WO2007110079A2 - Targeted delivery of fgfr ligands into the brain - Google Patents

Targeted delivery of fgfr ligands into the brain Download PDF

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Publication number
WO2007110079A2
WO2007110079A2 PCT/DK2007/000152 DK2007000152W WO2007110079A2 WO 2007110079 A2 WO2007110079 A2 WO 2007110079A2 DK 2007000152 W DK2007000152 W DK 2007000152W WO 2007110079 A2 WO2007110079 A2 WO 2007110079A2
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Prior art keywords
peptide sequence
cell
disease
seq
compound according
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PCT/DK2007/000152
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French (fr)
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WO2007110079A3 (en
WO2007110079A8 (en
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Elisabeth Bock
Vladimir Berezin
Vladislav V. Kiselyov
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Enkam Pharmaceuticals A/S
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Publication of WO2007110079A8 publication Critical patent/WO2007110079A8/en
Publication of WO2007110079A3 publication Critical patent/WO2007110079A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/825—Metallothioneins

Definitions

  • the present invention relates to a compound consisting of metallothionein (MT) and a peptide sequence, wherein said peptide sequence and metallotionein are bound to each other via a non-covalent bond.
  • a peptide sequence comprised by the compound is a biologically active short peptide sequence which comprises at most 25 amino acid residues.
  • the peptide sequence is capable of binding to and modulating activity of a functional cell receptor, in particular fibroblast growth factor receptor (FGFR).
  • FGFR fibroblast growth factor receptor
  • the invention relates to pharmaceutical compositions comprising such compound or such peptide sequence and use of the compound, peptide sequence and pharmaceutical composition for treatment of conditions and diseases where modulating activity of FGFR is beneficial for treatment.
  • the use is for treatment of conditions and diseases of the brain including conditions requiring stimulating brain cell proliferation, differentiation and/or survival, stimulating neural plasticity associated with learning and memory, modulating adhesion and motility of cells, in particular cancer cells.
  • the compound of the invention is capable of penetrating the blood brain barrier (BBB).
  • the blood brain barrier is one of the major problems in drug delivery to the brain.
  • the kinetics of brain penetration has two components, extent and rate. Achieving a high extent of brain penetration is an important focus for central nervous system (CNS) drug discovery.
  • Optimal brain penetration can be achieved by reducing efflux transport at the BBB, and it is critical to ensure that a high total brain/plasma ratio (the most commonly used parameter for measuring brain penetration) is due to efflux transport activity at the BBB and not related to high non-specific brain tissue binding or low plasma binding. Rapid brain penetration is essential for those drugs that require fast onset of action in the CNS.
  • a peptide drug which is to be delivered into the brain has to be a ligand of one of the receptors involved in transcytosis, or at least it has to have a capability to bind to the receptor and thereby induce receptor mediated endocytosis.
  • Megalin is a scavenger receptor due to its multifunctional binding properties. Among its ligands are lipoproteins, vitamin-binding and carrier proteins, drugs, hormones and enzymes as well as signalling molecules, (see for review May et al, 2005). Megalin is one of the receptors which are involved in transcytosis of proteins and peptides through the blood-brain barrier (BBB).
  • BBB blood-brain barrier
  • One of the best-characterized physiological functions of megalin is the proximal-tubular reuptake of low-molecular weight proteins (Zou et al., 2004).
  • MTs are metal binding proteins (61-68 amino acids), which normally bind seven zinc ions, although zinc/copper mixtures have been reported. Expression of some iso- forms of MT is rapidly induced in response to increases in zinc or copper levels, and also by a large number of hormones and cytokines, including glucocorticoids, inter- leukin 1 and 6, interferons and so on. The exact physiological role of MTs is not fully understood. Early suggestions that they act to prevent accumulation of toxic levels of heavy metals are no longer much favoured, and if their role is indeed in metal metabolism, it is more likely that they are involved in the intracellular homeostasis of zinc. However, MTs are efficient scavengers of free radicals and are able to protect DNA and other molecules from oxidation, suggesting that their function may be protective. MTs are also considered to be intracellular stress proteins which respond to a wide variety of stimuli.
  • MT1 and MT2 have been shown to prevent apoptotic cell death in the CNS (Giralt et al., 2002).
  • MT1 and MT2I also improve the clinical outcome and reduce mortality in different CNS disorders (Penkowa, 2002).
  • MT2 treatment has also been shown to significantly stimulate neurite extension from both dopaminergic and hippocampal neurons, significantly increase survival of dopaminergic neurons ex- posed to 6-hydroxydopamine (6-OHDA) and protects significantly hippocampal neurons from amyloid ⁇ -peptide-induced neurotoxicity (K ⁇ hler et al.,2003).
  • MTs has been shown to mediate neuroprotection in genetically engineered mouse model of Parkinson's disease (Ebadi et al., 2005). Treatment using MT2 and other MTs has been suggested for motor neuron disease, head injury, Alzheimer's and Parkinson's diseases (WO03105910).
  • megalin MT is known to bind to some poorly identified receptors in astrocytes (El Refaey et al., 1997), and it also binds to itself forming aggregates (WiI- helmsen et al. 2002). Other binding partners of MT of proteineous origin have not so far been described.
  • peptide sequences of a relatively short length derived from fibroblast growth factor receptor (FGFR) ligands are capable of binding to metallothionein protein (MT) or a fragment thereof forming thereby a compound consisting of MT and one or more such sequences where the
  • MT and sequence(s) are bound to each other via non-covalent bond and/or via sulf- hydryl moieties of the cysteine. It was further surprisingly found that such compound is stable in water solutions under physiological conditions in vivo and in vitro and it is capable of penetrating the BBB when administered in a subject in vivo.
  • the invention relates to a compound consisting of MT and a peptide sequence wherein the MT and peptide sequence are bound to each other via a non-covalent bond or via sulfhydryl moieties of the cysteine.
  • the peptide sequence of the compound is a relatively short peptide sequence which comprises at most 25 amino acid residues.
  • the sequence is a biologically active peptide.
  • the invention relates to biologically activity of the peptide which is associ- ated with activity of a functional cell receptor.
  • a peptide sequence of the compound executes its biological activity via binding to functional receptors involved in regulation of the mentioned physiological processes. Upon binding the sequence may either activate or inhibit activity of the receptor which it has affinity to.
  • the invention preferably relates to biologically active short peptide sequences which are capable of binding to fibroblast growth factor receptor (FGFR).
  • FGFR fibroblast growth factor receptor
  • the invention preferably relates to FGFR which is expressed by a cell of the brain and is involved in regulation of physiological processes occurred in the brain.
  • the invention concerns regulation of the processes of cell differentiation, cell survival and/or cell plasticity associated with learning and memory, tissue reparation due to the oxidative stress and/or inflammatory responses.
  • a biologically active sequence is delivered to the cells of the brain as a part of the compound of the invention where the sequence is non-covalently bound to a carrier protein.
  • the carrier protein of the invention is metallothionein protein (MT). Accordingly, another aspect of the invention relates to the use of MT as carrier protein for the delivery of a short biologically active peptide sequence into the brain.
  • MT metallothionein protein
  • a complex of MT with a short peptide sequence for the manufacturing of a medicament
  • said medicament is for therapeutic treatment involving stimulating brain cell proliferation, stimulating neurite outgrowth, stimulating brain cell survival, stimulating neural cell plasticity associated with learning and memory, and/or inhibiting inflammation, modulating cell adhesion and/or cell motility;
  • a pharmaceutical composition comprising MT with a peptide sequence of the invention; a method of treatment of a condition or a disease involving stimulating brain cells proliferation, stimulating neurite outgrowth, stimulating brain cell survival, stimulating neural cell plasticity associated with learning and memory, and/or inhibiting inflammation, modulating cell adhesion and/or cell motility comprising using a compound of the invention; a method of treatment of a condition or a disease involving modulating activity of FGFR comprising using a compound of the invention.
  • Figure 1 demonstrates binding of NCAM F3, 2 module derived peptide (FGL) to MT. Binding was studied by means of SPR analysis. Approximately 2000 resonance units (RU) of the MT2 protein (Sigma) were immobilized on the sensor chip. The binding is given as the response difference between the binding to the sensor chip with the immobilized MT2 and a blank sensor chip (unspecific binding). The peptide was injected into the sensor chip at indicated concentrations.
  • RU resonance units
  • Figure 2 demonstrates binding of NCAM F3, 1 module derived peptides (ABL and EFL) to MT. Binding was studied by means of SPR analysis. Approximately 2000 resonance units (RU) of the MT2 protein were immobilized on the sensor chip. The binding is given as the response difference between the binding to the sensor chip with the immobilized MT2 and a blank sensor chip (unspecific binding). The peptides were injected into the sensor chip at indicated concentrations.
  • RU resonance units
  • Figure 3 demonstrates binding of the peptides derived from FGF1 (2F1 /Dyo1 and 10F1/Deka1) and FGF17 (2F17/Dyo17 and 10F17/Deka17) to MT. Binding was studied by means of SPR analysis. Approximately 2000 resonance units (RU) of the MT2 protein were immobilized on the sensor chip. The binding is given as the response difference between the binding to the sensor chip with the immobilized MT2 and a blank sensor chip (unspecific binding). The peptides were injected into the sensor chip at indicated concentrations.
  • RU resonance units
  • Figure 4 demonstrates binding of the beta 10-beta11 loop region derived peptides (dekafins) of different FGF to the combined second and third Ig modules of FGFR1.
  • Binding was studied by means of SPR analysis. Approximately 2000 resonance units (RU) of the FGFR constructs were immobilized on the sensor chip. The binding is given as the response difference between the binding to the sensor chip with the immobilized FGFR modules and a blank sensor chip (unspecific binding). The peptides were injected into the sensor chip at a concentration of 1 ⁇ g/ml with the exception of dekafin ⁇ , which was tested in a concentration of 100 ⁇ g/ml.
  • Binding affinity (1/Kd) of dekafins was tested by means of SPR analysis. Approximately 2000 resonance units (RU) of the FGFR constructs were immobilized on the sensor chip. The binding is given as the response difference between the binding to the sensor chip with the immobilized FGFR modules and a blank sensor chip (unspecific binding). The
  • Results from four independent experiments are expressed as means ⁇ SEM.
  • FIG. 5 demonstrates phosphorylation of FGFR1 by dekafins and FGFs.
  • TREX- 293 cells transfected with FGFR containing a C-terminal Strepll tag, were stimulated with the dekafin peptides or FGFs. After stimulation, activated FGFR was im- munoprecipitated by anti-phosphotyrosine antibodies and then analyzed by western blotting by antibodies against the Strepll tag.
  • (a) Dose response of FGFR1 phosphorylation by FGF1 and FGF10. Quantification of FGFR1 phosphorylation was per- formed by densitomeric analysis of band intensity.
  • Results from at least four independent experiments are in all cases expressed as a percentage ⁇ SEM, with untreated controls set at 100%.
  • (b) Dose response of FGFR1 phosphorylation by dekafins. Quantification of FGFR1 phosphorylation was performed by densitomeric analysis of band intensity. Results from at least four independent experiments are in all cases expressed as a percentage ⁇ SEM, with untreated controls set at 100%. *P ⁇ 0.05, **P ⁇ 0.01 and * * *P ⁇ 0.001 , compared with controls.
  • Figure 6 demonstrates the effect of dekafins and FGFs on neurite outgrowth from CGN.
  • CGN cultures from 7-old-day rats were treated with the dekafin peptides or FGFs for 24 hr. The cultures were fixed and immunostained with rabbit anti-rat GAP- 43 primary antibodies, and then with secondary Alexa Fluor®488 goat anti rabbit antibodies, (a) The effect of the dekafin peptides at various doses on neurite extension. Results from four independent experiments are in all cases expressed as a percentage ⁇ SEM, with untreated controls set at 100%. *P ⁇ 0.05, * * P ⁇ 0.01 and ***P ⁇ 0.001 , compared with controls, (b) The effect of various doses of FGF1 and FGF10 on neurite extension.
  • Results from four independent experiments are in all cases expressed as a percentage ⁇ SEM, with untreated controls set at 100%.
  • Results from four independ- ent experiments are in all cases expressed as a percentage ⁇ SEM, with untreated controls set at 100%.
  • *P ⁇ 0.05 and **P ⁇ 0.01 compared with peptide-stimulated controls.
  • Figure 7 demonstrates the effect of dekafins on survival of CGN induced to undergo apoptosis.
  • the neurons were allowed to differentiate for 7 days in a high potassium (40 mM) medium before apoptosis was induced by changing the medium to a low- potassium (5mM) medium. Forty-eight hours later, survival was estimated, (a) High KCI: cells not induced to undergo apoptosis. Low KCI: cells induced to undergo apoptosis.
  • Low KCI + IGF-1 , FGF1 or FGF10 cells induced to undergo apoptosis in the presence of IGF-1, FGF1 or FGF10, respectively,
  • Figure 8 demonstrates the effect of dekafins on social recognition memory. Cognitive function was evaluated using the social recognition test as reflected by the recognition ratio (see Materials and Methods). From 10 to 12 animals were tested in each group. *P ⁇ 0.05, compared with untreated controls.
  • Figure 9 demonstrates binding of dyofins to the combined second and third Ig modules of FGFR1.
  • Binding was determined by means of SPR analysis. Approximately 2000 resonance units (RU) of the FGFR construct were immobilized on the sensor chip. The binding is given as the response difference between the binding of the individual dyofins to the sensor chip with the immobilized FGFR modules and to a blank sensor chip (unspecific binding).
  • the peptides were injected into the sensor chip at a concentration of 1 ⁇ g/ml with the exception of dyofini , 2 and 9.
  • Dyofini and 2 were tested in a concentration of 10 ⁇ g/ml
  • Dyofin9 was tested in a concentration of 200 ⁇ g/ml.
  • Figure 10 demonstrates the effects of dyofins on phosphorylation of FGFR1.
  • TREX- 293 cells transfected with FGFRIc containing a C-terminal Strepll tag, were stimulated with the dyofin peptides or FGF2. After stimulation, activated FGFR was im- munopurified by means of anti-phosphotyrosine antibodies and then analyzed by western blotting using antibodies against the Strepll tag.
  • Figure 11 demonstrates the effect of dyofins and FGF2 on neurite outgrowth from CGN.
  • CGN cultures were treated with the dyofin peptides or FGF2 for 24 hr.
  • the cultures were fixed and immunostained with rabbit anti-rat GAP-43 primary antibodies, and then with secondary Alexa Fluor®488 goat anti rabbit antibodies,
  • *P ⁇ 0.05, **P ⁇ 0.01 and ***P ⁇ 0.001 compared with untreated controls
  • Figure 12 demonstrates the effect of dyofins on survival of CGN induced to undergo apoptosis.
  • the neurons were allowed to differentiate for 7 days in a high potassium (40 mM) medium before apoptosis was induced by changing the medium to a low- potassium (5mM) medium. Forty-eight hours later, survival was estimated, (a) High KCI: cells not induced to undergo apoptosis. Low KCI: cells induced to undergo apoptosis.
  • Low KCI + IGF-1 or FGF2 cells induced to undergo apoptosis in the presence of IGF-1 or FGF2, respectively,
  • Figure 13 demonstrates the effect of dyofins on social recognition memory.(a) dyo- fin1; (b) dyofin2; (c) dyofin ⁇ ; (d) dyofin17. Cognitive function was evaluated using the social recognition test as reflected by the recognition ratio (see Materials and Methods). From 10 to 12 animals were tested in each group. **P ⁇ 0.01 , compared with untreated controls.
  • Figure 14 demonstrates the effect of the ABL, CDL and EFL peptides on phosphorylation of FGFR . TREX-293 cells transfected with FGFRIc were stimulated with different concentrations of the peptides or 100 ng FGF2 (not shown) for 20 min. After stimulation, activated FGFR was immunoprecipitated and immunoblotted using anti-phosphotyrosine antibodies. *P ⁇ 0.05, **P ⁇ 0.01, compared with untreated controls.
  • Figure 15 demonstrates the effect of the second F3 module of NCAM, the FGL pep- tide on phosphorylation of the FGFR1.
  • HEK293 cells transiently transfected with a His-tagged version of the FGFR1, were stimulated for 20 min with either 5 ⁇ g/ml F3,2 module or 50 ⁇ g/ml FG loop peptide.
  • the total amount of the FGF-receptor 1 and the amount of the FGF-receptor phosphorylation was estimated by immunoblot- ting using anti-pentahis (anti-His) and anti-phosphotyrosine (anti-P-tyr) antibodies, respectively. Quantification of the FGF-receptor phosphorylation by densitometric analysis of the band intensity.
  • Phosphorylation was estimated relative to the control (untreated cells), which has been normalized to 1.0. Error bar represents one standard deviation (SD). P ⁇ 0.05 by paired t test comparing treated cells with controls. The t test was performed on array of six independent sets of non-normalized data.
  • Figure 16 shows the effect of the FGL peptide on neurite outgrowth from dopaminergic (•), hippocampal (A) and cerebellar granule neurons ( ⁇ ).
  • Dopaminergic neurons were grown at a density of 100,000 cells/cm 2 on poly-D-lysine coated 24- well cell culture plates for 72 hours with various concentrations of FGL d . The cul- tures were subsequently immunostained for tyrosine hydroxylase. Hippocampal neurons and CGN were plated at a density of 10,000 cells/cm 2 on 8-well permanox chamber slides and incubated for 24 hours in the presence of various concentrations of FGL d . Subsequently the neurons were immunostained for GAP-43. Results from at least 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.
  • Figure 17 demonstrates the effect of the FGL peptide 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 2 for six days without or with various concentrations of peptide on 24-well cell culture plates coated with poly-D-lysine were exposed to 100 ⁇ M 6- OHDA for two hours. Medium was changed and various concentrations of FGL d were added. The neurons were grown for another 24 hours before the cultures they were fixed and immunostained for tyrosine hydroxylase.
  • Hippocampal neurons (HN) (c and d) from day 19 rat embryos were seeded at a density of 40,000 cells/cm 2 on poly-L-lysine coated 8-well permanox chamber slides and grown for 24 hours in medium containing 20 ⁇ M Amyloid- ⁇ 25-35 peptide (A ⁇ 25-35) at the presence of various concentrations of FGL d , before they were fixed and stained with Hoechst 33258.
  • Cerebella granular langerons (CGN) (e and f) from postnatal day 7 rats were grown at a density of 100,000 cells/cm 2 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 various concentrations of FGL d . After two days of incubation, the cultures were fixed and stained with Hoechst 33258.
  • Figure 18 demonstrates the results of TUNEL staining of CGN cultures induced to undergo apoptosis with and without treatment with the ABL, CDL or EFL peptides. The results are expressed as percentage ⁇ SEM of live neurons as compared to the total number of neurons. Control cultures induced to undergo cell death without peptide treatment were set at 100 %; * * * p ⁇ 0.001 when compared to the cultures induced to undergo cell death (low KCI).
  • Figure 19 shows a stimulatory effect of the FGL, ABL, CDL and EFL peptides on learning and memory in experimental animals: Social Recognition test (A ) and Fear Conditioning test (B and C).
  • Figure 20 demonstrates that the FGL peptide prevents development of neuropa- thological changes induced by i.c.v. administration of Ap 25-35 . Bars indicate IR quantified on the basis of average brightness. Open bars represent control animals; solid bars represent Ap 25- 35-treated animals.
  • FIG 21 Intranasal and subcutaneous administration of FGL 2 at day 7, 10 and 13 after injection of A ⁇ 25-35 prevents neuronal cell death and impairment of cognitive function induced by i.c.v. administration of A ⁇ 25 - 35 .
  • Open bars represent control animals; solid bars represent A ⁇ 25 - 35 -treated animals. Animals were sacrificed four weeks after A ⁇ 25 - 35 -injection.
  • Values in a, b, d, and e have been normalized to the values of control animals. Asterisks indicate significant differences when compared to control animals; plus signs indicate significant differences when com- pared to A ⁇ 25-35 -treated animals.
  • the present invention relates to a compound consisting of metallotionein protein (MT) or a fragment thereof and a peptide sequence, wherein the MT and peptide sequence are bound to each other via a non-covalent bond or via sulfhydryl moieties of the cysteine.
  • non-covalent bonds via which the MT and peptide sequence may be bound in the compound include hydrophobic interactions (Van- der Waals forces), hydrogen bonds, ionic bonds, hydrophilic interactions.
  • the invention relates to a compound where the MT and peptide sequence are bound to each other directly without an interconnecting grouping or linker. Throughout the specification of the invention the wording "bound" is used interchangeably with the wording "associated”.
  • the binding between MT and a peptide sequence in the compound is further characterised by a specific affinity binding constant (K 0 ) which has the value of about 10 "10 M to about 10 '3 M., such as of about 10 "8 M to about 10 "4 M or between about 10 "9 M and about 10 "5 M, such as about 10 "7 M or about 10 "6 M.
  • K 0 specific affinity binding constant
  • the compound is further characterized in that it is relatively stable in a water solution under physiological conditions both in vivo and in vitro.
  • physiological conditions includes conditions of the body including the body temperature, pH and ion strength of the body liquids and other factors which constitute the micro-environment of different body tissues and liquids in vivo, e.g. micro-environments of the blood, brain, muscles, etc..
  • the compound according to the invention is characterized in that it despite of its relative stability under such conditions the peptide sequence and MT of the compound dissociate from each other when the compound reaches an appropriate functional cell receptor, such as a receptor of a brain cell, which the peptide sequence is capable of binding to.
  • a peptide sequence which is comprised by the compound comprises at most 25 contiguous amino acid residues, such as from 3 to 25 amino acid residues, such as from 3 to 20, for example from 3 to 15, such as from 3 to 10, such as form 5 to 25, such as from 7 to 25, fro example from 8 to 25, or from 10 to 25, such as from 12 to 25 or from 14 to 25. Sequences comprising from 5 to 20 contiguous amino acid residues are preferred.
  • the invention relates to compounds comprising MT and any contiguous peptide sequence of at most 25 amino acid residues which are associated together in the compound via a non-covalent bond or via sulfhydryl covalent bond made of the moieties of the cysteins.
  • a preferred peptide sequence of at most 25 amino acid residues according to the invention is characterized in that it comprises the amino acid motif x1-x2-x3-x4-x5, wherein x1 is a hydrophobic, charged amino acid residue or G; x2 is a charged amino acid residue, T or S, x3 is any amino acid residue; x4 is a charged amino acid residue, T or S, x5 is a hydrophobic, charged amino acid residue T, S, N, Q or G.
  • x1 may be any hydrophobic amino acid residue, however residues L, P, M as the x1 residue of the sequence may in some embodiments be preferred. In other embodiments a charged amino acid residue as the x1 residue may be preferred. The charged amino acid residue may be selected from K, R, H, D or E. Still in other embodiments x1 may be G.
  • X2 in some embodiments may be a charged amino acid residue independently selected from K, R, H, D or E. In other embodiments it may be the T or S residue.
  • X3 may be any amino acid residue, however, in some embodiments a hydrophobic residue in this position may be preferred, preferably the V, L, or P residue, in other embodiments, it may be preferred a non-charged hydrophilic residue such as S, T, Q or N, still, in other embodiments it may be preferred a residue selected from G or C or a charged amino acid residue.
  • X4 may be a charged amino acid residue, S or T depending on the preferred embodiment.
  • X5 may be selected from a hydrophobic or charged amino acid residue, or selected from residues T, S, N, Q or G.
  • Amino acid residues of any of the positions x1 to x5 are according to the invention to be selected independently, however, in some preferred embodiments both x2 and x4 may be selected from charged amino acid residues.
  • the C-terminal amino acid of a peptide of the invention exists as the free carboxylic acid, this may also be specified as "-OH".
  • the C-terminal amino acid of a compound of the invention may be the amidated derivative, which is indicated as "-NH 2 ".
  • the N-terminal amino acid of a polypeptide comprise a free amino-group, this may also be specified as "H-”.
  • amino acid can be selected from any amino acid, whether naturally occurring or not, such as alfa amino acids, beta amino acids, and/or gamma amino acids. Accordingly, the group comprises but are not limited to: Ala, VaI, Leu, lie, Pro, Phe, Trp, Met, GIy, Ser, Thr, Cys, Tyr, Asn, GIn, Asp, GIu, Lys, Arg, His Aib, NaI, Sar, Orn, Lysine analogues, DAP, DAPA and 4Hyp.
  • 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 GIu and Asp.
  • Basic and acidic amino acid residues constitute a group of charged amino acid residues.
  • the group of hydrophobic amino acid residues is represented by the residues of amino acids Leu, lie, VaI 1 Phe, Trp, Tyr, Met, Ala and Pro.
  • the invention relates to naturally occurring, synthetically/recombinant prepared peptide sequences/fragments, and/or peptide sequences/fragments prepared by means of enzymatic/chemical cleavage of bigger polypeptides, wherein said peptide sequences/fragments are integral parts of said bigger polypeptides.
  • the invention relates to isolated individual peptide sequences.
  • isolated means that a peptide sequence exists as a separate individual compound, but not a part of a polypeptide which the peptide sequence is derived/originate from.
  • Non-limited examples of isolated individual peptide sequences which comprise the above identified motif may be amino acid sequences which set forth in SEQ ID NOs: 1-45.
  • a peptide sequence comprised by the compound of the invention may comprise or consists of any of these sequences, their fragments or variants, wherein said fragments and variants are capable of binding to MT and thereby forming a MT- peptide sequence compound which is stable in solution under physiological conditions.
  • a peptide sequence may comprise an amino acid sequence selected from SEQ ID NOs: 1-45, or a fragment or variant of said sequence.
  • a peptide sequence may consist of a sequence selected from SEQ ID NOs:1-45, or a fragment or variant of said sequence.
  • the peptide sequence may be present in the compound as a single copy, i.e. formulated as a monomer of the peptide sequence, or it may be present as several copies of the same sequence, e.g. as a multimer comprising two or more copies of a sequence selected from SEQ ID NOs: 1-45, or two or more copies of a fragment or a variant of said sequence.
  • a peptide sequence is a multimeric presentation, such as denrimer comprising four identical peptide sequence bound to the lysine core consisting of three lysine residues, or such as an LPA dimer described in WO2005014623. Still in another embodiment a sequence may be selected from SEQ ID NOs:46-51 , or it may be a fragment or variant or multimer of said sequence.
  • a sequence may be selected from SEQ ID NOs:1- 18, or from fragments or variants thereof. In other preferred embodiments a sequence may be selected from SEQ ID NOs:42-45, or from fragments or a variants thereof. Still in other preferred embodiments a sequence may be selected from SEQ ID NOs: 19-41, or from fragments or variants thereof, or it may be preferably selected from SEQ ID NOs:46-51 , or from fragments or variants thereof
  • the invention relates to variants of peptide sequences described in the application as well.
  • variant of a peptide sequence means that the peptides may be modified, for example by substitution of one or more of the amino acid residues. Both L-amino acids and D-amino acids may be used. Other modification may comprise derivatives such as esters, sugars, etc. Examples are methyl and acetyl esters.
  • variants may be understood as exhibiting amino acid sequences gradually differing from the preferred predetermined sequence, as the number and scope of insertions, deletions and substitutions including conservative substitutions increase. This difference is measured as a reduction in homology between the pre- determined sequence and the variant.
  • variants of the peptide fragments according to the invention may comprise, within the same variant, or fragments thereof or among different variants, or fragments thereof, at least one substitution, such as a plurality of substi- tutions introduced independently of one another.
  • Variants of the complex, or fragments thereof may thus comprise conservative substitutions independently of one another, wherein at least one glycine (GIy) of said variant, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of Ala, VaI, Leu, and lie, and independently thereof, variants, or fragments thereof, wherein at least one alanine (Ala) of said variants, or fragments thereof is substi- tuted with an amino acid selected from the group of amino acids consisting of GIy, VaI, Leu, and lie, and independently thereof, variants, or fragments thereof, wherein at least one valine (VaI) of said variant, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of GIy, Ala, Leu, and lie, and independently thereof, variants, or fragments thereof, wherein at least one leucine (Leu) of said variant, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of GIy,
  • the groups of conservative amino acids are as the following: P, A, G (neutral, weakly hydrophobic), S, T (neutral, hydrophilic) Q, N (hydrophilic, acid amine) E, D (hydrophilic, acidic) H, K, R (hydrophilic, basic) L, I, V, M, F, Y, W (hydrophobic, aromatic) C (cross-link forming)
  • a variant may be 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%, even more preferably 97%, 98% or 99% homology to an amino acid sequence selected from SEQ ID NOs:1-51 , or it may be 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%, even more preferably 97%, 98% or 99% positive amino acid matches compared to an amino acid sequence selected from SEQ ID NOs:1-51.
  • a positive amino acid match is defined herein 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 K to R, E to D, L to M, Q to E, I to V, I to L, A to S, Y to W, K to Q, S to T, N to S and Q to R.
  • the homology of one amino acid sequence with another amino acid is defined as a percentage of identical amino acids in the two collated sequences.
  • sequence homology is used herein synonymously with the term “sequence similarity”.
  • sequence homology may be routinely calculated using well known algorithms such as BLOSUM 30, BLOSUM 40, BLOSUM 45, BLOSUM 50, BLOSUM 55, BLO- SUM 60, BLOSUM 62, BLOSUM 65, BLOSUM 70, BLOSUM 75, BLOSUM 80, BLOSUM 85, or BLOSUM 90;
  • Substitution of amino acids in a peptide sequence of the invention which results in formation of the peptide sequence variants included in the scope of the invention may in one embodiment be made based upon their hydrophobicity and hydrophilicity values and the relative similarity of the amino acid side-chain substituents, including charge, size, and the like.
  • Exemplary amino acid substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • a variant which is an amino acid sequence of at least 6 amino acid residues having at least 65% sequence similarity with a sequence selected from the sequences of SEQ ID NOs:1-51, preferably an amino acid sequence of 6 to 20 contiguous amino acid residues, which has more then 70% sequence similarity with a sequence selected from the sequences of SEQ ID NOs:1-51 , such as from 71% to 80% similarity, preferably from 81% to 85%, more preferably from 86% to 90%, even more preferably from 91% to 95%, and even more preferably more then 95% of sequence similarity, such as 96-99% similarity.
  • a variant which consists of a sequence of SEQ ID NOs:1-51 , wherein said sequence comprising one or more amino acid residues which is/are covalently attached to a derivative of a sugar or lipid, or which comprise a chemical grouping such as for example a phosphoryl or acetyl residues, or which may comprise any other chemical moieties which do not prevent the sequence to bind to MT and form the compound of the invention.
  • the present invention does also relate to fragments of the peptide sequences described in the application.
  • a preferred fragment is a fragment of a sequence selected from SEQ ID NOs: 1-51 which has the length of at least 40% of the length of said sequence, more preferably at least 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.
  • the fragment may comprise from 3 to 13 amino acid residues. It is also preferred that the fragment comprises the amino acid motif described above.
  • the invention in another aspect relates to a short peptide sequence as above which possesses biological activity related to a physiological process which takes place in the brain, e.g. capability of stimulating neural plasticity or cell survival.
  • Biological activity of the peptide sequence is according to the invention is associated with the capability of said peptide sequence to modulate activity of a functional cell receptor.
  • a preferred functional cell receptor of the present invention is a receptor selected from the family of fibroblast growth factor receptors (FGFR).
  • FGFR1 , FGFR2, FGFR3, FGFR4 or another FGFR receptor of the family may be selected according to different embodiments.
  • the prototypical FGFR consists of three immunoglobulin-like modules (Ig1 - Ig3), a trans-membrane domain and a cytoplasmic tyrosine kinase domain.
  • the linker re- gion between the Ig1 and Ig2 modules is very long, consisting of 20-30 amino acid residues, including a stretch of acidic amino acids termed the acid box.
  • FGFRs also bind heparin/heparan sulphate, which is required for the high-affinity FGF-FGFR interaction.
  • FGFR isoforms lacking the Ig1 module
  • FGFR2 the Ig1 module combined with the Ig1-lg2 linker sequence
  • Ig1-lg2 linker alone in FGFR3
  • a short peptide sequence as described above may derive from a FGFR ligand, e.g. a fibroblast growth factor (FGF) or cell adhesion molecule, such as the neural cell adhesion molecule (NCAM).
  • FGF fibroblast growth factor
  • NCAM neural cell adhesion molecule
  • derived is the present context meant that the amino acid sequence of an isolated short peptide sequence represents a subsequence/fragment of the peptide sequence of an FGFR ligand, e.g. a subsequence of the FGF or NCAM protein sequence.
  • the invention relates to a fragment of a cell adhesion molecule which is a ligand of FGFR.
  • adhesion molecules may be NCAM or L1.
  • the invention preferably relates to NCAM fragments selected from the sequences set forth in SEQ ID NOs:42-45
  • the peptide sequence may be a fragment of L1 , e.g. selected from the following sequences: APEKWFSLGKV (SEQ ID NO: 46),
  • RFHILFKALPEGKVSPD SEQ ID NO: 50
  • LHHLAVKTNGTG SEQ ID NO: 51
  • Fragments and variants of the above peptide sequences which are capable of binding to MT and which are capable of binding to an FGFR, are also in the scope of the invention.
  • NCAM and L1 are in the scope of the invention as examples of the peptide sequence comprised by the compound of the invention, i.e. formulated as peptide fragments associated with MT.
  • the invention in another embodiment relates to an isolated fragment of an FGF which is capable of binding to MT and which is capable of binding to an FGFR.
  • An FGF of the invention may be selected from any members of the FGF family, i.e. FGF1-23. Examples of such fragments are be the sequences identified herein as SEQ ID NOs: 1-41.
  • the invention relates to isolated FGF fragments having the sequences identified as SEQ ID Nos: 4, 8, 10, 16, 17, 26, 33, 35, 39 and 41.
  • the invention relates to the latter sequences both as a part of the compound of the invention and as individual compounds, i.e. not bound to MT or any other moiety. In some embodiments these sequences may be preferred as separate individual peptide sequences, in the other embodiments they may be preferred as a part of the compound where they are associated with MT.
  • the invention includes these fragments in the scope of protection when they form a part of the compound of the invention, i.e. as the MT bound fragments.
  • the invention also relates to variants and fragments of the above peptide sequences.
  • the fragments and variants of these sequences are characterized by structural and functional features described above.
  • a peptide sequence as described above is associated with MT or a fragment thereof in a compound.
  • the compound of the invention consists of at least two peptide sequences, wherein one of the sequences is a peptide sequence of MT protein or the sequence of a fragment thereof and another peptide sequence is at least one short peptide sequence as described above.
  • the MT protein of the invention may be selected from metallothionein-1 A (MT1A), metallothionein-1 B (MT1B), metallothionein-1 E (MT1 E), metallothionein-1 F (MT1 F), metallothionein-1 G (MT1G), metallothionein-1 H (MT1 H), metallothionein-11 (MT1 I), metallothionein-1 K (MT1 K), metallothionein-1 L (MT1L), metallothionein-1 R
  • MT3 metallothionein-4
  • MT4 metallothionein-4
  • a short peptide sequence of the compound may be associated with a fragment of MT.
  • An example of such MT fragment may be a fragment which comprises a subsequence of MT which comprises at least one of the following amino acid sequences:
  • KKCCCSCCPVGCAK (SEQ ID NO:53)
  • KKSCCSCCPVGCAK (SEQ ID NO:54)
  • KKSCCSCCPVGCAK (SEQ ID NO:58)
  • KKSCCSCCPVGCAK (SEQ ID NO:59)
  • KKSCCSCCPVGCAK (SEQ ID NO:63)
  • KKSCCSCCPAECEK (SEQ ID NO:64)
  • AKDCVCKGGEAAEAEAEKCS SEQ ID NO:78
  • ARGCICKGGSDKCS SEQ ID NO:79
  • MDPNCSCAAGVSCT SEQ ID NO:83
  • MDPNCSCAAGVSCT SEQ ID NO:84
  • MDPNCSCEAGGSCA SEQ ID NO:85
  • MDPNCSCAAGDSCT SEQ ID NO:91
  • CAGSCKCKECKCTS (SEQ ID NO:95)
  • CASSCKCKECKCTS (SEQ ID NO:98)
  • CAGSCKCKKCKCTS (SEQ ID NO:99)
  • CAGSCKCKECKCTS (SEQ ID NO: 100)
  • CASSCKCKECKCTS (SEQ ID NO: 101)
  • CAGSCKCKECKCTS (SEQ ID NO:102)
  • CASSCKCKECKCTS (SEQ ID NO: 103) CAGSCKCKECKCTS (SEQ ID NO:104)
  • CAGSCKCKECKCTS (SEQ ID NO: 105)
  • CADSCKCEGCKCTS (SEQ ID NO:106)
  • CGDNCKCTTCNCKT SEQ ID NO: 107.
  • MT1A subsequence may be selected from SEQ ID NOs:52, 66, 80 or 94;
  • MT1B subsequence may be selected from SEQ ID NOs:53, 67, 81 or 95.
  • MT1 E subsequence may be selected from SEQ ID NOs:54, 68, 82 or 96;
  • MT1 F subsequence may be selected from SEQ ID NOs:55, 69, 83 or 97;
  • MT1G subsequence may be selected from SEQ ID NOs:56, 70, 84 or 98;
  • MT1 H subsequence may be selected from SEQ ID NOs:57, 71 , 85 or 99;
  • MT1 I subsequence may be selected from SEQ ID NOs:58, 72, 86 or 100;
  • MT1 K subsequence may be selected from SEQ ID NOs:59, 73, 87 or 101;
  • MT of the compound may be represented by MT protein or it may be a peptide fragment of said MT which may be selected from the fragments identified above.
  • MT ot a fragment thereof may be represented by a peptide sequence which is homologues to said MT sequence or said fragment, with the proviso that this homologues sequence is capable of binding to a short peptide sequence of the invention and together with the latter short peptide sequence form a compound of the invention which is capable of penetrating the BBB.
  • the present invention relates to a compound that comprises a peptide sequence which is capable of binding to a functional cell receptor and modulating activity of said receptor. It is preferred that the receptor is a cell surface receptor. Most preferred functional receptor is a receptor selected from of the family of fibroblast family growth factor receptors (FGFRs).
  • FGFRs fibroblast family growth factor receptors
  • the fibroblast growth factor receptor may be fibroblast growth factor receptor 1 (FGFR1 ), in another embodiment the fibroblast growth factor receptor may be fibroblast growth factor receptor 2 (FGFR2), in another embodiment the fibroblast growth factor receptor may be fibroblast growth factor receptor 3 (FGFR3), in another embodiment the fibroblast growth factor receptor may be fibroblast growth factor receptor 4 (FGFR4) or in still another embodiment it may be fibroblast growth factor receptor 5 (FGFR5).
  • the invention in particular relates to FGFR1, modulating FGFR1 activity and modulating FGFR1 activity associated physiological processes.
  • Binding of a peptide sequence of the invention to a FGFR results in a change in the receptor activation status which is reflected by an increase in tyrosine phosphorylation of the receptor or a change in the activation status of one or more of intracellular proteins involved in FGFR-associated signal transduction, for example STAT1, JNK, PLCY, ERK, STAT5, PI3K, PKC, FRS2 and/or GRB2 proteins activation status.
  • the result of modulating of FGFR signalling by a peptide sequence of the invention may also be reflected by a change in a physiological effect on the cellular or higher level, e.g. the body level.
  • the degree of phoshorylation is estimated as at least 20% above the control value, such as at least 20-200 %, for example at least 50-200%.
  • the control value in the present content is meant the degree of phosphorylation of FGFR in the medium where a peptide sequence capable of activating of FGFR is absent.
  • said concentration may be between 0.1-1000 ⁇ M, 1- 1000 ⁇ M, for example 1-200 ⁇ M, for example 10-200 ⁇ M, such as 20-180 ⁇ M, for example 30-160 ⁇ M, such as 40-140 ⁇ M, for example 50-130 ⁇ M, such as 60-120 ⁇ M, for example 70-110 ⁇ M, such as 80-100 ⁇ M.
  • FGFR tyrosine phosphorylation or activation of any of the FGFR-associated downstream molecules may be estimated by any conventional methods, such as for example immunocytochemistry, immunoblotting or immunoprecipitation, using commercially available antibody against the activated proteins.
  • the degree of activation is estimated as at least 20% above/below the control value, such as at least 20-200 %, for example at least 50-200%.
  • the control value is estimated as a degree of phosphorylation of the protein of interest in the medium where a compound capable of activation of FGFR is absent.
  • modulation of FGFR activation by a peptide sequence of the invention may result in modulating an FGFR related physiological process, for example modulating FGFR related cell differentiation, e.g. induction of differentiation of progenitor cells, stimulation of matu- ration of cells being on an early stage of differentiation, differentiation of cancer cells.
  • modulating FGFR related cell differentiation e.g. induction of differentiation of progenitor cells, stimulation of matu- ration of cells being on an early stage of differentiation, differentiation of cancer cells.
  • the invention relates to peptide sequences which are capable of activating
  • FGFR via direct binding to the receptor.
  • the peptide sequences of the invention may also modulate receptor activation induced by other FGFR ligands binding.
  • modulate means both stimulating and inhibiting activation of the receptor.
  • FGFR related physiological processes of interest which can be modulated by binding of a peptide sequence of the invention to FGFR may be selected from
  • - stem cell differentiation for example neuronal precursor cell differentiation
  • - neural cell differentiation and/or regeneration of nerves for example neurite outgrowth
  • - neural plasticity associated with memory and learning for example synaptic efficacy
  • oxidative stress response such as expression of scavenges of reactive oxygen species - activation of astrogliosis, such as expression of neuroprotective growth factors and proteins such as e.g. BDNF, NT-3, GDNF, neurturin, artemin, NGF, variety of fibroblast growth factors (FGFs), S100-proteins (S100A4, S100A6, S100A10, S100A12, S100B), IGF-2, neuregulin by astrocytes
  • scavenges of reactive oxygen species - activation of astrogliosis such as expression of neuroprotective growth factors and proteins such as e.g. BDNF, NT-3, GDNF, neurturin, artemin, NGF, variety of fibroblast growth factors (FGFs), S100-proteins (S100A4, S100A6, S100A10, S100A12, S100B), IGF-2, neuregulin by astrocytes
  • -inflammatory -inflammatory responses for example activation of microglia and macrophages, expression of pro-inflammatory cytokines and/or by stimulating anti-inflammatory responses;
  • angiogenesis in the lesioned area for example expression of growth factor promoting angiogenesis such as VEGF and FGF2;
  • - cell-cell and/or cell-extracellular matrix adhesion for example neural, glial or cancer cell adhesion to cellular environment and/or extracellular matrix
  • - morphological plasticity of cells for example neuronal plasticity associated with learning and memory
  • biological activity of a peptide sequence which is comprised by the compound of the invention is according to the invention associated with biological activity of
  • the invention preferably relates to cells of neural system, in particular brain cells, such as neuronal cells and/or glial cells.
  • FGFR which is expressed on the surface of brain cells is a preferred functional cell receptor of the invention.
  • the molecular processes involving a biological activity of the peptide are more preferably those which are related to a neuronal cell.
  • one of the preferred biological activities of the peptide sequence according to the invention is the capability of stimulating neuronal cell differentiation.
  • neural differentiation is understood herein both as differentiation of neural precursor cells, or neural stem cells, and further differentiation of neural cells, such as for example maturation of neuronal cells.
  • An example of such differentiation may be neurite outgrowth from immature neurons, branching of neurites, and also neuron regeneration.
  • the invention concerns biological activity of a peptide sequence associated with stimulating of differentiation of neural precursor/stem cells or immature neurons and/or stimulating neurite outgrowth from mature neurons, for examples neurons which were traumatizes but survived and are committed to regenerate damaged processes.
  • differentiation is related to the processes of maturation of neurons and extension of neurites, which take place after the last cell division of said neurons.
  • the compounds of the present invention may be capable of stopping neural cell division and initiating maturation said cells, such as initiating extension of neurites.
  • differentiation is related to initiation of the process of genetic, biochemical, morphological and physiological transformation of neuronal progenitor cells, immature neural cells or embryonic stem cells leading to formation of cells having functional characteristics of normal neuronal cell as such characteristics are defined in the art.
  • the invention defines "immature neural cell” as a cell that has at least one feature of neural cell accepted in the art as a feature characteristic for the neural cell.
  • Substances with the potential to promote neurite outgrowth as well as stimulate regeneration and/or differentiation of neuronal cells are prime targets in the search for compounds that facilitate for example neuronal regeneration and other forms of neuronal plasticity.
  • the ability to stimulate the neurite outgrowth related signalling, interfere with cell adhesion, stimulate neurite outgrowth, regeneration of nerves may be investigated.
  • the compound of the present invention comprising a biologically active peptide sequence of the invention is shown to be capable of promoting neurite outgrowth and is therefore considered to be a good pro- moter of regeneration of neuronal connections, and thereby of functional recovery after damages as well as a promoter of neuronal function in other conditions where such effect is required.
  • a compound comprising at least one of the above peptide sequences is capable of stimulating neurite outgrowth.
  • the invention concerns the neurite outgrowth improvement/stimulation such as about 75% improvement/stimulation above the value of neurite outgrowth of control/non- stimulated cells, for example 50%, such as about 150%, for example 100%, such as about 250, for example 200%, such as about 350 %, for example 300%, such as about 450%, for example 400%, such as about 500%.
  • Estimation of capability of a candidate compound to stimulate neurite outgrowth may be done by using any known method or assay for estimation of neurite outgrowth, such as for example as the described in Examples below.
  • the invention also concerns a method for stimulating neuronal cell differentiation comprising using the compound of the invention comprising a peptide sequence of the invention.
  • One of most preferred embodiments of the invention concerns the activity of the peptide sequences in connection with learning and memory, in particular, the capability of a peptide sequence to stimulate synaptic plasticity, spine formation, synaptic efficacy.
  • the invention also concerns a method for stimulating memory and/or learning comprising using a peptide sequence of the invention and/or compound comprising said sequence.
  • the invention relates to both short- term memory and long-term memory.
  • a peptide sequence of the invention capable of stimulating cell survival, in particular neuronal cell survival.
  • the invention concerns the capability of stimulating cell survival both due trauma and degenerative disease. Accordingly, the invention relates to a method for stimulating cell survival, preferably neuronal cell survival by using a peptide sequence of the invention and/or compound comprising said sequence.
  • Substances with the potential to enhance neuronal cells to survive due to damage as well as inhibit degeneration and/or apoptosis of neuronal cells in trauma and disease are prime targets in the search for candidate compounds for new medicine for treatment of neurodegenerative diseases such as for example Alzheimer's or Park- inson's diseases.
  • neurodegenerative diseases such as for example Alzheimer's or Park- inson's diseases.
  • the ability to stimulate survival related signalling, interfere with apoptosis related cellular reactions, stimulate regeneration of nerves may be investigated.
  • Compounds of the present invention are shown to promote neural cell survival and decrease the cell loss and therefore considered to be good candidates for promotion of regeneration of neural connections in brain and/or in peripheral neural system, and thereby of functional recovery after damages due trauma or disease as well as promoters of neuronal function in any other conditions where such effect is required.
  • survival is related to the processes associated with maintenance and/or recovery of cell function after the damage of the cell.
  • the compounds of the present invention may be capable of stopping or attenuating the processes committing the cell to death, such as inhibiting apoptosis of neural cells initiated by cell damage due trauma or disease.
  • “survival” is related to inhibition of the processes associated with the cell damage leading to cell death and initiation of the processes of genetic, biochemical, morphological and physiological transformation or reconstruction of cells, in particular neuronal cells, such as progenitor cells, immature neural cells or embryonic stem cells or mature neural cells having normal functional characteristics defined in the art.
  • the invention defines "immature neural cell” as a cell that has at least one feature of neural cell accepted in the art as a feature characteristic for the neural cell.
  • a compound comprising at least one of the above peptide sequences is capable of stimulating neural cell survival.
  • the invention concerns the neural cell survival stimulation such as about 75% stimulation above the value of survival of control/non-stimulated cells, for example 50%, such as about 150%, for example 100%, such as about 250, for example 200%, such as about 350 %, for example 300%, such as about 450%, for example 400%, such as about 500%.
  • Estimation of capability of a candidate compound to stimulate neural cell survival may be done by using any known method or assay for estimation of cell survival, such as for example the ones described in Examples of the present application.
  • the peptide sequence of the invention is also capable of inhibit- ing an inflammatory process, in particular an inflammatory process in the brain.
  • Inflammation is a defence reaction caused by tissue damage due to a mechanical injury or bacterial, virus or other organism infection.
  • the inflammatory response involves three major stages: first, dilation of capillaries to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; and third, leukocyte transmigration through endothelium and accumulation at the site of injury and infection.
  • the inflammatory response begins with a release of inflammatory mediators.
  • Inflammatory mediators are soluble, diffusible molecules that act locally at the site of tissue damage and infection, and at more distant sites, influencing consequent events of the inflammatory response.
  • Inflammatory mediators can be exogenous, e. g. bacterial products or toxins, or endogenous, which are produced within the immune system itself, as well as injured tissue cells, lymphocytes, mast cells and blood proteins.
  • Neuroinflammation plays a prominent role in the progression of Alzheimer's disease and may be responsible for degeneration in vulnerable regions such as the hippocampus. Neuroinflammation is associated with elevated levels of extracellular glu- tamate and potentially an enhanced stimulation of glutamate N-methyl-D-aspartate receptors.
  • Anti-inflammatory activity is another important biological activity of the peptide sequence of the invention.
  • the invention relates to anti-inflammatory peptide, which is capable of serving as an inhibitor of the sustained inflammatory response, in particular in the brain..
  • inflammatory mediators such as for example TNF alpha
  • TNF alpha inflammatory mediators
  • the sus- tained inflammatory response has been proven to be very harmful to the body. If the bacterial products or live bacteria become spread universally in the body from their local focus the inflammatory reaction becomes overwhelming and out of control and leads to sepsis which eventually progress further to severe sepsis and septic shock.
  • Anti-inflammatory peptides may be used to block or suppress the overwhelming sustained inflammatory response represented by a massive and harmful cytokine cascade in the blood and vital organs such as lung, liver intestine, brain and kidneys.
  • anti-inflammatory compound a com- pound which is capable of at least one of the following activities i) decrease or inhibit the gene expression in the immune cells, preferably monocytes/macrophages in response to bacterial products, live bacteria or trauma to produce endogenous inflammatory mediators including receptors for inflammatory mediators and transcription factors involved in the signal transduction of the inflammatory mediators, said mediators being preferably selected from the group comprising cytokines, selected from the group
  • the invention relates to a capability of a peptide sequence of the invention to stimulate expression of MT protein in vivo. This biological activity is among preferred biological activities of the peptide sequences of the invention as well.
  • Proteins and peptide sequences of the present invention may be prepared by a conventional synthetic method, recombinant DNA technology, enzymatic cleavage of full-length protein which a short peptide sequence is derived from, or any combination of said methods.
  • MT protein comprised by the compound the composition of the invention is preferably prepared using any of the recombinant technologies and a short peptide sequence of the invention is preferably prepared using a method of chemical synthesis described below.
  • the peptides of the invention are produced by use of recombinant 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.
  • oligonucleo- tides are synthesised, e.g. in an automatic DNA synthesiser, 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 I according to a standard protocol (Sambrook et al., Molecular cloning: A Laboratory manual. 2 rd ed., CSHL Press, Cold Spring Harbor, NY, 1989).
  • the present invention relates to full-length proteins selected from the groups of proteins identified above.
  • the DNA encoding the full-length proteins of the invention may al- ternatively be fragmented using specific restriction endonucleases.
  • the fragments of DNA are further purified using standard procedures described in Sambrook et al., Molecular 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 origin, for instance obtained by preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of the full-length protein by hybridisation using synthetic oligonucleotide probes in accordance with standard techniques (cf. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989).
  • the DNA sequence may also be prepared by polymerase chain reaction using specific primers, for instance as described in US 4,683,202 or Saiki et al., 1988, Science 239:487-491.
  • a recombinant expression vector which may be any vector, which may conveniently be subjected to recombinant DNA procedures.
  • the choice of vector will often depend on the host cell into which it is to be introduced.
  • the vector may be an autonomously replicating vector, i.e. a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid.
  • the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
  • 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 activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
  • suitable promoters for directing the transcription of the coding DNA sequence in mammalian cells are the SV 40 promoter (Subramani et al., 1981, MoI. Cell Biol. 1 :854-864), the MT-1 (metallothionein gene) promoter (Palmiter et al., 1983, Science 222: 809-814) or the adenovirus 2 major late promoter.
  • a suitable promoter for use in insect cells is the polyhedrin promoter (Vasu- vedan et al., 1992, FEBS Lett. 311:7-11).
  • Suitable promoters for use in yeast host cells include promoters from yeast glycolytic genes (Hitzeman et al., 1980, J. Biol. Chem. 255:12073-12080; Alber and Kawasaki, 1982, J. MoI. Appl. Gen.
  • Suitable promoters for use in filamentous fungus host cells are, for instance, the ADH3 promoter (McKnight et al., 1985, EMBO J. 4:2093-2099) or the 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 fungal hosts) the TPU (Alber and Kawasaki, op. cit.) or ADH3 (McKnight et al., op. cit.) promoters.
  • the vector may further comprise elements such as polyadenylation signals (e.g. from SV 40 or the adenovirus 5 EIb region), transcriptional enhancer sequences (e.g. the SV 40 enhancer) and translational enhancer sequences (e.g. the ones encoding adenovirus VA RNAs).
  • the recombinant expression vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.
  • a DNA sequence enabling the vector to replicate in the host cell in question.
  • An example of such a sequence is the SV 40 origin of replication.
  • the vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, such as the gene coding for dihydrofolate reductase (DHFR) or one which confers resistance to a drug, e.g. neomycin, hydromycin or methotrexate.
  • DHFR dihydrofolate reductase
  • the coding DNA sequences may be usefully fused with a second peptide coding sequence and a protease cleavage site coding sequence, giving a DNA construct encoding the fusion protein, wherein the protease cleavage site coding sequence positioned between the HBP fragment and second peptide coding DNA, inserted into a recombinant expression vector, and expressed in recombinant host cells.
  • said second peptide se- lected from, but not limited by the group comprising glutathion-S-reductase, calf thymosin, bacterial thioredoxin or human ubiquitin natural or synthetic variants, or peptides thereof.
  • a peptide sequence comprising a protease cleavage site may be the Factor Xa, with the amino acid sequence IEGR, en- terokinase, with the amino acid sequence DDDDK, thrombin, with the amino acid sequence LVPR/GS, or Acharombacter lyticus, with the amino acid sequence XKX, cleavage site.
  • the host cell into which the expression vector is introduced may be any cell which is capable of expression of the peptides or full-length proteins, and is preferably a eu- karyotic cell, such as invertebrate (insect) cells or vertebrate cells, e.g. Xenopus laevis oocytes or mammalian cells, in particular insect and mammalian cells.
  • suitable mammalian cell lines are the HEK293 (ATCC CRL-1573), COS (ATCC CRL-1650), BHK (ATCC CRL-1632, ATCC CCL-10) or CHO (ATCC CCL- 61) cell lines.
  • fungal cells may be used as host cells.
  • suitable yeast cells include cells of Saccharomyces spp. or Schizosaccharo- myces spp., in particular strains of Saccharomyces cerevisiae.
  • Other fungal cells are cells of filamentous fungi, e.g. Aspergillus spp. or Neurospora spp., in particular strains of Aspergillus oryzae or Aspergillus niger.
  • 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 for growing mammalian cells, such as a serum-containing or serum-free medium containing appropriate supplements, or a suitable medium for growing insect, yeast or fungal cells. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection).
  • the peptides or full-length proteins recombinantly produced by the cells may then be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, purification by a variety of chromatographic procedures, e.g. HPLC, ion exchange chromatography, affinity chromatography, or the like.
  • a salt e.g. ammonium sulphate
  • Short peptide sequences of the invention are preferably prepared by chemical syn- thesis.
  • Peptides may for example be synthesised by using Fmoc chemistry and with Acm- protected cysteins. After purification by reversed phase HPLC, peptides may be further processed to obtain for example cyclic or C- or N-terminal modified isoforms.
  • the methods for cyclization and terminal modification are well-known in the art and described in detail in the above-cited manuals.
  • the peptide sequences of the invention are produced synthetically, in particular, by the Sequence Assisted Peptide Synthesis (SAPS) method.
  • SAPS Sequence Assisted Peptide Synthesis
  • SAPS peptides may be synthesised either batchwise in a polyethylene vessel equipped with a polypropylene filter for filtration or in the continuous-flow version of the polyamide 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- fluorenylmethyloxycarbonyl (Fmoc) or tert. -Butyloxycarbonyl, (Boc) as N-a-amino protecting group and suitable common protection groups for side-chain functionality.
  • individual peptide sequences may then be formulated as mul- timers using well-known in the art techniques, for examples dimers of the sequences may be obtained by the LPA method described in WO 00/18791 , denrimeric poly- mers by the MAP synthesis described in PCT/US90/02039.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising one or more of the compounds defined above or one or more of peptide sequence as defined above, wherein the compound/peptide sequence is preferably capable of stimulating neurite outgrowth and/or neural cell differentiation, survival of neural cells and/or stimulating learning and/or memory.
  • the invention in one aspect concerns a pharmaceutical composition capable of stimulating differentiation of neuronal cells and/or stimulating regeneration of neuronal cells, and/or stimulating neuronal plas- ticity in connection with learning and memory, and/or stimulating survival of neural cells.
  • Another aspect of the invention relates to the use of a compound and/or peptide sequence as a medicament.
  • the medicament is preferably for the treatment or pro- phylaxis of any of the diseases and conditions indicated below.
  • the peptide sequences of the invention may be formulated both as comprising isolated individual peptide fragments, multimers or dimers thereof.
  • a further aspect of the invention is a process of producing a pharmaceutical compo- sition, comprising mixing an effective amount of one or more of the compounds of the invention, or a pharmaceutical composition according to the invention with one or more pharmaceutically acceptable additives or carriers, and administer an effective amount of at least one of said compound, or said pharmaceutical composition to a subject.
  • the compounds may be used in combination with a prosthetic device, wherein the device is a prosthetic nerve guide.
  • the present invention relates to a prosthetic nerve guide, characterised in that it comprises one or more of the compounds or the pharmaceutical composition as defined above. Nerve guides are known in the art.
  • the pharmaceutical composition may have the described above effects on cells in vitro or in vivo, wherein the composition is administered to a subject.
  • the medicament of the invention comprises an effective amount of one or more of the compounds as defined above, or a composition as defined above in combination with the pharmaceutically acceptable additives.
  • Such medicament may suitably be formulated for oral, percutaneous, intramuscular, intravenous, intracranial, intrathecal, intracerebroventricular, intranasal or pulmonal administration.
  • 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 example, water, saline, dextrose, glycerol, ethanol or the like, and combinations thereof.
  • 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 preparation.
  • 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, microcapsules, nanoparticles or the like.
  • 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.
  • 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 formulations 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, sustained release formulations or powders and generally contain 10-95% of the active ingredient(s), preferably 25-70%.
  • formulations are such suitable for nasal and pulmonal administration, e.g. inhalators and aerosols.
  • the active compound may be formulated as neutral or salt forms.
  • Pharmaceutically 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 example, 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 administered in a manner compatible with the dosage formulation, 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 and age of the subject, the disease to be treated and the stage of disease. Suitable dosage ranges are per kilo body weight normally of the order of several hundred ⁇ g active ingredient per administration with a preferred range of from about 0.1 ⁇ g to 5000 ⁇ g per kilo body weight.
  • the suitable dos- ages are often in the range of from 0.1 ⁇ g to 5000 ⁇ g per kilo body weight, such as in the range of from about 0.1 ⁇ g to 3000 ⁇ g per kilo body weight, and especially in the range of from about 0.1 ⁇ g to 1000 ⁇ g per kilo body weight.
  • the suitable dosages are often in the range of from 0.1 ⁇ g to 1000 ⁇ g per kilo body weight, such as in the range of from about 0.1 ⁇ g to 750 ⁇ g per kilo body weight, and especially in the range of from about 0.1 ⁇ g to 500 ⁇ g per kilo body weight such as in the range of from about 0.1 ⁇ g to 250 ⁇ g per kilo body weight.
  • 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.
  • a localised or substantially localised application is preferred.
  • the preparation further comprises pharmaceutically acceptable additives and/or carriers.
  • additives and carriers will be known in the art.
  • Administration may be a continuous infusion, 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 is administered within 8 hours from the factor onset, such as within 5 hours from the factor onset. Many of the compounds exhibit a long term effect whereby administration of the compounds may be conducted with long intervals, such as 1 week or 2 weeks.
  • the administration may be continuous or in small portions based upon controlled release of the active compound(s).
  • precursors may be used to control the rate 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.
  • the present invention relates to treatment of individuals for inducing differentiation, stimulating regeneration, plasticity and survival of neural cells in vitro or in vivo, said treatment involving administering an effective amount of one or more compounds as defined above.
  • Another strategy for administration is to implant or inject cells capable of expressing and secreting the compound in question. Thereby the compound may be produced at the location where it is going to act.
  • a compound of the invention comprises MT or a fragment thereof and a peptide sequence which possesses biological activity associated with the capability of the peptide sequence to bind to FGFR and modulate FGFR activity is capable of penetrating the BBB
  • FGFRs and their ligands has been shown to be important determinants of neuronal survival both during development and during adulthood, in particular in the brain (Reuss and von Bohlen und Halbach (2003) Cell tissue Res, 313:139-57).
  • Cell death plays a key role in normal neuronal development, where 50% of the developing neurons are eliminated through programmed cell death, and in the pathophysiology of neurodegenerative conditions, such as Alzheimer's and Parkinson's diseases. Therefore, a compound, which is capable to penetrate the BBB and promote neu- ronal cell survival by binding and activation FGFR in the brain is highly desirable.
  • the invention features compounds that promote survival of neural cells and can be used as medicaments for the treatment of conditions involving neural cell death.
  • a biologically active peptide sequence of the invention both in the MT-bound and free form, i.e. non-MT bound may also be used as a medicament for promotion of survival of another type of cells, e.g. different type of muscle cells, or, alternatively, for enhancing cell death of still another type of cells, e.g. cancer cells, as the FGFR signalling has been shown to be a survival factor for both muscle and cancer cells (Ozen et al. (2001) J Nat Cancer Inst. 93:1783-90; Miyamoto et al. (1998) J Cell Physiol. 177:58-67; Detilliux et al. (2003) Cardiovasc Res. 57:8-19).
  • the invention in particular concerns the peptide sequences of SEQ ID NOs: 4, 8, 10, 16, 17, 26, 33, 35, 39 and 41.
  • Another approach in the strategy aimed to achieve a compensation for functional cell loss is to create a new pool of said functional cells, for example by committing the progenitor (stem) cells to differentiate to a new population of differentiated cells, or to initiate regenerating processes in damaged cells.
  • FGFRs play an important role in the mechanisms triggering differentiation of a variety of progenitor cell types (Es- warakumar et al. (2005) Cytokine Growth Factor Rev. 16(2): 139-49), cancer cells (St Bernard et al. (2005) Endocrinology 146(3): 1145-53) and neural cells (Sapieha et al. (2003) MoI Cell Neurosci. 24(3):656-72).
  • the present invention provides compounds capable of modulating the activity of FGFRs, in particular stimulating activity of FGFRs. Consequently, said compounds are concerned by the invention for the production of a medicament for the treatment of diseases, wherein stimulating biological activity dependent on the activity of FGFRs is considered to be beneficial for treatment.
  • the medicament comprising or consisting of a biological sequence of the invention, such as SEQ ID NOs: 4, 8, 10, 16, 17, 26, 33, 35, 39 or 41 , and or a compound comprising MT and at least one of the short peptide sequences described above, may also used for prevention and/or treatment of
  • diseases or conditions of the central and peripheral nervous system such as postoperative nerve damage, traumatic nerve damage, impaired myelination of nerve fibers, postischaemic damage, e.g. resulting from a stroke, Parkinson'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; for treatment of diseases or conditions of the muscles including conditions with impaired function of neuro-muscular connections, such as after organ transplantation, or such as ge- netic 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 I and II, of the kidney such as nephrosis and of the heart, liver and bowel, and/or
  • postoperative nerve damage traumatic nerve damage, impaired myelination of nerve fibers, postischaemic, e.g. resulting from a stroke, Parkinson'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 as manic depression, and/or 4) cancer disease, and/or 5) prion diseases.
  • the invention concerns the cancer being any type of solid tumors requiring neoan- giogenesis. Cancers of neural system are of particular interest of the invention.
  • the invention concerns prion diseases selected from the group consisting of scrapie, Creutzfeldt-Jakob disease. It has been shown that FGFRs plays a distinct role in prion diseases (Castelnau et al. (1994) Exp Neurobiol. 130:407-10; Ye and Carp (2002) J MoI Neurosci. 18:179-88).
  • a compound of the invention and/or peptide sequence of the invention such as SEQ ID NOs: 4, 8, 10, 16, 17, 26, 33, 35, 39 and 41 , may be used for the manufacture of a medicament for 1 ) promotion of wound-healing, and/or 2) prevention of cell death of heart muscle cells, such as after acute myocardial infarction, or after angiogenesis, and/or
  • a peptide sequence of the invention has a capability to induce and/or stimulate the expression of MT when it is administered in vivo.
  • the invention relates to a medicament comprising SEQ ID NO:42.
  • Brain disorders like traumatic injury, pellagra dementia/toxicity; epilepsy, brain ischemia/stroke; EAE/MS (multiple sclerosis); and infectious encephalopathies; Amyotrophic Lateral Sclerosis, Parkinson's disease; peripheral nerve injury, cerebral malaria, ageing /age dementia, neuromuscular damage and diabetes, all, were associated with activity of MT.
  • the MT roles were partially or fully validated in human tissue or human patients during degenerative diseases such as AD, Pick's disease and ALS; and during MS, Binswanger's encephalopathy, and ischaemia and during neuromuscular damage and diabetes, and also during a number of common auto- immune, inflammatory and allergic diseases.
  • degenerative diseases such as AD, Pick's disease and ALS
  • MS Binswanger's encephalopathy
  • ischaemia during neuromuscular damage and diabetes, and also during a number of common auto- immune, inflammatory and allergic diseases.
  • the present invention relates to the above described peptides, fragments, or variants thereof, and compounds comprising at least one of said sequences and MT as medicaments for treatment of diseases wherein their capability of modulating - stem cell differentiation, for example stimulating neuronal precursor or
  • - cell survival in particular stimulating survival of neuronal and/or glial cells, for example inhibiting of apotosis of neural cells and/or glial, or stimulating apoptosis of cancer cells,
  • astrogliosis such as stimulating astrocytes to express neuroprotective growth factors and proteins such as e.g. BDNF, NT-3, GDNF, neurturin, artemin, NGF, variety of fibroblast growth factors (FGFs), S100-proteins (S100A4, S100A6, S100A10, S100A12, S100B), IGF-2, neuregulin; - inflammation, for example inhibitng activation of microglia and macrophages, inhibiting expression of pro-inflammatory cytokines and/or stimulating anti-inflammatory responses;
  • neuroprotective growth factors and proteins such as e.g. BDNF, NT-3, GDNF, neurturin, artemin, NGF, variety of fibroblast growth factors (FGFs), S100-proteins (S100A4, S100A6, S100A10, S100A12, S100B), IGF-2, neuregulin;
  • - inflammation for example inhibitng activation of microglia and macrophages
  • angiogenesis in the lesioned area for example by stimulating expression of growth factor promoting angiogenesis such as VEGF and FGF2;
  • growth factor promoting angiogenesis such as VEGF and FGF2;
  • - cell-cell and/or cell-extracellular matrix adhesion for example modulating neural or cancer cell adhesion to cellular environment and/or extracellular matrix;
  • - morphological plasticity of cells for example stimulating neuronal plasticity associated with learning and memory; - the effects related to FGFR ligands, for example stimulating or inhibiting these effects may be essential for the 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 which are resident cells or implanted or transplanted cells.
  • the treatment comprises treatment and/or prophylaxis of cell damage and/or cell death in relation to diseases or conditions of the central and peripheral nervous system, such as postoperative nerve damage, traumatic neuron damage, e.g. resulting from spinal cord injury, impaired myelination of nerve fibers, postischaemic damage, e.g. resulting from a stroke, multiinfarct dementia, multiple sclerosis, neuronal degeneration associated with diabetes mellitus, neuro-muscular degeneration, schizophrenia, Alzheimer's disease, Parkinson's disease, or Huntington's disease
  • diseases or conditions of the central and peripheral nervous system such as postoperative nerve damage, traumatic neuron damage, e.g. resulting from spinal cord injury, impaired myelination of nerve fibers, postischaemic damage, e.g. resulting from a stroke, multiinfarct dementia, multiple sclerosis, neuronal degeneration associated with diabetes mellitus, neuro-muscular degeneration, schizophrenia, Alzheimer's disease, Parkinson's disease, or Huntington'
  • the compounds according to the invention may be used for inducing differentiation, modulating proliferation, stimulate regeneration, neuronal plasticity and survival , i.e. stimulating survival.
  • the compound of the invention and/or pharmaceutical composition may be beneficially used for the stimulation of the ability to learn and/or of the short and/or long term memory.
  • the compound and/or pharmaceutical composition of the invention may be used in the treatment of clinical conditions, such as psychoses, such as senile and presenile organic psychotic conditions, alcoholic psychoses, drug psychoses, transient organic psychotic conditions, Alzheimer's disease, cerebral lipidoses, epilepsy, general paresis [syphilis], hepatolenticular degeneration, Huntington's chorea, Jakob-Creutzfeldt disease, multiple sclerosis, Pick's disease of the brain, polyarteriti nodosa, syphilis, schizophrenic disorders, affective psychoses, neurotic disorders, personality disorders, including character neurosis, nonpsychotic personality disorder associated with organic brain syndromes, paranoid personality disorder, fanatic personality, paranoid personality (disorder), paranoid traits, sexual deviations and disorders or dysfunctions, sleep disorders, depression and other mood disorders including manic or bipolar disorders, mental retardation, inherited or in relation with disease or trauma, disease in the nerve system and sense organs, cognitive anomalies
  • Inflammatory and toxic neuropathy including acute infective polyneuritis, Guillain-Barre syndrome, Postinfectious polyneuritis, polyneu- ropathy in collagen vascular disease, disorders affecting multiple structures of eye, purulent endophthalmitis, diseases of the ear and mastoid process, abnormality of organs and soft tissues in newborn, including in the nerve system, complications of the administration of anesthetic or other sedation in labor and delivery, diseases in the skin including infection, insufficient circulation problem, injuries, including after surgery, crushing injury, burns, atrophic dermatitis, psoriasis.
  • Injuries to nerves and spinal cord including division of nerve, lesion in continuity (with or without open wound), traumatic neuroma (with or without open wound), traumatic transient paralysis (with or without open wound), accidental puncture or laceration during medical procedure, injury to optic nerve and pathways, optic nerve injury, second cranial nerve, injury to optic chiasm, injury to optic pathways, injury to visual cortex, unspecified, poisoning by drugs, medicinal and biological substances, genetic or traumatic atrophic muscle disorders; 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 I and II, of the kidney, such as nephrosis, metabolic disorders, such as obscenity lipid disorders, diabetes type I and II, diseases of endocrine glands, such as diabetes mellitus I and II, pituitary gland tumour, disorders of amino acid transport and metabolism, disorders of purine and pyrimidine metabolism and gout, bone disorders, such as
  • CML and AML CML and AML
  • muscles sarcoma
  • cancers with dysfunction and/or over- or under-expression of specific receptors and/or expression of mutated receptors or associated with soluble receptors such as but not limited to Erb-receptors and FGF-receptors.
  • Encephalitis may occur as primary or secondary manifestation of TOGAVIRIDAE INFECTIONS; HERPESVIRIDAE INFECTIONS; ADENOVIRIDAE INFECTIONS; FLAVIVIRIDAE INFECTIONS; BUNYAVIRIDAE INFECTIONS; PICORNAVIRIDAE INFECTIONS; PARAMYXOVIRIDAE INFECTIONS; ORTHOMYXOVIRIDAE INFECTIONS; RETROVIRIDAE INFECTIONS; and ARENAVIRIDAE INFECTIONS.
  • a peptide, compound or a pharmaceutical composition of the invention may be used for treatment inflammation in the brain, e.g. inflammation associated with a viral infection.
  • a peptide sequence, a compound and pharmaceutical composition may be used for treatment of Guillain-Barre syndrome, its variant forms, such as Miller Fisher syndrome, and other complement dependent neuromuscular disorders.
  • Peptide sequences, compounds and pharmaceutical compositions of the invention may also be used for treatment children with autism.
  • Autism is a brain disorder that begins in early childhood and persists throughout adulthood; affects three crucial areas of development: communication, social interaction, and creative or imaginative play. It is estimated to afflict between 2 and 5 of every 1000 children and is four times more likely to strike boys than girls. Children with autism have difficulties in social interaction and communication and may show repetitive behaviour and have unusual attachments to objects or routines.
  • a further aspect the invention relates to a method of treating a disease or condition as discussed above comprising administering a compound or peptide sequence of the invention, or pharmaceutical composition comprising thereof to a subject in need.
  • a compound or peptide sequence of the invention or pharmaceutical composition comprising thereof to a subject in need.
  • FGF beta10-beta11 loop region derived peptide sequences (dekafins)
  • Deka6 TYIALS KYG RVKRG (SEQ ID NO: 6) derived from FGF6
  • DekalO MYVALNGKGAPRRG (SEQ ID NO: 10) derived from FGF10
  • Dyo2 ANRYLAMKEDGRLLAS (EQ ID NO:20) derived from FGF2
  • Dyo3 SGRYLAMNKRGRLYAS (SEQ ID NO:21 ) derived from FGF3
  • Dyo6 SALFVAMNSKGRLYAT (SEQ ID NO:24) derived from FGF6
  • Dyo9 SGLYLGMNEKGELYGS (SEQ ID NO:27) derived from FGF9
  • Dyo16 SGLYLGMNERGELYGS (SEQ ID NO:34) derived from FGF16
  • Dyo18 TEFYLCMNRKGKLVGK (SEQ ID NO:36) derived from FGF18
  • Dyo23 SRRYLCMDFRGNIFGS SEQ ID NO:41 derived from FGF23
  • NCAM Fn 3 1 and 2 modules derived peptide sequences: FGL EVYWAENQQGKSKA (SEQ ID NO:42)
  • peptides were purchased from Schafer-N (Copenhagen, Denmark). The peptides were synthesized as tetrameric dendrimers composed of four monomers coupled to a lysine backbone, purified by gel-filtration using SephadexTM G-10 (Amersham Bioscience, Sweden), and dissolved in sterile distilled water. Concentration was determined by spectrophotometry at the absorbance at 205 nm.
  • the FGFR inhibitor SU5402 was from Calbiochem (Bad Soden, Germany)
  • Trex293 cells (Invitrogen, Taastrup, Denmark) were stably transfected with human FGFRIc, splice variant HIc, with a C-terminal Strep Il tag (IBA Biotech, G ⁇ ttingen, Germany). The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with 200 ⁇ g/ml hygromycin (Invitrogen), 10% fetal calf serum (FCS), 1 % (v/v) glutamax, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin (all from Gibco BRL, Paisley, UK). For determination of phosphorylation, 2x1 O ⁇ cells were starved overnight in medium without serum.
  • DMEM Dulbecco's modified Eagle's medium
  • FCS 10% fetal calf serum
  • glutamax 100 U/ml penicillin
  • streptomycin all from Gibco BRL, Paisley, UK.
  • Purified proteins (25 ⁇ l from each sample) were separated by SDS- PAGE and transferred to a polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA), lmmunoblotting was performed using rabbit antibodies (diluted 1 :2000) against the recombinant Strepll tag (IBA Biotech) and swine anti-rabbit IgG horseradish peroxidase conjugate (diluted 1 :2000) (DakoCytomation, Glostrup, Denmark) in 5% (w/v) nonfat dry milk.
  • the immune complexes were developed by SuperSig- nal® West Dura extended duration substrate (Pierce, Rockford, IL, USA), and visualized and quantified using the SynGene Gene Tool image analysis software (Synoptics , Cambridge, UK).
  • CGNs cerebellar granule neurons
  • CGN CGN were prepared from 7 days old Wistar rats (Charles River, Sulzfeld, Germany or M ⁇ llegaard, Ejby, Denmark) essentially as previously described by (Schousboe I, Larsson OM, lnt J Dev Neurosci. 1989;7(1 ):115-21 ). Briefly, the cerebella were cleared of meninges and blood vessels, roughly homogenized by chopping and thereafter trypsinized. The neurons were washed in the presence of DNAse 1 and soybean trypsin inhibitor (Sigma-Aldrich), and cellular debris was pelleted by cen- trifugation.
  • CGN cultures were plated on uncoated eight-well Lab-Tek ® chamber slides (Nunc, Roskilde, Denmark) in Neurobasal-A medium (Gibco BRL) supplemented with 0.4%(w/v) bovine serum albumin (BSA), 2% (v/v) B27, 0.5% (v/v) glutamax, 100 U/mL penicillin and 100 ⁇ g/mL streptomycin at a density of 10,000 cells/well. Peptides or FGF2 were added to the medium immediately after plating and cells were maintained at 37°C, 5% CO2 for 24 h.
  • BSA bovine serum albumin
  • the neck of the rat was maximally flexed in the atlanto-occipital coupling, and 5.0 ⁇ l peptide solution (1.2 ⁇ g/ ⁇ l) or vehicle (PBS, 0.5% BSA) were administered by percutaneous injection into the cis- terna magna with a G25 needle connected with a syringe through a calibrated polyethylene tube.
  • 5.0 ⁇ l peptide solution (1.2 ⁇ g/ ⁇ l) or vehicle (PBS, 0.5% BSA) were administered by percutaneous injection into the cis- terna magna with a G25 needle connected with a syringe through a calibrated polyethylene tube.
  • 25 ⁇ l peptide solution (8 ⁇ g/ ⁇ l) or vehicle (sterile water) was administered through each nostril.
  • Animals exposed to intracistemal or intranasal administration were anesthetisized with 3% halothane in 30% O 2 /70% N 2 O.
  • a focal brain injury on the right fronto-parietal cortex was made by applying a piece of dry-ice (-78 0 C) directly onto the skull for 30 seconds in mice and 60 seconds in rats, as previously described in detail (Penkowa M, Moos T. GHa. 1995 Mar; 13(3):217-27.).
  • the rats were treated s.c. with the tetrameric form of the FGL peptide one day before lesion and one and two days after the lesion (10 mg/kg bodyweight/ injection). Three days after the lesion animals were fixed by transcardial perfusion with paraformaldehyde. Histochemistry and immunohistochemistry (IHC) were performed on sections cut from organs taken from fixated animals.
  • TdT Terminal deoxynucleotidyl transferase
  • dUTP deoxyuridine triphosphate
  • TUNEL Terminal deoxyuridine triphosphate
  • FragELTM Fragment End Labeling
  • Sections were also immunostained for markers of oxidative stress such as peroxynitrite-induced nitra- tion of tyrosine residues (NITT) and malondialdehyde (MDA) andfor markers of in- flammation such as interleukin (IL)-I, IL-12 and tumor necrosis factor (TNF) ⁇ , as described by Penkowa M, et al. (Glia. 2000 Dec;32(3):271-85).
  • markers of oxidative stress such as peroxynitrite-induced nitra- tion of tyrosine residues (NITT) and malondialdehyde (MDA)
  • markers of in- flammation such as interleukin (IL)-I, IL-12 and tumor necrosis factor (TNF) ⁇ , as described by Penkowa M, et al. (Glia. 2000 Dec;32(3):271-85).
  • Intracerebroventricular, Lev., Administration of the A ⁇ 25 . 35 Peptide Aggregates of A ⁇ 25 - 35 were prepared by incubating the peptides at a concentration of 3 ⁇ g/ ⁇ l in sterile water for 4 days at 37 0 C. Formation of aggregates were confirmed visually by microscope inspection. Subsequently, 5 ⁇ l of aggregated A ⁇ 25 - 35 were injected i.c.v. (1.2 ⁇ l/min) with a 10- ⁇ l Hamilton syringe using the following coordinates: 0.8 mm posterior to bregma; 1.5 mm lateral to the sagittal suture; 3.8 mm beneath the surface of the brain.
  • mice were housed individually for 7 days; the first 6 days in their home cage, the last day in the test cage.
  • a 3-week-old male Wistar rat was introduced into the test cage of the adult test rat for 4 minutes. After a period of 30 minutes, the same juvenile rat was re-introduced or, alternatively, as a control, an unfamiliar juvenile was introduced.
  • the Social Recognition Ratio was calculated as T 2 Z(T ⁇ T 2 ), T 1 and T 2 being the time spent on investigating the juvenile animal during the first and the second encounter, respectively.
  • a recognition ratio of 0.50 indicates that there was no difference between the first and second meeting indicating that there at the second meeting was no memory of the first meeting.
  • a decrease in investigation time during the second meeting might be the result of a nonspecific (not memory-connected) FGL-induced reduction of investigatory activity. This possibility was assessed by presenting a novel juvenile to FGL-treated rats during the second meeting.
  • FGL 2 FGL 2 is a dimeric form of the FGL peptide described in WO2005014623
  • scopolamine (0.01mg/kg; Sigma) were both given s.c. 24 hours and 30 min prior to the initial trial, respectively.
  • a juvenile rat was introduced into the test cage of the adult rat for 2 min during the first and second trial, with an intertrial interval of 15 minutes. Animals demonstrating aggressive behavior were excluded.
  • coronal segment of the brain from -0,92 to -4,8 mm relative to the bregma was post-fixed for 24 hours and then cryoprotected in phosphate-buffered 30% sucrose.
  • segments were systematically cut in a number of se- ries each constituting of one 80- ⁇ m thick section followed by four 40- ⁇ m thick sec- tions.
  • the 80- ⁇ m sections were used for histological analysis; 40- ⁇ m sections were used for immunohistochemistry.
  • the 80- ⁇ m sections were stained with 0.5% cresyl violet (Nissl) for routine histological examination and stereological evaluations.
  • the total number of neurons and the volume of the examined structures (the neocortex, including the anterior and posterior cingulate cortex and the motor cortex areas (bregma -0,92 to -2,80), and the CA3 area of the dorsal hippocampus (from -1.6 to -4.52), were measured by exam- ining an average of eight sections per structure.
  • V(ref) t - k - a(p) • ]TP , where V(re ⁇ is the total volume of the structure, t is the average section thickness, k is a constant, a ⁇ p) is the area per point on the counting grid, and ⁇ P is the total number of points hitting the structure of interest.
  • the concept "disector” is an imaginary rectangular box applied to the microscope slide.
  • the the height of the disector is employed by using 80- ⁇ m-thick sections in which the plane of focus is moved up or down (48) and the x- and y-axis are defined by a square (the counting frame) superimposed on the magnified digital image of the tissue.
  • the approximate number of particles counted in a disector, ⁇ Q ⁇ , the height, h, of the disector, and the area of the counting frame, ⁇ (fr ⁇ me) are parameters defined by the investigator.
  • the total number of particles, N(p ⁇ rt) in a specimen of a
  • N(p ⁇ rt) N(p ⁇ rt) .
  • Op- tical disectors were superimposed onto a color monitor at a final magnification of 3000X using the CAST-GRID software (Olympus, Denmark).
  • tissue sections were stained with rabbit polyclonal antibodies against A ⁇ i -4 o (Alfa Diagnostic Int.), Ap 37-42 , and Ap I-17 (Chemicon Int.) for detection of amyloid deposits, and GFAP (DAKO Cytomation) for detection of astrocytes, or with monoclonal antibodies against phospho ⁇ t.au, (clone AT-8, Innogenetics) or CD11b (Sero- tec) for detection of microglia, lmmunohistochemistry was performed according to standard procedures. In brief, sections were pre-treated with 3% H 2 O 2 , and incubated with a primary antibody. To enhance the detection of A ⁇ , sections were also pre-treated with 70% formic acid. Visualization of antigens was performed by the streptavidin-biotin-peroxidase method with diaminobenzidine as the chromogen ac- cording to the manufacturer's instructions (DAKO Cytomation).
  • Immunoreactivities were quantified on the basis of average brightness using the image analysis software PrAverB (Protein Laboratory, University of Copenhagen). For each staining the amount of antigen was defined as the percentage of area oc- cupied by positive immunoreactivity.
  • lysis buffer 100 mM Tris-HCI, pH 6.8, 5% SDS, 20% glycerol including protease inhibitors (Complete Protease Inhibitor Cocktail Tablets; Roche Diagnostics) and phosphatase inhibitors (Phosphatase Inhibitor Cocktail Set II; Calbiochem)
  • protease inhibitors Complete Protease Inhibitor Cocktail Tablets; Roche Diagnostics
  • phosphatase inhibitors Phosphatase Inhibitor Cocktail Set II; Calbiochem
  • the MT2 protein (from Sigma) was immobilized on a sensor chip CM5 using an amine coupling kit (Biosensor AB) as follows: the chip was activated by 20 ⁇ l activation solution; the protein was immobilized using 12 ⁇ l 20 ⁇ g/ml protein in 10 mM sodium phosphate buffer pH 6.0; the chip was blocked by 35 ⁇ l blocking solution.
  • Various peptides at the indicated concentrations were injected into the sensor chip. The curve corresponding to the difference between binding to MT2 and a blank chip was used for analysis.
  • FGLd the dendrimeric form of FGL
  • the FGL peptide is part of the second F3 module of NCAM, which is capable of binding and activating the FGF receptor (Kiselyov et al., 2003).
  • MT-2 ABL 4.71 ⁇ 1.18x10 5.88 ⁇ 1.97x10 ' 1.33 ⁇ 0.46x10 ' MT-2 : Dyo17 6.52 ⁇ 0.87x10 3 1.21 ⁇ 0.16x10 ": 2.11 ⁇ 0.65x10
  • the peptides used for binding were in denrimeric form (four copies of a peptide sequence (four monomers) built on the three-lysine backbone)
  • Dekafins binds to the combined lg2-3 modules ofFGFRI
  • Dekafins activate FGFR1
  • TREX-293 cells stably transfected with FGFR1 containing a C-terminal Strepll tag were stimulated with various concentrations of different dekafins, FGF1, FGF10 (used as positive controls), or nothing for various times. After stimulation, the degree of phosphorylation of FGFR1 was determined by immunoprecipitation of FGFR following by western blotting of the precipitates.
  • Dekafins induce neurite outgrowth
  • CGN cerebellar granular neurons
  • FGF1 and FGF10 Dissociated neurons from the cerebellum
  • Figure 6 The effect of all dekafins was quantified and is shown in Figure 6. The highest efficacy (the level of stimulation of neurite outgrowth with respect to the concentration of a peptide) was observed for Dekal, Deka2, Deka ⁇ , Deka ⁇ and Deka ⁇ followed by Deka3 and Dekal 7, DekalO and Deka9.
  • Dekafins promote neuronal cell survival
  • FGFs are known as neuroprotectants in the CNS. We therefore tested whether the dekafins which are derived from different FGFs are capable of promoting neuronal cell survival as well.
  • Dekafins enhance memory associated with social recognition
  • Dyofins bind to the combined lg2-3 modules ofFGFRI
  • the dyofins are capable of binding to FGFR1 lg2-3 modules although with different affinities, and basic residues (lysines/ arginines) in positions 9, 10 and 12 are important for a high binding efficacy of Dyo3, Dyo8, Dyo10 and Dyo17.
  • FGFR1 is activated by dyofins
  • Activation of FGFR by duofins was studied in a cell culture system described above. From Figure 10a it can be seen that all dyofins stimulate FGFR phosphorylation in a dose-dependent fashion. The highest efficacy was observed for Dyo3, Dyo8, Dyo9, Dyo10 and Dyo17. Treatment of cells with FGF2 resulted in an increase of FGFR phosphorylation which was four to five folds higher than that of dyofins ( Figure 10b).
  • Neuritogenic activity of dyofins is depended on FGFR activation. As can be seen from Figure 11c, treatment of neurons with increasing concentrations of the FGFR inhibitor SU5402 abrogates the neuritogenic response to both dyofins and FGF2.
  • Dyofins promote neuronal cell survival
  • Peptides FGL ABL, CDL and EFL bind to FGFR and stimulate FGFR-dependent neurite outgrowth and neuronal survival
  • NCAM derived peptides FGL, ABL, EFL and CDL are fragments of fibronectin type-3 module 1 and 2 (Fn3,1 and Fn3,2) of NCAM which have been shown to be involved in a direct interaction NCAM with FGFR.
  • EFL peptides to FGFR are shown in Table 2 below.
  • the peptides are capable of activating FGFR and FGFR-dependent neural cell differentiation and cell survival demonstrated that the peptides are capable of binding. From Figure 14 it can be seen that phosphorylation of FGFR1 after stimulation of Trex293 cells stably transfected with FGFR1 with the peptides is increased (with a maximum response after 30 minutes, which was within the same timeframe as the maximum response for FGF1 (not shown)). Cells were incubated with various concentrations of the ABL, CDL or EFL peptides or FGF1 (as a positive control) for various periods of time between 5 min and 1h.
  • Activation pf FGFR was estimated with regard to phosphorylation of FGFR1 by immunoprecipitation of the receptor and subsequent immunoblotting using the anti-phosphotyrosine antibody 4G10-AC (the procedure described above).
  • the FGL peptide is capable of activating FGFR receptor as well ( Figure 15).
  • the FGL, ABL, CDL and EFL all peptides are potent stimulators of neurite outgrowth ( Figures 16) and/or neuronal survival ( Figures 17-18).
  • the effect of the peptides was evaluated in the corresponding assays described above.
  • Contextual fear conditioning Contextual fear conditioning paradigm was used to study long term effects of FGL, ABL, CDL and EFL on memory.
  • Contextual fear conditioning is a classical conditioning paradigm in which the animal is placed in a box and is given time to explore the context. Thereafter the animal is subjected to a series of consecutive shocks (unconditioned stimuli, US) and then it is removed from the context. After different time intervals the animal is returned to the context and the freezing response (conditioned response, CR) is recorded.
  • the freezing response, activity level and the exploratory behaviour of the animals when the animal was introduced to a new environment (without receiving shock) was recorded aw well. It was found no differences between treated and non-treated animals (results not shown) indicating that the increased freezing response observed with ABL treated animals is due to an improved memory.
  • FGL, ABL, CDL and EFL all have a positive effect on short term memory as evaluated by the ability of an adult male to recognise a juvenile animal 2 h after they were first introduced.
  • the capability of stimulating retention of memory of animals for a specific context (fear conditioning) for a longer time (24 h) is more pronounced for the FGL and ABL peptides than for the CDL or EFL peptides.
  • Alzheimer's disease the effect of the FGL peptide in vivo
  • the Alzheimer's disease brain pathology was induces in rats as described above.
  • FGL was administered in a dimeric or a tetrameric version, referred to as FGL 2 and FGL 4 , respectively (these formulations of FGL are effective to induce dimerization of the FGFR).
  • FGL strongly induced expression of the MT proteins in asuble- sioned zone of the brain when compared to the lesioned animals received vehicle.
  • the only observed effect of FGL was an increased expression of MT-I+II, whilst expression levels of all other tested factors were identical after vehicle and FGL administration.
  • FGL induced increased levels of MT-I+II in the meninges, vascular cells, ependyma, the choroid plexus and in some scattered astroglial cells.
  • the MT-I+II isoproteins are antioxidants and anti-apoptotic neuroprotective factors, which significantly reduce delayed tissue damage.
  • the only factors induced by FGL were MT-I+II, and after the lesion, the increase in MT-I+II expression in FGL-treated animals was sustained throughout the 36 days of observation. Therefore, we investigated the role of FGL-induced MT-I+II expression by treating MT-I+II deficient mice (MTKO) and transgenic MT-I overexpressing mice (TgMT) with FGL or vehicle after brain lesion. The animals were sacrificed three dpi.
  • FGL-treatment clearly reduced the number of cells suffering from oxidative stress (8- OH-dG), apoptotic cell death (TUNEL) and neurodegeneration (NFT), whilst surviving NSE-positive neurons, FAP-positive astrocytes and MT-I+II expressing cells all were increased as compared to untreated controls.
  • FGL increased the number of surviving neurons around the lesion as well as the expres- sion of FGF-1, FGF-2, FGFR 1 TGF ⁇ , VEGF, NT-3, NT-4/5, BDNF, angiopoietin-1/4, EPO, PSA-NCAM, and S100A4.
  • TgMT mice clearly displayed reduced tissue damage and im- proved recovery relative to wildtype mice after the lesion, and FGL treatment led to a further decrease of these processes as judged by stainings for 8-OH-dG and TUNEL.
  • FGL increased the already high level of NSE, GFAP and MT-l+ll, whereas no effect on microglial activity could be seen.
  • Further confirmation of the described findings was obtained by stainings for MDA, NFT, APP, caspase-3 and cytochrome-c.
  • induction of expression of the MT-I+II isoproteins is necessary to achieve the neuroprotective effects observed with FGL-treatment.

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Abstract

The present invention relates to a compound consisting of metallothionein (MT) and a peptide sequence, wherein said peptide sequence and metallotionein are bound to each other via a non-covalent bond. A peptide sequence comprised by the compound is a biologically active short peptide sequence which comprises at most 25 amino acid residues. The peptide sequence is capable of binding to and modulating activity of a functional cell receptor, in particular fibroblast growth factor receptor (FGFR). The invention relates to pharmaceutical compositions comprising such compound or such peptide sequence and use of the compound, peptide sequence and pharmaceutical composition for treatment of conditions and diseases where modulating activity of FGFR is beneficial for treatment. In preferred embodiments the use is for treatment of conditions and diseases of the brain including conditions requiring stimulating brain cell proliferation, differentiation and/or survival, stimulating neural plasticity associated with learning and memory, modulating adhesion and motility of cells, in particular cancer cells. The compound of the invention is capable of penetrating the blood brain barrier (BBB).

Description

Targeted delivery of FGFR ligands into the brain
Field of invention
The present invention relates to a compound consisting of metallothionein (MT) and a peptide sequence, wherein said peptide sequence and metallotionein are bound to each other via a non-covalent bond. A peptide sequence comprised by the compound is a biologically active short peptide sequence which comprises at most 25 amino acid residues. The peptide sequence is capable of binding to and modulating activity of a functional cell receptor, in particular fibroblast growth factor receptor (FGFR). The invention relates to pharmaceutical compositions comprising such compound or such peptide sequence and use of the compound, peptide sequence and pharmaceutical composition for treatment of conditions and diseases where modulating activity of FGFR is beneficial for treatment. In preferred embodiments the use is for treatment of conditions and diseases of the brain including conditions requiring stimulating brain cell proliferation, differentiation and/or survival, stimulating neural plasticity associated with learning and memory, modulating adhesion and motility of cells, in particular cancer cells. The compound of the invention is capable of penetrating the blood brain barrier (BBB).
Background of invention
The blood brain barrier (BBB) is one of the major problems in drug delivery to the brain. The kinetics of brain penetration has two components, extent and rate. Achieving a high extent of brain penetration is an important focus for central nervous system (CNS) drug discovery. Optimal brain penetration can be achieved by reducing efflux transport at the BBB, and it is critical to ensure that a high total brain/plasma ratio (the most commonly used parameter for measuring brain penetration) is due to efflux transport activity at the BBB and not related to high non- specific brain tissue binding or low plasma binding. Rapid brain penetration is essential for those drugs that require fast onset of action in the CNS.
Rapid delivery of peptide drugs through the BBB could possibly been achieved via receptor mediated trancytosis. However, a peptide drug which is to be delivered into the brain has to be a ligand of one of the receptors involved in transcytosis, or at least it has to have a capability to bind to the receptor and thereby induce receptor mediated endocytosis.
There are several receptors involved in transcytosis known in the prior art. One of them is megalin/low-density lipoprotein receptor related protein 2 (LRP2). Megalin is a scavenger receptor due to its multifunctional binding properties. Among its ligands are lipoproteins, vitamin-binding and carrier proteins, drugs, hormones and enzymes as well as signalling molecules, (see for review May et al, 2005). Megalin is one of the receptors which are involved in transcytosis of proteins and peptides through the blood-brain barrier (BBB). One of the best-characterized physiological functions of megalin is the proximal-tubular reuptake of low-molecular weight proteins (Zou et al., 2004). It has also been shown that megalin mediates renal uptake of heavy metal metallothionein (MT) complexes (Klassen et al., 2004). A binding site of MT for megalin has been recently identified (Klassen et al., 2004; WO1005072270).
MTs are metal binding proteins (61-68 amino acids), which normally bind seven zinc ions, although zinc/copper mixtures have been reported. Expression of some iso- forms of MT is rapidly induced in response to increases in zinc or copper levels, and also by a large number of hormones and cytokines, including glucocorticoids, inter- leukin 1 and 6, interferons and so on. The exact physiological role of MTs is not fully understood. Early suggestions that they act to prevent accumulation of toxic levels of heavy metals are no longer much favoured, and if their role is indeed in metal metabolism, it is more likely that they are involved in the intracellular homeostasis of zinc. However, MTs are efficient scavengers of free radicals and are able to protect DNA and other molecules from oxidation, suggesting that their function may be protective. MTs are also considered to be intracellular stress proteins which respond to a wide variety of stimuli.
There are four MT subgroups that have been described, namely MT1 , MT2, MT3, and MT4. MT1 and MT2 have been shown to prevent apoptotic cell death in the CNS (Giralt et al., 2002). MT1 and MT2I also improve the clinical outcome and reduce mortality in different CNS disorders (Penkowa, 2002). MT2 treatment has also been shown to significantly stimulate neurite extension from both dopaminergic and hippocampal neurons, significantly increase survival of dopaminergic neurons ex- posed to 6-hydroxydopamine (6-OHDA) and protects significantly hippocampal neurons from amyloid β-peptide-induced neurotoxicity (Køhler et al.,2003). MTs has been shown to mediate neuroprotection in genetically engineered mouse model of Parkinson's disease (Ebadi et al., 2005). Treatment using MT2 and other MTs has been suggested for motor neuron disease, head injury, Alzheimer's and Parkinson's diseases (WO03105910).
Besides megalin MT is known to bind to some poorly identified receptors in astrocytes (El Refaey et al., 1997), and it also binds to itself forming aggregates (WiI- helmsen et al. 2002). Other binding partners of MT of proteineous origin have not so far been described.
References
Ebadi M, Brown-Borg H, El Refaey H, Singh BB, Garett S, Shavali S, and Sharma SK. Metallothionin-mediated neuroprotection in genetically engineered mouse mod- els of Parkinson's disease. Brain Res. MoI. Brain Res. 2005, 134:67-75.
El Refaey H, Ebadi M, Kuszynski CA, Sweeney J, Hamada FM, Hamed A. Identification of metallothionein receptors in human astrocytes. Neurosci. Lett. 1997, 231 :131-134.
Giralt M, Penkowa M, Lago N, Camats J, Hernandez J, Molinero A and Hidalgo J. Metallothionein-1+2 protect the CNS after a focal brain injury. Exp. Neurol. 2002, 173pp: 114-128.
Klassen RB, Crenshaw K, Kozyraki R, Verroust PJ, Tio L, Atrian S, Allen PL, Hammond TG Megalin mediates renal uptake of heavy metal metallothionein complexes. Am J Physiol Renal Physiol. 2004, 287:F393-403.
Køhler LB, Berezin V, Bock E and Penkowa M. The role of metallothionein Il in neu- ronal differentiation and survival. Brain Res. 2003, 992:128-136.
May P, Herz J, Bock HH. Molecular mechanisma of lipoprotein receptor signalling. Cell Moll Life Sci. 2005, 62:2325-2338. Penkowa M. Metallothionein expression and roles in the central nervous system. Biomed. Rev. 2002, 13:1-18.
Wilhelmsen TW, Olsvik PA, Hansen BH, Andersen RA. Evidence for oligomerization of metallothioneins in their functional state. 2002, J Chromatogr. A. 979:249-254.
Zou Z, Chung B, Nguyen T, Mentone S, Thompson B, and Biemesderfer D. Linking receptor-mediated endocytosis and cell signalling: evidence for regulated intramem- brane proteolisis of megalin in proximal tubule. J Biol Chem 2004, 179:34302- 34310.
Summary of the invention
The authors of the present invention surprisingly found that peptide sequences of a relatively short length derived from fibroblast growth factor receptor (FGFR) ligands are capable of binding to metallothionein protein (MT) or a fragment thereof forming thereby a compound consisting of MT and one or more such sequences where the
MT and sequence(s) are bound to each other via non-covalent bond and/or via sulf- hydryl moieties of the cysteine. It was further surprisingly found that such compound is stable in water solutions under physiological conditions in vivo and in vitro and it is capable of penetrating the BBB when administered in a subject in vivo.
Thus, in a first aspect the invention relates to a compound consisting of MT and a peptide sequence wherein the MT and peptide sequence are bound to each other via a non-covalent bond or via sulfhydryl moieties of the cysteine.
The peptide sequence of the compound is a relatively short peptide sequence which comprises at most 25 amino acid residues. The sequence is a biologically active peptide. The invention relates to biologically activity of the peptide which is associ- ated with activity of a functional cell receptor. According to the invention a peptide sequence of the compound executes its biological activity via binding to functional receptors involved in regulation of the mentioned physiological processes. Upon binding the sequence may either activate or inhibit activity of the receptor which it has affinity to. The invention preferably relates to biologically active short peptide sequences which are capable of binding to fibroblast growth factor receptor (FGFR). The invention preferably relates to FGFR which is expressed by a cell of the brain and is involved in regulation of physiological processes occurred in the brain. In particular, the invention concerns regulation of the processes of cell differentiation, cell survival and/or cell plasticity associated with learning and memory, tissue reparation due to the oxidative stress and/or inflammatory responses.
A biologically active sequence is delivered to the cells of the brain as a part of the compound of the invention where the sequence is non-covalently bound to a carrier protein. The carrier protein of the invention is metallothionein protein (MT). Accordingly, another aspect of the invention relates to the use of MT as carrier protein for the delivery of a short biologically active peptide sequence into the brain.
Further aspects of the invention include
- the use of a complex of MT with a short peptide sequence of the invention as a medicament;
- the use of a complex of MT with a short peptide sequence of the invention as a pharmaceutical composition;
- the use of a complex of MT with a short peptide sequence for the manufacturing of a medicament, said medicament is for therapeutic treatment involving stimulating brain cell proliferation, stimulating neurite outgrowth, stimulating brain cell survival, stimulating neural cell plasticity associated with learning and memory, and/or inhibiting inflammation, modulating cell adhesion and/or cell motility; a pharmaceutical composition comprising MT with a peptide sequence of the invention; a method of treatment of a condition or a disease involving stimulating brain cells proliferation, stimulating neurite outgrowth, stimulating brain cell survival, stimulating neural cell plasticity associated with learning and memory, and/or inhibiting inflammation, modulating cell adhesion and/or cell motility comprising using a compound of the invention; a method of treatment of a condition or a disease involving modulating activity of FGFR comprising using a compound of the invention.
Description of the drawings Figure 1 demonstrates binding of NCAM F3, 2 module derived peptide (FGL) to MT. Binding was studied by means of SPR analysis. Approximately 2000 resonance units (RU) of the MT2 protein (Sigma) were immobilized on the sensor chip. The binding is given as the response difference between the binding to the sensor chip with the immobilized MT2 and a blank sensor chip (unspecific binding). The peptide was injected into the sensor chip at indicated concentrations.
Figure 2 demonstrates binding of NCAM F3, 1 module derived peptides (ABL and EFL) to MT. Binding was studied by means of SPR analysis. Approximately 2000 resonance units (RU) of the MT2 protein were immobilized on the sensor chip. The binding is given as the response difference between the binding to the sensor chip with the immobilized MT2 and a blank sensor chip (unspecific binding). The peptides were injected into the sensor chip at indicated concentrations.
Figure 3 demonstrates binding of the peptides derived from FGF1 (2F1 /Dyo1 and 10F1/Deka1) and FGF17 (2F17/Dyo17 and 10F17/Deka17) to MT. Binding was studied by means of SPR analysis. Approximately 2000 resonance units (RU) of the MT2 protein were immobilized on the sensor chip. The binding is given as the response difference between the binding to the sensor chip with the immobilized MT2 and a blank sensor chip (unspecific binding). The peptides were injected into the sensor chip at indicated concentrations.
Figure 4 demonstrates binding of the beta 10-beta11 loop region derived peptides (dekafins) of different FGF to the combined second and third Ig modules of FGFR1.(a) Binding was studied by means of SPR analysis. Approximately 2000 resonance units (RU) of the FGFR constructs were immobilized on the sensor chip. The binding is given as the response difference between the binding to the sensor chip with the immobilized FGFR modules and a blank sensor chip (unspecific binding). The peptides were injected into the sensor chip at a concentration of 1 μg/ml with the exception of dekafinθ, which was tested in a concentration of 100μg/ml. (b) Binding affinity (1/Kd) of dekafins. Results from four independent experiments are expressed as means ± SEM. (c) Binding of dekafini and its variants to lg2-3 of FGFR. Results from four independent experiments are in all cases expressed as a percentage ± SEM1 with non-mutated dekafini set at 100%. Alanine substitutions are shown in bold. *P < 0.05, compared with non-mutated dekafini. +P<0.05, dekafini truncated from the N-terminus by six residues compared with dekafini truncated by only four residues.
Figure 5. demonstrates phosphorylation of FGFR1 by dekafins and FGFs.TREX- 293 cells, transfected with FGFR containing a C-terminal Strepll tag, were stimulated with the dekafin peptides or FGFs. After stimulation, activated FGFR was im- munoprecipitated by anti-phosphotyrosine antibodies and then analyzed by western blotting by antibodies against the Strepll tag. (a) Dose response of FGFR1 phosphorylation by FGF1 and FGF10. Quantification of FGFR1 phosphorylation was per- formed by densitomeric analysis of band intensity. Results from at least four independent experiments are in all cases expressed as a percentage ± SEM, with untreated controls set at 100%. (b) Dose response of FGFR1 phosphorylation by dekafins. Quantification of FGFR1 phosphorylation was performed by densitomeric analysis of band intensity. Results from at least four independent experiments are in all cases expressed as a percentage ± SEM, with untreated controls set at 100%. *P<0.05, **P<0.01 and ***P<0.001 , compared with controls.
Figure 6 demonstrates the effect of dekafins and FGFs on neurite outgrowth from CGN. CGN cultures from 7-old-day rats were treated with the dekafin peptides or FGFs for 24 hr. The cultures were fixed and immunostained with rabbit anti-rat GAP- 43 primary antibodies, and then with secondary Alexa Fluor®488 goat anti rabbit antibodies, (a) The effect of the dekafin peptides at various doses on neurite extension. Results from four independent experiments are in all cases expressed as a percentage ± SEM, with untreated controls set at 100%. *P<0.05, **P<0.01 and ***P<0.001 , compared with controls, (b) The effect of various doses of FGF1 and FGF10 on neurite extension. Results from four independent experiments are in all cases expressed as a percentage ± SEM, with untreated controls set at 100%. *P<0.05, compared with untreated controls, (e) Effect of an FGFR inhibitor, SU5402, on neurite outgrowth induced by the dekafin peptides. Results from four independ- ent experiments are in all cases expressed as a percentage ± SEM, with untreated controls set at 100%. *P<0.05 and **P<0.01 , compared with peptide-stimulated controls.
Figure 7 demonstrates the effect of dekafins on survival of CGN induced to undergo apoptosis. The neurons were allowed to differentiate for 7 days in a high potassium (40 mM) medium before apoptosis was induced by changing the medium to a low- potassium (5mM) medium. Forty-eight hours later, survival was estimated, (a) High KCI: cells not induced to undergo apoptosis. Low KCI: cells induced to undergo apoptosis. Low KCI + IGF-1 , FGF1 or FGF10: cells induced to undergo apoptosis in the presence of IGF-1, FGF1 or FGF10, respectively, (b) Cells induced to undergo apoptosis and exposed to various concentrations of the dekafin peptides. Results from at least four independent experiments are expressed as a percentage ± SEM, with the cultures induced to undergo apoptosis set at 100%. *P<0.05, **P<0.01 and ***P<0.001 , compared with untreated cultures induced to undergo apoptosis.
Figure 8 demonstrates the effect of dekafins on social recognition memory. Cognitive function was evaluated using the social recognition test as reflected by the recognition ratio (see Materials and Methods). From 10 to 12 animals were tested in each group. *P<0.05, compared with untreated controls.
Figure 9 demonstrates binding of dyofins to the combined second and third Ig modules of FGFR1.(a) Binding was determined by means of SPR analysis. Approximately 2000 resonance units (RU) of the FGFR construct were immobilized on the sensor chip. The binding is given as the response difference between the binding of the individual dyofins to the sensor chip with the immobilized FGFR modules and to a blank sensor chip (unspecific binding). The peptides were injected into the sensor chip at a concentration of 1 μg/ml with the exception of dyofini , 2 and 9. Dyofini and 2 were tested in a concentration of 10μg/ml, and Dyofin9 was tested in a concentration of 200μg/ml. (b) Binding affinity (1/Kd) of dyofins. Results from four independent experiments are expressed as means ± SEM. (c) Binding of dyofiniO and its variants to lg2-3 of FGFR. Results from three independent experiments are in all cases expressed as a percentage ± SEM, with non-mutated dyofiniO set at 100%. Alanine substitutions are shown in bold. **P < 0.01 , ***<0.001 , when compared with non- mutated dyofiniO.
Figure 10 demonstrates the effects of dyofins on phosphorylation of FGFR1. TREX- 293 cells, transfected with FGFRIc containing a C-terminal Strepll tag, were stimulated with the dyofin peptides or FGF2. After stimulation, activated FGFR was im- munopurified by means of anti-phosphotyrosine antibodies and then analyzed by western blotting using antibodies against the Strepll tag. (a) Dose response study of FGFR1 phosphorylation by dyofins. *P<0.05, **P<0.01 and ***P<0.001, compared with controls. (b) Phosphorylation of FGFR1 by 100ng/ml FGF2. ***P<0.001 , compared with controls. Quantification of FGFR1 phosphorylation was performed by densitomeric analysis of band intensity. Results from at least four independent ex- periments are in all cases expressed as a mean ± SEM, with untreated controls set at 100%.
Figure 11 demonstrates the effect of dyofins and FGF2 on neurite outgrowth from CGN. CGN cultures were treated with the dyofin peptides or FGF2 for 24 hr. The cultures were fixed and immunostained with rabbit anti-rat GAP-43 primary antibodies, and then with secondary Alexa Fluor®488 goat anti rabbit antibodies, (a) The effect of the dyofin peptides at various doses on neurite extension. *P<0.05, **P<0.01 and ***P<0.001 , compared with untreated controls, (b) The effect of various doses of FGF2 on neurite extention. *P<0.05, compared with untreated controls. (c) Effect of an FGFR inhibitor, SU5402, on neurite outgrowth induced by the dyofin peptides. *P<0.05 and **P<0.01 , compared with peptide-stimulated controls. Results from four independent experiments are expressed as a mean ± SEM, with untreated controls set at 100%.
Figure 12 demonstrates the effect of dyofins on survival of CGN induced to undergo apoptosis. The neurons were allowed to differentiate for 7 days in a high potassium (40 mM) medium before apoptosis was induced by changing the medium to a low- potassium (5mM) medium. Forty-eight hours later, survival was estimated, (a) High KCI: cells not induced to undergo apoptosis. Low KCI: cells induced to undergo apoptosis. Low KCI + IGF-1 or FGF2: cells induced to undergo apoptosis in the presence of IGF-1 or FGF2, respectively, (b) Cells induced to undergo apoptosis and exposed to various concentrations of the dyofin peptides. Results from at least four independent experiments are expressed as a mean ± SEM, with the cultures induced to undergo apoptosis set at 100%. *P<0.05, **P<0.01 and ***P<0.001 , compared with untreated cultures induced to undergo apoptosis.
Figure 13 demonstrates the effect of dyofins on social recognition memory.(a) dyo- fin1; (b) dyofin2; (c) dyofinθ; (d) dyofin17. Cognitive function was evaluated using the social recognition test as reflected by the recognition ratio (see Materials and Methods). From 10 to 12 animals were tested in each group. **P<0.01 , compared with untreated controls. Figure 14 demonstrates the effect of the ABL, CDL and EFL peptides on phosphorylation of FGFR . TREX-293 cells transfected with FGFRIc were stimulated with different concentrations of the peptides or 100 ng FGF2 (not shown) for 20 min. After stimulation, activated FGFR was immunoprecipitated and immunoblotted using anti-phosphotyrosine antibodies. *P<0.05, **P<0.01, compared with untreated controls.
Figure 15 demonstrates the effect of the second F3 module of NCAM, the FGL pep- tide on phosphorylation of the FGFR1. HEK293 cells, transiently transfected with a His-tagged version of the FGFR1, were stimulated for 20 min with either 5 μg/ml F3,2 module or 50 μg/ml FG loop peptide. The total amount of the FGF-receptor 1 and the amount of the FGF-receptor phosphorylation was estimated by immunoblot- ting using anti-pentahis (anti-His) and anti-phosphotyrosine (anti-P-tyr) antibodies, respectively. Quantification of the FGF-receptor phosphorylation by densitometric analysis of the band intensity. Phosphorylation was estimated relative to the control (untreated cells), which has been normalized to 1.0. Error bar represents one standard deviation (SD). P<0.05 by paired t test comparing treated cells with controls. The t test was performed on array of six independent sets of non-normalized data.
Figure 16 shows the effect of the FGL peptide on neurite outgrowth from dopaminergic (•), hippocampal (A) and cerebellar granule neurons (■). Dopaminergic neurons were grown at a density of 100,000 cells/cm2 on poly-D-lysine coated 24- well cell culture plates for 72 hours with various concentrations of FGLd. The cul- tures were subsequently immunostained for tyrosine hydroxylase. Hippocampal neurons and CGN were plated at a density of 10,000 cells/cm2 on 8-well permanox chamber slides and incubated for 24 hours in the presence of various concentrations of FGLd. Subsequently the neurons were immunostained for GAP-43. Results from at least 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.
Figure 17 demonstrates the effect of the FGL peptide 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/cm2 for six days without or with various concentrations of peptide on 24-well cell culture plates coated with poly-D-lysine were exposed to 100 μM 6- OHDA for two hours. Medium was changed and various concentrations of FGLd were added. The neurons were grown for another 24 hours before the cultures they were fixed and immunostained for tyrosine hydroxylase.
Hippocampal neurons (HN) (c and d) from day 19 rat embryos were seeded at a density of 40,000 cells/cm2 on poly-L-lysine coated 8-well permanox chamber slides and grown for 24 hours in medium containing 20 μM Amyloid-β 25-35 peptide (Aβ 25-35) at the presence of various concentrations of FGLd, before they were fixed and stained with Hoechst 33258.
Cerebella granular meurons (CGN) (e and f) from postnatal day 7 rats were grown at a density of 100,000 cells/cm2 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 various concentrations of FGLd. After two days of incubation, the cultures were fixed and stained with Hoechst 33258.
(a) - Effect of 10 ng/ml GDNF on survival of dopaminergic neurons treated with 6- OHDA.
(b) Effect of various concentrations of FGLd on survival of dopaminergic neurons treated with 6-OHDA.
(c) Effect of 50 ng/ml BDNF on hippocampal cultures treated with Aβ 25-35.
(d) Effect of various concentrations of FGLd on survival of hippocampal neurons treated with Aβ 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 undergo apoptosis.
Figure 18 demonstrates the results of TUNEL staining of CGN cultures induced to undergo apoptosis with and without treatment with the ABL, CDL or EFL peptides. The results are expressed as percentage ± SEM of live neurons as compared to the total number of neurons. Control cultures induced to undergo cell death without peptide treatment were set at 100 %; *** p<0.001 when compared to the cultures induced to undergo cell death (low KCI). Figure 19 shows a stimulatory effect of the FGL, ABL, CDL and EFL peptides on learning and memory in experimental animals: Social Recognition test (A ) and Fear Conditioning test (B and C).
Figure 20 demonstrates that the FGL peptide prevents development of neuropa- thological changes induced by i.c.v. administration of Ap25-35. Bars indicate IR quantified on the basis of average brightness. Open bars represent control animals; solid bars represent Ap25-35-treated animals. Animals were sacrificed four weeks after administration of Aβ25-35- a, c, e, and g show changes quantified in the cingulate cortex; b, d, f, and h show changes quantified in the hippocampus, a, b: Amyloid IRs identified with antibodies against Aβ1-40 (One-way ANOVA; a: F(4, 28) = 6.9; P < 0.0006, and b: F(4, 26) = 30.4; P < 0.0001 ). c, d: Tau phosphorylation (One-way ANOVA; c: F(3, 20) = 2.04; P < 0.14, and d: F(3, 20) = 3.64; P < 0.0304, respectively), e, f: Microglia (One-way ANOVA; e: F(3, 20) = 4.45; P < 0.015, and f: F(3, 20) = 11.0; P < 0.0002). g and h: Astrocytes (One-way ANOVA; g: F(3, 20) = 16.0; P < 0.0001 , and h: F(3, 20) = 10.7; P < 0.0002). All values have been normalized to the values of control animals. Asterisks indicate significant differences when compared to control animals; plus signs indicate significant differences when compared to Aβ25-3s-treated animals.
Figure 21 Intranasal and subcutaneous administration of FGL2 at day 7, 10 and 13 after injection of Aβ25-35 prevents neuronal cell death and impairment of cognitive function induced by i.c.v. administration of Aβ25-35. Open bars represent control animals; solid bars represent Aβ25-35-treated animals. Animals were sacrificed four weeks after Aβ25-35-injection. a-c: Intranasal administration; d-f: subcutaneous administration, a, d: The total number of neurons was quantified in the cingulate cortex (a: One-way ANOVA; F(2, 15) = 15.99; P < 0.0002, and d: One-way ANOVA; F(2, 20) = 3.06; P < 0.069). b, e: Number of neurons quantified in the hippocampus (b: One-way ANOVA; F(2, 12) = 6.30; P < 0.014, and e: One-way ANOVA; F(3, 20) = 6.10; P < 0.015, respectively), c, f: Cognitive function quantified using the recognition ratio (c: One-way ANOVA; F(2, 33) = 10.65; P < 0.0003, and f: One-way ANOVA; F(2, 28) = 9.64; P < 0.0007). Values in a, b, d, and e have been normalized to the values of control animals. Asterisks indicate significant differences when compared to control animals; plus signs indicate significant differences when com- pared to Aβ25-35-treated animals. Detailed description of the invention
1. Compound In a first aspect the present invention relates to a compound consisting of metallotionein protein (MT) or a fragment thereof and a peptide sequence, wherein the MT and peptide sequence are bound to each other via a non-covalent bond or via sulfhydryl moieties of the cysteine. Examples of non-covalent bonds via which the MT and peptide sequence may be bound in the compound include hydrophobic interactions (Van- der Waals forces), hydrogen bonds, ionic bonds, hydrophilic interactions. The invention relates to a compound where the MT and peptide sequence are bound to each other directly without an interconnecting grouping or linker. Throughout the specification of the invention the wording "bound" is used interchangeably with the wording "associated".
The binding between MT and a peptide sequence in the compound is further characterised by a specific affinity binding constant (K0) which has the value of about 10"10M to about 10'3 M., such as of about 10"8 M to about 10"4 M or between about 10"9M and about 10"5 M, such as about 10"7 M or about 10"6 M.
The compound is further characterized in that it is relatively stable in a water solution under physiological conditions both in vivo and in vitro. The wording "physiological conditions" includes conditions of the body including the body temperature, pH and ion strength of the body liquids and other factors which constitute the micro-environment of different body tissues and liquids in vivo, e.g. micro-environments of the blood, brain, muscles, etc.. The compound according to the invention is characterized in that it despite of its relative stability under such conditions the peptide sequence and MT of the compound dissociate from each other when the compound reaches an appropriate functional cell receptor, such as a receptor of a brain cell, which the peptide sequence is capable of binding to.
A peptide sequence which is comprised by the compound comprises at most 25 contiguous amino acid residues, such as from 3 to 25 amino acid residues, such as from 3 to 20, for example from 3 to 15, such as from 3 to 10, such as form 5 to 25, such as from 7 to 25, fro example from 8 to 25, or from 10 to 25, such as from 12 to 25 or from 14 to 25. Sequences comprising from 5 to 20 contiguous amino acid residues are preferred.
Thus, the invention relates to compounds comprising MT and any contiguous peptide sequence of at most 25 amino acid residues which are associated together in the compound via a non-covalent bond or via sulfhydryl covalent bond made of the moieties of the cysteins. A preferred peptide sequence of at most 25 amino acid residues according to the invention is characterized in that it comprises the amino acid motif x1-x2-x3-x4-x5, wherein x1 is a hydrophobic, charged amino acid residue or G; x2 is a charged amino acid residue, T or S, x3 is any amino acid residue; x4 is a charged amino acid residue, T or S, x5 is a hydrophobic, charged amino acid residue T, S, N, Q or G.
According to the invention, x1 may be any hydrophobic amino acid residue, however residues L, P, M as the x1 residue of the sequence may in some embodiments be preferred. In other embodiments a charged amino acid residue as the x1 residue may be preferred. The charged amino acid residue may be selected from K, R, H, D or E. Still in other embodiments x1 may be G.
X2 in some embodiments may be a charged amino acid residue independently selected from K, R, H, D or E. In other embodiments it may be the T or S residue.
X3 may be any amino acid residue, however, in some embodiments a hydrophobic residue in this position may be preferred, preferably the V, L, or P residue, in other embodiments, it may be preferred a non-charged hydrophilic residue such as S, T, Q or N, still, in other embodiments it may be preferred a residue selected from G or C or a charged amino acid residue.
X4 according to the invention may be a charged amino acid residue, S or T depending on the preferred embodiment. X5 may be selected from a hydrophobic or charged amino acid residue, or selected from residues T, S, N, Q or G.
Amino acid residues of any of the positions x1 to x5 are according to the invention to be selected independently, however, in some preferred embodiments both x2 and x4 may be selected from charged amino acid residues.
In the present application the standard one-letter code for amino acid residues is applied as well as the standard three-letter code. Abbreviations for amino acids are in accordance with the recommendations in the IUPAC-IUB Joint Commission on Biochemical Nomenclature Eur. J. Biochem, 1984, 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 understood 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.
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 "-NH2". Where nothing else is stated the N-terminal amino acid of a polypeptide comprise a free amino-group, this may also be specified as "H-".
Where nothing else is specified amino acid can be selected from any amino acid, whether naturally occurring or not, such as alfa amino acids, beta amino acids, and/or gamma amino acids. Accordingly, the group comprises but are not limited to: Ala, VaI, Leu, lie, Pro, Phe, Trp, Met, GIy, Ser, Thr, Cys, Tyr, Asn, GIn, Asp, GIu, Lys, Arg, His Aib, NaI, Sar, Orn, 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 and/or phosphorylation 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 GIu and Asp. Basic and acidic amino acid residues constitute a group of charged amino acid residues. The group of hydrophobic amino acid residues is represented by the residues of amino acids Leu, lie, VaI1 Phe, Trp, Tyr, Met, Ala and Pro.
The invention relates to naturally occurring, synthetically/recombinant prepared peptide sequences/fragments, and/or peptide sequences/fragments prepared by means of enzymatic/chemical cleavage of bigger polypeptides, wherein said peptide sequences/fragments are integral parts of said bigger polypeptides. The invention relates to isolated individual peptide sequences. The term "isolated" means that a peptide sequence exists as a separate individual compound, but not a part of a polypeptide which the peptide sequence is derived/originate from.
Non-limited examples of isolated individual peptide sequences which comprise the above identified motif may be amino acid sequences which set forth in SEQ ID NOs: 1-45. A peptide sequence comprised by the compound of the invention may comprise or consists of any of these sequences, their fragments or variants, wherein said fragments and variants are capable of binding to MT and thereby forming a MT- peptide sequence compound which is stable in solution under physiological conditions.
Thus, in one embodiment a peptide sequence may comprise an amino acid sequence selected from SEQ ID NOs: 1-45, or a fragment or variant of said sequence. In another embodiment a peptide sequence may consist of a sequence selected from SEQ ID NOs:1-45, or a fragment or variant of said sequence. The peptide sequence may be present in the compound as a single copy, i.e. formulated as a monomer of the peptide sequence, or it may be present as several copies of the same sequence, e.g. as a multimer comprising two or more copies of a sequence selected from SEQ ID NOs: 1-45, or two or more copies of a fragment or a variant of said sequence. The preferred presentation of a peptide sequence is a multimeric presentation, such as denrimer comprising four identical peptide sequence bound to the lysine core consisting of three lysine residues, or such as an LPA dimer described in WO2005014623. Still in another embodiment a sequence may be selected from SEQ ID NOs:46-51 , or it may be a fragment or variant or multimer of said sequence.
In some preferred embodiments a sequence may be selected from SEQ ID NOs:1- 18, or from fragments or variants thereof. In other preferred embodiments a sequence may be selected from SEQ ID NOs:42-45, or from fragments or a variants thereof. Still in other preferred embodiments a sequence may be selected from SEQ ID NOs: 19-41, or from fragments or variants thereof, or it may be preferably selected from SEQ ID NOs:46-51 , or from fragments or variants thereof
As it is mentioned above, the invention relates to variants of peptide sequences described in the application as well.
In one aspect the term "variant of a peptide sequence" means that the peptides may be modified, for example by substitution of one or more of the amino acid residues. Both L-amino acids and D-amino acids may be used. Other modification may comprise derivatives such as esters, sugars, etc. Examples are methyl and acetyl esters.
In another aspect "variants" may be understood as exhibiting amino acid sequences gradually differing from the preferred predetermined sequence, as the number and scope of insertions, deletions and substitutions including conservative substitutions increase. This difference is measured as a reduction in homology between the pre- determined sequence and the variant.
In still another aspect, variants of the peptide fragments according to the invention may comprise, within the same variant, or fragments thereof or among different variants, or fragments thereof, at least one substitution, such as a plurality of substi- tutions introduced independently of one another. Variants of the complex, or fragments thereof may thus comprise conservative substitutions independently of one another, wherein at least one glycine (GIy) of said variant, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of Ala, VaI, Leu, and lie, and independently thereof, variants, or fragments thereof, wherein at least one alanine (Ala) of said variants, or fragments thereof is substi- tuted with an amino acid selected from the group of amino acids consisting of GIy, VaI, Leu, and lie, and independently thereof, variants, or fragments thereof, wherein at least one valine (VaI) of said variant, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of GIy, Ala, Leu, and lie, and independently thereof, variants, or fragments thereof, wherein at least one leucine (Leu) of said variant, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of GIy, Ala, VaI, and lie, and independently thereof, variants, or fragments thereof, wherein at least one isoleucine (lie) of said variants, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of GIy, Ala, VaI and Leu, and independently thereof, variants, or fragments thereof wherein at least one aspartic acids (Asp) of said variant, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of GIu, Asn, and GIn, and independently thereof, variants, or fragments thereof, wherein at least one aspargine (Asn) of said variants, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of Asp, GIu, and GIn, and independently thereof, variants, or fragments thereof, wherein at least one glutamine (GIn) of said variants, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of Asp, GIu, and Asn, and wherein at least one phenyla- lanine (Phe) of said variants, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of Tyr, Trp, His, Pro, and preferably selected from the group of amino acids consisting of Tyr and Trp, and independently thereof, variants, or fragments thereof, wherein at least one tyrosine (Tyr) of said variants, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of Phe, Trp, His, Pro, preferably an amino acid selected from the group of amino acids consisting of Phe and Trp, and independently thereof, variants, or fragments thereof, wherein at least one arginine (Arg) of said fragment is substituted with an amino acid selected from the group of amino acids consisting of Lys and His, and independently thereof, variants, or fragments thereof, wherein at least one lysine (Lys) of said variants, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of Arg and His, and independently thereof, variants, or fragments thereof, and independently thereof, variants, or fragments thereof, and wherein at least one proline (Pro) of said variants, or fragments thereof is substituted with an amino acid se- lected from the group of amino acids consisting of Phe, Tyr, Trp, and His, and inde- pendently thereof, variants, or fragments thereof, wherein at least one cysteine (Cys) of said variants, or fragments thereof is substituted with an amino acid selected from the group of amino acids consisting of Asp, GIu, Lys, Arg, His, Asn, GIn, Ser, Thr, and Tyr.
It thus follows from the above that the same functional equivalent of a peptide fragment, or fragment of said functional equivalent may comprise more than one conservative amino acid substitution from more than one group of conservative amino acids as defined herein above. The term "conservative amino acid substitution" is used synonymously herein with the term "homologous amino acid substitution".
The groups of conservative amino acids are as the following: P, A, G (neutral, weakly hydrophobic), S, T (neutral, hydrophilic) Q, N (hydrophilic, acid amine) E, D (hydrophilic, acidic) H, K, R (hydrophilic, basic) L, I, V, M, F, Y, W (hydrophobic, aromatic) C (cross-link forming)
According to the invention, a variant may be 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%, even more preferably 97%, 98% or 99% homology to an amino acid sequence selected from SEQ ID NOs:1-51 , or it may be 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%, even more preferably 97%, 98% or 99% positive amino acid matches compared to an amino acid sequence selected from SEQ ID NOs:1-51. A positive amino acid match is defined herein 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 K to R, E to D, L to M, Q to E, I to V, I to L, A to S, Y to W, K to Q, S to T, N to S and Q to R. The homology of one amino acid sequence with another amino acid is defined as a percentage of identical amino acids in the two collated sequences. The wording "sequence homology" is used herein synonymously with the term "sequence similarity". The sequence homology may be routinely calculated using well known algorithms such as BLOSUM 30, BLOSUM 40, BLOSUM 45, BLOSUM 50, BLOSUM 55, BLO- SUM 60, BLOSUM 62, BLOSUM 65, BLOSUM 70, BLOSUM 75, BLOSUM 80, BLOSUM 85, or BLOSUM 90;
Substitution of amino acids in a peptide sequence of the invention which results in formation of the peptide sequence variants included in the scope of the invention may in one embodiment be made based upon their hydrophobicity and hydrophilicity values and the relative similarity of the amino acid side-chain substituents, including charge, size, and the like. Exemplary amino acid substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
In some embodiments the following variants may be preferred:
1. a variant which is an amino acid sequence of at least 6 amino acid residues having at least 65% sequence similarity with a sequence selected from the sequences of SEQ ID NOs:1-51, preferably an amino acid sequence of 6 to 20 contiguous amino acid residues, which has more then 70% sequence similarity with a sequence selected from the sequences of SEQ ID NOs:1-51 , such as from 71% to 80% similarity, preferably from 81% to 85%, more preferably from 86% to 90%, even more preferably from 91% to 95%, and even more preferably more then 95% of sequence similarity, such as 96-99% similarity.
2. a variant which consists of a sequence of SEQ ID NOs:1-51 , wherein said sequence comprising one or more amino acid residues which is/are covalently attached to a derivative of a sugar or lipid, or which comprise a chemical grouping such as for example a phosphoryl or acetyl residues, or which may comprise any other chemical moieties which do not prevent the sequence to bind to MT and form the compound of the invention.
As it is mentioned above, the present invention does also relate to fragments of the peptide sequences described in the application.
When referred to a fragment of a peptide sequence, according to the invention a preferred fragment is a fragment of a sequence selected from SEQ ID NOs: 1-51 which has the length of at least 40% of the length of said sequence, more preferably at least 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. In the present context the fragment may comprise from 3 to 13 amino acid residues. It is also preferred that the fragment comprises the amino acid motif described above.
In another aspect the invention relates to a short peptide sequence as above which possesses biological activity related to a physiological process which takes place in the brain, e.g. capability of stimulating neural plasticity or cell survival. Biological activity of the peptide sequence is according to the invention is associated with the capability of said peptide sequence to modulate activity of a functional cell receptor. A preferred functional cell receptor of the present invention is a receptor selected from the family of fibroblast growth factor receptors (FGFR). FGFR1 , FGFR2, FGFR3, FGFR4 or another FGFR receptor of the family may be selected according to different embodiments.
The prototypical FGFR consists of three immunoglobulin-like modules (Ig1 - Ig3), a trans-membrane domain and a cytoplasmic tyrosine kinase domain. The linker re- gion between the Ig1 and Ig2 modules is very long, consisting of 20-30 amino acid residues, including a stretch of acidic amino acids termed the acid box. FGFRs also bind heparin/heparan sulphate, which is required for the high-affinity FGF-FGFR interaction. Binding studies of several FGFs to various FGFR fragments and crystal structures of several FGFs in complex with fragments of FGFRs consisting of the Ig2 and Ig3 modules indicate that these modules and the Ig2-lg3 linker region are sufficient for the specific FGF-FGFR interaction. FGF-FGFR binding results in dimerisation of FGFR leading to auto-phosphorylation of the receptor tyrosine kinase domains. Regulation of the binding specificity of FGFs is primarily achieved by alternative splicing of FGFRs. Alternative splicing of exons encoding the C- terminal part of the Ig3 module in FGFR1-3 results in two isoforms (3b and 3c) possessing different FGF binding specificity. There are also FGFR isoforms lacking the Ig1 module (FGFR1 and 2), the Ig1 module combined with the Ig1-lg2 linker sequence (FGFR2), or the Ig1-lg2 linker alone (in FGFR3). A short peptide sequence as described above may derive from a FGFR ligand, e.g. a fibroblast growth factor (FGF) or cell adhesion molecule, such as the neural cell adhesion molecule (NCAM). By the term "derived" is the present context meant that the amino acid sequence of an isolated short peptide sequence represents a subsequence/fragment of the peptide sequence of an FGFR ligand, e.g. a subsequence of the FGF or NCAM protein sequence.
In one embodiment the invention relates to a fragment of a cell adhesion molecule which is a ligand of FGFR. Examples of such adhesion molecules may be NCAM or L1. The invention preferably relates to NCAM fragments selected from the sequences set forth in SEQ ID NOs:42-45
In another embodiment the peptide sequence may be a fragment of L1 , e.g. selected from the following sequences: APEKWFSLGKV (SEQ ID NO: 46),
DWNAPQIQYRYQWR (SEQ ID NO: 47), ' DLAQVKGHLRGYN (SEQ ID NO: 48),
RHVHSHMWPAN (SEQ ID NO: 49),
RFHILFKALPEGKVSPD (SEQ ID NO: 50) or LHHLAVKTNGTG (SEQ ID NO: 51 ).
Fragments and variants of the above peptide sequences which are capable of binding to MT and which are capable of binding to an FGFR, are also in the scope of the invention.
The above fragments of NCAM and L1 are in the scope of the invention as examples of the peptide sequence comprised by the compound of the invention, i.e. formulated as peptide fragments associated with MT.
In another embodiment the invention relates to an isolated fragment of an FGF which is capable of binding to MT and which is capable of binding to an FGFR. An FGF of the invention may be selected from any members of the FGF family, i.e. FGF1-23. Examples of such fragments are be the sequences identified herein as SEQ ID NOs: 1-41. In a particular embodiment the invention relates to isolated FGF fragments having the sequences identified as SEQ ID Nos: 4, 8, 10, 16, 17, 26, 33, 35, 39 and 41. The invention relates to the latter sequences both as a part of the compound of the invention and as individual compounds, i.e. not bound to MT or any other moiety. In some embodiments these sequences may be preferred as separate individual peptide sequences, in the other embodiments they may be preferred as a part of the compound where they are associated with MT.
Concerning the other isolated FGF fragments, such as SEQ ID NOs: 1-3, 5-7. 8,9, 11-15, 18-25, 27-32, 33, 34, 36-38 or 40, the invention includes these fragments in the scope of protection when they form a part of the compound of the invention, i.e. as the MT bound fragments.
The invention also relates to variants and fragments of the above peptide sequences. The fragments and variants of these sequences are characterized by structural and functional features described above.
According to the invention a peptide sequence as described above is associated with MT or a fragment thereof in a compound. Thus, the compound of the invention consists of at least two peptide sequences, wherein one of the sequences is a peptide sequence of MT protein or the sequence of a fragment thereof and another peptide sequence is at least one short peptide sequence as described above.
The MT protein of the invention may be selected from metallothionein-1 A (MT1A), metallothionein-1 B (MT1B), metallothionein-1 E (MT1 E), metallothionein-1 F (MT1 F), metallothionein-1 G (MT1G), metallothionein-1 H (MT1 H), metallothionein-11 (MT1 I), metallothionein-1 K (MT1 K), metallothionein-1 L (MT1L), metallothionein-1 R
(MT1R), metallothionein-1 X (MT1 X), metallothionein-2 (MT2), metallothionein-3
(MT3) or metallothionein-4 (MT4). The invention relates to sequences of these MT proteins which are identified in the Gene Bank as the following Ace. Nos: Q9BQN2,
P04731 , P07438, P04732, P04733, P13640, P80294, P80295, P80296, Q93083,
P80297, P02795, P25713, P47944, respectively.
As mentioned above a short peptide sequence of the compound may be associated with a fragment of MT. An example of such MT fragment may be a fragment which comprises a subsequence of MT which comprises at least one of the following amino acid sequences:
KKSCCSCCPMSCAK (SEQ ID NO:52)
KKCCCSCCPVGCAK (SEQ ID NO:53) KKSCCSCCPVGCAK (SEQ ID NO:54)
KKSCCSCCPVGCSK (SEQ ID NO:55)
KKSCCSCCPVGCAK (SEQ ID NO:56)
KKSCCSCCPLGCAK (SEQ ID NO:57)
KKSCCSCCPVGCAK (SEQ ID NO:58) KKSCCSCCPVGCAK (SEQ ID NO:59)
KKSCCSCCPVGCAK (SEQ ID NO:60)
KKSCCSCCPMGCAK (SEQ ID NO:61)
KKSCCSCCPVGCAK (SEQ ID NO:62)
KKSCCSCCPVGCAK (SEQ ID NO:63) KKSCCSCCPAECEK (SEQ ID NO:64)
RKSCCPCCPPGCAK (SEQ ID NO:65)
AQGCICKGASEKCS (SEQ ID NO:66)
AQGCVCKGSSEKCS (SEQ ID NO:67)
AQGCVCKGASEKCS (SEQ ID NO:68) AQGCVCKGASEKCS (SEQ ID NO:69)
AQGCICKGASEKCS (SEQ ID NO:70)
AQGCICKGASEKCS (SEQ ID NO:71)
AQGCICKGASEKCS (SEQ ID NO:72)
AQGCICKGASEKCS (SEQ ID NO:73) AQGCICKGTSDKCS (SEQ ID NO:74)
AQGCVCKGASEKCS (SEQ ID NO:75)
AQGCICKGTSDKCS (SEQ ID NO:76)
AQGCICKGASDKCS (SEQ ID NO:77
AKDCVCKGGEAAEAEAEKCS (SEQ ID NO:78) ARGCICKGGSDKCS (SEQ ID NO:79)
MDPNCSCATGGSCT (SEQ ID NO:80)
MDPNCSCTTGGSCA (SEQ ID NO:81)
MDPNCSCATGGSCT (SEQ ID NO:82)
MDPNCSCAAGVSCT (SEQ ID NO:83) MDPNCSCAAGVSCT (SEQ ID NO:84) MDPNCSCEAGGSCA (SEQ ID NO:85)
MDPNCSCAAGVSCT (SEQ ID NO:86)
MDPNCSCAAAGVSCT (SEQ ID NO:87)
MDPNCSCSPVGSCA (SEQ ID NO:88) MDPNCSCATGGSCS (SEQ ID NO:89)
MDPNCSCDPVGSCA (SEQ ID NO:90)
MDPNCSCAAGDSCT (SEQ ID NO:91 )
MDPETCPCPSGGSCT (SEQ ID NO:92)
MDPRECVCMSGGICM (SEQ ID NO:93) CTGSCKCKECKCNS (SEQ ID NO:94)
CAGSCKCKECKCTS (SEQ ID NO:95)
CAGSCKCKECKCTS (SEQ ID NO:96)
CAGSCKCKECKCTS (SEQ ID NO:97)
CASSCKCKECKCTS (SEQ ID NO:98) CAGSCKCKKCKCTS (SEQ ID NO:99)
CAGSCKCKECKCTS (SEQ ID NO: 100)
CASSCKCKECKCTS (SEQ ID NO: 101)
CAGSCKCKECKCTS (SEQ ID NO:102)
CASSCKCKECKCTS (SEQ ID NO: 103) CAGSCKCKECKCTS (SEQ ID NO:104)
CAGSCKCKECKCTS (SEQ ID NO: 105)
CADSCKCEGCKCTS (SEQ ID NO:106)
CGDNCKCTTCNCKT (SEQ ID NO: 107).
In particular, MT1A subsequence may be selected from SEQ ID NOs:52, 66, 80 or 94; MT1B subsequence may be selected from SEQ ID NOs:53, 67, 81 or 95. MT1 E subsequence may be selected from SEQ ID NOs:54, 68, 82 or 96; MT1 F subsequence may be selected from SEQ ID NOs:55, 69, 83 or 97; MT1G subsequence may be selected from SEQ ID NOs:56, 70, 84 or 98; MT1 H subsequence may be selected from SEQ ID NOs:57, 71 , 85 or 99; MT1 I subsequence may be selected from SEQ ID NOs:58, 72, 86 or 100; MT1 K subsequence may be selected from SEQ ID NOs:59, 73, 87 or 101; MT1 L subsequence may be selected from SEQ ID NOs:60, 74, 88 or 102; MT1 R subsequence may be selected from SEQ ID NOs:61 , 75, 89 or 103; MT1X subsequence may be selected from SEQ ID NOs:62, 76, 90 or 104; MT2 subsequence may be selected from SEQ ID NOs:63, 77, 91 or 105 MT3 subsequence may be selected from SEQ ID NOs:64, 78, 92 or 106; MT4 subsequence may be selected from SEQ ID NOs:65, 79, 93 or 107.
In different embodiments of the invention MT of the compound may be represented by MT protein or it may be a peptide fragment of said MT which may be selected from the fragments identified above. Further, in some embodiments MT ot a fragment thereof may be represented by a peptide sequence which is homologues to said MT sequence or said fragment, with the proviso that this homologues sequence is capable of binding to a short peptide sequence of the invention and together with the latter short peptide sequence form a compound of the invention which is capable of penetrating the BBB.
2. Biological activity The present invention relates to a compound that comprises a peptide sequence which is capable of binding to a functional cell receptor and modulating activity of said receptor. It is preferred that the receptor is a cell surface receptor. Most preferred functional receptor is a receptor selected from of the family of fibroblast family growth factor receptors (FGFRs). In one embodiment the fibroblast growth factor receptor may be fibroblast growth factor receptor 1 (FGFR1 ), in another embodiment the fibroblast growth factor receptor may be fibroblast growth factor receptor 2 (FGFR2), in another embodiment the fibroblast growth factor receptor may be fibroblast growth factor receptor 3 (FGFR3), in another embodiment the fibroblast growth factor receptor may be fibroblast growth factor receptor 4 (FGFR4) or in still another embodiment it may be fibroblast growth factor receptor 5 (FGFR5). The invention in particular relates to FGFR1, modulating FGFR1 activity and modulating FGFR1 activity associated physiological processes.
Binding of a peptide sequence of the invention to a FGFR, e.g. FGFR1 , results in a change in the receptor activation status which is reflected by an increase in tyrosine phosphorylation of the receptor or a change in the activation status of one or more of intracellular proteins involved in FGFR-associated signal transduction, for example STAT1, JNK, PLCY, ERK, STAT5, PI3K, PKC, FRS2 and/or GRB2 proteins activation status. The result of modulating of FGFR signalling by a peptide sequence of the invention may also be reflected by a change in a physiological effect on the cellular or higher level, e.g. the body level.
When FGFR activation is measured as the level of phosphorylation of the receptor, the degree of phoshorylation is estimated as at least 20% above the control value, such as at least 20-200 %, for example at least 50-200%. The control value in the present content is meant the degree of phosphorylation of FGFR in the medium where a peptide sequence capable of activating of FGFR is absent.
When estimating an efficient concentration of a peptide sequence with respect to modulating of FGFR activity, said concentration may be between 0.1-1000μM, 1- 1000 μM, for example 1-200 μM, for example 10-200 μM, such as 20-180 μM, for example 30-160 μM, such as 40-140 μM, for example 50-130 μM, such as 60-120 μM, for example 70-110 μM, such as 80-100 μM.
FGFR tyrosine phosphorylation or activation of any of the FGFR-associated downstream molecules, such as for example STST1, JNK, PLCy, ERK, STAT5, PI3K, PKC, FRS2 and/or GRB2 proteins, may be estimated by any conventional methods, such as for example immunocytochemistry, immunoblotting or immunoprecipitation, using commercially available antibody against the activated proteins. The degree of activation is estimated as at least 20% above/below the control value, such as at least 20-200 %, for example at least 50-200%. The control value is estimated as a degree of phosphorylation of the protein of interest in the medium where a compound capable of activation of FGFR is absent.
As FGFRs are involved in regulation of diverse physiological processes, modulation of FGFR activation by a peptide sequence of the invention may result in modulating an FGFR related physiological process, for example modulating FGFR related cell differentiation, e.g. induction of differentiation of progenitor cells, stimulation of matu- ration of cells being on an early stage of differentiation, differentiation of cancer cells.
Thus, the invention relates to peptide sequences which are capable of activating
FGFR via direct binding to the receptor. The peptide sequences of the invention may also modulate receptor activation induced by other FGFR ligands binding. In the present content the term "modulate" means both stimulating and inhibiting activation of the receptor.
FGFR related physiological processes of interest which can be modulated by binding of a peptide sequence of the invention to FGFR may be selected from
- stem cell differentiation, for example neuronal precursor cell differentiation
- cancer cells differentiation;
- neural cell differentiation and/or regeneration of nerves, for example neurite outgrowth; - neural plasticity associated with memory and learning, for example synaptic efficacy;
- cell survival, in particular survival of neuronal and/or glial cells,
- the oxidative stress response, such as expression of scavenges of reactive oxygen species - activation of astrogliosis, such as expression of neuroprotective growth factors and proteins such as e.g. BDNF, NT-3, GDNF, neurturin, artemin, NGF, variety of fibroblast growth factors (FGFs), S100-proteins (S100A4, S100A6, S100A10, S100A12, S100B), IGF-2, neuregulin by astrocytes
- inflammation, for example activation of microglia and macrophages, expression of pro-inflammatory cytokines and/or by stimulating anti-inflammatory responses;
- angiogenesis in the lesioned area, for example expression of growth factor promoting angiogenesis such as VEGF and FGF2;
- cell-cell and/or cell-extracellular matrix adhesion, for example neural, glial or cancer cell adhesion to cellular environment and/or extracellular matrix; - morphological plasticity of cells, for example neuronal plasticity associated with learning and memory;
- the effects related to FGFR ligands.
Thus, biological activity of a peptide sequence which is comprised by the compound of the invention is according to the invention associated with biological activity of
FGFR. It is preferred that biological activity of the peptide is associated with a physiological process occurring in the brain. Accordingly, the capabilities of the peptide sequence to modulate the processes of cell survival and/or cell differentiation and/or cell plasticity and/or inhibiting inflammation which take place in the brain are preferred. Accordingly, the invention preferably relates to cells of neural system, in particular brain cells, such as neuronal cells and/or glial cells. FGFR which is expressed on the surface of brain cells is a preferred functional cell receptor of the invention.
The molecular processes involving a biological activity of the peptide are more preferably those which are related to a neuronal cell.
As already mentioned above, one of the preferred biological activities of the peptide sequence according to the invention is the capability of stimulating neuronal cell differentiation.
The term "neuronal differentiation" is understood herein both as differentiation of neural precursor cells, or neural stem cells, and further differentiation of neural cells, such as for example maturation of neuronal cells. An example of such differentiation may be neurite outgrowth from immature neurons, branching of neurites, and also neuron regeneration.
Thus, one preferred embodiment the invention concerns biological activity of a peptide sequence associated with stimulating of differentiation of neural precursor/stem cells or immature neurons and/or stimulating neurite outgrowth from mature neurons, for examples neurons which were traumatizes but survived and are committed to regenerate damaged processes.
In the present context "differentiation" is related to the processes of maturation of neurons and extension of neurites, which take place after the last cell division of said neurons. The compounds of the present invention may be capable of stopping neural cell division and initiating maturation said cells, such as initiating extension of neurites. Otherwise, "differentiation" is related to initiation of the process of genetic, biochemical, morphological and physiological transformation of neuronal progenitor cells, immature neural cells or embryonic stem cells leading to formation of cells having functional characteristics of normal neuronal cell as such characteristics are defined in the art. The invention defines "immature neural cell" as a cell that has at least one feature of neural cell accepted in the art as a feature characteristic for the neural cell. Substances with the potential to promote neurite outgrowth as well as stimulate regeneration and/or differentiation of neuronal cells, such as certain endogenous trophic factors, are prime targets in the search for compounds that facilitate for example neuronal regeneration and other forms of neuronal plasticity. To evaluate the potential of the present compound, the ability to stimulate the neurite outgrowth related signalling, interfere with cell adhesion, stimulate neurite outgrowth, regeneration of nerves, may be investigated. The compound of the present invention comprising a biologically active peptide sequence of the invention is shown to be capable of promoting neurite outgrowth and is therefore considered to be a good pro- moter of regeneration of neuronal connections, and thereby of functional recovery after damages as well as a promoter of neuronal function in other conditions where such effect is required.
According to the present invention a compound comprising at least one of the above peptide sequences is capable of stimulating neurite outgrowth. The invention concerns the neurite outgrowth improvement/stimulation such as about 75% improvement/stimulation above the value of neurite outgrowth of control/non- stimulated cells, for example 50%, such as about 150%, for example 100%, such as about 250, for example 200%, such as about 350 %, for example 300%, such as about 450%, for example 400%, such as about 500%.
Estimation of capability of a candidate compound to stimulate neurite outgrowth may be done by using any known method or assay for estimation of neurite outgrowth, such as for example as the described in Examples below.
Accordingly, the invention also concerns a method for stimulating neuronal cell differentiation comprising using the compound of the invention comprising a peptide sequence of the invention.
One of most preferred embodiments of the invention concerns the activity of the peptide sequences in connection with learning and memory, in particular, the capability of a peptide sequence to stimulate synaptic plasticity, spine formation, synaptic efficacy. Thus, the invention also concerns a method for stimulating memory and/or learning comprising using a peptide sequence of the invention and/or compound comprising said sequence. The invention relates to both short- term memory and long-term memory.
In another preferred embodiment of the invention a peptide sequence of the invention capable of stimulating cell survival, in particular neuronal cell survival. The invention concerns the capability of stimulating cell survival both due trauma and degenerative disease. Accordingly, the invention relates to a method for stimulating cell survival, preferably neuronal cell survival by using a peptide sequence of the invention and/or compound comprising said sequence.
Substances with the potential to enhance neuronal cells to survive due to damage as well as inhibit degeneration and/or apoptosis of neuronal cells in trauma and disease, are prime targets in the search for candidate compounds for new medicine for treatment of neurodegenerative diseases such as for example Alzheimer's or Park- inson's diseases. To evaluate the potential of the present peptides, the ability to stimulate survival related signalling, interfere with apoptosis related cellular reactions, stimulate regeneration of nerves may be investigated. Compounds of the present invention are shown to promote neural cell survival and decrease the cell loss and therefore considered to be good candidates for promotion of regeneration of neural connections in brain and/or in peripheral neural system, and thereby of functional recovery after damages due trauma or disease as well as promoters of neuronal function in any other conditions where such effect is required.
In the present context "survival" is related to the processes associated with maintenance and/or recovery of cell function after the damage of the cell. The compounds of the present invention may be capable of stopping or attenuating the processes committing the cell to death, such as inhibiting apoptosis of neural cells initiated by cell damage due trauma or disease. Otherwise, "survival" is related to inhibition of the processes associated with the cell damage leading to cell death and initiation of the processes of genetic, biochemical, morphological and physiological transformation or reconstruction of cells, in particular neuronal cells, such as progenitor cells, immature neural cells or embryonic stem cells or mature neural cells having normal functional characteristics defined in the art. The invention defines "immature neural cell" as a cell that has at least one feature of neural cell accepted in the art as a feature characteristic for the neural cell. According to the present invention a compound comprising at least one of the above peptide sequences is capable of stimulating neural cell survival. The invention concerns the neural cell survival stimulation such as about 75% stimulation above the value of survival of control/non-stimulated cells, for example 50%, such as about 150%, for example 100%, such as about 250, for example 200%, such as about 350 %, for example 300%, such as about 450%, for example 400%, such as about 500%.
Estimation of capability of a candidate compound to stimulate neural cell survival may be done by using any known method or assay for estimation of cell survival, such as for example the ones described in Examples of the present application.
In another embodiment the peptide sequence of the invention is also capable of inhibit- ing an inflammatory process, in particular an inflammatory process in the brain.
Inflammation is a defence reaction caused by tissue damage due to a mechanical injury or bacterial, virus or other organism infection. The inflammatory response involves three major stages: first, dilation of capillaries to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; and third, leukocyte transmigration through endothelium and accumulation at the site of injury and infection. The inflammatory response begins with a release of inflammatory mediators. Inflammatory mediators are soluble, diffusible molecules that act locally at the site of tissue damage and infection, and at more distant sites, influencing consequent events of the inflammatory response. Inflammatory mediators can be exogenous, e. g. bacterial products or toxins, or endogenous, which are produced within the immune system itself, as well as injured tissue cells, lymphocytes, mast cells and blood proteins.
Neuroinflammation plays a prominent role in the progression of Alzheimer's disease and may be responsible for degeneration in vulnerable regions such as the hippocampus. Neuroinflammation is associated with elevated levels of extracellular glu- tamate and potentially an enhanced stimulation of glutamate N-methyl-D-aspartate receptors. Anti-inflammatory activity is another important biological activity of the peptide sequence of the invention. Thus, the invention relates to anti-inflammatory peptide, which is capable of serving as an inhibitor of the sustained inflammatory response, in particular in the brain..
The continuous presence of inflammatory mediators, such as for example TNF alpha in the body in response to sustained presence of bacterial products or even live bacteria locally during days or weeks following trauma and/or infection promotes the reactions to inflammation, such as, for example, heat, swelling, and pain. The sus- tained inflammatory response has been proven to be very harmful to the body. If the bacterial products or live bacteria become spread universally in the body from their local focus the inflammatory reaction becomes overwhelming and out of control and leads to sepsis which eventually progress further to severe sepsis and septic shock. Anti-inflammatory peptides may be used to block or suppress the overwhelming sustained inflammatory response represented by a massive and harmful cytokine cascade in the blood and vital organs such as lung, liver intestine, brain and kidneys.
In the present context by the term "anti-inflammatory compound" is meant a com- pound which is capable of at least one of the following activities i) decrease or inhibit the gene expression in the immune cells, preferably monocytes/macrophages in response to bacterial products, live bacteria or trauma to produce endogenous inflammatory mediators including receptors for inflammatory mediators and transcription factors involved in the signal transduction of the inflammatory mediators, said mediators being preferably selected from the group comprising cytokines, selected from the group
TNFalpha IL-1 , IL-6, G-CSF, GM-CSF, M-CSF. Chemokines selected from the group comprising IL-8, MCP-1 , receptors selected from the group Tissue factor and IL-2Ralpha, ii) decrease or inhibit the production bradykinin by the phase contact system, iii) decrease or inhibit the attractant potential for monocytes, and/or iv) decrease or inhibit the life-time of monocytes, neutrophils and other immune cells serving as an inducer of apoptosis, v) decrease or inhibit vascular endothelial cells to express the adhesion molecules, said adhesion molecules being preferably selected from the group comprising PECAM1 ICAM-1 , E-selectins, VCAM-1 vi) decrease or inhibit activation of the contact phase system to produce bra- dykinin leading to increased vascular permeability, vii) stimulate the synthesis of an anti-inflammatory mediator selected from the group of IL-10 and IL-12, . viii) inhibiting complement activation; ix) decreasing the risk of neural cell degeneration in the presence of chronic neuroinflammation, e.g. neurons which express glutamate N-methyl-D- aspartate receptors.
Additionally, the invention relates to a capability of a peptide sequence of the invention to stimulate expression of MT protein in vivo. This biological activity is among preferred biological activities of the peptide sequences of the invention as well.
3. Production of individual peptide sequences
Proteins and peptide sequences of the present invention may be prepared by a conventional synthetic method, recombinant DNA technology, enzymatic cleavage of full-length protein which a short peptide sequence is derived from, or any combination of said methods. MT protein comprised by the compound the composition of the invention is preferably prepared using any of the recombinant technologies and a short peptide sequence of the invention is preferably prepared using a method of chemical synthesis described below.
Recombinant preparation
Thus, in one embodiment the peptides of the invention are produced by use of recombinant 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 phosphoamidine method, oligonucleo- tides are synthesised, e.g. in an automatic DNA synthesiser, 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 I according to a standard protocol (Sambrook et al., Molecular cloning: A Laboratory manual. 2 rd ed., CSHL Press, Cold Spring Harbor, NY, 1989). The present invention relates to full-length proteins selected from the groups of proteins identified above. The DNA encoding the full-length proteins of the invention may al- ternatively be fragmented using specific restriction endonucleases. The fragments of DNA are further purified using standard procedures described in Sambrook et al., Molecular 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 origin, for instance obtained by preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of the full-length protein by hybridisation using synthetic oligonucleotide probes in accordance with standard techniques (cf. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989). The DNA sequence may also be prepared by polymerase chain reaction using specific primers, for instance as described in US 4,683,202 or Saiki et al., 1988, Science 239:487-491.
The DNA sequence is then inserted into a recombinant expression vector, which may be any vector, which may conveniently be subjected to recombinant DNA procedures. The choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which 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 activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of suitable promoters for directing the transcription of the coding DNA sequence in mammalian cells are the SV 40 promoter (Subramani et al., 1981, MoI. Cell Biol. 1 :854-864), the MT-1 (metallothionein gene) promoter (Palmiter et al., 1983, Science 222: 809-814) or the adenovirus 2 major late promoter. A suitable promoter for use in insect cells is the polyhedrin promoter (Vasu- vedan et al., 1992, FEBS Lett. 311:7-11). Suitable promoters for use in yeast host cells include promoters from yeast glycolytic genes (Hitzeman et al., 1980, J. Biol. Chem. 255:12073-12080; Alber and Kawasaki, 1982, J. MoI. Appl. Gen. 1: 419-434) or alcohol dehydrogenase genes (Young et al., 1982, in Genetic Engineering of Microorganisms for Chemicals, Hollaender et al, eds., Plenum Press, New York), or the TPH (US 4,599,311) or ADH2-4c (Russell et al., 1983, Nature 304:652-654) promoters. Suitable promoters for use in filamentous fungus host cells are, for instance, the ADH3 promoter (McKnight et al., 1985, EMBO J. 4:2093-2099) or the 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 fungal hosts) the TPU (Alber and Kawasaki, op. cit.) or ADH3 (McKnight et al., op. cit.) promoters. The vector may further comprise elements such as polyadenylation signals (e.g. from SV 40 or the adenovirus 5 EIb region), transcriptional enhancer sequences (e.g. the SV 40 enhancer) and translational enhancer sequences (e.g. the ones encoding adenovirus VA RNAs).
The recombinant expression vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question. An example of such a sequence (when the host cell is a mammalian cell) is the SV 40 origin of replication. The vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, such 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 DNA sequences coding the peptides or full-length proteins, the promoter and the terminator, respectively, and to insert them into suit- able vectors containing the information 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 the invention the coding DNA sequences may be usefully fused with a second peptide coding sequence and a protease cleavage site coding sequence, giving a DNA construct encoding the fusion protein, wherein the protease cleavage site coding sequence positioned between the HBP fragment and second peptide coding DNA, inserted into a recombinant expression vector, and expressed in recombinant host cells. In one embodiment, said second peptide se- lected from, but not limited by the group comprising glutathion-S-reductase, calf thymosin, bacterial thioredoxin or human ubiquitin natural or synthetic variants, or peptides thereof. In another embodiment, a peptide sequence comprising a protease cleavage site may be the Factor Xa, with the amino acid sequence IEGR, en- terokinase, with the amino acid sequence DDDDK, thrombin, with the amino acid sequence LVPR/GS, or Acharombacter lyticus, with the amino acid sequence XKX, cleavage site.
The host cell into which the expression vector is introduced may be any cell which is capable of expression of the peptides or full-length proteins, and is preferably a eu- karyotic cell, such as invertebrate (insect) cells or vertebrate cells, e.g. Xenopus laevis oocytes or mammalian cells, in particular insect and mammalian cells. Examples of suitable mammalian cell lines are the HEK293 (ATCC CRL-1573), COS (ATCC CRL-1650), BHK (ATCC 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 cells are described in e.g. Kaufman and Sharp, J. MoI. Biol. 159, 1982, pp. 601-621; Southern and Berg, 1982, J. MoI. Appl. Genet. 1:327- 341; Loyter et al., 1982, Proc. Natl. Acad. Sci. USA 79: 422-426; Wigler et al., 1978, Cell 14:725; Corsaro and Pearson, 1981 , in Somatic Cell Genetics 7, p. 603; Graham and van der Eb, 1973, Virol. 52:456; and Neumann et al., 1982, EMBO J. 1 :841-845.
Alternatively, fungal cells (including yeast cells) may be used as host cells. Examples of suitable yeast cells include cells of Saccharomyces spp. or Schizosaccharo- myces spp., in particular strains of Saccharomyces cerevisiae. Examples of other 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 for growing mammalian cells, such as a serum-containing or serum-free medium containing appropriate supplements, or a suitable medium for growing insect, yeast or fungal cells. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection).
The peptides or full-length proteins recombinantly produced by the cells may then be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, purification by a variety of chromatographic procedures, e.g. HPLC, ion exchange chromatography, affinity chromatography, or the like.
Synthetic preparation
Short peptide sequences of the invention are preferably prepared by chemical syn- thesis.
The methods for synthetic production of peptides are well known 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., Oxford University Press, 2002, or in: Pharmaceutical Formulation: Development of Peptides and Proteins, Frokjaer and Hovgaard eds., Taylor and Francis, 1999.
Peptides may for example be synthesised by using Fmoc chemistry and with Acm- protected cysteins. After purification by reversed phase HPLC, peptides may be further processed to obtain for example cyclic or C- or N-terminal modified isoforms. The methods for cyclization and terminal modification are well-known in the art and described in detail in the above-cited manuals. In a preferred embodiment the peptide sequences of the invention are produced synthetically, in particular, by the Sequence Assisted Peptide Synthesis (SAPS) method.
By SAPS peptides may be synthesised either batchwise in a polyethylene vessel equipped with a polypropylene filter for filtration or in the continuous-flow version of the polyamide 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- fluorenylmethyloxycarbonyl (Fmoc) or tert. -Butyloxycarbonyl, (Boc) as N-a-amino protecting group and suitable common protection groups for side-chain functionality.
When synthesised, individual peptide sequences may then be formulated as mul- timers using well-known in the art techniques, for examples dimers of the sequences may be obtained by the LPA method described in WO 00/18791 , denrimeric poly- mers by the MAP synthesis described in PCT/US90/02039.
4. Pharmaceutical composition
The invention also relates to a pharmaceutical composition comprising one or more of the compounds defined above or one or more of peptide sequence as defined above, wherein the compound/peptide sequence is preferably capable of stimulating neurite outgrowth and/or neural cell differentiation, survival of neural cells and/or stimulating learning and/or memory. Thus, the invention in one aspect concerns a pharmaceutical composition capable of stimulating differentiation of neuronal cells and/or stimulating regeneration of neuronal cells, and/or stimulating neuronal plas- ticity in connection with learning and memory, and/or stimulating survival of neural cells.
Another aspect of the invention relates to the use of a compound and/or peptide sequence as a medicament. The medicament is preferably for the treatment or pro- phylaxis of any of the diseases and conditions indicated below.
In the context of the invention the term "pharmaceutical composition" is used synonymously with the term "medicament".
In a pharmaceutical composition the peptide sequences of the invention may be formulated both as comprising isolated individual peptide fragments, multimers or dimers thereof.
A further aspect of the invention is a process of producing a pharmaceutical compo- sition, comprising mixing an effective amount of one or more of the compounds of the invention, or a pharmaceutical composition according to the invention with one or more pharmaceutically acceptable additives or carriers, and administer an effective amount of at least one of said compound, or said pharmaceutical composition to a subject.
In one embodiment of the process as mentioned above, the compounds may be used in combination with a prosthetic device, wherein the device is a prosthetic nerve guide. Thus, in a further aspect, the present invention relates to a prosthetic nerve guide, characterised in that it comprises one or more of the compounds or the pharmaceutical composition as defined above. Nerve guides are known in the art.
The pharmaceutical composition may have the described above effects on cells in vitro or in vivo, wherein the composition is administered to a subject.
The medicament of the invention comprises an effective amount of one or more of the compounds as defined above, or a composition as defined above in combination with the pharmaceutically acceptable additives. Such medicament may suitably be formulated for oral, percutaneous, intramuscular, intravenous, intracranial, intrathecal, intracerebroventricular, intranasal or pulmonal administration.
Strategies in formulation development of medicaments and compositions based on the compounds of the present invention generally correspond to formulation strategies 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 example, 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 preparation.
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, microcapsules, nanoparticles or the like.
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 formulations 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, sustained release formulations or powders and generally contain 10-95% of the active ingredient(s), preferably 25-70%.
Other formulations are such suitable for nasal and pulmonal administration, e.g. inhalators and aerosols.
The active compound may be formulated as neutral or salt forms. Pharmaceutically 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 example, 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 administered in a manner compatible with the dosage formulation, 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 and age of the subject, the disease to be treated and the stage of disease. Suitable dosage ranges are per kilo body weight normally of the order of several hundred μg active ingredient per administration with a preferred range of from about 0.1 μg to 5000 μg per kilo body weight. Using monomeric forms of the compounds, the suitable dos- ages are often in the range of from 0.1 μg to 5000 μg per kilo body weight, such as in the range of from about 0.1 μg to 3000 μg per kilo body weight, and especially in the range of from about 0.1 μg to 1000 μg per kilo body weight. Using multimeric forms of the compounds, the suitable dosages are often in the range of from 0.1 μg to 1000 μg per kilo body weight, such as in the range of from about 0.1 μg to 750 μg per kilo body weight, and especially in the range of from about 0.1 μg to 500 μg per kilo body weight such as in the range of from about 0.1 μg to 250 μg per kilo body weight. In particular when administering nasally smaller dosages are used 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 preferred.
Some of the compounds of the present invention are sufficiently active, but for some of the others, the effect will be enhanced if the preparation further comprises pharmaceutically acceptable additives and/or carriers. Such additives and carriers will be known in the art. In some cases, it will be advantageous to include a compound, which promotes delivery of the active substance to its target.
In many instances, it will be necessary to administrate the formulation multiple times. Administration may be a continuous infusion, 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 is administered within 8 hours from the factor onset, such as within 5 hours from the factor onset. Many of the compounds exhibit a long term effect whereby administration 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 in small portions based upon controlled release of the active compound(s). Furthermore, precursors may be used to control the rate 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.
As discussed above, the present invention relates to treatment of individuals for inducing differentiation, stimulating regeneration, plasticity and survival of neural cells in vitro or in vivo, said treatment involving administering an effective amount of one or more compounds as defined above.
Another strategy for administration is to implant or inject cells capable of expressing and secreting the compound in question. Thereby the compound may be produced at the location where it is going to act.
5. Treatment
A compound of the invention comprises MT or a fragment thereof and a peptide sequence which possesses biological activity associated with the capability of the peptide sequence to bind to FGFR and modulate FGFR activity is capable of penetrating the BBB
FGFRs and their ligands has been shown to be important determinants of neuronal survival both during development and during adulthood, in particular in the brain (Reuss and von Bohlen und Halbach (2003) Cell tissue Res, 313:139-57).Cell death plays a key role in normal neuronal development, where 50% of the developing neurons are eliminated through programmed cell death, and in the pathophysiology of neurodegenerative conditions, such as Alzheimer's and Parkinson's diseases. Therefore, a compound, which is capable to penetrate the BBB and promote neu- ronal cell survival by binding and activation FGFR in the brain is highly desirable. Thus, in one aspect the invention features compounds that promote survival of neural cells and can be used as medicaments for the treatment of conditions involving neural cell death. However, a biologically active peptide sequence of the invention both in the MT-bound and free form, i.e. non-MT bound, may also be used as a medicament for promotion of survival of another type of cells, e.g. different type of muscle cells, or, alternatively, for enhancing cell death of still another type of cells, e.g. cancer cells, as the FGFR signalling has been shown to be a survival factor for both muscle and cancer cells (Ozen et al. (2001) J Nat Cancer Inst. 93:1783-90; Miyamoto et al. (1998) J Cell Physiol. 177:58-67; Detilliux et al. (2003) Cardiovasc Res. 57:8-19). In these embodiments the invention in particular concerns the peptide sequences of SEQ ID NOs: 4, 8, 10, 16, 17, 26, 33, 35, 39 and 41.
Another approach in the strategy aimed to achieve a compensation for functional cell loss is to create a new pool of said functional cells, for example by committing the progenitor (stem) cells to differentiate to a new population of differentiated cells, or to initiate regenerating processes in damaged cells. FGFRs play an important role in the mechanisms triggering differentiation of a variety of progenitor cell types (Es- warakumar et al. (2005) Cytokine Growth Factor Rev. 16(2): 139-49), cancer cells (St Bernard et al. (2005) Endocrinology 146(3): 1145-53) and neural cells (Sapieha et al. (2003) MoI Cell Neurosci. 24(3):656-72).
Activity of cell-surface receptors is strictly regulated in a healthy organism. Mutations, 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 maintenance of various cellular processes. The latter is the manifestation of a disease. It has also been shown that attenuation of FGFR signalling leads to development of a number of different of pathologic condi- tions, e.g. diabetes (Hart et al., Nature 2000, 408:864-8). Activation of FGF receptors is involved as in normal, as in pathologic angiogenesis (Slavin, Cell Biol lnt 1995, 19:431-44). It is important for development, proliferation, functioning 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 struc- ture (Cancilla et al., Kidney lnt 2001 , 60:147-55), and it is implicated in mound healing and cancer disease (Powers et al., Endocr Relat Cancer. 2000, 7:165-97).
Activity of FGFRs has also be reported to be important for stimulation and mainte- nance of neural plasticity associated with memory and learning (Reuss et al. (2003) Cell Tissue Res. 313(2):139-57; Oomura et al. (1996) Ann N Y Acad Sci. 786:337- 47).
The present invention provides compounds capable of modulating the activity of FGFRs, in particular stimulating activity of FGFRs. Consequently, said compounds are concerned by the invention for the production of a medicament for the treatment of diseases, wherein stimulating biological activity dependent on the activity of FGFRs is considered to be beneficial for treatment.
Thus, the medicament comprising or consisting of a biological sequence of the invention, such as SEQ ID NOs: 4, 8, 10, 16, 17, 26, 33, 35, 39 or 41 , and or a compound comprising MT and at least one of the short peptide sequences described above, may also used for prevention and/or treatment of
1 ) diseases and conditions of the central and peripheral nervous system, or of the 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 myelination of nerve fibers, postischaemic damage, e.g. resulting from a stroke, Parkinson'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; for treatment of diseases or conditions of the muscles including conditions with impaired function of neuro-muscular connections, such as after organ transplantation, or such as ge- netic 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 I and II, of the kidney such as nephrosis and of the heart, liver and bowel, and/or
3) postoperative nerve damage, traumatic nerve damage, impaired myelination of nerve fibers, postischaemic, e.g. resulting from a stroke, Parkinson'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 as manic depression, and/or 4) cancer disease, and/or 5) prion diseases.
The invention concerns the cancer being any type of solid tumors requiring neoan- giogenesis. Cancers of neural system are of particular interest of the invention.
The invention concerns prion diseases selected from the group consisting of scrapie, Creutzfeldt-Jakob disease. It has been shown that FGFRs plays a distinct role in prion diseases (Castelnau et al. (1994) Exp Neurobiol. 130:407-10; Ye and Carp (2002) J MoI Neurosci. 18:179-88).
In another embodiment a compound of the invention and/or peptide sequence of the invention, such as SEQ ID NOs: 4, 8, 10, 16, 17, 26, 33, 35, 39 and 41 , may be used for the manufacture of a medicament for 1 ) promotion of wound-healing, and/or 2) prevention of cell death of heart muscle cells, such as after acute myocardial infarction, or after angiogenesis, and/or
3) revascularsation, and/or
4) stimulation of the ability to learn and/or of the short and/or long-term memory.
In still another embodiments a compound of the invention may be used for the manufacture of a medicament for
1 ) prevention of cell death due to ischemia;
2) prevention of body damages due to alcohol consumption;
A peptide sequence of the invention has a capability to induce and/or stimulate the expression of MT when it is administered in vivo. Thus, it is another aspect of the invention to provide a medicament for treatment of a MT associated disease or condition wherein the medicament comprises a short peptide sequence described above. In particular, the invention relates to a medicament comprising SEQ ID NO:42. Brain disorders like traumatic injury, pellagra dementia/toxicity; epilepsy, brain ischemia/stroke; EAE/MS (multiple sclerosis); and infectious encephalopathies; Amyotrophic Lateral Sclerosis, Parkinson's disease; peripheral nerve injury, cerebral malaria, ageing /age dementia, neuromuscular damage and diabetes, all, were associated with activity of MT. The MT roles were partially or fully validated in human tissue or human patients during degenerative diseases such as AD, Pick's disease and ALS; and during MS, Binswanger's encephalopathy, and ischaemia and during neuromuscular damage and diabetes, and also during a number of common auto- immune, inflammatory and allergic diseases.
Thus, the present invention relates to the above described peptides, fragments, or variants thereof, and compounds comprising at least one of said sequences and MT as medicaments for treatment of diseases wherein their capability of modulating - stem cell differentiation, for example stimulating neuronal precursor or
- cancer cells differentiation;
- neural cell differentiation and/or regeneration of nerves, for example stimulating- neurite outgrowth;
- neural plasticity associated with memory and learning, for example stimulating synaptic efficacy;
- cell survival, in particular stimulating survival of neuronal and/or glial cells, for example inhibiting of apotosis of neural cells and/or glial, or stimulating apoptosis of cancer cells,
- the oxidative stress response, for example stimulating expression of scavenges of reactive oxygen species
- activation of astrogliosis, such as stimulating astrocytes to express neuroprotective growth factors and proteins such as e.g. BDNF, NT-3, GDNF, neurturin, artemin, NGF, variety of fibroblast growth factors (FGFs), S100-proteins (S100A4, S100A6, S100A10, S100A12, S100B), IGF-2, neuregulin; - inflammation, for example inhibitng activation of microglia and macrophages, inhibiting expression of pro-inflammatory cytokines and/or stimulating anti-inflammatory responses;
- angiogenesis in the lesioned area, for example by stimulating expression of growth factor promoting angiogenesis such as VEGF and FGF2; - cell-cell and/or cell-extracellular matrix adhesion, for example modulating neural or cancer cell adhesion to cellular environment and/or extracellular matrix;
- morphological plasticity of cells, for example stimulating neuronal plasticity associated with learning and memory; - the effects related to FGFR ligands, for example stimulating or inhibiting these effects may be essential for the 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 which are resident cells or implanted or transplanted cells.
Thus, the treatment comprises treatment and/or prophylaxis of cell damage and/or cell death in relation to diseases or conditions of the central and peripheral nervous system, such as postoperative nerve damage, traumatic neuron damage, e.g. resulting from spinal cord injury, impaired myelination of nerve fibers, postischaemic damage, e.g. resulting from a stroke, multiinfarct dementia, multiple sclerosis, neuronal degeneration associated with diabetes mellitus, neuro-muscular degeneration, schizophrenia, Alzheimer'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 atrophic muscle disorders; or for the treatment of diseases or conditions of various or- gans, such as degenerative conditions of the gonads, of the pancreas, such as diabetes mellitus type I and II, of the kidney, such as nephrosis the compounds according to the invention may be used for inducing differentiation, modulating proliferation, stimulate regeneration, neuronal plasticity and survival , i.e. stimulating survival.
In yet a further embodiment the compound of the invention and/or pharmaceutical composition may be beneficially used for the stimulation of the ability to learn and/or of the short and/or long term memory.
In particular the compound and/or pharmaceutical composition of the invention may be used in the treatment of clinical conditions, such as psychoses, such as senile and presenile organic psychotic conditions, alcoholic psychoses, drug psychoses, transient organic psychotic conditions, Alzheimer's disease, cerebral lipidoses, epilepsy, general paresis [syphilis], hepatolenticular degeneration, Huntington's chorea, Jakob-Creutzfeldt disease, multiple sclerosis, Pick's disease of the brain, polyarteriti nodosa, syphilis, schizophrenic disorders, affective psychoses, neurotic disorders, personality disorders, including character neurosis, nonpsychotic personality disorder associated with organic brain syndromes, paranoid personality disorder, fanatic personality, paranoid personality (disorder), paranoid traits, sexual deviations and disorders or dysfunctions, sleep disorders, depression and other mood disorders including manic or bipolar disorders, mental retardation, inherited or in relation with disease or trauma, disease in the nerve system and sense organs, cognitive anomalies, inflammatory disease of the central nervous system, such as meningitis, encephalitis, cerebral degenerations such as Alzheimer's disease, Pick's disease, senile degeneration of brain, communicating hydrocephalus, obstructive hydrocepha- lus, Parkinson's disease including other extra pyramidal disease and abnormal movement disorders, spinocerebellar disease, cerebellar ataxia, Marie's, Sanger- Brown, Dyssynergia cerebellaris myoclonica, primary cerebellar degeneration, such as spinal muscular atrophy, familial, juvenile, adult spinal muscular atrophy, motor neuron disease, amyotrophic lateral sclerosis, motor neuron disease, progressive bulbar palsy, pseudobulbar palsy, primary lateral sclerosis, other anterior horn cell diseases, anterior horn cell disease, unspecified, other diseases of spinal cord, syringomyelia and syringobulbia, vascular myelopathies, acute infarction 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, myelopathy, drug-induced, radiation-induced myelitis, disorders of the autonomic nervous system, disorders of peripheral autonomic, sympathetic, parasympathetic, or vegetative system, familial dysautonomia [Riley- Day syndrome], idiopathic peripheral autonomic neuropathy, carotid sinus syncope or syndrome, cervical sympathetic dystrophy or paralysis; peripheral autonomic neu- ropathy in disorders classified elsewhere, amyloidosis, diseases of the peripheral nerve system, brachial plexus lesions, cervical rib syndrome, costoclavicular syndrome, scalenus anterior syndrome, thoracic outlet syndrome, brachial neuritis or radiculitis, including in newborn. Inflammatory and toxic neuropathy, including acute infective polyneuritis, Guillain-Barre syndrome, Postinfectious polyneuritis, polyneu- ropathy in collagen vascular disease, disorders affecting multiple structures of eye, purulent endophthalmitis, diseases of the ear and mastoid process, abnormality of organs and soft tissues in newborn, including in the nerve system, complications of the administration of anesthetic or other sedation in labor and delivery, diseases in the skin including infection, insufficient circulation problem, injuries, including after surgery, crushing injury, burns, atrophic dermatitis, psoriasis. Injuries to nerves and spinal cord, including division of nerve, lesion in continuity (with or without open wound), traumatic neuroma (with or without open wound), traumatic transient paralysis (with or without open wound), accidental puncture or laceration during medical procedure, injury to optic nerve and pathways, optic nerve injury, second cranial nerve, injury to optic chiasm, injury to optic pathways, injury to visual cortex, unspecified, poisoning by drugs, medicinal and biological substances, genetic or traumatic atrophic muscle disorders; 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 I and II, of the kidney, such as nephrosis, metabolic disorders, such as obscenity lipid disorders, diabetes type I and II, diseases of endocrine glands, such as diabetes mellitus I and II, pituitary gland tumour, disorders of amino acid transport and metabolism, disorders of purine and pyrimidine metabolism and gout, bone disorders, such as fracture, osteoporosis, osteo arthritis (OA), obesity, stem cell protection or maturation in vivo or in vitro, autoimmune disorders, such as rheumatoid arthritis, SLE, ALS, and MS, chronic rheumatic heart disease, ischaemic heart disease, arrhythmia, asthma and other allergic reactions, cancer in various organs, such as neoplasms, such as malignant neoplasms, benign neoplasms, carcinoma in situ, and neoplasms of uncertain behaviour, more specifically cancer in breast, thyroidal, pancreas, brain, lung, kidney, prostate, liver, heart, skin, blood organ, (incl. but not limited to CML and AML), muscles (sarcoma), cancers with dysfunction and/or over- or under-expression of specific receptors and/or expression of mutated receptors or associated with soluble receptors, such as but not limited to Erb-receptors and FGF-receptors.
Inflammation of the brain is often consequence of infection, autoimmune processes, toxins, and other conditions. Viral infections are a relatively frequent cause of this condition. Encephalitis may occur as primary or secondary manifestation of TOGAVIRIDAE INFECTIONS; HERPESVIRIDAE INFECTIONS; ADENOVIRIDAE INFECTIONS; FLAVIVIRIDAE INFECTIONS; BUNYAVIRIDAE INFECTIONS; PICORNAVIRIDAE INFECTIONS; PARAMYXOVIRIDAE INFECTIONS; ORTHOMYXOVIRIDAE INFECTIONS; RETROVIRIDAE INFECTIONS; and ARENAVIRIDAE INFECTIONS.
Accordingly, a peptide, compound or a pharmaceutical composition of the invention may be used for treatment inflammation in the brain, e.g. inflammation associated with a viral infection.
A large body of clinical and experimental data indicate that complement activation is an important mechanism for neuronal and glial injury in Guillain-Barre syndromes. Inhibition of complement activation therefore might be expected to limit the progression of the disease (Halstead et al.(2005) Annals of Neurology 58:203-21 ).
Thus, in another embodiment, a peptide sequence, a compound and pharmaceutical composition may be used for treatment of Guillain-Barre syndrome, its variant forms, such as Miller Fisher syndrome, and other complement dependent neuromuscular disorders.
Peptide sequences, compounds and pharmaceutical compositions of the invention may also be used for treatment children with autism.
Autism is a brain disorder that begins in early childhood and persists throughout adulthood; affects three crucial areas of development: communication, social interaction, and creative or imaginative play. It is estimated to afflict between 2 and 5 of every 1000 children and is four times more likely to strike boys than girls. Children with autism have difficulties in social interaction and communication and may show repetitive behaviour and have unusual attachments to objects or routines.
In recent years, there have been scientific hints of immune system irregularities in children with autism.
A further aspect the invention relates to a method of treating a disease or condition as discussed above comprising administering a compound or peptide sequence of the invention, or pharmaceutical composition comprising thereof to a subject in need. Examples Peptides
FGF beta10-beta11 loop region derived peptide sequences (dekafins)
Dekai WFVGLKKNGSCKRG (SEQ ID NO 1) derived from FGF1
Deka2 WYVALKRTGQYKLG (SEQ ID NO 2) derived from FGF2
Deka3 WYVSVN G KG RP RRG (SEQ ID NO 3) derived from FG F3
Deka4 M FIALSKNG KTKKG (SEQ ID NO: 4) derived from FGF4
Dekaδ WYVALN KRG KAKRG (SEQ ID NO: 5) derived from FGF5
Deka6 TYIALS KYG RVKRG (SEQ ID NO: 6) derived from FGF6
Deka7 MFVALNQKGIPVRG (SEQ ID NO 7) derived from FG F7
Dekaδ WYMAFTRKG RPRKG (SEQ ID NO: 8) derived from FG F8
Deka9 YYVALNKDGTPREG (SEQ ID NO: 9) derived from FGF9
DekalO MYVALNGKGAPRRG (SEQ ID NO: 10) derived from FGF10
Deka11 WYLGLDKEGQVMKG (SEQ ID NO: 11) derived from FGF11
Deka12 WFLGLNKEGQIMKG (SEQ ID NO: 12) derived from FGF12
Deka13 WYLGLNKEGEIMKG (SEQ ID NO: 13) derived from FGF13
Deka14 WFLGLNKEGQAMKG (SEQ ID NO: 14) derived from FGF14
Deka16 YYVALNKDGSPREG (SEQ ID NO: 15) derived from FGF16
Deka17 WFMAFTRQGRPRQA (SEQ ID NO: 16) derived from FGF17
Dekaiδ WYVGFTKKGRPRKG (SEQ ID NO: 17) derived from FGF18
Deka20 YFVALNKDGTPRDG (SEQ ID NO: 18) derived from FGF20
FGF beta6-beta7 loop region derived peptide sequences (dyofins)
Dyo1 TGQYLAMDTDGLLYGS SEQ ID NO: 19) derived from FGF1
Dyo2 ANRYLAMKEDGRLLAS (EQ ID NO:20) derived from FGF2
Dyo3 SGRYLAMNKRGRLYAS (SEQ ID NO:21 ) derived from FGF3
Dyo4 SRFFVAMSSKGKLYGS (SEQ ID NO:22) derived from FGF4
Dyo5 SNKFLAMSKKGKLHAS (SEQ ID NO:23) derived from FGF5
Dyo6 SALFVAMNSKGRLYAT (SEQ ID NO:24) derived from FGF6
Dyo7 SEFYLAMNKEGKLYAK (SEQ ID NO:25) derived from FGF7
Dyoδ TGLYICMNKKGKLIAK SEQ ID NO:26) derived from FGF8
Dyo9 SGLYLGMNEKGELYGS (SEQ ID NO:27) derived from FGF9
Dyo10 SNYYLAMNKKGKLYGS (SEQ ID NO:28) derived from FGF10
Dyo11 LGHYMAMNAEGLLYSS SEQ ID NO:29 derived from FGF11
Dyo12 ASLYVAMNGEGYLYSS (SEQ ID NO:30) derived from FGF12
Dyo13 TKLYLAMNSEGYLYTS (SEQ ID NO:31 ) derived from FGF13
Dyo14 TGLYIAMNGEGYLYPS (SEQ ID NO:32) derived from FGF14
Dyo15 SVRYLCMSADGKIYGL (SEQ ID NO:33) derived from FGF15
Dyo16 SGLYLGMNERGELYGS (SEQ ID NO:34) derived from FGF16
Dyo17 SEKYICMNKRGKLIGK SEQ ID NO:35) derived from FGF17
Dyo18 TEFYLCMNRKGKLVGK (SEQ ID NO:36) derived from FGF18
Dyo19 SVRYLCMGADGKMQGL (SEQ ID NO:37) derived from FGF19
Dyo20 SGLYLGMNDKGELYGS SEQ ID NO:38) derived from FGF20
Dyo21 TSRFLCQRPDGALYGS (SEQ ID NO:39) derived from FGF21
Dyo22 SGFYVAMNRRGRLYGS (SEQ ID NO:40) derived from FGF22
Dyo23 SRRYLCMDFRGNIFGS SEQ ID NO:41 ) derived from FGF23
NCAM Fn 3, 1 and 2 modules derived peptide sequences: FGL EVYWAENQQGKSKA (SEQ ID NO:42)
ABL SIDRVEPYSSTAQVQFD (SEQ ID NO:43)
EFL TIMGLKPETRYAVR (SEQ ID NO:44) CDL KAEWKSLGEEAWHSK (SEQ ID NO: 45),
All peptides were purchased from Schafer-N (Copenhagen, Denmark). The peptides were synthesized as tetrameric dendrimers composed of four monomers coupled to a lysine backbone, purified by gel-filtration using SephadexTM G-10 (Amersham Bioscience, Sweden), and dissolved in sterile distilled water. Concentration was determined by spectrophotometry at the absorbance at 205 nm. The FGFR inhibitor SU5402 was from Calbiochem (Bad Soden, Germany)
Methods 1. In vitro studies
1.1. Surface Plasmon Resonance (SPRl
Analysis of binding was performed employing a BIAcoreX instrument (Biosensor AB, Uppsala, Sweden) at 25°C using 10 mM pH 7.4 sodium phosphate containing 150 mM NaCI as running buffer (phosphate-buffered saline, PBS). The flow-rate was 5 μl/min. Data were analysed by non-linear curve-fitting using the manufacturer's software. The combined FGFR Ig modules 2 and 3 of FGFR1 , splice variant INc (prepared as described by (Kiselyov V, et al. Structure (Camb) 2003, 11 :691-701.), were immobilized on a sensor chip, CM5, using an amine coupling kit (Biosensor AB) as follows: the chip was activated by 20 μl activation solution; the protein was immobilized using 12 μl 20 μg/ml protein in 10 mM sodium phosphate buffer pH 6.0; the chip was blocked by 35 μl blocking solution. Various peptides at the indicated concentrations were injected into the sensor chip. The curve corresponding to the difference between binding to FGFR1 and a blank chip was used for analysis.
1.2. Phosphorylation of FGFR1
Trex293 cells (Invitrogen, Taastrup, Denmark) were stably transfected with human FGFRIc, splice variant HIc, with a C-terminal Strep Il tag (IBA Biotech, Gδttingen, Germany). The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with 200μg/ml hygromycin (Invitrogen), 10% fetal calf serum (FCS), 1 % (v/v) glutamax, 100 U/ml penicillin, 100μg/ml streptomycin (all from Gibco BRL, Paisley, UK). For determination of phosphorylation, 2x1 O^ cells were starved overnight in medium without serum. After treatment with peptides or FGF2 (R&D Sys- terns Europe, Abingdon, UK) for 20 min, the cells were lysed by 300 μl lysis buffer containing 1% (v/v) NP-40 (Sigma-Aldrich, Copenhagen, Denmark), complete protease inhibitors (Roche, Mannheim, Germany) (1 :50), phosphatase inhibitors (CaI- biochem inhibitor cocktail III) (1 :100) in PBS. Then protein concentration was deter- mined using the bicinchoninic acid assay (Pierce, Rockford, IL, USA). 500μg protein from each lysate was incubated with 15 μl agarose-coupled anti-phosphotyrosine antibodies (4G10-AC) (Upstate Biotechnologies, Lake Placid, NY, USA) for 6 hr at 4 0C. The bound proteins were washed and eluted with 180 mM phenylphosphate (Sigma-Aldrich). Purified proteins (25 μl from each sample) were separated by SDS- PAGE and transferred to a polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA), lmmunoblotting was performed using rabbit antibodies (diluted 1 :2000) against the recombinant Strepll tag (IBA Biotech) and swine anti-rabbit IgG horseradish peroxidase conjugate (diluted 1 :2000) (DakoCytomation, Glostrup, Denmark) in 5% (w/v) nonfat dry milk. The immune complexes were developed by SuperSig- nal® West Dura extended duration substrate (Pierce, Rockford, IL, USA), and visualized and quantified using the SynGene Gene Tool image analysis software (Synoptics , Cambridge, UK).
1.3. Primary cell culture: cerebellar granule neurons (CGNs)
CGN were prepared from 7 days old Wistar rats (Charles River, Sulzfeld, Germany or Møllegaard, Ejby, Denmark) essentially as previously described by (Schousboe I, Larsson OM, lnt J Dev Neurosci. 1989;7(1 ):115-21 ). Briefly, the cerebella were cleared of meninges and blood vessels, roughly homogenized by chopping and thereafter trypsinized. The neurons were washed in the presence of DNAse 1 and soybean trypsin inhibitor (Sigma-Aldrich), and cellular debris was pelleted by cen- trifugation. For neurite-outgrowth assays, CGN cultures were plated on uncoated eight-well Lab-Tek® chamber slides (Nunc, Roskilde, Denmark) in Neurobasal-A medium (Gibco BRL) supplemented with 0.4%(w/v) bovine serum albumin (BSA), 2% (v/v) B27, 0.5% (v/v) glutamax, 100 U/mL penicillin and 100 μg/mL streptomycin at a density of 10,000 cells/well. Peptides or FGF2 were added to the medium immediately after plating and cells were maintained at 37°C, 5% CO2 for 24 h.
1.4. lmmunocytochemistry Cell cultures were fixed in 4% (w/v) paraformaldehyde followed by blocking with 1%(w/v) BSA and then incubateded with polyclonal rabbit antibodies against rat GAP-43 (Chemicon, AH Diagnostics, Aarhus, Denmark) (1 :1000 dilution with 1 % BSA) followed by incubation with secondary Alexa Fluor®488 goat anti-rabbit anti- bodies (Molecular Probes, Eugene, Oregon, USA) (1:700 dilution with 1 % BSA).
1.4. Evaluation of neurite-outgrowth
Recording was done by computer-assisted microscopy on a Nikon Diaphot inverted microscope (Tokyo, Japan) equipped with an epifluorescence attachment and a Nikon Plan 2Ox objective. Images were grabbed with a CCD video camera (Grundig Electronics, Germany) using the software package "Prima" developed at the Protein Laboratory (University of Copenhagen, Denmark) and stored as 768x576 pixel, eight-bit, gray scale GIF-images. The length of neuronal processes per cell was estimated on the basis of a stereological approach using a software package "Process length" developed at the Protein Laboratory (Rønn et al. Nature Biotech. 1999, 17:1000-1005.). For estimation of neurite outgrowth 200±20 neurons were processed in each individual experiment.
1.5. Cell survival assay in vitro Primary CGN cultures from seven days old rats were plated at a density of 100,000 cells/cm2 in eight-well permanox chamber slides coated with poly-D-Lysine and grown in a medium supplemented with 40 mM KCI. After 24 hours cytosine-β-D- arabinofuranoside was added to the culture to inhibit proliferation of non-neuronal cells. Neurons were left to differentiate for further 6 days in vitro, before apoptosis was induced by shifting cells into a starving medium containing either 5 mM KCI alone (negative control), 4OmM KCI (positive control) or 5 mM KCI plus various concentrations of peptide. 48 hours after apoptosis induction cells were fixated and stained with Hoechst 33258. Neuronal viability was estimated by comparing the amount of live neurons with the total number of neurons based on nuclear morphol- ogy.
1.6. TUNEL assay (DNA fragmentation)
Primary cultures of seven days old rats were plated, cultured and induced to undergo apoptosis as described above for neuronal survival, however cells were fix- ated 24 hour after apoptosis induction. By using the Fluorescien FragEL DNA frag- mentation Kit, which label free DNA ends with a green fluorescent colour, the amount of neurons undergoing DNA fragmentation can be estimated and compared to the total amount of cells, which were stained with propidium idodide.
2. In vivo studies
2.1. Administration of peptides in vivo
For suboccipital intracistemal administration, the neck of the rat was maximally flexed in the atlanto-occipital coupling, and 5.0 μl peptide solution (1.2 μg/μl) or vehicle (PBS, 0.5% BSA) were administered by percutaneous injection into the cis- terna magna with a G25 needle connected with a syringe through a calibrated polyethylene tube. For intranasal administration, 25 μl peptide solution (8 μg/μl) or vehicle (sterile water) was administered through each nostril. Animals exposed to intracistemal or intranasal administration were anesthetisized with 3% halothane in 30% O2/70% N2O. Subcutaneous (s.c.) administration was performed by a 2 ml/kg (10.8 mg/kg) injection without anesthesia.
2.2. Cell survival in vivo
A focal brain injury on the right fronto-parietal cortex was made by applying a piece of dry-ice (-780C) directly onto the skull for 30 seconds in mice and 60 seconds in rats, as previously described in detail (Penkowa M, Moos T. GHa. 1995 Mar; 13(3):217-27.). The rats were treated s.c. with the tetrameric form of the FGL peptide one day before lesion and one and two days after the lesion (10 mg/kg bodyweight/ injection). Three days after the lesion animals were fixed by transcardial perfusion with paraformaldehyde. Histochemistry and immunohistochemistry (IHC) were performed on sections cut from organs taken from fixated animals. For immu- nohistochemical investigation, brains were dissected and postfixed in Zamboni's fixative for 2-3 hours, dehydraded in graded alcohol followed by xylol and subsequently embedded in paraffin before being cut in 3 μm frontal sections throughout the entire area of the lesion. Terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick end labeling (TUNEL) was performed using the Fragment End Labeling (FragEL™) Detection Kit (Calbiochem, USA, code QIA33). The FragEL kit contains all the materials used below and each step was performed according to the manufacturer's recommendations. Sections were also immunostained for markers of oxidative stress such as peroxynitrite-induced nitra- tion of tyrosine residues (NITT) and malondialdehyde (MDA) andfor markers of in- flammation such as interleukin (IL)-I, IL-12 and tumor necrosis factor (TNF) α, as described by Penkowa M, et al. (Glia. 2000 Dec;32(3):271-85).
2.3. Social recognition test Male Wistar rats (Charles River) were housed individually for one week, the first 6 days in their home cage and the last day in the test cage. On the day of testing, a three-week-old male rat was introduced into the test cage of the adult test rat (250 g) for 4 min and then removed. After a period of 120 min, the same juvenile rat was re-introduced. During the meeting, the explorative behavior of the adult towards the juvenile was recorded. The recognition ratio was calculated as T2/(T1+T2), T1 and T2 being the time spent on investigating the juvenile animal during the first and the second encounter, respectively (Kogan JH, et al. Hippocampus 2000, 10: 47-56).
2.4. Fear conditioning test Statistics Statistical evaluation of behavioral tests was performed using the paired t-test, employing the commercially available software package Fig-P, version 2.98 (Biosoft, Cambridge, UK). The results are given as mean values ± SEM from at least three independent experiments.
2.5. Alzheimer's disease model Animals
Male Wistar rats (-300 g) were housed, two in each cage, with free access to food and water. Juvenile male Wistar rats (3 weeks old, 40-50 g) were housed in groups of six. Animals were kept in a regulated environment (23 0C, 50% humidity, 12-h light/dark cycle), and experiments were carried out in the light phase (10 a.m.-2 p.m.).
Intracerebroventricular, Lev., Administration of the Aβ25.35 Peptide Aggregates of Aβ25-35 (Bachem AG) were prepared by incubating the peptides at a concentration of 3 μg/μl in sterile water for 4 days at 37 0C. Formation of aggregates were confirmed visually by microscope inspection. Subsequently, 5 μl of aggregated Aβ25-35 were injected i.c.v. (1.2 μl/min) with a 10-μl Hamilton syringe using the following coordinates: 0.8 mm posterior to bregma; 1.5 mm lateral to the sagittal suture; 3.8 mm beneath the surface of the brain. Sham operated rats received the same volume of sterile distilled water. Correct i.c.v. injection was confirmed by histological examination of the brain. The procedure was performed under anesthesia (Hyp- norm/Midazolam; 0.3 ml/100 g animal, Lp.; i.e. 23.6 μg fentanyl, 0.75 mg fluanisone, 375 μg midazolam/100 g aminal).
Social Recognition Test
Before tests involving Aβ-treatment, animals were housed individually for 7 days; the first 6 days in their home cage, the last day in the test cage. On the day of testing, a 3-week-old male Wistar rat was introduced into the test cage of the adult test rat for 4 minutes. After a period of 30 minutes, the same juvenile rat was re-introduced or, alternatively, as a control, an unfamiliar juvenile was introduced. During the meetings, the investigating behavior of the adult towards the juvenile (licking, sniffing, chewing the fur of the juvenile) was recorded. The Social Recognition Ratio was calculated as T2Z(T^T2), T1 and T2 being the time spent on investigating the juvenile animal during the first and the second encounter, respectively.
A recognition ratio of 0.50 indicates that there was no difference between the first and second meeting indicating that there at the second meeting was no memory of the first meeting. A decrease in investigation time during the second meeting might be the result of a nonspecific (not memory-connected) FGL-induced reduction of investigatory activity. This possibility was assessed by presenting a novel juvenile to FGL-treated rats during the second meeting.
For tests involving scopolamine-treatment, FGL2 (FGL2 is a dimeric form of the FGL peptide described in WO2005014623) (8 mg/kg) and scopolamine (0.01mg/kg; Sigma) were both given s.c. 24 hours and 30 min prior to the initial trial, respectively. A juvenile rat was introduced into the test cage of the adult rat for 2 min during the first and second trial, with an intertrial interval of 15 minutes. Animals demonstrating aggressive behavior were excluded.
Tissue preparation Animals were terminated by Lp. injection of an over-dose of pentobarbital (200 mg/kg) and perfused transcardially with PBS followed by 4% formaldehyde in PBS.
The coronal segment of the brain from -0,92 to -4,8 mm relative to the bregma was post-fixed for 24 hours and then cryoprotected in phosphate-buffered 30% sucrose.
Starting at a random position, segments were systematically cut in a number of se- ries each constituting of one 80-μm thick section followed by four 40-μm thick sec- tions. The 80-μm sections were used for histological analysis; 40-μm sections were used for immunohistochemistry.
Histology The 80-μm sections were stained with 0.5% cresyl violet (Nissl) for routine histological examination and stereological evaluations. The total number of neurons and the volume of the examined structures (the neocortex, including the anterior and posterior cingulate cortex and the motor cortex areas (bregma -0,92 to -2,80), and the CA3 area of the dorsal hippocampus (from -1.6 to -4.52), were measured by exam- ining an average of eight sections per structure.
Stereology
The volume of the macroscopic brain regions was estimated using Cavalieri's principle: V(ref) = t - k - a(p) • ]TP , where V(reή is the total volume of the structure, t is the average section thickness, k is a constant, a{p) is the area per point on the counting grid, and ∑P is the total number of points hitting the structure of interest.
Estimations of total numbers were obtained using optical disectors. The concept "disector" is an imaginary rectangular box applied to the microscope slide. The the height of the disector is employed by using 80-μm-thick sections in which the plane of focus is moved up or down (48) and the x- and y-axis are defined by a square (the counting frame) superimposed on the magnified digital image of the tissue. The approximate number of particles counted in a disector, ∑Q~ , the height, h, of the disector, and the area of the counting frame, α(frαme) , are parameters defined by the investigator. The volume of the disector, v(dis) , is given as: v(dis) = h • α(frαme) . The total number of particles, N(pαrt) , in a specimen of a
given volume, is: N(pαrt) . Cell counting was performed
Figure imgf000060_0001
using a BX-50 Olympus microscope equipped with a 100X oil-immersion objective (NA = 1.35), a motorized stage, and an electronic Heidenhain microcator with digital readout for measuring movements in the z-direction with a precision of 0.5 μm. Op- tical disectors were superimposed onto a color monitor at a final magnification of 3000X using the CAST-GRID software (Olympus, Denmark). Per brain, an average of 107 disector probes (82-140) were sampled with an average number of 250 neu- rons (196 - 305) counted per neocortex and an average of 280 neurons (210 - 363) per CA3. The coefficient of error (CE = SEM/mean) of the individual estimates was 6-7%.
lmmunohistochemistry
40-μm tissue sections were stained with rabbit polyclonal antibodies against Aβi-4o (Alfa Diagnostic Int.), Ap37-42, and ApI-17 (Chemicon Int.) for detection of amyloid deposits, and GFAP (DAKO Cytomation) for detection of astrocytes, or with monoclonal antibodies against phospho~t.au, (clone AT-8, Innogenetics) or CD11b (Sero- tec) for detection of microglia, lmmunohistochemistry was performed according to standard procedures. In brief, sections were pre-treated with 3% H2O2, and incubated with a primary antibody. To enhance the detection of Aβ, sections were also pre-treated with 70% formic acid. Visualization of antigens was performed by the streptavidin-biotin-peroxidase method with diaminobenzidine as the chromogen ac- cording to the manufacturer's instructions (DAKO Cytomation).
Immunoreactivities were quantified on the basis of average brightness using the image analysis software PrAverB (Protein Laboratory, University of Copenhagen). For each staining the amount of antigen was defined as the percentage of area oc- cupied by positive immunoreactivity.
Western blotting
For evaluation of the expression and phosphorylation of GSK3β, brain tissues were placed in Eppendorf tubes and frozen in liquid nitrogen. Samples were homogenized in lysis buffer (100 mM Tris-HCI, pH 6.8, 5% SDS, 20% glycerol including protease inhibitors (Complete Protease Inhibitor Cocktail Tablets; Roche Diagnostics) and phosphatase inhibitors (Phosphatase Inhibitor Cocktail Set II; Calbiochem)), homogenized by sonication, and precleaned by centrifugation. Protein concentrations were determined using the Pierce BCA assay (Pierce Biotech. Inc.) according to the manufacturer's instructions. Subsequently, samples were resolved on a 12.5% SDS- PAGE gel and transferred to a PVDF membrane. Total GSK3β was detected with a monoclonal antibody (clone GSK-3B, Sigma). GSK3β phosphorylated on Ser9 and Tyr216 was detected using polyclonal rabbit antibodies (Oncogene Research Products and Sigma, Respectively). For confirmation of correct loading, blots were stripped and reprobed with antibodies against actin (Sigma). Primary antibodies were detected with HRP-conjugated goat anti-mouse or goat anti-rabbit antibodies (DAKO Cytomation), and the blots were visualized with the ECL detection kit (Amer- sham Biosciences) using the chemiluminiscence capture and analysis system Gene Gnome (Syngene). Quantifications were performed using the GeneSnap software (Syngene).
Statistics
All data are expressed as mean values with SE indicated. All data except social recognition tests have been evaluated using one-way ANOVAs followed by Newman- Keuls multiple comparison post testing for detection of intergroup differences. Social recognition tests were evaluated by one-way ANOVAs or unpaired f-tests.
Results 1. Binding of peptide sequences to MT
Analysis of binding of peptides to MT was performed by SPA. The MT2 protein (from Sigma) was immobilized on a sensor chip CM5 using an amine coupling kit (Biosensor AB) as follows: the chip was activated by 20 μl activation solution; the protein was immobilized using 12 μl 20 μg/ml protein in 10 mM sodium phosphate buffer pH 6.0; the chip was blocked by 35 μl blocking solution. Various peptides at the indicated concentrations were injected into the sensor chip. The curve corresponding to the difference between binding to MT2 and a blank chip was used for analysis.
From Figure 1 it can be seen that the dendrimeric form of FGL (FGLd), binds to the inmmobilized on a sensor-chip MT in a dose-dependent manner. The FGL peptide (is part of the second F3 module of NCAM, which is capable of binding and activating the FGF receptor (Kiselyov et al., 2003).
We also tested whether other peptides derived from NCAM and various FGFs are capable of binding to MT2 as well. From Figure 2 it appears that the peptides derived from the first F3 module of NCAM, the ABL, EFL and CDL peptides, which are capable of binding and activating the FGF receptor (Fig. 14; see also WO2005123759), are also capable of binding to MT2. From Figure 3 it can be seen that peptides derived from different FGFs, 2FGF1 (Dyo1 ), 2FGF17 (Dyo17), 10FGF1 (Dekal) and 10FGF17 (Deka17), all are capable of binding to MT2 as well.
The affinity binding constants for all tested peptides are shown in Table 1 below. Table 1. Affinity binding constants
/fa (M"1 s"1) Ms"1) KD (M)
MT-2 : FGL 9.O1±3.43x1O 1.80*0.58x10 2.35±0.36x10 ,-6 MT-2 : EFL 5.55±0.69x102 3.24±0.32x10': 6.18±1.13x10"
-5
MT-2 : ABL 4.71±1.18x10 5.88±1.97x10' 1.33±0.46x10' MT-2 : Dyo17 6.52±0.87x103 1.21±0.16x10": 2.11±0.65x10
-6
MT-2 : Dekal 7 1.06±0.18x10 5.02±0.22x10"' 5.24±1.15x10 MT-2 : Dyo1 2.50±0.12x10 3.07±0.31x10" 1.25±0.19x10'!
-3 -7 MT-2 : Dekal 8.27±2.49x10 1.73±0.40x10 2.55±0.79x10
The peptides used for binding were in denrimeric form (four copies of a peptide sequence (four monomers) built on the three-lysine backbone)
Dekafins binds to the combined lg2-3 modules ofFGFRI
Binding of dekafins to FGFR1 was studied by SPR analysis. A recombinant protein consisting of the second and third Ig modules of FGFR1 was immobilized on the surface of a sensor chip, and the binding of the peptides in solution to the immobilized receptor was measured. Results of the binding of dekafins to immobilized recombinant lg2-3 of FGFR1 are shown in Figure 4a. The tested peptides were capable of binding to FGFR1 with apparent affinities ranging from 1.2x108 M"1 (Dekal) to 5.3x105 M-1 (Deka9) (Figure 4b).
To identify amino acid residues important for receptor binding dekafini was selected for analysis of alanine substitutions of different amino acid residues of the peptide and truncating of the sequence. The peptide variants were used at the same concentration (1 μg/ml), the binding equilibrium was determined for every peptide (Figure 4c). As it appears from Figure 4c, the binding efficacy of dekafini is substantially reduced when the basic amino acids (Lys6, Lys7 or Arg13) are substituted for alanine, indicating that these residues are important for binding to FGFR1. Truncation of four residues from the N-terminus by does not reduce the peptide binding affinity, whereas truncation of six residues (thereby including Lys6) inhibits, although only partially, the binding. Truncation two residues (thereby including Arg13) from the C- terminus completely abrogates of the ability of the peptide to bind the receptor.
Dekafins activate FGFR1 To study whether the binding of dekafins to FGFR results in FGFR activation in living cells, TREX-293 cells stably transfected with FGFR1 containing a C-terminal Strepll tag were stimulated with various concentrations of different dekafins, FGF1, FGF10 (used as positive controls), or nothing for various times. After stimulation, the degree of phosphorylation of FGFR1 was determined by immunoprecipitation of FGFR following by western blotting of the precipitates.
From Figure 5a it can be seen that dekafiniO and FGF1 (100 ng/ml) both induce FGFR phosphorylation in a time-dependent manner, the highest stimulation being observed 20 min after application of 20 μg/ml of the peptide.
The effect of the other dekafins and FGF10 (100 ng/ml) was tested using an exposure time of 20 min. Treatment of TREX-293 cells with FGF10 resulted in a significant increase of FGFR phosphorylation (Figure 5b). The dekafins also stimulated FGFR phosphorylation, but with different potency (Figure 5c,d). The highest potency was observed for Dekai (maximal effect at approximately 0.04-1.0 μg/ml) followed by Dekaδ, Dekaδ and Dekaδ (0.2-1 μg/ml), Deka2 (SEQ ID NO:2) and Deka17 (1 μg/ml), Deka3 (5 μg/ml), DekalO and Deka9 (20-100μg/ml).
Dekafins induce neurite outgrowth The neuritogenic activity of decafins was tested in cultures of cerebellar granular neurons (CGN) using as positive controls FGF1 and FGF10. Dissociated neurons from the cerebellum (CGN) were grown for 24 h in the presence of the individual peptides. The effect of all dekafins was quantified and is shown in Figure 6. The highest efficacy (the level of stimulation of neurite outgrowth with respect to the concentration of a peptide) was observed for Dekal, Deka2, Dekaδ, Dekaβ and Dekaδ followed by Deka3 and Dekal 7, DekalO and Deka9. The most pronounced neuritogenic response was observed for Dekal , Dekaδ, Dekaθ, Dekaδ, DekalO and Dekal 7 (500-700% stimulation) followed by Deka2, Deka3 and Deka9 (300-400% stimulation). Thus, this shows that high affinity binding of peptides to FGFR correlates with a high neuritogenic activity for Dekal, Dekaδ, Dekaθ and Dekaδ. Generally, dekafins which have a lower affinity binding to FGFR were less potent, however in some cases the efficacy of these peptides was comparable with the high affinity binders (e.g. DekalO and Dekal 7).
Interestingly, the cognate ligands of FGFR, FGF1 and FGF10, also stimulate neurite outgrowth from CGN in a dose-dependent fashion, however, the efficacy of the growth factors is much lower than that of the dekafins (Figure 6b). This indicates that a high affinity binding of FGFR ligands does not necessarily correlate with their potency to stimulate the FGFR-dependent neural cell differentiation.
The notion that the neuritogenic activity of dekafins depends on FGFR activation was proved in experiments were neurons were treated with the peptides simultane- ously with increasing concentrations of the FGFR inhibitor SU5402. As can be seen from Figure 6c, treatment of neurons with the inhibitor abrogates the neuritogenic response to Dekal , Deka2 and DekalO.
These results show that activation of FGFR by dekafins results in neuronal differen- tiation.
Dekafins promote neuronal cell survival
FGFs are known as neuroprotectants in the CNS. We therefore tested whether the dekafins which are derived from different FGFs are capable of promoting neuronal cell survival as well.
The effect of all nine dekafin peptides on cell survival was studied using in vitro systems described above. As appears from Figure 7a, cell death induced by reducing the potassium concentration in the medium can be prevented by treatment with IGF-1 and FGF1 (but not with FGF10). Treatment with Deka6, Dekaδ, Deka9 and Deka17 (but not with Dekal, Deka2, Deka3, Dekaδ or DekalO) promotes survival of CGN in cell culture as well (Figure 7b).
Dekafins enhance memory associated with social recognition
In the normal adult brain FGFs have been reported to be involved in processes associated with learning and memory. We therefore tested whether dekafins could modulate cognition. To address this question, the peptides were tested for a capability to improve social recognition memory retention.
Each test consisted of two exposures of an experimental adult rat to the same juvenile animal, with an interval of 120-min. The time spent on social investigation was measured cumulatively for 4 min in each session (T1 and 12, respectively), and the recognition ratio was calculated as T2/(T1+T2). When employing a 120-min time interval between the two sessions control animals did not demonstrate any retention of social memory resulting in a recognition ratio close to 0.5. The dekafin peptides were administered subcutaneously one or 24 h before the test. Statistical significant effects were only found for Dekal and DekalO. The results are shown in Figure 8.
From Figure 8 it appears that administration of any of the peptides one hour before the test has no effect on social memory performance, although a slight, but insignificant (p=0.07 as compared with untreated control) tendency to improve memory re- tention is seen for Dekaδ (Figure 8c). However, when the peptides are administered 24 h before the test, Dekal and DekalO induces a decrease in recognition ratio (Figure 8a and d), indicating that these two peptides has the potential to improve memory of social recognition. No effect was observed after administration of any other dekafins (exemplified by Deka2 and Dekaδ (Figure 8b and c)).
In a series of separate experiments the animals pre-treated with Dekal and DekalO were exposed to two different juvenile animals at the first and second sessions, rather that the same juvenile animal twice, and in these experiments, the treated animals correctly recognized the juvenile animal presented in the second meeting as unfamiliar, spending the same time investigating the juvenile animals in both the first and second meeting (Figure 8) This confirms that tDekal and DekalO specifically improve social memory, whereas they do not have an effect on general activity or social engagement of the treated animals.
Dyofins bind to the combined lg2-3 modules ofFGFRI
Binding of the dyofin peptides to FGFR1 was as described above. Figure 9a demonstrates. All tested peptides were capable of binding to FGFR1 with apparent affinities ranging from 2.45x1010 M"1 (Dyo8) to 1.96x105 M"1 (Dyo9) (Figure 9b).
Amino acid residues of importance for receptor binding were identified using Dyo10 comprising alanine substitutions of selected amino acid residues. Results of the binding are shown in Figure 9c. As appears from the figure, the binding efficacy of Dyo10 is significantly reduced when the basic amino acids (Lys9, Lys10 or Lys12) are substituted for alanine, indicating that these residues are important for binding to FGFR1. Truncations of Dyo10 from the N-terminus by eight residues and from the C-terminus by four residues does not reduce the affinity of binding, whereas truncations of six and eight residues from the C-terminus (thereby including Lys9, Lys10 or Lys12) results in abrogation of binding.
Thus, the dyofins are capable of binding to FGFR1 lg2-3 modules although with different affinities, and basic residues (lysines/ arginines) in positions 9, 10 and 12 are important for a high binding efficacy of Dyo3, Dyo8, Dyo10 and Dyo17.
FGFR1 is activated by dyofins Activation of FGFR by duofins was studied in a cell culture system described above. From Figure 10a it can be seen that all dyofins stimulate FGFR phosphorylation in a dose-dependent fashion. The highest efficacy was observed for Dyo3, Dyo8, Dyo9, Dyo10 and Dyo17. Treatment of cells with FGF2 resulted in an increase of FGFR phosphorylation which was four to five folds higher than that of dyofins (Figure 10b).
Dyofins nduce neurite outgrowth
The capability of dyofins to stimulate neurite outgrowth was estimated in primary neuron cell culture described above. The results of experiments presented in Figure 11a show that Dyo2, Dyo3, Dyoδ, Dyo10 and Dyo17 induces neurite extension in a dose-dependent manner, whereas Dyo1 and Dyo9 do not have the effect. In these experiments a high affinity binding to FGFR correlated with the potency of dyofins to induce neurite outgrowth. How- ever, the cognate high affinity ligand of FGFR, FGF2, which also stimulated neurite outgrowth from CGN in a dose-dependent fashion, did have much lower efficacy than Dyo2 which is derived from this growth factor (Figure 11b), and the threshold of the effect did not correlate with the potency of FGF2 to activate FGFR (with regard to FGFR phosphorylation by FGF2).
Neuritogenic activity of dyofins is depended on FGFR activation. As can be seen from Figure 11c, treatment of neurons with increasing concentrations of the FGFR inhibitor SU5402 abrogates the neuritogenic response to both dyofins and FGF2.
Dyofins promote neuronal cell survival
The effect of dyofins on cell survival was estimated in cell culture models describeed above. As appears from Figure 12a, cell death induced by reducing the potassium concentration in the medium can be prevented both by treatment with IGF-1 and FGF2 and with Dyo1 , Dyo2, Dyo3, Dyo9, Dyoδ, Dyo10 and Dyo17 (Figure 12b). At higher concentrations (5 μg/ml), Dyo3, Dyoδ and Dyo17 inhibit survival of neurons grown in a low potassium medium.
These results indicate that activation of FGFR in CGN under certain conditions may lead to either an increase or a decrease of neuronal survival depending on the par- ticular dyofin and the chosen concentration.
Dyofins enhance memory associated with social recognition
The effect of dyofins on social recognition memory retention was estimated in the social memory paradigm described above. Dyofins were administered subcutaneously 1h or 24 h before the test. Statistical significant effects were observed for Dyo1 , Dyo2 and Dyo9. The results are shown in Figure 6.
From the figure it appears that administration of the peptides 1 h before the test has no significant effect on social memory performance, although a slight improvement of memory retention can be observed (Figure 13). However, when peptides were administered 24 h before the test, Dyo1 , Dyo2 and Dyo9, induced a decrease in the recognition ratio (Figures 13a, b and c), indicating that these three peptides are capable of improving the retention of social recognition. No effect was observed after administration of some other dyofins (e.g.dyofin17 (Figure 13d)).
Animals treated with Dyo1 , Dyo2 and Dyo9 in separate experiments were exposed to two different juvenile animals at the first and second sessions, rather that the same juvenile animal twice, and in these experiments, the treated animals correctly recognized the juvenile animal presented in the second meeting as unfamiliar, spending the same time investigating the juvenile animals in both the first and second meeting (Figures 13a-c) This confirms that these three dyofins improve social memory retention.
Peptides FGL ABL, CDL and EFL bind to FGFR and stimulate FGFR-dependent neurite outgrowth and neuronal survival
NCAM derived peptides FGL, ABL, EFL and CDL are fragments of fibronectin type-3 module 1 and 2 (Fn3,1 and Fn3,2) of NCAM which have been shown to be involved in a direct interaction NCAM with FGFR. (Kiselyov et al 2003; WO03016351 ,
WO2004056865, WO2005014623, WO2005123759).
The equilibrium dissociation constants defined for the binding of the ABL, CDL and
EFL peptides to FGFR are shown in Table 2 below.
Table 2 .
Figure imgf000069_0001
The peptides are capable of activating FGFR and FGFR-dependent neural cell differentiation and cell survival demonstrated that the peptides are capable of binding. From Figure 14 it can be seen that phosphorylation of FGFR1 after stimulation of Trex293 cells stably transfected with FGFR1 with the peptides is increased (with a maximum response after 30 minutes, which was within the same timeframe as the maximum response for FGF1 (not shown)). Cells were incubated with various concentrations of the ABL, CDL or EFL peptides or FGF1 (as a positive control) for various periods of time between 5 min and 1h. Activation pf FGFR was estimated with regard to phosphorylation of FGFR1 by immunoprecipitation of the receptor and subsequent immunoblotting using the anti-phosphotyrosine antibody 4G10-AC (the procedure described above). The FGL peptide is capable of activating FGFR receptor as well (Figure 15).
The FGL, ABL, CDL and EFL all peptides are potent stimulators of neurite outgrowth (Figures 16) and/or neuronal survival (Figures 17-18). The effect of the peptides was evaluated in the corresponding assays described above.
Effect of peptides FGL, ABL, CDL and EFL on short- and long term memory
Social recognition test
To investigate whether the FGL, ABL, CDL and EFL peptides have an effect on short-term memory the social recognition test was employed. In this test a juvenile rat (4 weeks old) was introduced into the home cage of an adult rat for a period of 4 minutes. After a 2h time interval, the same juvenile rat was re-introduced into the adult's home cage. During both meetings, the investigating behaviour of the adult towards the juvenile rat (licking, sniffing, chewing the fur of the juvenile) was recorded and a decreased investigative behaviour at the second meeting signified that the adult animal recognized the juvenile. To minimise the stress imposed on both the juvenile and the adult animal by the procedure, we habituated the animals to the experimental set-up before the start of the experiment. Vehicle (water, n=9), ABL (n=9), CDL (n=8) or EFL (n=9) peptides were administrated subcutaneously 1h prior to the first meeting. Using this set-up it was found that administration of 4 mg/kg peptide 1h prior to the first meeting induces a significant decrease in time spent on investigative behaviour at the second meeting (paired t-test, ABL: p=0.04, CDL: p=0.01 , EFL: p=0,0008 when compared to vehicle treated animals) (Figure 19 A) indicating that the ABL, CDL and EFL peptides induce an improvement of short-term memory. To assess whether the decrease in investigation time from the first to the second exposure was linked to memory or it was due to non-specific effects, such as an inhibition of social interaction, the same test was performed but using another (different) juvenile animal presented at the second introduction. No any significant decrease in investigation time at the second meeting were observed (results are not shown), indicating that the effect of the ABL, CDL and EFL peptides is specific and due to stimulation of short-term memory by the peptides.
Contextual fear conditioning Contextual fear conditioning paradigm was used to study long term effects of FGL, ABL, CDL and EFL on memory.
Contextual fear conditioning is a classical conditioning paradigm in which the animal is placed in a box and is given time to explore the context. Thereafter the animal is subjected to a series of consecutive shocks (unconditioned stimuli, US) and then it is removed from the context. After different time intervals the animal is returned to the context and the freezing response (conditioned response, CR) is recorded. The animal to be tested received water (ctl, n=9-11 ), the FGL (n=9), ABL (n=7-9), CDL (n=8) or EFL (n=8-9) peptides administrated intranasally either prior (Figure 19 B) or immediately after fear conditioning (Figure 19 C). The animal was conditioned by placing in the contextual fear conditioning box and given 3 minutes to investigate the context. Thereafter it received three consecutive shocks (1 second each with 1 minute interval inbetween) and then it was removed from the box. 24h, 48h and 3 weeks after the first conditioning the animal was returned to the box for 8 minutes and the context conditioned freezing response was recorded. To minimise stress imposed on the animals by the procedure, the animals were habituated to the experimental set-up prior to the start of the experiment by subjecting the animal to be tested through the procedure several times without introduction to the context and shock. When the ABL, CDL or EFL peptides were administrated twice before the conditioning it was not observed any effect (in contrast to FGL the difference in freezing response was recorded (Figure 19B). However, when the peptides were administrated immediately after conditioning, an overall difference in freezing response 24h after training between the groups was apparent (one-way ANOVA. P= 0.009). Further, post-hoc analysis revealed that administration of the ABL peptide induced an increased freezing response compared to control treated animals (t-test: p= 0.02), indicating that the administration of this peptide immediately after conditioning improves retention of the conditioned freezing response 24h later. 8 days after conditioning, the freezing responses of ABL treated animals were comparable to control treated animals (water treated) indicating that the improved retention lasts for the period between 24h and 8 days. To ensure that the increased freezing response observed after treatment with the ABL peptide was due to an improved retention of memory and not due to an increased anxiety of the tested animals, the freezing response, activity level and the exploratory behaviour of the animals when the animal was introduced to a new environment (without receiving shock) was recorded aw well. It was found no differences between treated and non-treated animals (results not shown) indicating that the increased freezing response observed with ABL treated animals is due to an improved memory.
Thus, FGL, ABL, CDL and EFL, all have a positive effect on short term memory as evaluated by the ability of an adult male to recognise a juvenile animal 2 h after they were first introduced. The capability of stimulating retention of memory of animals for a specific context (fear conditioning) for a longer time (24 h) is more pronounced for the FGL and ABL peptides than for the CDL or EFL peptides.
Alzheimer's disease: the effect of the FGL peptide in vivo
The Alzheimer's disease brain pathology was induces in rats as described above.
FGL was administered in a dimeric or a tetrameric version, referred to as FGL2 and FGL4, respectively (these formulations of FGL are effective to induce dimerization of the FGFR).
Initially, the ability of FGL to prevent pathological changes induced by Aβ25-35 was examined by administering FGL intracistemally on day 7, 10 and 13 following Aβ25- 35-injection. As shown in Figure 20 a-b, administration of both FGL2 and FGL4 caused a statistically significant reduction in Aβ IR to control level. Administration of a control peptide had no effect on Aβ IR. Therefore, the majority of the subsequent experiments were performed with only vehicle as control.
In the hippocampus a significant two-fold increase of tau phosphorylation was observed in response to Aβ25-35-injection (Figure 2Od). However, after FGL-treatment this tau phosphorylation was not significantly different from control level. Aβ25-35- injection induced significant increases in microgliosis (Figure 20e-f) and astrocytosis (Figure 20g-h), and in all cases FGL-administration reduced these reactions to con- trol levels. Figure 21 presents effects obtained with FGL2, when administered intranasally (Figure 21a-c) and subcutaneously (Figure 21 d-f ). Aβ25.35-injection induced a significant decrease in the total number of neurons in the cortex (Figure 21a) and hippocampus (Figure 21b, e). Both intranasal and subcutaneous FGL-administration prevented this cell loss, resulting in neuronal cell numbers in the respective areas not significantly different from those seen in control animals. In addition, both intranasal and subcutaneous FGL-administration was able to prevent the Aβ25-35-induced impairment of cognitive function (Figure 21c, f). These results demonstrate that FGL is able to cross the blood-brain-barrier and subsequently exert its function within the nervous system.
Neuronal rescue, neurogenesis and tissue repair by the FGL peptide in vivo
In lesioned animals FGL strongly induced expression of the MT proteins in asuble- sioned zone of the brain when compared to the lesioned animals received vehicle. In the control group of unlesioned, healthy animals treated with vehicle or FGL, the only observed effect of FGL was an increased expression of MT-I+II, whilst expression levels of all other tested factors were identical after vehicle and FGL administration. In the brains of unlesioned animals, FGL induced increased levels of MT-I+II in the meninges, vascular cells, ependyma, the choroid plexus and in some scattered astroglial cells. Interestingly, increased MT-I+II levels in unlesioned FGL-treated animals could also be demonstrated in peripheral organs including spleen, liver, and bone marrow. Thus, induction of the neuroprotective MT-I+II isoproteins was the only effect of FGL treatment demonstrated in healthy controls, and this effect was observed both in the CNS and systemically.
The MT-I+II isoproteins are antioxidants and anti-apoptotic neuroprotective factors, which significantly reduce delayed tissue damage. In unlesioned healthy controls, the only factors induced by FGL were MT-I+II, and after the lesion, the increase in MT-I+II expression in FGL-treated animals was sustained throughout the 36 days of observation. Therefore, we investigated the role of FGL-induced MT-I+II expression by treating MT-I+II deficient mice (MTKO) and transgenic MT-I overexpressing mice (TgMT) with FGL or vehicle after brain lesion. The animals were sacrificed three dpi. We found that FGL-induced effects observed in brain-injured rats were absent in MTKO mice, but present in their wildtype controls (129sv strain). In the TgMT mice, FGL had a slight, but synergistic effect with the elevated MT levels. In the TgMT wildtype controls (C57/BL6 strain), addition of FGL had the same effects as in the 129sv controls. From Fig. 4 it appears, that in wildtype mice (129sv and C57/BL6) with brain injury, FGL treatment had the same effects as those described in rats, i.e. FGL-treatment clearly reduced the number of cells suffering from oxidative stress (8- OH-dG), apoptotic cell death (TUNEL) and neurodegeneration (NFT), whilst surviving NSE-positive neurons, FAP-positive astrocytes and MT-I+II expressing cells all were increased as compared to untreated controls. Also, in wildtype mice FGL increased the number of surviving neurons around the lesion as well as the expres- sion of FGF-1, FGF-2, FGFR1 TGFα, VEGF, NT-3, NT-4/5, BDNF, angiopoietin-1/4, EPO, PSA-NCAM, and S100A4. No anti-inflammatory effect of FGL three dpi was detected employing the tomato lectin (lectin) marker of microglial cells. As previously reported, lesioned MTKO animals exhibited a pronounced, delayed tissue damage. Thus, the MTKO mice exhibited an increase in oxidative stress (8-OH-dG), apop- tosis (TUNEL) and microglial activation (lectin), whereas the numbers of surviving NSE-positive neurons, GFAP-positive astrocytes, and MT-I+II expressing cells were reduced as compared to the wildtype controls. However, most importantly no effect of FGL treatment on any of these parameters could be demonstrated in the MTKO mice. In contrast, TgMT mice clearly displayed reduced tissue damage and im- proved recovery relative to wildtype mice after the lesion, and FGL treatment led to a further decrease of these processes as judged by stainings for 8-OH-dG and TUNEL. Moreover, FGL increased the already high level of NSE, GFAP and MT-l+ll, whereas no effect on microglial activity could be seen. Further confirmation of the described findings was obtained by stainings for MDA, NFT, APP, caspase-3 and cytochrome-c. Thus, induction of expression of the MT-I+II isoproteins is necessary to achieve the neuroprotective effects observed with FGL-treatment.

Claims

Claims
1. A compound consisting of metallothionein (MT), or a fragment thereof, and a peptide sequence comprising at most 25 contiguous amino acid residues, wherein the MT, or the fragment thereof, and the peptide sequence are bound to each other via a non-covalent bond and/or via sulfhydryl moieties of the cysteine present in the amino acid sequences of the peptide sequence and MT or the fragment thereof.
2. The compound according to claim 1, wherein affinity of the binding of the MT and the peptide sequence is characterized by affinity binding constant K0 having the value of about 10"8M to about 10"4 M.
3. The compound according to claim 1 or 2, wherein the peptide sequence comprises the amino acid motif x1-x2-x3-x4-x5, wherein x1 is a hydrophobic, charged amino acid residue or G; x2 is a charged amino acid residue, T or S, x3 is any amino acid residue; x4 is a charged amino acid residue, T or S, x5 is a hydrophobic, charged amino acid residue T, S or G.
4. The compound according to any of claims 1-3, wherein the peptide sequence is a biologically active peptide sequence.
5. The compound according to claim 4, wherein the peptide sequence is capable of binding to a functional cell receptor and modulating the activity of said receptor.
6. The compound according to claim 5, wherein the functional receptor is a receptor of a cell of the brain.
7. The compound according to claim 6, wherein the functional receptor is selected from of the family of fibroblast family growth factor receptors (FGFRs).
8. The compound according to claim 7, wherein the fibroblast growth factor receptor is selected from fibroblast growth factor receptor 1 (FGFR1), fibroblast growth factor receptor 2 (FGFR2), fibroblast growth factor receptor 3 (FGFR3), or fibroblast growth factor receptor 4 (FGFR4).
9. The compound according to claim 8, wherein the peptide sequence is capable of binding to and modulating activity of FGFR1.
10. The compound according to claims 8 or 9, wherein the peptide sequence is derived from a ligand of FGFR
11. The compound according to claim 10, wherein the peptide sequence is a peptide fragment of the neural cell adhesion molecule (NCAM), neural cell adhesion L1 or fibroblast growth factor (FGF).
12. The compound according to any of the claims 1 to 11 , wherein the peptide sequence comprises an amino acid sequence selected from SEQ ID NOs:1-51 , or a fragment or variant of said sequence.
13. The compound according to claim 12, wherein the peptide sequence is selected from SEQ ID NOs: 1-51 , or is a fragment or variant of said sequence.
14. The compound according to claim 12 or 13, wherein the peptide sequence is formulated as a multimer comprising two or more copies of a sequence selected from SEQ ID NOs:1-51 , or two or more copies of a fragment or a variant of said sequence.
15. The compound according to claim 14, wherein the peptide sequence is selected from SEQ ID NOs:42-45, or a fragment or a variant thereof.
16. The compound according to claim 14, wherein the peptide sequence is selected from SEQ ID NOs: 1-18, or a fragment or a variant thereof.
17. The compound according to claim 14, wherein the peptide sequence is selected from SEQ ID NOs: 19-41 , or a fragment or a variant thereof.
18. The compound according to claim 14, wherein the peptide sequence is selected from SEQ ID NOs:46-51 , or a fragment or a variant thereof.
19. The compound according to any of the preceding claims, wherein the peptide sequence is capable of stimulating cell survival and/or cell differentiation and/or cell plasticity associated with memory and learning and/or inhibiting inflammation and/or modulating cell adhesion.
20. The compound according to claim 19, wherein the cell is a cell of the neural system.
21. The compound according to claims 19 or 20, wherein the cell is a cell of the brain.
22. The compound according to claims 19 or 20, wherein the cell is a neuronal cell.
23. The compound according to claims 19 or 20, wherein the cell is a glial cell.
24. The compound according to any of the preceding claims, wherein the metallothionein is selected from metallothionein-1 A (MT1A), metallothionein-1B (MT1B), metallothionein-1 E (MT1E), metallothionein-1 F (MT1F), metallothionein-1 G (MT1G), metallothionein-1 H (MT1 H), metallothionein-11 (MT1 I), metallothionein-1 K (MT1K), metallothionein-1 L (MTI L), metallothionein-1 R (MT1 R), metallothionein-1X (MT1 X), metallothionein-2 (MT2), metallothionein-3 (MT3) or metallothionein-4 (MT4).
25. The compound according to claim 24, wherein the metallothionein is MT2.
26. A peptide sequence as identified in SEQ ID Nos: 4, 8, 10, 16, 17, 26, 33, 35, 39 or 41.
27. A pharmaceutical composition comprising a compound according to any of the claims 1 to 25 or a peptide sequence according to claim 26.
28. Use of a compound according to any of the claims 1 to 25 as a medicament.
29. Use a peptide sequence according to claim 26 as a medicament.
30. Use of a compound according to any of the claims 1 to 25 or a peptide sequence according to t claim 26 for the preparation of a medicament for treatment of a disease or condition associated with activity of FGFR or a FGFR ligand.
31. The use according to claim 30, wherein the treatment of disease or condition involves modulating neural cell survival, cell differentiation, cell proliferation and/or cell plasticity, such as plasticity associated with learning and memory.
32. The use according to claim 30, wherein the condition or disease is a condition or disease of the central and/or peripheral nervous system.
33. The use according to claim 32, wherein the condition or disease is selected from postoperative nerve damage, traumatic nerve damage, impaired myelination of nerve fibers, postischaemic damage, such as after stroke, nerve degeneration associated with diabetes mellitus, disorders affecting the circadian clock or neuro- muscular transmission.
34. The use according to claim 30, wherein the condition or disease selected from of conditions or diseases of the muscles including conditions with impaired function of neuro-muscular connections, such as after organ transplantation, or such as genetic or traumatic atrophic muscle disorders.
35. The use according to claim 30, wherein the condition or disease is cancer.
36. The use according to claim 35, wherein the cancer is a cancer of the central neural system.
37. The use according to claim 30, wherein the condition or disease is an impaired ability to learn and/or impaired memory.
38. The use according to claim 30, wherein the condition or disease is Parkinson's disease, Alzheimer's disease, Huntington's disease or dementia such as multiinfarct dementia.
39. The use according to claim 30, wherein the condition or disease is a mental disease, such as a disorder of thought and/or mood, neuropsychiatric disorders including bipolar (BPD), genetically related unipolar affective disorders, delusional disorders, paraphrenia, paranoid psychosis, schizophrenia, schizotypal disorder, schizoaffective disorder, schizoaffective bipolar and genetically related unipolar affective disorders, psychogenic psychosis, catatonia, periodic bipolar and genetically related unipolar affective disorders, cycloid psychosis, schizoid personality disorder, paranoid personality disorder, bipolar and genetically related unipolar affective disorders related affective disorders and subtypes of unipolar affective disorder.
40. The use according to claim 30, wherein the condition or disease is associated with brain damages due to alcohol consumption
41. The use according to claim 30, wherein the disease is a prion disease.
42. The use according to claim 30, wherein the condition or disease is characterised by sustained inflammation.
43. The use according to claim 30, wherein the condition or disease is brain inflammation due to microbial or viral infection or an autoimmune disease.
44. The use according to claim 43 wherein the condition or disease is Guillain-Barre syndrome or its variant form, such as Miller Fisher syndrome, or another complement dependent disorder.
45. Method of treatment of a condition or disease associated with activity of FGFR or a FGFR ligand comprising administering to an individual in need an effective amount of a compound according to claims 1-25, a peptide sequence according to claim 26, or a pharmaceutical composition according to claim 27.
46. Use of metallotionein as a carrier protein for delivery of a peptide sequence into the brain.
47. The use according to claim 30, wherein the peptide sequence is a ligand of a functional cell receptor.
48. The use according to claim 47, wherein the peptide sequence is selected from SEQ ID NOs:1-51, or a fragment or a variant thereof.
49. Use of a peptide sequence derived from an FGFR ligand for the preparation of a medicament for treatment of a disease or condition associated with or requiring activity of metallothionein.
50. The use according to claim 49, wherein the sequence is derived from NCAM.
51. The use according to claim 50, wherein the sequence is selected from the sequences identified as SEQ ID NOs:42-45, or a fragment or a variant thereof.
52. The use according to claim 51 , wherein the sequence is SEQ ID NO:42, or a fragment or a variant thereof.
53. Method of treatment of a condition or a disease associated with a deficiency of synthesis or function of metallothionein, said method comprising administering a peptide sequence according to claims 51 or 52, or a medicament comprising thereof to a subject in need.
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