JP7599684B2 - Pharmaceutical composition for treating skin cancer or its precancerous lesions - Google Patents

Description

The present invention relates to a pharmaceutical composition used in the treatment of skin cancer or its precancerous lesions, and a method for screening candidate compounds for therapeutic agents for skin cancer or its precancerous lesions.

Skin cancers are mainly classified into squamous cell carcinoma (squamous cell carcinoma of the skin), basal cell carcinoma, and malignant melanoma, depending on the cells from which the malignant cells originate. Melanoma is particularly highly malignant among skin tumors, and has a low 5-year survival rate. One of the reasons for the high malignancy of melanoma is that it metastasizes to other organs at a high rate. Melanomas are classified into acral lentigo, superficial spreading, nodular, and lentigo maligna, depending on the type of disease. Acral lentigo accounts for approximately 40% of all cases in Japan. Meanwhile, in the United States, approximately 60% of all cases are superficial spreading. Genetic abnormalities such as BRAF are involved in carcinogenesis, and BRAF gene mutations are found in less than 30% of Japanese patients, but 40-60% of Caucasian patients.

The basic treatment for melanoma is surgical therapy to remove the affected area, but if metastasis to other organs is observed, chemotherapy or radiation therapy is required. Melanoma has often shown resistance to conventional anticancer drugs, but in 2014, immune checkpoint inhibitors such as nivolumab were launched in addition to chemotherapy, and new molecular targeted drugs such as dabrafenib and trametinib were then put into practical use. However, immune checkpoint inhibitors can cause autoimmune diseases collectively known as immune-related adverse effects (irAEs). In addition, the molecular targeted drugs that have been put into practical use are only applicable to cases with BRAF gene mutations, so they cannot be used to treat melanomas in which the BRAF gene is not mutated. Therefore, there is a need to develop new therapeutic drugs that are effective through a different mechanism from these drugs and have fewer side effects.

Cholecystokinin (CCK) is a peptide hormone, and its specific receptors are of two types: CCKA receptor and CCKB receptor. CCK receptors are mainly present on the cell surface of the digestive tract and central nervous system, and CCK has the function of regulating the secretion of gastric juice, bile, and pancreatic juice. For this reason, CCK receptor antagonists can be the active ingredient of drugs for treating gastrointestinal disorders (Patent Document 1). In fact, oral preparations 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 ulcers. Animal experiments have demonstrated that proglumide exerts CCK receptor antagonistic effects not only when administered orally, but also when administered intravenously and intraperitoneally (Non-Patent Document 1).

CCK has a trophic effect on pancreatic acini, and the peptide hormone gastrin, which is a ligand for the CCKB receptor, has a trophic effect on the digestive epithelium. By utilizing these actions, proglumide exerts an antitumor effect on these digestive system tumors. In fact, a study using a mouse model has reported that the growth of pancreatic and colon cancers is suppressed by systemic administration of proglumide (Patent Document 2).

Special Publication No. 2009-539929 Special Publication No. 6-67832

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

The present invention aims to provide a novel pharmaceutical composition for use in the treatment of skin cancers, including melanoma, or precancerous lesions thereof.

In order to develop a new drug with a mechanism different from that of conventional drugs, the inventors searched for factors involved in the proliferation and survival of melanoma cells. As a result, they discovered that CCK receptor antagonists have the effect of inhibiting proliferation and inducing apoptosis in melanoma cells, and thus completed the present invention.

That is, the present invention provides the following.
[1] A drug comprising a cholecystokinin receptor antagonist as an active ingredient,
the cholecystokinin receptor antagonist is one or more selected from the group consisting of lorglumide, proglumide, loxiglumide, dexyloxiglumide, and KSG-504;
A pharmaceutical composition for use in treating skin cancer or its precancerous lesions.
[ 2 ] The pharmaceutical composition according to [1], which is used for the treatment of melanoma, squamous cell carcinoma, actinic keratosis, or basal cell carcinoma.
[ 3 ] A method for screening a candidate compound for a therapeutic agent for skin cancer or a precancerous lesion thereof, comprising:
A screening method comprising carrying out a binding test for a cholecystokinin A receptor on a low molecular weight compound library, a nucleic acid library, or a peptide library, and selecting as the candidate compound a substance having a dissociation constant (KD) with the cholecystokinin A receptor smaller than that of cholecystokinin.

The pharmaceutical composition according to the present invention contains a cholecystokinin receptor antagonist as an active ingredient, and is a new therapeutic agent with a mechanism of action different from that of conventional therapeutic agents for skin cancer such as melanoma or its precancerous lesions. Therefore, the pharmaceutical composition according to the present invention is also expected to be used as a therapeutic agent for melanoma, etc., resistant to conventional therapeutic agents.
Furthermore, by the screening method according to the present invention, candidate compounds for the active ingredient of the pharmaceutical composition can be screened efficiently.

1 shows a stained image obtained by immunostaining a tissue section from a lesion taken from a melanoma patient in Reference Example 1 using an anti-CCKA receptor antibody. FIG. 1 shows the results of staining mouse melanoma-derived B16 cells and human melanoma-derived A375 cells with an anti-CCKA receptor antibody or an anti-CCKB receptor antibody and a fluorescently labeled secondary antibody in Reference Example 1, and then analyzing the cells using a flow cytometer. In Reference Example 1, RT-PCR was performed on RNA extracted from mouse melanoma-derived B16 cells and human melanoma-derived A375 cells, and this is an electrophoretic image of the PCR amplified product of CCK mRNA. FIG. 1 shows the results of analysis using a flow cytometer after staining human squamous cell carcinoma-derived HSC-1 cells and A431 cells with an anti-CCKA receptor antibody or an anti-CCKB receptor antibody and a fluorescently labeled secondary antibody in Reference Example 1. FIG. 1 shows the results of measuring absorbance at a wavelength of 450 nm when assaying B16 cells (A) and A375 cells (B) treated with lorglumide using WST-8 reagent in Example 1. FIG. 1 shows the results of measuring absorbance at a wavelength of 450 nm when B16 cells treated with lorglumide, proglumide, or loxiglumide were assayed using WST-8 reagent in Example 1. FIG. 2 shows the results of measuring the mean fluorescence intensity of CFSE in B16 cells treated with lorglumide in Example 1. FIG. 1 shows the results of measuring the percentage (%) of the total number of early apoptotic cells and late apoptotic cells relative to the total number of cells in B16 cells (A) and A375 cells (B) treated with lorglumide and cultured for 48 hours in Example 2. FIG. 13 shows the results of measuring the relative value (%) of caspase 3/7 activity in B16 cells treated with lorglumide and cultured for 48 hours in Example 3. This figure shows the results of measuring the percentage (%) of the total number of early apoptotic cells and late apoptotic cells relative to the total number of cells in B16 cells that were pretreated with Z-DEVD-FMK, a caspase 3-specific inhibitor, and then treated with lorglumide and cultured for 48 hours in Example 3. In Example 4, a wound was made on a monolayer of B16 cells treated with lorglumide after cell proliferation was stopped, and then the area including the wound was observed over time under a microscope, showing transmitted light images obtained by this microscope. This figure shows the results of measuring the relative intercellular space area (%) of a wound in a monolayer of B16 cells treated with lorglumide after stopping cell proliferation in Example 4, and then culturing the wound for 24 hours. 13 is a graph showing the results of administering lorglumide intratumorally over time to mice subcutaneously implanted with B16 cells in Example 5, and measuring the change in tumor volume (mm ³ ) over time. FIG. 13 shows the results of measuring the tumor weight (mg) 14 days after the start of lorglumide administration when lorglumide was administered intratumorally over time to mice subcutaneously implanted with B16 cells in Example 5. FIG. 13 shows the results of administering lorglumide topically (applying to the skin) over time to mice subcutaneously implanted with B16 cells in Example 5, and measuring the change in tumor volume (mm ³ ) over time. FIG. 13 shows the results of administering lorglumide topically over time to mice subcutaneously implanted with B16 cells in Example 5, and measuring the tumor weight (mg) on day 14 after the start of lorglumide administration.

<Pharmaceutical Composition>
The pharmaceutical composition according to the present invention contains a CCK receptor antagonist as an active ingredient and is used for treating skin cancer or its precancerous lesion. 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, the precancerous lesion of skin cancer for which the pharmaceutical composition according to the present invention is used for treatment may be actinic keratosis, etc. The pharmaceutical composition according to the present invention is preferably used for treating melanoma, spinous cell carcinoma, actinic keratosis, or basal cell carcinoma, and is particularly preferably used for treating melanoma. Hereinafter, "skin cancer or its precancerous lesion" may be referred to as "skin cancer, etc."

As shown in the Examples below, CCK receptor antagonists have antiproliferative effects, apoptosis-inducing effects, and chemotaxis (migration)-inhibiting effects on skin cancer cells, including melanoma cells, or precancerous lesion cells. Unlike conventional therapeutic agents for melanoma, etc., CCK receptor antagonists exert an antitumor effect based on a novel mechanism of action that targets the CCK receptor. Therefore, the pharmaceutical composition of the present invention exerts an antitumor effect on skin cancer cells, including melanoma cells, regardless of the presence or absence of a BRAF gene mutation.

The CCK receptor antagonist used as an 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 or a substance having a non-competitive antagonistic effect. The substance may be a low molecular weight compound, a nucleic acid aptamer, a peptide aptamer, or a protein such as an antibody.

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

The CCK receptor antagonist used as an active ingredient in the present invention can be appropriately selected from known substances having CCK receptor antagonistic activity. Among the CCK receptor antagonists, examples of low molecular weight compounds include proglumide, lorglumide (CAS No.: 97964-56-2), loxiglumide (CAS No.: 107097-80-3), dexyloxiglumide (119817-90-2), spiroglumide (CAS No.: 137795-35-8), itriglumide (CAS No.: 201605-51-8), KSG-504 (CAS No.: 137005-17-5), YM022 (CAS No.: 145084-28-2), and derivatives thereof. Among these, proglumide is preferred because it has been widely used as an orally administered drug to treat gastritis and gastric ulcers and can be safely ingested.

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

The active ingredient of the pharmaceutical composition of the present invention may be a solvate of a free CCK receptor antagonist or a solvate of a pharmacologically acceptable salt of a CCK receptor antagonist. Examples of the solvent that forms the solvate include water and ethanol.

The amount of CCK receptor antagonist contained in the pharmaceutical composition of the present invention is not particularly limited as long as it is a therapeutically effective amount for skin cancer, etc., i.e., an amount sufficient to obtain a therapeutic effect for skin cancer, etc., and is appropriately determined taking into consideration the type of skin cancer, route of administration, form of administration (dosage form), number of doses per day, administration interval, biological species, sex, age, weight, etc. of the subject to be administered.

When the pharmaceutical composition of the present invention contains as an active ingredient a drug, such as proglumide, that is currently or has been approved as a pharmaceutical in the past, the administration route, dosage form, amount of active ingredient per administration, administration interval, and other dosage methods may be similar to those of drugs for other diseases for which the drug has 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 can be appropriately selected from various administration routes such as oral administration, intravenous administration, transdermal administration, enteral administration, and nasal administration. Taking into consideration the intended administration route, the pharmaceutical composition according to the present invention can be formulated by a conventional method into oral solid preparations such as powders, granules, capsules, tablets, chewable preparations, and sustained-release preparations; oral liquid preparations such as solutions and syrups; external preparations such as patches, ointments, gels, creams, sprays, lotions, and liniments; injections, suppositories, and the like.

Other components contained in the pharmaceutical composition of the present invention include, for example, pharmacologically acceptable additives such as excipients, binders, lubricants, disintegrants, flow agents, solvents, solubilizers, buffers, suspending agents, emulsifiers, isotonicity agents, stabilizers, preservatives, antioxidants, flavoring agents, and coloring agents. In addition, the pharmaceutical composition may contain other active ingredients other than the CCK receptor antagonist.

Examples of excipients 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 binders include pregelatinized starch, gelatin, gum arabic, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, crystalline cellulose, D-mannitol, trehalose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, and polyvinyl alcohol. Examples of lubricants include stearic acid, calcium stearate, talc, sucrose fatty acid esters, and polyethylene glycol. Examples of disintegrants include crospovidone (crosslinked polyvinylpyrrolidone), low-substituted hydroxypropylcellulose, starch, alginic acid, and sodium alginate. Examples of flow agents include silicic acid, anhydrous silicic acid, aluminum silicate, calcium silicate, magnesium aluminometasilicate compound, aluminum oxide, aluminum hydroxide, magnesium oxide, and magnesium hydroxide. 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 derivatives, etc. Examples of the buffer include sodium citrate hydrate, sodium acetate hydrate, sodium hydrogen carbonate, trometamol, boric acid, borax, sodium hydrogen phosphate hydrate, sodium dihydrogen phosphate, etc. Examples of the suspending agent or emulsifying agent include sodium lauryl sulfate, gum arabic, gelatin, lecithin, glycerin monostearate, polyvinyl alcohol, polyvinylpyrrolidone, celluloses such as sodium carboxymethylcellulose, polysorbates, polyoxyethylene hydrogenated castor oil, etc. Examples of the isotonicity agent include sugars such as lactose, glucose, D-mannitol, sodium chloride, potassium chloride, glycerin, propylene glycol, polyethylene glycol, urea, etc. Stabilizers include polyethylene glycol, sodium dextran sulfate, sodium sulfite, etc. Preservatives include paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, chlorocresol, dehydroacetic acid, sorbic acid, etc. Antioxidants include sulfites, ascorbic acid, etc. Flavoring agents include sweeteners and fragrances commonly used in the pharmaceutical and food fields. Coloring agents include coloring agents commonly used in the pharmaceutical and food fields.

The pharmaceutical composition of the present invention is administered to animals that have developed or are suspected of developing skin cancer or the like. The pharmaceutical composition is preferably administered to mammals, more preferably to humans, or livestock or laboratory animals such as mice, rats, rabbits, guinea pigs, hamsters, monkeys, sheep, horses, cows, pigs, donkeys, dogs, and cats, and even more preferably to humans.

<Method of screening candidate compounds for therapeutic agents for skin cancer, etc.>
CCK receptors are also expressed on the cell surface of skin cancer cells, including melanoma cells, and precancerous lesion cells, and downstream signaling of the receptors is involved in cell proliferation, apoptosis, chemotaxis, etc. Therefore, CCK receptor antagonists have proliferation inhibitory effects, apoptosis-inducing effects, and chemotaxis inhibitory effects on skin cancer cells, etc., and exert antitumor effects. Thus, substances having an antagonistic effect against CCK receptors can be selected as candidate compounds for therapeutic agents for skin cancer, etc.

CCK receptor antagonists can be selected, for example, by screening various low molecular weight compound libraries, nucleic acid libraries, peptide libraries, etc. using binding tests to the CCK receptor. Among the selected substances, those having a higher binding affinity to the CCK receptor than CCK, i.e., a smaller dissociation constant (K _D ), can more effectively competitively inhibit downstream cell signaling of the CCK receptor in skin cancer cells, including melanoma cells, and are promising candidate compounds for therapeutic agents for skin cancer, etc.

CCK receptor antagonists can also be screened using, for example, cells that express CCK receptors and secrete specific substances in response to CCK stimulation. For example, cells such as gastric body parietal cells can be used that express CCK receptors and secrete gastric acid in response to CCK stimulation. If the amount of gastric acid secreted when the cells are cultured in a culture medium containing CCK and the candidate compound is clearly less than the amount of gastric acid secreted when the cells are cultured in a culture medium containing only CCK, the candidate compound is selected as a CCK receptor antagonist.

The present invention will now 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 receptors in melanoma cells was examined.

(1) Expression of CCKA receptor in melanoma lesion tissues The expression of CCKA receptor in lesion tissues of melanoma patients collected for treatment purposes was examined by immunohistological techniques. Specifically, tissue sections of melanoma lesions collected from three patients with superficial spreading melanoma and three patients with acral lentigo melanoma were immunostained using rabbit anti-CCKA receptor antibody (Invitrogen). The stained images are shown in Figure 1. In the figure, the areas indicated by the arrows are areas stained with the anti-CCKA receptor antibody. As shown in Figure 1, expression of CCKA receptor was observed in superficial spreading and acral lentigo melanoma lesions.

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

RNA was extracted from the cells and the expression of CCK was examined by RT-PCR. As a control, the expression of β-actin was also examined. Figure 3 shows the agarose gel electrophoresis image of the amplified products obtained as a result of RT-PCR. As shown in Figure 3, the RT-PCR amplified product of CCK mRNA was confirmed in both B16 and A375 cells, confirming the expression of CCK in both cells. These results suggest that melanoma cells are regulated by CCK through an autocrine/paracrine mechanism.

(3) Expression of CCKA receptor and CCKB receptor in cultured cells derived from non-melanoma skin cancer CCK receptor expression in HSC-1 cells and A431 cells derived from human squamous cell carcinoma was examined in the same manner by flow cytometry. The analysis results are shown in Figure 4. As shown in Figure 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 examined. As CCK receptor antagonists, lorglumide, a selective CCK receptor antagonist, and proglumide and loxiglumide, non-selective CCK receptor antagonists, were used. The proliferation ability of cells was measured by WST assay using WST-8 reagent or assay using CFSE (Carboxyfluorescein Succinimidyl Ester).

(1) Effect of Lorglumide on the Growth of B16 and A375 Cells B16 and A375 cells were seeded on a 96-well plate and cultured overnight, after which Lorglumide was added and cultured for 48 hours. Then, 10 μL of WST-8 reagent (Dojindo) was added to each well and cultured for an additional 3 hours, after which the absorbance at a wavelength of 450 nm was measured using a plate reader. WST-8 is reduced by intracellular dehydrogenase to produce formazan, which is yellow in color. Therefore, in the WST assay, the absorbance and the number of viable cells are proportional to each other.

The results of measuring the absorbance at a wavelength of 450 nm for each well are shown in Figure 5 (**: p<0.01 vs. untreated control group). As shown in the figure, lorglumide reduced the number of viable cells in a concentration-dependent manner in both B16 and A375 cells.

(2) Effects of Lorgulumide, Proglumide, and Loxiglumide on the Proliferation of B16 Cells B16 cells were seeded onto a 96-well plate and cultured overnight, after which lorgulumide, proglumide, or loxiglumide was added and cultured for 48 hours, after which an assay was performed using WST-8 reagent in the same manner as in (1) above.

The results of measuring the absorbance at a wavelength of 450 nm for each well are shown in Figure 6 (*: p<0.01 vs. untreated control group, **: p<0.01). As shown in the figure, proglumide and loxiglumide, like lorglumide, reduced the number of live cells in a concentration-dependent manner, but lorglumide was the most effective in reducing the number of cells.

(3) Cell proliferation assay Lorglumide was added to the labeled B16 cells and the cells were cultured for 48 hours. The CFSE intensity (fluorescence intensity) of the cells after the culture was then measured using a flow cytometer. The CFSE intensity was halved with each cell division. CFSE labeling was performed using the CFSE Cell Division Assay Kit (Cayman).

The results of measuring the average CFSE fluorescence intensity are shown in Figure 7. In the lorglumide-added group, the average CFSE fluorescence intensity of the cells was significantly higher than in the untreated control group, meaning that the number of cell divisions was lower (**: p<0.01 vs. untreated control group). These results demonstrate that lorglumide inhibits the proliferation of melanoma cells.

[Example 2]
The apoptosis-inducing effect of CCK receptor antagonists on melanoma cells was examined. Lorglumide was used as the CCK receptor antagonist. The apoptosis-inducing effect was examined using an apoptosis detection assay using fluorescently labeled Annexin V. Annexin V binds to phosphatidylserine, a lipid constituting the cell membrane. Phosphatidylserine is present on the inner side of the cell membrane in living cells, and is exposed to the cell membrane surface when apoptosis is induced. Therefore, cells in which apoptosis has been induced emit fluorescence due to fluorescently labeled Annexin V.

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

Figure 8 shows the percentage (%) of the total number of early and late apoptotic cells relative to the total number of cells, based on the results of flow cytometer analysis. As shown in the figure, the percentage of early and late apoptotic cells significantly increased in B16 and A375 cells cultured with lorglumide (**: p<0.01 vs. untreated control group). These results demonstrate that lorglumide induces apoptosis in melanoma cells.

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

B16 cells were seeded in a 96-well plate, lorglumide (200 μM) was added, and the cells were cultured for 48 hours. Following the manual provided by the reagent supplier, a substrate solution was added to the cultured cells and the cells were cultured for an additional 2 hours, after which the relative luminescence (%) was measured as caspase 3/7 activity using a plate reader. The measurement results are shown in Figure 9. As shown in the figure, in the group cultured with lorglumide, caspase 3/7 activity was increased to about 1.7 times that of the untreated control group (**: p<0.01 vs. untreated control group).

Therefore, B16 cells were pretreated for 2 hours with Z-DEVD-FMK, a specific inhibitor of caspase 3, and then lorglumide was added and cultured for 48 hours, and apoptosis-positive cells were analyzed using a flow cytometer. The analysis results are shown in Figure 10. As shown in the figure, apoptosis was significantly suppressed by pretreatment with Z-DEVD-FMK. These results suggest that caspase 3 is involved in the signal transduction of apoptosis induced by lorglumide.

[Example 4]
The inhibitory effect of CCK receptor antagonists on the chemotaxis of melanoma cells was examined. Lorglumide was used as the CCK receptor antagonist.

To stop cell proliferation, B16 cells were pretreated with mitomycin C for 1.5 hours, then seeded in a monolayer on a 96-well plate and cultured overnight. After the culture, the B16 melanoma monolayer was wounded with a yellow tip, and lorglumide was added and cultured for 24 hours. The area including the wound was observed under a microscope over time. The observation results are shown in Figure 11. As shown in the figure, in the control group not treated with lorglumide, cells migrated to the wounded area and the area of the gap decreased over time. On the other hand, in the group to which lorglumide was added, cell migration was inhibited and the area of the gap hardly decreased. The area of the gap caused by the wound after 24 hours of culture after the wound was calculated using image analysis software Image J. Figure 12 shows the results of measuring the relative area of intercellular spaces after 24 hours of culture after wounding (%: [area of intercellular spaces 24 hours after wounding]/[area of intercellular spaces at the time of wounding] x 100). As shown in the figure, the higher the concentration of lorglumide, the larger the relative area of intercellular spaces after culture and the lower the migration ability of B16 cells. These results demonstrate that CCK receptor antagonists inhibit the migration of melanoma cells. Since cell migration is deeply involved in local tissue invasion and distant metastasis, it has been suggested that CCK receptor antagonists not only exert an antitumor effect by inducing apoptosis, but also inhibit tumor growth and metastasis by inhibiting cell migration.

[Example 5]
The antitumor effect of lorglumide was examined in a mouse model with subcutaneously implanted melanoma. The mouse model with subcutaneously implanted melanoma was prepared by implanting B16 cells (10 ⁵ cells) subcutaneously into the abdominal cavity of a C57BL/6 mouse.

(1) Effect of intratumoral administration of lorglumide When the volume of the tumor formed by subcutaneously implanted B16 cells reached 10-30 ^mm3 , lorglumide (100 μg, 4 mg/kg) was administered intratumorally. As a control, the same amount of saline was administered intratumorally instead of lorglumide. Thereafter, the same administration of lorglumide was repeated every 3-4 days. The results of measuring the tumor volume ([tumor volume ( ^mm3 )] = [longer diameter (mm)] × [minor diameter (mm)] ² × 1/2) of each mouse over time are shown in Figure 13. In addition, on the 14th day after the start of lorglumide administration, the tumor mass was harvested and the tumor weight (mg) was measured. The results of measuring the tumor weight are shown in Figure 14.

As shown in Figure 13, the increase in tumor volume was significantly suppressed in the lorglumide-treated group compared to the saline-treated group (**: p<0.01 vs. saline-treated group). In addition, as shown in Figure 14, administration of lorglumide also significantly reduced tumor weight (*: p<0.05 vs. saline-treated group).

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

The results of measuring the tumor volume ( ^mm3 ) of each mouse over time are shown in Figure 15, and the results of measuring the tumor weight (mg) 14 days after the start of lorglumide administration are shown in Figure 16. As shown in Figure 15, the increase in tumor volume was significantly suppressed by topical application of lorglumide. Also, as shown in Figure 16, the tumor weight 14 days after the start of topical application of lorglumide was significantly lower in the topical lorglumide group compared to the control group. These results demonstrated that tumor growth can be suppressed in mouse tumor models by intratumoral or topical administration of a CCK receptor antagonist.

Claims (3)

The active ingredient is a cholecystokinin receptor antagonist.
the cholecystokinin receptor antagonist is one or more selected from the group consisting of lorglumide, proglumide, loxiglumide, dexyloxiglumide, and KSG-504;
A pharmaceutical composition for use in treating skin cancer or its precancerous lesions. The pharmaceutical composition according to claim 1 , which is used for the treatment of melanoma, squamous cell carcinoma, actinic keratosis, or basal cell carcinoma. A method for screening a candidate compound for a therapeutic agent for skin cancer or a precancerous lesion thereof, comprising:
A screening method comprising carrying out a binding test for a cholecystokinin A receptor on a low molecular weight compound library, a nucleic acid library, or a peptide library, and selecting as the candidate compound a substance having a dissociation constant (KD) with the cholecystokinin A receptor smaller than that of cholecystokinin.

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