KR20120082262A - Pharmaceutical composition comprising peptide having inhibitory activities against transendothelial migration of leucocytes and metastasis of cancer cells - Google Patents

Abstract

PURPOSE: A pharmaceutical composition containing peptide fragments derived from CD166(ALCAM) is provided to suppress leukemia extravasation or treating inflammatory diseases. CONSTITUTION: A pharmaceutical composition for preventing or treating inflammatory diseases contains peptides having amino acids of sequence numbers 1-3 as an active ingredient and pharmaceutically acceptable carrier. The inflammatory diseases are caused by hypersensitive reaction. A pharmaceutical composition for suppressing cancer metastasis contains the peptides as an active ingredient and pharmaceutically acceptable carrier.

Description

Pharmaceutical composition comprising peptide having inhibitory activities against transendothelial migration of leucocytes and metastasis of cancer cells}

The present invention provides a pharmaceutical composition for preventing or treating an inflammatory disease comprising a peptide fragment (peptide fragment or small peptide) derived from CD166 (ALCAM); And it relates to a pharmaceutical composition for inhibiting cancer metastasis. The peptide has an extravasation inhibitory activity of leukocytes and a metastasis inhibitory activity of cancer cells.

The protective response of the living body to restore the structure and function of tissues damaged by infection, trauma, etc. are collectively called an inflammatory response. Mobilization of leukocytes to the site of inflammation is important for rapid resolution of infection and repairing tissue damage resulting from various traumas. However, faulty or persistent inflammatory reactions cause damage and disease of human tissue. For example, inflammatory diseases are caused by bacterial or viral infections such as meningitis, enteritis, dermatitis, uveitis, encephalitis, adult respiratory distress syndrome, or non-infectious factors such as trauma, autoimmune diseases, and organ transplant rejection. Inflammatory diseases are classified into acute and chronic inflammatory diseases in which symptoms or pathological features are distinguished. Local symptoms of acute inflammation, such as allergies or bacterial and viral infections, are manifested by changes in blood flow and blood vessel size, changes in vascular permeability, and infiltration of white blood cells. In contrast, rheumatoid arthritis, atherosclerosis. The main pathological features of chronic inflammation, including chronic nephritis and cirrhosis, are the infiltration of monocytes, neutrophils, lymphocytes, and plasma cells into the site of inflammation, resulting in chronic inflammation.

Infiltration of leukocytes into the site of inflammation is essential for an inflammatory response to occur, which involves the involvement of several cell adhesion molecules. That is, the leukocytes moved to the blood vessels of the inflammation site by the induction of chemokine secreted from the inflammation site rotates on the surface of the vascular endothelial cells while reducing the movement speed, and the white blood cells stop the rotation and endothelial cell wall. It can be divided into an adhesion stage and a transmigration stage of infiltration of capillaries and bottom membranes. This last step, or transmigration stage, is called diapedesis.

Cancer cells changed by carcinogens proliferate faster than normal cells, form tumor masses, penetrate surrounding tissues, and inhibit normal body function. By inducing angiogenesis, cancer cells are supplied with nutrients and oxygen, and cancer cells are metastasized by angiogenesis. Cancer cells not only grow indefinitely at specific sites, but also move away from existing growth sites and move to new sites to grow. This process is called metastasis. The metastasis process involves the steps of cancer cells turning into highly mobile mesenchymal cells, leaving the existing tissues, invading and moving surrounding connective tissues and capillaries, and moving out of blood vessels and out of blood vessels, within connective tissues. Migration and growth at new sites.

Regulation of cell surface expression and activation of adhesion molecules plays a very important role in the metastasis of tumor cells (Zetter, B. R. (1993). Adhesion molecules in tumor metastasis. Semin Cancer Biol. 4: 219). The process of metastasis of tumor cells is achieved through the regulation of cell surface expression and activity of cell adhesion materials, and understanding of cell adhesion molecules as well as substances that control their expression and function is essential for the full understanding of metastasis. to be. (Bailly, M., Yan, L., Whitesides, GM, Condeelis, JS, and Segall, JE (1998) .regulation of Protusion Shape and Adhesion to the sustratum during chemoacic esponses of mammalian carcinoma cells.Exp Cell Res. 241: 285; Frisch, SM, Vuori, K., Ruoslahti, E., and Chan-Hui., P. (1996) .Control of adhesion-dependent cell survival by focal adhesion kinase.J Cell Biol 134: 793; and Hannigan, GE, Leung-Hagesteijn, C., Fitz-Gibbon, L., Coppolino, MG, Radeva, G., Filmus, J., Bell, JC, and Dedhar, S. (1996) .Regulation of cell adhesion and anchorage- dependent growth by a new β1-integrin-linked protein kinase.Nature 379: 91).

On the other hand, CD166 (ALCAM) is a cell adhesion molecule consisting of an extracellular region, a transmembrane region and a short intracellular region containing five Ig regions. CD166 is known as the ligand of CD6, and its binding forms an immunosynapse between antigen-presenting cells and lymphocytes (Zimmerman AW et al. 2006. Long-term engagement of CD6 and ALCAM is essential for T- cell proliferation induced by dendritic cells.Blood 107: 3212). In addition to immune cells, they are expressed in various types of cells, including endothelial cells, and play an important role in extravasation of leukocytes, especially as components of endothelial cell junctions (Masedunskas A, King JA, Tan F, Cochran R, Stevens T, Sviridov D, Ofori-Acquah SF., 2006.Activated leucocyte cell adhesion molecule is a component of the endothelial junction involved in transendothelial monocyte migration.FEBS Lett.580: 2637.Cayrol R, Wosik K, Berard JL, Dodelet-Devillers A, Ifergan I, Kebir H, Haqqani AS, Kreymborg K, Krug S, Moumdjian R, Bouthillier A, Becher B, Arbor N, David S, Stanimirovic D, Prat A., 2008.Activated leukocyte cell adhesion molecule promotes leukocyte trafficking into the central nervous system.Nature Imm. 9: 137). It is also involved in the regulation of cell migration, such as neurogenesis, pterygium, neurogenesis, melanoma invasion, etc. (Swart GW. 2002. Activated leukocyte cell adhesion molecule (CD166 / ALCAM): developmental and mechanistic aspects of cell clustering and cell migration.Eur. J. Cell Biol. 81: 313). Recently, it has been reported to be involved in angiogenesis as a binding protein of galectin-8 (Cardenas Delgado VM, Nugnes LG, Colombo LL, Troncoso MF, Fernandez MM, Malchiodi EL, Frahm I, Croci DO, Compagno D, Rabinovich GA , Wolfenstein-Todel C, Elola MT. 2011. Modulation of endothelial cell migration and angiogenesis: a novel function for the "tandem-repeat" lectin galectin- 8.FASEB J. 25: 242).

The present inventors can inhibit the inflammatory response by inhibiting extravasation of leukocytes by peptide fragments having a specific sequence derived from CD166 (peptide fragment or small peptide), and also inhibit metastasis of cancer cells by inhibiting extravasation of cancer cells. It was found that it can be suppressed.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising a specific peptide derived from CD166 as an active ingredient.

In addition, an object of the present invention is to provide a pharmaceutical composition for inhibiting cancer metastasis (cancer metastasis) comprising a specific peptide derived from CD166 as an active ingredient.

According to an aspect of the present invention, a pharmaceutical composition for preventing or treating an inflammatory disease comprising a peptide selected from the group consisting of peptides consisting of amino acid sequences of SEQ ID NOs: 1 to 3 as an active ingredient, and comprising a pharmaceutically acceptable carrier This is provided.

The inflammatory disease may in particular be inflammation caused by hypersensitivity.

According to another aspect of the present invention, a pharmaceutical agent for inhibiting cancer metastasis comprising a peptide selected from the group consisting of peptides consisting of amino acid sequences of SEQ ID NOs: 1 to 3 as an active ingredient and comprising a pharmaceutically acceptable carrier A composition is provided.

In one embodiment, the peptide contained as an active ingredient in the pharmaceutical composition is a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

In another embodiment, the peptide contained as an active ingredient in the pharmaceutical composition is a peptide consisting of the amino acid sequence of SEQ ID NO: 2.

Peptides of the present invention, that is, peptides selected from the group consisting of peptides consisting of amino acid sequences of SEQ ID NOS: 1 to 3 can be usefully applied to pharmaceutical compositions for the prevention or treatment of inflammatory diseases by inhibiting extravasation of leukocytes. In addition, the peptides may be usefully applied to the pharmaceutical composition for inhibiting cancer cell metastasis, that is, inhibiting cancer metastasis, by inhibiting extravasation of cancer cells.

1 is a result of measuring the extravascular leakage of WEHI247.1 cells when the mouse monocyte cell line WEHI293 was treated with the peptide of the present invention.
Figure 2 is the result of measuring the extravasation of the LLC-1 cells when the peptide of the present invention in the mouse Lewis lung carcinoma cell line LLC-1.
Figure 3 is the result of measuring the change in ear thickness when the peptide of the present invention is injected into the mice induced IgE-mediated immediate hypersensitivity reaction.

As used herein, "inflammatory" or "inflammatory disease" includes acute and / or chronic inflammatory diseases, for example, rheumatoid arthritis, adhesive capsulitis, sinovitis ), Osteoarthritis, osteoarthritis, osteoporosis, periarthritis, multiple sclerosis, osteomyelitis, systemic lupus erythematosus, rheumatoid polyps rheumatica (PMR), Sjogren's Syndrome, progressive systemic sclerosis (scleroderma), ankylosing spondylitis, polymyositis, dermatomyositis, pemphigus (pemphigoid), Type I diabetes mellitus, myasthenia gravis, Hashimoto's thyroditis, Graves' disease, Gdfe Inflammatory bowel such as Goodpasture's disease, mixed connective tissue disease, sclerosing cholangitis, Crohn's disease or ulcerative colitis disease, inflammatory dermatoses, usual interstitial pneumonitis (UIP), lymphoid interstitial pneumonia, giant cell interstitial pneumonia, cellular interstitial pneumonia ), Desquamative interstitial pneumonia, asbestosis, silicosis, berylliosis, talcosis, pneumoconiosis, adult respiratory distress syndrome (Adult) Inflammatory respiratory diseases, asthma and atopic derm, including Respiratory Distress Syndrome and extrinsic allergic alveolitis immediate hypersensitivity reactions such as atitis, allergic rhinitis, food allergy, sarcoidosis, Wegener's granulomatosis, various angiitis, chronic hepatitis (chronic active hepatitis), delayed-type hypersensitivity reactions, such as poison ivy dermatitis, psoriatic arthritis, Reiter's syndrome, rheumatic fever, acute or Acute or chronic glomerulonephritis, acute exacerbations, pyelonephritis, cellulitis, cystitis, acute cholecystitis, inflammatory aortic aneurysm, atherosclerosis Atherosclerosis, Still's disease, Parkinson's disease, Alzheimer's disease, and the like. In addition, the peptides of the present invention can be used in patients with inflammatory diseases, such as reperfusion injury, autoimmune diseases, tissue transplantation rejection or tissue allograft organ rejection. As used herein, the term "inflammatory" or "inflammatory disease" should be understood to include diseases involving the above-described inflammatory disease.

The peptide of the present invention may be preferably applied to hypersensitivity reactions, autoimmune diseases, and / or rheumatoid arthritis among the above inflammatory diseases, and more preferably to hypersensitivity reactions and autoimmune diseases. Especially preferably, it may be inflammation caused by hypersensitivity, which may be delayed type such as immediate hypersensitivity reactions such as asthma and poison ivy dermatitis. And delayed-type hypersensitivity reactions.

We made fragments of various lengths from CD166 and searched for their activity. Surprisingly, as peptides derived from CD166, certain peptide fragments of short length consisting of three to four amino acids not only have anti-inflammatory activity by inhibiting endothelial transmembrane migration of leukocytes, but also inhibit extravasation of cancer cells. By doing so, it was found to have metastasis inhibitory activity of cancer cells. The peptide fragments are shown in Table 1 below.

Peptide Name SEQ ID NO: Amino acid sequence CD166-pep1 SEQ ID NO: 1 Cys-ser-leu CD166-pep2 SEQ ID NO: 2 Ser-Leu-His-Tyr CD166-pep3 SEQ ID NO: 3 Glu-gly-leu

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating an inflammatory disease comprising a peptide selected from the group consisting of peptides consisting of amino acid sequences of SEQ ID NOs: 1 to 3 as an active ingredient, and comprising a pharmaceutically acceptable carrier. . The inflammatory disease particularly includes inflammation caused by hypersensitivity.

In another aspect, the present invention provides a pharmaceutical composition for inhibiting cancer metastasis (cancer metastasis) comprising a peptide selected from the group consisting of peptides consisting of the amino acid sequence of SEQ ID NO: 1 to 3 as an active ingredient, a pharmaceutically acceptable carrier do.

In the pharmaceutical composition of the present invention, the peptide contained as the active ingredient is preferably a peptide consisting of the amino acid sequence of SEQ ID NO: 1 or a peptide consisting of the amino acid sequence of SEQ ID NO: 2.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier, and may include, for example, excipients such as lactose and corn starch, lubricants such as magnesium stearate, known and usable emulsifiers, suspensions, buffers, and isotonic agents. Topics, and the like. The pharmaceutical compositions of the invention may be formulated in oral or parenteral dosage forms, preferably parenteral dosage forms. In the case of intramuscular, intraperitoneal, subcutaneous and intravenous dosage forms, a sterile solution of the active ingredient is usually prepared and may comprise a buffer which can suitably adjust the pH of the solution, and for intravenous administration isotonic in the formulation. May be included to impart this. In addition, the pharmaceutical composition of the present invention may be in the form of an aqueous solution containing a pharmaceutically acceptable carrier such as saline having a pH of 7.4, and may be locally introduced into the patient's intramuscular blood flow in the form of a solution. have.

The pharmaceutical composition of the present invention is a patient suffering from various inflammatory diseases; Or to patients suffering from various solid or myeloid leukemias including breast cancer, stomach cancer, colon cancer, colon cancer, rectal cancer, pancreatic cancer, and the like at a dose of about 1 to 2000 mg / kg per day. Appropriate dosages will generally vary depending on the age, weight and symptoms of the patient.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrating the present invention, and the present invention is not limited to these examples.

Example 1 Synthesis of Peptides

Peptides of SEQ ID NOS: 1 to 3 (see Table 1 above) were synthesized by the FMOC solid-phase method using an automated synthesizer (PeptrEx-R48, Peptron, Daejeon, Korea). The synthesized peptide was purified and analyzed by reverse-phase HPLC using a C18 analytical RP column (Shiseido capcell pak) (Prominence LC-20AB, Shimadzu, Japan), and mass spectrometry (HP 1100 Series LC). / MSD, Hewlett-Packard, Roseville, USA).

Example 2. Preparation of a Composition Comprising a Peptide

Peptides of SEQ ID NO: 1 to 3 were dissolved in phosphate buffered saline (PBS) to prepare a concentration of 1 M. The obtained protein solution was diluted with PBS and used in the following test example.

Test Example 1. In vitro of monocytes ( in vitro Extravasation inhibition test

BEND3 (ATCC, USA), a mouse brain endothelial cell, was cultured in the Wikan of the Boyden chamber. The culture supernatant was removed and mouse monocytes WEHI247.1 (ATCC, USA) not pretreated or pretreated for 1 hour with the peptide solutions (100 μM) of SEQ ID NOs. x 10 ⁵ each was added. In the lower compartment, incubation of monocytes was performed by adding a culture solution containing the supernatant obtained by centrifugation of NIH / 3T3, a mouse fibroblast, in a DMEM serum-free medium containing 0.005% vitamin C and 0.1% bovine albumin. Induced. The number of cells infiltrated into the lower cell while incubating for 6 hours was measured. The test was repeated five times, and the results are shown in FIG. 1. In FIG. 1, Control added 5 x 10 ⁵ mouse monocytes WEHI247.1 not pretreated with peptide solution.

As can be seen in Figure 1, when treated with the peptides according to the present invention, the number of monocytes leaked out of the blood vessels significantly reduced to about 30-90% of the control group. Given that the leukocytes move along the blood vessels and then flow out of the blood vessels to move to the inflammatory site, the peptide of the present invention is expected to have an effective inflammatory inhibitory activity.

Test Example 2 In Vitro of Cancer Cells in vitro ) Extravasation inhibition test

BEND3, a mouse brain endothelial cell, was cultured in a Wikan in a Boyden chamber. The culture supernatant was removed and the mouse Lewis lung carcinoma cell line LLC-1 (ATCC, USA) was pretreated or untreated for 1 hour with the peptide solution (100 μM) of SEQ ID NOs. 1-3 prepared in Example 2, respectively. 5 x 10 ⁵ pieces were added to the treatment. In the lower compartment, cultured blood vessels of lung cancer cells were cultured with supernatant obtained by centrifugation of NIH / 3T3, a mouse fibroblast, incubated for 16 hours in DMEM serum-free medium containing 0.005% vitamin C and 0.1% bovine serum albumin. Outflow was induced. The number of cells infiltrated into the lower cell while incubating for 6 hours was measured. The test was repeated three more times, and the results are shown in FIG. 2. In Figure 2 Control (control) added 5 x 10 ⁵ mouse Lewis lung carcinoma cell line LLC-1 not pretreated with peptide solution.

As can be seen in Figure 2, when treated with the peptide of the present invention, extravasation of mouse lung cancer cells was inhibited to about 30-60% of the control group. Given that cancer cells infiltrating into blood vessels are flowed out of blood vessels in order to move along blood vessels and metastasize to organs, the peptides of the present invention are expected to have an effective cancer cell metastasis inhibiting activity.

Test Example 3 Inhibition of IgE-mediated immediate hypersensitivity reactions

Six-week-old male Balb / c mice were sensitized by injecting 5 μg of IgE antibody into the tail vein. After 24 hours, the peptide fragment of SEQ ID NO: 1 obtained in Example 1 was dissolved in 100 µl of saline and intraperitoneally administered at a concentration of 10 mg / kg per mouse. A solution of 2,4-dinitrofluorobenzene (DNFB) dissolved at a concentration of 0.15% in a mixture of acetone and olive oil (4: 1, v / v) was used as an antigen. IgE-mediated immediate hypersensitivity by applying the solution to the ear Induced. Negative control mice were injected with 100 μl of saline and not treated with DNFB, whereas positive control mice were injected with 100 μl of saline and treated with DNFB. After 3 hours and 6 hours, the change in ear thickness was measured using a digital caliper. From day 1 after peptide treatment, test mice were intraperitoneally injected with peptide fragment of SEQ ID NO: 1 at a concentration of 10 mg / kg, and control mice were intraperitoneally injected with saline, and the change in ear thickness was compared for 3 days.

3 is a result of measuring the change in ear thickness of the test group and the positive control mice increased by DNFB treatment compared to the negative control group. From the results of Figure 3, it can be seen that the ear thickness is significantly reduced compared to the positive control when the peptide fragment according to the present invention is injected. Therefore, the peptide of the present invention including the peptide of SEQ ID NO: 1 can effectively inhibit the IgE-mediated immediate hypersensitivity reaction, and is also expected to effectively inhibit the inflammation caused by the hypersensitivity reaction.

<110> KNU-Industry Cooperation Foundation <120> Pharmaceutical composition comprising peptide having inhibitory          activities against transendothelial migration of leucocytes and          metastasis of cancer cells <130> PN0453 <160> 3 <170> Kopatentin 1.71 <210> 1 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> peptide fragment <400> 1 Cys ser leu   One <210> 2 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> peptide fragment <400> 2 Ser Leu His Tyr   One <210> 3 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> peptide fragment <400> 3 Glu gly leu   One

Claims (5)

A pharmaceutical composition for preventing or treating an inflammatory disease comprising a peptide selected from the group consisting of peptides consisting of amino acid sequences of SEQ ID NOs: 1 to 3, and comprising a pharmaceutically acceptable carrier. The pharmaceutical composition of claim 1, wherein the inflammatory disease is inflammation caused by hypersensitivity. A pharmaceutical composition for inhibiting cancer metastasis, comprising a peptide selected from the group consisting of peptides consisting of amino acid sequences of SEQ ID NOs: 1 to 3 as an active ingredient, and comprising a pharmaceutically acceptable carrier. The pharmaceutical composition according to any one of claims 1 to 3, wherein the peptide is a peptide consisting of the amino acid sequence of SEQ ID NO: 1. The pharmaceutical composition according to any one of claims 1 to 3, wherein the peptide is a peptide consisting of the amino acid sequence of SEQ ID NO: 2.

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