HK1241732B - Pharmaceutical composition for inhibiting growth of cancer stem cells, containing aldehyde inhibitor and biguanide-based compound - Google Patents

Description

Composition of a drug for inhibiting the growth of cancer stem cells containing an aldehyde inhibitor and a biguanide compound

Technical Field

The present invention relates to a pharmaceutical composition for inhibiting the growth of cancer stem cells, comprising an aldehyde inhibitor and a biguanide compound.

Background Art

Cancer is one of the most common causes of death worldwide. With approximately 10 million new cases of cancer each year, cancer accounts for approximately 12% of all deaths, making it the third leading cause of death.

Among various cancers, brain cancer is particularly characterized by its characteristic of occurring regardless of age, with a higher frequency in infants than in other cancers. Brain cancer as a whole refers to primary brain cancers that arise in the brain tissue and surrounding meninges, as well as secondary brain cancers that arise from metastases from cancers in the skull or other parts of the body. This type of brain cancer differs from cancers that arise in other organs in many ways. Specifically, cancers that arise in organs such as the stomach, lungs, and breasts are limited to one or two types in each organ and generally share the same or similar characteristics. However, in the brain, a wide variety of cancers arise, including, for example, glioblastoma multiforme, malignant gliomas, lymphomas, germ cell tumors, metastatic tumors, and others.

Among these brain cancers, gliomas, particularly glioblastoma multiforme (GBM), are the most malignant and aggressive, and therefore a very deadly disease with a very poor prognosis, with an average survival of approximately one year or less after diagnosis. Because the boundary between brain cells and tumor cells is unclear, complete removal of GBM by surgery is nearly impossible.

Despite advances in cancer treatment, the mainstays of treatment currently include surgery, radiation, and chemotherapy. Chemotherapy is primarily used to treat metastatic, or particularly aggressive, cancers. Most cancer therapeutics currently used in clinical practice are cytotoxins. Cytotoxic agents work by destroying or killing cells that exhibit rapid growth.

An ideal cytotoxic agent would be specific for cancer and tumor cells, while leaving normal cells unaffected. Unfortunately, such an ideal cytotoxic agent has not yet been discovered, and instead agents that specifically target rapidly dividing cells (tumor and normal cells) are used. Therefore, there is a great need for materials that are cytotoxic to cancer cells but have only mild effects on normal cells. Indeed, much recent research has focused on developing alternative anticancer agents that can specifically inhibit the growth of tumor cells.

Therefore, there is an urgent need to develop chemotherapeutic agents other than surgical treatment, but effective treatments have not yet been developed, so research and development are needed.

Technical issues

The purpose of the present invention is to provide a pharmaceutical composition that can effectively inhibit the growth of cancer stem cells, thereby inhibiting the proliferation, invasion and metastasis of cancer cells, thereby preventing and/or treating cancer.

Technical Solution

The present inventors conducted extensive research and discovered that co-administration of an aldehyde inhibitor and a biguanide compound can inhibit the growth of cancer stem cells, thereby suppressing the proliferation, invasion, and metastasis of cancer cells, thereby preventing and/or treating cancer. Based on this discovery, the present invention was completed.

As used herein, the term "cancer stem cell" generally refers to a cancer cell with self-renewal or differentiation potential, which is a characteristic potential of stem cell characteristics. For example, cancer stem cells may include neurospheres of stem cells in the central nervous system of the brain. Unlike general cancer cells, under normal tumor growth conditions ("normal tumor growth conditions" refers to a state in which the nutrients (glucose) required for cell growth are sufficient and the tumor microenvironment growth conditions are rich, so there is no cell stress), cancer stem cells can proliferate at a slower rate, or can be maintained in a dormant state, and therefore may be resistant to anticancer agents. For example, unlike normal tumor cells, cancer stem cells can control the expression of transcriptional regulators such as PGC-1a, so the function of the main metabolic regulatory substances may be different from that of common cancer cells. Therefore, the term "cancer stem cell" generally refers to a cell that is resistant to apoptosis under nutrient deficiency conditions by regulating the cell signaling system connected to its mechanism, and has invasiveness and/or metastasis potential. However, cancer stem cells are not limited thereto, and may include any cell that can differentiate into a general cancer cell.

As used herein, the expression "inhibiting the growth of cancer stem cells" is intended to include inhibiting cancer stem cell maintenance, inhibiting cancer stem cell malignancy, and inhibiting cancer stem cell invasion.

Specifically, the present invention relates to a pharmaceutical composition for inhibiting the growth of cancer stem cells, comprising an aldehyde inhibitor and a biguanide compound. Preferably, the aldehyde inhibitor can be gossypol, and the biguanide compound can be phenformin.

Here, "gossypol" is a phenol derivative found in large quantities in cotton plants. In China, it has been found to inhibit male sperm function. Consequently, gossypol has been studied as a male oral contraceptive. Furthermore, "phenformin," commonly known as a diabetes treatment, physiologically regulates carbohydrate and lipid metabolism.

In the present invention, the combination of gossypol and phenformin preferably exhibits a very high synergistic effect in inhibiting the growth of cancer stem cells. Here, gossypol is preferably a compound represented by the following formula 1 or a derivative thereof, but is not limited thereto. Phenformin is preferably a compound represented by the following formula 2 or a derivative thereof, but is not limited thereto:

Formula 1

Formula 2

In the pharmaceutical composition of the present invention, the weight ratio of the aldehyde inhibitor to the biguanide compound can be 1:1-100, preferably 1:2-20.

Furthermore, in the pharmaceutical composition of the present invention, the content of the aldehyde inhibitor may be 0.5 to 50 μM.

In addition, in the pharmaceutical composition of the present invention, the content of the biguanide compound can be 10 to 1000 μM.

As described above, the compositions of the present invention can inhibit the growth of cancer stem cells, thereby preventing and/or treating cancers selected from the group consisting of uterine cancer, breast cancer, gastric cancer, brain cancer, rectal cancer, colorectal cancer, skin cancer, blood cancer, and liver cancer. Preferably, the compositions of the present invention can inhibit the proliferation, maintenance, malignancy, and invasiveness of neurospheres, thereby effectively preventing and/or treating brain cancer, particularly glioblastoma.

However, the pharmaceutical composition of the present invention can be used together with other additional anticancer agents to effectively treat not only cancer stem cells but also general cancer cells.

The anticancer agent that can be used in the present invention can be one or more selected from the following group: nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, neratinib, lapatinib, gefitinib, vandetanib, nilotinib, semaxanib, bosutinib, axitinib, cediranib, lestaurinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, sorafenib, bevacizumab, cisplatin, cetuximab, quercetin, Parasitic, asparaginase, retinoic acid, hydroxycoduride, dasatinib, estramustine, ozogamicin, ibritumomab tiuxetan, epazocine, aminolevulinic acid, amsacrine, alemtuzumab, procarbazine, alprostadil, holmium nitrate chitosan, gemcitabine, doxorubicin, pemetrexed, tegafur, capecitabine, gimeracil, oteracil, azacitidine, methotrexate, uracil, cytarabine, fluorouracil, fludarabine, enoxaparin Tadalafil, flutamide, decitabine, mercaptopurine, thioguanine, cladribine, carmofur, raltitrexed, docetaxel, paclitaxel, irinotecan, belotecan, topotecan, vinorelbine, etoposide, vincristine, vinblastine, teniposide, doxorubicin, epirubicin, mitoxantrone, mitomycin, bleomycin, daunorubicin, actinomycin D, pirarubicin, aclarubicin, peplomycin, sirolimus, temozolomide, Busulfan, ifosfamide, cyclophosphamide, melphalan, altretamine, dacarbazine, thiotepa, nimustine, chlorambucil, dulcitol, leucovorin, tretonine, exemestane, aminoglutethimide, anagrelide, vinorelbine, fadrozole, tamoxifen, toremifene, testolactone, anastrozole, letrozole, vorozole, bicalutamide, lomustine, and carmustine, but are not limited thereto.

In the present invention, the pharmaceutical composition can be in the form of capsules, tablets, granules, injectable solutions, ointments, powders or beverages. The pharmaceutical composition can be used for human administration.

For use, according to conventional methods, the pharmaceutical composition of the present invention can be formulated into oral preparations, including powders, granules, capsules, tablets, aqueous suspensions, etc., skin external preparations, suppositories and sterile injection solutions, but are not limited thereto. The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier of the present invention includes binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments and fragrances that can be used for oral administration, and buffers, preservatives, analgesics, solubilizers, isotonic agents and stabilizers that can be used for injection, and bases, excipients, lubricants, preservatives, etc. that can be used for topical administration. The pharmaceutical composition of the present invention can be formulated in various ways by mixing with the pharmaceutically acceptable carriers described above. For example, for oral administration, the pharmaceutical composition of the present invention can be formulated into tablets, lozenges, capsules, elixirs, suspensions, syrups and wafers, etc., and for injections, can be formulated into single-dose ampoules or multi-dose vials. In addition, the pharmaceutical composition of the present invention can be formulated into solutions, suspensions, tablets, capsules, and sustained-release preparations, etc.

At the same time, examples of carriers, excipients and diluents suitable for the preparation include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, the pharmaceutical composition of the present invention may also contain fillers, anticoagulants, lubricants, wetting agents, spices, emulsifiers and preservatives.

The routes of administration of the pharmaceutical composition of the present invention include, but are not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardial, transdermal, subcutaneous, intraperitoneal, intranasal, gastrointestinal, topical, sublingual, and rectal routes. Oral or parenteral administration is preferred. As used herein, the term "parenteral" refers to, including subcutaneous, transdermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intradural, intralesional, and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention can also be formulated into a suppository for rectal administration.

The pharmaceutical composition of the present invention can change according to various factors, including the activity of the specific compound used, the patient's age, body weight, general health, sex, diet, administration time, route of administration, excretion rate, drug content and the severity of the specific disease to be prevented or treated. The dosage of the pharmaceutical composition can be suitably selected by those skilled in the art according to the patient's condition, body weight, the severity of the disease, the form of the medicine and the route of administration and administration time, and can be 0.0001-50mg/kg/day or 0.001-50mg/kg/day. The pharmaceutical composition can be administered once or several times a day. Dosage is not intended to limit the scope of the present invention in any way. Pharmaceutical composition according to the present invention can be formulated into pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries or suspensions.

Beneficial effects

The pharmaceutical composition of the present invention contains a combination of an aldehyde inhibitor and a biguanide compound, which can effectively inhibit the growth of tumor stem cells such as neurosphere cells, and can also inhibit the proliferation, invasion and metastasis of cancer cells, thereby preventing and/or treating cancers such as brain cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows the results of MTT assay performed at different concentrations of gossypol in Reference Example 1.

FIG2 shows changes in the viability of U87 cells after treatment with each composition of Examples and Comparative Examples in Experimental Example 1.

FIG3 shows photographs of U87 neurosphere cells after various treatments in Experimental Example 2. FIG.

FIG. 4 shows the changes in the radius of U87 neurosphere cells after various treatments in Experimental Example 2. FIG.

FIG. 5 shows the extent of U87 neurosphere cell formation after various treatments in Experimental Example 2. FIG.

FIG6 shows photographs of U87 neurosphere cells after various treatments in Experimental Example 3.

FIG. 7 shows the extent of brain cancer development in orthotopic xenograft model mice after various treatments in Experimental Example 4. FIG.

FIG. 8 shows the survival rate of orthotopic xenograft model mice after various treatments in Experimental Example 4. FIG.

Best Mode

The present invention provides a pharmaceutical composition comprising an aldehyde inhibitor and a biguanide compound, which can effectively inhibit the growth of cancer stem cells such as neurospheres, thereby inhibiting the proliferation, invasion and metastasis of cancer cells, thereby preventing and/or treating cancers such as brain cancer.

Invention Mode

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these Examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example

Reference Example 1: Analysis of cell viability after treatment with gossypol

U87 cells (GBM cells) were treated with 0.5, 1, 5, 10, and 50 μM gossypol for 72 hours ( FIG. 1 ).

As shown in FIG1 , when cells were treated with various concentrations of gossypol for 72 hours, cell growth was inhibited.

Experimental Example 1: Analysis of cell viability after treatment with a combination of gossypol and phenformin

U87 cells were seeded onto 96-well plates and cultured at 37°C for 24 hours. The cells were then treated with the pharmaceutical compositions of Examples and Comparative Examples listed in Table 1 below. The cells were then treated with 20 μL/well of MTS reagent and incubated at 37°C for 4 hours. Next, the absorbance at 490 nm was measured, and the change in absorbance relative to the untreated control was calculated to determine cell viability. The results are shown in Figure 2.

Table 1

Composition Comparative Example 1 - Comparative Example 2 0.5 μM gossypol Comparative Example 3 1 μM gossypol Comparative Example 4 10 μM phenformin Example 1 10 μM phenformin + 0.5 μM gossypol Example 2 10 μM phenformin + 1 μM gossypol

As shown in FIG2 , when cells were treated with gossypol and phenformin in combination, cell viability was significantly reduced compared to cells treated with gossypol or phenformin alone.

Experimental Example 2: Neurosphere Formation Analysis

U87 cells were cultured in DMEM/F-12 medium containing 2 wt% 1xB27, 0.02 wt% bFGF (20 ng/ml), 0.02 wt% EGF (20 ng/ml), and 50 U/ml penicillin-50 mg/ml streptomycin (100x, Gibco, Invitrogen Korea, Seoul, South Korea) to form tumor spheres. Next, the cells were seeded into 96-well plates at a density of 10 cells/well and treated with 1 μM gossypol, 10 μM gossypol, 10 μM phenformin, a combination of 1 μM gossypol and 10 μM phenformin, or a combination of 10 μM gossypol and 10 μM phenformin. The cells were then cultured at 37°C for 3 weeks. To observe the morphology and size of U87 cells, the obtained cell cultures were observed using an inverted phase-contrast microscope (1x71 inverted microscope; Olympus, Tokyo, Japan) and photographed using a digital camera (DP70 digital microscope camera; Olympus). The photographs are shown in Figure 3. Furthermore, the changes in the radius of neurospheres following various treatments are shown in Figure 4, and the extent of neurosphere formation is shown in Figure 5.

In Figure 4 , the percentage change in neurosphere radius is expressed as the percentage of the average radius of neurospheres after various treatments relative to the average radius of neurospheres seeded on a 96-well plate. In Figure 5 , the degree of neurosphere formation is expressed as the percentage of the number of neurosphere cells after each treatment relative to the number of neurosphere cells seeded on a 96-well plate.

As can be seen from the cell photographs in FIG3 , even when the neurosphere cells were treated with gossypol or phenformin alone, the size of the neurosphere cells decreased, but when the neurosphere cells were treated with a combination of gossypol and phenformin, no neurosphere cells were observed.

Furthermore, as shown in Figures 4 and 5 , the radius of the neurosphere cells was significantly decreased when the cells were treated with the combination of gossypol and phenformin, and the number of neurosphere cells was also significantly decreased when treated with the gossypol and phenformin combination, compared to when the cells were treated with gossypol or phenformin alone.

Experimental Example 3: Transwell invasion assay

U87 cells (2×10 ⁵ cells/well) were suspended in 0.1 ml of growth medium and then treated with 1 μM gossypol, 10 μM gossypol, 10 μM phenformin, a combination of 1 μM gossypol and 10 μM phenformin, or a combination of 10 μM gossypol and 10 μM phenformin. The cells were then added to the upper well of a transwell chamber (8 mm pore size; Corning glass). Here, the upper portion of the transwell chamber was infused with 0.5 ml of growth medium, and the upper portion was pre-coated with 8.4 mg/ml of Matrigel (Corning Matrigel matrix) with reduced growth factors. The cells in the chamber were cultured at 37°C for 48 hours, after which non-invading cells on the upper surface of the filter were removed with a cotton swab. Cells that had migrated to the lower surface of the filter were fixed and stained with a Diff-Quick kit (Fisher), and the resulting cell culture was observed using a phase contrast microscope (Olympus). A photograph of the cell culture is shown in Figure 6. To determine cell invasion, the number of cells in 10 microscopic fields per well was counted.

As shown in Figure 6 , the radius of the neurosphere cells was significantly decreased when the cells were treated with the combination of gossypol and phenformin, and the number of neurosphere cells was also significantly decreased when treated with the gossypol and phenformin combination, compared to when the cells were treated with gossypol or phenformin alone.

This indicates that compared with the treatment with gossypol or phenformin alone, the combined treatment of gossypol and phenformin of the present invention can effectively inhibit the proliferation and metastasis of brain cancer stem cells, thereby significantly increasing the therapeutic effect of brain cancer.

Experimental Example 4: Evaluation of the Effects of Gossypol in an Orthotopic Xenograft Model

Gossypol and phenformin used in animal studies were dissolved in DMSO and PBS, respectively. Co-administered combinations of gossypol and phenformin were dissolved in PBS containing 10 wt% DMSO and 1 wt% hydrogenated castor oil.

For the construction of the orthotopic xenograft model, 4-8 week old male athymic nude mice (Central Laboratory, South Korea) were used. For stabilization, mice were kept in a sterile environment for at least one week before experimental use and given adequate diet. All protocols in the animal study were approved by the Yonsei University Institutional Animal Care and Use Committee. First, mice were anesthetized by intraperitoneal injection of 30 mg/kg of Zotai and 10 mg/kg of Xylazine, and 2×10 ⁵ U87-luc cells were transplanted into the right frontal lobe of the brain to a depth of 4.5 mm using a Hamilton syringe. U87-luci cells were injected simultaneously into 5 mice of the same group at a rate of 0.5 μl/min by a microinjection syringe pump. Thereafter, gossypol (40 mg/kg) and/or phenformin (100 mg/kg) were orally administered daily. According to the type of administration, the mouse groups are shown in Table 2 below.

Table 2

Type of medication Comparative Example 6 Distilled water was given after U87-leu cell transplantation Example 4 Gossypol alone after U87-leu cell transplantation Example 5 Phenformin alone after U87-leu cell transplantation Example 6 Co-administration of gossypol and phenformin after U87-leu cell transplantation

Body weight gain or loss was checked daily, and mice were euthanized according to approved protocols when their body weight decreased by 15 wt% compared with that before the start of the experiment.

Bioluminescence was collected and analyzed using an IVIS imaging system and image analysis program (Real-Time Imaging V4.2 software). To this end, 15 minutes before signal measurement, 100 μl of d-luciferin (30 mg/mL in PBS) was injected intraperitoneally into each mouse under 2.5% isoflurane anesthesia. Signal measurements were taken 1, 3, and 5 weeks after U87-luc cell transplantation, with each measurement lasting 5 seconds. The results are shown in Figure 7.

The experimental results obtained using an orthotopic xenograft model are shown in Figure 7. As can be seen, fluorescence was observed in Comparative Example 6, Example 4 (gossypol administered alone), and Example 5 (phenformin administered alone) five weeks after cell transplantation. These results indicate that U87-leu cells caused brain cancer, but Example 6 (co-administration of gossypol and phenformin) exhibited significantly lower levels of brain cancer compared to Comparative Example 6, Example 4, and Example 5. Furthermore, the results of mouse survival measurement ( Figure 8 ) indicate that the survival rate of the mouse group in Example 6 was higher than that in the mouse groups in Comparative Example 6 and Examples 4 and 5.

Although the embodiments of the present invention have been described in detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these embodiments and various changes and modifications may be made without departing from the technical spirit as defined in the appended claims.

Industrial Usability

As described above, the pharmaceutical composition of the present invention contains a combination of an aldehyde inhibitor and a biguanide compound, which can effectively inhibit the growth of cancer stem cells such as neurospheres, and can also inhibit the proliferation, invasion and metastasis of cancer cells, thereby preventing and/or treating cancers such as brain cancer.

Claims (3)

1. Use of gossypol and phenformin in the preparation of a pharmaceutical composition for inhibiting the growth of glioblastoma. 2. The use as described in claim 1, wherein the weight ratio of gossypol and phenformin is 1:1-10. 3. The use as described in claim 1, wherein the inhibition of glioblastoma growth is to inhibit glioblastoma maintenance, inhibit glioblastoma deterioration, or inhibit glioblastoma invasion.