Pharmaceutical composition for preventing or treating melanoma, comprising cimetidine as active ingredient

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating melanoma, comprising cimetidine as an active ingredient, and, more specifically, to a pharmaceutical composition for preventing or treating melanoma, comprising cimetidine and at least one antibody selected from the group consisting of an anti-CTLA-4 antibody and an anti-PD-1 antibody as active ingredients. The present invention can provide a pharmaceutical composition for treating melanoma in which a melanoma treatment effect is increased using cimetidine and an antibody in a combination therapy, and optimized antibody capable of maximizing the melanoma treatment effect during the combination therapy has been identified. In addition, the pharmaceutical composition of the present invention can be specifically applied to melanoma cells to maximize a melanoma treatment effect while minimizing immune-related side effects.

Description

Pharmaceutical composition for the prevention or treatment of melanoma containing cimetidine as an active ingredient

[0001] The present invention relates to a pharmaceutical composition for the prevention or treatment of melanoma comprising cimetidine as an active ingredient, and more specifically, to a pharmaceutical composition for the prevention or treatment of melanoma comprising cimetidine; and one or more antibodies selected from the group consisting of anti-CTLA-4 antibodies and anti-PD-1 antibodies as active ingredients.

[0002] Melanoma is a skin cancer originating from melanocytes. It typically begins as a small growth of pigment in normal skin and usually starts in areas exposed to ultraviolet radiation. About one in three cases of melanoma develops from a pre-existing mole, and it can also occur around and inside the eyes, inside the mouth, above the genitals, in the rectum, in the brain, and under the fingernails.

[0003] Recently, the incidence of melanoma has been rapidly increasing worldwide. While early-stage melanoma is generally treated through surgical excision, melanoma that has progressed beyond the initial stage often requires intensive treatment, including immunotherapy or targeted therapy.

[0004] Immune checkpoint inhibitors (ICIs) are highly effective in treating various types of cancer. Despite their excellent therapeutic efficacy, ICIs present a problem: they exhibit low response rates, failing to produce therapeutic effects in a large number of patients due to resistance issues. Consequently, research on new ICIs has been actively underway recently; more specifically, there is a growing focus on developing combination therapies that combine ICIs with additional treatments such as targeted therapy, chemotherapy, and radiation therapy.

[0005] However, immunotherapy using immune checkpoint inhibitors is accompanied by immune-related adverse events (irAEs), which increase the immune response against normal tissues as well as cancer cells. Furthermore, the concomitant use of immune checkpoint inhibitors exacerbates the frequency and severity of irAEs, leading to situations where treatment must be discontinued. Cutaneous irAEs are the most frequently observed and tend to appear early in treatment with immune checkpoint inhibitors. Symptoms of these cutaneous irAEs include rashes, psoriatic rashes, itching, and vitiligo.

[0006] Therefore, there is a growing need to minimize these cutaneous irAEs during chemotherapy for melanoma, and there is an increasing need for treatment methods that can selectively produce anticancer effects only on tumor cells without affecting normal skin cells.

[0007] Meanwhile, cimetidine is a histamine H2 receptor antagonist that inhibits gastric acid secretion in the stomach and is primarily used to treat gastrointestinal problems such as ulcers and gastroesophageal reflux disease. To date, effects of cimetidine such as T cell stimulation, modulation of immune responses, and antitumor activity have been reported. Most of these effects are auxiliary results achieved by modulating immune checkpoints, but the role of cimetidine in controlling immune checkpoints has not yet been reported.

[0008] The object of the present invention is to provide a pharmaceutical composition for treating melanoma that enhances the therapeutic effect of melanoma by using cimetidine and an antibody in combination therapy.

[0009] Another objective of the present invention is to provide a pharmaceutical composition for treating melanoma that is specifically applied to melanoma cells to maximize the therapeutic effect on melanoma while minimizing immune-related side effects.

[0010] The technical problems that the invention aims to solve are not limited to those mentioned above, and other unmentioned technical problems can be clearly understood by those skilled in the art from the description of the invention.

[0011] The present invention provides a pharmaceutical composition for the prevention or treatment of melanoma comprising cimetidine as an active ingredient.

[0012] In the present invention, the pharmaceutical composition is characterized by further comprising one or more antibodies selected from the group consisting of anti-CTLA-4 antibodies and anti-PD-1 antibodies.

[0013] In the present invention, cimetidine; and one or more antibodies selected from the group consisting of anti-CTLA-4 antibodies and anti-PD-1 antibodies are each formulated and administered simultaneously or sequentially.

[0014] The present invention can provide a pharmaceutical composition for treating melanoma that enhances the therapeutic effect of melanoma by using cimetidine and an antibody in combination therapy.

[0015] In addition, the present invention can provide a pharmaceutical composition for treating melanoma that is specifically applied to melanoma cells to maximize the therapeutic effect on melanoma while minimizing immune-related side effects.

[0016] The effects of the present invention are not limited to those mentioned above, and other unmentioned effects will be clearly understood by those skilled in the art from the description in the claims.

[0017] Figure 1 shows the results of analyzing the melanoma inhibitory effect in mice when cimetidine and anti-CTLA-4 antibody were treated alone or in combination.

[0018] Figure 2 shows the results of analyzing the cytotoxic cytokine production effect when cimetidine and anti-CTLA-4 antibody were treated alone or in combination.

[0019] Figure 3 shows the results of analyzing the melanoma inhibition effect in mice when cimetidine and anti-PD-1 antibodies were treated alone or in combination.

[0020] Figure 4 shows the results of analyzing the cytotoxic cytokine production effect when cimetidine and anti-PD-1 antibody were treated alone or in combination.

[0021] Figure 5 shows the results of analyzing the melanoma inhibition effect in mice when cimetidine and anti-PD-L1 antibody were treated alone or in combination.

[0022] Figure 6 shows the change in PD-L1 expression in melanoma cells by cimetidine treatment.

[0023] Figure 7 shows the change in PD-L1 expression in kidney cancer cells due to cimetidine treatment.

[0024] Figure 8 shows the change in PD-L1 expression in melanocytes and keratinocytes due to cimetidine treatment.

[0025] Figure 9 shows the results of analyzing protein expression of major signaling pathways by cimetidine.

[0026] The terms used in this specification have been selected based on currently widely used general terms whenever possible, taking into account their functions in the present invention; however, these terms may vary depending on the intent of those skilled in the art, case law, the emergence of new technologies, etc. Additionally, in specific cases, terms have been arbitrarily selected by the applicant, and in such cases, their meanings will be described in detail in the relevant description of the invention. Therefore, the terms used in this invention should be defined not merely by their names, but based on their meanings and the overall content of the invention.

[0027] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by those skilled in the art to which the present invention pertains. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and should not be interpreted in an ideal or overly formal sense unless explicitly defined in this application.

[0028] Numerical ranges include the values ​​defined in the above ranges. All maximum numerical limits given throughout this specification include all lower numerical limits as clearly written. All minimum numerical limits given throughout this specification include all higher numerical limits as clearly written. All numerical limits given throughout this specification will include all better numerical ranges within a wider numerical range, as clearly written.

[0029]

[0030] The present invention will be described in detail below.

[0031]

[0032] The present invention may provide a pharmaceutical composition for the prevention or treatment of melanoma comprising cimetidine as an active ingredient.

[0033] The term Cimetidine used in the present invention is a drug that inhibits the secretion of gastric acid in the stomach and may be a histamine H2 receptor antagonist used mainly to treat gastrointestinal problems such as ulcers and gastroesophageal reflux disease, and may be denoted as “CIM”.

[0034] The above pharmaceutical composition may be characterized by further including one or more antibodies selected from the group consisting of anti-CTLA-4 antibodies and anti-PD-1 antibodies, and preferably may be characterized by further including an anti-CTLA-4 antibody or an anti-PD-1 antibody.

[0035] The above pharmaceutical composition may be characterized by cimetidine; and one or more antibodies selected from the group consisting of anti-CTLA-4 antibodies and anti-PD-1 antibodies, each formulated and administered simultaneously or sequentially.

[0036] According to one embodiment of the present invention, the pharmaceutical composition may be characterized in that cimetidine and an anti-CTLA-4 antibody are each formulated and administered simultaneously or sequentially.

[0037] According to one embodiment of the present invention, the pharmaceutical composition may be characterized in that cimetidine and an anti-PD-1 antibody are each formulated and administered simultaneously or sequentially.

[0038] There are no restrictions on the method of administering the above cimetidine, anti-CTLA-4 antibody, or anti-PD-1 antibody to an individual, but preferably, cimetidine may be administered orally, and the anti-CTLA-4 antibody or anti-PD-1 antibody may be administered by intraperitoneal injection.

[0039] According to one embodiment of the present invention, the pharmaceutical composition is characterized by treating melanoma by increasing the production of cytotoxic cytokines in CD8+ T cells compared to a control group that was not administered the composition.

[0040] According to one embodiment of the present invention, the pharmaceutical composition can inhibit melanoma by specifically reducing PD-L1 expression in melanoma cells.

[0041]

[0042] Hereinafter, the experimental techniques used in the embodiments and experimental examples of the present invention will be described in detail.

[0043]

[0044] Patient skin biopsy

[0045] Skin biopsies were performed with informed consent from melanoma patients at Pusan ​​National University Yangsan Hospital. The Institutional Review Board of Pusan ​​National University Yangsan Hospital approved this study (IRB No. 05-2022-242). This study was conducted in accordance with the International Guidelines for Biomedical Research in Human Subjects (CIOMS).

[0046]

[0047] Mouse model preparation

[0048] Wild-type C57BL / 6(B6) mice were purchased from Koatech, South Korea. Mice were used between 6 and 12 weeks of age, and all animals were housed in a pathogen-free animal facility at Pusan ​​National University Yangsan Hospital. All animal experiments and protocols were approved by the Institutional Animal Care and Use Committee of Pusan ​​National University Yangsan Hospital (Approval No. 2022-003-A1C3).

[0049]

[0050] cell culture

[0051] B16F10 mouse melanoma cells, A375 and SK-MEL-28 human melanoma cells, and AHCN human renal carcinoma cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Biowest), and H358 human non-small cell lung cancer cells and Renca mouse renal carcinoma cells were cultured in RPMI-1640 (Wellgene) supplemented with 10% (v / v) FBS (Biowest) and 1% penicillin / streptomycin (P / S) (Gibco) in a 37°C incubator with high humidity containing 5% CO2. NHEM normal human epidermal melanocytes were cultured in melanocyte growth medium (PromoCell) according to the manufacturer's instructions. In some experiments, cells were seeded into 24-well plates and treated with 1 mM histamine (Sigma-Aldrich) and / or various concentrations of CIM (Sigma-Aldrich). Dimethyl sulfoxide (DMSO) was used as a medium for treatment. B16F10 cells were treated with 1 mM histamine and 5 mM CIM, followed by treatment with 10 µM forskolin (Fsk) (MCE) or 500 µM SQ22536 (MCE). After 16 hours of treatment, cells were washed with DPBS and trypsin was treated with TrypLE Express Enzyme (Gibco). Cells were harvested into cell culture medium and washed twice with DPBS. The cell suspension was then used for further experiments.

[0052]

[0053] Patient-derived melanoma cell culture

[0054] Tumor tissue was surgically excised from a melanoma patient and immediately placed in cold PBS. The excised tissue was cut into small pieces with scissors and transferred to tissue digestion buffer (1 mg / ml collagenase IV and 0.1 mg / ml DNase I in DMEM). The tissue suspension was incubated at 37°C for 30 minutes with gentle rotation. After incubation, the cell suspension was filtered through a 70 μm cell filter and washed twice with DMEM supplemented with 10% FBS and 1% P / S. Subsequently, the cells were cultured in a high-humidity 37°C incubator containing 5% CO2.

[0055]

[0056] Human and mouse keratinocyte cultures

[0057] To isolate normal human primary keratinocytes, human skin tissue was washed twice with DPBS containing 2% P / S, and then the tissue was cut into small pieces using scissors. The tissue pieces, containing both the epidermis and dermis, were carefully placed on the surface of a 100 mm cell culture dish. DMEM supplemented with 10% FBS and 1% P / S was added to the dish, and the cells were incubated at 37°C and 5% CO2 until a cell density of 70% was reached. To remove fibroblasts, the cells were rinsed twice with DPBS and then incubated for 3 minutes using TryPLE Enzyme Express. The isolated fibroblasts were washed away, and the remaining keratinocytes were obtained by further incubation with TryPLE Enzyme Express. The human primary keratinocytes were then stored in KBM-Gold™ Basal Medium (Lonza) supplemented with KBM Gold SingleQuots (Lonza) for further experiments. To isolate mouse primary keratinocytes, dorsal skin from WT mice was collected. Subcutaneous fat and connective tissue were removed, and the skin was placed in cold PBS. Then, 1 cm of skin was applied. 2The epidermis and dermis were separated by cutting into pieces and incubating in 5 mg / ml Dispase II solution (Sigma-Aldrich) at 37°C for 90 minutes. The epidermis was exfoliated and finely chopped, and keratinocytes were isolated by incubating in TryPLE Express enzyme at 37°C for 5–10 minutes. Trypsin was neutralized with Trypsin Neutralizing Solution (Lonza), and the cell suspension was filtered through a 70 µm cell filter followed by further filtration through a 40 µm cell filter. After centrifugation, the cells were suspended in KBM-Gold™ Basal Medium supplemented with KBM Gold SingleQuots. Cells were counted using Countess 3 FL (Invitrogen) and plated onto Collagen Type I coated plates (SPL). Keratinocytes were cultured at 37°C in a high-humidity incubator containing 5% CO2.

[0058]

[0059] Tumor inoculation

[0060] 1x10 on B6 mouse 6 WT B16 cells were injected subcutaneously into the right flank of each mouse and randomized to each treatment group. CIM was administered orally at a daily dose of 100 mg / kg five times a week. 250 μg anti-PD-1 antibody, 200 μg anti-PD-L1 antibody, or 200 μg anti-CTLA-4 antibody was injected intraperitoneally twice a week. All of these antibodies were purchased from BioXcell. Tumor size was measured three times a week using a digital caliper starting from day 7 after tumor inoculation. After two weeks, all mice were sacrificed, and tumor weights were measured using a scale.

[0061]

[0062] Flow cytometry analysis

[0063] To analyze tumor cells in the tumor microenvironment, solid tumor tissues isolated from tumor-bearing mice were physically cleaved with scissors. Red blood cells were removed using RBC lysis buffer (Sigma-Aldrich), and then the activation of the buffer was stopped by adding culture medium supplemented with 10% FBS and 1% P / S. The tumors were filtered through a 70 µm cell filter (SPL), washed with culture medium, and resuspended in culture medium. Subsequently, cell viability and cell count were evaluated using Countess 3 FL. To isolate tumor-infiltrating lymphocytes, the cell suspension was centrifuged in layers on a Ficoll gradient (cytiva). After centrifugation, the interfacial layer containing TILs was collected, washed, and resuspended in culture medium. The viability and cell count of the isolated TILs were determined using Countess 3 FL. For surface staining, cells were incubated with fluorescent conjugated antibodies at 4°C for 30 minutes. All staining was performed in the presence of CD16 / CD32 blocking antibodies (Tonbo biosciences). For intracellular staining, after surface staining, cells were fixed and permeated with intracellular fixation buffer (eBioscience), and then stained with fluorescent conjugated antibodies at room temperature for 45 minutes. Dead cells were excluded using DAPI staining or Fixable Viability Dye (BD). Antibodies with the following specificities were used for staining: CD8a (53-6.7), CD31 (MEC13.3), PD-L1 (10F.9G2), IFN-γ (XMG1.2), granzyme B (QA16A02), perforin (S16009A), and homozygous control antibodies, all of which were purchased from BioLegend. TCRβ (H57-597), CD4 (GK.15), CD45 (30-F11), and CD90.2 (30-H12) were all purchased from Tonbo Biosciences.For Melan-A or HRH2 staining, cells were incubated with Melan-A (Abcam) or HRH2 (Invitrogen) antibodies at room temperature for 45 minutes, and then with FITC-conjugated goat anti-mouse IgG antibody (Invitrogen) at room temperature for 30 minutes. Data were collected using BD CANTOII and analyzed using FlowJo software.

[0064]

[0065] Western Blot

[0066] To lyse cells and extract total protein, cells were lysed using RIPA lysis buffer (iNtRON) supplemented with 100X protease and phosphatase inhibitors (GenDEPOT), and total protein was extracted. Protein concentration was measured using a Bradford protein assay kit (Abbkine). Equal amounts of protein were separated by SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) and transferred to a polyvinylidene fluoride (PVDF) membrane (cytiva). The membrane was blocked for 1 hour at room temperature with 5% skim milk (BD) (TBS-T) prepared in TBS buffer (Biosesang) containing 0.1% Tween-20 (Sigma-Aldrich). The membranes were incubated overnight at 4°C with primary antibodies against PD-L1, PKA, p-PKA, CREB, p-CREB, ERK1 / 2, p-ERK1 / 2, AKT, p-AKT (S473 and T308), β-catenin, p-β-catenin (all Cell Signaling), and β-actin (Santa Cruz). After washing three times with TBS-T, the membranes were incubated with HRP-conjugated secondary antibodies at room temperature for 1 hour and then washed. Detection was performed using enhanced chemiluminescence (ECL, Advansta), and protein bands were visualized using FUSION Solo X (Vilber).

[0067]

[0068] Statistical analysis

[0069] Experiments were conducted independently and repeated at least three times. Statistical analysis was performed using GraphPad Prism 8 software (GraphPad Software, USA), and data were presented as mean ± SEM. Statistical differences were analyzed using unpaired or paired two-sided Student t-tests and two-way ANOVA. Statistical significance was indicated as follows: not significant (ns), *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0070] The embodiments of the present invention are described in detail below, but it is obvious that the present invention is not limited by the following embodiments.

[0071]

[0072] Example 1. Treatment of melanoma with combination therapy of cimetidine and anti-CTLA-4 antibody

[0073]

[0074] 1-1. Analysis of Melanoma Inhibitory Effects in Mice Treated with Cimetidine and Anti-CTLA-4 Antibody

[0075] B16F10 cells were subcutaneously inoculated into wild-type C57BL / 6 (B6) mice, followed by treatment with CIM or α-CTLA-4 alone, and treatment with CIM and α-CTLA-4 in combination, respectively. The methods for CIM and α-CTLA-4 antibody treatment were described in detail in the 'tumor inoculation' experimental technique described above, and the process is schematically illustrated in Fig. 1A. Next, mice inoculated with B16F10 cells were treated with monotherapy or combination therapy, and the volume and weight of melanoma tumors were measured to compare the anti-tumor effects. The measured tumor volumes and weights are shown in Figs. 1B and 1C.

[0076] Referring to Figures 1B and 1C, when treated with monotherapy using CIM or α-CTLA-4, the volume and weight of the melanoma decreased, but when treated with a combination of CIM and α-CTLA-4, the volume and weight of the melanoma decreased even more. Therefore, it was confirmed that treating melanoma with a combination of CIM and α-CTLA-4 has a superior therapeutic effect compared to monotherapy.

[0077]

[0078] 1-2. Analysis of PD-L1 expression in mice treated with cimetidine and anti-CTLA-4 antibody

[0079] In the same manner as in Example 1-1 above, B16F10 cells were subcutaneously inoculated into wild-type C57BL / 6(B6) mice, followed by treatment with CIM or α-CTLA-4 alone and treatment with CIM and α-CTLA-4 in combination, respectively. Next, the expression of PD-L1 in the tumor cells of each group was compared, and the results are shown in Figures 1D and 1E.

[0080] Referring to Figures 1D and 1E, unlike mice treated with CIM, α-CTLA-4 did not regulate the expression of PD-L1 in tumor cells.

[0081]

[0082] 1-3. Analysis of the Long-Term Suppressive Effect of Cimetidine and Anti-CTLA-4 Antibody in Mice

[0083] In Example 1-1 above, the melanoma inhibitory effect when cimetidine and anti-CTLA-4 antibody were administered alone or in combination was analyzed for up to 14 days. In Example 1-3 of this study, the melanoma inhibitory effect when cimetidine and anti-CTLA-4 antibody were administered alone or in combination was analyzed for up to 37 days. The experimental method was carried out in the same manner as in Example 1-1 above, and the results are shown in Fig. 1F, and the change in mouse body weight during the entire experimental period is shown in Fig. 1G.

[0084] Referring to Figure 1F, it was confirmed that the growth of melanoma was most effectively inhibited in the group administered with cimetidine and anti-CTLA-4 antibody, and in particular, tumor growth was completely inhibited in 2 out of 5 animals.

[0085] Referring to Figure 1G, no weight loss in mice was observed in any of the treatment groups throughout the entire experimental period.

[0086]

[0087] Example 2. Production of cytotoxic cytokines following combination therapy of cimetidine and anti-CTLA-4 antibody

[0088] In the same manner as in Example 1-1 above, B16F10 cells were subcutaneously inoculated into wild-type C57BL / 6(B6) mice, followed by treatment with CIM or α-CTLA-4 alone, and treatment with CIM and α-CTLA-4 in combination, respectively. Next, the production of cytotoxic cytokines by T cells after treatment in each group was confirmed.

[0089] More specifically, the percentage and number of CD8+ tumor infiltrating lymphocytes (TILs) producing Pfn, Grz B, and IFN-γ are shown in Figures 2A to 2D, and the percentage and number of CD4+ tumor infiltrating lymphocytes (TILs) producing Pfn, Grz B, and IFN-γ are shown in Figures 2E to 2H.

[0090] Referring to Figures 2A to 2D, cytotoxic cytokines in CD8+ T cells increased in the group treated with CIM or α-CTLA-4 monotherapy, and cytotoxic cytokines in CD8+ T cells increased further in the group treated with CIM and α-CTLA-4 combination therapy.

[0091] On the other hand, referring to Figures 2E to 2H, cytotoxic cytokines increased in CD4+ T cells in the group treated with CIM or α-CTLA-4 monotherapy, but the synergistic effect of the combination therapy was minimal compared to CD8+ T cells.

[0092] In summary, the combination of CIM and α-CTLA-4 antibodies effectively inhibited tumor formation due to increased CD8+ T cell infiltration and a significant increase in cytotoxic cytokines in CD8+ T cells.

[0093]

[0094] Example 3. Treatment of melanoma with combination therapy of cimetidine and anti-PD-1 antibody

[0095]

[0096] 3-1. Analysis of Melanoma Inhibitory Effects of Co-treatment with Cimetidine and Anti-PD-1 Antibody in Mice

[0097] B16F10 cells were subcutaneously inoculated into wild-type C57BL / 6 (B6) mice, followed by treatment with CIM or α-PD-1 alone, and treatment with CIM and α-PD-1 in combination, respectively. The methods for CIM and α-PD-1 antibody treatment were described in detail in the 'tumor inoculation' experimental technique described above, and the process is schematically illustrated in Fig. 3A. Next, mice inoculated with B16F10 cells were treated with monotherapy or combination therapy, and the volume and weight of melanoma tumors were measured to compare the anti-tumor effects. The measured tumor volumes and weights are shown in Figs. 3B and 3C.

[0098] Referring to Figures 3B and 3C, when treated with monotherapy using CIM or α-PD-1, the volume and weight of the melanoma decreased, but when treated with a combination of CIM and α-PD-1, the volume and weight of the melanoma decreased even more. Therefore, it was confirmed that treating melanoma with a combination of CIM and α-PD-1 has a superior therapeutic effect compared to monotherapy.

[0099]

[0100] 3-2. Analysis of PD-L1 Expression in Mice Treated with Cimetidine and Anti-PD-1 Antibody

[0101] B16F10 cells were subcutaneously inoculated into wild-type C57BL / 6(B6) mice in the same manner as in Example 1-1 above, followed by CIM or α-PD-1 alone and CIM and α-PD-1 combined treatment, respectively. Next, the expression of PD-L1 in the tumor cells of each group was compared, and the results are shown in Figures 3D and 3E.

[0102] Referring to Figures 3D and 3E, it was confirmed that CIM effectively inhibited PD-L1 expression in tumor cells, and that PD-L1 expression was further inhibited when CIM and α-PD-1 were administered together.

[0103]

[0104] Example 4. Production of cytotoxic cytokines following combination therapy of cimetidine and anti-PD-1 antibody

[0105] In the same manner as in Example 1-1 above, B16F10 cells were subcutaneously inoculated into wild-type C57BL / 6 (B6) mice, followed by treatment with CIM or α-PD-1 alone, and treatment with CIM and α-PD-1 in combination, respectively. Next, the production of cytotoxic cytokines by T cells after treatment in each group was confirmed.

[0106] More specifically, the percentage and number of CD8+ tumor infiltrating lymphocytes (TILs) producing Pfn, Grz B, and IFN-γ are shown in Figures 4A to 4D, and the percentage and number of CD4+ tumor infiltrating lymphocytes (TILs) producing Pfn, Grz B, and IFN-γ are shown in Figures 4E to 4H.

[0107] Referring to Figures 4A to 4D, cytotoxic cytokines in CD8+ T cells increased in the group treated with CIM or α-PD-1 monotherapy, and cytotoxic cytokines in CD8+ T cells increased further in the group treated with CIM and α-PD-1 combination therapy.

[0108] On the other hand, referring to Figures 4E to 4H, cytotoxic cytokines increased in CD4+ T cells in the group treated with CIM or α-PD-1 monotherapy, but no synergistic effect was observed with combination therapy.

[0109] In summary, the combination of CIM and α-PD-1 antibodies effectively inhibited melanoma tumor formation due to increased CD8+ T cell infiltration and a significant increase in cytotoxic cytokines in CD8+ T cells.

[0110]

[0111] Comparative Example 1. Treatment of melanoma with combination therapy of cimetidine and anti-PD-L1 antibody

[0112] B16F10 cells were subcutaneously inoculated into wild-type C57BL / 6 (B6) mice, followed by treatment with CIM or α-PD-L1 alone, and treatment with CIM and α-PD-L1 in combination, respectively. The methods for CIM and α-PD-L1 antibody treatment were described in detail in the 'tumor inoculation' experimental technique described above, and the process is schematically illustrated in Fig. 5A. Next, mice inoculated with B16F10 cells were treated with monotherapy or combination therapy, and the volume and weight of melanoma tumors were measured to compare the anti-tumor effects. The measured tumor volumes and weights are shown in Figs. 5B and 5C.

[0113] Referring to Figures 5B and 5C, when treated with monotherapy using CIM or α-PD-L1, the volume and weight of the melanoma decreased, but no synergistic effect was observed when treated with a combination of CIM and α-PD-L1.

[0114] Therefore, through the results of Examples 1 and 2 and Comparative Example 1 above, it was confirmed that in order to maximize the therapeutic effect of melanoma when treating melanoma with cimetidine, co-administering α-CTLA-4 or α-PD-1 with α-PD-L1 is more effective.

[0115]

[0116] Experimental Example 1. Regulation of melanoma cell-specific PD-L1 expression by cimetidine

[0117]

[0118] 1-1. Regulation of PD-L1 Expression by Cimetidine in Melanoma Cells

[0119] In Experimental Example 1-1, the expression of HRH2 (histamine receptor H2) was confirmed in mouse melanoma cell lines (B16-F10) and human melanoma cell lines (A375P and SK-MEL-28), and the expression of PD-L1 regulated by cimetidine treatment was confirmed in mouse melanoma cell lines (B16-F10), human melanoma cell lines (A375P and SK-MEL-28), and primary melanoma cells isolated from lesions of melanoma patients. The results of Experimental Example 1 are shown in Fig. 6.

[0120] Referring to Figure 6A, it was confirmed that HRH2 is expressed in both mouse melanoma cell lines (B16-F10) and human melanoma cell lines (A375P and SK-MEL-28). Histamine receptor expression is significantly increased in various cancers, such as melanoma and colorectal cancer, promoting tumor formation and metastasis. Additionally, melanoma is characterized by high levels of HRH2 expression.

[0121] Referring to Figures 6B to 6D, it was confirmed that PD-L1 expression was reduced by cimetidine in mouse melanoma cell lines (B16-F10), but PD-L2 was hardly expressed in B16-F10 cell lines.

[0122] Referring to Figures 6E to 6H, it was confirmed that the expression of PD-L1 was reduced by cimetidine in human melanoma cell lines (A375P and SK-MEL-28).

[0123] Referring to Figures 6I and 6J, it was confirmed that the expression of PD-L1 was reduced in primary melanoma cells isolated from melanoma patient lesions.

[0124]

[0125] 1-2. Regulation of PD-L1 Expression by Cimetidine in Renal Cancer Cells

[0126] In Experimental Example 1-2, the expression of PD-L1 regulated by cimetidine treatment in mouse renal cancer cell line (Renca) and human renal cancer cell line (ACHN) was confirmed, and the results are shown in Figure 7.

[0127] Referring to Figures 7A and 7B, it was confirmed that PD-L1 expression was not regulated by cimetidine in mouse renal carcinoma cell line (Renca), and referring to Figures 7C and 7D, it was confirmed that PD-L1 expression was not regulated by cimetidine in human renal carcinoma cell line (ACHN).

[0128]

[0129] 1-3. Regulation of PD-L1 Expression by Cimetidine in Melanocytes and Keratinocytes

[0130] The epidermis of the skin where melanoma occurs is mainly composed of keratinocytes and melanocytes, which play a very important role in the immune microenvironment of the skin. Accordingly, in Experimental Example 1-2, the expression of PD-L1 regulated by cimetidine treatment in mouse melanocytes (Melan-A), human melanocytes, mouse keratinocytes, and human keratinocytes was confirmed, and the results are shown in Fig. 8.

[0131] Referring to Figures 8A to 8D, it was confirmed that the expression of PD-L1 was not regulated by cimetidine in mouse melanocytes (Melan-A) and human melanocytes, and referring to Figures 8E to 8H, it was confirmed that the expression of PD-L1 was not regulated by cimetidine in mouse keratinocytes and human keratinocytes.

[0132]

[0133] According to the results of Experimental Example 1-1 above, cimetidine reduced the expression of PD-L1 in melanoma cells. On the other hand, according to the results of Experimental Examples 1-2 and 1-3 above, cimetidine did not regulate the expression of PD-L1 in renal carcinoma cells, keratinocytes, and melanocytes. That is, cimetidine selectively inhibits PD-L1 expression in melanoma without affecting PD-L1 expression in other cells, including skin cells. Therefore, by selectively inhibiting PD-L1 expression in melanoma, cimetidine can be used as a drug to alleviate side effects during the treatment of melanoma.

[0134]

[0135] Experimental Example 2. Analysis of Protein Expression in Major Signaling Pathways Induced by Cimetidine

[0136] B16F10 cells were treated with CIM at concentrations of 0, 0.5, or 5 mM for 1 or 16 hours with or without the addition of 10 μM PKA / CREB activator (Forskolin, Fsk), 1 μM AKT activator (Recilisib), or 1 μM ERK activator (C16-PAF). Subsequently, the expression of key proteins of the PKA / CREB, MAPK / ERK, PI3K-AKT, and Wnt signaling pathways was analyzed by Western blot, and the results are shown in Figure 9.

[0137] Referring to Figure 9A, the phosphorylation levels of p-PKA, p-CREB, p-ERK1 / 2, and p-AKT decreased in a concentration-dependent manner with cimetidine treatment, but the phosphorylation level of p-β-catenin did not show a change.

[0138] Referring to Figure 9B, B16F10 cells were treated with 5 mM cimetidine and either Fsk, a PKA / CREB signaling pathway activator, or C16-PAF, an ERK signaling pathway activator, for 16 hours. As a result, PD-L1 expression, which was reduced by cimetidine, was increased by Fsk and C16-PAF.

[0139] Referring to Figure 9C, B16F10 cells were treated with 5 mM cimetidine and either Fsk, a PKA / CREB signaling pathway activator, or Recilisib, an AKT signaling pathway activator, for 1 hour. As a result, PD-L1 expression, which was reduced by cimetidine, was increased by Fsk and Recilisib.

[0140] Therefore, combining the results of Figures 9A to 9C, it means that the PKA / CREB and AKT signaling pathways are involved in regulation during the early stages of PD-L1 expression, and the PKA / CREB and ERK signaling pathways are involved in regulation during the late stages of expression.

[0141]

[0142] From the foregoing description, those skilled in the art to which the present invention pertains will understand that the present invention may be implemented in other specific forms without altering its technical concept or essential features. In this regard, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. A pharmaceutical composition for the prevention or treatment of melanoma containing cimetidine as an active ingredient.

2. In Paragraph 1, The above pharmaceutical composition is A pharmaceutical composition for the prevention or treatment of melanoma, characterized by further comprising one or more antibodies selected from the group consisting of anti-CTLA-4 antibodies and anti-PD-1 antibodies.

3. In Paragraph 1, The above pharmaceutical composition is A pharmaceutical composition for the prevention or treatment of melanoma, characterized by the simultaneous or sequential administration of cimetidine; and one or more antibodies selected from the group consisting of anti-CTLA-4 antibodies and anti-PD-1 antibodies, each formulated.

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