JP2015024960A - Cxcr4 activity inhibiting peptide and an application thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a peptide and the like that can suppress or inhibit CXCR4 activity, and can suppress or inhibit the activity of both CXCL12 and CXCL14.SOLUTION: The peptide and the like related to the invention are a dimer peptide resulting from dimerizing via a linker bond of a peptide or a variant peptide thereof containing a region having the α-helix structure of the C-terminal side of a human CXCL14 chemokine protein, a derivative or a salt thereof.

Description

  The present invention relates to a peptide capable of inhibiting the activity of CXCR4, which is a CXCL12 chemokine receptor, and uses thereof. Specifically, the present invention relates to a peptide that can inhibit the activities of both CXCL12 and CXCL14 chemokines by inhibiting the activity of CXCR4, and uses thereof.

  CXCL14 was discovered in 1999 as a CXC type chemokine that disappears in epithelial cancer tissues. CXCL14 has the activity of attracting tissue macrophages, immature dendritic cells, and NK cells, but is not induced during tissue damage repair, suggesting that it plays a role different from a group of inflammatory chemokines (Non-Patent Document 1). Among the many chemokines, only the amino acid sequence of CXCL14 and its related CXCL12 is well conserved from fish to humans. In 2006, CXCL12 and its receptor CXCR4 proved to be essential for establishing hematopoietic stem cells in the bone marrow niche. AMD3100 (mozovir), a CXCL12-specific inhibitor, has been approved by the FDA as a hematopoietic stem cell collection agent and is used for the treatment of multiple myeloma and the like. Subsequent studies revealed that the CXCL12 / CXCR4 system plays an essential role in the malignant transformation and metastasis of cancer cells derived from various tissues.

On the other hand, research on CXCL14 has only just begun compared to CXCL12, where research is ongoing. The inventor has already proved that CXCL14 is an important metabolic regulator associated with the development of obesity and type 2 diabetes (Non-patent Documents 2 and 3). When CXCL14 knockout mice (CXCL14-KO mice) were fed with a high-fat diet, visceral white fat accumulated as much as wild-type mice (control), and enlargement of individual adipocytes was observed. However, CXCL14-KO mice fed a high-fat diet had a reduced number of macrophages infiltrating internal fat compared to wild-type mice, and the chronic inflammatory response was alleviated. As a result, CXCL14-KO mice fed with a high fat diet improved fatty liver and retained the insulin sensitivity of the individuals. CXCL14 was also expressed in the hypothalamus and was involved in the induction of the appetite-promoting peptide NPY / AgRP during fasting. CXCL14-KO mice showed low body weight due to reduced food intake, which was more prominently observed in double mutant mice with ob / ob and ^Ay obese mice. Therefore, CXCL14 was speculated to be a unique chemokine that works in the direction of storing energy (facilitating obesity) in both peripheral tissues and the central nervous system. Interestingly, although CXCL14 is not upregulated in acute inflammatory reactions, it increases the level of expression in obese individuals with visceral fat and affected areas of autoimmune rheumatoid arthritis, contributing to the development of insulin resistance and arthritis, respectively. Has been revealed. This suggests that CXCL14 may be a worsening factor for various diseases caused by chronic inflammation as well as obese diabetes.

In contrast to the above phenomenon, it has been reported that when CXCL14 is forcibly expressed in an epithelial cancer cell line derived from oral cancer or gastric cancer and transplanted into nude mice, the solid cancer formation rate decreases. In CXCL14 transgenic mice, the tumor formation rate of cancer cells transplanted subcutaneously was significantly reduced. CXCL14 is strongly transcriptionally induced by administration of EGF receptor kinase inhibitors such as Gefitinib (Iressa) and Erlotinib (Terseva), so it may function as an effector of these anticancer agents. However, the molecular mechanism of anticancer activity exhibited by CXCL14 has not yet been elucidated.
CXCL12 and CXCL14 are important chemokines that are closely related to the development of cancer and diabetes.

T. Hara and Y. Nakayama. CXCL14 and insulin action. Vitamins and Hormones, 80: 107-123, 2009. N. Nara, Y. Nakayama, S. Okamoto, H. Tamura, M. Kiyono, M. Muraoka, K. Tanaka, C. Taya, H. Shitara, R. Ishii, H. Yonekawa, Y. Minokoshi, and T Hara. Disruption of CXC motif chemokine ligand-14 in mice ameliorates obesity-induced insulin resistance. J. Biol. Chem., 282: 30794-30803, 2007. K. Tanegashima, S. Okamoto, Y. Nakayama, C. Taya, H. Shitara, R. Ishii, H. Yonekawa, Y. Minokoshi, and T. Hara. CXCL14 deficiency in mice attenuates obesity and inhibits feeding behavior in a novel environment. PLoS ONE, 5: e10321, 2010.

  Under such circumstances, it has been desired to identify a CXCL14 receptor-constituting molecule and develop a new type of CXCR4 activity modulator based thereon.

  The present invention has been made in view of the above situation, and the dimer peptide, its derivative or a salt thereof, and an inhibitor of CXCR4 activity, an inhibitor of cell chemotaxis, treatment or prevention of diabetes shown below. The pharmaceutical composition for cancer, the pharmaceutical composition for cancer treatment, etc. are provided.

(1) A dimer peptide, a derivative thereof, or a dimer peptide obtained by dimerizing a peptide containing a region having an α-helix structure on the C-terminal side of human CXCL14 chemokine protein or a mutant peptide thereof via a linker bond salt.

(2) A dimer peptide, a derivative thereof or a salt thereof obtained by dimerizing the following peptide (a) or (b) via a linker bond.
(a) a peptide comprising any one of the amino acid sequences represented by SEQ ID NOs: 2 to 5
(b) A dimer peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in any one of the amino acid sequences represented by SEQ ID NOs: 2 to 5 and dimerized. Occasionally peptides with inhibitory or inhibitory activity on CXCR4 activity

(3) A dimer peptide, a derivative thereof or a salt thereof obtained by dimerizing the following peptide (a) or (b) via a linker bond.
(a) a peptide comprising any one of the amino acid sequences represented by SEQ ID NOs: 2 to 5
(b) A dimer peptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in any one of the amino acid sequences represented by SEQ ID NOs: 2 to 5 and dimerized. Occasionally peptides with inhibitory or inhibitory activity on CXCR4 activity

In the dimer peptide of the above (1) to (3), a derivative thereof or a salt thereof, examples of the linker bond include a disulfide bond or an oxime bond.
Examples of the dimer peptides of (1) to (3) include those represented by the following formula (I) or formula (II).

  Examples of the dimer peptides, derivatives thereof or salts thereof of (1) to (3) above include those having antagonistic activity against CXCR4.

(4) A CXCR4 activity inhibitor or inhibitor comprising the dimer peptide of (1) to (3), a derivative thereof, or a salt thereof.
(5) A cell chemotaxis inhibitor comprising the dimer peptide of (1) to (3), a derivative thereof, or a salt thereof.
(6) A pharmaceutical composition for treating or preventing diabetes, comprising the dimer peptide of the above (1) to (3), a derivative thereof, or a salt thereof.
(7) A pharmaceutical composition for treating cancer comprising the dimer peptide of the above (1) to (3), a derivative thereof, or a salt thereof.

According to the present invention, it is possible to provide a peptide (dimer peptide) or the like that can inhibit the activities of both CXCL12 and CXCL14 by inhibiting the activity of CXCR4.
The peptide of the present invention is extremely useful in that it can be used as an active ingredient such as a cell chemotaxis inhibitor, a pharmaceutical composition for treating or preventing diabetes, and a pharmaceutical composition for treating cancer. is there.

It is a schematic diagram which shows the structure and component factor of the receptor which bear the biological activity of CXCL12 and CXCL14. It is a figure which shows that CXCL14 inhibits CXCL12 chemotaxis of a THP-1 cell. It is a figure which shows that CXCL14 inhibits the activity and receptor binding of CXCL12 in a concentration-dependent manner (and thus CXCL14 is a natural antagonist of CXCL12). It is a schematic diagram which shows the three-dimensional structure of CXCL14 and the structure of each dimer peptide (N2C, CG2O) derived from CXCL14. It is a figure which shows that N2C and CG2O inhibit the binding to the receptor of CXCL14 in a concentration-dependent manner. It is a figure which shows that N2C and CG2O inhibit CXCL12 / CXCL14 chemotaxis of THP-1 cells. It is a figure which shows the body weight change of the high fat diet obesity mouse | mouth which administered N2C and CG2O. It is a figure which shows the insulin sensitivity of the high fat diet obesity mouse | mouth which administered N2C and CG2O.

  Hereinafter, the present invention will be described in detail. The scope of the present invention is not limited to these descriptions, and other than the following examples, the scope of the present invention can be appropriately changed and implemented without departing from the spirit of the present invention. It should be noted that all publications cited in the present specification, for example, prior art documents, publications, patent publications and other patent documents are incorporated herein by reference.

1. Summary of the present invention
CXCL14 is a non-inflammatory chemokine that allows tissue macrophages and immature dendritic cells to remain in the subcutaneous and surrounding organs, but also worsens obesity diabetes, develops rheumatoid arthritis, and suppresses malignant growth of epithelial cancer cells In recent years it has become clear that it is involved.
In contrast, the present inventors have found that CXCL14 receptor contains CXCR4, which is CXCL12 receptor, and 7-transmembrane protein Lphn2 whose function is unknown (submitted in the paper). Furthermore, CXCL14 has been shown to inhibit CXCL12 chemokine activity in a concentration-dependent manner by binding to CXCR4 (submitted). While CXCL12 attracts hematopoietic stem cells to the bone marrow niche, it is also a chemokine that promotes malignant growth and metastasis of cancer cells from various tissues. CXCL14 is presumed to play an important role in the construction and maintenance of normal tissues as a natural antagonist of CXCL12 as well as transmitting intracellular signals specific to CXCL14 by activating receptors including CXCR4-Lphn2. The

  This time, the present inventor has expected that CXCR4 antagonists and agonists will become novel therapeutic agents for diabetes and cancer, and has performed structural modification of CXCL14 based on molecular structure prediction. As a result, a peptide containing 27 amino acid residues on the C-terminal side in the amino acid sequence (SEQ ID NO: 1) of the CXCL14 chemokine protein (the 51st to 77th amino acid residues of the amino acid sequence represented by SEQ ID NO: 1) or a mutation thereof Dimer peptides (specifically, N2C and CG2O (see formula (I) and formula (II) described later)), which are dimerized from each other via a linker bond at the N-terminal side. ) Was found to significantly inhibit the activity of both CXCL12 and CXCL14. In addition, the above inhibitory activity is observed for monomeric peptides that have not been dimerized and dimer peptides that have been dimerized via a linker bond (chemical bond used for the linker) on the C-terminal side. I couldn't.

2. CXCR4 activity-inhibiting peptide The peptide of the present invention is a peptide that can inhibit CXCR4 activity. Specifically, a peptide containing a region having an α-helix structure on the C-terminal side of human CXCL14 chemokine protein or a mutant peptide thereof is used. , A dimer peptide obtained by dimerization via a linker bond.
In the present invention, the term “inhibition” includes the concept of “suppression” and means to completely eliminate (ie, inactivate) the activity as well as to reduce the activity.

  In the present invention, the “peptide” is not limited, and may be any peptide composed of at least two amino acids linked by peptide bonds, and includes oligopeptides and polypeptides. Furthermore, a polypeptide in which a certain three-dimensional structure is formed is called a protein. In the present invention, such a protein is also included in the “peptide”. Therefore, the peptide used for the dimer peptide of the present invention can mean any of oligopeptides, polypeptides, and proteins.

  Here, the amino acid sequence of the human CXCL14 chemokine protein (also simply referred to as CXCL14) is the amino acid sequence represented by SEQ ID NO: 1 (77 amino acid residues) as shown below, of which the α-helix structure is located on the C-terminal side. Is preferably a region containing the 51st to 77th amino acids of the amino acid sequence (underlined portion), more preferably the 51st to 77th amino acids of the amino acid sequence. It is a region consisting of amino acids and a region consisting of the 50th to 77th amino acids.

Amino acid sequence of CXCL14 (SEQ ID NO: 1):
SKCKCSRKGPKIRYSDVKKLEMKPKYPHCEEKMVIITTKSVSRYRGQEHC LHPKLQSTKRFIKWYNAWNEKRRVYEE

The mutant peptide of the peptide containing the region having the α helix structure is not limited, but, for example, a peptide (specifically, a sequence having an appropriate amino acid substitution to stabilize the α helix structure) Peptides including the amino acid sequences represented by Nos. 3 and 5) are preferred.
The peptide of the present invention is a dimer peptide (preferably a homodimer peptide) obtained by dimerizing a peptide containing a region having the α-helix structure or a mutant peptide thereof, and each peptide has a linker bond. It is a dimerized through. Here, the linker bond is not limited, and examples thereof include a disulfide bond and an oxime bond. Moreover, it is preferable that the linker bond is bonded between the N-terminal sides of each peptide chain.

More specifically, the peptide of the present invention is preferably a dimer peptide obtained by dimerizing the peptide of the following (a) through a linker bond as described above.
(a) Peptide containing any one of the amino acid sequences represented by SEQ ID NO: 2 to 5 below SEQ ID NO: 2: LHPKLQSTKRFIKWYNAWNEKRRVYEE
Sequence number 3: LHPKL E STK K FI E WYN K WNEKRR K YEE
Sequence number 4: CLHPKLQSTKRFIKWYNAWNEKRRVYEE
Sequence number 5: CLHPKL E STK K FI E WYN K WNEKRR K YEE

The peptide (a) is more preferably a peptide consisting of any one of the amino acid sequences represented by SEQ ID NOs: 2 to 5.
The amino acid sequence represented by SEQ ID NO: 2 is a sequence consisting of the 51st to 77th amino acids in the amino acid sequence of CXCL14 represented by SEQ ID NO: 1, and the amino acid sequence represented by SEQ ID NO: 3 is 6th, 10th, 13th, 17th and 24th amino acids of the amino acid sequence represented by SEQ ID NO: 2 are substituted with glutamic acid (E) or lysine (K) (in the amino acid sequence represented by SEQ ID NO: 3) (See underlined amino acid residues).

  The amino acid sequence represented by SEQ ID NO: 4 is a sequence consisting of the 50th to 77th amino acids in the amino acid sequence of CXCL14 represented by SEQ ID NO: 1, and the amino acid sequence represented by SEQ ID NO: 5 is , Wherein the seventh, eleventh, fourteenth, eighteenth, and twenty-fifth amino acids of the amino acid sequence represented by SEQ ID NO: 4 are substituted with glutamic acid (E) or lysine (K) (the amino acid sequence represented by SEQ ID NO: 5) (See amino acid residues underlined in the middle).

As described above, the peptide of the present invention may be a dimer peptide obtained by dimerizing the following peptide (b) through a linker bond.
(b) A dimer peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in any one of the amino acid sequences represented by SEQ ID NOs: 2 to 5 and dimerized. Sometimes peptides with inhibitory activity on CXCR4 activity

  More preferably, the peptide of (b) consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in any one of the amino acid sequences represented by SEQ ID NOs: 2 to 5, and It is a peptide having CXCR4 activity inhibitory activity when it is a dimerized dimer peptide.

Here, the “amino acid sequence in which one or several amino acids are deleted, substituted or added” is, for example, about 1 to 5, preferably about 1 to 3, more preferably 1 to An amino acid sequence in which about two amino acids have been deleted, substituted, or added can be mentioned, and is not limited. The introduction of mutations such as the above-mentioned deletion, substitution, addition and the like can be carried out using a mutation-introducing kit using site-directed mutagenesis, such as GeneTailor ^™ Site-Directed Mutagenesis System (Invitrogen) and TaKaRa Site-Directed Mutagenesis System. (Mutan-K, Mutan-Super Express Km, etc .: manufactured by Takara Bio Inc.) and the like can be used. In addition, whether or not the peptide has a deletion, substitution or addition mutation introduced therein can be confirmed using various amino acid sequencing methods and structural analysis methods such as X-ray and NMR.

Furthermore, in addition to the peptide consisting of the amino acid sequence represented by SEQ ID NOs: 2 to 5, about 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% with respect to the amino acid sequence As mentioned above, peptides having an amino acid sequence having 98% or more homology or 99% or more homology and having a CXCR4 activity inhibitory activity when dimerized into dimer peptides are also included. In general, the larger the homology value, the better.
In the present invention, “having an inhibitory activity of CXCR4 activity when it is a dimerized dimer peptide” specifically means having an activity of inhibiting the binding of CXCL14 and CXCL12 to the CXCR4 receptor. The activity can be measured by, for example, a binding assay using CXCL14 or CXCL12 labeled with ¹²⁵ I.

As long as the peptide of the above (a) or (b) is a peptide comprising any one of the amino acid sequences represented by SEQ ID NOs: 2 to 5, or a peptide comprising the amino acid sequence deleted, substituted or added, The number of constituent amino acid residues is not limited, and may be 22 or more (in the case of the deleted amino acid, etc.) or 27 or more, for example, 27 to 100 residues. It may be 27 to 50 residues, 27 to 40 residues, or 27 to 30 residues.
The dimer peptide obtained by dimerizing the peptide of the above (a) or (b) is also dimerized via a linker bond as described above, and the linker bond includes a disulfide bond and An oxime bond etc. are mentioned preferably. Moreover, it is preferable that the linker bond is bonded between the N-terminal sides of each peptide chain.

  More specifically, the peptide of the present invention is preferably a dimer peptide represented by the following formula (I) or formula (II). The dimer peptide of the formula (I) is a peptide having the amino acid sequence represented by SEQ ID NO: 4 obtained by disulfide bond (S—S bond) between N-terminal cysteine (C) residues. The dimer peptide represented by the formula (II) is a peptide having the amino acid sequence represented by SEQ ID NO: 3 bound by oxime bonds between N-terminal leucine (L) residues.

  The peptide of (a) or (b) may be a peptide derived from a natural product, or may be obtained by artificial chemical synthesis, and is not limited. Peptides obtained by artificial chemical synthesis are preferred because they are easy to produce and can be prepared in large quantities at a low cost. A peptide derived from a natural product is preferred because it often has no adverse effects such as cytotoxicity.

  Chemically synthesized peptides can be obtained using known peptide synthesis methods. Examples of the synthesis method include an azide method, an acid chloride method, an acid anhydride method, a mixed acid anhydride method, a DCC method, an active ester method, a carboimidazole method, and a redox method. In addition, the solid phase synthesis method and the liquid phase synthesis method can be applied to the synthesis. A commercially available peptide synthesizer may be used. After the synthesis reaction, the peptide can be purified by combining known purification methods such as chromatography.

Examples of peptides derived from natural products include naturally occurring oligopeptides, polypeptides and proteins, or fragments of these. A peptide derived from a natural product may be obtained directly from a natural product by a known recovery method and purification method, or a gene encoding the peptide is incorporated into various expression vectors by a known gene recombination technique. After being introduced into cells and expressed, they may be obtained by known recovery and purification methods. Alternatively, cell-free protein synthesis using commercially available kits such as reagent kits PROTEIOS ^™ (Toyobo), TNT ^™ System (Promega), PG-Mate ^™ (Toyobo) and RTS (Roche Diagnostics) etc. The peptide may be produced by a system and obtained by a known recovery method and purification method, and is not limited.

  In the present invention, as a CXCR4 activity-inhibiting peptide, a derivative of the peptide can be included together with or in place of the various dimer peptides described above. The derivative is meant to include all those that can be prepared from the dimer peptide (or various monomer peptides used in the dimer peptide). For example, a part of the constituent amino acids is substituted with an unnatural amino acid. And those in which a part of the constituent amino acids (mainly their side chains) are chemically modified.

  Furthermore, in the present invention, as the CXCR4 activity-inhibiting peptide, the dimer peptide and / or a salt of the derivative can be included together with or in place of the various dimer peptides described above and derivatives thereof. The salt is preferably a physiologically acceptable acid addition salt or basic salt. Acid addition salts include, for example, salts with inorganic acids such as hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, or acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, apple And salts with organic acids such as acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and trifluoroacetic acid. Examples of basic salts include salts with inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide and magnesium hydroxide, and salts with organic bases such as caffeine, piperidine, trimethylamine and pyridine. .

Salts can be prepared using a suitable acid such as hydrochloric acid or a suitable base such as sodium hydroxide. For example, it can be prepared by treatment using standard protocols in water or in a liquid containing an inert water-miscible organic solvent such as methanol, ethanol or dioxane.
The above-described dimer peptide, derivative thereof and salts thereof of the present invention are all peptides that can inhibit CXCR4 activity, and more specifically, preferably have antagonist activity against CXCR4.

3. Inhibitor of CXCR4 activity and inhibitor of cell chemotaxis The present invention provides an inhibitor of CXCR4 activity comprising the dimer peptide of the present invention described above, a derivative thereof, or a salt thereof.
Moreover, this invention also provides the inhibitor of cell chemotaxis containing the dimer peptide of this invention mentioned above, its derivative (s), or these salts.

  The inhibitor of CXCR4 activity and the inhibitor of cell chemotaxis may be composed of the dimer peptide of the present invention described above, a derivative thereof, or a salt thereof, or the dimer peptide and other components. May be included and is not limited. Examples of other components include, but are not limited to, buffer solutions such as PBS and Tris-HCl, and additives such as sodium azide and glycerol. When other components are included, the content ratio can be appropriately set within a range in which the inhibitory activity of CXCR4 activity and the suppressive activity of cell chemotaxis by the dimer peptide or the like are not significantly hindered. Specifically, when used in the above peptide solution, the peptide concentration is not limited, but is preferably 10 nM or more, more preferably 50 to 1000 nM, and still more preferably 100 to 1000 nM.

  The present invention also provides a kit for inhibiting CXCR4 activity and a kit for inhibiting cell chemotaxis, characterized in that it comprises the dimer peptide of the present invention, a derivative thereof, or a salt thereof as a constituent component. The

4). Pharmaceutical composition The dimer peptide of the present invention described above, a derivative thereof or a salt thereof is useful as an active ingredient contained in a pharmaceutical composition. In particular, it is useful as an active ingredient of a pharmaceutical composition for treating or preventing diabetes and a pharmaceutical composition for treating cancer.
The pharmaceutical composition of the present invention is preferably provided in the form of a pharmaceutical composition comprising the inhibitor of the present invention as an active ingredient and further comprising a pharmaceutically acceptable carrier.

  "Pharmaceutically acceptable carrier" refers to excipients, diluents, extenders, disintegrants, stabilizers, preservatives, buffers, emulsifiers, fragrances, colorants, sweeteners, thickeners, taste masking Agents, solubilizers or other additives. By using one or more of such carriers, pharmaceutical compositions in the form of injections, solutions, capsules, suspensions, emulsions or syrups can be prepared. These pharmaceutical compositions can be administered orally or parenterally. Other forms for parenteral administration include injections that contain one or more active substances and are prescribed by conventional methods. In the case of an injection, it can be produced by dissolving or suspending it in a pharmaceutically acceptable carrier such as physiological saline or commercially available distilled water for injection. Moreover, when administering the dimer peptide of this invention mentioned above etc. in the living body, a colloid dispersion system can also be used. The colloidal dispersion system is expected to increase the in vivo stability of the peptide or to efficiently transport the compound to a specific organ, tissue or cell. The colloidal dispersion system is not particularly limited as long as it is usually used, but polyethylene glycol, polymer composite, polymer aggregate, nanocapsule, microsphere, bead, and oil-in-water emulsifier, micelle, mixed micelle And lipid-based dispersion systems including liposomes, preferably liposomes or artificial membrane vesicles that are effective in efficiently transporting compounds to specific organs, tissues or cells.

  The dosage of the pharmaceutical composition of the present invention varies depending on the patient's age, sex, weight and symptoms, therapeutic effect, administration method, treatment time, type of dimer peptide of the present invention contained in the pharmaceutical composition, and the like. May be. Usually, it can be administered in a range of 100 μg to 5000 mg per adult per person, but is not limited.

  For example, when administered by injection, the amount of 100 μg to 100 mg per kg body weight can be administered to a human patient once to several times per day on an average per day. Examples of the administration form include intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, and intraperitoneal injection, and intravenous injection is preferred. In addition, injections can be prepared as non-aqueous diluents (eg, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol), suspensions, and emulsions. Such sterilization of the injection can be performed by filtration sterilization using a filter, blending of a bactericide, and the like. The injection can be produced as a form prepared at the time of use. That is, it can be used as a sterile solid composition by lyophilization, etc., and dissolved in sterile water for injection or other solvent before use.

The present invention also provides use of the dimer peptide of the present invention, a derivative thereof, or a salt thereof for the manufacture of a medicament (drug) for treating and / or preventing diabetes. The present invention also provides the dimer peptide of the present invention, a derivative thereof, or a salt thereof for the treatment and / or prevention of diabetes.
Furthermore, the present invention provides a method for treating and / or preventing diabetes characterized by using the dimer peptide of the present invention, a derivative thereof or a salt thereof (that is, administering to a patient), The present invention also provides use of the dimer peptide of the present invention, a derivative thereof or a salt thereof for treating and / or preventing diabetes.

Here, in the present invention, as diabetes, diabetes that develops in close association with obesity, particularly “type 2 diabetes” that exhibits insulin resistance is preferable as the target disease.
The present invention also provides use of the dimer peptide of the present invention, a derivative thereof, or a salt thereof for producing a medicament (drug) for treating cancer. The present invention also provides the dimer peptide of the present invention, a derivative thereof or a salt thereof for cancer treatment.

Furthermore, the present invention provides a method for treating cancer, characterized by using the dimer peptide of the present invention, a derivative thereof, or a salt thereof (that is, administering to a patient), The present invention also provides the use of the dimer peptide of the present invention, a derivative thereof or a salt thereof for the treatment.
Here, in the present invention, examples of the cancer to be treated include lung cancer, oral cancer, and breast cancer, but are not particularly limited.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

<Method>
In vitro activity evaluation method of CXCL14 and CXCL12
For the in vitro bioassay of CXCL12 and CXCL14, a chemotaxis test using human monocytic leukemia cell line THP-1 and a binding assay using ¹²⁵ I-labeled ligand were used. CXCL14 exhibits strong THP-1 cell attracting activity only in the presence of anti-CXCL14 monoclonal antibody MAB730 (R & D) (Tanegashima, K. Suzuki, Y. Nakayama, and T. Hara. Antibody-assisted enhancement of biological activities of CXCL14 in human monocytic leukemia-derived THP-1 cells and high fat diet-induced obese mice. Exp. Cell Res., 316: 1263-1270, 2010.), 100 nM human CXCL14 (Peprotech) + MAB730 for CXCL14 chemotaxis studies In the CXCL12 chemotaxis test, 10 nM human CXCL12 (Peprotech) was placed in a 24-well-plate (lower chamber). THP-1 cells (2 x 10 ⁵ ) were placed in Kurabo Industries' 5μ Chemotaxicell chamber (upper chamber), reacted for 2 hours at 37 ° C, and then the cells that passed through the membrane pore were Diff-quick stained and then counted. . Various CXCL14 mutant peptides (1 to 1000 nM) were added only in the Chemotaxicell chamber (upper chamber) to quantify the effect of inhibiting cell migration.

In the binding assay, 10 nM ¹²⁵ I-labeled CXCL14 or 10 nM ¹²⁵ I-labeled CXCL12 and THP-1 cells were mixed, reacted at 4 ° C for 2 hours, separated by centrifugation, and the radiation dose was measured using a γ counter. Was measured. After adding 100 times the amount of unlabeled CXCL14 or various CXCL14 mutant peptides to the above assay system and reacting them, the radiation dose of the cells was measured to calculate the ligand binding inhibition rate.

<Result>
Although the structure of the CXCL14 receptor has not been elucidated for a long time, the present inventors recently discovered that the receptor contains CXCR4 and a 7-transmembrane protein Lphn2 whose function is unknown (FIG. 1). CXCL14 binds with high affinity to CXCR4 but does not signal alone. It is speculated that the intracellular signal by CXCL14 stimulation is transmitted through Lphn2. Lphn2 expression sites in brain, lung, skeletal muscle, macrophages, miso, etc. were almost consistent with the expression pattern of ligand CXCL14. Interestingly, because CXCL14 is a non-signaling ligand of CXCR4, it almost completely blocked chemotactic migration of the THP-1 cell line via CXCL12 (FIG. 2). Therefore, the physiological role of CXCL14 in an individual could be distinguished from the direct role of CXCL14 alone or indirect by suppression of CXCL12 function (FIG. 1). Since the suppression of CXCL12 activity by CXCL14 was observed at a lower concentration than the inhibition of receptor binding (FIG. 3), it was speculated that CXCL14 caused allosteric structural change by binding to CXCR4.

<Method>
Synthesis method of CXCL14 mutant peptide
CXCL14 consists of 77 amino acid residues (SEQ ID NO: 1), but the C-terminal side has a typical α-helix structure. This helix region exists in the range of the 51st to 77th amino acid residues in 77 amino acid residues, and the present inventor believes that this helix structure plays a very important role in the interaction with the receptor. I guessed. Therefore, it was decided to synthesize a peptide containing the C-terminal side (the 51st to 77th amino acid residues) in the amino acid sequence of CXCL14 by the Fmoc type solid phase synthesis method and to examine its activity. Monomer peptides were synthesized by a known method (specifically, a paper on total synthesis of CXCL14 by the present inventors (K. Tsuji, A. Shigenaga, Y. Sumikawa, K. Tanegashima, K. Sato, K. Aihara, T. Hara, and A. Otaka.Application of N to C- or C to N-directed sequential native chemical ligation to the preparation of CXCL14 analogs and their biological evaluation.Bioorg. Med. Chem., 19: 4014-4020 , 2011.). For dimer peptides, cysteine (Cys: equivalent to the natural sequence) or oxyaminoacetic acid (H ₂ NO-CH ₂ COOH) is condensed on the N-terminal side of the 51st amino acid, and disulfide (SS) is used for the Cys transductant. The bond and the oxyaminoacetic acid introduced substance were dimerized by forming an oxime bond using an aldehyde linker. In addition, homodimers utilizing oxime bond formation are substituted with amino acids for the purpose of stabilizing the α-helix structure. This amino acid substitution is based on the functional differentiation strategy of α-helix peptides, and the usefulness of the amino acid substitution has been demonstrated through the creation of anti-HIV peptides (A. Otaka, M. Nakamura, D. Nameki, E. Kodama, S. Uchiyama, S. Nakamura, H. Nakano, H. Tamamura, Y. Kobayashi, M. Masuoka, and N. Fujii.Remodeling of gp41-C34 Peptide Leads to Highly Effective Inhibitors of the Fusion of HIV- 1 with Target Cells. Angew. Chem. Int. Ed., 41: 2937-2940, 2002.). The dimer peptide with disulfide bond is named N2C, and the dimer peptide with oxime bond is named CG2O. The structures of these dimer peptides are shown in the following formulas (I) (N2C) and (II) (CG2O), respectively, and FIG. Indicated. Each dimer peptide was synthesized, purified by HPLC, and subjected to a physiological activity verification experiment. For HPLC analysis and HPLC fractionation, Cosmosil 5C ₁₈ -AR-II analytical column (Nacalai Tesque, 4.6 × 250 mm, flow rate 1.0 mL / min), a 5C ₁₈ -AR-II semi-preparative column (Nacalai Tesque , 10 × 250 mm, flow rate 3.0 mL / min) or a 5C ₁₈ -AR-II preparative column (Nacalai Tesque, 20 × 250 mm, flow rate 10 mL / min) was used. At this time, as an eluent, a mixed solvent system consisting of 0.1% TFA aqueous solution (v / v, solvent A) and 0.1% TFA in MeCN (v / v, solvent B) was used. Was detected. In addition, accurate mass spectrometry was performed by measurement with Waters MICROMASS (registered trademark) LCT PREMIER.

・ N2C (CXCL14 (50-77) SS dimmer)
Analytical HPLC condition: linear gradient of solvent B in solvent A, 5-45% over 30 min, retention time = 22.0 min.
Preparative HPLC condition: linear gradient of solvent B in solvent A, 20-30% over 30 min.
MS (ESI-TOF) m / z calcd for ([M + 8H] ⁸⁺ ) 906.2, found 906.3.

・ CG2O (CXCL14 (51-77, 56Glu, 60Lys, 63Glu, 67Lys, 74Lys) Oxime dimer)
Analytical HPLC condition: linear gradient of solvent B in solvent A, 5-45% over 30 min, retention time = 23.6 min.
Preparative HPLC condition: linear gradient of solvent B in solvent A, 20-30% over 30 min.
MS (ESI-TOF) m / z calcd for ([M + 8H] ⁸⁺ ) 970.0, found 969.8.

<Result>
As described above, the C-terminal side of CXCL14 has a characteristic α-helix structure, which may be a target site for a specific inhibitor. Therefore, we synthesized various peptides in which the C-terminal side of human CXCL14 was structurally modified, and performed a chemotaxis test using THP-1 cells to examine the activity inhibitory effect of CXCL14. As a result, a dimer peptide (N2C) obtained by dimerizing a peptide containing the 27th amino acid residues 51 to 77 of the C-terminal side of human CXCL14 with an SS bond, and a dimer obtained by dimerization with an oxime bond In both of the peptides (CG2O), an activity specifically blocking the receptor binding of ¹²⁵ I-labeled CXCL14 was detected (FIG. 5). Furthermore, when these compounds were subjected to a chemotaxis test using THP-1 cells, both of them significantly suppressed chemotactic migration of THP-1 cells by CXCL12 and CXCL14 at a concentration of 100 nM (FIG. 6). . From the above results, it was found that N2C and CG2O are effective inhibitors against both CXCL12 and CXCL14.

<Method>
In vivo administration experiment to obese mice A 6-week-old C57BL / 6 female mouse (Japan SLC) was fed with High fat diet-32 (Crea Japan High Fat Diet) and bred for 20 weeks. Nine obese mice whose body weight reached around 46 g were transferred to individual cages and acclimatized for one week, and then N2C (1 μg / g body weight) or CG2O (1 μg / g body weight) prepared in Example 2 and PBS as a solvent control. Each of the three animals was injected intraperitoneally. Injections were made daily at 18:00 for 7 days. During this time, the weight of each mouse was measured daily. Fasted on the evening of the last day of peptide administration, the fasting blood glucose level was measured at 10 am the next day using Glutest Ace R (Sanwa Chemical Laboratory). Next, human insulin (0.75 mU / g body weight) was intraperitoneally injected into each mouse, and blood glucose levels after 30, 60, 90 and 120 minutes were measured over time.

<Result>
Since CXCL14 is a worsening factor for obesity diabetes, a compound that inhibits its activity is expected to be a novel therapeutic agent. Therefore, N2C and CG2O, in which CXCL14 inhibitory activity was detected by an in vitro assay, were administered to obese mice, and body weight changes and insulin sensitivity were compared with control mice. As a result, it was shown that the body weight tends to decrease in the CG2O-administered mouse group (FIG. 7), and insulin sensitivity can be significantly improved (FIG. 8). On the other hand, the body weight change and insulin sensitivity of the group of mice administered with N2C were almost the same as the group of control mice administered with PBS (FIGS. 7 and 8). Therefore, it was shown that CG2O is a compound having an effect of improving the pathological condition of obese diabetes.

<Consideration on this example>
CXCR4 plays an important role as a receptor for the chemokine CXCL12 in bone marrow homing of hematopoietic stem cells and malignant growth and metastasis of cancer cells, but is also used as an entry receptor for T cell infectious HIV. Therefore, development of inhibitors that target CXCR4 as a molecular target is underway in various parts of the world. In this example, an attempt was made to create a new type of CXCR4 activity inhibitor (CXCR4 antagonist) based on the structural information of CXCR4 and its ligand CXCL12 and its natural antagonist CXCL14. As a result, we succeeded in developing novel peptide compounds N2C and CG2O that suppress chemotaxis cell migration by both CXCL14 and CXCL12. Among them, CG2O was extremely useful, showing an in vivo effect of improving insulin resistance in obese mice. Since CG2O is dimerized by an oxime bond that does not exist in nature, it is assumed that CG2O is highly stable in the body.
CXCL14 is a factor that exacerbates obese diabetes, but also has an activity of suppressing malignant growth of squamous cell carcinoma. Therefore, the CXCR4 activity-inhibiting peptide of the present invention has the potential to develop into a new therapeutic agent that overcomes cancer and metabolic syndrome, which have a huge number of patients around the world.

Sequence number 3: Recombinant peptide Sequence number 5: Recombinant peptide

Claims (10)

  A dimer peptide, a derivative thereof, or a salt thereof obtained by dimerizing a peptide containing a region having an α-helix structure on the C-terminal side of human CXCL14 chemokine protein or a mutant peptide thereof via a linker bond. A dimer peptide, a derivative thereof or a salt thereof obtained by dimerizing the following peptide (a) or (b) through a linker bond.
(a) a peptide comprising any one of the amino acid sequences represented by SEQ ID NOs: 2 to 5
(b) A dimer peptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in any one of the amino acid sequences represented by SEQ ID NOs: 2 to 5 and dimerized. Occasionally peptides with inhibitory or inhibitory activity on CXCR4 activity A dimer peptide, a derivative thereof or a salt thereof obtained by dimerizing the following peptide (a) or (b) through a linker bond.
(a) a peptide comprising any one of the amino acid sequences represented by SEQ ID NOs: 2 to 5
(b) A dimer peptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in any one of the amino acid sequences represented by SEQ ID NOs: 2 to 5 and dimerized. Occasionally peptides with inhibitory or inhibitory activity on CXCR4 activity   The dimer peptide, derivative thereof, or salt thereof according to any one of claims 1 to 3, wherein the linker bond is a disulfide bond or an oxime bond. The dimer peptide, a derivative thereof, or a salt thereof according to any one of claims 1 to 4, wherein the dimer peptide is represented by the following formula (I) or formula (II).
  The dimer peptide, derivative thereof, or salt thereof according to any one of claims 1 to 5, which has antagonist activity against CXCR4.   The inhibitor or inhibitor of CXCR4 activity containing the dimer peptide of any one of Claims 1-6, its derivative (s), or these salts.   The inhibitor of cell chemotaxis containing the dimer peptide of any one of Claims 1-6, its derivative (s), or these salts.   A pharmaceutical composition for treating or preventing diabetes comprising the dimer peptide according to any one of claims 1 to 6, a derivative thereof, or a salt thereof.   The pharmaceutical composition for the treatment of cancer containing the dimer peptide of any one of Claims 1-6, its derivative (s), or these salts.