JP2022041479A - Medicinal composition for treatment of skin cancer or precancerous lesions thereof - Google Patents

Abstract

To provide a pharmaceutical composition for the treatment of skin cancer or its precancerous lesions.SOLUTION: Provided is a pharmaceutical composition comprising a cholecystokinin receptor antagonist as an active ingredient and used for the treatment of skin cancer or precancerous lesions thereof. Also provided is the pharmaceutical composition in which the cholecystokinin receptor antagonist is a CCKA receptor antagonist. Further provided is a method for screening a candidate compound for a therapeutic agent for skin cancer or precancerous lesions thereof. In the screening method, a substance having an antagonistic effect on cholecystokinin receptor is selected as the candidate compound.SELECTED DRAWING: None

Description

The present invention relates to a pharmaceutical composition used for treating skin cancer or its precancerous lesion, and a method for screening a candidate compound for a therapeutic agent for skin cancer or its precancerous lesion.

Skin cancer is mainly classified into spinous cell carcinoma (squamous cell carcinoma of the skin), basal cell carcinoma, and malignant melanoma (melanoma), depending on the cells that produce malignant cells. In particular, melanoma is a tumor with a high degree of malignancy and a low 5-year survival rate among skin tumors. One of the causes of high malignancy of melanoma is that it metastasizes to other organs at a high rate. Melanoma is classified into terminal mole type, superficial enlarged type, nodular type, and malignant mole type according to the disease type. In Japan, the terminal mole type accounts for about 40% of the total. On the other hand, in the United States, about 60% of the total is superficial expansion type. Genetic abnormalities such as BRAF are involved in carcinogenesis, and less than 30% of Japanese patients have BRAF gene mutations, but 40-60% of Caucasians.

Treatment of melanoma is basically surgical treatment to remove the lesion, but if metastasis to other organs is observed, drug therapy or radiation therapy in addition to it is required. Melanoma was often resistant to conventional anticancer drugs, but in 2014, in addition to chemotherapy, immune checkpoint inhibitors such as nivolumab were launched, followed by molecular targets such as dabrafenib and trametinib. A new drug has been put into practical use. However, immune checkpoint inhibitors can develop autoimmune disorders that are summarized as immune-related adverse effects (irAEs). In addition, the practically used molecular target drug cannot be used in the treatment of melanoma in which the BRAF gene is not mutated because its application is limited when it has a BRAF gene mutation. Therefore, it is desired to develop a new therapeutic drug that exerts its effect by a mechanism different from these drugs and has few side effects.

Cholecystokinin (CCK) is a peptide hormone, and there are two types of specific receptors thereof, the CCKA receptor and the CCKB receptor. CCK receptors are mainly present on the cell surface of the gastrointestinal tract and the central nervous system, and CCK has an action of regulating the secretion of gastric juice, bile, and pancreatic juice. Therefore, the CCK receptor antagonist (CCK receptor antagonist) can be an active ingredient of a therapeutic agent for gastrointestinal disorders (Patent Document 1). In fact, oral formulations of proglumide (CAS No .: 6620-60-6), a non-selective CCK receptor antagonist for both CCKA and CCKB receptors, have been used to treat gastritis and gastric ulcer. Was there. It has been proved by animal experiments that proglumide exerts a CCK receptor antagonistic action not only by oral administration but also by intravenous administration and intraperitoneal administration (Non-Patent Document 1).

CCK has a nourishing effect on the pancreatic acinus, and the peptide hormone gastrin, which is a ligand for the CCKB receptor, has a nourishing effect on the digestive epithelium. Utilizing these effects, proglumide exerts an antitumor effect on these gastrointestinal tumors. In fact, studies using a mouse model have reported that systemic administration of proglumide suppresses the growth of pancreatic cancer and colon cancer (Patent Document 2).

Special Table 2009-539929 Special Fair 6-67832 Gazette

Makovec, et al., Arzneimittel Forschung Drug research, 1987, vol.37, p.1265-1268.

It is an object of the present invention to provide a novel pharmaceutical composition used for the treatment of skin cancer including melanoma or its precancerous lesion.

As a result of searching for factors involved in the growth and survival of melanoma cells in order to develop a new drug having a mechanism different from that of conventional drugs, the present inventors have found that a CCK receptor antagonist suppresses the growth of melanoma cells. The present invention has been completed by finding that it has an action and an apoptosis-inducing action.

That is, the present invention provides the following.
[1] A pharmaceutical composition containing a cholecystokinin receptor antagonist as an active ingredient and used for the treatment of skin cancer or precancerous lesions thereof.
[2] The pharmaceutical composition according to the above [1], wherein the cholecystokinin receptor antagonist is a CCKA receptor antagonist.
[3] The pharmaceutical composition according to the above [1], wherein the cholecystokinin receptor antagonist is at least one selected from the group consisting of lorglumide, proglumide, and loxyglumide.
[4] The pharmaceutical composition according to any one of the above [1] to [3], which is used for the treatment of melanoma, spinous cell carcinoma, actinic keratosis, or basal cell carcinoma.
[5] A screening method for screening a candidate compound for a therapeutic agent for skin cancer or a precancerous lesion thereof, wherein a substance having an antagonistic effect on a cholecystokinin receptor is selected as the candidate compound.

The pharmaceutical composition according to the present invention contains a cholecystokinin receptor antagonist as an active ingredient, and is a new therapeutic agent having a different mechanism of action from conventional therapeutic agents for skin cancer such as melanoma or precancerous lesions thereof. be. Therefore, the pharmaceutical composition according to the present invention is also expected as a therapeutic agent for melanoma and the like resistant to conventional therapeutic agents.
In addition, the screening method according to the present invention can efficiently screen candidate compounds for the active ingredient of the pharmaceutical composition.

In Reference Example 1, it is a stained image obtained by performing immunostaining with an anti-CCKA receptor antibody on a tissue section of a lesion portion collected from a melanoma patient. In Reference Example 1, mouse melanoma-derived B16 cells and human melanoma-derived A375 cells were stained with an anti-CCKA receptor antibody or an anti-CCKB receptor antibody and a fluorescently labeled secondary antibody, and then using a flow cytometer. It is a figure which showed the analysis result. In Reference Example 1, RT-PCR was performed on RNA extracted from mouse melanoma-derived B16 cells and human melanoma-derived A375 cells, and it is an electrophoretic image of a PCR amplification product of CCK mRNA. In Reference Example 1, HSC-1 cells and A431 cells derived from human squamous cell carcinoma were stained with an anti-CCKA receptor antibody or an anti-CCKB receptor antibody and a fluorescently labeled secondary antibody, and then a flow cytometer was used. It is a figure which showed the result of the analysis using. In Example 1, the assay using the WST-8 reagent was performed on B16 cells (A) and A375 cells (B) treated with rolglumid, and the measurement result of the absorbance at a wavelength of 450 nm is shown. In Example 1, it is the figure which performed the assay using the WST-8 reagent with B16 cells treated with lorglumide, proglumide, or loxyglumide, and showed the measurement result of the absorbance at a wavelength of 450 nm. It is a figure which showed the measurement result of the average fluorescence intensity of CFSE of the B16 cell treated with lorglumid in Example 1. FIG. In Example 2, the measurement result of the ratio (%) of the total number of early apoptotic cells and late apoptotic cells to the whole cells of B16 cells (A) and A375 cells (B) treated with rolglumid and cultured for 48 hours was obtained. It is a figure shown. It is a figure which showed the measurement result of the relative value (%) of the caspase 3/7 activity of the B16 cell which was treated with lorglumid and cultured for 48 hours in Example 3. FIG. In Example 3, the total number of early-apoptotic cells and late-apoptotic cells for the entire cell of B16 cells pretreated with the caspase 3-specific inhibitor Z-DEVD-FMK, then treated with rolglumid and cultured for 48 hours. It is a figure which showed the measurement result of the ratio (%) of. In Example 4, after the monolayer of B16 cells treated with rolglumid after stopping cell proliferation was injured, the region containing the injuries was observed over time under a microscope and obtained by a transmitted light image. be. In Example 4, the measurement result of the relative area (%) of the intercellular space of the wound after injuring the monolayer of B16 cells treated with rolglumid after stopping cell proliferation and culturing for 24 hours is shown. It is a figure. FIG. 5 is a diagram showing the results of intratumoral administration of lorglumid to mice subcutaneously transplanted with B16 cells in Example 5 and measurement of changes in tumor volume (mm ³ ) over time. In Example 5, a mouse subcutaneously transplanted with B16 cells was intratumorally administered with lorglumid over time, and the measurement result of the tumor weight (mg) on the 14th day from the start of rolglumid administration is shown. FIG. 5 is a diagram showing the results of external administration (applied to the skin) of lorglumid over time to mice subcutaneously transplanted with B16 cells and measuring changes in tumor volume (mm ³ ) over time. In Example 5, it is the figure which showed the measurement result of the tumor weight (mg) on the 14th day from the start of the administration of lorglumid by externally administering lorglumid to the mouse subcutaneously transplanted with B16 cells.

<Medicinal composition>
The pharmaceutical composition according to the present invention contains a CCK receptor antagonist as an active ingredient and is used for the treatment of skin cancer or precancerous lesions thereof. The skin cancer for which the pharmaceutical composition according to the present invention is used for treatment may be any of spinous cell carcinoma, basal cell carcinoma, and melanoma. In addition, examples of precancerous lesions of skin cancer for which the pharmaceutical composition according to the present invention is used for treatment include actinic keratosis and the like. The pharmaceutical composition according to the present invention is preferably used for the treatment of melanoma, spinous cell carcinoma, actinic keratosis, or basal cell carcinoma, and is particularly preferably used for the treatment of melanoma. Hereinafter, "skin cancer or its precancerous lesion" may be referred to as "skin cancer or the like".

As shown in Examples below, the CCK receptor antagonist has a growth inhibitory effect, an apoptosis-inducing effect, and a chemotaxis (migratory ability) inhibitory effect on skin cancer cells including melanoma cells or precancerous lesion cells thereof. Have. Unlike conventional therapeutic agents such as melanoma, CCK receptor antagonists exhibit antitumor action based on a novel mechanism of action targeting CCK receptors. Therefore, the pharmaceutical composition according to the present invention exerts an antitumor effect on skin cancer cells such as melanoma cells regardless of the presence or absence of BRAF gene mutation.

The CCK receptor antagonist substance used as the active ingredient in the present invention is not particularly limited as long as it has an antagonistic effect on the CCK receptor, and may be a substance having a competitive antagonistic effect, and is non-competitive. It may be a substance having a target antagonistic action. The substance may be a small molecule compound, a nucleic acid aptamer, a peptide aptamer, or a protein such as an antibody.

The CCK receptor antagonist used as the active ingredient in the present invention may be a selective antagonist for the CCKA receptor or a selective antagonist for the CCKB receptor, and may be a CCKA receptor and a CCKB receptor. It may be an antagonist that acts non-selectively on both. As the CCK receptor antagonist substance used as the active ingredient in the present invention, a substance having an antagonistic effect on the CCKA receptor is preferable.

As the CCK receptor antagonist substance as an active ingredient in the present invention, a known substance having a CCK receptor antagonistic action can be appropriately selected and used. Among the CCK receptor antagonists, low molecular weight compounds include, for example, proglumide, lorglumide (CAS No.: 97964-56-2), loxyglumide (CAS No.: 107097-80-3), and dexyloxyglumide (119817). -90-2), Spilogulmid (CAS No .: 137795-35-8), Itrigrumid (CAS No .: 201605-51-8), KSG-504 (CAS No .: 137005-17-5), YM022 (CAS) No .: 145084-28-2), and derivatives thereof and the like. Among them, proglumide has been widely used as an orally administered drug for treating gastritis and gastric ulcer, and is preferable because it can be safely ingested.

The pharmaceutical composition according to the present invention may contain a CCK receptor antagonist as a pharmacologically acceptable salt thereof. The pharmacologically acceptable salt may be an inorganic acid salt, an organic acid salt having a basic moiety such as an amine, an alkaline salt, or an organic salt. May be.

The active ingredient of the pharmaceutical composition according to the present invention may be a solvate of a free CCK receptor antagonist or a solvate of a pharmacologically acceptable salt of the CCK receptor antagonist. .. Examples of the solvent for forming the solvate include water, ethanol and the like.

The amount of the CCK receptor antagonist contained in the pharmaceutical composition according to the present invention is particularly effective as long as it is a therapeutically effective amount for skin cancer and the like, that is, an amount sufficient to obtain a therapeutic effect for skin cancer and the like. It is not limited, and is appropriately determined in consideration of the type of skin cancer, administration route, administration form (drug type), number of administrations per day, administration interval, species to be administered, sex, age, body weight, etc. Will be done.

When the pharmaceutical composition according to the present invention contains a drug such as proglumide, which has already been approved as a drug in the present or in the past, as an active ingredient, the route of administration, dosage form, dose of the active ingredient in one administration, The dosage such as the dosing interval can be the same as the therapeutic agent for other diseases for which these agents have already been approved, and these may be improved.

The administration route of the pharmaceutical composition according to the present invention is not particularly limited, and is appropriately selected from various administration routes such as oral administration, intravenous administration, transdermal administration, enteral administration, and nasal administration. be able to. The pharmaceutical composition according to the present invention is an oral solid agent such as a powder, a granule, a capsule, a tablet, a chewable agent, a sustained-release agent, or a solution by an ordinary method in consideration of a target administration route or the like. , Oral solutions such as syrups; external preparations such as patches, ointments, gels, creams, sprays, lotions, liniments; injections, suppositories and the like.

Other components contained in the pharmaceutical composition according to the present invention include, for example, excipients, binders, lubricants, disintegrants, fluidizers, solvents, solubilizers, buffers, suspending agents. , Emulsifiers, tonicity agents, stabilizers, preservatives, antioxidants, flavoring agents, colorants and other pharmacologically acceptable additives. In addition, the pharmaceutical composition may contain an active ingredient other than the CCK receptor antagonist.

Examples of the excipient include sugars such as lactose, glucose and D-mannitol, celluloses such as starch and crystalline cellulose, sugar alcohols such as erythritol, sorbitol and xylitol, dicalcium phosphate, calcium carbonate and kaolin. .. Examples of the binder include pregelatinized starch, gelatin, gum arabic, methyl cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose, crystalline cellulose, D-mannitol, trehalose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinylpyrrolidone, polyvinyl alcohol and the like. Examples of the lubricant include stearic acid, calcium stearate, talc, sucrose fatty acid ester, polyethylene glycol and the like. Examples of the disintegrant include crospovidone (crosslinked polyvinylpyrrolidone), low-degree-of-substitution hydroxypropyl cellulose, starch, alginic acid, sodium alginate and the like. Examples of the fluidizing agent include silicic acid, silicic acid anhydride, aluminum silicate, calcium silicate, magnesium aluminometasilicate compound, aluminum oxide, aluminum hydroxide, magnesium oxide, magnesium hydroxide and the like. Examples of the solvent include purified water and physiological saline. Examples of the solubilizing agent include dextran, polyvinylpyrrolidone, sodium benzoate, ethylenediamine, salicylic acid amide, nicotinic acid amide, polyoxyethylene hydrogenated castor oil derivative and the like. Examples of the buffer include sodium citrate hydrate, sodium acetate hydrate, sodium hydrogen carbonate, trometamol, boric acid, borosand, sodium hydrogen phosphate hydrate, sodium dihydrogen phosphate and the like. Examples of the suspending agent or emulsifier include celluloses such as sodium lauryl sulfate, gum arabic, gelatin, lecithin, glycerin monostearate, polyvinyl alcohol, polyvinylpyrrolidone, and sodium carboxymethyl cellulose, polysorbates, and polyoxyethylene hydrogenated castor oil. Can be mentioned. Examples of the tonicity agent include lactose, glucose, sugars such as D-mannitol, sodium chloride, potassium chloride, glycerin, propylene glycol, polyethylene glycol, urea and the like. Examples of the stabilizer include polyethylene glycol, sodium dextran sulfate, sodium sulfite and the like. Examples of the preservative include paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, chlorocresol, dehydroacetic acid, sorbic acid and the like. Examples of the antioxidant include sulfites, ascorbic acid and the like. Examples of the flavoring agent include sweeteners and flavors commonly used in the pharmaceutical and food fields. Colorants include colorants commonly used in the pharmaceutical and food fields.

The pharmaceutical composition according to the present invention is administered to an animal having or suspected of developing skin cancer or the like. The pharmaceutical composition is preferably administered to mammals, and includes humans, mice, rats, rabbits, guinea pigs, hamsters, monkeys, sheep, horses, cows, pigs, donkeys, dogs, cats and the like. It is more preferably administered to domestic animals and laboratory animals, and even more preferably to be administered to humans.

<Method of screening candidate compounds for therapeutic agents such as skin cancer>
CCK receptors are also expressed on the cell surfaces of skin cancer cells such as melanoma cells and cells with precancerous lesions thereof, and the signaling downstream of the receptors is involved in cell proliferation, apoptosis, motility, etc. ing. Therefore, the CCK receptor antagonist has a growth inhibitory action, an apoptosis-inducing action, and a chemotaxis-suppressing action on skin cancer cells and the like, and exerts an antitumor effect. Therefore, a substance having an antagonistic effect on the CCK receptor can be selected as a candidate compound for a therapeutic agent for skin cancer and the like.

The CCK receptor antagonist can be selected, for example, by screening various small molecule compound libraries, nucleic acid libraries, peptide libraries and the like using a binding test for the CCK receptor. Among the selected substances, substances having a higher binding affinity for the CCK receptor than CCK, that is, a substance having a small dissociation constant ( _KD ), are downstream of the CCK receptor in skin cancer cells such as melanoma cells. It can more effectively competitively inhibit cell signaling and is promising as a candidate compound for therapeutic agents such as skin cancer.

CCK receptor antagonists can also be screened using cells that express the CCK receptor, for example, and secrete a particular substance upon CCK stimulation. For example, cells expressing the CCK receptor and secreting gastric acid by CCK stimulation can be used, such as gastric corpus parietal cells. When the amount of gastric acid secretion when the cells are cultured in the culture medium containing CCK and the candidate compound is clearly smaller than the amount of gastric acid secretion when the cells are cultured in the culture medium containing only CCK, the candidate compound is CCKed. Selected as a receptor antagonist.

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[Reference Example 1]
The expression of CCKA receptor in melanoma cells was examined.

(1) Expression of CCKA receptor in melanoma lesion tissue The expression of CCKA receptor in the lesion tissue of a melanoma patient collected for therapeutic purposes was examined by an immunohistological method. Specifically, a rabbit anti-CCKA receptor antibody (manufactured by Invitrogen) was used for tissue sections of melanoma lesions collected from 3 superficial enlarged melanoma patients and 3 terminal mole type melanoma patients, respectively. Immunostaining was performed. The stained image is shown in FIG. In the figure, the sites indicated by arrows are the sites stained with the anti-CCKA receptor antibody. As shown in FIG. 1, expression of CCKA receptor was observed in superficial enlarged type and terminal mole type melanoma lesions.

(2) Expression of CCKA receptor and CCKB receptor in melanoma-derived cultured cells The CCK receptor expression of mouse melanoma-derived B16 cells and human melanoma-derived A375 cells was examined by flow cytometry. Specifically, each cell is incubated with a rabbit anti-CCKA receptor antibody (manufactured by Bioss) for 1 hour, and then incubated with a fluorescently labeled secondary antibody (anti-rabbit IgG antibody, manufactured by Biolegend) for 30 minutes for staining. did. In addition, B16 cells and A375 cells were stained in the same manner except that a rabbit anti-CCKB receptor antibody (manufactured by Alomone labs) was used instead of the rabbit anti-CCKA receptor antibody. The stained cells were analyzed using a flow cytometer. As a control, cells stained only with fluorescently labeled secondary antibody were also analyzed using a flow cytometer in the same manner. The analysis results are shown in FIG. As shown in FIG. 2, expression of CCKA receptor was confirmed in both B16 cells and A375 cells. Regarding the CCKB receptor, in A375 cells, the histogram of the cells stained with the anti-CCKB receptor antibody was shifted to the right from the histogram of the cells stained with the secondary antibody alone. Was confirmed to be expressed.

RNA was extracted from the cells and the expression of CCK was examined by the RT-PCR method. As a control, β-actin expression was also examined. The agarose gel electrophoresis image of the amplification product obtained as a result of RT-PCR is shown in FIG. As shown in FIG. 3, RT-PCR amplification products of CCK mRNA were confirmed in both B16 cells and A375 cells, and it was confirmed that CCK was expressed in both cells. These results suggest that melanoma cells are regulated by CCK by the autocrine / paracrine mechanism.

(3) Expression of CCKA receptor and CCKB receptor in cultured cells derived from non-melanoma skin cancer The CCK receptor expression of HSC-1 cells and A431 cells derived from human squamous cell carcinoma was examined in the same manner by the flow cytometry method. .. The analysis results are shown in FIG. As shown in FIG. 4, expression of both CCKA receptor and CCKB receptor was confirmed in both HSC-1 cells and A431 cells.

[Example 1]
The growth inhibitory effect of CCK receptor antagonists on melanoma cells was investigated. As CCK receptor antagonists, rolglumide, which is a CCKA receptor selective antagonist, and proglumide and loxyglumide, which are non-selective CCK receptor antagonists, were used. Cell proliferation ability was measured by WST assay using WST-8 reagent or assay using CFSE (Carboxyfluorescein Succinimidyl Ester).

(1) Effect of rolglumid on the proliferation of B16 cells and A375 cells B16 cells and A375 cells were seeded on a 96-well plate and cultured overnight, then lolglumid was added and cultured for 48 hours. Then, 10 μL of WST-8 reagent (manufactured by Dojindo) was added to each well, and the cells were further cultured for 3 hours, and then the absorbance at a wavelength of 450 nm was measured using a plate reader. WST-8 is reduced by intracellular dehydrogenase to produce yellow formazan. Therefore, in the WST assay, the absorbance and the number of living cells are in a proportional relationship.

The measurement result of the absorbance of each well at a wavelength of 450 nm is shown in FIG. 5 (**: p <0.01 vs. untreated control group). As shown in the figure, in both B16 cells and A375 cells, lorglumid reduced the number of viable cells in a concentration-dependent manner.

(2) Effect of rolglumide, proglumide and loxyglumide on the proliferation of B16 cells B16 cells were seeded on a 96-well plate and cultured overnight, then lorglumide, proglumide or loxyglumide was added and cultured for 48 hours, and then the above-mentioned. An assay using the WST-8 reagent was performed in the same manner as in (1).

The measurement result of the absorbance of each well at a wavelength of 450 nm is shown in FIG. 6 (*: p <0.01 vs. untreated control group, **: p <0.01). As shown in the figure, proglumide and loxyglumide also decreased the number of viable cells in a concentration-dependent manner like lorglumide, but the effect of reducing the number of cells was highest in lorglumide.

(3) Cell proliferation assay Lolglumid was added to labeled B16 cells and cultured for 48 hours. Then, the brightness (fluorescence intensity) of CFSE of the cells after culturing was measured using a flow cytometer. The brightness of CFSE is halved at each cell division. CFSE labeling was performed using the CFSE Cell Division Assay Kit (manufactured by Cayman).

The measurement result of the average fluorescence intensity of CFSE is shown in FIG. It was revealed that the mean fluorescence intensity of CFSE of cells was significantly higher, that is, the number of cell divisions was lower in the rolglumid-added group than in the untreated control group (**: p <0.01 vs. no). Treatment control group). From these results, it was clarified that lorglumid suppresses the proliferation of melanoma cells.

[Example 2]
The effect of CCK receptor antagonists on melanoma cells was investigated. Lolglumid was used as a CCK receptor antagonist. Apoptosis-inducing effects were investigated using an apoptosis detection assay with fluorescently labeled Annexin V. Annexin V binds to phosphatidylserine, a cell membrane-constituting lipid. Since phosphatidylserine is present inside the cell membrane in living cells and is exposed on the cell membrane surface when apoptosis is induced, the cells in which apoptosis is induced emit fluorescence by the fluorescent label Annexin V.

After adding lorglumid to B16 cells or A375 cells and culturing for 48 hours, the cells were stained with Annexin V labeled with the fluorescent substance PE (R-Phycoerythrin) and the dead cell nuclear stain 7AAD (7-Amino-Actinomycin D). The stained cells were analyzed with a flow cytometer. Cells in the early stages of apoptosis are not stained with 7AAD, whereas late apoptotic cells are stained with 7AAD. That is, early apoptotic cells appear in the fraction of 7AAD- / AnnexinV +, late apoptotic cells appear in the fraction of 7AAD + / AnnexinV +, and living cells appear in the fraction of 7AAD- / AnnexinV-.

Based on the analysis result of the flow cytometer, the measurement result of the ratio (%) of the total number of cells of early apoptotic cells and late apoptotic cells to the whole cells is shown in FIG. As shown in the figure, the proportion of early and late apoptotic cells was significantly increased in B16 cells and A375 cells cultured with the addition of rolglumid (**: p <0.01 vs. untreated control group). From these results, it was clarified that lorglumid induces apoptosis in melanoma cells.

[Example 3]
When an apoptosis-inducing signal is transmitted to cells, a complex cascade of cysteine proteases called caspases is initiated to perform apoptosis. Caspases are classified into three types: induced, executive, and inflammatory, and caspases 3, 6 and 7, which are classified as executive caspases, play a particularly important role in the signaling pathway of apoptosis execution. Therefore, the caspase 3/7 activity in apoptosis induced by CCK receptor antagonists was investigated. Caspase 3/7 activity was examined using the Caspase-Glo 3/7 Assay System (manufactured by Promega).

B16 cells were seeded on 96-well plates, rolglumid (200 μM) was added, and the cells were cultured for 48 hours. According to the manual of the reagent provider, the substrate solution was added to the cultured cells and the cells were cultured for another 2 hours, and then the relative luminescence amount (%) was measured as the caspase 3/7 activity using a plate reader. The measurement results are shown in FIG. As shown in the figure, in the group cultured with the addition of rolglumid, the caspase 3/7 activity was enhanced to about 1.7 times that of the untreated control group (**: p <0.01 vs. no). Treatment control group).

Therefore, B16 cells were pretreated with Z-DEVD-FMK, which is a specific inhibitor of caspase 3, for 2 hours, then rolglumid was added and cultured for 48 hours, and apoptosis-positive cells were analyzed by a flow cytometer. The analysis result is shown in FIG. As shown in the figure, pretreatment with Z-DEVD-FMK significantly suppressed apoptosis. These results suggest that caspase 3 is involved in rolglumid-induced apoptosis signaling.

[Example 4]
The chemotaxis-suppressing effect of CCK receptor antagonists on melanoma cells was investigated. Lolglumid was used as a CCK receptor antagonist.

To stop cell growth, B16 cells were pretreated with mitomycin C for 1.5 hours, then seeded in a single layer on 96-well plates and cultured overnight. After scratching the B16 melanoma monolayer after culturing with a yellow chip, rolglumid was added and the cells were cultured for 24 hours. The area containing the scratch was observed under a microscope over time. The observation results are shown in FIG. As shown in the figure, in the rolglumid-untreated control group, cells migrated to the injured area and the area of the gap decreased over time. On the other hand, in the group to which rolglumid was added, cell migration was suppressed and the area of the gap was hardly reduced. The area of the gap created by the wound after being injured and cultured for 24 hours was calculated using the image analysis software Image J. Relative area of intercellular space after 24 hours of culture after injury (%: [area of gap at the time of culturing for 24 hours after injury] / [area of gap at the time of injury] × 100 ) Is shown in FIG. As shown in the figure, the higher the concentration of rolglumid, the wider the relative area of the gap after culturing, and the lower the migration ability of B16 cells. From these results, it was clarified that the CCK receptor antagonist suppresses the migration of melanoma cells. Since cell migration ability is deeply involved in local tissue invasion and distant metastasis, CCK receptor antagonists not only exert antitumor effects by inducing apoptosis, but also suppress cell migration. It was suggested that it may suppress tumor growth and metastasis.

[Example 5]
The antitumor effect of lorglumid was investigated in melanoma subcutaneous transplantation model mice. The melanoma subcutaneous transplantation model mouse was prepared by transplanting B16 cells ⁽ 105 cells) subcutaneously into the abdomen of a C57BL / 6 mouse.

(1) Effect of intratumoral administration of lorglumid When the volume of the tumor by subcutaneously transplanted B16 cells reached 10 to ³⁰ mm3, lorglumid (100 μg, 4 mg / kg) was administered intratumorally. As a control, the same amount of saline was administered intratumorally instead of lorglumid. Then, the same rolglumid administration was repeated every 3 to 4 days. FIG. 13 shows the results of measuring the tumor volume ([tumor volume (mm ³ )] = [major diameter (mm)] × [minor diameter (mm)] ² × 1/2) of each mouse over time. In addition, on the 14th day from the start of administration of rolglumid, a tumor mass was collected and the tumor weight (mg) was measured. The measurement result of the tumor weight is shown in FIG.

As shown in FIG. 13, in the lorglumid-administered group, the increase in tumor volume was significantly suppressed as compared with the saline-administered group (**: p <0.01 vs. saline-administered group). In addition, as shown in FIG. 14, the tumor weight was significantly reduced by the administration of rolglumid (*: p <0.05 vs. saline administration group).

(2) Effect of external therapy of rolglumid For melanoma subcutaneous transplant model mice in which tumors caused by subcutaneously transplanted B16 cells can be observed with the naked eye, lorglumid (400 μg) dissolved in a hydrophilic ointment was added to 2, 5, and 9. And every day except the 12th day, it was applied to the tumor once a day. The control group was coated with the same amount of hydrophilic ointment instead of the rolglumid-containing hydrophilic ointment. In addition, in each mouse, acetone (30 μL) was applied to the tumor tissue immediately before the application of the rolglumid-containing hydrophilic ointment in order to enhance the skin permeability. The tumor volume of each mouse and the tumor weight 14 days after the start of rolglumid administration were measured in the same manner as in the mice administered intratumorally with lolglumid.

The results of measuring the tumor volume (mm ³ ) of each mouse over time are shown in FIG. 15, and the results of measuring the tumor weight (mg) on the 14th day from the start of lorglumid administration are shown in FIG. As shown in FIG. 15, the increase in tumor volume was significantly suppressed by external application of rolglumid. In addition, as shown in FIG. 16, the tumor weight 14 days after the start of external use of rolglumid was significantly lower in the external use of rolglumid group than in the control group. From these results, it was clarified that in a mouse tumor model, tumor growth can be suppressed by intratumoral administration or external application of a CCK receptor antagonist.

Claims (5)

A pharmaceutical composition containing a cholecystokinin receptor antagonist as an active ingredient and used for the treatment of skin cancer or precancerous lesions thereof. The pharmaceutical composition according to claim 1, wherein the cholecystokinin receptor antagonist is a CCKA receptor antagonist. The pharmaceutical composition according to claim 1, wherein the cholecystokinin receptor antagonist is at least one selected from the group consisting of lorglumide, proglumide, and loxyglumide. The pharmaceutical composition according to any one of claims 1 to 3, which is used for the treatment of melanoma, spinous cell carcinoma, actinic keratosis, or basal cell carcinoma. A method for screening candidate compounds for the treatment of skin cancer or precancerous lesions thereof.
A screening method for selecting a substance having an antagonistic effect on a cholecystokinin receptor as the candidate compound.

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