RU2588143C2 - Peptide compound, useful for inhibiting formation of amyloid plaques - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to peptide compound R^a-R¹-R²-R³-R⁴-R^b (I) or R^a-R⁴-R³-R²-R¹-R^b (II), where R^a is N-end primary amine group of amino acid R¹ or R⁴, or free, or substituted with an amino protective group, R^b is a hydroxyl group of C-end carboxyl group of amino acid R¹ or R⁴, or free, or substituted with hydroxy protective group, and R¹-R²-R³-R⁴ or R⁴-R³-R²-R¹ represent HADD, KADD, DDAK, RADD, DDAR, KAED, NEAR, 5, DEAR, HADE, EDAH, KADE, EDAK, HAMBURG, EDAR, HAEE, EEAH, KAEE, EEAK, WEEE and EEAR. Present invention also relates to use of said useful peptide compounds, in particular, for binding with β-amyloid peptides and inhibiting polymerisation in form of amyloid plaques.

EFFECT: compounds can be used as a drug or for preparing or determining preparations for treating neurodegenerative diseases, particularly Alzheimer's disease.

21 cl, 9 dwg, 2 tbl, 4 ex

Description

The present invention relates to a novel peptide compound mainly used to bind to β-amyloid peptides and / or to prevent or reduce the formation of amyloid plaques, mainly by inhibiting the polymerization of the aforementioned amyloid peptide as amyloid plaques.

The present invention also relates to the use as a medicine or for the preparation or determination of drugs for the treatment of neurodegenerative diseases, in particular Alzheimer's disease.

Alzheimer's disease is a neurodegenerative disease, mainly in the elderly, characterized by a prolonged, from 5 to 15 years from the onset of the disease, and irreversible decrease in mental abilities and, in the later stages of the disease, loss of psychomotor functions.

Alzheimer's disease is one of the most common causes of dementia in older people over 55, regardless of gender and lifestyle [1].

In some patients, the disease may be associated with hereditary disorders, but in most patients the disease occurs sporadically, without any specific reason [2].

To this day, there is no therapeutic method for treating sporadic or hereditary forms of this disease. At present, it is well known that Alzheimer's disease is not an inevitable consequence of the aging process, but is induced by an abnormal phenomenon of aggregation of β-amyloid peptides (hereinafter “Aβ”) [3, 4].

Aβ is a peptide of 39 to 43 amino acids (SwissProt ID P05067), which is formed after sequential cleavage of the amyloid precursor protein (hereinafter referred to as "APP"). An Aβ peptide is a compound that is usually found in body fluids, such as blood or cerebrospinal fluid, in which it is present in low concentrations of 0.5 to 5 nM, and this is the same in healthy people as in patients with the disease Alzheimer's [5, 6].

On the other hand, the progression of Alzheimer's disease is characterized by extracellular accumulation or deposition of so-called amyloid plaques in the patient’s brain. These plaques are the result of the conformation of the Aβ peptide monomer into the form of dimers, then large soluble oligomers, and then their aggregation into the form of insoluble fibrillar β layered aggregates, which ultimately give rise to amyloid plaques [7].

However, the exact molecular mechanism of the formation of amyloid plaques and the initiation of Alzheimer's disease remains partially unexplained to this day.

Some studies essentially attribute the high amyloidogenic potential to fragments consisting of amino acids 17–42 from the COOH end of the Aβ peptide [9]. Other studies do not exclude that fragments consisting of amino acids 1-16 of the Aβ peptide from the region of the NH ₂ end of the Aβ peptide are involved in the pathogenesis of Alzheimer's disease [10].

At the moment, according to all these available data, it can be concluded that the 1-16 N-terminal region of the Aβ peptide is a region that undoubtedly participates in the most convincing and decisive path for the formation of amyloid plaques, for the following reasons.

First of all, this region in rats contains three mutations that distinguish rat Aβ peptide from it in other mammals, even if the rat does not suffer from Alzheimer's disease.

It has been determined that the 1-16 N-terminal region is a binding domain where zinc (Zn) and copper (Cu) are attached to the Aβ peptide [11-14]. At present, it has been revealed that amyloid plaques are characterized by an abnormally high concentration of divalent metals and, in particular, zinc [15]. A number of studies and experiments, both in vivo and in vitro, have shown that high concentrations of zinc cause precipitation of the Aβ peptide, leading to the formation of amyloid plaques. In addition, it was shown that amyloid plaques are more significantly formed in the environment of neurons with high zinc concentrations [16–20].

That is why it is currently believed that blocking the interaction of the Aβ peptide with zinc should lead to a decrease in the aggregation of the Aβ peptide in the form of amyloid plaques and thereby prevent the pathologies associated with this. Thus, in US 6001852, as a medicine for the treatment of Alzheimer's disease, Clioquinol, which is a zinc chelating agent, is proposed. However, the use of a zinc chelating agent requires special targets in the region of the binding domain where zinc is attached to the Aβ peptide in order to avoid interference with normal zinc homeostasis in the body.

Other studies [21-30] show that nicotinic acetylcholine receptors play the role of the main goal of neurotoxicity in connection with disorders in the cholinergic and cognitive system caused by the formation of amyloid plaques. That is why in US 7018797, a method of inhibiting the binding of β-amyloid peptides to the α-7 nicotinic acetylcholine receptor was proposed for the treatment of neurodegenerative diseases. In US 7018797, compounds derived from naphthalene, in particular 5,8-dihydroxy-trans-2-di (N-propylamino) -3-methyl-1,2,3,4-tetrahydronaphthalene, were more specifically proposed as compounds which can inhibit the binding of the Aβ peptide to the α-7 nicotinic acetylcholine receptor.

Other studies have shown that the interaction of the 13-1 battle region of the amino acid Aβ peptide (HHQK, SEQ.ID.NO.27) with brain microglia cells is carried out by binding to heparan sulfate, which is a binding molecule for these cells, entering the glycosaminoglycan category. The binding of the Aβ peptide to the surface molecules of glycosaminoglycans of microglia cells induces the formation of Aβ peptide oligomers. The Tramiprosate molecule imitates glycosaminoglycans and inhibits the oligomerization of Aβ peptides [31–36].

In US 7314724, the use of soluble forms of laminins and fragments thereof (> 10 kDa) that are capable of binding to Aβ and keeping it in a monomeric state was proposed as a medicine against Alzheimer's disease.

More specific mechanisms of interaction between Aβ peptides and nicotinic acetylcholine receptors, as well as with laminins, have not yet been disclosed.

SmithKline Beecham publication WO 93/13789 describes compounds with a complex formula comprising oligopeptides protected on both sides, the general formula of which encompasses two hexa- or heptapeptides joined together by chemical bonding. The use of the compounds described herein relates to the stimulation of hematopoiesis to protect the body against infections, in particular to the stimulation of the myelopoietic system, and does not explain the mechanism of action of these compounds.

US 2004/214165 describes a protein export system in mycobacteria. The 646 sequence of 4-HAED amino acids is simply the sequence of the terminal portion of one of the mycobacterial proteins that is involved in the protein export process. But this HAED sequence is not described in US 2004/214165 as a formula for an individual isolated oligopeptide, and even less, so to speak, especially as a therapeutically active peptide.

EP 2130550 describes the HAED sequence (SEQ.ID.NO.39) as a RNA helicase protein pattern that is active in the production of cytokines by mononuclear blood cells. But EP 2130550 does not describe the HAED tetrapeptide as an isolated compound, and moreover, as a protected compound and, moreover, moreover, as an active compound per se.

In an article by Gomez-Ruiz et al. In J. Dairy Sci. 90: 4966-4973 from 2007 describes the EDAK peptide and a product that is found in cheese resulting from the degradation of milk proteins caused by fermentation due to lactic acid bacteria enzymes or enzymes that have been artificially added to cheeses. This article neither describes nor suggests that this isolated, purified tetrapeptide and, especially, protected by protective groups, i.e. outside its mixture with cheese, it can be a compound of interest taken separately or, moreover, it can be an active compound in itself.

In an article by Chu et al. in J. Am. Chem. Sco. 1995, 117, 5419-54205 describes a method for determining tetrapeptides spectrometrically. These tetrapeptides are defined as binding to receptors, i.e. vancomycin, only the N-terminus of the tetrapeptide is protected. The DDAH tetrapeptide is only one of the peptides used in the experiment, selected to demonstrate the results of manipulations. The article does not describe the features of the activity of this tetrapeptide DDAH.

An object of the present invention is to provide a new compound that can be used in the determination or manufacture of a preparation for the treatment or prophylaxis of diseases associated with neurodegenerative disorders and / or the formation of amyloid plaques by polymerization or aggregation of β-amyloid peptides.

In particular, it is an object of the present invention to provide a novel compound capable of inhibiting the interaction of β-amyloid peptides with Zn (II) ions. Indeed, it is known that the formation of amyloid plaques due to aggregation or polymerization of a β-amyloid peptide is induced by the formation of complexes of zinc with a β-amyloid peptide, zinc to some extent acts as a cofactor of this aggregation or polymerization.

In particular, it is an object of the present invention to provide a new compound capable of inhibiting the formation of β-amyloid plaques and making it possible to treat or prevent Alzheimer's disease or any other degenerative diseases associated with the formation of amyloid plaques.

или $${\text{R}}^{\text{a}}{\text{-R}}^{\text{4}}{\text{-R}}^{\text{3}}{\text{-R}}^{\text{2}}{\text{-R}}^{\text{1}}{\text{-R}}^{\text{b}}\text{ (II)}$$

, гдеTo this end, the present invention provides a peptide compound with the following general formula (I) or (II): $${\text{R}}^{\text{a}}{\text{-R}}^{\text{one}}{\text{-R}}^{\text{2}}{\text{-R}}^{\text{3}}{\text{-R}}^{\text{four}}{\text{-R}}^{\text{b}}\text{(I)}$$

or $${\text{R}}^{\text{a}}{\text{-R}}^{\text{four}}{\text{-R}}^{\text{3}}{\text{-R}}^{\text{2}}{\text{-R}}^{\text{one}}{\text{-R}}^{\text{b}}\text{(Ii)}$$

where

- R ¹ represents the amino acid residue of H, R and K;

- R ² represents the amino acid residue A;

- R ³ represents an amino acid residue of E or D;

- R ⁴ represents an amino acid residue of E or D;

- R ^a represents the N-terminal basic amine function of amino acids R ¹ and R ⁴ , either free or substituted by a protected group, preferably an acetyl group (A _C ),

- R ^b represents the hydroxyl function of the C-terminal carboxyl group of amino acids R ¹ and R ⁴ , either free or substituted by a protected group, preferably an NH ₂ , NH _R or NR _R group with R representing a C1-C4 alkyl chain, i.e. e. corresponding to an amide type group, preferably an amide group (NH ₂ ).

It is understood that the peptide compounds in accordance with the present invention are intended to be individually isolated and purified compounds, with a purity of preferably more than 98%, preferably at least 99%, residues can be formed with other peptides, among other things.

In a known manner and preferably, amino acids R ¹ , R ² , R ³ , R ⁴ must be configured in the form of their natural isomers L. But amino acids R ¹ , R ² , R ³ and R ⁴ can also be analogous to the D configuration.

However, it is preferred that the peptide compound of the present invention have a purity of the L isomer of greater than 98%, preferably at least 99% of the L isomers.

The inventors determined these tetrapeptides by characterizing various amino acids by binding the tetrapeptide to the EVHH site (SEQ.ID.NO.28) of the 11-14 β-amyloid region, while the characterized amino acids R ¹ , R ³ and R ⁴ , are of the opposite sign, those. able to form strong ionic bonds with amino acid E (glutamate) at position 11 for R ¹ and, accordingly, with H (histidine) at positions 13 and 14 for R ³ and, accordingly, R ⁴ , i.e. with 3 of the potentially participating binding sites of Zn (II) ions with β-amyloid, and the hydrophobic amino acid A (alanine) can form a strong hydrophobic effect with V.

Thus, the characterized tetrapeptides form parallel (formula I) and antiparallel (formula II) non-covalent interactions with the EVHH target of the 11-14 β-amyloid region. Given the fact that three of the four amino acids in the target EBHH region have highly charged groups, it is preferable that the amino acids have ionic complementarity with these three amino acids, while the choice of amino acid R ² was made depending on the consideration of steric complementarity between the side chains of alanine and valine, even further stabilization due to hydrophobic contacts between V and A, ionic bonds with E and H.

The inventors experimentally showed that tetrapeptides, as described above, bind to 3 chelating agents of the Zn (II) ion, namely amino acids at positions 11, 13 and 14, which represent an optimal compromise, compared to other corresponding targets of various sizes containing 1-4 chelating agents Zn (II) selected from positions 6, 11, 13 and 14, especially from regions 9-11, 10-12, 11-13, 12-14, 13-15, 14-16 , 11-16, 5-14 and 9-14, and according to other determination criteria, in particular, according to the size of the peptide, namely, the optimal compromise is the possibility l use this compound in vivo to inhibit the binding of Zn (II) ions to a β-amyloid peptide (Aβ) and to inhibit the oligomerization of a β-amyloid peptide by binding the tetrapeptide to the domain for binding of Zn (II) ions of a β-amyloid peptide (Aβ )

It seems that the strength of the interaction can be undermined by the bond at position 6. Conversely, a target that overlaps only two sites at positions 6 and 11 or 13 and 14 is insufficient to inhibit the binding of Zn (II) ions.

Another advantage of tetrapeptides according to this invention is that they are highly electrically charged, which contributes to the possibility of their passage through the blood-brain barrier.

Peptide compounds include, in particular, the sequences R ¹ -R ² -R ³ -R ⁴ and R ⁴ -R ³ -R ² -R ¹ defined by formulas I and II in Table 1 below, and are repeated in sequences SEQ.ID.NO. from 1 to 24 from the list of sequences attached to the description.

Table 1 SEQ.ID.NO.1 = HADD SEQ.ID.NO.2 = DDAH SEQ.ID.NO.3 = KADD SEQ.ID.NO.4 = DDAK SEQ.ID.NO.5 = RADD SEQ.ID.NO.6 = DDAR SEQ.ID.NO.7 = HAED SEQ.ID.NO.8 = DEAH SEQ.ID.NO.9 = KAED SEQ.ID.NO.10 = DEAK SEQ.ID.NO.11 = RAED SEQ.ID.NO.12 = DEAR SEQ.ID.NO.13 = HADE SEQ.ID.NO.14 = EDAH SEQ.ID.NO.15 = KADE SEQ.ID.NO.16 = EDAK SEQ.ID.NO.17 = RADE SEQ.ID.NO.18 = EDAR SEQ.ID.NO.19 = HAEE SEQ.ID.NO.20 = EEAH SEQ.ID.NO.21 = KAEE SEQ.ID.NO.22 = EEAK SEQ.ID.NO.23 = RAEE SEQ.ID.NO.24 = EEAR

The affinity of these tetrapeptides with ligands corresponding to fragment 1-16 of the β-amyloid peptide region was verified by calculating the dissociation constant, Kd, in Examples 1 to 3. Their dissociation constants are lower than those of other peptides studied: tetrapeptides, tripeptides, hexapeptides or decapeptides not included in this definition by a factor of 10 ⁴ .

In a known manner, these protective groups are compatible with pharmaceutical use in vivo. The function of the protective groups is to neutralize the electric charge of the amino and carboxyl ends of the peptide. In particular, the N-terminus may be protected by a formyl group (HCO-) or the acetyl group (CH ₃ CO-), and the C-terminus may be protected by the groups -NH ₂ , -NHR, -NRR, where R is C ₁ -C _{4 by} alkyl or hereinafter -OR, where R is C ₁ -C ₄ alkyl or alkylamine, such as -OCH ₃ , -OCH ₂ CH ₃ or -OCH ₂ CH ₂ NH ₂ .

It should be recalled that:

hydrophobic amino acids with a non-polar group are alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), phenylalanine (F), proline (P) and tryptophan (W),

- hydrophobic amino acids with a polar group are glycine (G), series (S), threonine (T), cysteine (C), tyrosine (Y), asparagine (N) and glutamine (Q),

- amino acids with a negatively charged group in a neutral (acidic) pH are aspartic acid (D) and glutamic acid (E), and

- amino acids with a positively charged group in a neutral (basic) pH are lysine (K), arginine (R) and histidine (H).

All natural amino acids are chiral, therefore optically active, with the exception of glycine, and all are in the L configuration, in accordance with the FISHER configuration, or in the S configuration, in accordance with the Kahn-Ingold-Prelog notation.

There are three important types of interaction in the bonds between peptides or in secondary structures of peptides, such as α helices or type β folded layers resulting from the formation of hydrogen bonds between the CO and NH groups of the main skeleton of the peptide, namely:

- hydrophobic effect: amino acids for which the radicals are hydrophobic have a greater affinity for each other than for the water molecules surrounding the protein. Conversely, hydrophilic amino acids are usually arranged in such a way as to be in contact with water,

- ionic bonds: radicals that are positively charged form ionic bonds with those that are negatively charged, and

- hydrogen bonds that are present in interactions that are less strong than those formed as a result of ionic bonds and hydrophobic effects.

Preferably, a peptide compound of the invention selected from compounds of formula (I) or (II), wherein R ¹ -R ² -R ³ -R ⁴ or, respectively, R ⁴ -R ³ -R ² -R ¹ is HAEE , EDAR, EEAH, EEAK, EEAR, HADE, KADD, KAEE, RADD, RADE, RAEE, HADD, DDAK, KAED, DEAR or KADE. These peptides correspond in different order to the sequence of SEQ.ID.NO.1, 3, 4, 5, 9, 12, 13, 15, 17, 18, 19, 20, 21, 22, 23, and 24.

The tetrapeptides mentioned above have dissociation constants of their interaction with a fragment 1-16 of the β-amyloid region, Kd, as calculated in Examples 1-3 below, which are lower than for the other peptides studied.

More preferably, the peptide compound of the invention is among the compounds of formula (I) or (II), wherein R ¹ -R ² -R ³ -R ⁴ or R ⁴ -R ³ -R ² -R ¹ is one of tetrapeptides: HADD, DDAK, RADD, KAED, DEAR, KADE, HAEE and RAFE. These peptides correspond to the sequences SEQ.ID.NO.1, 4, 5, 9, 12, 15, 19 and 23, respectively. They have even lower dissociation constants, Kd, than the other peptides studied, as calculated in Examples 1 to 3.

More preferably, the peptide compound of the invention selected from compounds of formula (I) or (II), wherein R ¹ -R ² -R ³ -R ⁴ or R ⁴ -R ³ -R ² -R ¹ is one of the two tetrapeptides HAEE and RADD.

- R ¹ represents an amino acid residue of H,

- R ² represents the amino acid residue A,

- R ³ represents the amino acid residue E,

- R ⁴ is an amino acid residue of E.

The most preferred peptide compounds correspond to the formulas Ac-HAEE-NH ₂ and Ac-RADD-NH ₂ .

The dissociation constants of the interaction, Kd, of both of these peptides with the ligand corresponding to fragment 1-16 of the β-amyloid region, in Example 1, were the lowest of all peptides tested.

Both of these peptide compounds also appear as the most representative under conditions of inhibiting the binding of Zn (II) ions to a β-amyloid peptide and inhibiting the polymerization or aggregation of a β-amyloid peptide in the form of a plaque, in the presence of physiological concentrations of Zn (II) ions, in particular 100 to 400 μM, by binding the specified β-amyloid peptide, but, above all, both of these peptide compounds have the properties of inhibiting the formation of amyloid plaques in vivo in mouse models, the most representative flax are described in the Examples below.

The object of the present invention is the use of a peptide compound according to this invention for reducing or preventing the binding of Zn (II) ions to a β-amyloid peptide by binding the specified peptide compound to the specified β-amyloid peptide.

Even more specifically, it is an object of the present invention to use a peptide compound of the invention to reduce or prevent the polymerization and / or aggregation of a β-amyloid peptide in the presence of Zn (II) ions, more specifically at physiological concentrations of Zn (II) ions, features from 100 to 400 μM, by binding the specified peptide compound to the specified β-amyloid peptide.

Therefore, the present invention relates to the use of a peptide compound of the invention for the treatment of diseases associated with the formation of amyloid plaques.

The object of the present invention is the use of a peptide compound according to this invention as a medicine, in particular for the treatment of neurodegenerative diseases and, preferably, for the treatment of Alzheimer's disease.

The object of the present invention is also a suitable composition for use in accordance with this invention, in which the active substance contains at least one residue R ¹ -R ² -R ³ -R ⁴ or R ⁴ -R ³ -R ² -R ¹ peptide compounds according to this invention.

The active substance can be a multimer consisting of several monomers with the sequence R ¹ -R ² -R ³ -R ⁴ or R ⁴ -R ³ -R ² -R ¹ linked through bonds capable of degrading over time in the body and thereby release tetrapeptide, an active monomer.

In a known manner, the peptide compound of the invention may be formulated in the form of an ester or their physiologically acceptable derivative salts.

More specifically, an object of the present invention is a pharmaceutical composition comprising a compound according to this invention or an active substance containing at least one R ¹ -R ² -R ³ -R ⁴ or R ⁴ -R ³ -R ² -R ¹ residue of a peptide compound of the invention with a pharmaceutically acceptable carrier, preferably passing through a parenteral, transdermal or transmucosal pathway.

More specifically, the pharmaceutical composition according to this invention will be formulated to be injectable, especially for the intravascular, intramuscular, subcutaneous, intraspinal or cerebrospinal tract.

Other routes of administration, such as the oral, buccal, nasal, rectal or local route, may be provided provided that the peptide compound of the invention is formulated to withstand degradation until brain target cells are reached.

More preferably, said composition is in lyophilized form.

More specifically, in a composition according to the present invention, the concentration of said peptide compound is from 20 to 2000 μM. The following examples describe and demonstrate the effectiveness of the peptide compound of the invention, and are given by way of illustration only and should not be construed as limiting the invention.

In the light of the examples below, other features and advantages of the present invention will become apparent.

Figure 1 shows kinetic curves for the binding of the Ac-HAEE-NH ₂ peptide to a ligand corresponding to the 1-16 region of β-amyloid in various concentrations, i.e. curve A = 2 μM, curve B = 5 μM, curve C = 10 μM, curve D = 15 μM, curve E = 20 μM, curve F = 25 μM and curve G = 0 μM, with equipment and in accordance with the procedure of Example one.

FIG. from 2 to 9. Kinetic curves are presented for the binding of peptides of the sequence SEQ.ID.NO.1, 4, 5, 9, 12, 15, 19, and 23, respectively, with the equipment and according to the procedure of Example 3, the specified tetrapeptide should be more purified, than in Example 1.

Variations of the response units of the spectrometer on the ordinate axis (EO = response unit) over time make it possible to calculate the dissociation constant, Kd, bonds of the tetrapeptide with a fragment of region 1-16 of the β-amyloid of the peptide, as described in Examples 1 and 3.

Example 1: binding of Ac-HAEE-NH ₂ tetrapeptide (SEQ.ID.NO.19.) To an immobilized peptide fragment corresponding to the 1-16 region of the β-amyloid peptide.

The analysis was carried out using surface plasmon resonance spectroscopy (SPR) of the bonds of the Ac-HAEE-NH ₂ tetrapeptide with an immobilized peptide fragment corresponding to region 1-16 of the β-amyloid peptide.

Using this analysis, it is possible to calculate the dissociation constant, Kd, the interaction of the peptide with the immobilized ligand, it was clear that the Kd value is less, the higher the interaction with the ligand and, therefore, the stability of the resulting interaction complex is high. In these methods, it is believed that a significant specific interaction is present if the Kd is less than or equal to 10 ^-4 M. On the other hand, if the Kd greater than or equal to 10 ^-3 M, it is considered that there is no significant binding or interaction.

Surface plasmon resonance is a physical phenomenon, mainly known for its use as a method of measuring the ligand bond with a receptor adsorbed on the surface of a metal layer. The SPR detection system measures the variation of the refractive index in the immediate vicinity of the boundary when the ligand binds to the receptor.

The principles of surface plasmon resonance spectroscopy are described in Nagata, K. and Handa, H. (eds), Springer-Verlag, Tokyo, 2000.

More specifically, in an SPR spectroscopy system, for example, using biosensors from BIACORE, after ligands are immobilized on the surface of a chip-type sensor, in this case on the indicated β-amyloid fragment, the analytical reagent is introduced onto the indicated surface at a constant flow rate through the microfluidic channel system. Polarized light emitted from a light source is reflected from the gold-plated surface of the chip, and then detected by a diode reader. Accordingly, the binding of the ligand and the analytical molecule differs in the intensity of the reflected light. Changing the response of the spectrometer (EO = response unit) allows you to calculate the dissociation constant of the interaction.

Biosensor chips were obtained from GE Healthcare (USA). SPR spectroscopic experiments were performed on a BIACORE 3000 apparatus. Reagents for immobilizing a fragment of a β-amyloid peptide (EDC, NHS, PDEA and cysteine) were purchased from GE Healthcare (USA). The buffer used to immobilize the peptide ligand due to the formation of thiol bonds was 10 mM sodium acetate at pH 4.5. The regeneration buffer was an HBS buffer containing medium with 10 mM HEPES and 3 mM EDTA, 0.005% P20 surfactant and 150 mM NaCl at pH 7.4. The immobilization buffer was HBS buffer at pH 7.4 and HEPES buffer at pH 6.8, 50 mM, with 100 μM Zn (II) ions for the reactive tetrapeptide Ac-HAEE-NH ₂ . All buffers were filtered before use.

The ligand peptide fragment was: Ac-DAEFRHDSGYEVHHQKGGGGC-NH ₂ corresponding to the sequence SEQ.ID.NO.26. This 21 amino acid peptide contains 16 amino acids of the N-terminal amyloid β-binding domain, i.e. DAEFRHDSGYEVHHQK (SEQ.ID.NO.25) followed by 4 amino acids glycine and C-terminus cysteine. Cysteine is necessary to immobilize the ligand on the surface of the chip using thiol bonds, according to the supplier’s procedure. The 4 amino acids of glycine therefore form a binding tetrapeptide (“linker”) between the chip and the ligand. In addition, the immobilized ligand thus has sufficient flexibility with respect to the surface, so legitimately simulating the interaction conditions between the tetrapeptide reagent and the ligand. This ligand was bound to a CM5 biosensor chip following the standard procedure via thiol bonds under the conditions described in the sensor manual provided by GE Healthcare supplier (USA).

The flow rate used at all stages was 5 μl / min. The carboxymethyl dextran matrix was activated by adding a 1: 1 mixture of 1-ethyl-3 (3-dimethylaminopropyl) -carbodiimide (EDC) and N-hydroxysuccinimide (NHS), i.e. 30 μl of EDC per 400 mM and NHS per 100 mM, followed by 80 mM PDEA (2 (2 pyridinyl dithio) ethane amine) in a solution of 0.1 M sodium borate at pH 8.5. Then a peptide ligand was added in a buffer solution of sodium acetate in an amount of 0.05 mg / ml. Thiol groups that did not react on the surface of the CM5 chips were removed with a 50 mM cysteine solution in 0.1 M sodium acetate buffer solution at pH 4, so as to provide a surface that would give a variation of response units (EO) of about 700 conventional units with a control fluid sample. The control fluid interacts with the surface of the chip in accordance with the same procedure.

The Ac-HAEE-NH ₂ reagent was prepared by diluting 7 concentrations, i.e. 0, 2, 5, 10, 15, 20 and 25 μM, and introduced in multi-channel mode (20 μl QUICKINJECT, 5 μl / min). The surface of the chip was opened for 330 seconds for a reagent buffer solution in order to track its binding. The surface of the chip was then regenerated by introducing a regeneration buffer (20 μl). The data of the control strip was subtracted from the raw data for the bands corresponding to the immobilized peptides of the reagent. Further, the dissociation constant, Kd, was calculated in a known manner from the curves established for the equilibrium response (REQ) from the concentration (M) in the evaluation by the BIA 4.1 program. Then, 7 reagent samples at various concentrations were introduced into channels with immobilized ligands on the surface of the chip.

The kinetic binding curves of Ac-HAEE-NH ₂ to immobilized peptides, after subtracting control responses, are described in FIG.

The curves in FIG. 1 make it possible to calculate the dissociation constant, Kd, of the interactions, which were estimated at 2.2 × 10 ⁻⁸ M.

In the presence of physiological concentrations of Zn (II) ions (100-400 μM), the dissociation constant was almost unchanged. However, high concentrations of Zn (II) ions (10 mM) suppressed the interaction of the ligand with the reagent. If it is believed that the binding of Zn (II) ions to an Aβ peptide is characterized by micromolar (μM) Kd values, it can be assumed that the Ac-HAEE-NH ₂ reagent competes with Zn (II) ions for binding to the Aβ peptide. However, the affinity of the reagent with the Aβ peptide is significantly higher than that of Zn (II) ions, so that the Ac-HAEE-NH ₂ peptide prevents the binding of Zn (II) ions to the Aβ peptide at physiological concentrations.

More specifically, the affinity of the Ac-HAEE-NH ₂ reagent and Aβ amyloid is greater than that of Zn (II) ions.

The experiments showed that in the presence of physiological concentrations of Zn (II) ions at the micromolar level (less than 500 μM), HAEE binding to the 1-16 region of β-amyloid practically does not change in the presence or absence of Zn (II) ions. On the other hand, higher concentrations of Zn (II) ions, of the order of 1 mmol (mM), suppress the interaction between HAEE and the 1-16 region of Aβ. These data show that HAEE has a greater affinity for the target region than Zn (II) ions and is able to block the interaction between Zn (II) and Aβ ions when Zn (II) ions are in physiological concentrations.

Example 2: Comparative Example

The following describes the experiments for binding of the peptide target ligand to the corresponding 1-16 region of β-amyloid, carried out with other peptides in accordance with the procedure described in Example 1.

1 - All 23 other tetrapeptides listed in Table 1 correspond to the SEQ sequence of SEQ.ID.NO. from 1 to 18 and from 20 to 24 with the same protective groups R ^a = Ac and R ^b = NH ₂ were tested, and their dissociation constants, Kd, were from 10 ^-5 to 10 ^-6 M. These dissociation constants are practically did not change in the presence of zinc at physiological concentrations from 100 to 400 μM. The tetrapeptides of the invention having a low dissociation constant, Kd, (with the exception of HAEE) were: EDAR, EEAH, EEAK, EEAR, HADE, KADD, KAEE, RADD, RADE, RAEE, HADD, DDAK, KAED, DEAR and KADE .

These values correspond to strong bonds with the target ligand of the 1-16 region of the β-amyloid peptide.

2 - Other comparative examples with 10 different tetrapeptides, similar to the tetrapeptides from Table 1, but where one amino acid was replaced with alanine (A), i.e. The following tetrapeptides are indicated: A AEE , HAE A, HA A E , A ADD , HA A D , HAD A, A ADE , RAD A, RA A E , KA A E. the corresponding sequences are SEQ.ID.NO. from 29 to 38, respectively, had a significantly larger dissociation constant, i.e. Kd from 10 ^-3 to 10 ^-4 M, which corresponds to a nonspecific or insignificant interaction with the target ligand.

SEQ.ID.NO.29 taken from SEQ.ID.NO.23, in which the first amino acid R was replaced by A.

SEQ.ID.NO.30 is taken from SEQ.ID.NO.19, where the last amino acid E has been replaced by A.

SEQ.ID.NO.31 is taken from SEQ.ID.NO.19, where the third amino acid E has been replaced by A.

SEQ.ID.NO.32 is taken from SEQ.ID.NO.5, in which the first amino acid R was replaced by A.

SEQ.ID.NO.33 is taken from SEQ.ID.NO.7, in which the third amino acid E has been replaced by A.

SEQ.ID.NO.34 is taken from SEQ.ID.NO.1, in which the last amino acid D was replaced by A.

SEQ.ID.NO.35 is taken from SEQ.ID.NO.13, in which the first amino acid H was replaced by A.

SEQ.ID.NO.36 is taken from SEQ.ID.NO.5, in which the last amino acid D was replaced by A.

SEQ.ID.NO.37 is taken from SEQ.ID.NO.23, where the third amino acid E has been replaced by A.

SEQ.ID.NO.38 is taken from SEQ.ID.NO.21, in which the third amino acid E has been replaced by A.

3-10 different tripeptides corresponding to the tetrapeptide SEQ.ID.NO.19 (HAEE) were tested, where 1, 2 or 3 amino acids were deleted in accordance with the following sequences of SEQ.ID.NO. 59 to 68: AEE , HAO , S HA , G HA , EE Q, A HA , IA H , FS H , E SD, IS H. It should be noted that the AEE tripeptide corresponding to the complementary tripeptide from the 12-14 region in accordance with the criteria for complementarity has been previously described. These tripeptides are complementary to the regions of Aβ peptides 9-11, 10-12, 11-13, 12-14, 13-15 and 14-16.

These 10 different tripeptides have a dissociation constant from 10 ^-3 to 10 ^-4 M, which reflects the absence of significant or specific interaction with the target.

4 - In addition, 10 different hexapeptides were tested, including the tetrapeptide SEQ.ID.NO.19 (HAEE), preferably according to the invention, with the addition of 2 amino acids at their N-terminus or C-terminus, namely the following hexapeptides: HAEE SD, HAEE AD, HAEE SE, HAEE GE, HAEE QE, LA HAEE , IA HAEE , FS HAEE , LG HAEE , IS HAEE . These hexapeptides are complementary to the Aβ regions of peptide 11-16 and 9-14.

These are 10 hexapeptides that are repeated in the sequence list in SEQ.ID.NO. from 39 to 48, also have relatively high dissociation constants, Kd, from 10 ^-3 to 10 ^-4 M, which reflect the absence of significant or specific interaction with ligand 1-16 of the β-amyloid region.

All these 10 hexapeptides correspond to sequences for which 2 additional amino acids can interact with amino acids 15-16 (QK) of β-amyloid N-terminal extension and with amino acids GY of region 9-10 (GY) in β-amyloid, in accordance with ionic or hydrophobic interactions as previously described.

5 - Finally, 10 decapeptides taken from the sequence listing in the following sequence were tested SEQ.ID.NO. 49 to 58: HSLA HAEE SD, KNIA HAEE AD, RNFS HAEE SE, KQLG HAEE GE, RQIS HAEE QE, DDHSLA HAEE , EEKNIA HAEE , EERNFS HAEE , DEKQLG HAEE , DERQIS HAEE , and they have dissociation constants, Kd, from 10 ^-3 to 10 ^-4 M, which reflect the absence of a significant or specific interaction with the target.

These decapeptides again took the preferred tetrapeptide of the invention, SEQ.ID.NO.19 (HAEE), with 2 additional amino acids at the N-terminus and 4 additional amino acids at the C-terminus for SEQ.ID.NOS. from 49 to 53, and 6 additional amino acids for decapeptides SEQ.ID.NO. 54 to 58. All decapeptides of SEQ.ID.NO. from 49 to 58, containing additional amino acids, are defined as capable of interacting with amino acids of regions 7-10 and 15-16 of β-amyloid in SEQ.ID.NO. from 49 to 53, and regions 5-10 of β-amyloid in SEQ.ID.NO. 54 to 58, which target the 5-14 Aβ peptide region.

All peptides tested in Sections 1- and 5- above included a protective group for the function of the N-terminal amine, R ^a = Ac and a protective group of the hydroxyl function of the C-terminal carboxyl group, R ^b = NH ₂ .

Example 3: Binding of tetrapeptides of the invention to an immobilized peptide fragment corresponding to region 1-16 of a 6-amyloid peptide.

The binding tests described in Example 1 were reproduced with the same materials, reagents and procedures, with the exception of the following significant differences:

- The BIACORE apparatus used was the BIACORE T100 apparatus. This BIACORE T100 gives more accurate results than the BIACORE 3000 because it includes a degassing system during analyte injection on the sensor and because the method for detecting the intermolecular complexes of the immobilized ligand / analyte on the CM5 chip is more accurate.

- the used tetrapeptides according to this invention were purified to at least 99% of the L-isomers in accordance with this HPLC, prevented the formation of the D-isomers and / or removed them during the synthesis. In Example 1, tetrapeptides included only 98% of the L. isomers.

- the applied concentrations of tetrapeptides according to this invention were 50, 100, 200, 500, 1000, 1500 and 2000 μM.

- These experiments were carried out in the absence of zinc.

- the surface of the CM5 chips provided a surface that gave variations in response units (EO) of about 1023 conventional units with the test control fluid.

The kinetics curves in FIG. from 2 to 9, allow us to calculate the dissociation constants, Kd, in accordance with the method described in previous publications (in particular, see reference [39]), i.e. the curves allow you to calculate kon and koff constants by the formula:

dR / dt = kon C (Rmax-R) -koff R, where:

- R represents the answer on the ordinate

- C represents the concentration of the analyte

- Rmax is theoretically the maximum value of R,

Kd dissociation constant was given by the formula Kd = koff / kon.

Table 2 shows the values of dissociation, Kd calculated for tetrapeptides from the sequences SEQ.ID.NO 1, 4, 5, 9, 12, 15, 19 and 23, which have the lowest values.

table 2 Analit Kd × 10 ⁴ M k _on × M ^-1 s ^-1 k _off × 10 ^-3 s ^-1 SEQ.ID.NO.1 32.5 ± 0.9 3.20 ± 0.08 10.40 ± 0.02 SEQ.ID.NO.4 13.7 ± 0.5 4.57 ± 0.09 6.2 ± 0.1 SEQ.ID.NO.5 0.13 ± 0.02 18.5 ± 0.1 e1 0.24 ± 0.04 SEQ.ID.NO.9 33 ± 7 5.7 ± 0.9 19 ± 1 SEQ.ID.NO.12 19.8 ± 0.7 3.58 ± 0.08 7.09 ± 0.09 SEQ.ID.NO.15 45 ± 3 2.2 ± 0.1 9.6 ± 0.2 SEQ.ID.NO.19 0.9 ± 0.3 0.37 ± 0.05 0.035 ± 0.008 SEQ.ID.NO.23 77 ± 6 1.5 ± 0.1 11.20 ± 0.08

In this case, two analytes containing the sequences SEQ.ID.NO.5 and 19 had the lowest dissociation constants, Kd, of between about 10 ^-4 and 10 ^-5 M.

The remaining six tetrapeptides from the sequences SEQ.ID.NO.4, 9, 12, 15, and 23 gave Kd values between 10 ⁻³ and 10 ⁻² M.

Other tetrapeptides, according to this model, showed dissociation constants, Kd, of more than 10 ^-2 M.

Example 4: Suppression of the formation of β-amyloid plaques by polymerization or aggregation.

The aggregation of Aβ peptides as amyloid plaques was demonstrated in vitro using the SYNTHALOID SCREENING PLATE ® kit from QUALITY CONYTROLLED BIOCHEMICALS Inc. USA, including the use of plaques coated with crystallization centers of Aβ peptides and peptides labeled with a fluorescent marker to identify aggregates (37, 38).

The complete β peptide 1-40, labeled with a fluorescent marker, was used in a buffer containing 50 mM HEPES, pH 7.4, 0.1% BSA and 10% FCS, this buffer contained physiological concentrations of Zn (II) ions, and inhibitors protease. These plaques were in the form of plates, including 96-well plates, into which 100 μl of Aβ-labeled peptides (1-40) were poured at a concentration of 100 nM. They were left to incubate for 3 hours at room temperature in the wells.

In some wells, the tetrapeptides AC-GAEE-NH ₂ and Ac-RADD-NH ₂ , respectively, of this invention, were introduced in different concentrations depending on the wells (0, 5, 10, 20, 40, 80, 100 and 150 μM), to test the effect of tetrapeptides of the invention on the aggregation of Aβ peptides.

To do this, at the end of the incubation, unbound proteins were removed from the wells using three washes using the buffer solution indicated above. The amount of bound protein in the form of aggregates associated with the wells was measured by measuring the fluorescence intensity.

The HAEE and RADD tetrapeptides of the invention, in fact, inhibit the aggregation of the Aβ peptide, with inhibitory concentrations of IC _{50 of} 20 μM. It should be recalled that the IC ₅₀ concentration is the concentration at which the aggregation of 50% of the Aβ peptide introduced into the wells can be prevented.

According to the tests described in the SYNTHALOID SCREENING PLATE ® Board User Guide, it is believed that the compound inhibits protein binding on a solid support if its IC ₅₀ concentration is less than 100 μM.

Studies over time show that prolonged incubation for at least 1 hour of amyloid aggregates with HAEE and RADD tetrapeptides at a concentration of 20, 40, 80, 100 and 150 μM further leads to irreversible disaggregation of amyloid aggregates.

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Claims (21)

1. The peptide compound of General formula (I) or (II)
R ^a -R ¹ -R ² -R ³ -R ⁴ -R ^b (I) or R ^a -R ⁴ -R ³ -R ² -R ¹ -R ^b (II),
Where
- R ^a represents the N-terminal primary amine group of the amino acid R ¹ or R ⁴ , either free or substituted by an amino protecting group,
- R ^b represents a hydroxyl group of the C-terminal carboxyl group of amino acid R ¹ or R ⁴ , either free or substituted with a hydroxy protecting group, and
- R ¹ -R ² -R ³ -R ⁴ or R ⁴ -R ³ -R ² -R ¹ are HADD, KADD, DDAK, RADD, DDAR, KAED, DEAK, RAED, DEAR, HADE, EDAH, KADE, EDAK, RADE, EDAR, HAEE, EEAH, KAEE, EEAK, RAEE and EEAR,
wherein R ^{a of the} EDAK peptide contains said amino protecting group and R ^{b of the} EDAK peptide contains said hydroxy protecting group. 2. The peptide compound of claim 1, wherein the hydroxy group of the C-terminal carboxyl group of the peptide is substituted with a protecting group selected from: —NH ₂ , —NHR, —NRR, where R is C ₁ -C ₄ alkyl, and —OR, where R represents C ₁ -C ₄ alkyl or alkylamine. 3. The peptide compound according to claim 1, wherein the N-terminal amino group of the peptide is substituted with a protecting group selected from the formyl group (HCO-) and the acetyl group (CH ₃ CO-). 4. The peptide compound of claim 1, wherein the protecting groups are compatible with in vivo pharmaceutical use. 5. The peptide compound according to claim 1, selected from compounds of formula (I) or (II), where R ¹ -R ² -R ³ -R ⁴ or R ⁴ -R ³ -R ² -R ¹ are HAEE, EDAR , EEAN, EEAK, EEAR, HADD, HADE, KADD, KAEE, RADD, RADE, RAEE, DDAK, KAED, DEAR or KADE. 6. The peptide compound according to claim 5, selected from compounds of formula (I) or (II), where R ¹ -R ² -R ³ -R ⁴ or R ⁴ -R ³ -R ² -R ¹ are one of the tetrapeptides HADD, DDAK, RADD, KAED, DEAR, KADE, HAEE and RAEE. 7. The peptide compound according to claim 5, selected from compounds of formula (I) or (II), where R ¹ -R ² -R ³ -R ⁴ or R ⁴ -R ³ -R ² -R ¹ are one of two tetrapeptides NAEE and RADD. 8. The peptide compound according to claim 5, where the amino acids R ¹ , R ² , R ³ and R ⁴ are in the form of natural isomers L and R ^a and R ^b contain protective groups for all tetrapeptides R ^a -R ¹ -R ² -R ³ -R ⁴ -R ^b (I) or R ^a -R ³ -R ⁴ -R ¹ -R ² -R ^b (II). 9. The peptide compound according to claim 1, corresponding to the formula Ac-HAEE-NH ₂ or Ac-RADD-NH ₂ . 10. A medicament for reducing or preventing the binding of Zn (II) ions to a β-amyloid peptide, containing the peptide compound of claim 1 as an active substance. 11. A composition for reducing or preventing the binding of Zn (II) ions to a β-amyloid peptide, containing an active substance containing at least the peptide compound of claim 1, the purity of which is at least 98%. 12. A pharmaceutical composition for reducing or preventing the binding of Zn (II) ions to a β-amyloid peptide, comprising an effective amount of the drug of claim 10, with a pharmaceutically acceptable carrier. 13. The pharmaceutical composition according to p. 12, containing a pharmaceutically acceptable carrier for parenteral, transdermal or transmucosal administration. 14. The composition of claim 13, wherein the carrier is suitable for intravascular, intramuscular, subcutaneous, intraspinal or cerebrospinal administration. 15. A method of reducing or preventing the binding of Zn (II) ions to a β-amyloid peptide, comprising binding the peptide compound of claim 1 to said β-amyloid peptide. 16. A method of reducing or preventing the polymerization and (or) aggregation of a β-amyloid peptide in the presence of Zn (II) ions, comprising binding the peptide compound of claim 1 to said β-amyloid peptide. 17. A method for the therapeutic treatment of diseases in which the formation of amyloid plaques is established, comprising administering to the patient a pharmaceutical composition according to claim 12. 18. The method according to p. 17 for the treatment of neurodegenerative diseases. 19. The method according to p. 18 for the treatment of Alzheimer's disease. 20. The method of claim 17, wherein said peptide compound comprises HAEE or RADD. 21. The method of claim 20, wherein the peptide compound is Ac-HAEE-NH ₂ or Ac-RADD-NH ₂ .

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