Polypeptides for the treatment of stress syndrome, immune response syndrome and stroke syndrome - Patents.com

Abstract

The amino acid sequence, written in one-letter code, is represented by the following general formula: Z 1 X 1 X 2 X 3 X 4 X 5 QX 7 APX 10 X 11 SZ 3 A polypeptide having the formula: X 1 = V, M or L, in particular L; X 2 = V, L or M, in particular V; X 3 =R, K or C, in particular R; X 4 = Y or W, in particular Y, X 5 = S or T, in particular T, X 7 =K, C or R, in particular K; X 10 =Q or C, in particular Q, X 11 = V, M or F, in particular V, and, Z 3 =0 or Z 4 and Z 4 = 0, or the C-terminal carboxyl group of the peptide chain is modified, and this modification, together with the carboxyl group of the C-terminal amino acid of the peptide, is -C(O)-OR 1 Structure or -C(O)-NR 2 R 3 forming a moiety having the structure R 1 is a substituted or unsubstituted alkyl group, aryl group, aralkyl group, cycloalkyl group, and heterocycloalkyl group.

Description

[Technical Field]

[0001] The present invention relates to polypeptides and therapeutic uses of polypeptides, especially for the treatment of stress syndromes, immune response syndromes and stroke syndromes. [Background technology]

[0002] Cells are guided by chemokines as they migrate through the body. Chemokine-attracted cells follow the signal of increasing chemokine concentrations toward the source of the chemokine. There are currently four distinct known families of chemokines, which differ in the spacing of their first two cysteine ​​residues. The so-called CXC chemokines, or α-chemokines, have a spacer amino acid between the two cysteines, designated by the symbol X.

[0003] Thus, chemokines affect cells via chemokine receptors on the cell surface. Chemokine receptors are integral membrane proteins. CXC chemokine receptors, especially CXC chemokine receptor 4 (CXCR4), activate cancer cell proliferation and migration, leading to the formation of metastases throughout the body (preferentially in the lungs, bones, and liver). HIV can also use CXCR4 to infect CD4+ T cells.

[0004] CXCR4 is involved in multiple developmental and physiological processes, including stem cell homing (Mohle and Drost, 2012) and immune cell migration (Campbell et al., 2003). Artificial CXCR4 antagonists can mobilize hematopoietic stem cells (HSCs), which are used for immune reconstitution after organ transplantation or chemotherapy (Ratajczak and Kim, 2012; Schroeder and DiPersio, 2012). Furthermore, CXCR4 is the primary coreceptor for HIV-1 entry into target cells (Feng et al., 1996; Bleul et al., 1996). CXCR4 co-receptor utilization is highly effective; a high percentage of CD4+ T cells express this GPCR in vivo in lymphoid tissues. Nevertheless, HIV-1 mutants that utilize CC chemokine receptor 5 (CCR5) are found transmitted almost exclusively during chronic HIV-1 infection (Alkhatib et al., 1996; Deng et al., 1996; Dragic et al., 1996). Multiple factors have been proposed to contribute to the inefficient transmission of CXCR4-tropic (X4) HIV-1 strains (Margolis and Shattock, 2006). However, the mechanisms underlying the effective control of X4 HIV-1 in immunocompetent individuals remain poorly understood.

[0005] Research into CXCR4 antagonists has become an extensive field of study in recent years due to their diverse applications. In particular, efforts to discover strategies to intervene in cancer cell proliferation, differentiation, and metastasis have not yet met with the expected success in clinical studies. The development of one class of compounds, a CXCR4 antagonist called AMD3100 (a bicyclame compound: Hendrix and Flexner 2000), had to be discontinued for long-term treatment due to toxic side effects. Although AMD3100 has been registered for short-term, single-dose applications in stem cell mobilization, finding a suitable antagonist for the target CXCR4 remains a challenge.

[0006] Zirafi, O. et al. (J. Leukoc. Biol. 99:663-868; 2016) reported the development of an endogenous peptide inhibitor of CXCR4 as a CXCR4 antagonist. This peptide, a 16-residue fragment of human serum albumin, was isolated from a blood-derived peptide library as an inhibitor of CXCR4-tropic human immunodeficiency virus type 1 (HIV-1) receptors. The endogenous peptide inhibitor binds to the second extracellular loop of CXCR4, thereby preventing CXCL12 engagement and antagonizing the receptor. Consequently, the endogenous peptide inhibitor inhibits CXCR4-expressing cell migration via CXCL12 in vitro, mobilizes hematopoietic stem cells, and suppresses inflammatory responses in vivo. The authors discuss the development of endogenous peptide inhibitors of CXCR4, their suitability as disease biomarkers, and their role in the pathogenesis of human immunodeficiency virus / acquired immunodeficiency syndrome (HIV / AIDS) and cancer.

[0007] WO 2009 / 0045054 discloses a peptide having the amino acid sequence Z1-LVRYTKKVPQVSTPTL-Z2 (ALB-408), as well as biologically active fragments and / or variants and / or derivatives thereof, in particular amidated, acetylated, sulfated, phosphorylated and / or glycosylated derivatives, and peptides obtained by multiple synthesis and having the biological activity of ALB408-423, where Z represents the number of amino acid residues from 0 to 10.

[0008] WO 2014 / 198834 discloses peptides, particularly dimeric peptides, that are effective in inhibiting CXC-chemokine receptor 4 (CXCR4)-mediated HIV-1 NL4-3 (X4-tropic) infection with IC50 values ​​of less than 50 μM. Summary of the Invention [Problem to be solved by the invention]

[0009] Therefore, the object of the present invention is to provide peptides with antagonistic activity against native CXCR4, which in particular should inhibit the proliferation and metastasis of cancer cells and exhibit anti-inflammatory and allergic responses, as demonstrated by different analogues of different cancer types.

[0010] Another object of the present invention is to provide a compound capable of reducing the receptor activity of CXCR4.

[0011] Another object of the present invention is to provide compounds that can affect the proliferation of cancer cells.

[0012] Another object of the present invention is to provide compounds that can affect the migration or homing of cancer cells.

[0013] It is a further object of the present invention to provide compounds that are capable of influencing the formation of metastases.

[0014] Another object of the present invention is to provide compounds that can treat aggressive tumors so that the cancer is significantly inhibited or becomes a chronic disease.

[0015] It is yet another object of the present invention to provide compounds that can regulate and treat various diseases such as immune and allergic diseases, tissue growth regulation, and nervous system regulation. [Means for solving the problem]

[0016] The object of the present invention is achieved by any of the polypeptides of the present invention. The polypeptides of the present invention show great therapeutic potential.

[0017] The polypeptide of the present invention has an amino acid sequence represented by the following general formula (written in one-letter code): Z 1 X 1 X 2 X 3 X 4 X 5 X6 X 7 APX 10 X 11 SZ 3 Including, X 1 = V, M or L, in particular L; X 2 = V, L or M, in particular V; X 3 =R, K or C, in particular R; X 4 = Y or W, in particular Y, X 5 = S or T, in particular T, X 6 =Q or C, in particular Q, X 7 =K, C or R, in particular K; X 10 =Q or C, in particular Q, X 11 = V, M or F, in particular V, and, Z 1 =0, Z 2 or pyroglutamic acid, Z 2 = 0, or the N-terminal nitrogen atom of the peptide chain is modified, and this modification, together with the amino group of the N-terminal amino acid of the peptide, is -NR 2 R 3 forming a moiety having the structure R 2 and / or R 3 are each independently H or a substituted or unsubstituted acyl, alkyl, aryl, aralkyl, cycloalkyl, or heterocycloalkyl group, Z 3 =0 or Z 4 and Z 4 = 0, or the C-terminal carboxyl group of the peptide chain is modified, and this modification, together with the carboxyl group of the C-terminal amino acid of the peptide, is -C(O)-OR 1 Structure or -C(O)-NR 2 R 3 forming a moiety having the structure R 1is a substituted or unsubstituted alkyl group, aryl group, aralkyl group, cycloalkyl group, and heterocycloalkyl group.

[0018] one or more groups: Z 1 and / or Z 2 and / or Z 3 and / or Z 4 When it is stated that =0, it is meant that the respective group is not included in this particular embodiment.

[0019] Surprisingly, one of the chain ends (X 1 Polypeptides with a non-polar protein at the first chain end (S) and a polar protein at the other chain end (S) were found to exhibit antagonistic activity against native CXCR4.

[0020] In a preferred embodiment, X 6 is Q, and therefore the present invention relates to an amino acid sequence (written in one-letter code) having the general formula: Z 1 X 1 X 2 X 3 X 4 X 5 QX 7 APX 10 X 11 SZ 3 In the above polypeptide of the present invention, the peptide is selected from the following: X 1 = V, M or L, in particular L; X 2 = V, L or M, in particular V; X 3 =R, K or C, in particular R; X 4 = Y or W, in particular Y, X 5 = S or T, in particular T, X 7 =K, C or R, in particular K; X 10 =Q or C, in particular Q, X 11=V, M or F, especially V.

[0021] In a preferred embodiment of the present invention, the peptide of the present invention comprises at least one of the following amino acid sequences: LVRYTQKAPQVS, MVRYTQKAPQVS, VVRYTQKAPQVS, LMRYTQKQPQVS, LLRYTQKQPQVS, LVKYTQKQPQVS, LVCYTQKQPQVS, LVRWTQKAPQVS, LVRYSQKQPQVS, LVRYTCKAPQVS, LVRYTQCAPQVS, LVRYTQRAPQVS, LVRYTQKAPCVS, LVRYTQKAPQMS or LVRYTQKAPQFS. The sequence: LVRYTQKAPQVS is a particularly preferred embodiment.

[0022] According to the present invention, one or more amino acid residues may be exchanged, deleted or added in the amino acid sequence, or chemical modifications may be introduced into a single amino acid of the polypeptide, resulting in an improvement in the biological or pharmacological activity of the unmodified polypeptide of the present invention. The particular methods for modification are known to those skilled in the art.

[0023] Furthermore, at least one side chain of an amino acid of the polypeptide may be chemically modified, in particular phosphorylated, amidated, acetylated, glycosylated, PEGylated, HESylated, or a combination thereof.

[0024] The selection of a suitable functional group for the PEG derivative is based on the type of reactive group available on the molecule to which PEG is to be attached. For proteins, representative reactive amino acids include lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, and tyrosine. The N-terminal amino group and the C-terminal carboxylic acid can also be used as specific sites for conjugation with aldehyde-functional macromolecules.

[0025] According to the invention, the polypeptides of the invention may comprise at least one D-amino acid. In particular, the polypeptides of the invention may be composed of a chain of D-amino acids in a retro-inverso configuration of the polypeptide chain of the invention.

[0026] Therefore, retro-inverso peptides of the peptides of the invention are also included within the scope of the invention, as are other derivatives that stabilize the peptide bonds against peptidases. Within the scope of this application, the term derivatives refers to fragments of any length, including truncations at the N- and C-terminus, peptides of the invention with amino acid residue substitutions, including D-amino acid residues and modified amino acid residues, as well as peptides with disulfide bonds and N- and C-terminal extensions.

[0027] A further subject of the present invention is a medicament comprising at least one peptide of the invention and a pharmaceutically acceptable carrier. In one of the simplest embodiments, the polypeptide of the invention can be administered in water for infusion, physiological saline or an aqueous buffer solution. Other dosage forms are also possible, such as encapsulation in liposomes, which form nanoparticles of various sizes, for example, between 20 and 2,000 nm.

[0028] Typically, the peptides of the invention are administered at 10-1000 mg / kg body weight for a period sufficient to halt tumor growth. The administration period is usually 2 to 10 weeks. The agents of the invention are preferably suitable for oral, intravenous, intramuscular, intradermal, subcutaneous, or intrathecal administration, or are present in an aerosol form suitable for pulmonary and nasal administration, particularly encapsulated in liposomes or for use in aqueous solutions or liposomal packaging.

[0029] The subject of the present invention is also polynucleotides encoding the polypeptides of the invention and / or their fragments, variants, derivatives and analogs. The polynucleotides of the invention may consist of DNA, RNA, genomic DNA or PNA. Polynucleotides that hybridize to the polynucleotides of the invention encoding the polypeptides of the invention are also subject of the present invention.

[0030] Further subject matter of the present invention are vectors comprising a polynucleotide according to the invention, and genetically engineered host cells comprising a vector according to the invention.

[0031] A further subject of the present invention is an antibody directed against at least one of the polypeptides of the invention.

[0032] On the other hand, another subject of the present invention are antagonist / inhibitory compounds directed against the polypeptides of the invention.

[0033] The subject of the present invention is also For the treatment of nervous system diseases, especially stroke, Parkinson's disease, Alzheimer's disease, and multiple sclerosis; In the field of immunology, in particular for the treatment of WHIM syndrome, systemic lupus erythematosus and rheumatoid arthritis, In the field of oncology, in particular for the treatment of cancers that exhibit CRCX receptors, such as liver cancer, pancreatic cancer, prostate cancer, breast cancer, etc. Treatment of stem cell mobilization, proliferation, and migration defects, T cell activation, and immunoblast support such as CTL / PD-1. For the treatment of wounds caused by burns, For the treatment of antifibrosis, For the treatment or prevention of scars, For the treatment of heart disease, especially heart failure, For the treatment of metabolic diseases, especially diabetes, For the treatment of viral diseases, particularly infections with HIV-1, HIV-2, cytomegalovirus, herpes simplex virus (type 1 and type 2), varicella-zoster virus, hepatitis A and B virus, influenza virus, poliovirus, rhinovirus, rubella virus, measles virus, rabies virus, Rous sarcoma virus, and Epstein-Barr virus, For the treatment of infections caused by bacteria and fungi, especially Pseudomonas, Candida, and Staphylococcus aureus, For the treatment of infectious and abnormal infectious processes, for use in the treatment of growth disorders, neuronal diseases, disorders of the blood coagulation cascade and hematopoiesis, vascular diseases, diseases of the immune system, and The peptide of the present invention for use in wound and bone healing, pulmonary disorders and allergies.

[0034] Other processes for producing the polypeptides of the present invention are solid phase synthesis by Merrifield synthesis or liquid phase synthesis using protected amino acids and subsequent purification by methods known to those skilled in the art.

[0035] Furthermore, other processes for producing the polypeptides of the present invention may employ heterologous expression methods known to those skilled in the art using conventional biotechnological vectors, followed by post-translational or chemical modifications as required.

[0036] A subject of the present invention is a diagnostic agent comprising a polyclonal or monoclonal antibody according to the invention or comprising a nucleic acid or mRNA encoding a polypeptide according to the invention.

[0037] A further subject of the present invention is A diagnostic agent comprising a polypeptide of the present invention or a polynucleotide of the present invention, for use in a test system for assaying tissue, plasma, urine, and cerebrospinal fluid levels of the substance; and a diagnostic agent and test system for detecting the polypeptide of the present invention, for assaying tissue, plasma, urine, and cerebrospinal fluid levels of the substance by mass spectrometry, such as MALDI-MS or ESI-MS, in combination with sample preparation by RP-HPLC, protein precipitation, and / or solid phase extraction. Mass spectrometry methods are preferably used to detect trace amounts of molecules in the femtomolar or attomole range.

[0038] Also subject of the present invention are diagnostic agents comprising the polypeptides according to the invention as markers for viral diseases, bacterial and fungal infections, inflammatory and tumorigenic processes, as markers in inflammatory processes, disturbed inflammatory responses, neoplastic diseases, growth disorders, diseases of the immune system, WHIM syndrome, systemic lupus erythematosus, and as markers in bone diseases, etc.

[0039] The present invention will be further explained in more detail using the peptide LVRYTQKAPQVS (SEQ ID NO: 1) as a representative peptide of the present invention. [Example]

[0040] <Peptide> The peptide of SEQ ID NO: 1 and its various derivatives were synthesized by conventional solid-phase synthesis using Fmoc chemistry on a Peptide Synthesizer 9050 (Applied Biosystems). The peptides were purified by RP chromatography, and their identity and purity were confirmed by analytical RP-HPLC, MALDI-MS, and LC-ESI-MS.

[0041] <Cancer cell invasion assay> Cancer cell invasion assays were performed in humanized rats (Eyol, E. et al., Oncology Reports, 28:2177-2187, 2012). Pancreatic cancer cells were transplanted. Successful transplantation was monitored by luminescence imaging and increased CXCR4 expression.

[0042] After successful implantation of the cancer cells, the rats were treated with the peptide according to SEQ ID NO: 1. The results after 1 week of treatment (1 week treatment), after 2 weeks of treatment (2 week treatment) and 2 weeks after the end of treatment are shown in Table 1 below.

[0043] <Results for peptide of SEQ ID NO: 1> [Table 1]

[0044] Untreated rats died within a few days of tumor implantation.

[0045] Rats treated with the peptides of the present invention survived for at least two weeks. Tumor growth was arrested, and partial or even complete tumor regression was observed depending on the peptide concentration. No toxic effects of the peptides were observed.

[0046] <Sequence list> SEQ ID NO: 1 Leu Val Arg Tyr Thr Gln Lys Ala Pro Gln Val Ser SEQ ID NO: 2 Met Val Arg Tyr Thr Gln Lys Ala Pro Gln Val Ser SEQ ID NO: 3 Val Val Arg Tyr Thr Gln Lys Ala Pro Gln Val Ser SEQ ID NO:4 Leu Met Arg Tyr Thr Gln Lys Gln Pro Gln Val Ser SEQ ID NO:5 Leu Leu Arg Tyr Thr Gln Lys Gln Pro Gln Val Ser SEQ ID NO:6 Leu Val Lys Tyr Thr Gln Lys Gln Pro Gln Val Ser SEQ ID NO:7 Leu Val Cys Tyr Thr Gln Lys Gln Pro Gln Val Ser SEQ ID NO:8 Leu Val Arg Trp Thr Gln Lys Ala Pro Gln Val Ser SEQ ID NO:9 Leu Val Arg Tyr Ser Gln Lys Gln Pro Gln Val Ser SEQ ID NO: 10 Leu Val Arg Tyr Thr Cys Lys Ala Pro Gln Val Ser SEQ ID NO: 11 Leu Val Arg Tyr Thr Gln Cys Ala Pro Gln Val Ser SEQ ID NO: 12 Leu Val Arg Tyr Thr Gln Arg Ala Pro Gln Val Ser SEQ ID NO: 13 Leu Val Arg Tyr Thr Gln Lys Ala Pro Cys Val Ser SEQ ID NO: 14 Leu Val Arg Tyr Thr Gln Lys Ala Pro Gln Met Ser SEQ ID NO: 15 Leu Val Arg Tyr Thr Gln Lys Ala Pro Gln Phe Ser

Claims

1. Amino acid sequence: A polypeptide selected from the group consisting of LVRYTQKAPQVS, MVRYTQKAPQVS, VVRYTQKAPQVS, LVRWTQKAPQVS, LVRYTQCAPQVS, LVRYTQRAPQVS, LVRYTQKAPCVS, LVRYTQKAPQMS and LVRYTQKAPQFS.

2. The polypeptide of claim 1, wherein at least one side chain of an amino acid of said polypeptide is chemically modified.

3. The polypeptide of claim 2, wherein at least one side chain of an amino acid of the polypeptide is phosphorylated, amidated, acetylated, glycosylated, PEGylated, HESylated, or a combination thereof.

4. The polypeptide according to any one of claims 1 to 3, wherein the polypeptide comprises at least one D-amino acid.

5. A medicament comprising at least one of the polypeptides according to any one of claims 1 to 4 and a pharma- ceutically acceptable carrier.

6. 6. The agent according to claim 5, suitable for oral, intravenous, intramuscular, intradermal, subcutaneous or intrathecal administration, or in an aerosol form suitable for pulmonary administration, or for use in an aqueous solution or liposomal packaging.

7. The agent of claim 6 which is encapsulated in a liposome.

8. A polynucleotide encoding the polypeptide of claim 1.

9. The polynucleotide according to claim 8, which is composed of DNA, RNA, genomic DNA or PNA.

10. A vector comprising the polynucleotide of claim 8.

11. A genetically engineered host cell comprising the vector of claim 10.

12. An antibody against a polypeptide described in any one of claims 1 to 4.

13. A polypeptide according to any one of claims 1 to 4, For the treatment of nervous system diseases, In the field of immunology, In the field of oncology, For the treatment of stem cell mobilization, proliferation and migration defects, T cell activation, and support of immunoblasts such as CTL / PD-1; For the treatment of wounds caused by burns, For anti-fibrotic treatment, For the treatment or prevention of scarring, To treat heart disease, For the treatment of metabolic diseases, For the treatment of viral diseases, For the treatment of infections caused by bacteria and fungi, For the treatment of infectious and abnormal infectious processes, for use in the treatment of growth disorders, neuronal diseases, disorders of the blood coagulation cascade and hematopoiesis, vascular diseases, diseases of the immune system, and A polypeptide for use in improving wound and bone healing.

14. A polypeptide for use according to claim 13, comprising For the treatment of stroke, Parkinson's disease, Alzheimer's disease, and multiple sclerosis. For the treatment of WHIM syndrome, systemic lupus erythematosus and rheumatoid arthritis, For the treatment of cancer, To treat heart failure, For the treatment of diabetes, for the treatment of HIV-1, HIV-2, cytomegalovirus, herpes simplex virus (type 1 and type 2), varicella zoster virus, hepatitis A and B viruses, influenza virus, poliovirus, rhinovirus, rubella virus, measles virus, rabies virus, Rous sarcoma virus, Epstein-Barr virus infections, and A polypeptide for use in the treatment of infections caused by Pseudomonas, Candida, and Staphylococcus aureus.

15. 15. The polypeptide for use according to claim 14, wherein said cancer is liver cancer, pancreatic cancer, prostate cancer or breast cancer which expresses the CRCX receptor.

16. A process for preparing a polypeptide according to any one of claims 1 to 4, comprising the steps of: A process for preparation by cation exchange extraction, followed by elution of absorbed material, regeneration of the extract containing said polypeptide from blood filtrate by cation exchange chromatography and fractional reversed phase chromatography.

17. A process for preparing a polypeptide according to any one of claims 1 to 4, comprising preparation by solid phase synthesis in the Merrifield synthesis or by liquid phase synthesis using protected amino acids and purification thereof.

18. A process for preparing a polypeptide according to any one of claims 1 to 4 by the method of heterologous expression using conventional biotechnological vectors and subsequent post-translational or chemical modification, if required.

19. A diagnostic agent comprising a polyclonal or monoclonal antibody according to claim 12, or a nucleic acid or mRNA encoding a polypeptide according to any one of claims 1 to 4.

20. A diagnostic agent comprising a polypeptide according to any one of claims 1 to 4 or a polynucleotide according to claim 8 for use in a test system for assaying tissue, plasma, urine and cerebrospinal fluid levels of the substance.

21. 5. Diagnostics and test systems for detecting a polypeptide according to any one of claims 1 to 4 and for assaying tissue, plasma, urine and cerebrospinal fluid levels of this substance by mass spectrometry such as MALDI-MS or ESI-MS in combination with sample preparation by RP-HPLC, protein precipitation and / or solid phase extraction.

22. A diagnostic agent comprising a polypeptide according to any one of claims 1 to 4 as a marker for viral diseases, bacterial and fungal infections, inflammatory and tumorigenic processes, as a marker for inflammatory processes, disturbed inflammatory responses, neoplastic diseases, growth disorders, diseases of the immune system and as a marker for bone diseases.

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