KR102203334B1 - Combination therapy of anticancer drugs and pimozide - Google Patents

Abstract

The present invention relates to an anticancer adjuvant comprising pimozide as an active ingredient, wherein pimozide has an effect of enhancing sensitivity to an anticancer agent, so when administered in combination with an anticancer agent, apoptosis of cancer cells or multiple drug-resistant cancer cells By increasing the, it can be particularly useful in the treatment of anticancer drug-resistant cancer.

Description

Combination therapy of anticancer drugs and pimozide}

The present invention is an anticancer adjuvant comprising pimozide as an active ingredient; And it relates to a composition for anticancer comprising pimozide and an anticancer agent as an active ingredient.

Among the mechanisms that cause anticancer drug-resistant cancer, the most representative method is to overexpress the P-gp (P-glycoprotein) protein in the cell membrane of cancer cells. In other words, resistant cancer cells can be regarded as cells that have evolved as a defense mechanism that overexpresses P-gp and discharges (pumping-out or efflux) anticancer drugs that are toxic to them that have entered the cell. Resistant cancer cells overexpressing P-gp do not die even if the existing anticancer drugs are administered, and because the amount of the anticancer drugs must be increased, the toxicity of the human body due to the anticancer drugs is inevitably increased. Accordingly, there is a need for concurrent administration of a drug capable of maintaining an anticancer agent for a long time in resistant cancer cells by inhibiting overexpression of P-gp or reducing its excretion ability.

Many studies on coadministration drugs that can target resistant cancers that overexpress P-gp have been conducted over the past 10-20 years. Verapamil, a first-generation drug, was developed, and various attempts have been made since then to the development of a third-generation drug, a non-competitive inhibitor, through computer simulation. Although co-administration drugs are continuously being developed, a clinical protocol has not yet been made to prevent overexpression or activity of P-gp and increase the efficiency of anticancer drugs. In particular, when administering a P-gp inhibitor developed because of P-gp, which is also expressed in normal cells, it is becoming a major problem that it affects normal cells in the human body. Therefore, researchers have tried to find drugs that are harmless to normal cells or that specifically act on resistant cancers that overexpress P-gp while maintaining the function of normal cells. With this effort, among plant extracts, substances having an excellent inhibitory effect on P-gp have been reported. For example, curcumin, fumagillin, piperine, etc. are examples.

Korean Patent Publication No. 10-2004-0045882

It is an object of the present invention to provide an anticancer adjuvant comprising pimozide as an active ingredient.

Another object of the present invention is to provide an anticancer composition comprising pimozide and an anticancer agent as an active ingredient.

Another object of the present invention is to provide a method for preventing or treating cancer comprising administering pimozide and an anticancer agent to an individual.

In order to achieve the above object, the present invention provides an anticancer adjuvant comprising pimozide as an active ingredient.

In one embodiment of the present invention, the pimozide may be one to enhance sensitivity to an anticancer agent.

In one embodiment of the present invention, the anticancer agent is Eribulin (Halaven), vincristine (Vincristine), vinblastine (Vinblastine), vinorelbine (Vinorelvine), paclitaxel (Paclitaxel), docetaxel (Docetaxel) ), Etoposide, Topotecan, Irinotecan, Dactinomycin, Doxorubicin, Daunorubicin, Mitomycin, or Bleomycin or Bleomycin Can be.

In one embodiment of the present invention, the pimozide may increase apoptosis of cancer cells or multiple drug-resistant cancer cells.

In one embodiment of the present invention, the pimozide may inhibit the activity of P-gp (P-glycoprotein), which is overexpressed in cancer cells or multidrug resistant cancer cells.

In one embodiment of the present invention, the pimozide may be administered in combination with an anticancer agent, and may be administered simultaneously with the anticancer agent (simultaneous), separately (separate) or sequentially (sequential), but is limited thereto. no.

In one embodiment of the present invention, the pimozide may be administered at a concentration of 1 to 10 μM, preferably at a concentration of 5 to 10 μM, more preferably at a concentration of 5 μM It may be administered as.

In one embodiment of the present invention, the cancer may be an anticancer drug-resistant cancer, and specifically, breast cancer, ovarian cancer, colon cancer, liver cancer, thyroid cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, prostate cancer, esophageal cancer, cervical cancer, colon cancer , Bladder cancer, central nervous system tumor, oral cancer, and may be selected from the group consisting of brain tumors.

In addition, the present invention provides an anticancer composition comprising pimozide and an anticancer agent as active ingredients.

In addition, the present invention provides a method for preventing or treating cancer comprising administering a pimozide and an anticancer agent to an individual.

The composition according to the present invention has an effect of enhancing sensitivity to an anticancer agent, and when administered in combination with an anticancer agent, by increasing apoptosis of cancer cells or multidrug resistant cancer cells, it can be particularly useful for the treatment of anticancer drug-resistant cancer.

1A shows KBV20C cells were treated with 5 μM pimozide (PIM-5), 10 μM or 20 μM verapamil (VER-10, VER-20, positive control) for 24 hours, and then stained with rhodamine 123. This is the result of FACS analysis (Con: control, 0.1% DMSO treatment group).
Figure 1B is a KBV20C cell for 24 hours 50 ng / ml halaben (HAL), 10 μM verapamil (VER), 10 μM pimozide (PIM), halaben + verapamil (HAL + VER) and halaben + After treatment with pimozide (HAL+PIM), annexin V analysis results are shown (Con: control group, 0.1% DMSO treatment group).
Figure 2A is a KBV20C cell for 24 hours 50 ng / ml of halaben (HAL), 5 μM, 10 μM of pimozide (PIM-5, PIM-10) or halaben + pimozide (HAL + PIM-5, HAL+PIM-10), and then cell cycle analysis through FACS (Con: control group, 0.1% DMSO treatment group).
2B and 2C show that KBV20C cells were treated with 50 ng/ml of halaben (HAL), 5 μM of pimozide (PIM-5) or halaben+pimozide (HAL+PIM-5) for 24 hours, Results of analyzing the expression levels of CDK4 (Cyclin-dependent kinase 4), p21, pRb, Cyclin E, CDC2, Cyclin B1, Survivin, pH2AX (phosphorylated-H2A histone family member X), pAkt, pJnk, and p38 through Western blot It is (Con: control, 0.1% DMSO treatment group).
Figure 3A is a KBV20C cell for 24 hours 5 nM of vincristine (VIC), 10 μM of verapamil (VER), 10 μM of pimozide (PIM), vincristine + verapamil (VIC + VER) or vincristine + pimozide After treatment with (VIC+PIM), the results of Annexin V analysis are shown (Con: control group, 0.1% DMSO treatment group).
Figure 3B is a KBV20C cell for 24 hours 5 nM vincristine (VIC), 10 μM verapamil (VER), 10 μM pimozide (PIM), vincristine + verapamil (VIC + VER) or vincristine + pimozide It is the result of cell cycle analysis through FACS after treatment with (VIN+PIM) (Con: control group, 0.1% DMSO treatment group).
Figure 4A shows KBV20C cells treated with 5 μM, 10 μM fluphenazine (FLU-5, FLU-10) or 5 μM, 10 μM pimozide (PIM-5, PIM-10) for 24 hours, This is the result of staining with rhodamine 123 and analyzed by FACS (Con: control group, 0.1% DMSO treatment group).
Figure 4B is a KBV20C cell for 24 hours 5 μM fluphenazine (FLU), 5 μM pimozide (PIM), 50 ng / ml halaben (HAL), halaben + fluphenazine (HAL + FLU) Alternatively, after treatment with halaben+pimozide (HAL+PIM), cells were observed under a microscope (x4) (Con: control group, 0.1% DMSO treated group; and scale bar=100 μm).

The term of the present invention, "drug-resistance" refers to a symptom that exhibits extremely low sensitivity to an anticancer treatment regimen, and thus the symptoms of cancer are not improved, alleviated, alleviated or treated by the treatment regimen. Anticancer drug resistance may appear because cancer may be resistant to a specific anticancer treatment regimen from the beginning, and initially did not show resistance, but due to a long period of treatment, the properties of cancer cells change and thus no longer show susceptibility to the same treatment. .

The term "anti-cancer adjuvant" of the present invention is an agent capable of improving, enhancing or enhancing the anti-cancer effect of an anti-cancer agent, and does not exhibit anti-cancer activity by itself, but when used together with an anti-cancer agent, it improves or enhances the anti-cancer effect of the anti-cancer agent. Or it may be a formulation that can be increased. In addition, when an agent exhibiting concentration-dependent anticancer activity is used with an anticancer agent at a level that does not exhibit anticancer activity by itself, it may be an agent capable of improving, enhancing or increasing the anticancer effect of the anticancer agent.

The administration route of the anticancer adjuvant may be administered through any general route as long as it can reach the target tissue. The anticancer adjuvant of the present invention may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, intranasally, intrapulmonary, or rectal administration as desired, but is not limited thereto. Does not. In addition, the anticancer adjuvant may be administered by any device capable of moving the active substance to target cells.

The composition according to the present invention may further comprise a suitable carrier, excipient or diluent commonly used in the preparation of the composition.

Carriers, excipients or diluents that can be used in the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, Methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil.

The composition according to the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. have.

In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations include at least one excipient, such as starch, calcium carbonate, and sucrose ( sucrose) or lactose (lactose), gelatin, etc. are mixed to prepare.

In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, and preservatives may be included. .

The composition of the present invention may be administered in a pharmaceutically effective amount, and the term "pharmaceutically effective amount" of the present invention means an amount sufficient to treat or prevent a disease at a reasonable benefit/risk ratio applicable to medical treatment or prevention. The effective dose level is the severity of the disease, the activity of the drug, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the time of administration of the composition of the present invention used, the route of administration and the rate of excretion of the treatment period, It can be determined according to factors including drugs used in combination or coincidental with the composition of the present invention and other factors well known in the medical field.

The dosage of the composition of the present invention can be determined by those skilled in the art in consideration of the purpose of use, the degree of addiction to the disease, the patient's age, weight, sex, history, or the type of substance used as an active ingredient. For example, the pharmaceutical composition of the present invention may be administered in an amount of about 0.1 ng to about 100 mg/kg, preferably 1 ng to about 10 mg/kg per adult, and the frequency of administration of the composition of the present invention is specifically Although not limited, it may be administered once a day or divided into several doses. The above dosage does not in any way limit the scope of the present invention.

Hereinafter, the present invention will be described in more detail through examples. These examples are for explaining the present invention more specifically, and the scope of the present invention is not limited to these examples.

Example 1. Experimental method

1.1. Resistant cell line used

In the present invention, KB cells, a human oral squamous carcinoma cell line, and KBV20C cells (Dr. Yong Kee Kim, College of Pharmacy, Sookmyung Women's University), a multidrugresistant subline thereof, were used. . KBV20C cells are a cell line that has become resistant to anticancer agents through overexpression of P-gp (P-glycoprotein) by treating KB cells with anticancer agents for a long time. The cells were cultured in RPMI 1640 medium (WelGENE, Daegu, South Korea) containing 10% FBS (fetal bovine serum), 100 U/ml penicillin and 100 μg/ml streptomycin.

1.2. Rhodamine 123 staining

KBV20C cells were dispensed in a 60-mm dish with 30-40% confluence, and 0.1% DMSO (Con, control), pimozide, PIM) or verapamil (VER, positive control). Thereafter, the cells were treated with 2 μg/ml of rhodamine 123 for about 1 hour at 37° C., the medium was removed, and then washed with PBS. Stained cells were analyzed using a Guava EasyCyte Plus Flow Cytometer (Merck Millipore, Burlington, MA, USA).

1.3. Annexin V staining

KBV20C cells were dispensed with 30-40% confluence in a 60-mm dish, 0.1% DMSO (Con, control), 10 μM of pimozide, PIM), 10 μM of verapamil (VER, positive control), 50 ng/ml of Halaven (HAL) alone or a combination thereof were treated. Thereafter, the cells were detached with trypsin, centrifuged to obtain a pellet, and washed with PBS. Cells were reacted at room temperature for 15 minutes by adding 100 μl of binding buffer to which 5 μl of Annexin V-FITC and 5 μl of PI were added. The stained cells were analyzed for the degree of apoptosis using a Guava EasyCyte Plus Flow Cytometer (Merck Millipore, Burlington, MA, USA).

1.4. Western Blot

KBV20C cells were dispensed in a 60-mm dish with 30-40% confluence, 0.1% DMSO (Con, control), 5 μM of pimozide, for 24 hours. PIM), 50 ng/ml of Halaven (HAL) alone or a combination thereof. Thereafter, the cells were washed twice with cold PBS and removed with a scraper. For total protein isolation, cells were suspended in PRO-PREP™ protein extract solution (iNtRON, Seongnam, South Korea) and placed on ice for 30 minutes. Then, it was centrifuged at 4° C. and 15,000× g for 5 minutes. The amount of protein was measured according to the manufacturer's instructions using a protein assay kit (Bio-Rad, Hercules, CA, USA). Thereafter, SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was performed, followed by blotting with a membrane, and protein expression levels were analyzed using antibodies specific for each protein.

1.5. Cell cycle analysis

KBV20C cells were dispensed with 30-40% confluence in a 60-mm dish, and 0.1% DMSO (Con, control), 5 μM or 10 μM of pimozide (pimozide, PIM), 50 ng/ml of Halaven (HAL) alone or a combination thereof. Thereafter, the cells were detached with trypsin and centrifuged to obtain a pellet, washed with PBS, and then suspended by adding 75% ethanol at 4° C. for 1 hour. Then, it was washed with PBS and resuspended for 30 minutes in a cold PI (propidium iodide) solution (100 μg/ml RNase A and 50 μg/ml PI in PBS). Stained cells were analyzed for cell cycle using a Guava EasyCyte Plus Flow Cytometer (Merck Millipore, Billerica, MA, USA).

Example 2. Inhibition of P-gp (P-glycoprotein) activity of pimozide

The present inventors performed rhodamine staining (Rhodamine 123 staining) to evaluate whether the drug inhibits P-gp (P-glycoprotein) activity. Rhodamine is a substrate of P-gp, and when the activity of P-gp is inhibited, rhodamine accumulates in cells, so it is possible to quantitatively analyze the inhibition of P-gp activity through the amount of rhodamine.

As a result, it was confirmed that when 5 μM of pimozide was treated on KBV20C cells, a resistant cell line, for 24 hours, it showed similar degree of inhibition of P-gp activity as when treated with 10 μM of verapamil, a positive control (Fig. 1A).

Accordingly, the present inventors confirmed that the inhibitory effect of P-gp activity was remarkable in resistant cell lines even when pimozide was treated at a low concentration.

Example 3. Effect of increasing apoptosis of resistant cell lines by pimozide and anticancer agent (Eribulin, Halaven)

The present inventors performed annexin V staining, which can measure the increase in early and late apoptosis, in order to confirm the apoptosis effect after co-treatment with pimozide and an anticancer agent on KBV20C cells, which are resistant cell lines. Performed.

As a result, when the resistant cell line, KBV20C cells, were treated with pimozide and halaben in combination, the apoptosis effect was increased compared to the case of treatment with halaben alone, which is similar to that of the combination treatment of halaben and verapamil. It was confirmed that it exhibited the apoptosis effect of (Fig. 1B).

Accordingly, the present inventors confirmed that the effect of increasing the apoptosis of resistant cell lines even at low concentrations when pimozide and an anticancer agent are co-treated.

Example 4. Results of cell cycle analysis of resistant cell lines by pimozide and anticancer agent (Eribulin, Halaven)

The present inventors performed cell cycle analysis in order to confirm whether they affect the cell cycle after treatment with pimozide and an anticancer agent in combination with KBV20C cells, which are resistant cell lines. As a result, when the resistant cell line KBV20C cells were treated in combination with pimozide and halaben, the G2 phase was significantly increased compared to the case of treatment alone (FIG. 2A). In particular, when 5 μM of pimozide was co-treated with halaben, the G2 phase was 42%, and when 10 μM of pimozide was co-treated with halaben, the G2 phase was 56%, which significantly increased G2 arrest. It was confirmed that it was lost.

Accordingly, the present inventors confirmed that G2 arrest increased when pimozide and an anticancer agent were treated in combination.

Example 5. Analysis results of cell cycle-related factors or apoptosis-related factors by pimozide and anticancer agents (Eribulin, Halaven)

The present inventors described cell cycle-related factors such as CDK4 (Cyclin-dependent kinase 4), p21, pRb, Cyclin E, CDC2 and Cyclin B1, and apoptosis-related factors such as Survivin, pH2AX (phosphorylated-H2A histone family member X), pAkt, pJnk And Western blot was performed to confirm the expression level of the p38 protein.

As a result, when the resistant cell line KBV20C cells were treated in combination with pimozide and halaben, the expression level of pRB was significantly increased, and the expression level of Cdc2 was significantly decreased (Fig. 2B and 2C), it was confirmed that this was the result consistent with G2 arrest in the cell cycle analysis.

Accordingly, the present inventors confirmed that the apoptosis of resistant cell lines can be increased through G2 arrest when pimozide and an anticancer agent are co-treated.

Example 6. Effect of pimozide and anticancer agent (vincristine)

The present inventors conducted an experiment to analyze whether pimozide has an effect on cell proliferation or apoptosis in combination treatment with not only halaben but also other anticancer agents. Briefly, KBV20C cells were dispensed with 30-40% confluence in a 60-mm dish, and 0.1% DMSO (Con, control), 10 μM of pimozide, for 24 hours. PIM), 10 μM of verapamil, 5 nM of Vincristine (VIC) alone or a combination thereof were treated. Then, Annexin V analysis and cell cycle analysis were performed.

As a result, when the anticancer agent vincristine was treated alone or pimozide or verapamil was treated alone, there was no difference from the control group, but when pimozide or verapamil was treated in combination with vincristine, apoptosis was significantly increased. In particular, pimozide had an effect of significantly increasing apoptosis than verapamil (FIG. 3A).

In addition, in the cell cycle, when vincristine was treated alone or pimozide or verapamil was treated alone, there was no difference from the control group, but when pimozide or verapamil was treated in combination with vincristine, G2 arrest was remarkable. In particular, pimozide had the effect of increasing G2 arrest more remarkably than verapamil (Fig. 3B).

Accordingly, the present inventors have confirmed that the combination treatment of pimozide and various anticancer agents has the effect of remarkably increasing apoptosis through G2 arrest of resistant cell lines at a low concentration.

Example 7. Comparison of the effects of pimozide and fluphenazine

Through a relative comparison with fluphenazine, which the present inventors reported in the paper 3 years ago, it was analyzed to what extent pimozide has an excellent effect. That is, in order to compare the effects of pimozide and fluphenazine, the degree of inhibition of P-gp activity was measured through Rhodamine123 staining. Briefly, KBV20C cells were dispensed with 30-40% confluence in a 60-mm dish, and 0.1% DMSO (Con, control), 5 μM, 10 μM of pimozide (pimozide, PIM) or 5 μM, 10 μM of fluphenazine (FLU). Then, after staining with rhodamine, it was analyzed by FACS.

As a result, it was confirmed that pimozide had an excellent inhibitory effect on P-gp activity than fluphenazine at a high concentration even at a low concentration of 5 μM (FIG. 4A).

In addition, the present inventors analyzed the cell proliferation or apoptosis effect of pimozide and fluphenazine. Briefly, KBV20C cells were dispensed with 30-40% confluence in a 60-mm dish, 0.1% DMSO (Con, control), 5 μM of pimozide, for 24 hours. PIM), 5 μM of Fluphenazine (FLU), 50 ng/ml of Halaven (HAL) alone or a combination thereof were treated. Thereafter, the effect of cell proliferation or apoptosis was analyzed under a microscope. As a result, it was confirmed that apoptosis was significantly increased when 5 μM of pimozide and halaben were treated in combination, compared to the case where 5 μM of fluphenazine and halaben were treated in combination (FIG.

Accordingly, the present inventors have found that pimozide has a more remarkable P-gp inhibitory effect than fluphenazine, and when pimozide is treated in combination with an anticancer agent, it can significantly induce apoptosis of resistant cell lines even at a lower concentration than the existing fluphenazine. It was confirmed that it can be.

Claims (15)

Pimozide; And as an anticancer adjuvant comprising Halaven or Vincristine as an active ingredient,
The anticancer adjuvant is used in anticancer drug-resistant cancer, cancer in which p-gp is overexpressed,
The cancer in which p-gp is overexpressed is a cancer in which susceptibility to anticancer drugs is reduced by overexpression of p-gp,
The anticancer adjuvant, characterized in that to increase the sensitivity to the anticancer agent in cancer cells. delete delete The method of claim 1,
The anticancer adjuvant, characterized in that to increase the apoptosis of cancer cells or multi-drug resistant cancer cells. delete delete The method of claim 1,
The anticancer adjuvant, characterized in that the pimozide is administered simultaneously with halaben or vincristine (simultaneous), separately (separate) or sequentially (sequential). The method of claim 1,
The anticancer adjuvant, characterized in that the pimozide is administered at a concentration of 5 to 10 μM. delete The method of claim 1,
The cancer is selected from the group consisting of breast cancer, ovarian cancer, colon cancer, liver cancer, thyroid cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, prostate cancer, esophageal cancer, cervical cancer, colon cancer, bladder cancer, central nervous system tumor, oral cancer, and brain tumor. Anticancer adjuvant. Pimozide; And as an anticancer composition comprising halaven or vincristine as an active ingredient,
The anticancer composition is used for cancers in which p-gp is overexpressed in anticancer drug-resistant cancer,
The cancer in which p-gp is overexpressed is a cancer in which susceptibility to anticancer drugs is reduced by overexpression of p-gp,
The anticancer composition is an anticancer composition, characterized in that it increases the sensitivity to anticancer agents in cancer cells. delete The method of claim 11,
The composition, characterized in that the pimozide is administered at a concentration of 5 to 10 μM. delete The method of claim 11,
The cancer is selected from the group consisting of breast cancer, ovarian cancer, colon cancer, liver cancer, thyroid cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, prostate cancer, esophageal cancer, cervical cancer, colon cancer, bladder cancer, central nervous system tumor, oral cancer, and brain tumor. Composition made by.

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