KR20030005860A - An Anticancer Agent Comprising Mepacrine as Active Ingredient - Google Patents

Abstract

PURPOSE: Provided is an anticancer agent containing mepacrine known as phospholipase A2 inhibitor as an effective ingredient and a pharmaceutically acceptable carrier. The mepacrine can selectively inhibit the proliferation of cancer cells when applied to treatment of the cancer cells, it is thus widely used as a safe anticancer agent. CONSTITUTION: The anticancer agent contains mepacrine, together with a pharmaceutically acceptable carrier such as a binding agent, a disintegrating agent, a diluent, a lubricant, a sweetening agent, a stabilizer, a preservative, a flavoring and a mixture thereof. The binding agent is polyvinyl pyrrolidone or hydroxypropyl cellulose, the disintegrating agent is calcium carboxymethyl cellulose or sodium starch glycolate, the diluent is corn starch, lactose, soybean oil, crystalline cellulose or mannitol, the lubricant is magnesium stearate or talc, the sweetening agent is white sugar, fructose, sorbitol or aspartame, the stabilizer is sodium carboxymethyl cellulose, cyclodextrin, vitamin C, citrin acid or white wax, the preservative is methylparaben sodium or propylparaben sodium.

Description

An anticancer agent containing mepacrine as an active ingredient {An Anticancer Agent Comprising Mepacrine as Active Ingredient}

The present invention relates to an anticancer agent comprising mepacrine as an active ingredient. More specifically, the present invention relates to an anticancer agent comprising meparcline known as an inhibitor of phospholipase A2 as an active ingredient and a pharmaceutically acceptable carrier.

Ras is a 21kDa molecular weight protein that is present in all cells and binds to guanine triphosphate (GTP) or guanine diphosphate (GDP) to activate GTP and to deactivate GDP, depending on the type of binding nucleic acid. Depending on the conditions, binding between the lower proteins is determined to activate or inactivate the signaling pathway. Ras proteins are involved in regulating cell proliferation and cell cycle activity through their role as molecular switches, and it is already known to mediate cancerous traits by inducing continuous division of cells upon overactivity. Indeed, in 50% of patients with colon carcinoma and 90% of patients with bladder carcinoma and pancreatic carcinoma, a form of RasV12, an already activated carcinogenic Las mutant gene, is found. .

When Ras receptors located on the cell surface are activated by an external stimulus, this signal is transferred to the GDP-binding inactive Ras protein on the cell membrane, which transfers GDP to GTP and activates the Ras protein. The activated Ras protein transmits signals through well-known MAP kinase chains such as Raf, Mek, and Erk1 / 2, or Rac1 protein, which induces the expression of transcriptional regulators such as fos and increases cell growth and growth. And differentiation. Currently, signaling of Ras protein requires both the MAP kinase chain or Rac protein chain, and it is assumed that it will play a cooperative role.

Signaling of Ras protein in cancer cells induced by Ras protein is expected to be an important part to control the progress and development of cancer, and efforts to carry out cancer therapy by controlling them are continuously performed. However, the treatment of a substance that inhibits electrical signaling is positive because it inhibits not only Ras signaling but also other signaling important to cell metabolism, resulting in the destruction of normal cell metabolism in vivo. Results are not reported.

Thus, there is a constant need to develop a method for selectively inhibiting the signaling of Ras proteins.

Accordingly, the present inventors have made intensive studies to develop a method of selectively inhibiting the signaling of the Ras protein, and as a result, the meparine, which is an inhibitor of phospholipase A2, is located in the Ras protein signaling system. When processed to the generated cancer cells, it was confirmed that the proliferation of cancer cells can be selectively suppressed, the present invention was completed.

After all, the main object of the present invention is to provide an anticancer agent comprising meparkline as an active ingredient.

1 is a photograph showing the effect of mepacline on the growth of cancer cells.

Figure 2 is a graph showing the effect of inhibiting the expression of the Ras gene by mepacline.

Figure 3 is a graph showing the cancer cell proliferation inhibitory effect by meparkak.

The anticancer agent of the present invention is mepacakine as an active ingredient, and includes a pharmaceutically acceptable carrier. Mephacline, also called atbrin, is a quinacrine family of compounds developed for use as an antiparasitic agent for various parasites, and is known to have specific effects on malaria as well as various parasites. It is known as an inhibitor of phospholipase A2, which is known to be involved in signaling through the Rac1 protein, which carries the signal of the protein.

Since electric mepacacine was developed as an insect repellent to be applied to humans, it is known to be safe for the human body. Therefore, in order to determine toxicity against cancer cells rather than normal cells, the fiber of rat transformed with cancer cells in laboratory conditions The blast cells were cultured in a solid medium and treated with mepacline, and the transformed cancer cells were found to effectively inhibit the overexpression of the Ras gene and the proliferation of cancer cells. In addition, cisplastin, which is about 10 times more sensitive to cells transformed into cancerous traits by Ras protein and is administered to mice causing cancer, is cisplastin conventionally used as an anticancer agent. The survival rate was almost similar to that of. Therefore, apart from the activity of normal Ras protein, it can be seen that it is possible to safely and specifically control the proliferation of cancer cells caused by abnormally activated Ras over-activated.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

Example 1 Preparation of Transgenic Cancer Cells

By known methods, rat Rat cells were cultured in 6-well plates in DMEM medium containing 10% (v / v) FBS (890 ml / L DMEM, 10 ml / L non-essential amino acids (100 X), 2 ml / L gentamycin, 1 ml / L fungizon, 100 ml / L FBS) and cultured for 2 days, and injected the intracellular expression vector (pSPORT) containing luciferase cDNA as a marker gene by calcium phosphate precipitation. Rat2-N cells as a control group were prepared, and rat 2-HRas ^V12 cells as experimental groups were prepared by injecting a carcinogenic Ras gene expression vector (pSPORT-HRas ^V12 ) containing luciferase cDNA as a marker gene instead of pSPORT. (Cancer Research, 52: 6877-6884, 1992).

In order to confirm the transformation of the previously prepared Rat2-HRas ^V12 cells, a soft agar assay was performed: That is, the cells were transformed into cancer cells although they had attachment requirements in normal mammalian cell culture. Using a characteristic that changes to anchorage independent growth, a DMEM medium containing 0.6% (w / v) agar is poured into a 100 mm plate to make a solid medium, and 10,000 Rat2-HO6 are placed on top of the electric solid medium. After the culture medium containing the cells (DMEM medium, 10% (v / v) FBS, 0.3% (w / v) agar) was added and cooled to solidify the culture medium, and then incubated for 10 days at 37 ℃, A control group was prepared using Rat2-N cells instead of Rat2-HRas ^V12 cells, and colonies generated in the control group and the experimental group were stained with p-iodonitrotetrazolium violet dye, and colonies were obtained. The number of was counted. As a result, the colonies could not be generated in the control group, but because the colonies were generated in a large amount in the experimental group, transformation to cancer cells was normally performed.

Example 2 Effect of Meparkline

Example 2-1 : Effect of meparkline in solid medium culture

Except for the addition of 1 μM mepacane to the solid medium, using the same method as in Example 1, the soft infants were analyzed to determine the number of colonies generated in the control group and the experimental group (see FIG. 1). ). 1 is a photograph showing the effect of mepacline on the growth of cancer cells. As shown in FIG. 1, it was found that colonization of Rat2-HRas ^V12 cells was inhibited when meparkin was added.

Example 2-2 : Inhibitory Effect of Marker Gene Expression

Cultured Rat-2 cells in the same manner as in Example 1, and the expression vector (pSPORT) was added to calcium phosphate under each DMEM medium to which 0.5% (v / v) FBS and 0, 1 or 2.5 μM mefaqueline were added. (Calcium phosphate) was injected into the cells by precipitation method to prepare Rat2-N as a control group, and injected with a normal Ras gene expression vector (pSPORT-HRas ^WT ) containing luciferase cDNA as a marker gene instead of pSPORT. Phosphorus Rat2-HRas ^WT cells were prepared, and rat 2-HRas ^V12 cells of Experimental Group 2 were prepared by injecting a carcinogenic Ras gene expression vector (pSPORT-HRas ^V12 ) instead of pSPORT. After 6 hours, each control and experimental groups were incubated for 6 hours by exchanging each DMEM medium with 0.5% (v / v) FBS and 0, 1 or 2.5 μM mepaqueline, and each control. And the experimental groups were observed with a fluorescence microscope, and the expression rate of luciferase gene (luciferase) included as a marker gene in the expression vector pSPORT was measured (see Figure 2). Figure 2 is a graph showing the inhibitory effect of Ras gene expression by mepacline, (□) represents a control group, (◆) represents experimental group 1, (■) represents experimental group 2. As shown in Figure 2, when mepacline is treated, since the activity of luciferase does not decrease in cells not containing the Ras gene, it is determined that the introduced expression vector is normally expressed, but in the cells containing the Ras gene, Lucifer It was determined that the activity of Lase decreased in the concentration-dependent manner of mepacline, so that the expression rate of the expression vector was decreased. In particular, in the case of Experimental Group 2 containing carcinogenic Ras gene, the expression rate decreased rapidly.

Example 2-3 : Effect of mepacline on cancer cell proliferation

Inoculated with 1 × 10 ⁵ each of the experimental group and the control group prepared in Example 1 in the solid medium, incubated for 6 hours at 37 ℃, and then added to the 10 μM mefacline, the control group not added mepacline After the experimental group was added to mefacline and incubated for 5 days, using a hemocytometer (hematocytometer) to measure the number of cells surviving in the plate every day (see Figure 3). Figure 3 is a graph showing the effect of inhibiting cancer cell proliferation by mepacline, (■) represents Rat2-HRas ^V12 cells not added mepacline, (◇) is Rat2-HRas ^V12 added mepacline Cells are shown, (□) represents Rat2-N cells to which mepacline is not added, and (◆) represents Rat2-N cells to which mepacline is added. As shown in FIG. 3, when mepacline was not added, rapid proliferation of cancer cells appeared, but when mepacline was treated, the proliferation of cancer cells was gradually suppressed to maintain normal cell levels. It was found that the cells were not significantly affected by mephacline.

Example 2-4 : Measurement of Cell Proliferation Inhibitory Amount of Mepacline

Except for changing the concentration of mepacacin, normal cells and cancer cells were cultured in the same manner as in Example 2-3, and killed and surviving cells were collected using trypan blue staining and a cell counter. By counting separately, the amount of mepacline added (ED ₅₀ ) that inhibits the proliferation of each cell by 50% was measured. As a result, the ED ₅₀ value of normal cells was 6.43mM, whereas the ED ₅₀ value of cancer cells was 0.82mM. Since the mepacline was able to inhibit the proliferation of cancer cells even with a small amount, selective cancer cells It suggested that it can be used as a therapeutic agent.

Example 2-5 : Mepacline's cancer treatment effect

Intraperitoneal of C57BL / 6 male mice were injected with 1 × 10 ⁶ cancer cells p388 leukemia caused by the Ras gene, and control group which was not administered at all, and 1 day later, 100 μg / kg cisplatin, a known anticancer agent. Prepare the control group 1, 2, 3, and 4 administered with 1 μg / kg, 10 μg / kg, 100 μg / kg or 200 μg / kg of mepacline after 1 day and the control group once The survival time of each mouse was measured (see Table 1).

Survival Measurement of Mice Experimental group Survival (days) % Survival rate increase Control 9.8 100 Comparison 16.0 163 Experiment group 1 10.1 104 Experiment group 2 10.9 111 Experiment group 3 12.1 124 Experimental Group 4 14.1 144

As shown in Table 1, it can be seen that mepacline shows a similar level of activity as cisplatin, a known anticancer agent.

Formulation

The anticancer agent of the present invention is mepacline as an active ingredient, and includes a pharmaceutically acceptable carrier, which may be a binder (eg, polyvinylpyrrolidone, hydroxypropyl cellulose), disintegrant (E.g. carboxymethylcellulose calcium, sodium starch glycolate), diluents (e.g. corn starch, lactose, soybean oil, crystalline cellulose, mannitol), glidants (e.g. magnesium stearate, talc), sweeteners (e.g. white sugar, fructose, Sorbitol, aspartame), stabilizers (e.g. sodium carboxymethylcellulose, alpha or beta cyclodextrin, vitamin C, citric acid, white lead), preservatives (e.g. methyl paraoxybenzoate, propyl paraoxybenzoate, sodium benzoate), flavorings (e.g. Ethyl vanillin, masking flavor, menthol flavono, herbal flavor) or mixtures thereof, and tablets, capsules, soft capsules, liquids, ointments, or injections It can be prepared in formulations.

Dosage

The dosage of mepacacine of the present invention is different depending on the age, weight, and the degree of symptoms of the patient, but in the case of adults (based on 60 kg body weight), it is preferable to administer 10-20 mg once or three times daily parenterally. In addition, it may be appropriately determined by the experience of those skilled in the art.

Acute Toxicity Test

In order to determine the acute toxicity of mepacline in the present invention, mepacline was intraperitoneally injected to male C57BL / 6 mice, and LD ₅₀ values were determined by observing the number of deaths of mice over 7 days after administration. _{The 50} value was about 1500 mg / kg. Therefore, in the range of the effective amount indicated above, it was found that the anticancer agent containing the mepacline of the present invention as an active ingredient is a sufficiently safe drug.

As described and demonstrated in detail above, the present invention provides an anticancer agent comprising meparcline known as an inhibitor of phospholipase A2 as an active ingredient and a pharmaceutically acceptable carrier. According to the present invention, when treated with cancer cells mepacline known as an inhibitor of phospholipase A2, it can selectively inhibit the proliferation of cancer cells, it can be widely used in safe chemotherapy.

Claims (10)

An anticancer agent comprising mepacakine as an active ingredient and a pharmaceutically acceptable carrier. The method of claim 1, Pharmaceutically acceptable carriers include binders, disintegrants, diluents, glidants, Sweeteners, stabilizers, preservatives, flavors or mixtures thereof Characterized Anticancer drugs. The method of claim 2, The binder is polyvinylpyrrolidone or hydroxypropylcellulose Characterized by Anticancer drugs. The method of claim 2, The disintegrant is carboxymethyl cellulose calcium or sodium starch glycolate. Characterized by Anticancer drugs. The method of claim 2, Diluents include corn starch, lactose, soybean oil, crystalline cellulose or Characterized in that it is mannitol Anticancer drugs. The method of claim 2, Glidants are characterized in that magnesium stearate or talc Anticancer drugs. The method of claim 2, The sweetener is characterized in that it is sucrose, fructose, sorbitol or aspartame Anticancer drugs. The method of claim 2, Stabilizers include sodium carboxymethylcellulose, cyclodextrin, vitamin C, Characterized in that it is citric acid or white lead Anticancer drugs. The method of claim 2, Preservatives are methyl paraoxybenzoate, propyl paraoxybenzoate or Sodium benzoate, characterized in that Anticancer drugs. The method of claim 2, The fragrance may be ethyl vanillin, masking flavor, menthol flavor or herbal flavor. Characterized Anticancer drugs.