WO2022114919A2 - Integrin-binding peptide and pharmaceutical composition containing same - Google Patents

Integrin-binding peptide and pharmaceutical composition containing same Download PDF

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Publication number
WO2022114919A2
WO2022114919A2 PCT/KR2021/017875 KR2021017875W WO2022114919A2 WO 2022114919 A2 WO2022114919 A2 WO 2022114919A2 KR 2021017875 W KR2021017875 W KR 2021017875W WO 2022114919 A2 WO2022114919 A2 WO 2022114919A2
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cancer
peptide
integrin
structural formula
integer
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PCT/KR2021/017875
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French (fr)
Korean (ko)
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WO2022114919A3 (en
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오명열
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(주)한국바이오셀프
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

  • the present invention relates to an integrin-binding peptide and a pharmaceutical composition comprising the same.
  • Intratumoral injection is a method of directly injecting an anticancer agent into a tumor tissue, and is one of the most direct and effective anticancer therapies.
  • most of the administered anticancer agents are hydrophobic chemicals having a molecular weight of less than 500 kDa, and there is a problem in that it is difficult to penetrate into the target cancer cells and is released quickly even if absorbed. As such, since the anticancer drug remaining outside is toxic to surrounding normal cells, the actual anticancer effect is lowered and the side effects are increased.
  • Integrin acts as a heterodimer in which ⁇ and ⁇ subunits are non-covalently bonded, and ⁇ and ⁇ subunits are paired to form 22 integrin families.
  • Integrin mainly binds to extracellular matrix proteins such as vibronectin, fibronectin, collagen, laminin, von Willebrand factor (vWF), fibrinogen, etc. There is a difference in ligand specificity, and one type of integrin can bind to several ligands at the same time.
  • Integrins are cell surface receptors that regulate important physiological functions of cells, such as cell adhesion and migration, differentiation and proliferation, and participate in angiogenesis and tumor metastasis through interactions that link intracellular cytoskeletal proteins with extracellular matrix molecules. .
  • the present inventors discovered a novel integrin-binding peptide during research on integrin-targeting therapy to solve the above problems, and confirmed that the therapeutic effect is significantly increased when the drug is administered in combination with the present invention completed.
  • Another object of the present invention is to provide an anticancer adjuvant.
  • Another object of the present invention is to provide a pharmaceutical composition for combined administration for the prevention or treatment of cancer.
  • Another object of the present invention is to provide a method for treating cancer.
  • Another object of the present invention is to provide a food composition for adjuvant cancer treatment.
  • the present invention provides an integrin-binding peptide represented by the following structural formula 1:
  • A consists of the amino acid sequence of VG; wherein B is a targeting moiety; wherein C is an elastin-like peptide; Wherein n is an integer of 4 to 8, and m is an integer of 1 to 10.
  • the present invention provides an anticancer adjuvant comprising the peptide according to the present invention.
  • the present invention provides a pharmaceutical composition for co-administration for the prevention or treatment of cancer comprising the peptide according to the present invention and an anticancer agent.
  • the present invention provides a method for treating cancer, comprising administering the peptide and anticancer agent according to the present invention to a subject.
  • the present invention provides a food composition for adjuvant cancer treatment comprising the peptide according to the present invention.
  • the novel integrin-binding peptide of the present invention when administered in combination with an anti-cancer agent in an animal model of cancer, the therapeutic effect of the anti-cancer agent is significantly enhanced, so it can be usefully used as a new effective treatment strategy for cancer treatment. .
  • FIG. 1 is a schematic diagram showing the concept of the integrin-binding peptide of the present invention.
  • FIG. 2 is a diagram showing the results of purifying the integrin-binding peptide.
  • FIG. 3 is a diagram showing the results of confirming the mouse tumor after three administrations (day 7) in the negative control group, the positive control group and the experimental group.
  • FIG. 4 is a view showing the results of confirming the change in the individual mouse body weight for each experimental group.
  • 5 is a diagram showing the results of confirming the change in the average mouse weight for each experimental group.
  • FIG. 6 is a view showing the results of confirming the size change of individual mouse tumors for each experimental group.
  • FIG. 7 is a diagram showing the results of confirming the change in the size of the mouse average tumor size for each experimental group (Significantly different between G1 vs G2, G3 at 7 th day (*p ⁇ 0.5, **p ⁇ 0.05)).
  • the present invention provides an integrin-binding peptide represented by the following structural formula 1:
  • A consists of the amino acid sequence of VG
  • B is a targeting moiety
  • C is an elastin-like peptide
  • n is an integer from 4 to 8
  • m is an integer from 1 to 10.
  • the integrin-binding peptide of the present invention is a temperature-sensitive peptide, and may be referred to as “Uptake Inducible Protein (UIP)”.
  • UIP Uptake Inducible Protein
  • the UIP according to the present invention is an elastin-like polypeptide (ELP) containing an amino acid RGD sequence, and more preferably, the total amino acid sequence may be represented by VGRGD(VGVPG) 6 (SEQ ID NO: 1).
  • VGRGDVGVPGVGVPGVGVPGVGVPGVGVPGVGVPG (SEQ ID NO: 1)
  • Elastin is a major component of the extracellular matrix and is involved in the elasticity of connective tissue, and is formed by self-aggregation of precursor tropoelastin.
  • Tropoelastin contains hydrophobic valine and glycine and proline that interfere with the formation of alpha helices and beta sheets.
  • UIP which is an integrin-binding peptide according to the present invention, has an amino acid sequence RGD that binds to integrin, a cell membrane protein, based on the tropoelastin sequence, thereby enabling targeting to cancer cells.
  • elastin-like polypeptides exhibit reversible phase separation, remaining soluble below the transition temperature, but forming amorphous coacervates above the transition temperature. Using these characteristics, it can be expected that when directly injected near a tumor in combination with an existing anticancer agent, it stays in the body as a coacervate for a long time and acts on the target site.
  • the targeting moiety is any one selected from the group consisting of RGD peptide, ⁇ -helical peptide, mitochondrial targeting peptide, Fc receptor binding peptide, and beta endorphin receptor ligand It may be more than one peptide.
  • the elastin-like peptide may consist of an amino acid sequence of VGVPG, preferably, the amino acid sequence of VGVPG is repeated 4 to 8 times, more preferably, an amino acid sequence repeated 6 times may be made of
  • amino acid variations are made based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, and the like.
  • amino acid side chain substituents such as hydrophobicity, hydrophilicity, charge, size, and the like.
  • arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine and histidine; alanine, glycine and serine; And phenylalanine, tryptophan and tyrosine can be said to be biologically functional equivalents.
  • the hydropathic index of amino acids may be considered.
  • Each amino acid is assigned a hydrophobicity index according to its hydrophobicity and charge: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • the hydrophobic amino acid index is very important in conferring an interactive biological function of a peptide. It is a known fact that amino acids having a similar hydrophobicity index must be substituted to retain similar biological activity. When introducing a mutation with reference to the hydrophobicity index, the substitution is made between amino acids showing a difference in the hydrophobicity index, preferably within ⁇ 2, more preferably within ⁇ 1, and even more preferably within ⁇ 0.5.
  • hydrophilicity values are assigned to each amino acid residue: arginine (+3.0 ); lysine (+3.0); asphaltate (+3.0 ⁇ 1); glutamate (+3.0 ⁇ 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ⁇ 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); Tryptophan (-3.4).
  • the substitution is made between amino acids exhibiting a difference in the hydrophilicity value within preferably ⁇ 2, more preferably within ⁇ 1, and even more preferably within ⁇ 0.5.
  • Amino acid exchanges in peptides that do not entirely alter the activity of the molecule are known in the art.
  • the most commonly occurring exchanges are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/ It is an exchange between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly.
  • the integrin-binding peptide of the present invention also includes a sequence exhibiting substantial identity with the sequence described in the sequence listing.
  • the substantial identity is at least 80% or higher when the peptide sequence of the present invention and any other sequences are aligned as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. It refers to a sequence that is homologous, more preferably 90% or more homologous.
  • the alignment method for sequence comparison may be any method known in the art without limitation.
  • the integrin-binding peptide of the present invention is characterized in that it enhances the therapeutic effect of the drug.
  • the drug is not limited thereto, but may be a drug that requires targeted therapy, for example, an anticancer agent that can act as a target on cancer cells.
  • it may be an anticancer agent that can be substituted with a carboxy group or a hydroxyl group residue. More preferably, it may be 5-fluorouracil (5-FU) or paclitaxel, but is not limited thereto.
  • the integrin-binding peptide of the present invention may be used in combination with a drug or in a form combined with a drug.
  • the integrin-binding peptide of the present invention may be characterized in that the drug is conjugated to the N-terminus or C-terminus of the peptide of the present invention.
  • the form to be bound may be any form known in the art without limitation, but may preferably be one in which a drug is bound to the N-terminus or C-terminus of the peptide, or may be bound by a linker peptide.
  • linker peptide that can be used in the present invention does not inhibit the effects of the integrin-binding peptide and the drug, any linker peptide known in the art can be used without limitation.
  • the integrin-binding peptide of the present invention may be characterized in that it is hydrogelled based on the transition temperature as a temperature-sensitive peptide, for example, it may be hydrogelled at a viscosity suitable for achieving sustained release of the drug at a temperature of 30° C. or higher.
  • the present invention also provides an anticancer adjuvant comprising an integrin-binding peptide represented by the following structural formula 1:
  • A consists of the amino acid sequence of VG
  • B is a targeting moiety
  • C is an elastin-like peptide
  • n is an integer from 4 to 8
  • m is an integer from 1 to 10.
  • Structural Formula 1 is the same as described above.
  • anticancer adjuvant refers to an agent capable of improving, enhancing or enhancing the anticancer effect of an anticancer agent.
  • the present invention provides a pharmaceutical composition for co-administration for the prevention or treatment of cancer comprising an integrin-binding peptide represented by the following structural formula 1 and an anticancer agent:
  • A consists of the amino acid sequence of VG
  • B is a targeting moiety
  • C is an elastin-like peptide
  • n is an integer from 4 to 8
  • m is an integer from 1 to 10.
  • Structural Formula 1 is the same as described above.
  • the cancer is not limited thereto, but may be one or more selected from the group consisting of breast cancer, brain tumor, skin cancer, pancreatic cancer, liver cancer, lung cancer, bone marrow cancer, esophageal cancer, colorectal cancer, stomach cancer, cervical cancer, prostate cancer, ovarian cancer, and head and neck cancer and preferably solid cancer including breast cancer.
  • the integrin-binding peptide according to the present invention may be administered simultaneously or sequentially with the anticancer agent.
  • the anticancer agent may be an anticancer agent for targeted therapy, preferably an anticancer agent that can be substituted with a carboxyl group or a hydroxyl group residue. More preferably, it may be 5-FU or paclitaxel, but is not limited thereto.
  • the integrin-binding peptide of the present invention can induce sustained release of an anticancer agent administered in combination based on the property of staying in the target site for a long time in a coacervate state. Therefore, the integrin-binding peptide of the present invention can act as an adjuvant to help delayed release and action of a drug.
  • the pharmaceutical composition of the present invention may contain the integrin-binding peptide and the anticancer agent alone, or may additionally contain one or more pharmaceutically acceptable carriers, excipients or diluents.
  • the pharmaceutically acceptable carrier may further include, for example, a carrier for parenteral administration.
  • the carrier for parenteral administration may include water, a suitable oil, saline, aqueous glucose and glycol, and the like, and may further include a stabilizer and a preservative.
  • Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid.
  • Suitable preservatives are benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol.
  • the pharmaceutical composition of the present invention may be administered to mammals including humans by any method. Preferably, it can be administered parenterally.
  • the parenteral administration method is not limited thereto, but may be administered intramuscularly, intramedullary, intrathecal, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal administration. More preferably, the pharmaceutical composition of the present invention may be administered by intratumoral injection.
  • composition of the present invention may be formulated as a preparation for parenteral administration according to the administration route as described above.
  • the formulation for parenteral administration it can be formulated in the form of injections, creams, lotions, external ointments, oils, moisturizers, gels, aerosols and nasal inhalants by methods known in the art.
  • the total effective amount of the pharmaceutical composition of the present invention may be administered to a patient as a single dose, and may be administered by a fractionated treatment protocol in which multiple doses are administered for a long period of time.
  • the pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the severity of the disease. For parenteral administration, based on the integrin-binding peptide and anticancer agent, it is preferably administered in an amount of 0.01 to 50 mg, more preferably 0.1 to 30 mg per 1 kg of body weight per day, divided into 1 to several times administered. .
  • the dose of the integrin-binding peptide and anticancer agent is determined by considering various factors such as the age, weight, health status, sex, severity of disease, diet and excretion rate of the patient, as well as the administration route and number of treatments of the pharmaceutical composition. Since the effective dosage is determined, one of ordinary skill in the art will be able to determine an appropriate effective dosage of the composition of the present invention in consideration of this point.
  • the pharmaceutical composition is not particularly limited in its formulation, administration route and administration method as long as the effect of the present invention is exhibited.
  • the pharmaceutical composition may further include one or more active ingredients exhibiting the same or similar medicinal effect in anticancer effect or anticancer effect enhancing effect in addition to the integrin-binding peptide and anticancer agent according to the present invention.
  • the present invention provides a method of treating cancer, comprising administering to a subject an integrin-binding peptide represented by the following structural formula 1 and an anticancer agent:
  • A consists of the amino acid sequence of VG
  • B is a targeting moiety
  • C is an elastin-like peptide
  • n is an integer from 4 to 8
  • m is an integer from 1 to 10.
  • Structural Formula 1 is the same as described above.
  • the term “individual” refers to an individual having a cancer disease, preferably a mammal including a human having a cancer disease, such as horse, sheep, pig, goat, dog, etc., preferably means human.
  • the integrin-binding peptide may be administered simultaneously or sequentially with an anticancer agent.
  • the administration is not limited, but may preferably be an intratumoral injection.
  • the present invention also provides a food composition for adjuvant cancer treatment comprising an integrin-binding peptide represented by the following structural formula 1:
  • A consists of the amino acid sequence of VG
  • B is a targeting moiety
  • C is an elastin-like peptide
  • n is an integer from 4 to 8
  • m is an integer from 1 to 10.
  • Structural Formula 1 is the same as described above.
  • the food composition according to the present invention includes all types of functional food, nutritional supplement, health food, health functional food and food additives. .
  • the term "health functional food” refers to a food manufactured and processed by extracting, concentrating, refining, mixing, etc., a specific ingredient as a raw material or a specific ingredient contained in a food raw material for the purpose of health supplementation, It refers to a food designed and processed to sufficiently exert biological control functions such as biological defense, regulation of biological rhythm, prevention and recovery of disease, etc., with the above ingredients, and performs functions related to prevention of disease or recovery of health, etc. say what you can do
  • the food composition according to the present invention can be prepared in various forms according to conventional methods known in the art.
  • the integrin-binding peptide according to the present invention may be ingested by granulating, encapsulating and powdering itself, or it may be prepared in the form of tea, juice, or drink for drinking. In addition, it can be prepared in the form of a composition by mixing the integrin-binding peptide of the present invention with a known substance or active ingredient known to have a cancer-preventing or therapeutic effect.
  • functional foods include beverages (including alcoholic beverages), fruits and their processed foods (eg, canned fruit, bottled, jam, marmalade, etc.), fish, meat, and their processed foods (eg, ham, sausage corn beef, etc.) , Breads and noodles (eg udon noodles, soba noodles, ramen, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, syrup, dairy products (eg butter, cheese, etc.), edible vegetable oil, margarine, vegetable protein, retort It can be prepared by adding the integrin-binding peptide of the present invention to food, frozen food, various seasonings (eg, soybean paste, soy sauce, sauce, etc.).
  • fruits and their processed foods eg, canned fruit, bottled, jam, marmalade, etc.
  • fish, meat, and their processed foods eg, ham, sausage corn beef, etc.
  • Breads and noodles eg udon noodles, soba noodles, ramen, spaghetti, macaroni, etc.
  • the preferred content of the integrin-binding peptide of the present invention in the food composition of the present invention is not limited thereto, but may be, for example, 0.01 to 80% by weight of the finally prepared food, preferably in the finally prepared food. It may be 0.01 to 50% by weight.
  • the food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients.
  • natural carbohydrates monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and natural sweeteners such as dextrin and cyclodextrin, synthetic sweeteners such as saccharin and aspartame may be used.
  • the proportion of the natural carbohydrate may be generally about 0.01 to 0.4 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention.
  • the food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol , a carbonation agent used in carbonated beverages, and the like.
  • the food composition of the present invention may contain fruit for the production of natural fruit juice, fruit juice beverage, and vegetable beverage. These components may be used independently or in combination. The proportion of these additives is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention, but is not limited thereto.
  • Uptake inducible protein (UIP) protein which is an integrin-binding peptide according to the present invention, was prepared as follows.
  • the sequence of UIP is synthesized by adding different restriction enzyme sequences to both ends of the 5' and 3' ends of the DNA nucleotide sequence encoding the amino acid sequence VGRGD(VGVPG) 6 , and pET-29a through a transformation method known in the art. (+) (Novagen, Merck Millipore) was inserted into the vector. After confirming the UIP sequence through sequencing, the UIP protein was finally obtained through protein expression and purification. The results of UIP protein purification according to the present invention are shown in FIG. 2 , and it was confirmed that a UIP protein containing a tag, of 8.4 kDa, was obtained through this.
  • Mouse breast cancer cell line 4T1 to be administered to mice was cultured by adding 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin to RPMI-1640 medium.
  • 4T1 cells 1x10 6 cells/head prepared as above were subcutaneously transplanted into mice (BALB/c, 6 weeks old, female) to evaluate the anticancer efficacy of UIP and 5-FU co-administration.
  • the experimental group was classified (PBS administration group, positive control group 5-FU administration group, experimental group 5-FU and UIP combination administration group), and PBS, 5-FU or UIP and 5 for each experimental group -FU was administered three times at intervals of 3 days (1 day, 4 days, 7 days) subcutaneously near the tumor, and then the size of the tumor and the weight of the mouse were measured.
  • the experimental groups and conditions used are shown in Table 1 below.
  • Figure 3 shows the mouse tumor photos after administration 3 times (day 7) for each experimental group, the changes in individual mouse body weight are shown in Tables 2 and 4 below, and the changes in the average body weight for each experimental group are shown in Tables 3 and 5 below. shown in
  • Tumor size reduction rates were compared in the control group and the experimental group.
  • the positive control group G2
  • the p-value was 0.5 or less, indicating statistical significance.
  • the experimental group G3
  • the p-value was less than 0.5 from the 2nd day after administration of the test substance, indicating statistical significance
  • the p-value was less than 0.05, indicating strong statistical significance.
  • the tumor size change according to drug administration was evaluated based on the negative control group, and the size of the tumor compared to the negative control group
  • the reduction rate is shown in Table 6 below.
  • the positive control group did not show a significant difference from the negative control group until the 4th day, and the tumor size decreased by 7.6% on the 7th day, and statistical significance was observed.
  • the UIP combination administration group statistical significance was observed from the 2nd day, and the tumor size decreased by 20.6% on the 7th day. That is, the experimental group showed statistical significance from the 2nd and 4th days, and the 7th day showed strong statistical significance, confirming that the size of the breast cancer tumor was reduced more effectively than the positive control group, G2. Therefore, it was clearly confirmed that the UIP, which is the integrin-binding peptide according to the present invention, can enhance the effect of the drug administered together.

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Abstract

The present invention relates to an integrin-binding peptide and a combined use thereof with an anti-cancer agent. It has been identified that when an integrin-binding peptide according to the present invention is administered to a cancer animal model in combination with an anti-cancer agent, therapeutic effects of the anti-cancer agent are remarkably increased, and thus the present invention can be effectively used as an effective novel therapeutic strategy in cancer treatment.

Description

인테그린 결합성 펩타이드 및 이를 포함하는 약학적 조성물Integrin-binding peptide and pharmaceutical composition comprising same
본 발명은 인테그린 결합성 펩타이드 및 이를 포함하는 약학적 조성물에 관한 것이다. The present invention relates to an integrin-binding peptide and a pharmaceutical composition comprising the same.
종양 내 직접주사 요법(intratumoral injection)은 항암제를 종양 조직 내에 직접 주사하는 방법으로, 가장 직접적이고 효과적인 항암요법 중 하나이다. 하지만, 투여하는 항암제의 대부분은 분자량 500 kDa 미만의 소수성 화학물로, 표적 암세포 내에 침투가 어렵고 흡수되더라도 금방 방출되어 버리는 문제점이 있다. 이처럼 외부에 남아있는 항암제는 주변의 정상세포에 독성을 나타내기 때문에 실질적 항암 효과가 낮아지고 부작용은 높아지는 문제가 산재하고 있는 실정이다.Intratumoral injection is a method of directly injecting an anticancer agent into a tumor tissue, and is one of the most direct and effective anticancer therapies. However, most of the administered anticancer agents are hydrophobic chemicals having a molecular weight of less than 500 kDa, and there is a problem in that it is difficult to penetrate into the target cancer cells and is released quickly even if absorbed. As such, since the anticancer drug remaining outside is toxic to surrounding normal cells, the actual anticancer effect is lowered and the side effects are increased.
이에 따라 표적 부위에 머무르며 약물을 서서히 방출하여 암세포 내로 약물을 전달시켜 항암 효과를 증대시킬 수 있는 새로운 기술의 개발이 요구되고 있다. Accordingly, there is a demand for the development of a new technology capable of increasing the anticancer effect by staying at the target site and slowly releasing the drug to deliver the drug into the cancer cells.
한편, 인테그린(Integrin)은 α와 β 서브유닛(subunit)이 비공유 결합으로 이루어진 헤테로다이머(heterodimer)로 작용하며, α와 β 서브유닛이 쌍을 이루어 22가지의 인테그린 패밀리를 구성하고 있다. 인테그린은 주로 비브로넥틴(Vibronectin), 피브로넥틴(Fibronectin), 콜라겐(collagen), 라미닌(laminin), vWF(von Willebrand factor), 피브리노겐(fibrinogen) 등의 세포외 메트릭스 단백질에 결합하나, 인테그린의 종류별로 리간드 특이성에 차이가 있으며, 한 종류의 인테그린이 여러 가지 리간드에 동시에 결합할 수 있다. 인테그린은 세포 부착 및 이동, 분화, 증식 등과 같은 세포의 중요한 생리작용을 조절하는 세포 표면 수용체이며, 세포 내의 세포 골격 단백질과 세포 외의 기질 분자를 연결하는 상호 작용을 통해 혈관 생성과 종양 전이에 참여한다.On the other hand, integrin acts as a heterodimer in which α and β subunits are non-covalently bonded, and α and β subunits are paired to form 22 integrin families. Integrin mainly binds to extracellular matrix proteins such as vibronectin, fibronectin, collagen, laminin, von Willebrand factor (vWF), fibrinogen, etc. There is a difference in ligand specificity, and one type of integrin can bind to several ligands at the same time. Integrins are cell surface receptors that regulate important physiological functions of cells, such as cell adhesion and migration, differentiation and proliferation, and participate in angiogenesis and tumor metastasis through interactions that link intracellular cytoskeletal proteins with extracellular matrix molecules. .
따라서 현재 다양한 암의 치료에 있어서, 인테그린을 타겟으로 한 효과에 대하여 연구되고 있으나, 지속적으로 많은 연구가 필요한 실정이다.Therefore, in the current treatment of various cancers, the effect of targeting integrins is being studied, but a lot of continuous research is needed.
본 발명자들은 상기와 같은 문제를 해결하기 위하여 인테그린을 타겟하는 치료법에 대하여 연구하던 중, 신규한 인테그린 결합성 펩타이드를 발굴하고, 이와 약제를 병용투여하는 경우 치료 효과가 현저히 증가됨을 확인하여 본 발명을 완성하였다. In order to solve the above problems, the present inventors discovered a novel integrin-binding peptide during research on integrin-targeting therapy to solve the above problems, and confirmed that the therapeutic effect is significantly increased when the drug is administered in combination with the present invention completed.
따라서 본 발명의 목적은 인테그린 결합성 펩타이드를 제공하는 것이다. Accordingly, it is an object of the present invention to provide an integrin-binding peptide.
본 발명의 다른 목적은 항암 보조제를 제공하는 것이다.Another object of the present invention is to provide an anticancer adjuvant.
본 발명의 또 다른 목적은 암의 예방 또는 치료를 위한 병용 투여용 약학적 조성물을 제공하는 것이다.Another object of the present invention is to provide a pharmaceutical composition for combined administration for the prevention or treatment of cancer.
본 발명의 또 다른 목적은 암을 치료하는 방법을 제공하는 것이다.Another object of the present invention is to provide a method for treating cancer.
본 발명의 또 다른 목적은 암 치료 보조용 식품 조성물을 제공하는 것이다.Another object of the present invention is to provide a food composition for adjuvant cancer treatment.
상기 목적을 달성하기 위하여, 본 발명은 하기 구조식 1로 표시되는 인테그린 결합성 펩타이드를 제공한다: In order to achieve the above object, the present invention provides an integrin-binding peptide represented by the following structural formula 1:
[구조식 1][Structural Formula 1]
[A-B-Cn]m[A-B-Cn]m
여기서 상기 A는 VG의 아미노산 서열로 이루어지며; 상기 B는 표적지향성 모이어티이고; 상기 C는 엘라스틴-유사 펩타이드이며; 상기 n은 4 내지 8의 정수이고, 상기 m은 1 내지 10의 정수이다.wherein A consists of the amino acid sequence of VG; wherein B is a targeting moiety; wherein C is an elastin-like peptide; Wherein n is an integer of 4 to 8, and m is an integer of 1 to 10.
또한 상기 다른 목적을 달성하기 위하여, 본 발명은 본 발명에 따른 펩타이드를 포함하는 항암 보조제를 제공한다.In addition, in order to achieve the above other object, the present invention provides an anticancer adjuvant comprising the peptide according to the present invention.
또한 상기 또 다른 목적을 달성하기 위하여, 본 발명은 본 발명에 따른 펩타이드 및 항암제를 포함하는 암의 예방 또는 치료를 위한 병용 투여용 약학적 조성물을 제공한다.In addition, in order to achieve the above another object, the present invention provides a pharmaceutical composition for co-administration for the prevention or treatment of cancer comprising the peptide according to the present invention and an anticancer agent.
또한 상기 또 다른 목적을 달성하기 위하여, 본 발명은 본 발명에 따른 펩타이드 및 항암제를 개체에 투여하는 단계를 포함하는, 암을 치료하는 방법을 제공한다.In addition, in order to achieve the above another object, the present invention provides a method for treating cancer, comprising administering the peptide and anticancer agent according to the present invention to a subject.
또한 상기 또 다른 목적을 달성하기 위하여, 본 발명은 본 발명에 따른 펩타이드를 포함하는 암 치료 보조용 식품 조성물을 제공한다.In addition, in order to achieve the above another object, the present invention provides a food composition for adjuvant cancer treatment comprising the peptide according to the present invention.
본 발명에 따르면, 본 발명의 신규한 인테그린 결합성 펩타이드를 암 동물 모델에 항암제와 병용 투여한 경우, 항암제의 치료 효과가 현저히 증진되었으므로, 이를 암 치료에 있어 새로운 효과적인 치료 전략으로 유용하게 활용할 수 있다.According to the present invention, when the novel integrin-binding peptide of the present invention is administered in combination with an anti-cancer agent in an animal model of cancer, the therapeutic effect of the anti-cancer agent is significantly enhanced, so it can be usefully used as a new effective treatment strategy for cancer treatment. .
도 1은 본 발명의 인테그린 결합성 펩타이드에 대한 개념을 나타낸 모식도이다. 1 is a schematic diagram showing the concept of the integrin-binding peptide of the present invention.
도 2는 인테그린 결합성 펩타이드를 정제한 후 확인한 결과를 나타낸 도이다. 2 is a diagram showing the results of purifying the integrin-binding peptide.
도 3은 음성 대조군, 양성대조군 및 실험군에서 3회 투여(7일째) 후 마우스 종양을 확인한 결과를 나타낸 도이다.3 is a diagram showing the results of confirming the mouse tumor after three administrations (day 7) in the negative control group, the positive control group and the experimental group.
도 4는 실험군별 마우스 개별 몸무게의 변화를 확인한 결과를 나타낸 도이다. 4 is a view showing the results of confirming the change in the individual mouse body weight for each experimental group.
도 5는 실험군별 마우스 평균 몸무게의 변화를 확인한 결과를 나타낸 도이다. 5 is a diagram showing the results of confirming the change in the average mouse weight for each experimental group.
도 6은 실험군별 마우스 개별 종양의 크기 변화를 확인한 결과를 나타낸 도이다. 6 is a view showing the results of confirming the size change of individual mouse tumors for each experimental group.
도 7은 실험군별 마우스 평균 종양의 크기 변화를 확인한 결과를 나타낸 도이다(Significantly different between G1 vs G2, G3 at 7th day (*p<0.5, **p<0.05)).7 is a diagram showing the results of confirming the change in the size of the mouse average tumor size for each experimental group (Significantly different between G1 vs G2, G3 at 7 th day (*p<0.5, **p<0.05)).
이하, 본 발명에 대해 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 하기 구조식 1로 표시되는 인테그린 결합성 펩타이드를 제공한다:The present invention provides an integrin-binding peptide represented by the following structural formula 1:
[구조식 1][Structural Formula 1]
[A-B-Cn]m [A-B-Cn]m
상기 A는 VG의 아미노산 서열로 이루어지며;wherein A consists of the amino acid sequence of VG;
상기 B는 표적지향성 모이어티이고;wherein B is a targeting moiety;
상기 C는 엘라스틴-유사 펩타이드이며;wherein C is an elastin-like peptide;
상기 n은 4 내지 8의 정수이고, 상기 m은 1 내지 10의 정수임.Wherein n is an integer from 4 to 8, and m is an integer from 1 to 10.
본 발명의 인테그린 결합성 펩타이드는 온도 감응성 펩타이드이며, “UIP(Uptake Inducible Protein)”로 지칭될 수 있다. 본 발명에 따른 UIP는 아미노산 RGD 서열을 함유한 엘라스틴 유사 폴리펩타이드(Elastin-like polypeptide, ELP)로, 보다 바람직하게 총 아미노산 서열은 VGRGD(VGVPG)6로 표시될 수 있다(서열번호 1).The integrin-binding peptide of the present invention is a temperature-sensitive peptide, and may be referred to as “Uptake Inducible Protein (UIP)”. The UIP according to the present invention is an elastin-like polypeptide (ELP) containing an amino acid RGD sequence, and more preferably, the total amino acid sequence may be represented by VGRGD(VGVPG) 6 (SEQ ID NO: 1).
VGRGDVGVPGVGVPGVGVPGVGVPGVGVPGVGVPG(서열번호 1)VGRGDVGVPGVGVPGVGVPGVGVPGVGVPGVGVPG (SEQ ID NO: 1)
엘라스틴은 세포외기질의 주요성분으로 결합조직의 탄성에 관여하며, 전구체인 트로포엘라스틴(Tropoelastin)의 자가응집(self-aggregation)에 의하여 형성된다. 트로포엘라스틴은 소수성인 발린과 알파나선 및 베타시트 형성을 방해하는 글리신 및 프롤린을 포함하고 있다. 본 발명에 따른 인테그린 결합성 펩타이드인 UIP는 도 1과 같이, 트로포엘라스틴 서열을 기반으로 세포막 단백질인 인테그린과 결합하는 아미노산 서열 RGD를 추가함으로써, 암세포에 대한 표적화가 가능하도록 하였다. 이와 별개로, 엘라스틴 유사 폴리펩티드는 가역적인 상 분리를 나타내는데, 전이 온도 이하에서는 용해성을 유지하지만 그 이상에서는 무정형 코아세르베이트(coacervate)를 형성한다. 이러한 특성을 이용하여, 기존의 항암제와 병용하여 종양 부근에 직접적으로 주입했을 시에 체내에서 코아세르베이트 상태로 표적 장소에 오래 머물러 작용하는 것을 기대할 수 있다.Elastin is a major component of the extracellular matrix and is involved in the elasticity of connective tissue, and is formed by self-aggregation of precursor tropoelastin. Tropoelastin contains hydrophobic valine and glycine and proline that interfere with the formation of alpha helices and beta sheets. As shown in FIG. 1, UIP, which is an integrin-binding peptide according to the present invention, has an amino acid sequence RGD that binds to integrin, a cell membrane protein, based on the tropoelastin sequence, thereby enabling targeting to cancer cells. Separately, elastin-like polypeptides exhibit reversible phase separation, remaining soluble below the transition temperature, but forming amorphous coacervates above the transition temperature. Using these characteristics, it can be expected that when directly injected near a tumor in combination with an existing anticancer agent, it stays in the body as a coacervate for a long time and acts on the target site.
본 발명에 있어서, 표적 지향성 모이어티는 RGD 펩타이드, α-나선 펩타이드, 미토콘드리아 표적 펩타이드, Fc 수용체 결합 펩타이드(Fc receptor binding peptide) 및 베타 엔돌핀 수용체 리간드(beta endorphin receptor ligand)로 이루어진 군으로부터 선택된 어느 하나 이상의 펩타이드일 수 있다. 또한 상기 엘라스틴-유사 펩타이드는 VGVPG의 아미노산 서열로 이루어진 것일 수 있고, 바람직하게는 상기 VGVPG의 아미노산 서열이 4 내지 8번 반복된 아미노산 서열로 이루어진 것일 수 있으며, 보다 바람직하게는 6번 반복된 아미노산 서열로 이루어진 것일 수 있다.In the present invention, the targeting moiety is any one selected from the group consisting of RGD peptide, α-helical peptide, mitochondrial targeting peptide, Fc receptor binding peptide, and beta endorphin receptor ligand It may be more than one peptide. In addition, the elastin-like peptide may consist of an amino acid sequence of VGVPG, preferably, the amino acid sequence of VGVPG is repeated 4 to 8 times, more preferably, an amino acid sequence repeated 6 times may be made of
본 발명의 인테그린 결합성 펩타이드 범위에 포함될 수 있는 생물학적 기능 균등물은 본 발명의 펩타이드와 유사 및 동일한 생물학적 활성을 발휘하는 아미노산 서열의 변이를 포함하는데에 한정될 것이라는 것은 당업자에게 명확하다.It is clear to those skilled in the art that biological function equivalents that can be included in the scope of the integrin-binding peptide of the present invention will be limited to include variations in amino acid sequence that exhibit similar and identical biological activities to the peptides of the present invention.
이러한 아미노산 변이는 아미노산 곁사슬 치환체의 상대적 유사성, 예컨대, 소수성, 친수성, 전하, 크기 등에 기초하여 이루어진다. 아미노산 곁사슬 치환체의 크기, 모양 및 종류에 대한 분석에 의하여, 아르기닌, 라이신과 히스티딘은 모두 양전하를 띤 잔기이고; 알라닌, 글라이신과 세린은 유사한 크기를 가지며; 페닐알라닌, 트립토판과 타이로신은 유사한 모양을 갖는다는 것을 알 수 있다. 따라서 이러한 고려 사항에 기초하여, 아르기닌, 라이신과 히스티딘; 알라닌, 글라이신과 세린; 그리고 페닐알라닌, 트립토판과 타이로신은 생물학적으로 기능 균등물이라 할 수 있다.Such amino acid variations are made based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, and the like. According to the analysis of the size, shape and type of amino acid side chain substituents, arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine and histidine; alanine, glycine and serine; And phenylalanine, tryptophan and tyrosine can be said to be biologically functional equivalents.
변이를 도입하는 데 있어서, 아미노산의 소수성 인덱스(hydropathic index)가 고려될 수 있다. 각각의 아미노산은 소수성과 전하에 따라 소수성 인덱스가 부여되어 있다: 아이소루이신 (+4.5); 발린 (+4.2); 루이신(+3.8); 페닐알라닌(+2.8); 시스테인/시스타인 (+2.5); 메티오닌 (+1.9); 알라닌 (+1.8); 글라이신 (-0.4); 트레오닌 (-0.7); 세린 (-0.8); 트립토판 (-0.9); 타이로신 (-1.3); 프롤린 (-1.6); 히스티딘 (-3.2); 글루타메이트 (-3.5); 글루타민 (-3.5); 아스파르테이트 (-3.5); 아스파라긴 (-3.5); 라이신 (-3.9); 및 아르기닌 (-4.5).In introducing the mutation, the hydropathic index of amino acids may be considered. Each amino acid is assigned a hydrophobicity index according to its hydrophobicity and charge: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
펩타이드의 상호적인 생물학적 기능(interactive biological function)을 부여하는 데 있어서 소수성 아미노산 인덱스는 매우 중요하다. 유사한 소수성 인덱스를 가지는 아미노산으로 치환하여야 유사한 생물학적 활성을 보유할 수 있다는 것은 공지된 사실이다. 소수성 인덱스를 참조하여 변이를 도입시키는 경우, 바람직하게는 ±2 이내, 보다 바람직하게는 ±1 이내, 보다 더 바람직하게는 ±0.5 이내의 소수성 인덱스 차이를 나타내는 아미노산 사이에 치환을 한다.The hydrophobic amino acid index is very important in conferring an interactive biological function of a peptide. It is a known fact that amino acids having a similar hydrophobicity index must be substituted to retain similar biological activity. When introducing a mutation with reference to the hydrophobicity index, the substitution is made between amino acids showing a difference in the hydrophobicity index, preferably within ±2, more preferably within ±1, and even more preferably within ±0.5.
한편, 유사한 친수성 값(hydrophilicity value)을 가지는 아미노산 사이의 치환이 유사 및 동일한 생물학적 활성을 갖는 펩타이드를 초래한다는 것도 잘 알려져 있으며, 다음의 친수성 값이 각각의 아미노산 잔기에 부여되어 있다: 아르기닌 (+3.0); 라이신 (+3.0); 아스팔테이트(+3.0±1); 글루타메이트 (+3.0±1); 세린 (+0.3); 아스파라긴 (+0.2); 글루타민 (+0.2); 글라이신 (0); 쓰레오닌 (-0.4); 프롤린 (-0.5±1); 알라닌 (-0.5); 히스티딘 (-0.5); 시스테인 (-1.0); 메티오닌 (-1.3); 발린 (-1.5); 루이신 (-1.8); 아이소루이신 (-1.8); 타이로신 (-2.3); 페닐알라닌 (-2.5); 트립토판 (-3.4). On the other hand, it is also well known that substitution between amino acids with similar hydrophilicity values results in peptides with similar and identical biological activities, and the following hydrophilicity values are assigned to each amino acid residue: arginine (+3.0 ); lysine (+3.0); asphaltate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); Tryptophan (-3.4).
친수성 값을 참조하여 변이를 도입시키는 경우, 바람직하게는 ±2 이내, 보다 바람직하게는 ±1 이내, 보다 더 바람직하게는 ±0.5 이내의 친수성 값 차이를 나타내는 아미노산 사이에 치환을 한다.When a mutation is introduced with reference to the hydrophilicity value, the substitution is made between amino acids exhibiting a difference in the hydrophilicity value within preferably ±2, more preferably within ±1, and even more preferably within ±0.5.
분자의 활성을 전체적으로 변경시키지 않는 펩타이드에서의 아미노산 교환은 당해 분야에 공지되어 있다. 가장 통상적으로 일어나는 교환은 아미노산 잔기 Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly 간의 교환이다.Amino acid exchanges in peptides that do not entirely alter the activity of the molecule are known in the art. The most commonly occurring exchanges are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/ It is an exchange between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly.
상술한 생물학적으로 유사 및 동일한 활성을 갖는 변이를 고려한다면, 본 발명의 인테그린 결합성 펩타이드는 서열목록에 기재된 서열과 실질적인 동일성(substantial identity)을 나타내는 서열도 포함하는 것으로 해석된다. 상기의 실질적인 동일성은, 본 발명의 펩타이드 서열과 임의의 다른 서열을 최대한 대응되도록 얼라인하고, 당업계에서 통상적으로 이용되는 알고리즘을 이용하여 얼라인된 서열을 분석한 경우에, 최소 80% 이상의 상동성, 보다 바람직하게는 90% 이상의 상동성을 나타내는 서열을 의미한다. 서열 비교를 위한 얼라인먼트 방법은 당업계에 공지되어 있는 어떤 방법이라도 제한없이 사용할 수 있다. Considering the above-mentioned mutations having biologically similar and identical activities, it is construed that the integrin-binding peptide of the present invention also includes a sequence exhibiting substantial identity with the sequence described in the sequence listing. The substantial identity is at least 80% or higher when the peptide sequence of the present invention and any other sequences are aligned as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. It refers to a sequence that is homologous, more preferably 90% or more homologous. The alignment method for sequence comparison may be any method known in the art without limitation.
본 발명의 인테그린 결합성 펩타이드는 약제의 치료 효과를 증진시키는 것을 특징으로 한다. 상기 약제는 이에 제한되는 것은 아니나 표적 치료가 필요한 약제일 수 있고, 예컨대 암 세포에 표적으로 작용할 수 있는 항암제일 수 있다. 바람직하게는 카르복시기(carboxy group)나 히드록시기(hydroxyl group) 잔기로 치환할 수 있는 항암제일 수 있다. 보다 더 바람직하게는 5-fluorouracil(5-FU) 또는 파클리탁셀(paclitaxel)일 수 있으나 이에 제한되는 것은 아니다.The integrin-binding peptide of the present invention is characterized in that it enhances the therapeutic effect of the drug. The drug is not limited thereto, but may be a drug that requires targeted therapy, for example, an anticancer agent that can act as a target on cancer cells. Preferably, it may be an anticancer agent that can be substituted with a carboxy group or a hydroxyl group residue. More preferably, it may be 5-fluorouracil (5-FU) or paclitaxel, but is not limited thereto.
본 발명의 인테그린 결합성 펩타이드는 약제와 병용 또는 약제와 결합된 형태로 사용될 수 있다. 본 발명의 인테그린 결합성 펩타이드가 약제와 결합된 형태로 사용되는 경우, 본 발명의 펩타이드의 N 말단 또는 C 말단에 약제가 결합(conjugated)된 것을 특징으로 할 수 있다. 이때 결합되는 형태는 당 분야에 알려진 형태를 제한없이 사용할 수 있으나, 바람직하게는 펩타이드의 N 말단 또는 C 말단에 약제가 결합된 것일 수 있고, 링커 펩타이드(linker peptide)에 의해 결합된 것일 수 있다. The integrin-binding peptide of the present invention may be used in combination with a drug or in a form combined with a drug. When the integrin-binding peptide of the present invention is used in a form bound to a drug, it may be characterized in that the drug is conjugated to the N-terminus or C-terminus of the peptide of the present invention. At this time, the form to be bound may be any form known in the art without limitation, but may preferably be one in which a drug is bound to the N-terminus or C-terminus of the peptide, or may be bound by a linker peptide.
본 발명에서 사용될 수 있는 링커 펩타이드의 종류는 인테그린 결합성 펩타이드와 약제의 효과를 저해시키는 것이 아닌 한, 당 분야에 공지된 링커 펩타이드를 제한없이 사용할 수 있다. As long as the type of linker peptide that can be used in the present invention does not inhibit the effects of the integrin-binding peptide and the drug, any linker peptide known in the art can be used without limitation.
본 발명의 인테그린 결합성 펩타이드는 온도 감응성 펩타이드로 전이 온도를 기준으로 하이드로겔화되는 것을 특징으로 할 수 있으며, 예컨대 30℃ 이상의 온도에서 약물의 서방출을 달성하기에 적절한 점도로 하이드로겔화될 수 있다.The integrin-binding peptide of the present invention may be characterized in that it is hydrogelled based on the transition temperature as a temperature-sensitive peptide, for example, it may be hydrogelled at a viscosity suitable for achieving sustained release of the drug at a temperature of 30° C. or higher.
또한 본 발명은 하기 구조식 1로 표시되는 인테그린 결합성 펩타이드를 포함하는 항암 보조제를 제공한다:The present invention also provides an anticancer adjuvant comprising an integrin-binding peptide represented by the following structural formula 1:
[구조식 1][Structural Formula 1]
[A-B-Cn]m[A-B-Cn]m
상기 A는 VG의 아미노산 서열로 이루어지며;wherein A consists of the amino acid sequence of VG;
상기 B는 표적지향성 모이어티이고;wherein B is a targeting moiety;
상기 C는 엘라스틴-유사 펩타이드이며;wherein C is an elastin-like peptide;
상기 n은 4 내지 8의 정수이고, 상기 m은 1 내지 10의 정수임.Wherein n is an integer from 4 to 8, and m is an integer from 1 to 10.
상기 구조식 1에 대해서는 앞서 설명한 바와 같다. Structural Formula 1 is the same as described above.
본 발명에서, 용어 “항암 보조제”는 항암제의 항암 효과를 개선, 향상 또는 증대시킬 수 있는 제제를 의미한다.In the present invention, the term “anticancer adjuvant” refers to an agent capable of improving, enhancing or enhancing the anticancer effect of an anticancer agent.
또한 본 발명은 하기 구조식 1로 표시되는 인테그린 결합성 펩타이드 및 항암제를 포함하는 암의 예방 또는 치료를 위한 병용 투여용 약학적 조성물을 제공한다:In addition, the present invention provides a pharmaceutical composition for co-administration for the prevention or treatment of cancer comprising an integrin-binding peptide represented by the following structural formula 1 and an anticancer agent:
[구조식 1][Structural Formula 1]
[A-B-Cn]m[A-B-Cn]m
상기 A는 VG의 아미노산 서열로 이루어지며;wherein A consists of the amino acid sequence of VG;
상기 B는 표적지향성 모이어티이고;wherein B is a targeting moiety;
상기 C는 엘라스틴-유사 펩타이드이며;wherein C is an elastin-like peptide;
상기 n은 4 내지 8의 정수이고, 상기 m은 1 내지 10의 정수임.Wherein n is an integer from 4 to 8, and m is an integer from 1 to 10.
상기 구조식 1에 대해서는 앞서 설명한 바와 같다. Structural Formula 1 is the same as described above.
상기 암은 이에 제한되는 것은 아니나, 유방암, 뇌종양, 피부암, 췌장암, 간암, 폐암, 골수암, 식도암, 대장암, 위암, 자궁경부암, 전립선암, 난소암 및 두경부암으로 이루어진 군으로부터 선택된 1종 이상일 수 있고, 바람직하게는 유방암을 비롯한 고형암일 수 있다.The cancer is not limited thereto, but may be one or more selected from the group consisting of breast cancer, brain tumor, skin cancer, pancreatic cancer, liver cancer, lung cancer, bone marrow cancer, esophageal cancer, colorectal cancer, stomach cancer, cervical cancer, prostate cancer, ovarian cancer, and head and neck cancer and preferably solid cancer including breast cancer.
본 발명의 약학적 조성물에 있어서, 본 발명에 따른 인테그린 결합성 펩타이드는 항암제와 동시 또는 순차적으로 투여될 수 있다. 상기 항암제는 표적 치료용 항암제일 수 있고, 바람직하게는 카르복시기나 히드록시기 잔기로 치환할 수 있는 항암제일 수 있다. 보다 더 바람직하게는 5-FU 또는 파클리탁셀일 수 있으나 이에 제한되는 것은 아니다. 본 발명의 인테그린 결합성 펩타이드는 코아세르베이트 상태로 표적 장소에 오래 머물러 작용하는 특성을 기반으로, 병용 투여되는 항암제의 서방출을 유도할 수 있다. 따라서 본 발명의 인테그린 결합성 펩타이드는 약제의 지연된 방출 및 작용을 도와주는 보조제로 작용할 수 있다.In the pharmaceutical composition of the present invention, the integrin-binding peptide according to the present invention may be administered simultaneously or sequentially with the anticancer agent. The anticancer agent may be an anticancer agent for targeted therapy, preferably an anticancer agent that can be substituted with a carboxyl group or a hydroxyl group residue. More preferably, it may be 5-FU or paclitaxel, but is not limited thereto. The integrin-binding peptide of the present invention can induce sustained release of an anticancer agent administered in combination based on the property of staying in the target site for a long time in a coacervate state. Therefore, the integrin-binding peptide of the present invention can act as an adjuvant to help delayed release and action of a drug.
본 발명의 약학적 조성물은 인테그린 결합성 펩타이드 및 항암제를 단독으로 함유하거나, 또는 하나 이상의 약학적으로 허용되는 담체, 부형제 또는 희석제를 추가로 함유할 수 있다.The pharmaceutical composition of the present invention may contain the integrin-binding peptide and the anticancer agent alone, or may additionally contain one or more pharmaceutically acceptable carriers, excipients or diluents.
약학적으로 허용되는 담체로는 예컨대, 비경구 투여용 담체를 추가로 포함할 수 있다. 상기 비경구 투여용 담체는 물, 적합한 오일, 식염수, 수성 글루코스 및 글리콜 등을 포함할 수 있으며, 안정화제 및 보존제를 추가로 포함할 수 있다. 적합한 안정화제로는 아황산수소나트륨, 아황산나트륨 또는 아스코르브산과 같은 항산화제가 있다. 적합한 보존제로는 벤즈알코늄 클로라이드, 메틸- 또는 프로필-파라벤 및 클로로부탄올이 있다.The pharmaceutically acceptable carrier may further include, for example, a carrier for parenteral administration. The carrier for parenteral administration may include water, a suitable oil, saline, aqueous glucose and glycol, and the like, and may further include a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives are benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol.
본 발명의 약학적 조성물은 인간을 비롯한 포유동물에 어떠한 방법으로도 투여할 수 있다. 바람직하게는 비경구적으로 투여할 수 있다. 비경구적인 투여방법으로는 이에 한정되지는 않으나, 근육내, 골수내, 경막내, 경피, 피하, 복강내, 비강내, 장관, 국소, 설하 또는 직장내 투여될 수 있다. 보다 바람직하게는 본 발명의 약학적 조성물은 종양 내 직접주사(intratumoral injection)로 투여될 수 있다.The pharmaceutical composition of the present invention may be administered to mammals including humans by any method. Preferably, it can be administered parenterally. The parenteral administration method is not limited thereto, but may be administered intramuscularly, intramedullary, intrathecal, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal administration. More preferably, the pharmaceutical composition of the present invention may be administered by intratumoral injection.
본 발명의 약학적 조성물은 상술한 바와 같은 투여 경로에 따라 비경구 투여용 제제로 제형화할 수 있다.The pharmaceutical composition of the present invention may be formulated as a preparation for parenteral administration according to the administration route as described above.
상기 비경구 투여용 제제의 경우에는 주사제, 크림제, 로션제, 외용연고제, 오일제, 보습제, 겔제, 에어로졸 및 비강 흡입제의 형태로 당업계에 공지된 방법으로 제형화할 수 있다. In the case of the formulation for parenteral administration, it can be formulated in the form of injections, creams, lotions, external ointments, oils, moisturizers, gels, aerosols and nasal inhalants by methods known in the art.
본 발명의 약학적 조성물의 총 유효량은 단일 투여량(single dose)으로 환자에게 투여될 수 있으며, 다중 투여량(multiple dose)으로 장기간 투여되는 분할 치료 방법(fractionated treatment protocol)에 의해 투여될 수 있다. 본 발명의 약학 조성물은 질환의 정도에 따라 유효성분의 함량을 달리할 수 있다. 비경구 투여시는 상기 인테그린 결합성 펩타이드 및 항암제를 기준으로 하루에 체중 1 kg당 바람직하게 0.01 내지 50 mg, 더 바람직하게는 0.1 내지 30 mg의 양으로 투여되도록 1 내지 수회에 나누어 투여할 수 있다. 그러나 상기 인테그린 결합성 펩타이드 및 항암제의 용량은 약학적 조성물의 투여 경로 및 치료 횟수뿐만 아니라 환자의 연령, 체중, 건강 상태, 성별, 질환의 중증도, 식이 및 배설율 등 다양한 요인들을 고려하여 환자에 대한 유효 투여량이 결정되는 것이므로, 이러한 점을 고려할 때 당 분야의 통상적인 지식을 가진 자라면 본 발명의 조성물의 적절한 유효 투여량을 결정할 수 있을 것이다. 상기 약학적 조성물은 본 발명의 효과를 보이는 한 그 제형, 투여 경로 및 투여 방법에 특별히 제한되지 아니한다. 더불어 상기 약학적 조성물은 본 발명에 따른 인테그린 결합성 펩타이드 및 항암제 외에 항암 효과 또는 항암 효과 증진 효과에 있어서 동일 또는 유사한 약효를 나타내는 유효 성분을 1종 이상 더 포함할 수 있다.The total effective amount of the pharmaceutical composition of the present invention may be administered to a patient as a single dose, and may be administered by a fractionated treatment protocol in which multiple doses are administered for a long period of time. . The pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the severity of the disease. For parenteral administration, based on the integrin-binding peptide and anticancer agent, it is preferably administered in an amount of 0.01 to 50 mg, more preferably 0.1 to 30 mg per 1 kg of body weight per day, divided into 1 to several times administered. . However, the dose of the integrin-binding peptide and anticancer agent is determined by considering various factors such as the age, weight, health status, sex, severity of disease, diet and excretion rate of the patient, as well as the administration route and number of treatments of the pharmaceutical composition. Since the effective dosage is determined, one of ordinary skill in the art will be able to determine an appropriate effective dosage of the composition of the present invention in consideration of this point. The pharmaceutical composition is not particularly limited in its formulation, administration route and administration method as long as the effect of the present invention is exhibited. In addition, the pharmaceutical composition may further include one or more active ingredients exhibiting the same or similar medicinal effect in anticancer effect or anticancer effect enhancing effect in addition to the integrin-binding peptide and anticancer agent according to the present invention.
더불어 본 발명은 하기 구조식 1로 표시되는 인테그린 결합성 펩타이드 및 항암제를 개체에 투여하는 단계를 포함하는, 암을 치료하는 방법을 제공한다:In addition, the present invention provides a method of treating cancer, comprising administering to a subject an integrin-binding peptide represented by the following structural formula 1 and an anticancer agent:
[구조식 1][Structural Formula 1]
[A-B-Cn]m[A-B-Cn]m
상기 A는 VG의 아미노산 서열로 이루어지며;wherein A consists of the amino acid sequence of VG;
상기 B는 표적지향성 모이어티이고;wherein B is a targeting moiety;
상기 C는 엘라스틴-유사 펩타이드이며;wherein C is an elastin-like peptide;
상기 n은 4 내지 8의 정수이고, 상기 m은 1 내지 10의 정수임.Wherein n is an integer from 4 to 8, and m is an integer from 1 to 10.
상기 구조식 1에 대해서는 앞서 설명한 바와 같다. Structural Formula 1 is the same as described above.
본 발명에서 사용된 용어, "개체"는 암 질환을 가지고 있는 개체로서, 바람직하게는 암 질환을 가지고 있는 인간을 포함한 말, 양, 돼지, 염소, 개 등의 포유동물을 의미하나, 바람직하게는 인간을 의미한다. As used herein, the term “individual” refers to an individual having a cancer disease, preferably a mammal including a human having a cancer disease, such as horse, sheep, pig, goat, dog, etc., preferably means human.
본 발명의 암을 치료하는 방법에 있어서, 상기 인테그린 결합성 펩타이드는 항암제와 동시 또는 순차적으로 투여될 수 있다. 더불어 상기 투여는 제한되는 것은 아니나, 바람직하게는 종양내 직접주사(intratumoral injection)일 수 있다.In the method of treating cancer of the present invention, the integrin-binding peptide may be administered simultaneously or sequentially with an anticancer agent. In addition, the administration is not limited, but may preferably be an intratumoral injection.
또한 본 발명은 하기 구조식 1로 표시되는 인테그린 결합성 펩타이드를 포함하는 암 치료 보조용 식품 조성물을 제공한다:The present invention also provides a food composition for adjuvant cancer treatment comprising an integrin-binding peptide represented by the following structural formula 1:
[구조식 1][Structural Formula 1]
[A-B-Cn]m[A-B-Cn]m
상기 A는 VG의 아미노산 서열로 이루어지며;wherein A consists of the amino acid sequence of VG;
상기 B는 표적지향성 모이어티이고;wherein B is a targeting moiety;
상기 C는 엘라스틴-유사 펩타이드이며;wherein C is an elastin-like peptide;
상기 n은 4 내지 8의 정수이고, 상기 m은 1 내지 10의 정수임.Wherein n is an integer from 4 to 8, and m is an integer from 1 to 10.
상기 구조식 1에 대해서는 앞서 설명한 바와 같다. Structural Formula 1 is the same as described above.
본 발명에 따른 식품 조성물은 기능성 식품(functional food), 영양 보조제(nutritional supplement), 건강식품(health food), 건강기능식품(health functional food) 및 식품 첨가제(food additives) 등의 모든 형태를 포함한다.The food composition according to the present invention includes all types of functional food, nutritional supplement, health food, health functional food and food additives. .
본 발명에 있어서, 용어 "건강기능식품"이란 건강보조의 목적으로 특정성분을 원료로 하거나 식품 원료에 들어있는 특정성분을 추출, 농축, 정제, 혼합 등의 방법으로 제조, 가공한 식품을 말하며, 상기 성분에 의해 생체 방어, 생체리듬의 조절, 질병의 방지와 회복 등 생체조절기능을 생체에 대하여 충분히 발휘할 수 있도록 설계되고 가공된 식품을 말하는 것으로서, 질병의 예방 또는 건강의 회복 등과 관련된 기능을 수행할 수 있는 것을 말한다.In the present invention, the term "health functional food" refers to a food manufactured and processed by extracting, concentrating, refining, mixing, etc., a specific ingredient as a raw material or a specific ingredient contained in a food raw material for the purpose of health supplementation, It refers to a food designed and processed to sufficiently exert biological control functions such as biological defense, regulation of biological rhythm, prevention and recovery of disease, etc., with the above ingredients, and performs functions related to prevention of disease or recovery of health, etc. say what you can do
본 발명에 따른 식품 조성물은 당업계에 공지된 통상적인 방법에 따라 다양한 형태로 제조할 수 있다.The food composition according to the present invention can be prepared in various forms according to conventional methods known in the art.
예를 들면, 건강식품으로는 본 발명에 따른 인테그린 결합성 펩타이드 자체를 과립화, 캡슐화 및 분말화하여 섭취하거나 차, 쥬스 및 드링크의 형태로 제조하여 음용하도록 할 수 있다. 또한, 본 발명의 인테그린 결합성 펩타이드를 암 예방 또는 치료 효과가 알려진 공지의 물질 또는 활성 성분과 함께 혼합하여 조성물의 형태로 제조할 수 있다.For example, as a health food, the integrin-binding peptide according to the present invention may be ingested by granulating, encapsulating and powdering itself, or it may be prepared in the form of tea, juice, or drink for drinking. In addition, it can be prepared in the form of a composition by mixing the integrin-binding peptide of the present invention with a known substance or active ingredient known to have a cancer-preventing or therapeutic effect.
또한, 기능성 식품으로는 음료(알콜성 음료 포함), 과실 및 그의 가공식품(예: 과일통조림, 병조림, 잼, 마멀레이드 등), 어류, 육류 및 그 가공식품(예: 햄, 소시지콘비이프 등), 빵류 및 면류(예: 우동, 메밀국수, 라면, 스파게티, 마카로니 등), 과즙, 각종 드링크, 쿠키, 엿, 유제품(예: 버터, 치이즈 등), 식용식물유지, 마아가린, 식물성 단백질, 레토르트 식품, 냉동식품, 각종 조미료(예: 된장, 간장, 소스 등) 등에 본 발명의 인테그린 결합성 펩타이드를 첨가하여 제조할 수 있다.In addition, functional foods include beverages (including alcoholic beverages), fruits and their processed foods (eg, canned fruit, bottled, jam, marmalade, etc.), fish, meat, and their processed foods (eg, ham, sausage corn beef, etc.) , Breads and noodles (eg udon noodles, soba noodles, ramen, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, syrup, dairy products (eg butter, cheese, etc.), edible vegetable oil, margarine, vegetable protein, retort It can be prepared by adding the integrin-binding peptide of the present invention to food, frozen food, various seasonings (eg, soybean paste, soy sauce, sauce, etc.).
본 발명의 식품 조성물 중 상기 본 발명의 인테그린 결합성 펩타이드의 바람직한 함유량으로는 이에 한정되지 않지만 예를 들어 최종적으로 제조된 식품 중 0.01 내지 80 중량%일 수 있으며, 바람직하게는 최종적으로 제조된 식품 중 0.01 내지 50 중량%일 수 있다.The preferred content of the integrin-binding peptide of the present invention in the food composition of the present invention is not limited thereto, but may be, for example, 0.01 to 80% by weight of the finally prepared food, preferably in the finally prepared food. It may be 0.01 to 50% by weight.
본 발명의 식품 조성물은 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물은 포도당, 과당과 같은 모노사카라이드, 말토스, 슈크로스와 같은 디사카라이드, 및 덱스트린, 사이클로덱스트린과 같은 천연 감미제나, 사카린, 아스파르탐과 같은 합성 감미제 등을 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 조성물 100㎖ 당 일반적으로 약 0.01 내지 0.4g, 바람직하게는 약 0.02 내지 0.03g일 수 있다.The food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients. As the above-mentioned natural carbohydrates, monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and natural sweeteners such as dextrin and cyclodextrin, synthetic sweeteners such as saccharin and aspartame may be used. . The proportion of the natural carbohydrate may be generally about 0.01 to 0.4 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention.
상기 외에 본 발명의 식품 조성물은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다. 그 밖에 본 발명의 식품 조성물은 천연 과일쥬스, 과일쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 본 발명의 조성물 100중량부 당 0.01 내지 0.1 중량부의 범위에서 선택되는 것이 일반적이나, 이에 제한되는 것은 아니다.In addition to the above, the food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol , a carbonation agent used in carbonated beverages, and the like. In addition, the food composition of the present invention may contain fruit for the production of natural fruit juice, fruit juice beverage, and vegetable beverage. These components may be used independently or in combination. The proportion of these additives is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention, but is not limited thereto.
상술한 본 발명의 내용은 상호 모순되지 않는 한, 서로 동일하게 적용되며, 당해 기술분야의 통상의 기술자가 적절한 변경을 가해 실시하는 것 또한 본 발명의 범주에 포함된다.The above-described contents of the present invention are equally applied to each other as long as they do not contradict each other, and those skilled in the art to implement with appropriate changes are also included in the scope of the present invention.
이하 본 발명을 실시예를 통해 상세하게 설명하나 본 발명의 범위가 하기 실시예로만 한정되는 것은 아니다. Hereinafter, the present invention will be described in detail through Examples, but the scope of the present invention is not limited only to the Examples below.
실시예 1. 인테그린 결합성 펩타이드 준비Example 1. Preparation of integrin-binding peptides
본 발명에 따른 인테그린 결합성 펩타이드인 UIP(Uptake Inducible Protein) 단백질을 다음과 같이 준비하였다. UIP의 서열은 아미노산 서열 VGRGD(VGVPG)6를 코딩하는 DNA 염기서열의 5' 및 3' 양 말단에 각각 다른 제한효소 서열을 추가하여 합성하고, 당분야에 공지된 형질전환 방법을 통하여 pET-29a(+) (Novagen, Merck Millipore) 벡터에 삽입하였다. 시퀀싱을 통해 UIP 서열을 확인한 후, 단백질 발현 및 정제를 통하여 최종적으로 UIP 단백질을 얻었다. 본 발명에 따른 UIP 단백질 정제 결과는 도 2에 나타내었으며, 이를 통해 8.4kDa의, 태그(tag)를 포함하는 UIP 단백질을 수득한 것을 확인하였다. Uptake inducible protein (UIP) protein, which is an integrin-binding peptide according to the present invention, was prepared as follows. The sequence of UIP is synthesized by adding different restriction enzyme sequences to both ends of the 5' and 3' ends of the DNA nucleotide sequence encoding the amino acid sequence VGRGD(VGVPG) 6 , and pET-29a through a transformation method known in the art. (+) (Novagen, Merck Millipore) was inserted into the vector. After confirming the UIP sequence through sequencing, the UIP protein was finally obtained through protein expression and purification. The results of UIP protein purification according to the present invention are shown in FIG. 2 , and it was confirmed that a UIP protein containing a tag, of 8.4 kDa, was obtained through this.
실시예 2. UIP 및 항암제 병용투여에 따른 효과 확인Example 2. Confirmation of effects according to UIP and anticancer drug combination administration
2-1. 유방암 동물모델 준비 및 UIP와 항암제 병용 투여 2-1. Preparation of breast cancer animal model and administration of UIP and anticancer drug in combination
UIP를 항암제에 병용투여하면 항암제 효과가 증진될 수 있는지를 확인하기 위하여 마우스를 이용한 동물 실험을 수행하였다. 마우스에 투여할 마우스 유방암(mouse breast cancer) 세포주 4T1를 RPMI-1640 배지에 10% FBS(Fetal Bovine Serum)와 1% 페니실린(Penicillin)/스트렙토마이신(Streptomycin)를 첨가하여 배양하였다. 마우스(BALB/c, 6주령, 암컷)에 상기와 같이 준비한 4T1 세포 1x106 cells/head를 피하에 이식하여 UIP와 5-FU 병용투여에 대한 항암 효능을 평가하였다. In order to confirm whether the anticancer effect can be enhanced when UIP is administered in combination with an anticancer agent, an animal experiment using a mouse was performed. Mouse breast cancer cell line 4T1 to be administered to mice was cultured by adding 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin to RPMI-1640 medium. 4T1 cells 1x10 6 cells/head prepared as above were subcutaneously transplanted into mice (BALB/c, 6 weeks old, female) to evaluate the anticancer efficacy of UIP and 5-FU co-administration.
2-2. 몸무게 변화 측정2-2. Weight change measurement
마우스에 발생한 종양이 적정 크기가 되었을 때 실험군을 분류하고(PBS 투여군, 양성대조군인 5-FU 투여군, 실험군인 5-FU와 UIP의 병용 투여군), 각 실험군별로 PBS, 5-FU 또는 UIP와 5-FU를 종양 근처 피하에 3일 간격(1일, 4일, 7일)으로 3회 투여한 후 종양의 크기와 마우스 몸무게를 측정하였다. 사용한 실험군 및 조건을 하기 표 1에 나타내었다. When the tumor generated in the mouse reached an appropriate size, the experimental group was classified (PBS administration group, positive control group 5-FU administration group, experimental group 5-FU and UIP combination administration group), and PBS, 5-FU or UIP and 5 for each experimental group -FU was administered three times at intervals of 3 days (1 day, 4 days, 7 days) subcutaneously near the tumor, and then the size of the tumor and the weight of the mouse were measured. The experimental groups and conditions used are shown in Table 1 below.
샘플명sample name 농도density 마우스 (수)mouse (number)
군분리military separation 종양크기tumor size
음성대조군 (G1)Negative control group (G1) Vehicle (PBS)Vehicle (PBS) 10 μL/g10 μL/g 77 77
양성대조군 (G2)Positive control (G2) 5-FU15-FU1 30 mg/kg30 mg/kg 77 77
실험군 (G3) Experimental group (G3) 5-FU1 + UIP5-FU1 + UIP 30 mg/Kg + 10 μL/g30 mg/Kg + 10 μL/g 77 77
결과의 통계 분석은 음성대조군을 기준으로 t-test를 수행하였으며, p<0.05인 경우 통계학적 유의성이 있는 것으로 판정하였다. 이때 실험군 중 사망한 동물이나 이상 증상은 관찰되지 않았다. 각 실험군별 3회 투여(7일째) 후의 마우스 종양 사진을 도 3에 나타내었으며, 마우스 개별 몸무게의 변화를 하기 표 2 및 도 4에 나타내었고, 실험군별 평균 몸무게의 변화를 하기 표 3 및 도 5에 나타내었다. For statistical analysis of the results, t-test was performed based on the negative control group, and it was determined that there was statistical significance when p<0.05. At this time, no animals or abnormal symptoms were observed in the experimental group. Figure 3 shows the mouse tumor photos after administration 3 times (day 7) for each experimental group, the changes in individual mouse body weight are shown in Tables 2 and 4 below, and the changes in the average body weight for each experimental group are shown in Tables 3 and 5 below. shown in
Figure PCTKR2021017875-appb-img-000001
Figure PCTKR2021017875-appb-img-000001
Figure PCTKR2021017875-appb-img-000002
Figure PCTKR2021017875-appb-img-000002
표 2 및 표 3, 도 4 및 도 5에 나타낸 바와 같이, 실험 중 실험군의 몸무게에 있어서, 통계학적 유의성은 관찰되지 않았다. 구체적으로 음성대조군(G1)의 군별 평균 몸무게를 기준으로 양성대조군(G2) 및 실험군(G3) 군별 평균 몸무게로 각각 t-test 검정을 수행한 결과, 시험물질 투여 후 1일차, 2일차, 4일차, 7일차 모두 p값이 0.5이상으로 통계학적 유의성을 나타내지 않았다.As shown in Tables 2 and 3, and 4 and 5, no statistical significance was observed in the body weight of the experimental group during the experiment. Specifically, as a result of performing a t-test test with the average weight of each group of the positive control group (G2) and the experimental group (G3) based on the average weight of each group of the negative control group (G1), the first day, the second day, and the fourth day after administration of the test substance , and on the 7th day, the p-value was 0.5 or higher, indicating no statistical significance.
2-3. 종양 크기 감소율 확인2-3. Check tumor size reduction rate
대조군과 실험군에서 종양 크기 감소율을 비교하였다. 양성대조군(G2)의 경우, 시험물질 투여 후 1일차, 2일차, 4일차까지는 통계학적 유의성이 나타나지 않았으나, 7일차에 p값이 0.5 이하로 통계학적 유의성을 나타내었다. 반면 실험군(G3)의 경우, 시험물질 투여 후 2일차부터 p값이 0.5이하로 통계학적 유의성을 나타내었으며, 7일차에는 p값이 0.05이하로 강한 통계학적 유의성을 나타내었다. 각 실험군 별 마우스 개별 종양의 크기 변화는 표 4 및 도 6에, 군별 마우스 평균 종양의 크기 변화는 표 5 및 도 7에 나타내었다. Tumor size reduction rates were compared in the control group and the experimental group. In the case of the positive control group (G2), there was no statistical significance until the 1st, 2nd, and 4th days after administration of the test substance, but on the 7th day, the p-value was 0.5 or less, indicating statistical significance. On the other hand, in the case of the experimental group (G3), the p-value was less than 0.5 from the 2nd day after administration of the test substance, indicating statistical significance, and on the 7th day, the p-value was less than 0.05, indicating strong statistical significance. Changes in the size of individual mouse tumors for each experimental group are shown in Tables 4 and 6, and the changes in the average tumor size of each group are shown in Tables 5 and 7.
Figure PCTKR2021017875-appb-img-000003
Figure PCTKR2021017875-appb-img-000003
Figure PCTKR2021017875-appb-img-000004
Figure PCTKR2021017875-appb-img-000004
(Significantly different between G1 vs G2, G3 at 7th day (*p<0.5, **p<0.05))(Significantly different between G1 vs G2, G3 at 7 th day (*p<0.5, **p<0.05))
1일, 2일, 4일, 7일째에 각 군별 7마리의 종양 크기를 측정한 평균값을 토대로, 음성대조군을 기준으로 약물투여에 따른 종양크기 변화를 평가하였으며, 음성대조군과 비교한 종양의 크기 감소율을 하기 표 6에 나타내었다. On the 1st, 2nd, 4th, and 7th days, on the basis of the average value of the measurement of the size of 7 tumors in each group, the tumor size change according to drug administration was evaluated based on the negative control group, and the size of the tumor compared to the negative control group The reduction rate is shown in Table 6 below.
종양 크기 측정일 Tumor Sizing Date 1일1 day 2일2 days 4일4 days 7일7 days
음성대조군 (G1)Negative control group (G1) -- -- -- --
양성대조군 (G2)Positive control (G2) 1.4%1.4% 6.1%6.1% -1.8%-1.8% 7.6%7.6%
실험군 (G3)Experimental group (G3) 1.6%1.6% 7.7%7.7% 13.2%13.2% 20.6%20.6%
상기 결과에서 확인할 수 있는 바와 같이, 양성 대조군은 4일째까지 음성 대조군과 큰 차이를 보이지 않았으며, 7일째에서 종양 크기가 7.6% 감소하였고 통계학적 유의성이 관찰되었다. 반면, 실험군인 UIP 병용 투여군은 2일째부터 통계학적 유의성이 관찰되었으며, 7일째 종양 크기가 20.6% 감소하였다. 즉, 실험군에서는 2일째와 4일째부터 통계학적 유의성을 나타내었고 7일째는 강한 통계학적 유의성을 나타내어, 양성대조군인 G2보다 효과적으로 유방암 종양의 크기를 감소시켰음을 확인하였다. 따라서 이를 통해 본 발명에 따른 인테그린 결합성 펩타이드인 UIP가 함께 투여되는 약제의 효과를 증진시킬 수 있음을 명확히 확인하였다. As can be seen from the above results, the positive control group did not show a significant difference from the negative control group until the 4th day, and the tumor size decreased by 7.6% on the 7th day, and statistical significance was observed. On the other hand, in the experimental group, the UIP combination administration group, statistical significance was observed from the 2nd day, and the tumor size decreased by 20.6% on the 7th day. That is, the experimental group showed statistical significance from the 2nd and 4th days, and the 7th day showed strong statistical significance, confirming that the size of the breast cancer tumor was reduced more effectively than the positive control group, G2. Therefore, it was clearly confirmed that the UIP, which is the integrin-binding peptide according to the present invention, can enhance the effect of the drug administered together.
종합적으로 유방암 동물모델에 본 발명에 따른 신규한 인테그린 결합성 펩타이드인 UIP와 항암제를 병용 투여시 몸무게에 영향을 주지 않으면서도 종양의 크기를 현저히 감소시킨 바, 본 발명에 따른 인테그린 결합성 펩타이드, UIP는 항암제의 치료 효과를 증진시킬 수 있음을 확인하였다.Comprehensively, when UIP, a novel integrin-binding peptide according to the present invention, and an anticancer agent are administered in combination in an animal model of breast cancer, the size of the tumor is significantly reduced without affecting body weight. confirmed that it can enhance the therapeutic effect of anticancer drugs.

Claims (15)

  1. 하기 구조식 1로 표시되는 인테그린 결합성 펩타이드:An integrin-binding peptide represented by the following Structural Formula 1:
    [구조식 1][Structural Formula 1]
    [A-B-Cn]m[A-B-Cn]m
    상기 A는 VG의 아미노산 서열로 이루어지며;wherein A consists of the amino acid sequence of VG;
    상기 B는 표적지향성 모이어티이고;wherein B is a targeting moiety;
    상기 C는 엘라스틴-유사 펩타이드이며;wherein C is an elastin-like peptide;
    상기 n은 4 내지 8의 정수이고, 상기 m은 1 내지 10의 정수임.Wherein n is an integer from 4 to 8, and m is an integer from 1 to 10.
  2. 제 1항에 있어서,The method of claim 1,
    상기 표적지향성 모이어티는 RGD 펩타이드, α-나선 펩타이드, 미토콘드리아 표적 펩타이드, Fc 수용체 결합 펩타이드(Fc receptor binding peptide) 및 베타 엔돌핀 수용체 리간드(beta endorphin receptor ligand)로 이루어진 군으로부터 선택된 어느 하나 이상의 펩타이드인 것을 특징으로 하는, 펩타이드.The targeting moiety is any one or more peptides selected from the group consisting of RGD peptides, α-helical peptides, mitochondrial targeting peptides, Fc receptor binding peptides, and beta endorphin receptor ligands. Characterized by the peptide.
  3. 제 1항에 있어서,The method of claim 1,
    상기 엘라스틴-유사 펩타이드는 VGVPG의 아미노산 서열로 이루어진 것인, 펩타이드.The elastin-like peptide is a peptide consisting of the amino acid sequence of VGVPG.
  4. 제 1항에 있어서,The method of claim 1,
    상기 인테그린 결합성 펩타이드는 서열번호 1로 표시되는 아미노산 서열을 포함하는 것을 특징으로 하는, 펩타이드.The integrin-binding peptide is characterized in that it comprises the amino acid sequence represented by SEQ ID NO: 1, a peptide.
  5. 제 1항에 있어서,The method of claim 1,
    상기 인테그린 결합성 펩타이드는 약제의 치료 효과를 증진시키는 것을 특징으로 하는, 펩타이드.The integrin-binding peptide is characterized in that it enhances the therapeutic effect of the drug, the peptide.
  6. 제 5항에 있어서,6. The method of claim 5,
    상기 약제는 5-fluorouracil(5-FU) 또는 파클리탁셀(paclitaxel)인 것을 특징으로 하는, 펩타이드. The drug is 5-fluorouracil (5-FU) or paclitaxel (paclitaxel), characterized in that, the peptide.
  7. 하기 구조식 1로 표시되는 인테그린 결합성 펩타이드를 포함하는 항암 보조제:An anticancer adjuvant comprising an integrin-binding peptide represented by the following Structural Formula 1:
    [구조식 1][Structural Formula 1]
    [A-B-Cn]m[A-B-Cn]m
    상기 A는 VG의 아미노산 서열로 이루어지며;wherein A consists of the amino acid sequence of VG;
    상기 B는 표적지향성 모이어티이고;wherein B is a targeting moiety;
    상기 C는 엘라스틴-유사 펩타이드이며;wherein C is an elastin-like peptide;
    상기 n은 4 내지 8의 정수이고, 상기 m은 1 내지 10의 정수임.Wherein n is an integer from 4 to 8, and m is an integer from 1 to 10.
  8. 하기 구조식 1로 표시되는 인테그린 결합성 펩타이드 및 항암제를 포함하는 암의 예방 또는 치료를 위한 병용 투여용 약학적 조성물:A pharmaceutical composition for co-administration for the prevention or treatment of cancer comprising an integrin-binding peptide represented by the following Structural Formula 1 and an anticancer agent:
    [구조식 1][Structural Formula 1]
    [A-B-Cn]m[A-B-Cn]m
    상기 A는 VG의 아미노산 서열로 이루어지며;wherein A consists of the amino acid sequence of VG;
    상기 B는 표적지향성 모이어티이고;wherein B is a targeting moiety;
    상기 C는 엘라스틴-유사 펩타이드이며;wherein C is an elastin-like peptide;
    상기 n은 4 내지 8의 정수이고, 상기 m은 1 내지 10의 정수임.Wherein n is an integer from 4 to 8, and m is an integer from 1 to 10.
  9. 제 8항에 있어서,9. The method of claim 8,
    상기 암은 유방암, 뇌종양, 피부암, 췌장암, 간암, 폐암, 골수암, 식도암, 대장암, 위암, 자궁경부암, 전립선암, 난소암 및 두경부암으로 이루어진 군으로부터 선택된 1종 이상인 것을 특징으로 하는, 약학적 조성물.The cancer is one or more selected from the group consisting of breast cancer, brain tumor, skin cancer, pancreatic cancer, liver cancer, lung cancer, bone marrow cancer, esophageal cancer, colorectal cancer, stomach cancer, cervical cancer, prostate cancer, ovarian cancer, and head and neck cancer, pharmaceutical composition.
  10. 제 8항에 있어서,9. The method of claim 8,
    상기 인테그린 결합성 펩타이드는 항암제와 동시 또는 순차적으로 투여되는 것을 특징으로 하는, 약학적 조성물.The integrin-binding peptide is characterized in that it is administered simultaneously or sequentially with the anticancer agent, the pharmaceutical composition.
  11. 제 10항에 있어서,11. The method of claim 10,
    상기 항암제는 5-fluorouracil(5-FU) 또는 파클리탁셀(paclitaxel)인 것을 특징으로 하는, 약학적 조성물.The anticancer agent is 5-fluorouracil (5-FU) or paclitaxel (paclitaxel), characterized in that, the pharmaceutical composition.
  12. 제 8항에 있어서,9. The method of claim 8,
    상기 약학적 조성물은 종양 내 직접주사(intratumoral injection)로 투여되는 것을 특징으로 하는, 약학적 조성물.The pharmaceutical composition is characterized in that administered by intratumoral injection (intratumoral injection), the pharmaceutical composition.
  13. 하기 구조식 1로 표시되는 인테그린 결합성 펩타이드 및 항암제를 개체에 투여하는 단계를 포함하는, 암을 치료하는 방법:A method of treating cancer, comprising administering to an individual an integrin-binding peptide represented by the following Structural Formula 1 and an anticancer agent:
    [구조식 1][Structural Formula 1]
    [A-B-Cn]m[A-B-Cn]m
    상기 A는 VG의 아미노산 서열로 이루어지며;wherein A consists of the amino acid sequence of VG;
    상기 B는 표적지향성 모이어티이고;wherein B is a targeting moiety;
    상기 C는 엘라스틴-유사 펩타이드이며;wherein C is an elastin-like peptide;
    상기 n은 4 내지 8의 정수이고, 상기 m은 1 내지 10의 정수임.Wherein n is an integer from 4 to 8, and m is an integer from 1 to 10.
  14. 제 13항에 있어서,14. The method of claim 13,
    상기 투여는 종양 내 직접주사(intratumoral injection)인 것을 특징으로 하는, 암을 치료하는 방법.The method of treating cancer, characterized in that the administration is an intratumoral injection.
  15. 하기 구조식 1로 표시되는 인테그린 결합성 펩타이드를 포함하는 암 치료 보조용 식품 조성물:A food composition for adjuvant cancer treatment comprising an integrin-binding peptide represented by the following Structural Formula 1:
    [구조식 1][Structural Formula 1]
    [A-B-Cn]m[A-B-Cn]m
    상기 A는 VG의 아미노산 서열로 이루어지며;wherein A consists of the amino acid sequence of VG;
    상기 B는 표적지향성 모이어티이고;wherein B is a targeting moiety;
    상기 C는 엘라스틴-유사 펩타이드이며;wherein C is an elastin-like peptide;
    상기 n은 4 내지 8의 정수이고, 상기 m은 1 내지 10의 정수임.Wherein n is an integer from 4 to 8, and m is an integer from 1 to 10.
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