muscle disorders; or for trezatment of diseases or conditions of various organs, such as degenerative conditions of the AMENDED SHEET

¢ 115 P&CT/DK2004/000527 gonads, of the pancreas such as diabetes mellitus type | and Ill, of the kidney such as nephrosis and of the heart, live r and bowel, said subsstance or composition comprising a compound aas defined in any of the claims 1-20, and said method comprising administering said subsstance or composition.

36. A substance or composition for use in a method for the treatment of postoperative nerve damage, traumatic nerve damage, impaired mye: lination of nerve fibers, postischaemic, e.g. resulting from a stroke, Parkinson's disease, Alzheimer's disease, Hurtington's disease, demuentias such as multiinfarct dementia, sclerosis, nerve degeneration associated with diabetes mellitus, disorders affectingg the circadian clock or meuro-muscular transmission, and schizophreniaa, mood disorders, such as manic depression, said substance or compeosition comprising a commpound as defined in any of the claims 1-20 , and said method comprising administering said substance or compositi on.

37. A substance or composition for use in a method for the promotion of wound-healing, said substance or composition com prising a compound as defined in any of the claims 1-20, and said method comprising, administering said substance or composition.

38. A substance or composition for use in a method for the treatment of can cer, said substance or composition comprisimg a compound as defi ned in any of the claims 1-20, and said @method comprising, administering said substance or composition.

39. A smubstance or composition for use in a method of treatment according to claim 38, wherein the cancer is any type of so lid tumors requiring neangiogenesis. AMENDED SHEET

Ki 116 PCT/DK2004/000527

40. A substance or composition for use in a mexthod for the prevention of death of heart muscle cells, such as after acurte myocardial infarction, or after angiogenesis, said substance or composition comprising a compound as defined in any of the claims 1-20, and said method comprising administering said substance or co mposition.

41. A substance or composition for use in a method for revascularsation, said substance or composition comprising a compound as defined in any of claims 1-20, and said method comprising administering said substance or composition.

42 . A substance or composition for use in a meth od for the stimulation of the ability to learn and/or the short and/or long-term memory, said substance or composition comprising a compound as de=fined in any of claims 1-20, and said method comprising administering said substance or composition.

43. A substance or composition for use in a method for the prevention of cell death due to ischemia, said substance or composition comprising a compound as defined in any of claims 1-20, and said method comprising administering said substance or composition.

44. A substance or composition for use in a m ethod for the prevention of body damages due to alcohol consumption, said substance or composition comprising a compound as defined in any of claims 1-20, and said method comprising administering said substance or composition. AMENDED SHEET r 117 PCT/DK22004/000527

45. A sub stance or composition for use in a method for the treatement of prion diseases, said substance or composition comprising a compound as define=d in any of claims 1-20, and said method comprising &dministering said s ubstance or composition.

46. A sulostance or composition for use in a method of treatment of an indivicdual in need, said substance or composition comprising a compound as defined in any of claims 1-20, and said method comprising administering said substance or composition to said individual.

47. A cormpound according to any one of claims 1 to 20, substantially as hereim described and illustrated.

48. A cormposition according to claim 21, substantially as herein described and illustrated.

49. Use according to any one of claims 22 to 34, substantially as herein descr ibed and illustrated.

50. A submstance or composition for use in a method of treatment according to any one of claims 35 to 46, substantially as herein described and illustrated.

51. A new compound, a new composition, a new use of a compound as claimed in any one of claims 1-20, or a substance or com position for a new wise in a method of treatment, substantially as herein desscribed. AMENDED SHEET