WO2023099725A1 - Pharmaceutical preparation containing a mixture of a natriuretic peptide (anp) and a peptidic inhibitor of the cxcr4 receptor - Google Patents

Abstract

The invention relates to a pharmaceutical preparation containing a mixture of at least one natriuretic peptide (ANP) with at least one peptidic inhibitor of the CXCR4 receptor for treating vascular diseases, organ infarctions, peripheral circulatory disturbances, renal insufficiency, muscle degeneration, rheumatic disorders, tumor diseases, degenerative-genetic or acquired diseases, dermatological diseases or inflammatory skin syndromes and bone diseases.

Description

Pharmaceutical preparation containing a mixture of a natriuretic peptide (ANP) and a peptide inhibitor of the CXCR4 receptor

The subject matter of the present invention is a pharmaceutical preparation containing a mixture of at least one natriuretic peptide (ANP) with at least one peptide inhibitor of the CXCR4 receptor as effective components for the treatment of diseases.

The pharmaceutical composition according to the invention enables a surprisingly effective treatment of vascular diseases, organ infarctions, peripheral circulatory disorders, renal insufficiency, muscular degeneration, rheumatic diseases, tumor diseases, degenerative-genetic or acquired diseases, dermatological diseases or inflammatory skin syndromes and bone diseases due to a synergistic effect of its active components. The active components of the pharmaceutical composition according to the invention are at least one natriuretic peptide (ANP) and at least one peptidic inhibitor of the CXCR4 receptor, which are present in a mixture in the pharmaceutical composition according to the invention.

In one embodiment of the invention, vascular diseases selected from the group consisting of acute and chronic heart failure (ADHF, CHF) can be treated with the pharmaceutical composition according to the invention.

In another embodiment of the invention, organ infarctions selected from the group consisting of infarctions of the heart and/or the brain can be treated with the pharmaceutical composition according to the invention.

In a further embodiment of the invention, rheumatic disorders selected from the group consisting of juvenile rheumatoid arthritis can be treated with the pharmaceutical composition according to the invention.

In yet another embodiment of the invention, the pharmaceutical composition according to the invention can be used to treat tumors selected from the group consisting of CXCR4-dependent cancers and blood cancers.

In another embodiment of the invention, degenerative-genetic or acquired diseases selected from the group consisting of diseases of the nervous system and the sensory organs (AML, MS) can be treated with the pharmaceutical composition according to the invention.

In yet another embodiment of the invention, dermatological diseases selected from the group consisting of skin tumors can be treated with the pharmaceutical composition according to the invention.

In another embodiment of the invention, inflammatory skin syndromes and bone diseases selected from the group consisting of osteoporosis or tumor metastases can be treated with the pharmaceutical composition according to the invention.

The pharmaceutical preparation according to the invention can contain the natriuretic peptide (ANP) in an amount from 2.0 ng/kg/min to 64.0 ng/kg/min or from 0.4 mg/75 kg/d mg to 6.4 mg/75 kg /d mg per dose unit and/or the peptidic inhibitor of the CXCR4 receptor in an amount of 50 mg/75kg/d to 1000 dose units. The ANP and the peptidic inhibitor of the CXCR4 receptor are preferably present in the stated ranges of amounts.

The natriuretic peptide (ANP) present in the preparation according to the invention can be selected from the group consisting of ANF, ANP, urodilatin and their variants, mutants and derivatives.

In particular, such peptides with the following sequences should be mentioned as natriuretic peptides:

ANF 4-23 (SEQ ID No 1):

Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys

ANP-28 (SEQ ID No 2):

Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser- Phe-Arg-Tyr (SEQ ID No. 3) :

Thr-Ala-Pro-Arg-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-

Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr

The peptide inhibitor of the CXCR4 receptor present in the preparation according to the invention can be selected from the group consisting of peptides with the peptides mentioned in SEQ ID No. 4 to SEQ ID No. 19 and their variants, mutants and derivatives.

LVRYTKKVPQVS (SEQ ID No 4)

IVRYTKKVPQVS (SEQ ID No 5)

IVRWTKKVPQVS (SEQ ID No 6)

IVRWTCKVPQVS (SEQ ID No 7)

IVRWSKKVPCVS (SEQ ID No 8)

IVRWSKKVPQVS (SEQ ID No 9)

IMRWSRKMPCVS (SEQ ID No. 10)

ILRWSRKLPCVS (SEQ ID No. 11)

ILRWSRKMPCVS (SEQ ID No. 12)

ILRWTRKMPCVS (SEQ ID No. 13)

ILRWSRKMPCMS (SEQ ID No. 14)

ILRWSRKFPCVS (SEQ ID No. 15)

ILRWSRKMPCFS (SEQ ID No. 16)

ILRWSRKMPQFS SEQ ID No 17)

IVRWSKKMPQVS (SEQ ID No. 18)

LVRYTQKAPQVS (SEQ ID No 19)

In another embodiment of the invention, the natriuretic peptide (ANP) or peptide inhibitor of the CXCR4 receptor is a therapeutically active fragment, mutant and/or derivative of the natriuretic peptide or peptide inhibitor of the CXCR4 receptor.

Variants of the CXCR4 receptor natriuretic peptide or peptide inhibitor also include modified forms of the polypeptide, as well as its mutants or derivatives. Modified natriuretic peptides or peptidic inhibitors of the CXCR4 receptor are polypeptides in which one or more amino acids of the native sequence have been altered such that a non- naturally occurring amino acid residue is present in the polypeptide chain. In principle, such modifications can arise or be undertaken during or after protein translation and include, for example, phosphorylation, glycosylation, sulfonation, crosslinking, acylation or also proteolytic cleavage.

In addition to fragments of the natriuretic peptide or the peptide inhibitor of the CXCR4 receptor, its derivatives, variants and fragments are also suitable as agents for the diseases mentioned, provided they cause an inhibition of the infection with coronavirus that is comparable to the native natriuretic peptide or peptide inhibitor of the CXCR4 receptor .

Typically, amino acid substitutions can be made in a conservative manner. A conservative replacement is understood to mean a mutation in which one codon is replaced by another. This encodes a different amino acid that is chemically related to the original amino acid, such as glycine with alanine or threonine with serine. On the other hand, a non-conservative exchange occurs when the original codon is replaced by a codon that codes for an amino acid with different chemical properties, for example glycine with lysine.

The expression "substantially identical" or similar expressions used in connection with two polypeptides refers to two or more sequences or subsequences which have at least a 70%, in particular at least 80%, in particular at least 90%, in particular have at least 95% sequence identity if they are compared for maximum agreement using a sequence comparison algorithm and a-ligned. Substantial identity exists in particular if a sequence region that is at least 40-60 amino acids in length, in particular 60 -80 amino acids, in particular more than 90-100 amino acid residues and in particular is essentially identical over the entire length of the amino acid sequence of the native polypeptide.

The terms "identical" or percent "identical" in the context of two or more polypeptides refers to two or more sequences or subsequences that are the same or a specified percentage of amino acids that are identical are exhibited if they are compared for maximum agreement and "aligned" using a sequence comparison algorithm. Optimized alignment for comparing sequences can be carried out, for example, using the following algorithms: local homology alignment by Smith & Waterman, Adv. Appl. Math. 2:482 (1981), Homology Alignment Algorithm of Needleman & Wunsch, J Mol Biol 48:443 (1970), Pearson & Lipman, Proc Nat'l Acad Sci USA 85:2444 (1988), (GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), but also visual examination [compare, Current Protocols in Molecular Biology, (Ausubel, FM et al., eds.) John Wiley & Sons, Inc., New York (1987-1999), including Supplement 46 (April 1999)].

Fragments of the polypeptide can typically be obtained by cleaving the polypeptide chain of the natriuretic peptide or peptidic inhibitor of the CXCR4 receptor. Methods for fragmentation by cleaving the polypeptide chain are known to those skilled in the art.

In this way, polypeptides can be split by enzymes, so-called proteases. Depending on whether the proteases split polypeptides (proteins) within the amino acid chain or at the end, a distinction is made between proteinases (also called endopeptidases). They break down proteins within the amino acid chain. They usually recognize specific sequence sections within a protein that they can attack and cleave there.

On the other hand, exoproteases (also called exopeptidases) split off individual amino acids from the ends of the protein. In contrast to the proteinases, they mainly break down smaller peptides and not proteins. One distinguishes:

Carboxypeptidases, which cleave the amino acids from the carboxyl end (C-terminus), and aminopeptidases, which cleave the amino acids from the amino end (N-terminus).

Specific proteases cleave the peptide bond between two specific amino acids; in some cases, they also recognize more than one substrate. These proteases are used, for example, to examine the relationship between proteins, to prepare proteins for sequencing or to isolate active domains of a protein. Table 1 below shows some examples of such proteases: Table 1: Specific proteases and their properties

In contrast to the specific proteases, non-specific proteases cleave the peptide chain before or after a whole series of amino acids and thus generate much smaller cleavage fragments. Table 2 below shows some examples of such non-specific proteases:

Tab. 2: Non-specific proteases and their properties

In addition to proteases, certain chemicals can also be used for the proteolytic analysis of peptides. Cyanogen bromide (BrCN) is the most important reagent of this type and cleaves proteins on the C-terminal side of methionine residues. This produces peptidyl homoserine lactone. The resulting peptide fragments can then be separated in the polyacrylamide gel and visualized by staining. Tab. 3: Typical reagents for chemical proteolysis and their properties

A special case of proteolysis is the so-called limited proteolysis. In contrast to total hydrolysis, the protein digestion in this method is not complete, but takes place under precisely defined reaction conditions (proteolysis is therefore limited). In the globular regions of a native protein, the peptide bonds are far less exposed than the peptide bonds on the protein's surface. This means that if a protease acts for a short time or if the protease concentration is very low, the individual protein domains may first be separated before the protease also reaches cleavage sites located further inside. The limited proteolysis is thus carried out with a protease dilution and/or suboptimal reaction conditions and stopped after a short time. In this method, the protein is proteolytically cleaved in its native form (and not after denaturation); this sometimes has consequences for the choice of protease. For example, if the protein requires EDTA for optimal stability, a metalloprotease cannot be used for digestion.

Further information on protein analysis and the procedure for the proteolytic cleavage of polypeptides can be found in the textbook "Working methods of biochemistry" by Alfred Pingoud, Claus Urbanke, Walter de Gruyter, for example chapter 5.1.6.

According to the invention, the pharmaceutical preparation according to the invention can be liquid or solid.

According to the invention, the pharmaceutical preparation according to the invention can be formulated in such a way that it can be administered intravascularly, lymphatically, intracardially, epicutaneously, intracutaneously and subcutaneously, intranasally, locally and into the liquor space.

The natriuretic peptide or the peptidic inhibitor of the CXCR4 receptor can be present in an essentially aqueous solution, in particular in an aqueous solution with pharmaceutical excipients. The excipients can be added individually or in combination not only to the natriuretic peptide or peptidic inhibitor of the CXCR4 receptor but also to a final formulation. The excipients can be added at various points in the pharmaceutical preparation of the medicinal product.

It may be advisable to stabilize the natriuretic peptide or peptide inhibitor of the CXCR4 receptor against aggregation or oligomerization. If necessary, a bacteriostatic agent such as benzyl alcohol, a surfactant such as Tween 20, an isotonic agent such as mannitol, one or more stabilizing amino acids such as lysine or arginine, and an antioxidant may also be included in the formulation.

In another embodiment, the polypeptide is formulated for parenteral, intravenous, intramuscular, intranasal, inhaled, topical, or buccal administration. Infusion solutions, ointments, sprays and other forms of application of polypeptides known to the pharmaceutical chemist are particularly suitable as galenic formulations. It can be useful to administer the polypeptide bound to a suitable carrier or contained in liposomes.

sequence listing

SEQ ID No 1

Arg Ser Ser Cys Phe Gly Gly Arg Met Asp Arg Ile Gly Ala Gin Ser Gly Leu Gly Cys

SEQ ID No 2

Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Met Asp Arg Ile Gly Ala Gin Ser Gly

Leu Gly Cys Asn Ser Phe Arg Tyr

SEQ ID No 3

Thr Ala Pro Arg Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Met Asp Arg Ile Gly

Ala Gin Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr

SEQ ID No 4

Leu Val Arg Tyr Thr Lys Lys Val Pro Gin Val Ser

SEQ ID No 5

Ile Val Arg Tyr Thr Lys Lys Val Pro Gin Val Ser

SEQ ID No 6

Ile Val Arg Trp Thr Lys Lys Val Pro Gin Val Ser

SEQ ID No 7

Ile Val Arg Trp Thr Cys Lys Val Pro Gin Val Ser

SEQ ID No 8

Ile Val Arg Trp Ser Lys Lys Val Pro Cys Val Ser

SEQ ID No 9

Ile Val Arg Trp Ser Lys Lys Val Pro Gin Val Ser

SEQ ID No 10

Ile Met Arg Trp Ser Arg Lys Met Pro Cys Val Ser

SEQ ID No 11

Ile Leu Arg Trp Ser Arg Lys Leu Pro Cys Val Ser

SEQ ID No 12

Ile Leu Arg Trp Ser Arg Lys Met Pro Cys Val Ser

SEQ ID No 13

Ile Leu Arg Trp Thr Arg Lys Met Pro Cys Val Ser SEQ ID No 14

Ile Leu Arg Trp Ser Arg Lys Met Pro Cys Met Ser

SEQ ID No 15

Ile Leu Arg Trp Ser Arg Lys Phe Pro Cys Val Ser SEQ ID No 16

Ile Leu Arg Trp Ser Arg Lys Met Pro Cys Phe Ser

SEQ ID No 17

Ile Leu Arg Trp Ser Arg Lys Met Pro Gin Phe Ser

SEQ ID No 18 Ile Val Arg Trp Ser Lys Lys Met Pro Gin Val Ser

SEQ ID No 19

Leu Val Arg Tyr Thr Gin Lys Ala Pro Gin Val Ser

Claims

patent claims 1. Pharmaceutical preparation containing a mixture of at least one natriuretic peptide (ANP) with at least one peptidic inhibitor of the CXCR4 receptor for use in the treatment of vascular diseases, organ infarctions, peripheral circulatory disorders, renal insufficiency, muscle degeneration, rheumatic diseases, tumor diseases, degenerative-ge- genetic or acquired diseases, dermatological diseases or inflammatory skin syndromes and bone diseases. 2. Pharmaceutical preparation according to claim 1, wherein the vascular disease is selected from the group consisting of acute and chronic heart failure (ADHF, CHF). 3. Pharmaceutical preparation according to claim 1 or 2, wherein the organ infarctions are selected from the group consisting of infarctions of the heart and/or brain. 4. Pharmaceutical preparation according to at least one of claims 1 to 3, wherein the rheumatic condition is selected from the group consisting of juvenile rheumatoid arthritis. 5. Pharmaceutical preparation according to at least one of claims 1 to 4, wherein the tumor is selected from the group consisting of CXCR4-dependent types of cancer and blood cancer. 6. Pharmaceutical preparation according to at least one of claims 1 to 5, wherein the degenerative-genetic or acquired diseases is selected from the group consisting of diseases of the nervous system and the sensory organs (AML, MS). 7. Pharmaceutical preparation according to at least one of claims 1 to 6, wherein the dermatological disease is selected from the group consisting of skin tumors. 8. Pharmaceutical preparation according to at least one of claims 1 to 7, wherein the inflammatory skin syndromes and bone diseases are selected from the group consisting of osteoporosis or tumor metastases. 9. Pharmaceutical preparation according to at least one of claims 1 to 8, wherein the natriuretic peptide (URO) in an amount of 2.0 ng / kg / min to 64.0 ng / kg / min and the peptidic inhibitor EPI of the CXCR4 eceptors in an amount of 50 mg/75 kg/d to 1000 mg/75 kg/d. 10. Pharmaceutical preparation according to at least one of claims 1 to 9, wherein the natriuretic peptide (ANP) is selected from the group consisting of peptides with the amino acid sequences given in SEQ ID No 1 to SEQ ID No 3 and their variants, mutants and derivatives. 11. Pharmaceutical preparation according to at least one of claims 1 to 10, wherein the peptidic inhibitor of the CXCR4 receptor is selected from the Group consisting of peptides with the amino acid sequences given in SEQ ID No. 4 to SEQ ID No. 19 and their variants, mutants and derivatives. 12. Pharmaceutical preparation according to at least one of claims 1 to 11, wherein the preparation is liquid or solid. 13. Pharmaceutical preparation according to claim 12 for intravascular, lymphatic, intracardial application, epi-, intra- and subcutaneous, intranasal, local injection and in the liquor space.