HK1127069B - Antagonists against interaction of pf4 and rantes - Google Patents

Abstract

The invention relates to polypeptides of amino acid sequence SEQ ID NO: 1 according to formula (1), the use thereof for producing a medicament, and medicaments for the treatment of diseases related to monocyte recruitment.

Description

Antagonists for inhibiting the interaction of PF4 and RANTES

The invention relates to a polypeptide, a pharmacologically acceptable salt, a derivative and/or a conjugate thereof, and an application thereof in preparing medicines. The polypeptides can be used for the preparation of medicaments for diseases associated with monocyte recruitment.

Arteriosclerosis of arterial vessels constitutes the morphological background of cardiovascular diseases. In this case, the primary monocyte recruitment is decisive for the development of early lesions in arteriosclerosis. The immobilization of monocytes on the endothelium, the so-called monocyte blockade, manifests symptoms such as arteriosclerosis, vascular stenosis and thrombosis in the early stages of the pathology of cardiovascular diseases. Chemokines such as RANTES (which regulate the activation of normal T cell expression and secretion) are known to be involved in this process as signaling molecules.

Primary and secondary prevention known in the art can primarily function to lower blood lipids and inhibit platelet aggregation and activation through the use of drugs such as aspirin and clopidogrel. The disadvantage of the therapeutic methods using these drugs is that, on the one hand, they show only little specificity, and, on the other hand, they have serious side effects, such as myopathy and a high risk of bleeding.

Furthermore, it is known in the prior art to use RANTES-polypeptides as antagonists. For example, DE 10014516 a1 discloses the use of metRANTES as an antagonist for inhibiting RANTES receptor CCR 1. The disadvantage of using such antagonists is that chemokines are involved in a large number of physiological processes as signaling molecules, so that the use of such antagonists can have unpredictable effects on physiological processes and can exhibit a large number of side effects and serious consequences.

It is an object of the present invention to provide a therapeutic agent which overcomes at least one of the many disadvantages of the prior art. The invention aims to provide a therapeutic drug with better specificity.

The object of the present invention can be achieved by a polypeptide, a pharmacologically acceptable salt, derivative and/or conjugate thereof, wherein the polypeptide has an amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence of which is:

C-X₁-X₂-YFYTS-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-X₁₅-C

(1)(SEQ IDNO：1)

wherein:

X₁selected from the group comprising lysine, glutamine, arginine, histidine and/or asparagine, or an amino acid deletion;

X₂selected from the group comprising glutamic acid, aspartic acid and/or glutamine, or an amino acid deletion;

X₃selected from the group comprising glycine, serine and/or alanine;

X₄selected from the group comprising lysine, leucine and/or arginine;

X₅selected from the group comprising serine, cysteine, glycine and/or threonine;

X₆selected from the group comprising serine, glycine and/or threonine;

X₇selected from the group consisting of asparagineGroups of amines and/or glutamines;

X₈selected from the group comprising proline, tyrosine and/or glycine;

X₉selected from the group comprising glycine, alanine and/or serine;

X₁₀selected from the group comprising isoleucine, valine and/or asparagine;

X₁₁selected from the group comprising valine, isoleucine and/or asparagine;

X₁₂selected from the group comprising phenylalanine, tyrosine, isoleucine, valine, leucine and/or methionine;

X₁₃selected from the group comprising isoleucine, valine, leucine, methionine and/or phenylalanine;

X₁₄selected from the group comprising threonine, glycine, alanine, serine and/or tyrosine;

X₁₅selected from the group comprising arginine, lysine, glutamine, histidine and/or asparagine, or an amino acid deletion.

Advantageously, the polypeptides of the invention are suitable as antagonists for inhibiting the interaction of platelet factor 4 and RANTES.

The concept of "antagonist for inhibiting the interaction between platelet factor 4 and RANTES" can be understood by the polypeptide, protein or other compound of the invention, which acts as an antagonist to inhibit the interaction between platelet factor 4 and the chemokine RANTES.

It has surprisingly been found that the polypeptides of the invention have a particular effect on monocyte recruitment associated with the interaction of platelet factor 4(PF4) and the chemokine RANTES. The polypeptide of the invention has the advantage, in particular, that it has no or only a small effect on the numerous functions of the chemokines. The polypeptide of the invention is particularly advantageous in that it selectively blocks the recruitment of monocytes, for example on the endothelium.

The concept of "monocyte recruitment" in the present invention means the engraftment and explantation of monocytes on the endothelium, the fixation of monocytes and the spread of monocytes, for example, by division in the endothelium. Similarly, the fixation of monocytes is likewise manifested in the form of adhesion of monocytes or in the form of monocyte blockade, which in physiological cases can lead to shear flow, for example in capillaries, capillaries or arterial vessels.

The polypeptides of the invention have the great advantage of high specificity and of having no or only minor side effects on numerous metabolic processes, such as the immune or coagulation system, which are associated with the chemokines RANTES and PF 4. In particular, the risk of bleeding which occurs in conventional pharmacological treatment of cardiovascular diseases can be avoided by pharmacological therapy with the polypeptides of the invention.

In the present invention, "C" represents an amino acid letter code of cysteine which is generally used, and the corresponding "Y" represents tyrosine, "F" represents phenylalanine, "T" represents threonine, and "S" represents serine.

The polypeptide of the invention has a cysteine residue at both the amino-terminus and the carboxy-terminus, which may form a loop of the polypeptide. The advantage of the invention is, inter alia, that the cyclized polypeptide has a better stability. The polypeptides of the invention have a longer onset of action and are therefore used in smaller amounts.

By "polypeptide" is meant the use of synthetic or non-synthetic peptidic compounds, such as pure or modified natural protein fragments, natural forms or recombinant peptides or proteins. Also within the meaning of "polypeptide" according to the invention are pharmacologically acceptable salts, pharmacologically acceptable derivatives and/or conjugates of the corresponding polypeptide.

Preferred pharmacologically acceptable derivatives are, for example, esters, amides, N-acyl-and/or O-acyl derivatives, carboxylated polypeptides, acetylated polypeptides, phosphorylated polypeptides and/or glycosylated polypeptides. Preferred conjugates are e.g. sugar-or polyethylene glycol-conjugates, biotinylated, radioactive or fluorescently labeled polypeptides.

Advantageously, the length of the polypeptide of the invention does not exceed a maximum of 25 amino acids. The number of amino acids of the polypeptides of the invention is preferably 15 to 25 amino acids, particularly preferably 15 to 22. It was experimentally determined that the length of the polypeptide had an effect on its effect. It is particularly surprising that amino acid sequences having such a length have an inhibitory effect on monocyte arrest.

In a preferred embodiment the number of amino acids of the polypeptide of the invention is from 18 to 23, further preferred from 18 to 22, in a very preferred embodiment the number of amino acids of the polypeptide of the invention is from 19 to 22, in a further preferred embodiment the number of amino acids is from 20 to 21. The polypeptide of the invention is particularly preferably 22 amino acids. The meaning of "number of amino acids" in the polypeptide of the invention of course means the length of the amino acid sequence of the polypeptide.

In a preferred embodiment of the polypeptide of the invention X₁Is lysine, or X in the amino acid sequence₁Amino acid deletions in position, particularly preferred is X₁Is lysine. Furthermore, in a preferred embodiment of the polypeptides of the invention glutamic acid is suitable for X₂Or an amino acid deletion at that position. Particularly preferred is X₂Is glutamic acid. Surprisingly in X₁And/or X₂Polypeptides with amino acid deletions in position are capable of exerting an antagonistic effect. X₁And X₂The position is suitably arranged as an amino acid deletion.

X₃Small, neutral and flexible amino acids are preferred. In a preferred embodiment of the polypeptide of the invention X₃Selected from the group comprising glycine and/or serine, glycine being particularly preferred. It was determined that this amino acid had a positive effect on the structural stability of the polypeptide. A particular advantage of such an increased stability is that it may allow for an increased stability of the polypeptide such that the interaction time of the polypeptide and the chemokine PF4 may be such thatTo be lengthened. Thus, the improved stability of the polypeptide may enhance the antagonism of the polypeptide.

In a preferred embodiment of the polypeptide of the invention X₄Is lysine.

X₅Preferably from the group comprising serine, glycine and/or threonine. In a preferred embodiment of the polypeptide of the invention X₅Is serine. Embodiments of such polypeptides may improve the solubility of the polypeptide in an advantageous manner. The improved solubility of the polypeptide may in particular improve the applicability of the polypeptide in water. This makes it possible to simplify the administration of the polypeptide, i.e. the administration usually based on water. Furthermore, the dispersibility of the polypeptide in an aqueous system in vivo, particularly in blood, can be improved by increasing the solubility of the polypeptide.

X₆Preferably serine. In a further preferred embodiment of the polypeptide, X₇Is asparagine.

X₈Are preferably amino acids from the group comprising proline and/or tyrosine. X₈Proline is particularly preferred.

In a particularly preferred embodiment of the polypeptide of the invention X₉Is glycine. It was determined that glycine at this position of the amino acid sequence can make the polypeptide ligation stability very surprising.

X₁₀And X₁₃Each independently preferably from the group comprising valine and/or isoleucine. In a preferred embodiment of the polypeptide of the invention X₁₀Is isoleucine. In a further preferred embodiment of the polypeptide of the invention, X₁₃Is isoleucine. In a particularly preferred embodiment of the polypeptide of the invention, X₁₀And X₁₃Are all isoleucine. It can be confirmed that X is present in the polypeptide₁₀And X₁₃The isoleucine in (b) helps to improve the stability of the conformation of the polypeptide.

X₁₁Valine is preferred. X₁₂Preferably from the group comprising phenylalanine and/or tyrosine,X₁₂phenylalanine is particularly preferred. X₁₄Threonine is preferred.

X₁₅Preferably arginine, furthermore in X₁₅Amino acid deletions may also be present at positions. X₁、X₂And/or X₁₅Preferably amino acid deletions. Preferred is X₁、X₂Or X₁₅One of the amino acids is removed, likewise X₁And X₂All positions may be provided as amino acid deletions. In a further embodiment of the polypeptide of the invention, X₁、X₂And X₁₅Can be arranged as amino acid deletions. In a preferred embodiment of the polypeptide of the invention, from X₃To X₁₄Is set to have no amino acid deletion.

In a preferred embodiment of the polypeptide of the invention, X₅Is serine, X₉Is glycine, X₁₀Is isoleucine, and/or X₁₃Is isoleucine. In a preferred embodiment of the polypeptide of the invention, X₅Is serine, X₉Is glycine, X₁₀Is isoleucine, X₁₃Is isoleucine. It can be confirmed that X₅Is serine, X₉Is glycine, X₁₀Is isoleucine, X₁₃The polypeptide which is isoleucine has a very excellent antagonistic ability.

In a further preferred embodiment of the polypeptide of the invention, X₅Is serine, X₉Is glycine, X₁₀Is isoleucine, X₁₃Is isoleucine, X₁Is an amino acid deletion, and/or X₁₅Is an amino acid deletion. In a very preferred embodiment of the polypeptide of the invention, X₅Is serine, X₉Is glycine, X₁₀Is isoleucine, X₁₃Is isoleucine, X₁Is an amino acid deletion, X₁₅Is an amino acid deletion. The polypeptide thus arranged also has very excellent antagonistic ability.

In another preferred embodiment of the polypeptide of the invention, X₅Is a filamentAmino acid, X₈Is tyrosine, X₉Is glycine, X₁₀Is isoleucine, X₁₃Is isoleucine, X₁Is an amino acid deletion, and/or X15 is an amino acid deletion. In a very preferred embodiment of the polypeptide of the invention, X₅Is serine, X₈Is tyrosine, X₉Is glycine, X₁₀Is isoleucine, X₁₃Is isoleucine, X₁Is an amino acid deletion, X₁₅Is an amino acid deletion.

In another preferred embodiment of the polypeptide of the invention, X₃Is serine, X₅Is serine, X₈Is tyrosine, X₉Is glycine, X₁₀Is isoleucine, X₁₃Is isoleucine, X₁Is an amino acid deletion, and/or X₁₅Is an amino acid deletion. In a very preferred embodiment of the polypeptide of the invention, X₃Is serine, X₅Is serine, X₈Is tyrosine, X₉Is glycine, X₁₀Is isoleucine, X₁₃Is isoleucine, X₁Is an amino acid deletion, X₁₅Is an amino acid deletion.

In another preferred embodiment of the polypeptide of the invention, X₃Is serine, X₅Is serine, X₈Is proline, X₉Is glycine, X₁₀Is isoleucine, X₁₃Is isoleucine, X₁Is an amino acid deletion, X₂Is an amino acid deletion, and/or X₁₅Is an amino acid deletion. In a very preferred embodiment of the polypeptide of the invention, X₃Is serine, X₅Is serine, X₈Is proline, X₉Is glycine, X₁₀Is isoleucine, X₁₃Is isoleucine, X₁Is an amino acid deletion, X₂Is an amino acid deletion, X₁₅Is an amino acid deletion.

In a further preferred embodiment of the polypeptide of the invention. X₃Is serine, X₅Is serine, X₈Is cheeseAmino acid, X₉Is glycine, X₁₀Is isoleucine, X₁₂Is tyrosine, X₁₃Is isoleucine, X₁Is an amino acid deletion, X₂Is an amino acid deletion, and/or X₁₅Is an amino acid deletion. In a very preferred embodiment of the polypeptide of the invention, X₃Is serine, X₅Is serine, X₈Is tyrosine, X₉Is glycine, X₁₀Is isoleucine, X₁₂Is tyrosine, X₁₃Is isoleucine, X₁Is an amino acid deletion, X₂Is an amino acid deletion, X₁₅Is an amino acid deletion.

In a further preferred embodiment of the polypeptide of the invention, it may be so arranged that the polypeptide of the invention has the amino acid sequence shown in SEQ ID NO: 2:

CKEYFYTSGKCSNPAVVFVTRC(2)(SEQ ID NO：2)

and/or relative to SEQ ID NO: 2, according to SEQ ID NO of structural formula (2): 2 has at least one or several amino acid deletions, amino acid substitutions and/or amino acid insertions.

Amino acid substitutions preferably relate to the range of sequences selected from SEQ ID NOs: 2, i.e., a sequence ranging from glycine at position 9 to threonine at position 20. It is preferred that one or several amino acids selected from the group comprising glycine, cysteine, proline, valine, phenylalanine and/or alanine are replaced by amino acids selected from the group comprising serine, tyrosine, isoleucine and/or glycine. One advantage of such amino acid substitutions is that the stability of the polypeptide can be enhanced and/or the antagonistic effect of the polypeptide can be improved.

Such amino acid substitutions are preferably selected from those comprising glycine for serine, non-terminal cysteine for serine, proline for tyrosine, valine for isoleucine, phenylalanine for tyrosine, and/or alanine for glycine. Such amino acid substitutions may be arbitrarily mixed with each other.

Particularly preferred is at least one selected from the group consisting of SEQ ID NOs: 2 by substituting serine for the non-terminal cysteine at position 11 of the amino acid sequence of seq id No. 2. Such an alternative has the advantage that the solubility of the polypeptide, in particular in water, can be improved.

Particularly preferred is a peptide selected from the group consisting of SEQ ID NO: 2, in an embodiment thereof, the amino acid substitution is preferably selected from the group consisting of amino acid substitutions at positions 9 to 14 of glycine to serine, cysteine to serine, and/or proline to tyrosine, i.e. SEQ ID NO: 2 from glycine to proline. Preferably at least one such substitution is included in the sequence range, preferably at least the amino acid sequence of SEQ ID NO: 2 by a serine. Furthermore in a further preferred embodiment proline is replaced by tyrosine and/or glycine is replaced by serine. The substitution of cysteine in position 11 of the amino acid sequence can be combined with any other amino acid substitution, in particular with the substitution of proline by tyrosine and/or glycine by serine.

Also preferred is an embodiment of the invention selected from the group consisting of SEQ ID NOs: 2, the amino acid substitution of which involves an amino acid at position 15 to 20, i.e., from alanine to threonine. Amino acid substitutions in this amino acid range are preferably selected from the group comprising alanine for glycine, valine for isoleucine and/or phenylalanine for tyrosine. It is particularly preferred that alanine is replaced by glycine. Preferably, at least one selected from the group consisting of SEQ ID NOs of structural formula (2): 2 by isoleucine, particularly preferably at least two valines, wherein preferably the valines at positions 16 and 19 are replaced by isoleucine. It may also be preferred that each is selected from the group consisting of SEQ ID NOs of structural formula (2): 2 is replaced with isoleucine. In a further preferred embodiment of the invention phenylalanine may be replaced by tyrosine.

In a further embodiment of the invention it is preferred that the amino acid sequence selected from SEQ ID NOs of structural formula (2): 2 wherein the alanine at position 15 is replaced with glycine, the valine at position 16 is replaced with isoleucine, and the valine at position 19 is replaced with valine. It was determined that such amino acid substitutions resulted in very surprising stability of the linkage of the polypeptide.

The amino acid substitutions and/or amino acid deletions may be in any combination with each other, in particular the amino acid substitutions may be in any combination with each other.

In addition, the polypeptides of the invention may also have embodiments with amino acid deletions. Preferred amino acid deletions include lysine, glutamic acid and/or arginine. The amino acid deletion preferably relates to SEQ ID NO: 2, lysine at position 2, glutamic acid at position 3, and/or arginine at position 21. The amino acid deletions may be in any combination with each other. Preferably, one amino acid is deleted, and it may be so arranged that both lysine at position 2 and arginine at position 21 are deleted. In further embodiments, lysine at position 2, glutamic acid at position 3, and arginine at position 21 may be deleted.

Selected from the group consisting of SEQ ID NOs of structural formula (2): 2, and the amino acid range from 4 th to 20 th is preferably set to have no amino acid deletion. In a particularly preferred embodiment, there is neither amino acid deletion nor amino acid substitution in the amino acid range from position 4 to 8. Selected from the group consisting of SEQ ID NOs of structural formula (2): 2, preferably 3 amino acid substitutions, more preferably 4 amino acid substitutions, and even more preferably 5 amino acid substitutions in the amino acid range from 9 th to 20 th positions, and it can be confirmed through studies that such an amino acid sequence has excellent antagonistic properties.

In such embodiments, the number of amino acids of the polypeptide is advantageously from 15 to 25, preferably from 18 to 23, particularly preferably from 19 to 22, further preferably from 20 to 21, and especially preferably 22 amino acids.

The polypeptide of the present invention, a physiologically acceptable salt, derivative and/or conjugate thereof, a very suitable embodiment thereof, has an amino acid sequence represented by the following structural formula (3): 3, and the amino acid sequence of:

CKEYFYTSGKSSNPGIVFITRC(3)(SEQ ID NO：3).

it was confirmed by the study that the peptide has the amino acid sequence shown in SEQ ID NO: 3 has a very high antagonistic potential. In particular, it was confirmed that such a polypeptide had a very excellent antagonistic effect. It can be determined in particular that the peptide having the amino acid sequence of SEQ ID NO according to structural formula (3): 3 has a significant and reproducible inhibitory effect on the interaction between RANTES and PF4 which leads to an enhanced monocyte blockade.

Of particular advantage, has the amino acid sequence according to SEQ ID NO: 3 is particularly effective for the interaction of RANTES and PF 4. Further interference with chemokine function can thus be reduced or even eliminated. This makes it possible to make the polypeptides of the invention very useful for targeting diseases associated with monocyte recruitment or RANTES-dependent recruitment of other leukocyte species, such as eosinophils, in particular cardiovascular diseases.

Another preferred embodiment of the polypeptide of the invention, its physiologically acceptable salts, derivatives and/or conjugates, is a polypeptide having the amino acid sequence of SEQ ID NO: 4 in the sequence:

CEYFYTSGKSSNPGIVFITC(4)(SEQ ID NO：4).

another preferred embodiment of the polypeptide of the invention, its physiologically acceptable salts, derivatives and/or conjugates, is a polypeptide having the amino acid sequence of SEQ ID NO: 5 in the sequence:

CEYFYTSGKSSNYGIVFITC(5)(SEQ ID NO：5).

another preferred embodiment of the polypeptide of the invention, its physiologically acceptable salts, derivatives and/or conjugates, is a polypeptide having the amino acid sequence of SEQ ID NO: 6 in sequence:

CEYFYTSSKSSNYGIVFITC(6)(SEQ ID NO：6).

another preferred embodiment of the polypeptide of the invention, its physiologically acceptable salts, derivatives and/or conjugates, is a polypeptide having the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of:

CYFYTSSKSSNPGIVFITC(7)(SEQ ID NO：7).

an advantage of the polypeptides of the preferred embodiments is that such polypeptides preferably have enhanced stability. This makes it possible to allow a large number of polypeptides to reach the target site and stably interact with the protein or peptide conjugate. The high stability of the polypeptide makes it possible in particular to use the polypeptide both in vivo and in vitro. Another advantage of the polypeptides of the preferred embodiments is that the polypeptides have a higher solubility in water. The higher solubility in particular makes the polypeptide simple and convenient to use.

The following can also be provided: each L-amino acid is replaced by D-lysine. This may further improve the stability of the polypeptide.

The polypeptides of the invention are prepared according to the general synthetic methods of polypeptides.

Advantageously, the polypeptides of the invention are suitable as antagonists for inhibiting the interaction of platelet factor 4 and RANTES. The polypeptide of the invention can particularly inhibit the interaction of RANTES and platelet factor 4.

The polypeptides of the invention, their physiologically acceptable salts, derivatives and/or conjugates are suitable as antagonists for inhibiting the interaction of RANTES and platelet factor 4.

Based on the excellent properties of the polypeptide of the present invention, it is suitable for use as a medicament.

Another object of the invention relates to the use of a polypeptide according to the invention, i.e.in the manufacture of a medicament, especially in a preferred embodiment.

The present invention may be administered in the usual manner, preferably parenterally, for example orally, transdermally, subcutaneously and/or intravenously. For example, the polypeptides of the invention may be administered in vitro, such as prior to vascular bypass grafting, or intravenously, such as prior to or following catheter intervention or stent implantation.

For such applications, it is of great advantage to have very good solubility in water.

In addition to temporary or acute treatment, the polypeptides of the invention are also advantageous in that they can be administered over a longer period of time. The polypeptides of the invention may likewise be administered in one of extended release or delayed release forms. For example in the form of a long-acting injection or osmotic pump.

The polypeptides of the invention may likewise be administered in the form of nucleic acids encoding each polypeptide. The nucleic acid molecules are contained in the usual genetic vectors. Preferably in the form of a DNA sequence encoding each polypeptide. It may also be arranged to administer the polypeptide in the form of an RNA sequence encoding the polypeptide of the invention.

Another subject of the invention is a nucleic acid encoding a polypeptide of the invention, comprising a nucleic acid sequence preferably comprising a nucleotide sequence encoding a polypeptide having the sequence of SEQ ID NO: 1. SEQ ID NO according to structural formula (2): 2. SEQ ID NO according to structural formula (3): 3. SEQ ID NO according to structural formula (4): 4. SEQ ID NO according to structural formula (5): 5. SEQ ID NO according to structural formula (6): 6. SEQ ID NO according to structural formula (7): 7, or a polypeptide of the amino acid sequence of seq id No. 7. As nucleic acids for use in the present invention, they relate to DNA and RNA. The skilled person is well aware of DNA sequences encoding polypeptides according to the above formulae or sequences.

Encoding a polypeptide having the amino acid sequence of SEQ ID NO according to structural formula (3): 3 has the amino acid sequence of SEQ ID NO: 8, DNA sequence:

5′-TGCAAGGAATATTTCTACACTTCCGGGAAATCCTCCAATCCTGGAATTGTGTTCATCACTAGATGT-3′(8)(SEQ ID NO：8).

the skilled person is familiar with another encoding sequence having the sequence of SEQ ID NO according to structural formula (3): 3 amino acid sequence of the polypeptide. It is also known that changes in the sequence of a nucleic acid are permissible, for example, by the degeneracy of the genetic code.

Encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2, an example of a nucleic acid molecule having a polypeptide of amino acid sequence has the amino acid sequence shown in SEQ ID NO: 9, DNA sequence:

5′-TGCAAGGAATATTTCTACACTTCCGGGAAATGTTCCAATCCTGCCGTGGTGTTCGTCACTAGATGT-3′(9)(SEQ ID NO：9).

encoding a polypeptide having the amino acid sequence of SEQ ID NO according to structural formula (4): 4 has the amino acid sequence shown in the following structural formula (10): 10, DNA sequence:

5′-TGCGAATATTTCTACACTTCCGGGAAATCCTCCAATCCTGGAATTGTGTTCATCACTTGT-3′(10)(SEQ ID NO：10).

encoding a polypeptide having the amino acid sequence of SEQ ID NO according to structural formula (5): 5, an example of a nucleic acid molecule having a polypeptide of amino acid sequence has the amino acid sequence shown in SEQ ID NO: 11, DNA sequence:

5′-TGCGAATATTTCTACACTTCCGGGAAATCCTCCAATTACGGAATTGTGTTCATCACTTGT-3′(11)(SEQ ID NO：11).

encoding a polypeptide having the amino acid sequence of SEQ ID NO according to structural formula (6): 6 has the amino acid sequence of the following structural formula (12): 12, the DNA sequence:

5′-TGCGAATATTTCTACACTTCCTCTAAATCCTCCAATTACGGAATTGTGTTCATCACTTGT-3′(12)(SEQ ID NO：12).

encoding a polypeptide having the amino acid sequence of SEQ ID NO according to structural formula (7): 7 has the amino acid sequence of SEQ ID NO: 13, the DNA sequence:

5′-TGCTATTTCTACACTTCCTCTAAATCCTCCAATCCTGGAATTGTGTTCATCACTTGT-3′(13)(SEQ ID NO：13).

the skilled person is familiar with other nucleic acids encoding SEQ ID NO according to structural formula (1): 1. SEQ ID NO according to structural formula (2): 2. SEQ ID NO according to structural formula (3): 3. SEQ ID NO according to structural formula (4): 4. SEQ ID NO according to structural formula (5): 5. SEQ ID NO according to structural formula (6): 6. SEQ ID NO according to structural formula (7): 7, or a DNA sequence of a polypeptide of the amino acid sequence of (1). Likewise, the skilled artisan is familiar with the coding sequences of SEQ ID NOs according to structural formula (1): 1. SEQ ID NO according to structural formula (2): 2. SEQ ID NO according to structural formula (3): 3. SEQ ID NO according to structural formula (4): 4. SEQ ID NO according to structural formula (5): 5. SEQ ID NO according to structural formula (6): 6. SEQ ID NO according to structural formula (7): 7, or a pharmaceutically acceptable salt thereof.

Preferred dosages for the individual administration of the polypeptides of the invention range from 10 mg/day/75 kg body weight to 1000 mg/day/75 kg body weight, very preferably from 50 mg/day/75 kg body weight to 200 mg/day/75 kg body weight, and especially preferably 150 mg/day/75 kg body weight.

The polypeptides, their physiologically acceptable salts, derivatives and/or conjugates and/or the nucleic acids according to the invention are suitable for the therapeutic and/or prophylactic treatment, diagnosis and/or therapy of diseases which are associated with monocyte recruitment. Such as cardiovascular diseases and/or inflammations, in particular arteriosclerosis, stenosis, hypertension and/or graft rejection reactions, belong to this group of diseases.

The polypeptides and/or nucleic acids of the invention are suitable for use in the treatment of mammals, particularly humans.

The polypeptides of the invention have an effect on the adhesion of monocytes to the endothelium. It has surprisingly been found that particularly preferred embodiments of the polypeptides of the invention are able to inhibit the enhancement of monocyte arrest caused by the heterophilic interaction of RANTES and PF 4. Advantageously, a particularly preferred embodiment shows in the experiments a stronger inhibition of the increase in monocyte arrest caused by the heterophilic interaction of RANTES and PF4 than the hitherto known protein or peptide conjugates.

A particular advantage of the polypeptides of the invention can be achieved by administration of the polypeptides of the invention in such a way that the development of arteriosclerosis, postoperative or post-administration restenosis, for example after balloon dilatation, atherectomy or bypass surgery, can be reduced or eliminated. A particular advantage of the polypeptides of the invention is that they can reduce or eliminate further recruitment of monocytes to the endothelium at end-stage, or clinical disease or changes in morphological arteriosclerosis.

A particular advantage of the polypeptides of the invention is that they are particularly suitable for the prophylactic treatment of patients threatened by, for example, hypertension. It would be advantageous to use a prophylactic treatment which makes it possible to obtain polypeptides of the invention which have no or only a small side effect on all chemokine-related processes.

Another subject of the present invention accordingly relates to the use of a polypeptide according to the invention, of a physiologically acceptable salt, derivative and/or conjugate thereof, i.e. in the manufacture of a medicament for the treatment and/or prevention of a disease selected from the group comprising:

diseases associated with monocyte recruitment, such as cardiovascular diseases and/or inflammation, in particular arteriosclerosis, atherosclerosis, atheromatous plaque, stenosis, restenosis, hypertension, arthritis, myocarditis, autoimmune diseases including encephalomyelitis, enteritis, reperfusion after an infarction, for example a myocardial or cerebral infarction, transplant rejection and/or skin diseases, such as psoriasis, and/or

Diseases associated with RANTES-dependent recruitment of other leukocyte species such as eosinophils, especially allergic diseases such as asthma or pneumonia.

Especially when used for treating human atherosclerotic lesions, the polypeptide of the invention has good effect on the course of the disease. The polypeptides of the invention can attenuate the worsening of arteriosclerotic lesions, in particular by monocyte blockade. Another advantage of the polypeptides of the invention is that rejection reactions after organ and/or tissue transplantation can be reduced or even eliminated.

The polypeptides of the invention are particularly advantageous in that they have no or only very few side effects. This makes it possible to use the polypeptides of the invention for disease prevention. The polypeptides of the invention are furthermore advantageous, inter alia, in that, owing to their specificity, effects on further metabolic processes can be avoided, so that, for example, prophylactic administration to patients at risk of hypertension or to the prevention of arteriosclerotic lesions is possible.

Another subject of the invention relates to a medicament comprising a polypeptide of the invention, preferably comprising the polypeptide of SEQ ID NO: 1, a physiologically acceptable salt, derivative and/or conjugate thereof. The medicament of the invention particularly preferably comprises a nucleotide sequence selected from the group consisting of SEQ ID NO according to structural formula (3): 3. SEQ ID NO according to structural formula (4): 4. SEQ id no according to structural formula (5): 5. SEQ ID NO according to structural formula (6): 6. SEQ ID NO according to structural formula (7): 7, or a pharmaceutically acceptable salt thereof. The medicament of the invention particularly preferably comprises the amino acid sequence of SEQ ID NO according to structural formula (3): 3. The inventive medicament preferably has a polypeptide according to one of the above structural formulae or SEQ ID numbers, and the inventive medicament can be configured to have a polypeptide according to several structural formulae or SEQ ID numbers.

Medicaments comprising a polypeptide of the invention are particularly suitable for in vivo treatment of e.g. humans. A preferred use of a medicament comprising a polypeptide of the invention is in the treatment and/or prevention of diseases associated with monocyte recruitment, such as cardiovascular disease and/or inflammation, in particular arteriosclerosis, atherosclerosis, atheroma, stenosis, restenosis, hypertension, arthritis, myocarditis, autoimmune diseases including encephalomyelitis, enteritis, reperfusion following infarction, e.g. myocardial or cerebrovascular infarction, transplant rejection and/or skin diseases, such as psoriasis, and/or diseases associated with the recruitment of other leukocyte species, such as eosinophil-dependent RANTES, in particular allergic diseases, such as asthma or pneumonia.

Also subject of the invention are medicaments comprising a nucleic acid encoding a polypeptide of the invention. The nucleic acid molecules are contained in the usual genetic vectors.

Another subject of the present invention relates to a medicament for inhibiting monocyte blockade comprising a polypeptide of the invention, a physiologically acceptable salt, derivative and/or conjugate thereof, preferably comprising the amino acid sequence of SEQ ID NO: 3.

By "a drug inhibiting monocyte blockade" is meant in the present invention that the drug has a positive effect on the disease associated with the blockade of monocytes, e.g. monocytes fixed to the endothelium. In particular, the formation of atherosclerotic plaques may be reduced or even eliminated. The use of the polypeptides of the invention may preferably reduce and/or completely or almost completely eliminate monocyte recruitment and/or its fixation to active endothelium, in particular to atherosclerotic plaques or endocardium.

Examples for illustrating the invention are as follows:

materials and methods

Cell culture

Endothelial cells from human umbilical cord (HUVEC, human umbilical vessel endothiapells, promoCell, Hamburg, Germany) were cultured in endothelial cell growth medium (promoCell, Hamburg, Germany) using cells from 2 to 4 passages later.

Monocyte Mono Mac 6-cells (MM6, DSMZ) were cultured in RPM 11640 medium (PAA laboratories, Pascheng (Pasching), Austria) supplemented with 10% fetal bovine serum, 2mML glutamine (Biowhittaker), 1mM sodium pyruvate, 50. mu.g/ml gentamicin and 9. mu.g/ml insulin (MM6 medium). The concentration is 2 x 10⁵ml of cells were seeded in 2ml MM6 medium in 24-well plates and at 37 ℃ with 5% CO before the cells were used in the experiment₂For 3 to 4 days in a humid atmosphere.

Polypeptides

SEQ ID NO according to structural formula (3): 3 according to SEQ ID NO of structural formula (15): 15 and a mouse-ortholog of an amino acid sequence according to SEQ ID NO: 14 by a t-Boc-based solid-phase chemical synthesis method of a polypeptide, using a 4-benzylhydroxylamine resin. The molecular weight of the polypeptide was determined by electrospray mass spectrometry (Dawson PE, Kent SB. (2000) Annu RevBiochem.69: 923-.

Example 1

Plasmon resonance studies can be used to analyze a sample comprising SEQ ID NO according to structural formula (3): 3 on the formation of heterogeneous aggregation of RANTES and PF 4.

The plasmon resonance studies can be performed using HBS-EP buffer (10mM HEPES, 150mM NaCl, 0.005% Tween 20, pH 7.4).

The two cell flow chambers of the C1 chip (Biacore AB, Uppsala, Sweden) were activated by injection of 50. mu.l ethyl (dimethylaminopropyl) carbodiimide/N-hydroxy-succinimide (0.2M/0.05M, Pierce) and immediately 20. mu.l streptavidin was perfused onto its surface. Next, 20. mu.l of ethylenediamine (1M, pH8, Sigma-Aldrich) was injected onto the cell surface 4 consecutive times to inactivate it.

N-terminally biotinylated human platelet factor 4(bPF4) was synthesized by the t-Boc-based peptide solid phase chemical synthesis method and native chemical ligation of PF4 (Dawson PE, KentSB. (2000) Annu Rev biochem.69: 923-. The bPF4 was immobilized on the dextran surface of the C1 sensor chip, where bPF4 at 200. mu.g/ml in HBS-EP buffer was injected onto the flow cell surface and a nuclear magnetic unit (RU) was used at 240. The second flow chamber, which was not treated with bPF4, served as a control.

RANTES (0.5 μ M, recombinant human RANTES, Peprotech, Rocky Hill, NJ, USA), or RANTES (0.5 μ M) at various concentrations of 0 μ M, 10 μ M, 50 μ M and 100 μ M and a nucleic acid molecule comprising SEQ ID NO according to structural formula (3): 3 amino acid sequence was preincubated in HBS-EP buffer overnight at room temperature, 15 μ l each of the peptide/RANTES mixture was injected into the flow chamber at bPF4, and binding of RANTES was observed for 180 seconds. The binding process and the test were both carried out by means of a Biacore2000 apparatus (Biacore AB) at a flow rate of 5. mu.l/min. The RANTES-bound sensorgrams were corrected for non-specific background signals by BIAevaluation 3.0 software (Biacore AB) and equilibrium nuclear magnetic units (RU) were determined for each injection.

The results show that SEQ ID NO: 3 has a concentration-dependent inhibitory effect on the interaction of RANTES and PF4, wherein the combination of RANTES on immobilized PF4 is found in SEQ ID NO: 3 by a concentration of 100 μ M in the presence of the polypeptide of amino acid sequence 3.

Example 2

Plasmon resonance studies can be used to analyze the molecular mass of a polypeptide having the sequence of SEQ ID NO according to structural formula (3): 3, SEQ ID NO according to structural formula (2): 2 and a regulatory peptide for its inhibitory effect on the formation of heterogeneous aggregations of RANTES and PF 4.

In a further experiment, the combination of RANTES (0.5. mu.M) was combined under the conditions described in example 1 with different concentrations of the peptide comprising SEQ ID NO according to formula (3) of either 0. mu.M, 10. mu.M, 50. mu.M and 100. mu.M: 3. SEQ ID NO according to structural formula (2): 2, or SEQ ID NO according to structural formula (14) as shown below: 14 for the study:

KEYFYTSGK(14)(SEQ ID NO：14)

it was found that in this experiment, at polypeptide concentrations of 10. mu.M, 50. mu.M and 100. mu.M, the peptide has the amino acid sequence of SEQ ID NO according to structural formula (3): 3 compared to a polypeptide having the amino acid sequence of SEQ ID NO according to structural formula (2): 2 has more obvious and effective inhibition effect on the interaction of RANTES and PF 4. Having the amino acid sequence of SEQ ID NO according to structural formula (14): 14 at a concentration of 100. mu.M, showed no demonstrable inhibitory effect.

Example 3

Inhibition of monocyte blockade on active endothelium.

The interaction of the monocyte Mono Mac 6-cells on the active endothelial cells was studied.

A petri dish containing a confluent HUVEC-cell layer activated by IL-1 β (interleukin 1 β, Peprotech, 10ng/ml, 12 hours) was placed in the flow chamber. Mono Mac 6 cells (0.5X 10)⁶Cells/ml) were resuspended in pH 7.3 standard Hank solution (HBSS solution with 10mM Hepes) containing 0.5% bovine serum albumin (Serva) and kept on ice. Ca was added to MM6 monocytes 5 minutes before the experiment²⁺And Mg²⁺And the chemokines RANTES (Peprotech, Rocky Hill, NJ, USA) and PF4(ChromaTec, Greifswald) at final concentrations of 1mM and 60nM, respectively, and 6. mu.MHaving the amino acid sequence of SEQ ID NO according to structural formula (3): 3. SEQ ID NO according to structural formula (2): 2, a polypeptide having an amino acid sequence according to SEQ ID NO: 14, and warmed to 37 ℃. The cells thus pretreated were then incubated at 1.5dyn/cm²Is perfused onto the endothelium under shear force by means of a microscope of type IX50 from the company Olympus. The number of monocytes that adhere by interacting with endothelial cells will be determined by using a video camera (3CCD, JVC) and a recorder under multiple viewing angles. The obtained data were averaged (n ═ 5) ± (p < 0.02) to evaluate the control effect.

It can be confirmed that SEQ ID NO: 3, has a significant inhibitory effect, for example up to 80%, on the enhancement of the heterophilic interaction of RANTES and PF4, whereas the polypeptide of SEQ ID NO: 2 is weak in the inhibitory effect of the polypeptide. SEQ ID NO of structural formula (14): 14 has no significant inhibitory effect compared to the control peptide.

Example 4

In vivo study of arteriosclerotic mouse model

Female ApoE-/-littermates of 9 to 12 weeks old (The Jackson Lab, Bar Harbor, Maine, USA) were used as a model for arteriosclerosis. These mice were fed high fat (21% Fett; Altromin) for 12 weeksC1061) In that respect Two groups of mice were injected intraperitoneally three times a week during this period with 50 μ g of a saline solution of SEQ id no: 15, or a polypeptide of the amino acid sequence of seq id no,

CKEYFYTSSKSSNLAVVFVTRC(15)(SEQ ID NO：15)

(n-12 mice) or SEQ ID NO according to structural formula (14) as shown below: 14 of the amino acid sequence of seq id no:

KEYFYTSGK(14)(SEQ ID NO：14)

(n-7 mice). A group of untreated mice (n-12) served as controls.

Mice were sacrificed for histological studies. Mice were healthy and showed no signs of disease during the experiment. Blood samples were drawn from the start of the experiment to the end of the experiment. White blood cell counts were determined by cytometry and serum was collected and cholesterol levels were determined by the infinite cholesterol instrument (ThermoElectron, Melbourne, australia).

The degree of arteriosclerosis was determined at the aortic root and thoracoabdominal aorta, where lipid deposits were quantified by oil red-O-dye staining (Veillard NR, Kwak B, Pelli G, Mulhaupt F, James RW, Proudfoot AE, Mach F. antaginism of RANTES receiver reduction in microbiological research Res. 2004; 94: 253-61) and with the aid of computer image analysis (Dikus software, Hilgers, Aachen). Atherosclerotic lesion areas were identified in 5 μm transverse sections through the heart and aorta. For each aortic root, the atherosclerotic lesion area was determined by means of lipid stained areas of 6 sections at a distance of 50 μm from each other. The atherosclerotic lesion areas are distributed on the stained surface of each section. The thoraco-abdominal aorta was opened along the abdominal midline and the lesion area was stained with oil red-O-dye under frontal dissection. The amount of lipid deposits was calculated, wherein the area of the stained area was estimated by averaging the total area of thoracic abdominal aorta staining.

It was confirmed that the amino acid sequence represented by SEQ ID NO of structural formula (15): 15 and SEQ ID NO of structural formula (3): 3, the progression of atherosclerotic lesions is significantly reduced compared to mice treated with the regulatory peptide. It was also confirmed that the aortic root region, which showed atherosclerotic plaques, had a significant attenuation effect relative to the total valve surface in the treated mice. It was also confirmed that macrophages in atherosclerotic lesions were also significantly reduced.

It can thus be shown that the amino acid sequence of SEQ ID NO according to structural formula (3): 3 can slow the progression of atherosclerosis in vivo, and thus the polypeptides of the invention can be used in clinical therapy.

Claims (5)

1. A polypeptide consisting of SEQ ID NO: 15, amino acid sequence composition:

CKEYFYTSSKSSNLAVVFVTRC(15)(SEQ ID NO.15)。

2. the polypeptide of claim 1, wherein the polypeptide is an ester or an amide.

3. A medicament comprising a polypeptide according to claim 1 or a pharmacologically acceptable salt thereof.

4. Use of a polypeptide according to claim 1 or a pharmacologically acceptable salt thereof in the manufacture of a medicament for the treatment of atherosclerosis.

5. A nucleic acid encoding the polypeptide according to claim 1.