KR101575882B1 - Use of pinitol and d-chiro inositol in cancer treatment and prevention of cancer relapse - Google Patents

Abstract

The present invention relates to anti-cancer use of pinitol or D-chiroinositol, or a combination administration of pinitol or D-chiroinositol and an anticancer agent. (i) Pinitol and D-chiroinositol have anticancer activities that inhibit tumor growth and inhibit the proliferation of cancer stem cells. (Ii) Since pinitol and D-chiroinositol have an activity of inhibiting the proliferation of cancer stem cells in the tumor, they have an inhibitory effect on cancer recurrence and cancer metastasis as well as tumor growth inhibition. (Iii) When the combination of pinitol and D-chiroinositol is combined with an anticancer agent such as a known chemotherapeutic agent or cytotoxic agent, the aforementioned anticancer activity is synergistically increased. Thus, pinitol and D-chiroinositol can be developed as co-agents that can increase the effectiveness or decrease the dose of conventional anticancer agents.

Description

USE OF PINITOL OR D-CHYRO INOSITOL IN CANCER TREATMENT AND PREVENTION OF CANCER RELAPSE IN THE TREATMENT OF CANCER AND INHIBITING RECURRENCE < RTI ID = 0.0 >

The present invention relates to anti-cancer use of pinitol or D-chiroinositol, and use of concomitant administration of pinitol or D-chiroinositol with an anticancer agent.

Cancer stem cells are cancer cells with an unlimited regenerative capacity. When injected into immunosuppressed mice, they can produce tumors with high efficiency, and the inherent heterogeneity of primary tumors is well represented in the formed tumors (Al-Hajj M, Clarke MF, Self-renewal and solid tumor stem cells, Oncogene 2004; 23: 7274-82). The hypothesis that stem cells originate from a cell has already been proposed by Cohnheim in 1875 (Sell S. Stem cell origin of cancer and differentiation therapy. Crit Rev Oncol Hematol 2004; 51: 1-28) It has not been until recently that biology has developed. In Bonnet and Dick (Bonnet D, Dick JE, 1997), human acute myeloid leukemia is organized as a hierarchy from a primitive hematopoietic cell (Nat Med 1997; 3: 730-737) (Al-Hajj M. et al., Oncogene 2004; 23: 23-23). In this study, we investigated the expression of human leukemia in mice by immunohistochemistry, 7274-82) demonstrated the presence of stem cells in solid carcinoma, demonstrating the presence of stem cells in breast cancer.

Interest in cancer stem cells has been attributed to the failure of chemotherapy to effectively target and treat these cancer stem cell lines in the tumor, resulting in failure of chemotherapy and the recurrence of tumors (Al-Hajj M, Becker MW, Wicha M , Weissman I, Clarke MF, Therapeutic implications of cancer stem cells, Curr Opin Genet Dev 2004; 14: 43-7). Many cytotoxic anticancer drugs target rapidly proliferating cells, so perhaps the response to the drug would actually be a response to transfected cells, and a cancer stem cell with a slow proliferating ability could survive cytotoxic chemotherapy . One of the common features of normal stem cells and cancer stem cells is the ability to export drugs out of cells, which makes cancer stem cells more likely to be resistant to chemotherapy (Dean M, Fojo T, and Bates S. Tumour stem cells and drug resistance. Nat Rev Cancer 2005; 5: 275-84).

Pinitol (D-pinitol) is known to be contained in pine (pinewood) and legumes. When injected with insulin-deficient mice at a minimum dose of O.Olg / kg, the structure-unidentified pinitol-like substances extracted from Bougainvillea spectabilis were administered to normal and alloxan-treated insulin-deficient mice (P <0.05). In addition, it was reported that low blood sugar levels could be achieved (Narayanan, CR, Joshi, DD, Mujumdar, AM, Dhekne, VV 1987. Pinitol-A new antidiabetic compound from the leaves of Bougainvillea spectabilis. Current Science 56: 139141). D-chiro inositol is a stereoisomer of myo-inositol in which the hydroxyl group 3 is epimerized. D-cairo-inositol is a major component of inositol phosphoglycans (IPG) and has been reported to be an important mediator of insulin signaling and is known to be effective in the treatment of type 2 diabetes. D-chiroinositol is found mainly in eukaryotes and is biosynthesized by epimerization of myo-inositol. U.S. Patent No. 5,827,896 (PCT / IB96 / 00333) proposes to use D-pinitol and its derivatives for the treatment of diabetes, a metabolic disease. However, synergistic effects of anticancer activity of pinitol and D-chiro-inositol when combined with chemotherapeutic agents for anti-cancer therapies have not been reported.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

Al-Hajj M, Clarke MF. Self-renewal and solid tumor stem cells. Oncogene 2004; 23: 7274-82. Sell S. Stem cell origin of cancer and differentiation therapy. Crit Rev Oncol Hematol 2004; 51: 1-28. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 1997; 3: 730-737. Al-Hajj M, Becker MW, Wicha M, Weissman I, Clarke MF. Therapeutic implications of cancer stem cells. Curr Opin Genet Dev 2004; 14: 43-7. Brenton JD, Carey LA, Ahmed AA, Caldas C. Molecular classification and molecular prediction of breast cancer: ready for clinical application? J Clin Oncol 2005; 23: 7350-60. Dean M, Fojo T, Bates S. Tumor stem cells and drug resistance. Nat Rev Cancer 2005; 5: 275-84.

The present inventors have made efforts to find a novel cancer candidate substance which has excellent anticancer efficacy, has few side effects and can reduce the possibility of cancer recurrence. As a result, it was found that pinitol and D-chiroinositol inhibit the growth of tumor and inhibit the proliferation of cancer stem cells and exhibit synergistic anticancer activity when pinitol or D-chiro-inositol is used in combination with an anticancer agent. The present invention has been completed.

Accordingly, an object of the present invention is to provide an anticancer composition comprising pinitol or D-chiroinositol as an active ingredient.

Another object of the present invention is to provide a combination administration composition for anti-cancer comprising pinitol or D-chiroinositol and an anticancer agent.

Another object of the present invention is to provide a kit for anti-cancer combined use comprising pinitol or D-chiroinositol and an anticancer agent.

The objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided an anticancer composition comprising (i) a pharmaceutically effective amount of pinitol or D-chiroinositol and (ii) a pharmaceutically acceptable carrier.

The pinitol or D-chiroinositol of the present invention has antitumor activity. The pinitol or D-chiroinositol of the present invention has an activity of inhibiting proliferation of cancer cells as well as inhibiting proliferation of cancer stem cells. Accordingly, the antitumor activity of pinitol or D-chiroinositol includes an activity of inhibiting cancer cell proliferation (tumor growth or growth) inhibiting activity, as well as inhibiting cancer recurrence, diffusion or metastasis by cancer stem cell proliferation inhibition.

The formula of "pinitol" of the present invention is as follows.

The term "pinitol" of the present invention is meant to include both a suitable derivative or metabolite of the pinitol, a compound containing the pinitol, or a prodrug of the pinitol.

As used herein, the term " derivative of a pinitol "or" metabolite of a pinitol "includes pinitol glycosides, pinitol phospholipids, esterified pinitol, lipid-binding pinitol, pinitol phosphate and pinitol phytate pinitol phytates), and mixtures thereof.

As used herein, "pinitol containing compound" means any compound that contains a pinitol moiety as part of a larger structure. The "pinitol containing compound" includes a complex or chelate compound of at least one metal ion with a polysaccharide and a pinacol comprising at least one additional sugar (glucose, galactose, mannose, glucosamine, galactosamine and mannitol) , But is not limited thereto.

As used herein, the term "pro-drug of pinitol" refers to a derivative of pinitol that is converted in vivo to an authentic pinolide by an enzymatic or chemical process and exhibits improved delivery properties and / . Methods for preparing prodrugs of saccharides, such as prodrugs in the form of methylated or acetylated hydroxyl groups, and methods of administering them, are known in the art (Baker et al., J. Am. Med. Chem. , 27: 270-274 (1984)).

The pinolide of the present invention can be obtained from a variety of natural sources such as pine needles, chick peas, Bougainvillea leaves, alfalfa, soy beans and other legume plants Or can be obtained through a synthesis process. More preferably from the soybean fraction.

In the present invention, D-chiro inositol (DCI) is a stereoisomer of the myo-inositol in which the hydroxyl group 3 is epimerized and represented by the formula (2).

D-chiro-inositol (DCI) is a major component of inositol phosphoglycans (IPG) and is an important mediator of insulin signaling, mainly found in eukaryotes and biosynthesized by epimerization of myo-inositol. D-chiro-inositol (DCI) can be obtained mainly by hydrochloric acid hydrolysis of pinitol or kasugamycin (US Pat No. 5827896, US Pat No. 5091596, US Pat No. 5463142, US Pat No .5714643).

&Quot; Cancer &quot;, which is a disease to be treated by the composition of the present invention, refers to an aggressive characteristic of a cell that disregards normal growth limits and cleavage and growth, an invasive characteristic of penetrating into surrounding tissues, Is a generic term for a disease caused by a cell having a characteristic.

According to a preferred embodiment of the present invention, the cancer to be treated is selected from breast cancer, lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or ocular melanoma, uterine sarcoma, ovarian cancer, But are not limited to, colon cancer, colon cancer, tubal cancer, endometrial cancer, cervical cancer, small bowel cancer, endocrine cancer, thyroid cancer, pituitary cancer, kidney cancer, soft tissue tumor, urethral cancer, prostate cancer, Do not.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition comprising (i) a pharmaceutically effective amount of pinitol or D-chiroinositol (DCI); (Ii) a pharmaceutically effective amount of an anti-cancer drug; And (iii) a pharmaceutically acceptable carrier.

The pinitol or D-chiroinositol of the present invention exhibits a synergistic anticancer effect when it is co-administered with an anticancer agent. In the present invention, administration of pinitol or D-chiroinositol in combination with an anticancer agent has a synergistic effect in inhibiting proliferation (tumor growth or growth) of cancer cells as well as cancer stem cell proliferation inhibitory activity in tumors. Accordingly, in the present invention, when the combination of pinitol or D-chiro-inositol and an anticancer agent is administered, the anticancer activity is inhibited not only by the proliferation (tumor growth or growth) inhibitory activity of cancer cells, but also by the cancer recurrence, Lt; / RTI &gt; activity.

In the present invention, an anti-cancer drug refers to a chemical substance or drug used in the treatment of a tumor, preferably a chemotherapeutic agent or a cytotoxic agent. A chemotherapeutic agent or cytotoxic agent refers to a substance used to treat abnormal and uncontrolled progressive cell proliferation. The anticancer agent of the present invention includes, for example, taxanes, nitrogen mustards, ethyleneimine derivatives, alkylsulfonates, nitroso ureas, triazenes; Folic acid analogs, pyrimidine analogs, purine analogs, vinca alkaloids, enzymes, platinum coordination complexes, substituted ureas, methylhydrazine derivatives, corticosteroid inhibitors, or antagonists. More specifically, anticancer agents include steroids, progestins, estrogens, antiestrogens, and androgens. More specifically, anticancer agents include azaribine, bleomycin, bryostatin-1, busulfan, carmustine, chlorambucil, carboplatin, , Cisplatin, CPT-11, cyclophosphamide, cytarabine, dacarbazin, dactinomycin, daunorubicin, dexamethasone, diethylstilbestrol, doxorubicin , Ethinyl estradiol, etoposide, fluorouracil, fluoxymesterone, gemcitabine, hydroxyprogesterone caproate, hydroxyurea, and the like. hydroxyurea, L-asparaginase, leucovorin, lomustine, mechlorethamine, medroprogesterone acetate, megastar, But are not limited to, megestrol acetate, melphalan, mercaptopurine, methotrexate, mithramycin, mitomycin, mitotane, paclitaxel, phenyl butyrate, prednisone, Procarbazine, semustine streptozocin, tamoxifen, taxane, taxol, testosterone propionate, thalidomide, thioguanine, thiotepa, uracil Uracil mustard, vinblastine, or vincristine.

According to one embodiment of the present invention, the anticancer agent is selected from the group consisting of cyclophosphamide, ifosfamide, cytarabine, 6-mercaptopurine, 6-thioguanine (6- thioguanine, vincristine, doxorubicin, daunorubicin, chlorambucil, carmustine, vinblastine, methotrexate, and paclitaxel paclitaxel).

Anticancer agents suitable for use in the present invention are described in REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed. (Mack Publishing Co. 1995) and Goodman and Gilman's The Pharmacological Basis of Therapeutics (Goodman et al., Eds. Macmillan Publishing Co., New York, 1980 and 2001 editions), which are incorporated herein by reference.

The "pharmaceutically effective amount" of pinitol or D-chiroinositol in the pharmaceutical composition of the present invention means an amount sufficient to induce the enhancement of the anticancer activity of the anticancer drug to be coadministered. The enhancement of the anticancer activity of the anticancer agent can be measured through various known methods of the prior art and can be measured, for example, by a statistically significant reduction in the dosage or amount of the anticancer agent required for exhibiting the anticancer effect . For example, it can be measured in comparison with a suitable control which has been administered with a reference amount of an anticancer drug not administered with pinitol or D-chiroinositol.

A "pharmaceutically effective amount" of an anticancer agent in the present invention means an effective and sufficient amount for the treatment of cancer or tumor.

In the present invention, since the anticancer effect of the anticancer agent is increased by the concomitant administration of pinitol or D-chiroinositol, the pharmacologically effective amount of the anticancer agent can be reduced as compared with the standard dose of the anticancer agent. The reduction in the dosage of the anticancer drug has the advantage of not only achieving the same or improved treatment results, but also reducing side effects in the subject to be treated. A decrease in dosage can be a decrease in the individual dose, a decrease in the frequency of administration, or a combination thereof. The standard dose of the anticancer agent and the method of administration and schedule are known to those skilled in the art, for example, those skilled in the art, for example, in the prior art Physicians' Desk Reference, 56. sup. (2002) Publisher Edward R. Barnhart, New Jersey ("PDR"). In the present invention, the pharmacologically effective amount of the anticancer agent is usually significantly lower than the standard dose used for therapeutic administration. For example, to about 90%, 80%, or 70% of the standard capacity. More preferably, the therapeutic effect on cancer in the present invention can be obtained by dosing a reduced dose to less than 75% of the standard dose, even more preferably by a reduction in the range of about 75% to 25% &Lt; / RTI &gt; An effective anti-cancer effect can be achieved, for example, by a reduced dosage of less than 75%, less than 65%, less than 55%, less than 45%, less than 35%, or less than 25%.

The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above-mentioned components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The appropriate dosage of the pharmaceutical composition of the present invention may be determined by various methods depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient It can be prescribed. On the other hand, the dosage of the pharmaceutical composition of the present invention is preferably 0.001 to 1000 mg / kg (body weight) per day.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and when administered parenterally, it can be administered by intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, transdermal administration, or the like.

The concentration of the active ingredient contained in the composition of the present invention is determined in consideration of the purpose of the treatment, the condition of the patient, the period of time required, the severity of the disease, and the like.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

According to a preferred embodiment of the present invention, the cancer to be treated of the pharmaceutical composition of the present invention is a breast cancer, lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or ocular melanoma, Cancer, ovarian cancer, prostate cancer, bronchogenic carcinoma, or bone marrow carcinoma in a mammal, including a human, a colon, a rectum, a rectum, an anal cancer, a colon cancer, a fallopian tube cancer, an endometrial cancer, a cervical cancer, a small bowel cancer, But is not limited thereto.

According to another aspect of the present invention, the present invention provides a vial comprising (i) a vial comprising pinitol or D-chiroinositol, and (ii) a vial comprising one or more anti-cancer drugs, The present invention provides a kit for anti-cancer combined use.

In the above combination kit, the content of pinitol, D-chiro-inositol, anticancer agent, and anticancer activity is the same as described in the above anti-cancer pharmaceutical composition, and thus is not described in duplicate.

According to one embodiment of the present invention, the present invention further includes instructions for the use of the combination of pinitol or D-chiroinositol and one or more anticancer agents.

The present invention relates to the anticancer use of pinitol or D-chiroinositol, or the combined administration of pinitol or D-chiroinositol and an anticancer agent. The advantages and effects of the present invention are as follows.

(i) Pinitol and D-chiroinositol have anticancer activities that inhibit tumor growth and inhibit the proliferation of cancer stem cells.

(Ii) Since pinitol and D-chiroinositol have an activity of inhibiting the proliferation of cancer stem cells in the tumor, they have an inhibitory effect on cancer recurrence and cancer metastasis as well as tumor growth inhibition.

(Iii) When the combination of pinitol and D-chiroinositol is combined with an anticancer agent such as a known chemotherapeutic agent or cytotoxic agent, the aforementioned anticancer activity is synergistically increased. Thus, pinitol and D-chiroinositol can be developed as co-agents that can increase the effectiveness or decrease the dose of conventional anticancer agents.

FIG. 1 is a graph showing changes in body weight in the case of administration of pinitol, D-chiroinositol or doxorubicin alone or in combination to a human breast cancer cell (MDA-MB-231 cell) transplantation model using a specific pathogen member (SPF) nude mouse of the BALB / .
FIG. 2 is a graph showing the effect of the combination of pinitol and D-chiro-inositol alone or with doxorubicin on human breast cancer cell (MDA-MB-231 cell) transplantation model using a specific pathogen member (SPF) nude mouse of BALB / The results are the results of measuring the size over time.
FIG. 3A shows the results of the administration of pinitol and D-chiroinositol alone or in combination with doxorubicin to a human breast cancer cell (MDA-MB-231 cell) transplantation model using a specific pathogen member (SPF) nude mouse of the BALB / The size of the tumor was measured. Dox: doxorubicin, Pin: pinitol, DCI: D-chiroinositol.
FIG. 3B is a photograph showing the results of immunohistochemical staining of a human breast cancer cell (MDA-MB-231 cell) transplantation model using a specific pathogen member (SPF) nude mouse of BALB / C system on day 53 after administration of pinitol and D-chiroinositol alone or in combination with doxorubicin The size of the tumor was measured. Dox: doxorubicin, Pin: pinitol, DCI: D-chiroinositol.
FIG. 4A is a graph showing the effect of the combination of pinitol and D-chiroinositol alone or in combination with doxorubicin in a human breast cancer cell (MDA-MB-231 cell) transplantation model using a specific pathogen member (SPF) nude mouse of the BALB / The ratio of cancer stem cells in tumor was analyzed by flow cytometry. Dox: doxorubicin, Pin: pinitol, DCI: D-chiroinositol.
FIG. 4B shows the results obtained after administration of pinitol and D-chiro-inositol alone or in combination with doxorubicin in a human breast cancer cell (MDA-MB-231 cell) transplantation model using a specific pathogen member (SPF) nude mouse of the BALB / The ratio of cancer stem cells in tumor was analyzed by flow cytometry. Dox: doxorubicin, Pin: pinitol, DCI: D-chiroinositol.
FIG. 5A is a graph showing the results of the measurement of the effect of these test substances on the formation of mammoth peers in the case of treatment with pinitol, D-chiroinositol and doxorubicin.
FIG. 5B is a graphical representation of the experimental results of FIG. 5A.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Materials and Methods

1. Culture of cancer cells

Human breast cancer cell line MDA-MB-231 cells were used for the experiments. The breast cancer cell lines were prepared by dissolving the cancer cells that were being kept frozen in liquid nitrogen, followed by cell culture. Cell cultures were incubated for an appropriate period in a CO ₂ incubator (Forma, USA) at a temperature of 37 ° C and a CO ₂ concentration of 5%.

2. Experimental animals

Specific pathogen member (SPF) nude mice from BALB / C line (ORIENT BIO) were used as test animals.

3. Cancer cell implantation

On the last day of culture of human breast cancer cells (MDA-MB-231), all breast cancer cells were collected and counted and the cell concentration was adjusted to 3 x 10 ⁷ cells / ml using serum free medium. 0.3 ml (9 x 10 ⁶ cells / mouse) per mouse was injected into the subclavian space between the right clavicular and chest wall.

4. Test substances and control substances

D-pinitol (SIGMA-ALDRICH) and D-chiroinositol (SIGMA-ALDRICH) were used as test substances. As a control, doxorubicin hydrochloride (SIGMA-ALDRICH) was used. Pinitol was dissolved in distilled water at a concentration of 50 mg / ml and then orally administered to mice at a dose of 10 ml / kg repeatedly (final dose: 300, 500, 1000 mg / kg). D-chiro-inositol was dissolved in distilled water at a concentration of 50 mg / ml and then repeatedly orally administered at a dose of 10 ml / kg in mice (final dose: 300, 500, 1000 mg / kg). Doxorubicin was dissolved in saline at a concentration of 0.4 mg / ml and then administered intratumorally (20 times) to the mice repeatedly (4 times; 0, 5, 10, 15 days).

5. Measurement of anticancer effect in experimental animals transplanted with cancer cells

5.1. General symptom and weight change measurement

All animals were monitored for general symptoms and changes in body weight immediately before administration at the initiation and during the study period.

5.2. Measurement of tumor size change

After the tumor cells were transplanted, the tumor size was measured from the time of 53.0 mm ³ to 63 days using a vernier caliper (vernier caliper) for a total of 28 times, and then the length was measured by the length x width x height The tumor size was measured by the formula of height / 2.

5.3. Tumor regression and recurrence

As a test substance, pinitol or D-chiro-inositol was orally administered five times (Mon-Fri) orally, and the control substance, doxorubicin, was administered directly to the affected area of the tumor four times (0, 5, 10 and 15 days) We observed regression and relapse of the tumor.

5.4. Dead animal

As a test substance, death animals due to natural history were observed while continuously administering pinitol and D-chiroinositol.

6. In vitro phenotype analysis

6.1. Flow cytometry

Each drug was treated with about 1 x ¹⁰⁶ cells of breast cancer cells (MDA MB 231) and the experiment was performed after 48 hours. Cells isolated from the culture plate were washed once with PBS (BSA / PBS) supplemented with 1% BSA (bovine serum albumin), and suspended in 50 μl of PBS / BSA. The cells were reacted at 4 ° C for 20 minutes, washed twice with PBS, resuspended with 500 μl of PBS and analyzed with a flow cytometer (FACSCanto II, BD). Anti-AKT antibody, anti-p-AMPK antibody, anti-mTOR antibody and anti-p-mTOR antibody were used to confirm the anticancer mechanism. Antibodies to proteins involved in cell signaling to be. Anti-CD24 antibody and anti-CD44 antibody (BD pharmingen) were used for the measurement of cancer stem cells.

7. Culture of mammalian mammosphere

Cultures of mammalian mammosphere were incubated with 2% B-27 supplement (B-27 supplement, Invitrogen), 20 ng / mL EGF (epidermal growth factor, Invitrogen), 0.48 μg / mL hydrocortisone (SIGMA) Was performed using MEBM medium (Lonza) containing penicillin / streptomycin (Gibco, 15140). Breast cancer cell line MCF-7 cells were induced to mammothian pear formation through suspension culture at a concentration of 1000 cells / plate in an ultra-low attachment 6-well plate.

8. Statistical analysis method

The values of all the measurement items were measured by the t-test statistical method using the experimental control group and the drug administration group

And statistical significance was tested.

Experiment result

1. General symptom and weight change measurement result

In the case of repeated oral administration of pinitol and D-chiroinositol to nude mice transplanted with human breast cancer cells MDA-MB-231 at doses of 300, 500 and 1000 mg / kg, respectively, and doxorubicin (4 mg / kg, To investigate the degree of toxicity in combination with the general symptoms and weight changes of the animals during the administration period was observed. As a result, there were no specific general symptoms during the test period (see Table 1), and the results of the 46 days before observation of the dead animals showed that the administration of pinitol and D-chiro-inositol alone (500 mg / kg) and doxorubicin There was no statistically significant weight loss in the co-administered group and no statistically significant weight loss in the control (doxorubicin) treated group (see Table 2 and Figure 1).

2. Suppression of tumor growth

The effects of pinitol or D-chiro-inositol alone or in combination with doxorubicin on tumor growth were measured in nude mice transplanted with human breast cancer cells MDA-MB-231.

At the 46th day before the observation of the dead animals, the size of the tumor grew to 3349 ± 204.6 mm ³ in the control group, but the tumor size was 2320.2 ± 134.6 mm ³ ( p <0.001) in the group treated with 300 mg / kg of pinitol alone The tumor growth inhibition effect of 30.72% was inhibited by 35.94% in the tumor size of 2145.5 ± 135.8 mm ³ ( p <0.001) in the 500 mg / kg administration of pinitol alone, Was 1458.6 ± 210.5 mm ³ ( p <0.001), indicating a tumor growth inhibition effect of 56.45%. In addition, the tumor size was 210 ± 68.4 mm ³ ( p <0.001), 93.73% tumor growth inhibition effect in the group treated with 300 mg / kg of pinolol combined with doxorubicin 4 mg / kg, to ^{167.5 ± 73.5mm 3 (p <0.001} ) inhibited 95% of tumor growth effect, in the 1000 mg / kg administration group showed a tumor ^{102.8 ± 32.5mm 3 (p <0.001} ) with a 96.93% tumor growth inhibition .

In addition, the tumor size was 2014.2 ± 211.5 mm ³ ( p <0.001) in the group treated with D-chiro-inositol alone at a dose of 39.86%, and the tumor growth was inhibited by D-cairo administered with doxorubicin 4 mg / kg In the case of inositol 500 mg / kg, the tumor size was 162.5 ± 32.5 mm ³ ( p <0.001), indicating 95.15% tumor growth inhibitory effect.

There was a statistically significant tumor growth inhibitory effect of 85.15% (p < 0.001) in the control substance doxorubicin treated group (4 mg / kg) (see Table 3, Fig.

The control group 53 days after administration of the test substance, but the size of the tumor growth with 4420.2 ± 132mm ^3, pinitol single 300 mg / kg administration group the tumor size is ^{3102.8 ± 52.4 mm 3 (p <} 0.001) indicated by a 29.8% tumor growth inhibition the effects, pinitol single 500 mg / kg administration group the tumor size is ^{2936.4 ± 55.6mm 3 (p <0.001} ) in the tumor growth inhibitory effect of 33.57%, pinitol single 1000 mg / kg group the tumor size 1988.8 ± 261mm ³ ( p <0.001), indicating a tumor growth inhibitory effect of 55.01%. In addition, the tumor size was 445.2 ± 126.5 mm ³ ( p <0.001), 89.93% tumor growth inhibition effect in the group treated with 300 mg / kg of pinolol combined with doxorubicin 4 mg / kg, to ^{365.2 ± 140.5mm 3 (p <0.001} ) suppression in 91.74% of tumor growth effect, in the 1000 mg / kg administration group showed a tumor ^{225.8 ± 60.2mm 3 (p <0.001} ) with a 94.89% tumor growth inhibition .

In addition, in the group administered with 500 mg / kg of D-chiro-inositol alone, the tumor size was 2860.4 ± 259.3 mm ³ ( p <0.001), indicating a tumor growth inhibitory effect of 35.29%. In addition, D-cairo administered with doxorubicin 4 mg / In the case of inositol 500 mg / kg, tumor size was 368 ± 62.5 mm ³ ( p <0.001), indicating 91.67% tumor growth inhibitory effect.

There was a statistically significant tumor growth inhibitory effect of 74.66% (p <0.001) in the doxorubicin treated group (4 mg / kg) (see Table 3, Fig. 2, and Fig. 3b).

3. Tumor regression and recurrence

The effects of pinitol or D-chiro-inositol alone or in combination with doxorubicin on nude mice transplanted with human breast cancer cells MDA-MB-231 on tumor regression and tumor relapse Respectively. The tumor regression occurred in the combination of pinitol 300, 500, 1000 mg / kg plus doxorubicin 4 mg / kg, and D-chiro-inositol 500 mg / kg plus doxorubicin 4 mg / kg. No tumor recurrence was observed for days. The control substance, doxorubicin treated group, did not show tumor recurrence for 30 days after cancer cell transplantation (see Table 3 and FIG. 2).

4. Influence on Cancer Stem Cells (CSCs)

To examine the effect of pinitol on CSCs in human breast cancer cell (MDA-MB-231 cell) transplantation model using nude mice, the ratio of cancer stem cells on the 25th day and 46th day cancer cells was analyzed by flow cytometry Respectively. As a result, there was a decrease of 17.2% in the control group, 14.2%, 11.4%, and 9.5% in the administration group of pinitol 300, 500 and 1000 mg / kg on the 25th day and 5.04%, 4.2% and 3.5% in the group treated with the combination of pinitol and doxorubicin Respectively. In the case of D-chiro-inositol, it was found to be 10.8% when administered alone and decreased to 3.9% in the combination administration of D-chiro-inositol and doxorubicin (see FIG.

In the group treated with pinitol and doxorubicin, the risk of death was decreased by 20%, 17.5%, and 15.6%, respectively. In the group treated with pinitol alone, As shown in Fig. In the case of D-chiro-inositol, it was confirmed to be 16.8% when administered alone and decreased to 8.2% when D-chiro-inositol was administered in combination with doxorubicin (see FIG.

5. Measuring the impact on the formation of mammoth peers (Mammosphere)

When the stem cells are removed from the serum-free culture medium under the condition that the growth factors such as EGF and bFGF are treated, the differentiated cells are removed and the stem cells are grown to remove the cell clumps sphere. When the cancer cells are grown under the same conditions, the differentiated cancer cells are removed and only the tumor stem cells survive to form tumorsphere. As a result of measuring the effect of the test substance on formation of mammoth peers, it was found that the number of mammosphere mammosphere when treated with 300, 500 and 1000 mg / kg of pinitol was 20.5 x 10 ³ , 17.5 x 10 ³ , 13.5 x was measured 10 ^{to three,} the case treated with D- Cairo inositol ^{19.2 x 10 3, 16.5 x 10} 3, 16 x 10 is measured ^{to three,} reducing the mammoth peer with increasing the concentration of D- and pinitol Cairo inositol also (See Figs. 5A and 5B).

6. Dead animals

From the 51st day after the drug administration, the number of deaths due to natural history in the experimental control group began to appear. On the 53rd day, three deaths occurred at 2 and 56 days. In the 300 mg / kg of pinitol, At day 1, at 500 mg / kg, two deaths occurred at day 53, three deaths occurred at day 56, and one death occurred at day 63. In the group treated with 1000 mg / kg of pinitol, 1 death occurred on day 53, 3 deaths on day 56, and 1 death on day 60. In the group administered with D-chiro-inositol 500 mg / kg, there were two deaths on day 53 and day 56 on day 63. In the group treated with doxorubicin and pinitol 1000 mg / kg, 1 day on day 56 and 1 day on day 58 There were several deaths (see Table 1 and Table 2).

Table 1 below shows the results of measuring general symptoms after administration of test substance to nude mice transplanted with breast cancer cells (MDA-MB-231).

Tables 2 to 5 below show the results of measurement of weight change (%) after administration of the combination of pinitol alone and doxorubicin to nude mice transplanted with breast cancer cells (MDA-MB-231).

Tables 6 to 9 below show the results of measurement of changes in tumor size in nude mice transplanted with breast cancer cells by the combination of pinitol alone and doxorubicin.

Significant figures (t-test): * p <0.05, ** p <0.01, *** p <0.001 (vs Vehicle Control) ^† : Δt = Vt-Vo, Vt (Measurement of the tumor volume) Initial tumor volume

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (11)

(i) a pharmaceutically effective amount of pinitol or D-chiroinositol; And (ii) a pharmaceutical composition for inhibiting the recurrence of cancer by inhibiting cancer stem cell proliferation after chemotherapeutic treatment comprising a pharmaceutically acceptable carrier.
The method of claim 1, wherein the cancer is selected from the group consisting of breast cancer, lung cancer, gastric cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or ocular melanoma, uterine sarcoma, ovarian cancer, rectal cancer, Wherein the cancer is selected from the group consisting of cancer, endometrial cancer, cervical cancer, small bowel cancer, endocrine cancer, thyroid cancer, pituitary cancer, kidney cancer, soft tissue tumor, urethral cancer, prostate cancer, bronchial cancer and bone cancer. Composition.
delete (i) a pharmaceutically effective amount of pinitol or D-chiroinositol; (Ii) a pharmaceutically effective amount of an anti-cancer drug; And (iii) a pharmaceutical acceptable carrier for inhibiting tumor recurrence through inhibition of cancer stem cell proliferation after chemotherapy.
The method of claim 4, wherein the cancer is selected from the group consisting of breast cancer, lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or ocular melanoma, uterine sarcoma, ovarian cancer, rectal cancer, Wherein the cancer is selected from the group consisting of cancer, endometrial cancer, cervical cancer, small bowel cancer, endocrine cancer, thyroid cancer, pituitary cancer, kidney cancer, soft tissue tumor, urethral cancer, prostate cancer, bronchial cancer and bone cancer. Composition.
delete 5. The pharmaceutical composition according to claim 4, wherein the anticancer agent is a cytotoxic agent or a chemotherapeutic agent.
(i) a vial containing pinitol or D-chiroinositol, and (ii) a vial containing one or more anti-cancer drugs, the cancer recurrence through inhibition of cancer stem cell proliferation after chemotherapy Combination kits for inhibition.
delete The kit according to claim 8, wherein the anticancer agent is a cytotoxic agent or a chemotherapeutic agent.
9. The kit of claim 8, further comprising instructions for the combination of pinitol or D-chiroinositol and at least one anticancer agent.

Images