KR102528732B1 - Pharmaceutical composition for preventing or treating cancer comprising poziotinib and calcium channel blocker - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cancer containing poziotinib and a calcium channel inhibitor as active ingredients. It is useful as a pharmaceutical composition for inhibiting the growth or proliferation of cancer stem cells, as it can more effectively inhibit the signal transduction pathway and stem cell function related to the self-renewal of stem cells, and as a pharmaceutical composition for preventing or treating cancer recurrence and metastasis. The composition has a useful effect. In addition, the pharmaceutical composition of combination therapy further comprising a calcium channel blocker in addition to poziotinib of the present invention shows a synergistic effect that is greater than or equal to that of a single treatment by targeting different signal transduction pathways, compared to conventional chemotherapy. It is useful as a pharmaceutical composition for treating intractable ovarian cancer, and has a useful effect as a pharmaceutical composition for preventing or treating cancer recurrence and metastasis.

Description

Pharmaceutical composition for preventing or treating cancer comprising poziotinib and calcium channel inhibitor {Pharmaceutical composition for preventing or treating cancer comprising poziotinib and calcium channel blocker}

The present invention relates to a pharmaceutical composition for preventing or treating cancer containing poziotinib and a calcium channel inhibitor as active ingredients.

Epithelial ovarian cancer (EOC) is the most lethal gynecological cancer in women, with the highest mortality rate among all gynecological malignancies, with a 5-year survival rate of less than 30%. The low survival rate of ovarian cancer is related to the persistence of cancer stem cells (CSCs) even after chemotherapy.

Chemotherapy for ovarian cancer is generally treated with platinum-based drugs such as cisplatin, and also PARP inhibitors such as olaparib and niraparib are used as targeted therapy. Despite these treatment options, 50 to 70% of patients experience relapse after treatment. Recurrence is due to cancer stem cells (CSCs), which are responsible for the development, recurrence and metastasis of cancer. Moreover, cancer stem cells have the ability to self-renew and are a major factor in acquiring resistance to chemotherapy and radiation therapy.

Therefore, removal of ovarian cancer stem cells is important for treatment of intractable ovarian cancer, prevention of recurrence and metastasis of ovarian cancer, and prolongation of survival of ovarian cancer patients.

Chemotherapy is one of the comprehensive treatments used primarily to slow tumor development and prolong survival after surgery. In general, ovarian cancer patients may respond to taxane and platinum-based chemotherapy as the disease progresses, but , resistant cancer cells can persist at the metastatic site, remain dormant for a long period of time after initial treatment, and recur. Despite continuous efforts to develop combined surgery and chemotherapy treatments, long-term clinical outcomes in patients with advanced ovarian cancer have not improved significantly in recent decades. Clinically, ovarian cancer stem cells have been shown to survive conventional chemotherapy, and in this regard, cancer cells resistant to chemotherapy from ovarian cancer stem cells play an important role in the emergence and recurrence. Therefore, new strategies are needed to target and block cancer stem cell properties and stem cell signaling pathways.

Meanwhile, poziotinib is an EGFR/HER1, 2, 4 inhibitor under development by Hanmi Pharmaceutical, and is currently in phase 2 clinical trials as a non-small cell lung cancer (NSCLC) treatment.

Under the above background, the present inventors have sought to provide a novel therapeutic agent targeting cancer stem cell properties and stem cell signaling pathways for the treatment of intractable ovarian cancer, prevention of recurrence and metastasis of ovarian cancer, and prolongation of survival of ovarian cancer patients. HER4 was overexpressed in ovarian cancer stem cells, and poziotinib effectively blocked the sphere formation, viability and proliferation of ovarian cancer stem cells, and poziotinib also inhibited stem cells related to self-renewal of cancer stem cells. function, inhibiting the Wnt/beta-catenin, Notch and Hedgehog pathways, stopping the G1 cell cycle and inducing apoptosis,

Furthermore, as a result of efforts to develop a combination therapy that exhibits a synergistic effect in combination with poziotinib, the present inventors have found that a combination treatment of poziotinib and a calcium channel blocker (CCB) has a synergistic effect. It was confirmed using the Talalay method, and each drug inhibits stem cell activity by targeting different signaling pathways, inhibits the expression of stem cell markers, especially CD133, NANOG and KLF4, and inhibits self-renewal and The present invention was completed by confirming the transfer of beta-catenin to the nucleus and inhibition of phosphorylation of STAT5, AKT and ERK.

An object of the present invention is to provide a novel strategy for treatment of intractable ovarian cancer, prevention of recurrence and metastasis of ovarian cancer, and prolongation of survival of ovarian cancer patients, specifically, effectively blocking sphere formation, viability and proliferation of ovarian cancer stem cells. In addition, by effectively inhibiting the signal transduction pathway and stem cell function related to the self-renewal of cancer stem cells, it is effective in treating intractable ovarian cancer compared to conventional chemotherapy and providing a drug effective in preventing cancer recurrence and metastasis. In addition, it is to provide a combination therapy treatment strategy that can exhibit more than additive synergistic effects.

In order to achieve the above purpose,

The present invention provides a pharmaceutical composition for inhibiting the growth or proliferation of cancer stem cells, containing a compound represented by Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer recurrence and metastasis, containing the compound represented by Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

Furthermore, the present invention relates to a compound represented by Formula 1, a stereoisomer thereof or a pharmaceutically acceptable salt thereof; And it provides a pharmaceutical composition for preventing or treating cancer containing a calcium channel blocker as an active ingredient.

In addition, the present invention relates to a compound represented by Formula 1, a stereoisomer thereof or a pharmaceutically acceptable salt thereof; and a pharmaceutical composition for inhibiting cancer stem cell growth or proliferation containing a calcium channel blocker as an active ingredient.

Furthermore, the present invention relates to a compound represented by Formula 1, a stereoisomer thereof or a pharmaceutically acceptable salt thereof; And it provides a pharmaceutical composition for preventing or treating recurrence and metastasis of cancer containing a calcium channel blocker as an active ingredient.

The pharmaceutical composition containing poziotinib of the present invention can more effectively suppress the signal transduction pathway and stem cell function related to self-renewal of cancer stem cells compared to conventional chemotherapy, and thus cancer stem cell growth or proliferation. It is useful as a pharmaceutical composition for inhibition, and has a useful effect as a pharmaceutical composition for preventing or treating cancer recurrence and metastasis. In addition, the pharmaceutical composition of combination therapy further comprising a calcium channel blocker in addition to poziotinib of the present invention shows a synergistic effect that is greater than or equal to that of a single treatment by targeting different signal transduction pathways, compared to conventional chemotherapy. It is useful as a pharmaceutical composition for treating intractable ovarian cancer, and has a useful effect as a pharmaceutical composition for preventing or treating cancer recurrence and metastasis.

1 is a view showing the expression of EGFR (HER 1), ERBB2 (HER 2), and ERBB4 (HER 4) measured in ovarian cancer cells (A2780) and ovarian cancer stem cells (A2780-SP).
2 is a diagram showing the expression of EGFR (HER 1), ERBB2 (HER 2), and ERBB4 (HER 4) measured in ovarian cancer cells (SKOV3) and ovarian cancer stem cells (SKOV3-SP).
3 is a graph and a photograph showing inhibition of proliferation, GI50, sphere size and viability of stem cells in ovarian cancer cells (A2780) and ovarian cancer stem cells (A2780-SP) treated with cisplatin and poziotinib.
4 is a graph and a photograph showing inhibition of proliferation, GI50, spherical size and viability of stem cells in ovarian cancer cells (SKOV3) and ovarian cancer stem cells (SKOV3-SP) treated with cisplatin and poziotinib.
Figure 5 shows the survival rate according to the treatment of cisplatin and poziotinib for ovarian cancer cells (A2780), ovarian cancer stem cells (A2780-SP), and cancer stem cells isolated with cancer stem cell markers ALDH, CD117, and CD133 antibodies. it is a graph
6 is a graph illustrating changes in proliferation of ovarian cancer stem cells (A2780-SP) treated with poziotinib at different concentrations over time.
7 is a photograph showing changes in the expression of cancer stem cell markers (ALDH, CD133, OCT4, NANOG, and KLF4) in ovarian cancer stem cells (A2780-SP) according to poziotinib treatment at different concentrations by Western blotting.
FIG. 8 is a graph showing the metastasis of beta-catenin into the nucleus of ovarian cancer stem cells (A2780-SP) by treatment with poziotinib by concentration by Western blot and quantification thereof.
FIG. 9 is a graph showing photos of beta-catenin phosphorylation in ovarian cancer stem cells (A2780-SP) treated with poziotinib at different concentrations by western blot and quantification thereof.
10 is a cancer stem cell marker Notch, a sub-marker c-myc, GLI-1, HIP-1, PTCH-1 mRNA of the Hedgehog signaling pathway according to poziotinib treatment of ovarian cancer stem cells (A2780-SP) at different concentrations. It is a graph showing expression changes measured.
11 is a graph showing changes in the cell cycle of ovarian cancer stem cells (A2780-SP) treated with poziotinib at different concentrations and quantified.
12 is a graph showing changes in apoptosis in ovarian cancer stem cells (A2780-SP) treated with poziotinib at different concentrations and quantified.
13 is a photograph showing, by western blot, the changes in the expression of the apoptosis marker Cleaved PARP and the expression of the anti-apoptosis markers BCL2 and MCL-1 in ovarian cancer stem cells (A2780-SP) according to poziotinib treatment at different concentrations.
14 is a photograph showing the phosphorylation changes of STAT5, AKT, and ERK, which are sub-signaling markers of cancer stem cells, by western blotting and transcriptional changes of p85a and p110a in ovarian cancer stem cells (A2780-SP) treated with poziotinib at different concentrations. is a graph showing
Figure 15 is an isobologram of poziotinib and a calcium channel blocker (manidipine, lacidipine, lomerizine HCl or benidipine HCl) combined treatment in ovarian cancer cells (A2780) and ovarian cancer stem cells (A2780-SP) ( It is a graph showing an isobologram).
Figure 16 is an isobologram of poziotinib and a calcium channel blocker (manidipine, lacidipine, lomerizine HCl or benidipine HCl) combined treatment in ovarian cancer cells (SKOV3) and ovarian cancer stem cells (SKOV3-SP) ( It is a graph showing an isobologram).
Figure 17 is a combination treatment of poziotinib and a calcium channel blocker (manidipine, lacidipine, lomerizine HCl or benidipine HCl) in ovarian cancer cells (A2780, SKOV3) and ovarian cancer stem cells (A2780-SP, SKOV3-SP) This is a table showing the CI analysis (Combination index assay) values to confirm the synergistic effect by
18 is a photograph and a graph showing the effect of the combined treatment of poziotinib and manidipine at each concentration on the sphere size and sphere viability of ovarian cancer stem cells (A2780-SP).
19 shows expression of cancer stem cell markers (ALDH, CD133, OCT4, NANOG, and KLF4) in ovarian cancer stem cells (A2780-SP) by treatment with poziotinib or manidipine alone and combined treatment with poziotinib and manidipine. It is a photograph showing the change by Western blotting and a graph showing it by quantifying it.
20 is a photograph showing the phosphorylation changes of STAT5, AKT, and ERK, which are lower signaling markers of cancer stem cells, by the combined treatment of poziotinib and manidipine in ovarian cancer stem cells (A2780-SP) by western blotting, and quantifying them is the graph shown.
FIG. 21 is a western blot image showing changes in nuclear metastasis of beth-catenin by the combined treatment of poziotinib and manidipine in ovarian cancer stem cells (A2780-SP), a graph showing quantification thereof, and phosphorylation of beta-catenin. It is a graph showing pictures observed by Western blotting and quantifying them.
22 is a graph showing changes in apoptosis observed in ovarian cancer stem cells (A2780-SP) by treatment with poziotinib or manidipine alone and combined treatment with poziotinib and manidipine.
Figure 23 is a Western blot showing changes in the expression of anti-apoptosis markers BCL2, MCL-1 and SURVIVIN in ovarian cancer stem cells (A2780-SP) by treatment with poziotinib or manidipine alone and combined treatment with poziotinib and manidipine. It is a picture shown in and a graph showing it by quantifying it.
24 is a conceptual diagram showing a series of processes in which stemness is achieved in ovarian cancer stem cells. ErbB4 receptor tyrosine kinase and STAT5 cooperate to promote transcription of PI3K subunits p85a and p110a. AKT is phosphorylated downstream of PI3K signaling, and ERK phosphorylation is enhanced by overexpression of the ErbB4 receptor. On the other hand, overexpressed L/T-type calcium channels increase the flow of calcium into the cytoplasm and induce phosphorylation of AKT and ERK through PI3K and MAPK signals. As a result, elevated phosphorylation of AKT and ERK stabilizes phosphorylated beta-catenin moving from the cytoplasm to the nucleus, increasing the transcription of CD133, NANOG, SOX2, and KLF4, and maintaining the stem cell potential of definitive ovarian cancer stem cells.
25 is a conceptual diagram showing a process in which stem cell activity of ovarian cancer stem cells is suppressed and apoptosis is induced by the combined treatment of poziotinib and manidipine according to the present invention.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for inhibiting the growth or proliferation of cancer stem cells, containing a compound represented by Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

The compound represented by Formula 1 is podiotinib, IUPAC name 1-[4-[4-(3,4-dichloro-2-fluoroanilino)-7-methoxyquinazolin-6-yl]oxy It is a compound of piperidin-1-yl] prop-2-phen-1-one.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer recurrence and metastasis, containing the compound represented by Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

According to the present invention, poziotinib inhibits the stem cell function of cancer stem cells and induces apoptosis. Specifically, poziotinib inhibits ERBB4 (HER 4), effectively inhibits the sub-signal transduction system of cancer stem cells, inhibits stem cell function, and induces apoptosis.

In one embodiment of the present invention, ovarian cancer stem cells are confirmed to have high expression of ERBB4 (HER 4) among the EGFR family (see FIGS. 1 and 2), compared to platinum-based cisplatin, a conventional chemotherapeutic agent. It is confirmed that Otinib more effectively inhibits the proliferation of ovarian cancer and ovarian cancer stem cells, and more effectively lowers the sphere size and viability of stem cells (see FIGS. 3 and 4 ).

In another embodiment of the present invention, poziotinib was found to have superior survival rate inhibitory activity compared to cisplatin against ovarian cancer stem cells isolated with ALDH, CD117, and CD133 antibodies, which are cancer stem cell markers (see FIG. 5), and over time Poziotinib inhibits the proliferation of cancer stem cells in a concentration-dependent manner (see FIG. 6).

In another embodiment of the present invention, poziotinib inhibits the expression of cancer stem cell stem cell function markers ALDH, CD133, OCT4, NANOG and KLF4 in a concentration-dependent manner (see FIG. 7), and transmits cancer stem cell signals. It effectively inhibits the transfer of beta-catenin into the nucleus and the phosphorylation of beta-catenin in the system (see FIGS. 8 and 9), the cancer stem cell marker Notch, the sub-markers of the Hedgehog signaling system c-myc, GLI-1, Suppresses HIP-1 and PTCH-1 mRNA expression (see FIG. 10).

In another embodiment of the present invention, poziotinib arrests the G1 cell cycle of cancer stem cells and induces apoptosis in a concentration-dependent manner (see FIGS. 11 and 12), increases the expression of the apoptosis marker Cleaved PARP, and increases anti-apoptosis It was confirmed that the expression of markers BCL2 and MCL-1 was reduced (see FIG. 13 ).

In another embodiment of the present invention, poziotinib inhibits the phosphorylation of STAT5, AKT, and ERK, which are sub-signaling markers of cancer stem cells, in a concentration-dependent manner, and inhibits the transcription of p85a and p110a (see FIG. 14), thereby preventing cancer It effectively inhibits the signal transduction pathway and stem cell function related to the self-renewal of stem cells.

Therefore, the pharmaceutical composition containing poziotinib of the present invention can effectively inhibit the signal transduction pathway and stem cell function related to the self-renewal of cancer stem cells compared to conventional chemotherapy, thereby inhibiting cancer stem cell growth or proliferation. It is useful as a pharmaceutical composition for the prevention or treatment of recurrence and metastasis of cancer.

The cancers include benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, cerebral lymphoma, oligodendroglioma, intracranial race, ependymoma, brainstem tumor, laryngeal cancer, oropharyngeal cancer, nasal cavity/sinus cancer, nasopharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, thyroid cancer , oral cancer, chest tumor, small cell lung cancer, non-small cell lung cancer, thymus cancer, mediastinal tumor, esophageal cancer, breast cancer, abdominal tumor, stomach cancer, liver cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, small intestine cancer, colorectal cancer, anal cancer, bladder cancer, It is at least one selected from the group consisting of renal cancer, penile cancer, prostate cancer, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, female external genital cancer, and skin cancer.

The cancer may be difficult to treat with conventional chemotherapeutic agents or may be resistant to cancer, for example, refractory cancer, metastatic cancer, or recurrent cancer.

Preferably, the cancer is ovarian cancer, and more preferably, it may be a cancer that is difficult to treat or resistant to conventional chemotherapeutic agents, such as taxane or platinum-based therapies, such as refractory ovarian cancer, metastatic ovarian cancer or recurrent ovarian cancer.

The cancer stem cells are stem cells of one or more types of cancer among the above-mentioned carcinomas, and are preferably ovarian cancer stem cells. Preferably, the cancer stem cells may be cancer stem cells that are difficult to treat with conventional chemotherapeutic agents or resistant to them. More preferably, the cancer stem cells are ovarian cancer stem cells, and conventional chemotherapeutic agents; For example, it may be cancer stem cells that are difficult to treat with taxane- or platinum-based therapeutics or resistant to, for example, refractory ovarian cancer stem cells, metastatic ovarian cancer stem cells, or recurrent ovarian cancer stem cells.

In the present invention, "treatment" refers to all activities in which symptoms are improved or beneficially changed by administration of the composition according to the present invention.

In the present invention, "prevention" refers to any action that suppresses or delays the onset of disease by administering the composition according to the present invention.

The compound represented by Formula 1 of the present invention can be used in the form of a pharmaceutically acceptable salt, and an acid addition salt formed by a pharmaceutically acceptable free acid is useful as the salt. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid, etc., aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. It is obtained from non-toxic organic acids such as dioate, aromatic acids, aliphatic and aromatic sulfonic acids, etc., organic acids such as acetic acid, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid and the like. Such pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, i. Odide, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, sube rate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitro benzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate and the like. The acid addition salt according to the present invention can be prepared by a conventional method. For example, a precipitate formed by dissolving a derivative of Formula 1 in an organic solvent such as methanol, ethanol, acetone, methylene chloride, acetonitrile, etc. and adding an organic or inorganic acid thereto It can be prepared by filtering and drying, or by distilling the solvent and excess acid under reduced pressure, drying it, and crystallizing it in an organic solvent.

In addition, a pharmaceutically acceptable metal salt may be prepared using a base. An alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare a sodium, potassium or calcium salt as the metal salt. In addition, the corresponding salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable negative salt (eg, silver nitrate).

Furthermore, the compound represented by Formula 1 may be used in the form of not only pharmaceutically acceptable salts thereof, but also solvates, stereoisomers, and hydrates prepared therefrom.

The pharmaceutical composition of the present invention can be used as a single agent, and can be prepared and used as a combination preparation by further including an approved drug. It can be formulated into a pharmaceutical unit dosage form by adding a pharmaceutically acceptable carrier, excipient, or diluent.

In the present invention, "pharmaceutically acceptable" means that it does not significantly stimulate living organisms and does not inhibit the biological activity and properties of the active substance administered.

In the present invention, the pharmaceutical composition comprising a pharmaceutically acceptable carrier is a tablet, pill, powder, granule, capsule, suspension, internal solution, emulsion, syrup, sterilized aqueous solution, non-aqueous solvent, suspension, emulsion, It may have any one formulation selected from the group consisting of lyophilized preparations and suppositories.

The pharmaceutical composition may be in various oral or parenteral formulations. When formulated, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in one or more compounds, for example, starch, calcium carbonate, sucrose or lactose ( lactose) and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions for oral administration, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is.

Formulations for parenteral administration may include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, etc. may be used, but is not limited thereto.

In the composition of the present invention, the compound of Formula 1 may be included in a pharmaceutically effective amount. "Pharmaceutically effective amount" means an amount that is sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects, and the effective dose level is the patient's health condition, type of disease, severity, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration and excretion rate, duration of treatment, factors including drugs used in combination or concurrently, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

Furthermore, the present invention relates to a compound represented by Formula 1, a stereoisomer thereof or a pharmaceutically acceptable salt thereof; And it provides a pharmaceutical composition for preventing or treating cancer containing a calcium channel blocker as an active ingredient.

In one aspect of the present invention, the compound represented by Formula 1 is the aforementioned poziotinib, and the calcium channel blocker is preferably an L/T-type calcium channel blocker, for example manidipine, lacidipine, lomerizine HCl or Benidipine HCl.

[Manidipine]

[Lacidipine]

[Lomerizine HCl, Lomerizine HCl]

[Benidipine HCl, Benidipine HCl]

L/T-type calcium channels are overexpressed in ovarian cancer stem cells, and the calcium channel blocker targeting the L/T-type calcium channels of the present invention can inhibit sphere formation, viability and proliferation of cancer stem cells and induce apoptosis. It inhibits stem cell function (stemness) and AKT and ERK signaling pathways in ovarian cancer stem cells, and shows synergistic effects of more than additive when combined with poziotinib.

According to the present invention, the combined treatment of poziotinib and a calcium channel blocker exhibits an additive or higher synergistic effect, inhibits the stem cell function of cancer stem cells, and induces apoptosis. Specifically, poziotinib inhibits ERBB4 (HER 4), and calcium channel blocker inhibits the L/T-type channel to inhibit the sub-signal transduction system of cancer stem cells with a synergistic effect above the above, thereby suppressing stem cell activity. , induce apoptosis.

In one embodiment of the present invention, poziotinib did not show an additive or higher synergistic effect when treated in combination with platinum-based cisplatin, but poziotinib and a calcium channel blocker (manidipine, lacidipine, lomerizine HCl or beni Dipine HCl) combined treatment was investigated through CI analysis (Combination index assay) and Isobologram. As a result, a synergistic effect of more than additive was confirmed in both ovarian cancer and ovarian cancer stem cells, especially in ovarian cancer stem cells at all concentrations. In the combined treatment, an additive or higher synergistic effect was confirmed (see FIGS. 15, 16 and 17).

In another embodiment of the present invention, it was confirmed that the concentration-dependent combined treatment of poziotinib and a calcium channel blocker (manidipine) effectively suppressed the sphere size and sphere viability of ovarian cancer stem cells (see FIG. 18).

In another embodiment of the present invention, the combined treatment of poziotinib and a calcium channel blocker (manidipine) shows a synergistic effect that is greater than or equal to the treatment of each drug alone, while cancer stem cell markers ALDH, CD133, OCT4, NANOG and KLF4 suppresses the expression of (see FIG. 19), inhibits the phosphorylation of STAT5, AKT, and ERK, which are lower signaling markers of cancer stem cells (see FIG. 20), and inhibits the nuclear translocation and phosphorylation of beth-catenin (see FIG. 21). ).

In another embodiment of the present invention, the combined treatment of poziotinib and a calcium channel blocker (manidipine) induces apoptosis of cancer stem cells while showing an additive or higher synergistic effect compared to the treatment of each drug alone (see FIG. 22), It was found to inhibit the expression of anti-apoptotic markers BCL2, MCL-1 and SURVIVIN (see FIG. 23).

Therefore, the pharmaceutical composition of combination therapy further comprising a calcium channel blocker in addition to poziotinib of the present invention shows a synergistic effect that is greater than or equal to that of a single treatment by targeting different signaling pathways. It is a pharmaceutical composition for the prevention or treatment of cancer that is useful in contrast, and can effectively suppress the signal transduction pathway and stem cell function related to the self-renewal of cancer stem cells, compared to conventional chemotherapy, and cancer resistant to conventional chemotherapy. , It is useful as a pharmaceutical composition for the prevention or treatment of refractory cancer, metastatic cancer or recurrent cancer.

Although the present invention is not limited to a specific theory, as an example for explanation, as shown in FIG. 24 , the stemness of ovarian cancer stem cells has the following series of processes, specifically the ErbB4 receptor Tyrosine kinase and STAT5 cooperate to promote the transcription of PI3K subunits p85a and p110a, AKT is phosphorylated downstream of PI3K signaling, and ERK phosphorylation is enhanced by overexpression of the ErbB4 receptor, meanwhile in cancer stem cells. Overexpressed L/T-type calcium channels increase calcium flux into the cytoplasm, inducing phosphorylation of AKT and ERK through PI3K and MAPK signals, and phosphorylated beta- It stabilizes catenin and increases the transcription of CD133, NANOG, SOX2 and KLF4, thereby maintaining the stem cell function of the final ovarian cancer stem cell.

As shown in FIG. 25, the pharmaceutical composition of the combination therapy of poziotinib and calcium channel blocker of the present invention targets ErbB4 (HER 4) overexpressed in cancer stem cells, and the calcium channel blocker is L/ By inhibiting each of the different pathways by targeting the T channel, it suppresses the stem cell function of ovarian cancer stem cells and induces apoptosis while exhibiting synergistic effects that are more than additive.

Therefore, the pharmaceutical composition of the combination therapy of poziotinib and a calcium channel blocker of the present invention is for the prevention or treatment of cancer resistant to conventional chemotherapy, refractory cancer, metastatic cancer or recurrent cancer, compared to conventional chemotherapy. Provide more effective treatment strategies.

In addition, the present invention relates to a compound represented by Formula 1, a stereoisomer thereof or a pharmaceutically acceptable salt thereof; and a pharmaceutical composition for inhibiting cancer stem cell growth or proliferation containing a calcium channel blocker as an active ingredient.

Furthermore, the present invention relates to a compound represented by Formula 1, a stereoisomer thereof or a pharmaceutically acceptable salt thereof; And it provides a pharmaceutical composition for preventing or treating recurrence and metastasis of cancer containing a calcium channel blocker as an active ingredient.

Regarding the pharmaceutical composition for inhibiting the growth or proliferation of cancer stem cells and the pharmaceutical composition for preventing or treating cancer recurrence and metastasis, it can be seen from the above description that the practice of the present invention is obvious, and redundant descriptions are omitted. do.

Furthermore, the pharmaceutical composition disclosed in the present invention can be used in a combination therapy further comprising an additional anti-cancer therapy. The additional anti-cancer therapy is not limited as long as it is a therapy for inhibiting proliferation and killing the disclosed cancer, and may be, for example, radiation therapy or chemotherapy, and the therapeutic agent (anti-cancer agent) used in the chemotherapy is one of the anti-cancer agents listed below. There may be more than one species.

The term "anti-cancer agent" refers to known drugs used in conventional cancer treatment that exhibit cytotoxicity or growth inhibitory effects on cancer cells by acting on various metabolic pathways of cancer cells. It includes antimetabolites, plant alkaloids, topoisomerase inhibitors, alkylating agents, anticancer antibiotics, hormones and other drugs.

In one aspect of the present invention, the anticancer agent is doxorubicin, paclitaxel, vincristine, daunorubicin, vinblastine, actinomycin-D, docetaxel, etoposide, tenyne teniposide, bisantrene, homoharringtonine, Gleevec (STI-571), cisplatin, 5-fluorouracil, adriamycin, methotrexate, busulfan, chlorambucil ( chlorambucil), cyclophosphamide, melphalan, nitrogen mustard, and nitrosourea.

Preferably, the anticancer agent used in combination with the pharmaceutical composition of the present invention may be cisplatin or paclitaxel.

All combination therapies, combination preparations, and combination treatment methods disclosed herein can apply known combination therapies without limitation, and for example, the combination treatment (administration) can treat (administer) drugs simultaneously or sequentially. there is.

In addition, another aspect of the present invention, a pharmaceutical composition comprising the compound represented by Formula 1, a stereoisomer thereof or a pharmaceutically acceptable salt thereof or a pharmaceutical composition for combination therapy further comprising a calcium channel inhibitor is required. It provides a method for preventing or treating cancer, including administering to a subject, and also provides a method for inhibiting the growth or proliferation of cancer stem cells and a method for preventing or treating cancer recurrence and metastasis.

Furthermore, another aspect of the present invention, in the preparation of the pharmaceutical composition, the use of the compound represented by Formula 1, its stereoisomer or its pharmaceutically acceptable salt and the use of a combination preparation further comprising a calcium channel inhibitor provides

Hereinafter, the present invention will be described in detail by examples and experimental examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following Examples and Experimental Examples.

<Example 1> Preparation of Poziotinib

The chemical structure and purchase source of poziotinib used in Example 1 are shown in Table 1 below.

<Example 2> Preparation of Poziotinib and Manidipine Combination Formulation

Poziotinib and manidipine prepared in Example 1 were used for combined administration at the concentrations indicated in the following experimental examples.

<Example 3> Preparation of Poziotinib and Lacidipine Combination Formulation

Poziotinib and lacidipine prepared in Example 1 were used for combined administration at the concentrations indicated in the following experimental examples.

<Example 4> Preparation of Poziotinib and Lomerizine HCL Combination Formulation

Poziotinib and lomerizine HCL prepared in Example 1 were used for combined administration at the concentrations shown in each experimental example below.

<Example 5> Preparation of Poziotinib and Benedipin HCL Combination Formulation

Poziotinib and benedipine HCL prepared in Example 1 were used for combined administration at the concentrations indicated in the following experimental examples.

<Example 6> Preparation of Poziotinib, Manidipine and Cisplatin Triple Combination Formulation

A triple combination formulation further containing cisplatin in addition to the combination formulation prepared in Example 2 was used for combined administration at the concentrations shown in each of the experimental examples below.

The material name, chemical structure, and purchase location of the test materials used in Examples 1-6 are shown in Table 1 below.

Substance name chemical structure where to buy Poziotinib
(Poziotinib)

MedChemExpress manidipine
(Manidipine)

Selleckchem lacidipine
(Lacidipine)

Sigma Lomerizine HCl
(Lomerizine HCl)

Selleckchem Benidipine HCl
(Benidipine HCl)

Tocris Cisplatin
(Cisplatin)

MedChemExpress

<Experiment protocol>

Experimental Examples 1 to 7 of the present invention were conducted according to the following experimental protocol.

1. Cell viability assay

Ovarian cancer cells (A2780 and SKOV3) were plated in white bottom 96-well plates (corning) at a density of 3,000 viable cells per well. After 24 hours, example compounds were added to the cultures at the indicated concentrations. After 3 days, cell viability was assessed with CellTiter-Glo　 (Promega), and luciferase activity was detected using a Tecan plate reader (Biocompare).

Ovarian cancer stem cells (A2780-SP and SKOV3-SP) were plated in white-bottom 96-well plates (corning) at a viable cell density of 3,000 per well. Afterwards, the samples were centrifuged at 3,000 rpm for 3 min. After 24 hours, example compounds were added at the indicated concentrations. After 3 days, cells were suspended in fresh medium containing the same concentrations of compounds. After 3 days, sphere cells were observed using the EVOS Cell Imaging System (Thermo Fisher Scientific). Sphere size was measured using ImageJ software (NIH Image). After imaging, sphere cell viability was assessed using CellTiter-Glo　 (Promega), and luciferase activity was detected using a Tecan plate reader (Biocompare).

2. CI analysis (combination index assay)

CIs were analyzed using the Chou-Talalay method (Chou TC. Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer Res 2010; 70: 440-446) using CompuSyn software (COMBOSYN, Paramus, NJ, USA). was calculated according to First, the intercept and slope were estimated from the dose-response curve, and the median effective dose (ED50) was calculated. The ED50 was used to evaluate the CI, an essential measure for determining combination effects. Afterwards, we used ratios influenced by CI plots and isobologram analysis to determine whether the interaction between the two compounds was additive, synergistic, or antagonistic. . Affected ratios and isobolograms of CI plots yielded the same conclusion for synergy or antagonism. CI<1 indicates a synergistic action, CI=1 indicates an additive effect, and CI>1 indicates an antagonistic action (Chou TC. Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer Res 2010; 70 : 440-446. and Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 1984; 22: 27-55.).

3. Western blotting

Whole-cell lysates were obtained using protein extraction solution (radioimmunoprecipitation assay buffer containing phosphatase inhibitor and protease inhibitor cocktail). Nuclear and cytoplasmic fractions were prepared using a nuclear and cytoplasmic isolation kit (Thermo Fisher Scientific).

Cell lysates were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes for western blot analysis. After blocking with 5% skim milk, the membrane was incubated overnight at 4°C with primary antibody in blocking buffer, and then incubated with HRP (horseradish peroxidase)-conjugated secondary antibody for 2 hours at room temperature.

The primary antibodies against the specific antigens used in the experiments were as follows:

CD133 (Abcam, ab19898), ALDH (Santa Cruz Biotechnology, sc-166362), NANOG (Cell Signaling Technology, #3580S), SOX2 (Cell Signaling Technology, #2748S), KLF4 (GeneTex, GTX101508), vinculin (Santa Cruz Biotechnology , sc-73614), phospho-STAT5 (Y694) (Cell Signaling Technology, #9351S), STAT5 (Cell Signaling Technology, #94205S), phospho-AKT (Ser473) (Cell Signaling Technology, #3787S), AKT (Cell Signaling Technology, #9272S), phospho-ERK (Thr202/Tyr204) (Cell Signaling Technology, #9101S), ERK (Cell Signaling Technology, #9102S), beta-catenin (Cell Signaling Technology, #8480S), lamin B (Abbkine, #A01090), GAPDH (Santa Cruz Biotechnology, sc-47724), phospho-beta-catenin (S552) (Cell Signaling Technology, #9566S), BCL2 (Santa Cruz Biotechnology, sc-7382), MCL1 (Cell Signaling Technology, # 4572S), and survivin (Cell Signaling Technology, #2803S).

The secondary antibodies used were goat anti-mouse IgG-HRP (R&D Systems) and goat anti-rabbit IgG-HRP (R&D Systems).

Signals were amplified with an enhanced chemiluminescent HRP substrate (Bio-Rad Laboratories) and detected using a LAS-3000 Mini (Fujifilm). Signal intensities were calculated using Image J software (NIH Image).

4. Apoptosis assay (AV/PI staining)

A2780-SP cells (ovarian cancer stem cells) were plated on an Ultra-Low Attachment 6-well plate (corning) at a viable cell density of 1x10 ⁶ per well and cultured in a cancer stem cell medium. After 24 hours, A2780-SP cells were treated with the example compounds at the indicated concentrations, and incubated for 24 hours at 37°C, 5% CO ₂ , 95% humidity, and the cells were harvested using a centrifuge. . Spheres were dissociated into single cell suspensions by treatment with Accutase. The supernatant was decanted and the cells were gently resuspended and washed once with PBS. The cell pellet was resuspended in 0.1 mL of annexin V binding buffer (BD Biosciences). 5 μL of FITC annexin V and 5 μL of PI (BD Biosciences) were added and the cells were gently vortexed. Cells were incubated for 15 minutes at room temperature in the dark, then 400 μL of annexin V binding buffer was added to each tube. Flow cytometry was performed within 1 hour.

5. Real-time polymerase chain reaction (Quantitative Real-time polymerase chain reaction, Quantitative RT-PCR)

Total RNA from the samples was extracted using the Trizol RNA extraction kit (Invitrogen) according to the manufacturer's instructions. 1 μg of RNA was reverse transcribed into cDNA using a cDNA reverse transcription kit (Applied Biosystems). The synthesized cDNA was subjected to quantitative RT-PCR using SYBR™ Green PCR Master Mix and StepOne™ Real-Time PCR system (Applied Biosystems) using the indicated primers. GAPDH was used as a reference gene, and the results were expressed as relative expression to the control group using the 2-ddCt method.

The primers used in the experiment were as follows:

GAPDH (forward [F]: 5′-GGAGCCAAAAGGGTCATCAT-3′, reverse [R]: 5′-GTGATGGCATGGACTGTGGT-3′), CD133 (F: 5′-AGTCGGAAACTGGCAGATAGC-3′, R: 5′-GGTAGTGTTGTACTGGGCCAAT-3′ ), ALDH1 (F: 5′-CTCGAAATTAAGTACACCAA-3′, R: 5′-TCAGTAGACCCTGTGAATGC-3′), OCT3/4 (F: 5′-GACAACAATGAAAATCTTCAGGAGA-3′, R: 5′-TTCTGGCGCCGGTTACAGAACCA-3′), NANOG (F: 5′-TGCCTCACACGGAGACTGTC-3′, R: 5′-TGCTATTCTTCGGCCAGTTG-3′), SOX2 (F: 5′-GGGAAATGGGAGGGGTGCAAAAGAGG-3′, R: 5′-TTGCGTGAGTTGTGGATGGGGATTGGTG-3′), KLF4 (F: 5′-CCTACCTCGGAGAGAGACCG-3′, R: 5′-GGACTCCCTGCCATAGAGGA-3′), EGFR (F: 5′-AGGCACGAGTAACAAGCTCAC-3′, R: 5′-ATGAGGACATAACCAGCCACC-3′), ERBB2 (F: 5′-TGTGACTGCCTGTCCCTACAA -3′, R: 5′-CCAGACCATAGCACACTCGG-3′), and ERBB4 (F: 5′-GCAGATGCTACGGACCTTACG-3′, R: 5′-GACACTGAGTAACACATGCTCC-3′).

6. Combination effect test

A2780-SP ovarian cancer stem cells were co-treated with 4 μM of manidipine, 8 μM of lacidipine, 10 μM of lomerizine HCl or 10 μM of benicipin HCl, respectively, together with poziotinib. The combined effects of the example compounds and poziotinib were confirmed using the sphere formation assay and sphere proliferation assay.

7. Statistics

Each data was expressed as the mean ± standard deviation (SD) of at least three independent experiments. Statistically significant differences were determined using 1-way ANOVA with GraphPad Prism 5 (CA, USA). A p value of 0.05 or less was considered statistically significant.

<Experimental Example 1> Characteristic analysis of ovarian cancer cells and ovarian cancer stem cells

Compared to the parental cell lines, A2780 and SKOV3 ovarian cancer cells, A2780-SP and SKOV3-SP ovarian cancer stem cells exhibit CSC characteristics. Results are shown in Figures 1 and 2.

1 and 2, A2780-SP cells had higher mRNA levels of ERBB4 (HER4) than A2780 cells, and SKOV3-SP cells had higher mRNA levels of ERBB2 (HER2) and ERBB4 (HER4) than SKOV3 cells. It was high.

<Experimental Example 2> Poziotinib alone treatment effect on ovarian cancer and ovarian cancer stem cells

<2-1> Growth inhibitory effect analysis

First, the cell viability analysis of ovarian cancer cells (A2780 and SKOV3) and ovarian cancer stem cells (A2780-SP and SKOV3-SP) was performed using poziotinib of Example 1 and cisplatin as a positive control as in 1. In addition, the GI ₅₀ value of each compound was measured and displayed for each cell, and the results are shown in FIGS. 3 and 4.

3 and 4, in the case of cisplatin, it was confirmed that the effect of inhibiting the proliferation of ovarian cancer stem cells compared to the inhibition of ovarian cancer cells was low, whereas in the case of poziotinib of Example 1 of the present invention, the ovarian In both cancer cells and ovarian cancer stem cells, not only does it show an excellent anti-proliferation effect compared to cisplatin, but it is also confirmed that it has an anti-proliferation effect that is more than 10 times greater than the anti-proliferation effect seen in ovarian cancer, especially for ovarian cancer stem cells.

<2-2> Analysis of proliferation inhibitory effect in cancer stem cells

In addition, for the A2780 cancer stem cell group isolated with the cancer stem cell markers ALDH1, CD117, and CD133 antibodies, cell viability analysis was performed as described in 1. of the above experimental protocol using poziotinib of Example 1 and cisplatin as a positive control. Proliferation inhibition was observed, and the results are shown in FIG. 5 .

Referring to FIG. 5, in the case of cisplatin, the effect of inhibiting the proliferation of ovarian cancer stem cells compared to the inhibition of ovarian cancer cells was confirmed to be significantly lower, whereas in the case of poziotinib of Example 1 of the present invention, the effect of inhibiting the growth of ovarian cancer cells The anti-proliferation effect shown in ovarian cancer stem cells compared to the anti-proliferation effect is about 6 to 20 times or more, and the excellent anti-proliferation effect is confirmed.

<2-3> Analysis of cancer stem cell proliferation inhibitory effect by concentration

In addition, after treating A2780-SP with poziotinib at different concentrations (0, 1, 10, and 20 μM), changes in the proliferation of cancer stem cells were observed over time, and the results are shown in FIG. 6 .

Referring to FIG. 6 , it is confirmed that poziotinib suppresses the proliferation of ovarian cancer stem cells in a concentration-dependent manner even in the result of observing changes in the proliferation of ovarian cancer stem cells over time.

Therefore, poziotinib exhibits an excellent anti-proliferative effect on ovarian cancer stem cells in particular, and can therefore be used as an anti-cancer agent superior to existing anti-cancer agents for ovarian cancer, and has excellent anti-proliferative activity against ovarian cancer stem cells. In particular, it can be seen that it can be used as a more useful and effective anticancer agent for refractory ovarian cancer and recurrent ovarian cancer.

<2-4> Expression analysis of factors related to cancer stem cell activity (stemness), survival and growth

In order to confirm changes in stem cell function markers and signal transduction systems by treatment of ovarian cancer stem cells (A2780-SP) with different concentrations of poziotinib (0, 1, and 10 μM), ALDH1, CD133, OCT4, NANOG, and KLF4 Changes in the expression of cancer stem cell markers were confirmed by Western blotting, and the results are shown in FIG. 7 .

Referring to FIG. 7 , it is confirmed that the expression of cancer stem cell markers is decreased in a concentration-dependent manner according to poziotinib treatment.

In addition, nuclear protein expression and phosphorylation of beta-catenin were confirmed by Western blotting, and the results are shown in FIGS. 8 and 9.

Referring to FIGS. 8 and 9 , it was confirmed that nuclear translocation and phosphorylation of beta-catenin were reduced in a concentration-dependent manner according to poziotinib treatment.

Furthermore, changes in mRNA expression of c-myc, GLI-1, HIP-1, and PTCH-1, which are submarkers of the Notch and Hedgehog signaling pathways, were measured and shown in FIG. 10 .

Referring to FIG. 10, it is confirmed that c-myc, GLI-1, HIP-1, and PTCH-1 mRNA expressions are decreased in a concentration-dependent manner according to poziotinib treatment.

In addition, changes in phosphorylation of STAT5, AKT, and ERK, which are EGFR (HER2, 4) sub-signaling markers, and transcription of p85a and p110a were observed, and are shown in FIG. 14 .

Referring to FIG. 14, STAT5, AKT, and ERK phosphorylation were decreased in a concentration-dependent manner according to poziotinib treatment, and transcriptional decreases of p85a and p110a were confirmed.

Therefore, it was confirmed that the treatment with poziotinib according to the present invention reduced cancer stem cell capacity in a concentration-dependent manner and reduced the expression of factors related to self-renewal and cancer stem cell survival and growth, especially in ovarian cancer stem cells. It can be seen that it can be used as a more useful and effective anticancer agent especially for refractory ovarian cancer and recurrent ovarian cancer because of its excellent antiproliferative activity.

<2-5> Cell cycle and apoptosis analysis of ovarian cancer stem cells

Cell cycle changes in ovarian cancer stem cells (A2780-SP) treated by poziotinib concentrations (0, 1 and 10 μM) were observed and shown in FIG. 10 μM), the expression of the apoptosis marker Cleaved PARP and the expression of the anti-apoptosis markers BCL2 and MCL-1 were observed and shown in FIG. 13 .

Referring to FIG. 12, it is confirmed that the G1 cell cycle increases in a concentration-dependent manner with poziotinib treatment, and with reference to FIG. 13, expression of apoptosis markers increases in a concentration-dependent manner with poziotinib treatment, and anti-apoptotic markers It is confirmed that the expression of is decreased.

<Experimental Example 3> Combination treatment effect of poziotinib and calcium channel blocker on ovarian cancer and ovarian cancer stem cells

<3-1> CI (combination index) analysis

First, based on the results of 2-1 above, the concentration of poziotinib capable of reducing the viability of ovarian cancer stem cells by 10 to 20% was determined. Specifically, the concentration of poziotinib for A2780 and A2780-SP was 20 nM, the concentration of poziotinib for SKOV3 and SKOV3-SP was 10 nM, and the combined calcium channel blocker (manidipine, lacidipine, The concentrations of lomerizine HCl and benidipine HCl) were 0 to 30 μM. Performed as in the method of 2. of the above experimental protocol, CI values are shown in FIG. 17, and normalized isobolograms are shown in FIGS. 15 and 16.

CI values below the sloped line in each graph shown in FIGS. 15 and 16 represent a synergistic effect, values close to the line represent an additive effect, and values above the line represent an antagonistic effect.

Looking at Figures 15, 16 and 17, the combined treatment of the present invention showed a strong synergistic effect at all concentrations of the calcium channel blocker in A2780-SP and SKOV3-SP cells. In addition, although a synergistic effect was observed in A2780 and SKOV3 cells, it was confirmed that the synergistic effect was weakened when the calcium channel blocker was used at a certain concentration or higher.

From the above results, antiproliferative activity exhibiting a synergistic effect was confirmed, particularly in ovarian cancer stem cells, and it can be seen that the combined treatment strategy of poziotinib and a calcium channel blocker is effective for refractory ovarian cancer and recurrent ovarian cancer.

<3-2> Sphere formation and proliferation inhibition assay

In order to analyze the degree of growth of cancer stem cells according to the combination treatment of Example 2 of the present invention, A2780-SP cells were seeded to form spheres, and then manidipine at a specified concentration was administered alone or together with 20 nM poziotinib. combined treatment. After 6 days of treatment, sphere size and viability were measured, and the results are shown in FIG. 18 .

As shown in FIG. 18, the combined treatment of poziotinib and manidipine inhibited the sphere size and proliferation more effectively than the manidipine alone treatment.

<3-3> Expression analysis of factors related to cancer stem cell activity (stemness), survival and growth

In order to confirm the anticancer effect through changes in the expression of markers related to cancer stem cell capacity (stemness) and proteins related to survival and proliferation of cancer stem cells according to the combination treatment of Example 2 of the present invention, A2780-SP cells were seeded to form spheres, and then treated with manidipine at the designated concentration (1 μM) alone or in combination with 5 μM poziotinib for 24 hours. Changes in the expression of stem cell markers were confirmed by Western blotting, and the results are shown in FIG. 19 .

Referring to FIG. 19 , it is confirmed that the expression of cancer stem cell markers is decreased in a concentration-dependent manner according to the combination treatment of poziotinib and manidipine.

In addition, changes in phosphorylation of STAT5, AKT, and ERK, which are EGFR (HER2, 4) sub-signaling markers, and transcription of p85a and p110a were observed, and are shown in FIG. 20 .

Referring to FIG. 20 , it is confirmed that phosphorylation of STAT5, AKT, and ERK is decreased in a concentration-dependent manner according to the combination treatment of poziotinib and manidipine.

Furthermore, changes in nuclear protein expression and phosphorylation of beta-catenin were observed and shown in FIG. 21 .

Referring to FIG. 21 , it was confirmed that nuclear translocation and phosphorylation of beta-catenin were reduced in a concentration-dependent manner according to the combination treatment of poziotinib and manidipine.

From the above results, it can be seen that the combination treatment of poziotinib and manidipine significantly down-regulates the stem cell activity-related signaling pathway of A2780-SP cells through simultaneous inhibition of ErbB4 and L-type calcium channels.

Accordingly, the pharmaceutical composition of the present invention can be usefully used for cancer treatment by reducing the expression of markers related to stemness of cancer stem cells that induce resistance to conventional anticancer drugs.

<3-4> Cell cycle and apoptosis analysis of ovarian cancer stem cells

Ovarian cancer stem cells (A2780-SP) were treated with 1 μM manidipine or 5 μM poziotinib alone or in combination for 24 hours, and flow cytometry showed that early apoptosis (annexin V [AV] ⁺ and propidium iodide [PI] ^- ) and late apoptosis (AV ⁺ /PI ⁺ ), the cell ratio was measured, the results are shown in FIG. 22, and anti-apoptosis markers BCL2, MCL-1 and SURVIVIN expression changes were observed, and shown in FIG. 23 did

Referring to FIG. 22 , treatment with poziotinib or manidipine alone showed a low level of apoptosis, and in the case of combined treatment, apoptosis of ovarian cancer stem cells was significantly improved. 23, it is confirmed that the expression of anti-apoptosis markers 3BCL2, MCL-1 and SURVIVIN is lower in the combined treatment compared to the single treatment, and in particular, the expression of BCL2 is significantly reduced in the control group or the combined treatment compared to the single treatment. .

Claims (18)

delete delete delete delete delete delete delete a compound represented by Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof; And a pharmaceutical composition for preventing or treating cancer containing a calcium channel blocker as an active ingredient,
The calcium channel blocker is at least one selected from the following formulas A to D,
Characterized in that the cancer is ovarian cancer, a pharmaceutical composition for preventing or treating cancer:
[Formula 1]

[Formula A]

[Formula B]

[Formula C]

[Formula D]

.
According to claim 8,
The pharmaceutical composition inhibits ERBB4 (HER 4), inhibits the signal transduction pathway and stem cell function related to self-renewal of cancer stem cells, inhibits sphere formation, viability and proliferation of cancer stem cells, and A pharmaceutical composition characterized in that it induces apoptosis.
According to claim 8,
The pharmaceutical composition is from the group consisting of ALDH, CD133, OCT4, NANOG, KLF4 and SOX2 cancer stem cell markers and c-myc, GLI-1, HIP-1 and PTCH-1 submarkers of Notch and Hedgehog signaling pathways. A pharmaceutical composition characterized in that it inhibits the expression of one or more selected.
According to claim 8,
The pharmaceutical composition is characterized by inhibiting at least one phosphorylation selected from the group consisting of beta-catenin, STAT5, AKT and ERK.
delete delete delete delete delete a compound represented by Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof; And a pharmaceutical composition for preventing or treating cancer containing a calcium channel blocker as an active ingredient,
The calcium channel blocker is at least one selected from the following formulas A to D,
A pharmaceutical composition for preventing or treating recurrence and metastasis of cancer, characterized in that the cancer is ovarian cancer:
[Formula 1]

[Formula A]

[Formula B]

[Formula C]

[Formula D]

.

delete

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