JP6889458B2 - Cell proliferation inhibitor - Google Patents

Description

The present invention relates to a cell proliferation inhibitor and its use.

The black pigment melanin (eumelanin) has an indole skeleton (Fig. 1A) of 5,6-Dihydroxyindole (DHI) (Fig. 1B) and its quinone form, Indole-5,6-quinone (IQ) (Fig. 1C). Or, a high amount of randomly polymerized 5,6-Dihydroxyindole-2-carboxylic acid (DHICA) (Fig. 1D) and its quinone form, Indole-5,6-quinone-2-carboxylic acid (IQCA) (Fig. 1E). It has been reported as a molecule (Non-Patent Document 1). In the study of chemically synthesized melanin, single bonds are formed at the 2, 4 and 7 positions of the indole unit to form a sheet-like polymer having up to 11 units (Fig. 1F, G). Furthermore, it has been reported that this sheet-like structure has 4 to 5 layers and forms a basic structural unit (protomolecule) having a thickness of about 6 to 10 nm (Non-Patent Document 2). In the animal body, it is polymerized and produced in pigment cells called melanocytes. Melanin is a type of natural pigment that is widely distributed in all living organisms. It is biosynthesized from the amino acid tyrosine by the action of tyrosinase, and becomes eumelanin in the absence of cysteine (Fig. 2), and pheomelanin in the presence of cysteine. In the skin, exposure to ultraviolet rays activates melanocytes, synthesizes melanin, releases it extracellularly as melanosomes, and delivers it to keratinocytes, where melanin appears. Melanin that remains in keratinocytes absorbs ultraviolet rays there and protects deep cells from ultraviolet damage (Non-Patent Document 3). It is also known that melanocytes are also present in the choroid and retinal pigment epithelium of the eye and contribute to the regulation of light intensity in the eye. Furthermore, although it is also present in the inner ear, there are many unclear points about its significance.

Normally, melanin secreted from melanocytes is excreted from the body by turnover. The biosynthetic pathway of melanin has been clarified, and chemical synthesis is also possible industrially. However, artificially synthesized melanin and melanin extracted from living organisms are rarely used in the fields of life science and medicine. In particular, there are no reports of artificially adjusting its function by treating cultured cells or individual animals. This is because, whether it is natural melanin or synthetic melanin possessed by living organisms, there are physical problems that it is a polymer with a heterogeneous molecular weight and that it is difficult to dissolve in any solvent, so the biological activity of melanin is tested. This is thought to be because it is difficult to measure.

It should be noted that synthetic soluble melanin has been reported to have a vinegar action (Patent Document 1) that inhibits the replication of human immunodeficiency virus, an action that suppresses cytokine production (Patent Document 2), and an anti-inflammatory action (Patent Document 3).

U.S. Pat. No. 5,057,325 Japanese Patent Application Laid-Open No. 2001-512446 Special Table 2003-509529

Swift, J. A. 2009. Speculations on the molecular structure of eumelanin. Int J Cosmet Sci 31: 143-150. Littrell, K. C., J. M. Gallas, G. W. Zajac, and P. Thiyagarajan. 2003. Structural studies of bleached melanin by synchrotron small-angle X-ray scattering. Photochem Photobiol 77: 115-120. Ham, W. T., Jr., J. J. Ruffolo, Jr., H. A. Mueller, and D. Guerry, 3rd. 1980. The nature of retinal radiation damage: dependence on wavelength, power level and exposure time. Vision Res 20: 1105-1111. Izumi Nakajima. 2015. Introduction to Medicine-The Concept of Medicine and the Essence of Medicine. Nankodo.

The task is to discover a new action of melanin and to apply the action (provide applications).

In order to find a new use of melanin, the present inventors have focused on the solubility of melanin and examined its action in detail. Normally, melanin does not dissolve in any hydrophobic or hydrophilic solvent with stirring alone, but we have 500 mM Hepes (2- [4- (2-Hydroxyethyl) -1-piperazinyl] ethanesulfonic acid), pH 7. It was found that it easily dissolves up to 50 mg / ml in the physiological buffer solution of 5 (hereinafter referred to as solubilized melanin). Phosphate buffered solution (PBS), which is another commonly used buffer solution, is still poorly soluble, but if uniform dispersion treatment is performed by laser ablation, a uniform "particle dispersion solution" with a particle size of 100 nm can be easily obtained. It is obtained (hereinafter, fine particle dispersed melanin).

As a result of detailed studies focusing on cell proliferation (see Examples below), micronized or solubilized melanin does not cause serious cell death to cancer cells or hyperproliferative cells. It has been found to have a surprising effect of suppressing only cell proliferation.

By the way, there are cancers that develop congenitally due to genes, and those that develop due to acquired gene mutations due to various environmental factors, whether biological or abiotic. To do. In both cases, cells threaten the life of an individual by repeating disorderly proliferation indefinitely. For the treatment of cancer, surgical treatment by surgery, radiation treatment by rotating irradiation such as X-ray, cobalt, heavy particle beam, hormone therapy, chemotherapy, immunotherapy, molecular target therapeutic drug therapy as medical treatment are carried out. (Non-Patent Document 4). Antineoplastic drugs used in medical chemotherapy act on the DNA of cancer cells, block the action of proteins required for DNA replication and cell division, and lead to cell death. In recent years, molecular-targeted therapeutic drug therapy has attracted attention, and high therapeutic effects are being recognized. However, these treatment methods have many problems to be solved, such as high treatment cost, poor versatility, and side effects. The new action of melanin found by the present inventors, that is, suppression of cell proliferation, enables a new and unique therapeutic strategy for cancer, and its significance is great.

The following inventions are based on the above findings, achievements and considerations.
[1] A cell growth inhibitor containing solubilized melanin or dispersed melanin as an active ingredient.
[2] The growth inhibitor according to [1], wherein the solubilized melanin is Hepes-dissolved melanin.
[3] The growth inhibitor according to [1], wherein the average particle size of dispersed melanin is 70 nm to 130 nm.
[4] The growth inhibitor according to [3], wherein dispersed melanin is obtained by laser ablation.
[5] The growth inhibitor according to any one of [1] to [4], wherein the cell is a tumor cell.
[6] The growth inhibitor according to [5], which is used for the treatment of cancer.
[7] The growth inhibitor according to [6], which is used in combination with an anticancer agent.

Single bond mode of indole units in eumelanin. When 5,6-Dihydroxyindole (DHI) and Indole-5,6-quinone (IQ) are involved (F), 5,6-Dihydroxyindole-2-carboxylic acid (DHICA) and Indole-5,6-quinone- When 2-carboxylic acid (IQCA) is involved (G). Synthetic pathway of eumelanin. Eumelanin is synthesized in vivo using Tyrosine as a starting material. In vitro, melanin can be artificially synthesized by the method described in Patent Document 1. A. Preparation of micronized dispersion solution by laser ablation technology. Commercially available synthetic melanin was added to PBS to a concentration of 1 mg / ml, and laser irradiation was performed under certain conditions. B. Examination of the solubility of melanin dissolved in various biochemical neutral buffers (0.5M). Synthetic melanin was added to each buffer to a concentration of 50 mg / ml, and after centrifugation at 10,000 × g for 30 minutes, 10 μl of the solution was applied to the filter paper (a). The solution was then vigorously stirred and immediately 10 μl of the solution was applied to the filter paper (b). Eumelanin, a synthetic pathway for melanin-like substances. The soluble melanin or melanin-like substance used in this study was synthesized by the following method. That is, 1 g of L-dopa or L-dopamine (WAKO) was dissolved in 400 ml of 0.025N NaOH and incubated for 2 days at room temperature with constant air passing through a solution in 1N NaOH. 2 ml of concentrated hydrochloric acid was added to obtain a precipitate, which was centrifuged at 1,000 × g for 5 minutes, and then the precipitate was dissolved in 400 ml of ultrapure water. 1 ml of concentrated hydrochloric acid was added thereto, and the mixture was centrifuged at 1,000 × g for 5 minutes to obtain a precipitate again. This operation was carried out a total of 4 times to purify melanin or a melanin-like substance. Finally, dissolve in 20 ml of 0.025N NaOH (pH is neutral at this point, but if not neutral, neutralize with 1N NaOH), desalinate overnight with a dialysis membrane, and then freeze. It was dry. Cytotoxicity (A) and cell proliferation suppression test (B) on rat basophilic leukemia cells RBL-2H3 cells by fine particle-dispersed melanin using a laser ablation method. Cytotoxicity and proliferation suppression test of synthetic soluble melanin against HepG2. A. Cytotoxicity test with synthetic soluble melanin. B. Cytotoxicity test with synthetic soluble melanin-like substances. C. Cell proliferation inhibition test by synthetic soluble melanin. D. Cell proliferation inhibition test with synthetic soluble melanin-like substance. Cytotoxicity test of Hepes-dissolved melanin against human cancer cell line (left, white bar graph) and cell proliferation suppression test (right, black bar graph). A. Cell proliferation suppression test results of Hepes-lysed melanin against mouse melanoma cell line B16F10. B. Volume change of B16F10 cells transplanted into nude mice. The square plot shows changes in tumor volume in mice transplanted with an osmotic pump filled with RPMI1640 medium. Round plots show tumor volume changes in mice transplanted with an osmotic pump filled with Hepes-dissolved melanin. Analysis of the effect of Hepes-dissolved melanin treatment on the cell cycle using MCF-7 cells. The cell cycle was analyzed using a flow cytometer. The peak areas of G0 / G1 phase, S phase, and G2 / M phase were analyzed using FlowJo software and graphed as relative frequencies. The white bar graph represents the control, and the diagonal bar graph represents the melanin treatment. Inhibition of histone H3 phosphorylation by Hepes-lysed melanin treatment in HepG2 cell lines.

The present invention relates to a cell proliferation inhibitor. The two terms "inhibition" and "inhibition" have overlapping meanings and are often used interchangeably. Therefore, in the present specification, the term "suppression" is used in a unified manner unless it is particularly necessary to distinguish it from the context.

The active ingredient of the cell proliferation inhibitor of the present invention is dispersed melanin or solubilized melanin. "Dispersed melanin" refers to melanin in which particles having high particle size uniformity are dispersed in a solvent. The average particle size of dispersed melanin is, for example, 70 nm to 130 nm, preferably 90 nm to 110 nm. According to the studies by the present inventors, it has been found that the laser ablation technique can be used to prepare melanin in a state of being dispersed with a highly uniform particle size. Therefore, preferably, dispersed melanin is prepared by treatment by laser ablation. For example, a solution containing dispersed melanin (dispersed melanin) by laser ablation treatment with melanin suspended in a physiological buffer solution (typically PBS or physiological saline) or pure water. Solution) can be prepared.

"Solubilized melanin" is melanin in a dissolved state. According to the study by the present inventors, melanin, which is said to be difficult to understand for all solvents regardless of whether it is hydrophobic or hydrophilic, is a Hepes buffer solution (500 mM Hepes (2- [] as a typical example in the study. The surprising fact was revealed that it dissolves easily in 4- (2-Hydroxyethyl) -1-piperazinyl] ethanesulfonic acid), using pH 7.5). Therefore, solubilized melanin is preferably prepared by dissolving it in Hepes buffer. A partial modification of the composition by changing the content of some components of the Hepes buffer, excluding some components, or adding components (modified Hepes buffer). ) May be dissolved to prepare solubilized melanin. The molecular weight of the solubilized melanin is not particularly limited, but if the molecular weight is too small, the cell proliferation inhibitory activity decreases. Similarly, if the molecular weight is too large, it is considered that homogeneous cell treatment becomes difficult. The molecular weight of solubilized melanin is, for example, in the range of 50 kDa to 100 kDa.

The term "melanin" is used as a term that includes various melanins having similar structures (eumelanin, pheomelanin, neuromelanin, allomelanin, phytomelanin, etc.). Melanin is a pigment found in animals, plants, fungi, eubacteria and the like. The origin of melanin in the present invention is not particularly limited. Melanin is derived from chemical synthesis (see, for example, JP-A-2003-509529), enzyme synthesis (see, eg, JP-A-07-313155), and natural products (eg, squid, tacosumi, mushrooms, bananas, asagao seeds, etc.). Can be prepared by extraction / purification of melanin-producing cells (mouse B16, B16F10, Syrian hamster RPMI 1846, human HMY-1, MNT-1, HM3KO, A375, SK-Mel-28, etc.). .. In addition, various melanins are commercially available (for example, melanin oxidatively synthesized by hydrogen peroxide treatment of tyrosine provided by Sigma-Aldrich, and oxidatively synthesized by persulfate treatment of tyrosine provided by MP-Biochemicals. Melanin), such a commercially available product may be used. The purity or degree of purification of melanin is not particularly limited. Therefore, as long as it exhibits the expected effects, that is, cell proliferation inhibitory activity, melanin having a relatively low degree of purification, such as crudely purified melanin derived from a natural product, may be used.

In a preferred embodiment, eumelanin, ie 5,6-dihydroxyindole (DHI), indole-5,6-quinone (IQ), 5,6-dihydroxyindole-2-carboxylic acid (DHICA), indole-5,6 -A polymer containing quinone-2-carboxylic acid (IQCA) as a monomer is the active ingredient. The active ingredient may be a mixture of two or more types of melanin, which differ in structure and / or molecular weight. The melanin in the present invention does not contain a protein component, that is, it is distinguished from so-called melanin protein.

Typically, the cell proliferation inhibitor of the present invention is used to suppress proliferation of abnormally or overgrown cells. Examples of applicable cells include benign or malignant tumor cells, hyperplastic or polyps (eg, colon polyps, gastric polyps, laryngeal polyps, vocal cord polyps, cervical polyps, endometrial polyps, endometrial polyps). It is a cell that forms. Suppressing the proliferation of these cells can be a therapeutic or prophylactic treatment of the corresponding disease (ie, tumor, hyperplasia, polyp). Therefore, the cell proliferation inhibitor of the present invention can be used for the treatment or prevention of tumors (particularly malignant tumors / cancers), hyperplasia, polyps and the like. That is, it is possible to construct a therapeutic agent for these diseases by using the cell proliferation inhibitor of the present invention as an active ingredient. Here, the "therapeutic agent" refers to a drug showing a therapeutic or prophylactic effect on a target disease / pathological condition. Therapeutic effects include alleviating (mitigating) the symptoms or associated symptoms characteristic of the target disease / condition, preventing or delaying the exacerbation of the symptoms, and the like. The latter can be regarded as one of the preventive effects in terms of preventing aggravation. In this way, the therapeutic effect and the preventive effect are partially overlapping concepts, so it is difficult to clearly distinguish them, and the practical benefit of doing so is small. In addition, as long as it shows some therapeutic effect, preventive effect, or both with respect to the target disease / pathological condition, it corresponds to the therapeutic agent in the present specification.

"Cancer" that can be a target of the therapeutic agent of the present invention is broadly interpreted and includes carcinoma, sarcoma, hematological malignancies and the like. It may include benign tumors, benign malignant borderline lesions, and malignant tumors as a whole before the pathological diagnosis is confirmed, that is, before either benign or malignant tumor is confirmed. .. Specific examples of "cancer" include esophageal cancer, oral cancer, maxillary cancer, laryngeal cancer, pharyngeal cancer, gastric cancer, duodenal cancer, colon cancer, hepatocellular carcinoma, bile duct cell cancer, lung cancer, prostate cancer, renal cancer, and bladder papilla. Cancer, prostate cancer, urinary squamous epithelial cancer, osteosarcoma, chondrosarcoma, synovial sarcoma, mucinosarcoma, liposarcoma, multiple myeloma, malignant lymphoma, squamous cell carcinoma, malignant melanoma, glioma, Meningitis, neuroblastoma, breast cancer, breast sarcoma, intrauterine epithelial cancer, cervical squamous epithelial cancer, uterine adenocarcinoma, uterine sarcoma, ovarian cancer, malignant melanoma, thyroid papillary adenocarcinoma, thyroid follicle Cancer, acute myeloid leukemia, acute promedullary leukemia, acute myeloid monocytic leukemia, acute monocytic leukemia, acute lymphocytic leukemia, acute undifferentiated leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, adult T Cellular leukemia can be mentioned.

The cell proliferation inhibitor of the present invention exhibits an extremely unique property of suppressing only cell proliferation without causing serious cell death. In view of this characteristic, when applied to the treatment of cancer, the combined use with other anticancer agents exerts a synergistic effect, which brings about an improvement in the therapeutic effect and treatment results, or the treatment period. It can be expected to realize treatments that reduce the physical burden and economic cost of patients, such as shortening, reducing the concentration of anticancer drugs, suppressing side effects, and reducing costs. Therefore, in one aspect of the present invention, an anticancer agent is used in combination when used for the treatment of cancer. Antineoplastic agents to be used in combination include tyrosine kinase inhibitors (imatinib, nirotinib, dasatinib, gefitinib / elrotinib, lapatinib, sorafenib, snitinib, crizotinib, axitinib), antibody drugs (trastuzumab, sesutsumab, cetuximab, cetuximab, paclitaxel)・ Chiuxetan, gemtuzumab, ozogamycin, denosumab), mTOR inhibitor (temsirolimus, everolims, rapamycin), proteasome inhibitor (voltezomib), vitamin A derivative (tretinoin, tamibarotene), PD-1 immune checkpoint inhibitor (nibolumab), pyrimidin Antagonists (fluorouracil, tegafur uracil, tegafur gimeracil oteracil potassium, capecitabin, citarabin, gemcitabine, enocitabine, carmofur), purine antagonists (mercaptopurine, fludarabin, cladribine), folic acid antagonists (methotrexate, pemethrexate) Antagonists (triflulysine tipiracil hydrochloride, nelarabine, pentostatin), platinum preparations (cisplatin, carboplatin, oxaliplatin, nedaplatin), alkylating agents (cyclophosphamide, ifofamide, melfaran, dacarbazine, temozoromide, nimustin, busulfan) ), Antineoplastic agents (doxorubicin, doxorubicin hydrochloride liposome injection, epirubicin, amurubicin, idarubicin, daunorubicin, mitoxanthrone, bleomycin, mitomycin C, actinomycin D, L-asparaginase, acralubicin, pyrarubicin, pepromycin), topoisomerase inhibitors ( Irinotecan, nogitecan, etopocid), microtube blockers (vincristin, bindecin, vinblastin, binorerbin, elibrin, paclitaxel, paclitaxel albumin suspension, docetaxel), hormonal agents (tamoxyphene, tremiphen, anastrosol, retrozol, exemestane, gocerelin) , Leuprorelin, ethynyl estradiol, chlormaginone, bicartamide, flutamide, prednisolone) and the like can be exemplified.

The cell proliferation inhibitor of the present invention can be formulated according to a conventional method. When formulated, other components (eg, carriers, excipients, disintegrants, buffers, emulsifiers, suspensions, soothing agents, stabilizers, preservatives, preservatives, physiology) that are acceptable in the formulation. (Saline, etc.) can be contained. As the excipient, lactose, starch, sorbitol, D-mannitol, sucrose and the like can be used. As the disintegrant, starch, carboxymethyl cellulose, calcium carbonate and the like can be used. As the buffer, phosphate, citrate, acetate and the like can be used. As the emulsifier, gum arabic, sodium alginate, tragant and the like can be used. As the suspending agent, glycerin monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used. As the pain-relieving agent, benzyl alcohol, chlorobutanol, sorbitol and the like can be used. Propylene glycol, ascorbic acid and the like can be used as the stabilizer. As the preservative, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben and the like can be used. As the preservative, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.

The dosage form for formulation is not particularly limited, and for example, the present as nasal drops, eye drops, tablets, powders, fine granules, granules, capsules, syrups, injections, external preparations, suppositories, etc. The cell proliferation inhibitor of the present invention can be provided.

The cell proliferation inhibitor of the present invention contains an amount of an active ingredient necessary for obtaining the expected therapeutic effect or preventive effect (that is, a therapeutically effective amount). The amount of the active ingredient contained in the cell proliferation inhibitor of the present invention generally varies depending on the dosage form and form, but the amount of the active ingredient is, for example, in the range of about 0.1% by weight to about 95% by weight so as to achieve a desired dose. Set.

The cell proliferation inhibitor of the present invention is orally or parenterally (intravenously, intraarterial, subcutaneous, muscle, or intraperitoneal injection, transdermal, nasal, transmucosal, application, etc.) depending on its dosage form and morphology. Applies to. The “subject” here is not particularly limited, and includes humans and non-human mammals (including pet animals, livestock, and experimental animals. Specifically, for example, mice, rats, guinea pigs, hamsters, monkeys, cows, pigs, goats. , Sheep, dogs, cats, chickens, quail, etc.). In a preferred embodiment, the subject of application is a human.

The dose and amount of the cell proliferation inhibitor of the present invention are set so as to obtain the expected effect. In setting an effective dose, the symptomatology, age, gender, body weight, etc. of the target are generally taken into consideration. Those skilled in the art can set an appropriate dose in consideration of these matters. As the administration schedule, for example, once to several times a day, once every two days, once every three days, etc. can be adopted. In creating the administration / use schedule, the symptoms to be applied and the duration of effect of the active ingredient can be taken into consideration.

As is clear from the above description, the present application also provides a method for treating / preventing cancer, which comprises administering a therapeutically effective amount of the cell proliferation inhibitor of the present invention to a cancer patient.

The following research was conducted to find new uses for melanin (especially for medical applications). First, we focused on the poor solubility, which is an obstacle in evaluating the characteristics or activity of melanin, and aimed to create a method for dissolving or dispersing melanin.

1. 1. Materials and methods (1) Cells and culture conditions Rat basotropic leukemia cell line (RBL-2H3), human cancer cell line, cervical epithelial cancer (HeLa), breast cancer (MCF-7), liver Cellular cancer (HepG2), acute T-cell leukemia cells (Jurkat), and mouse melanoma cell line (B16F10) were used.

10% FBS, 100U penicillin, and 100 μg / ml streptomycin (nacalai tesque) were added to E-MEM medium (Wako Pure Chemical Industries) and cultured.

(2) Reagent As melanin, commercially available synthetic melanin (MP biomedicals, Sigma) was used. Cell counting kit 8 (Dojindo) was used as a reagent for measuring cell viability.

(3) Preparation of soluble melanin
(i) Laser ablation-treated melanin (fine particle-dispersed melanin)
Synthetic melanin was suspended in sterilized PBS to 0.1 wt%, and a dispersed melanin solution was obtained under the following laser irradiation conditions.
Wavelength 1064nm, FWHM 5ns, 1.2J / cm ²・ pulse, 10Hz, 2.5mmφ, 60 minutes

(ii) Hepes dissolved melanin
Hepes (Sigma-Aldrich) was dissolved in ultrapure water and filtered and sterilized using a hydrophilic PVDF membrane (Merck Millipore Corporation) with a pore size of 0.22 μm (Hepes buffer). Synthetic melanin was dissolved in this to 50 mg / ml and stored at room temperature.

(iii) The soluble melanin or melanin-like substance used in this study was synthesized by the following method. That is, 1 g of L-dopa or L-dopamine (WAKO) was dissolved in 400 ml of 0.025N NaOH and incubated for 2 days at room temperature with constant air passing through a solution in 1N NaOH. 2 ml of concentrated hydrochloric acid was added to obtain a precipitate, which was centrifuged at 1,000 × g for 5 minutes, and then the precipitate was dissolved in 400 ml of ultrapure water. 1 ml of concentrated hydrochloric acid was added thereto, and the mixture was centrifuged at 1,000 × g for 5 minutes to obtain a precipitate again. This operation was carried out a total of 4 times to purify melanin or a melanin-like substance. Finally, it was dissolved in 20 ml of 0.025N NaOH (pH is neutral at this point, but if not neutral, neutralized with 1N NaOH) and lyophilized.

(4) Cytotoxicity test
Cells were seeded in 96-well plates to 2 × 10 ^{5 cells / well.} After culturing at 37 ° C for 24 hours, it was confirmed that the cells were in a confluent state, and various concentrations of melanin were administered. Staurosporine (STPO) or arsenic was administered as a cell death inducer. After further culturing at 37 ° C. for 24 hours, the cell viability was measured with 8 cell counting kits.

(5) Cell proliferation suppression test
The cells were seeded at a low density in a 96-well plate at 2 × 10 ⁴ cells / well, and at the same time, various concentrations of melanin were administered. STPO or arsenic was used as the cell death inducer. After dosing, the cells were cultured at 37 ° C. for 24 hours, and the cell viability was measured with 8 reagents of the cell counting kit.

(6) Transplant tumor suppression test in a mouse model Hepes-dissolved melanin was injected at a concentration of 18.9 mg / ml into an osmotic pump (Alzet osmotic pump, Muromachi Machinery) that can continuously inject the drug solution into the body. Since the osmotic pump discharges the drug solution at 0.11 μl / hr, the melanin dose is 50 μg / mouse / day. An osmotic pump infused with melanin or Hepes as a control was surgically implanted in nude mice (BALB / c Slc-nu / nu, 9 weeks old, SLC Japan). ^{The next day, 2 × 10 5} mouse melanoma cell lines B16F10 were injected into a site approximately 1 cm near the osmotic pump, and the tumor volume was measured over time up to 20 days thereafter. Tumor volume was calculated using the following approximation formula (Takashi Sakagami et al. 1990. Rat RF warmer and tumor growth inhibitory effect using it: Experimental study on RF warming / hyperthermia chemotherapy (1st report) ). Journal of the Japanese Society of Hyperthermia 6: 473-490.).

(Approximate formula)
V = (W ² x L) / 2 (V: volume, W: minor axis, L: major axis)

(7) Cell cycle analysis
A human breast cancer cell line (MCF-7) was seeded on a 24-well microplate at 1 × 10 ^{6 cells / well.} Hepes solubilized tyrosine was administered, and 24 hours later, cells were fixed with 70% ethanol. DNA was stained with Propidium Iodide stain, and data were acquired using a flow cytometer (FACS Canto, BD Biosciences). The cell cycle was analyzed using analysis software (FlowJo).

(8) Western blotting
HepG2 cells of 3 × 10 ⁶ / ml (E-MEM medium) were seeded in a seed culture dish and cultured for 24 hours. Cells were treated with various concentrations of melanin diluted in E-MEM medium at 37 ° C. for 24 hours. Washed 3 times with PBS and contains 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 mM PMSF, Protease Inhibitor Cocktail (Sigma) and Phosphate Inhibitor Cocktail (Sigma). The protein concentration of the cell lysate lysed in 100 μl of the cell lysis buffer and clarified by centrifugation at 15,000 × g was measured, and then boiled together with the 4 × SDS sample buffer. A sample having a protein amount of 10 to 20 μg was separated by SDS-PAGE, transferred to a PVDF membrane, and then the target protein band was detected with a specific antibody.

2. Results (1) Creation of fine particle dispersion and completely solubilized melanin solution Since it was considered that melanin may be the main body that suppresses cell proliferation, an experiment was devised in which commercially available synthesized purified melanin was administered to RBL-2H3 cells. did. However, commercially available synthetic melanin is known to be a pigment that is insoluble in various organic solvents other than water and has pigment-like properties (Takeru Sato. 1998. Recent applications of Ikasumi pigments (special feature pigments) Health--The Secret of Food Active Ingredients). Food style 21 2: 80-81.). Therefore, a uniform dispersion solution was obtained by laser ablation technology. The obtained fine particle-dispersed melanin solution was found to have a particle size of 70 to 130 nm by electron microscopic observation (FIG. 3A). On the other hand, when we tried to dissolve it in various biochemical neutral buffers, we found for the first time that commercially available synthetic melanin was almost completely dissolved in Hepes buffer at 500 mM and pH 7.5 at a concentration of 50 mg / ml (Fig.). 3B). Further, a method for chemically synthesizing soluble melanin has been published (Patent Document 1). With reference to this method, when the initial reaction substance is L-Dopa, melanin (eumelanin) is synthesized, and when the initial reaction substance is L-Dopamine, a melanin-like substance can be prepared (FIG. 4). In the case of eumelanin, a carboxyl group is present at the 2-position of the polymerized indole unit, and in the case of a melanin-like substance, a polymer without a carboxyl group is formed. After synthesizing soluble melanin, melanin _{completely dissolved in Hepes or H 2} O was obtained. As described above, the melanin or the melanin-like substance solution having high uniformity in the aqueous solution thus obtained was used in the subsequent experiments.

(2) Cytotoxicity and cell proliferation suppression test by fine particle-dispersed melanin using laser ablation method Various concentrations of fine particle-dispersed melanin or cell death induction were applied to rat basophilic leukemia cells RBL-2H3, which became confluent. As a positive control, 2.5 μg / ml Staulosporin (STPO) was treated and cultured for 24 hours. Live cells were measured with Cell Counting Kit 8 and the cell viability was measured with the negative control value as 100%. As a control sample, 50% PBS EMEM medium was used. While most cells were killed by STPO treatment, fine particle-dispersed melanin did not induce cell death up to a maximum treatment concentration of 0.5 mg / ml (Fig. 5A). On the other hand, RBL-2H3 cells were seeded in a dish at a low density, and at the same time, various concentrations of fine particle-dispersed melanin or STPO were treated, and after culturing for 24 hours, the cell viability was measured by Cell Counting Kit 8. As a result, cell proliferation was significantly suppressed in a treatment concentration-dependent manner at treatment concentrations of 0.062 mg / ml to 0.5 mg / ml (Fig. 5B).

(3) Cytotoxicity and cell proliferation suppression test using synthetic soluble melanin Synthetic soluble melanin or synthetic soluble melanin-like substance was dissolved in 500 mM Hepes to prepare a stock solution of 50 mg / ml. The confluent human liver cancer-derived cell line HepG2 was treated with various concentrations (0.062 to 0.5 mg / ml) of synthetically soluble melanin or synthetically soluble melanin-like substance, and cultured for 24 hours. 1 mM arsenic was treated as a positive control for cell death induction. Live cells were measured with Cell Counting Kit 8 and the cell viability was measured with the negative control value as 100%. As a control sample, 5 mM EMEM medium containing Hepes was used. While most cells were killed by arsenic treatment, neither soluble melanin nor soluble melanin-like substance induced cell death up to a maximum treatment concentration of 0.5 mg / ml (FIGS. 6A and 6B). On the other hand, HepG2 cells were seeded in a dish at a low density, and at the same time, various concentrations of soluble melanin or soluble melanin-like substance were treated, and after culturing for 24 hours, the cell viability was measured by Cell Counting Kit 8. As a result, cell proliferation was significantly suppressed in a treatment concentration-dependent manner at a treatment concentration of 0.125 mg / ml to 0.5 mg / ml for soluble melanin and at a treatment concentration of 0.25 mg / ml to 0.5 mg / ml for soluble melanin-like substances. (Fig. 6C, D).

(4) Cytotoxicity and cell proliferation suppression test using Hepes-dissolved melanin Commercially available melanin was dissolved in 500 mM Hepes to prepare a stock solution of 50 mg / ml. Using this melanin, the cytotoxicity and cell proliferation inhibitory effect on human cervical cancer cell line (HeLa), human breast cancer cell line (MCF-7), and human T lymphocytic leukemia cell line (Jurkat) were investigated. Various cancer cell lines that became confluent were treated with various concentrations (0.031 to 0.5 mg / ml) of Hepes-dissolved melanin and cultured for 24 hours. Live cells were measured with Cell Counting Kit 8 and the cell viability was measured with the negative control value as 100%. As a result of the experiment, no cytotoxicity was shown in any of the cancer cell lines at the administered Hepes-dissolved melanin concentration. On the other hand, these cell lines were seeded in a dish at a low density, and at the same time, various concentrations of Hepes-dissolved melanin were treated, and after culturing for 24 hours, the cell viability was measured by Cell Counting Kit 8. As a result, in the HeLa and MCF-7 cell lines, the treatment concentration of Hepes-dissolved melanin was 0.125 mg / ml to 0.5 mg / ml, and in the Jurkat cell line, the treatment concentration of Hepes-dissolved melanin was 0.031 mg / ml to 0.5 mg / ml. Up to ml, cell proliferation was significantly suppressed in a treatment concentration-dependent manner (Fig. 7).

(5) Suppression test of transplanted tumor by Hepes-dissolved melanin using mice Commercially available melanin was dissolved in 500 mM Hepes to prepare a stock solution of 50 mg / ml. First, in vitro, the cell proliferation inhibitory effect of Hepes-dissolved melanin on the mouse melanoma cell line B16F10 was confirmed. B16F10 was seeded on a dish at a low density, and at the same time, various concentrations of Hepes-dissolved melanin were treated, and after culturing for 24 hours, the cell viability was measured by cell counting kit 8. As a result of the experiment, cell proliferation was significantly suppressed in a treatment concentration-dependent manner at treatment concentrations of Hepes-dissolved melanin from 0.25 mg / ml to 0.5 mg / ml (Fig. 8A). Next, Hepes-dissolved melanin was injected into an osmotic pump and transplanted into the subcutaneous ventral side of nude mice. The next day, the mouse melanoma cell line B16F10 was subcutaneously injected and the subsequent tumor volume was measured. As a result of the experiment, the tumor growth was significantly suppressed in the group to which Hepes melanin was administered (Fig. 8B).

(6) Cell cycle analysis of Hepes-lysed melanin-treated cancer cells Cell cycle analysis was performed to investigate the mechanism of cell proliferation suppression by melanin. Human breast cancer cell line MCF-7 was treated with Hepes-dissolved melanin at a concentration of 0.25 mg / ml for 48 hours. As a result of cell cycle analysis by flow cytometry, the proportion of cells in G0 / G1 phase was significantly increased and the proportion of cells in S phase was relatively decreased by Hepes-lysed melanin treatment (Fig.). 9). This result suggests that melanin treatment may arrest the cell cycle in the G0 / G1 phase and suppress its progression to the DNA synthesis phase.

(7) Effect of Hepes-dissolved melanin on histone phosphorylation
Since it was suggested that cell cycle progression may be inhibited in the G0 / G1 phase, the phosphorylation of histone H3, which is an indicator of mitotic progression, was investigated. The human liver cancer cell line HepG2 was treated with Hepes-dissolved melanin at a concentration of 0.25 or 0.5 mg / ml and cultured for 24 hours. Soluble proteins were extracted and histone H3 and phosphorylated histone H3 were detected by Western blotting, respectively. As a result of the experiment, phosphorylation of histone H3 was suppressed in a Hepes-dissolved melanin treatment concentration-dependent manner (Fig. 10). These results strongly suggest that melanin is involved in the inhibition of the mitotic process.

3. 3. Discussion Melanin is widely distributed in the biological world and is used in various ways depending on the species. There are very few attempts to use melanin pharmaceutically and biologically, and there are previous studies such as inhibition of HIV replication by artificially synthesized melanin (Japanese Patent Publication No. 2001-512437) and cytokine preparation ability (Japanese Patent Publication No. 2001-512446). However, reports on the use and application of melanin for the purpose of suppressing cell proliferation have not been made both in Japan and overseas. In this study, we mainly used cell lines of cancer cells, and solubilized and finely dispersed melanin did not induce obvious cell death in all the cell lines examined, including solid tumors and blood cell line cancers. At the concentration, cell proliferation was effectively suppressed. This means that the regulated melanin can suppress the growth of abnormally proliferating cancer cells without inducing cell death and prolong the life of cancer patients, as long as the regulated melanin does not seriously damage normal cells. Suggest that. A wide variety of anti-cancer agents have been developed so far, and many of them also damage normal cells and have side effects, but in recent years, molecular-targeted molecular-targeted therapeutic agents that cause cancer cell-specific disorders, and An antibody drug that induces reactivation of the immune surveillance mechanism has been developed and has been successful.

Although the present invention can be expected to have an effective effect even when used alone, by appropriately using it in combination with an existing anticancer drug, the treatment period can be shortened, the concentration of the anticancer drug can be reduced, side effects can be suppressed, and costs can be reduced. It can lead to effective cancer treatment that reduces the physical burden and financial cost of the patient.

Malignant melanoma is a cancerous form of melanocytes that produce melanin. There are cancerous tumors that retain melanin-producing ability and non-pigmented malignant melanoma that does not produce melanin. The melanin that retains the ability to produce melanin actually repeats cell proliferation, and at first glance, it seems that cell proliferation is not suppressed despite the release of melanin to the surroundings, but it is constant in the closed system in vitro. When an amount of melanin accumulates in a cell culture dish, it appears to suppress its own cell proliferation. It is possible that melanoma in an individual metastasizes before the concentration of melanin released by the cells themselves reaches a concentration that suppresses cell proliferation, and repeats new cell proliferation. B16F10, a mouse melanoma cell line, produces melanin. In an in vitro experimental system, although resistance to the proliferation inhibitory effect of melanin can be seen compared to other cancer cell lines, solubilized melanin added from the outside The cell proliferation inhibitory effect dependent on the treatment concentration was confirmed. Furthermore, in an in vivo experimental system that continuously releases solubilized melanin using an osmotic pump, the proliferation of B16F10 cells transplanted to the ventral part of nude mice was significantly suppressed. From this, it is considered that even if the melanoma produces melanin, cell proliferation is suppressed if the concentration of solubilized melanin around the cells is sufficient.

As a result of cell cycle analysis, treatment with solubilized melanin increased the proportion of cells in G0 / G1 phase and decreased the proportion of S phase, which is the relative DNA replication phase. From this, it can be assumed that G0 / G1-arrest was caused by the solubilized melanin treatment. The melanin treatment markedly suppressed the phosphorylation of histone H3, demonstrating the arrest of mitosis.

From the above results, it was found that melanin suppresses cell proliferation for the first time by solubilizing and dispersing fine particles. Even with solubilization and fine particle dispersion, melanin is a natural product that is originally biosynthesized in vivo and has the function of protecting tissues from ultraviolet rays, so it makes sense that cytotoxicity is extremely low. Exposure of cells to melanin triggers a defensive response to exposure to UV radiation, which is harmful to the cell, that is, to stop cell proliferation and prevent DNA replication errors due to UV radiation. Maybe.

The mechanism of cell proliferation suppression by melanin is awaiting detailed elucidation, but the property of suppressing abnormally proliferating cell division is not only the improvement of the therapeutic effect when used in combination with the above-mentioned anticancer drug, but also the improvement of the therapeutic effect. It can also be applied as an inhibitor of hyperplasia such as polyps, warts, and psoriasis. In addition, natural products containing melanin such as squid ink, octopus ink, bananas, mushrooms, and asagao seeds are thought to lead to the development of safer supplements with the expectation of cancer prevention.

The cell proliferation inhibitor of the present invention can be used alone or in combination with other anticancer agents for the treatment of "cancer". It is also expected to be applied to the treatment and prevention of benign tumors, hyperplasia, polyps, etc.

The present invention is not limited to the description of the embodiments and examples of the above invention. Various modifications are also included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims. The contents of the papers, published patent gazettes, patent gazettes, etc. specified in this specification shall be cited by reference in their entirety.

Claims (7)

Hepes A cell growth inhibitor containing dissolved melanin or dispersed melanin as an active ingredient. The growth inhibitor according to claim 1, wherein the Hepes-dissolved melanin is a solubilized esoteric melanin containing no protein component with Hepes. The growth inhibitor according to claim 1 or 2, wherein the molecular weight of Hepes-dissolved melanin is 50 kDa to 100 kDa. The growth inhibitor according to claim 1, wherein the dispersed melanin has an average particle size of 70 nm to 130 nm. The growth inhibitor according to any one of claims 1 to 4 , wherein the cell is a tumor cell. The growth inhibitor according to claim 5 , which is used for treating cancer. The growth inhibitor according to claim 6 , wherein an anticancer agent is used in combination.

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