TW201313227A - Use of isothiocyanates for treating cancer - Google Patents

Abstract

Novel Uses of small molecules, particularly, 6-methylsulfinylhexyl isothiocyanate and 6-methylsulfonylhexyl isothiocyanate, are disclosed herein. The two isothiocyanates are useful as lead compounds for manufacturing a medicament or a pharmaceutical composition for treating cancer, particularly drug-resistant cancer, in a patient.

Description

Method and use for treating cancer using isothiocyanates

The present disclosure is a novel use of certain small molecules, particularly novel uses of isothiocyanate compounds, which can be used to make pharmaceutical or pharmaceutical compositions that can treat cancer.

In most countries, cancer has become the leading cause of death. The current standard methods for treating cancer are as follows: surgical resection, chemotherapy, radiation therapy, immunotherapy, and biological therapy; in these methods, surgical resection and chemotherapy are most commonly used.

The primary goal of treatment is to completely remove cancer cells without harming other parts of the body. However, because cancer cells are easily invaded by surrounding tissues or transferred to other distal parts of the body through micromigration, the application level of surgical removal of cancer cells is limited. "Chemotherapy", as its name suggests, is treated with drugs that destroy cancer cells. At this stage, "chemical drug therapy" usually refers to the treatment with toxic drugs that can quickly affect cell division. However, in some cases, cancer cells develop resistance or become less sensitive to one or more of these cytotoxic drugs. Therefore, many related studies hope to find successful treatment of cancer, especially drug resistance. Or other effective treatments or means that are not sensitive to anticancer drugs. In addition, some cancers are inherently poorly predisposed or lack chemical agents with high specificity, such as pancreatic cancer, non-small-cell lung carcinoma (NSCLC) or esophageal cancer.

In view of this, there is a need in the art for an effective pharmaceutical compound that can be used to treat such cancers that are resistant or insensitive to current chemical treatments.

The present disclosure is based, at least in part, on the discovery of isothiocyanate compounds, particularly 6-methylsulfinylhexyl isothiocyanate, which is isolated from Wasabia japonica MATSUM. I7457) and its derivative, 6-methylsulfonylhexyl isothiocyanate (hereinafter referred to as I7557), respectively, are permeable to at least 45% of cancer cell growth arrested in the G ₂ /M cycle. And to achieve the purpose of delaying the growth of cancer cells. These findings suggest that the compounds of the present invention are useful as lead compounds for the treatment of cancer, including cancers resistant to cancer.

Accordingly, a first aspect of the present disclosure is to provide a method of treating cancer in a body. The method comprises administering to the individual a therapeutically effective amount of I7457 or I7557 or a pharmaceutically acceptable salt thereof. A cancer suitable for treatment with a compound of the invention is selected from the group consisting of pancreatic cancer, chronic myelogenous leukemia (CML), non-small-cell lung carcinoma (NSCLC), and esophageal cancer. . In a preferred embodiment, the CML is a CML cancer cell which is resistant to imatinib (the commercial name of the active anti-cancer drug is known as the Kilek film ingot). The above individual may be a mammal, preferably a human.

In certain embodiments, the method further comprises subjecting the cancer to radiation treatment after administering the I7457 or I7557 to the individual. In a preferred embodiment, the cancer is pancreatic cancer.

In certain embodiments, the method further comprises administering to the individual another agent known to improve the therapeutic effect of the cancer, simultaneously with, before, or after administration of the I7457 or I7557 to the individual. Examples of such agents include, but are not limited to, anticancer drugs, anti-angiogenic drugs, antiviral drugs, antibiotics, pain relieving drugs, anti-anemia drugs, cytokines, granulocyte colony-stimulating factors, G -CSF), anti-emetic drugs and other analogues.

Accordingly, a second aspect of the present disclosure is to provide a novel use of the above I7457 or I7557 which can be used to manufacture a pharmaceutical or pharmaceutical composition for treating cancer. The pharmaceutical or pharmaceutical composition produced will include a pharmaceutically effective amount of I7457 or I7557 or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable adjuvant.

The compound of the present invention, particularly I7457 or I7557, comprises from about 0.1% to about 99% by weight based on the total weight of the pharmaceutical composition, based on the total weight of the pharmaceutical composition. In certain embodiments, the amount of the compound of the invention is at least about 1% by weight based on the total weight of the pharmaceutical composition. In a particular embodiment, the amount of the compound of the invention is at least about 5% by weight based on the total weight of the pharmaceutical composition. In other embodiments, the amount of the compound of the invention is at least about 10% by weight based on the total weight of the pharmaceutical composition. In further embodiments, the amount of the compound of the invention is at least about 25% by weight of the total weight of the pharmaceutical composition.

In certain embodiments, the pharmaceutical or pharmaceutical composition of the present invention described above further comprises another agent known to improve the therapeutic effect of cancer. Examples of such agents include, but are not limited to, anticancer drugs, anti-angiogenic drugs, antiviral drugs, antibiotics, pain relieving drugs, anti-anemia drugs, cytokines, G-CSF, anti-emetic drugs, and the like.

These and other features of the present disclosure will be more apparent from the following detailed description and appended claims. The above summary and the following detailed description are merely illustrative, and are not intended to limit the scope of the disclosure.

The embodiments and the proper nouns are used to illustrate the invention and are not intended to limit the scope of the disclosure. The scope of the disclosure is also not specifically disclosed herein, but other embodiments that can be readily inferred by those skilled in the art after reading this disclosure.

At least part of the disclosure is due to an isothiocyanate compound isolated from Wasabia japonica MATSUM, especially 6-methylsulfinylhexyl isothiocyanate (I7457) and The derivative, 6-methylsulfonylhexyl isothiocyanate (I7557), has been developed with the discovery that it inhibits the growth activity of cancer cells (especially cancer cells having drug resistance). Therefore, these two isothiocyanate compounds can be used as lead compounds for medical agents for treating cancer.

The isothiocyanate compound is a compound containing an easily identifiable isothiocyanate (-NCS-) group. The following is the structural formula of the isothiocyanate compound found by the inventors of the present invention having novel uses:

6-methylsulfinylhexyl isothiocyanate (I7457)

6-methylsulfonylhexyl isothiocyanate (I7557)

The 6-methylsulfinylhexyl isothiocyanate (or I7457) of the present invention can be isolated from plants, seeds or plant extracts by conventional methods. Plants containing high amounts of isothiocyanate compounds include, but are not limited to, broccoli, sprouts, cabbage, broccoli, mustard seeds, wasabi, white radish, papaya seeds, and the like. The 6-methylsulfinium hexyl isothiocyanate compound of the present invention is preferably isolated from wasabi. As a derivative thereof, 6-methylsulfolylhexyl isothiocyanate (or I7557) can be purchased from commercial laboratories (e.g., LKT Laboratories, Inc (St. Paul, Minnesota, USA)).

Accordingly, the present disclosure provides a method for treating cancer in a body. The method comprises administering to the individual a therapeutically effective amount of one of the two compounds described above or a pharmaceutically acceptable salt thereof. The two compounds of the present invention can be effective to at least 45% of growth arrest in G ₁ / M cycle, to prevent continued proliferation of these cancer agents. A cancer suitable for treatment with a compound of the invention is selected from the group consisting of pancreatic cancer, chronic myelogenous leukemia (CML), and non-small-cell lung carcinoma (NSCLC). Preferably, any of the compounds disclosed herein are used to treat cancer cells that are resistant.

As noted, drug resistance refers to a state of cancer cells, particularly to the development of resistance to a single therapeutic agent. For example, cancer cells may have vinca alkaloids such as vinblastine, vincristine, and vinolate (such as doxorubicin), Anthracyclines such as daunorubicin and idarubicin; tubulin-dependent drugs (paclitaxel) or inhibitors of tyrosine kinase activity (eg, dasatinib ( Dasatinib), nilotinib and imatinib developed resistance.

In a preferred embodiment, the cancer that can be treated by either of the two compounds of the invention is CML, particularly resistant to the US FDA approved Kilek film ingot (active ingredient is imatinib). CML cancer cells. In another embodiment, the cancer that can be treated by either of the two compounds of the invention is pancreatic cancer, which is poorly premature and is useful for patients with locally advanced pancreatic cancer or metastatic cancer of the pancreas. The average survival time was only about 10 months and 6 months respectively. In another embodiment, a cancer that is treatable by any of the two compounds of the invention is an NSCLC that is insensitive to chemotherapy. In another example, the cancer is an esophageal cancer that is still the most important treatment with surgical resection.

In certain embodiments, an effective amount of a compound of the invention that can be administered to an individual via oral, intramuscular or intravenous injection is from about 1 to 100 mg/kg of body weight. An effective amount of a compound of the invention which can be administered to an individual per day is about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 mg/kg of body weight; preferably about 30-70 mg/kg. Individual body weight, for example about 30, 40, 50, 60, 70 mg/kg of individual body weight. These doses can be administered in a single administration or divided into multiple administrations within one day.

In certain embodiments, the method further comprises pre-treating the cancer prior to administration of the compound of the invention. In one embodiment, the cancer is pancreatic cancer.

In certain embodiments, the method further comprises administering to the individual another agent known to improve the therapeutic effect of the cancer, concurrently with, before, or after administration of the compound of the invention. Examples of such drugs include, but are not limited to, anticancer drugs, anti-angiogenic drugs, antiviral drugs, antibiotics, pain relieving drugs, anti-anemia drugs (such as erythrocyte auxin), cytokines (eg, granulosa cells - macrophages) Granulocyte-macrophage colony-stimulating factor, G-CSF or anti-emetic drug.

Examples of anticancer drugs include, but are not limited to, paclitaxel, docetaxel, camptothecin (CPT), topotecan (TPT), irinotecan, CPT-11), Doxorubicin, daunorubicin, epirubicin, fluorouracil, cis-platin, cyclophosphamide, vinblastine (vinblastine), vincristine, ifosfamide, melphalan, mitomycin, methotrexate, mitoxantrone, ghost Teniposide, etoposide, bleomycin, leucovorin, cytarabine, dactinomycin, streptozotocin Streptozocin), cobbutin A4-phosphate, SU5416 and others. Examples of anti-angiogenic drugs include, but are not limited to, DS 4152, TNP-470, SU6668, endostatin, 2-methoxyestradiol, cancer cell vasopressin ( Angiostatin), thalidomide, tetrathiomolybdate, linomide, IL-12 and others. Examples of antiviral drugs include, but are not limited to, amantadine, rimantadine, and the like. Examples of pain alleviating drugs include, but are not limited to, acetaminophen (e.g., p-amylaminophenol), non-sterol anti-inflammatory drugs (NSAID) (e.g., salicylate), and opioid drugs. (eg, morphine and opium). Examples of anti-anemia drugs include, but are not limited to, erythrocyte auxin.

The present disclosure also provides a pharmaceutical composition for treating cancer. The pharmaceutical composition comprises a therapeutically effective amount of a compound of the invention described above; and a pharmaceutically acceptable adjuvant.

Generally, the compounds of the invention will comprise from about 0.1% to about 99% by weight of the total pharmaceutical composition. In certain embodiments, the compound of the invention is present in an amount of at least 1% by weight of the total pharmaceutical composition. In a particular embodiment, the compound of the invention is present in an amount of at least 5% by weight of the total pharmaceutical composition. In other embodiments, the compound of the invention is present in an amount of at least 10% by weight based on the total weight of the pharmaceutical composition. In still other embodiments, the compound of the invention is present in an amount of at least 25% by weight of the total pharmaceutical composition.

In certain embodiments, the pharmaceuticals and pharmaceutical compositions of the present invention further comprise another agent known to improve the therapeutic effect of cancer. Examples of such drugs include, but are not limited to, anticancer drugs, anti-angiogenic drugs, antiviral drugs, antibiotics, pain relieving drugs, anti-anemia drugs (such as erythrocyte auxin), cytokines (such as granulosa cells - macrophages) Granulocyte-macrophage colony-stimulating factor, G-CSF or anti-emetic drug.

Can be prepared by the above-described agents or pharmaceutical compositions according to all accepted pharmaceutical process, the medium, such as Remington's Pharmaceutical Sciences ((1985) 17 th edition, ed. Alfonoso R. Gennaro, Mack Publishing Company, Easton, Pa) process. A pharmaceutically acceptable adjuvant means one that is compatible with the other ingredients of the formulation and that is compatible with the organism.

The compound of the present invention (i.e., I7457 or I7557) can be administered by a suitable method such as oral administration (e.g., oral administration of a capsule, suspension or tablet), or parenterally. Parenterally administered means include systemic administration such as intramuscular injection, intravenous injection, subcutaneous injection or intraperitoneal injection. Alternatively, it may be applied by means of a film, such as topical skin application or inhalation (such as intrabronchial, intranasal, intraoral or nasal drops); or intrarectal administration. Administration may be carried out alone or in combination with conventional pharmaceutically acceptable adjuvants. In a preferred embodiment, the compounds of the invention can be administered to an individual via oral means (e.g., through food).

If administered orally, the compound of the present invention can be formulated to contain various adjuvants (such as microcrystalline cellulose, calcium carbonate, dicalcium phosphate, and glycine); various disintegrating agents (such as starch, alginic acid, and specific hydrazine). Salts; and tablets of particulate binders (eg, polyvinylpyrrolidone, sucrose, gelatin, and acacia). In addition, lubricants such as magnesium stearate, sodium lauryl sulfate, and talc may also be included. This solid formulation can also be used as a filler in gelatin capsules, and preferred materials associated therewith include sugars in lactose or milk as well as high molecular weight polyethylene glycols. When used in the form of oral suspensions and / or special effects (elixirs), the active ingredients may be combined with various sweeteners or flavors, colorants or dyes, and emulsifiers and/or suspensions may be added if desired. And diluents such as water, alcohol, propylene glycol, glycerin.

If administered parenterally, the compound of the invention may be formulated as a liquid pharmaceutical composition which may be sterile solution or suspension which can be administered by intravenous, intramuscular, subcutaneous or intraperitoneal injection. liquid. Diluents which can be used in the manufacture of such sterile injectable solutions or suspensions include, but are not limited to, 1,3-butanediol, mannitol, water, Ringer's solution, isotonic sodium chloride solution. Fatty acids (e.g., oleic acid) and their glyceride derivatives, or natural pharmaceutically acceptable oils (e.g., olive oil or rapeseed oil) can also be used to make solutions or suspensions for injectable use. Alcohols or carboxymethylcellulose or similar dispersing agents for dilution may also be included in such oily solutions or suspensions. Other commonly used surfactants (e.g., Tweens or Spans series) or emulsifiers, or agents commonly used in the pharmaceutical arts to enhance bioavailability can be used.

The above-described pharmaceutical or pharmaceutical compositions of the present invention can be formulated into a variety of dosage forms for topical application. A wide variety of dermatologically acceptable inert adjuvants known in the art can be used in such dosage forms. Formulations suitable for topical application to the skin include liquids, creams, lotions, creams, gels, sprays, aerosol sprays, dermal patches, and the like. Commonly used inert adjuvants such as water, ethanol, polyvinylpyrrolidine, propylene glycol, mineral oil, stearyl alcohol and other materials which form a gel. All of the above dosage forms and adjuvants are well known in the pharmaceutical arts. The choice of dosage form is not critical to the effects of the compositions described herein.

The above pharmaceutical or pharmaceutical composition of the present invention may also be formulated into a variety of dosage forms suitable for mucosal application, such as buccal and/or sublingual pharmaceutical dosage units for delivery of a drug. Through the oral mucosa. A wide variety of biodegradable and pharmaceutically acceptable polymeric adjuvants can be used which provide the pharmaceutical compositions with acceptable adsorption and desired drug release patterns, as well as buccal and/or sublingual drugs. The active ingredient or other ingredients to be administered in the dosage unit are compatible. In general, the above polymeric adjuvants comprise a hydrophilic polymer that adheres to the wetted surface of the oral mucosa. Examples of polymeric adjuvants include, but are not limited to, acrylic acid polymers and copolymers; hydrolyzed polyvinyl alcohol; polyethylene oxides; polyacrylates; Vinyl polymers and copolymers; polyvinylpyrrolidine; dextran; guar gum; pectins; starch; and cellulosic polymers.

Accordingly, the present invention also provides a method for treating a cancer of a mammal, particularly a human, comprising administering to the individual the pharmaceutical or pharmaceutical composition comprising the active compound of the present invention. Such pharmaceutical or pharmaceutical compositions are preferably administered to a mammal, preferably a human, in a manner effective to deliver the active compound of the composition, suitably or to produce a desired effect. Suitable administration can be, for example, active or passive oral, nasal, transpulmonary or percutaneous membrane; or parenteral, such as rectal suppository, subcutaneous injection, intravenous injection, intramuscular injection, nasal cavity Internal or ophthalmic solution or ointment. In addition, the compounds of the invention may also be administered with other active agents.

It will be understood that the dosage of the compound of the invention will vary from patient to patient, not only because of the particular compound or composition employed, the route of administration, the compound (either alone or in combination with one or more drugs) The factors such as the response may also be affected by other factors, such as the state or severity of the disease to be treated; the age, sex or weight of the patient, the health of the patient; the severity of the pathological condition to be treated, and the patient's Other medical or special dietary content that is concurrently performed; and other factors conceivable by those of ordinary skill in the art; and the medical personnel responsible for care may ultimately determine the appropriate dosage based on these factors. The dosage and form of administration can be adjusted to provide a preferred therapeutic response. A therapeutically effective amount also refers to a toxic or detrimental effect of a compound or composition that is less than the therapeutic benefit it brings. In preferred instances, the compounds or compositions of the invention, when administered, should be administered in an appropriate dosage for a period of time such that the number and/or severity of symptoms are reduced.

The following is a description of the specific nouns used in this specification:

As used herein, the term "treating or treating" means administering the compound to a cancer cell or a body such that at least 45%, 50%, 55%, 60% or 65% of the cancer cell growth is arrested at The G ₂ /M cycle delays the growth of cancer cells, so that the tumor volume shrinks. Therefore, the term "treatment" is also used herein to mean killing cancer cells or inducing apoptosis in cancer cells.

The term "an effective amount" means that the dosage can achieve the desired therapeutic effect of treating cancer after an appropriate period of administration.

The words "compounds", "compositions", "active compounds", "agents" or "medicines" are interchangeable and refer to each other. A compound or composition capable of inducing a desired pharmaceutical and/or physiological response through local and/or systemic action when administered to a subject (human or animal).

The term "administered, administered" or "administration" as used herein refers to the direct administration of the compound or composition, or the administration of a prodrug, derivative, or analog of the active compound. The equivalent amount of one of the active compounds can be formed in the body of the subject.

The words "subject" or "patient" as used herein mean an animal, including a human, that is treatable by the compounds and/or methods. "Individual" or "patient" is used herein to encompass both male and female genders unless otherwise specified. Thus "individual" or "patient" encompasses any mammal, preferably a human, which may benefit from treatment with the compound.

The following examples are presented to illustrate the specific aspects of the disclosure and to assist those skilled in the art to understand and implement the present disclosure. However, the scope of the disclosure is not limited to these embodiments.

Example Materials and Methods

Cell culture In this study, human chronic myelogenous leukemia (CML) cell line K562R, human pancreatic cancer-like epithelial-like cell line PANC-1, human bark esophageal adenocarcinoma ( Barrett's-associated esophageal adenocarcinoma) cell line SEG-1, human esophageal squamous cell carcinoma line 81T/VGH and human esophageal adenocarcinoma line BE-3 3). Each cell lines were grown in Dulbecco's modified via over-the Eagle's medium (Dulbecco 's modified Eagle' s medium, DMEM) and added 10% fetal calf serum (fetal calf serum, FCS), 100 units /ml of penicillin, 100 μg/ml of streptomycin (Invitrogen, Carlsbad, CA) and 2 mM L-glutamic acid, and maintained at 37 ° C in a humid environment (5% CO ₂ and 95% air).

Induction of drug-resistant CML cell line CML cell line K562R was cultured in an environment with low dose of imatinib mesylate (also known as STI-571) to obtain resistance to imatinib. Cell line. During the operation, 0.2 μM of imatinib mesylate was added to the above DMEM medium, and K562R was cultured in this medium and maintained at 37 ° C in a humid environment (5% CO ₂ and 95% air).

MTT analysis MTT assay is a colorimetric assay that quantifies the enzyme (ie, reductase) in a living cell with a yellow tetrazolium compound - 3-(4,5-dimethylthiazole)-2,5- Reduction of 3-[4,5-dimethylthiazol]-2,5-diphenyltetrazolium bromide (MTT) into a (formazane) activity. This reduction can only be carried out when the cells are alive; therefore, MTT assays are often used to assess the ability of cells to survive and proliferate. Briefly, cells are contacted with 10 or 20 μM of test compound (eg, I7457 or I7557) for 48 or 72 hours; then MTT dye (500 μg/ml) is added and the reduction is allowed to proceed for 4 hours; then 500 μl is added Isopropyl alcohol to terminate the reduction reaction. The absorbance of the sample at a wavelength of 570 nm was measured using a spectrophotometer.

Cell cycle analysis Obtained cultured cells from the culture medium (whether or not pretreated with the compound of the invention (i.e., I7457 or I7557)), followed by treatment of the cells with 75% ice-cold alcohol at 4 ° C for at least one night, The cells are fixed. Thereafter, the cells were treated with A-type RNase (RNAase A) for about 30 minutes at room temperature, and after centrifugation, the cell pellet was resuspended in an appropriate amount of propidium iodide (PI) (20 μg/ml). Cells of different cycles were then measured by flow cytometry and counted.

Immunoblot analysis The incubated cells were lysed using a known lysis buffer and the total amount of protein was measured using the Bradford method. The extracted crude white (about 50-100 μg) was added to 0.15% of sodium dodecyl sulfate polyacrylamide gel, and the protein was separated by electrophoresis. Thereafter, the separated protein was transferred to a nitrocellulose membrane, and TBST buffer (0.8% sodium chloride, 0.02% potassium chloride, 25 mM Tris-HCl/pH 8.0, and 0.1%) containing 4% skim milk was used. Tween-20) to remove non-specific combinations. Various antibodies (including p-Plk-1, p-Chk-1, p-Chk-2, p-Cdc2, GAPDH, α-tubulin, β-tubulin) And p-histone H3 (purchased from Transduction Laboratories, Lexington, KY, USA) was separately dissolved in TRST buffer containing 2% skim milk, and separately separated protein bands. Incubate at 4 ° C for at least one night. After washing, the cells were incubated with horseradish conjugated secondary antibodies for about 30 minutes at room temperature, and then the protein bands were detected using ECL chemical luminescence reagent (Amersham Pharmacia Biotech, US). The film was exposed by X-ray to facilitate visual observation of the expressed protein band.

Liu staining The cells were transferred to glass slides and covered with solution A (0.5 g of methylene blue and 1.7 g of Eosin yellow, all dissolved in 1,000 ml of methanol). After 45 seconds, solution B (1.3 g of azure, 1.4 g of methyl blue, 23.38 g of disodium hydrogen phosphate (Na ₂ HPO ₄ ) and 6.5 g of potassium dihydrogen phosphate (KH ₂ PO _{4 ) were added.} ), all dissolved in 1,000 ml of distilled water), wherein the addition amount of solution B and solution A is 2:1. The surface of the solution was lightly blown to make the two solutions homogeneously mixed. The slide was allowed to stand for about 90 seconds and then the dye was quickly washed away with running water. The microscope was then used to observe the appearance of the stained cells.

Radiographic treatment and cell formation capacity analysis Approximately 100 cancer cells were implanted in a 6-well culture dish approximately 35 mm in diameter and added to DMEM medium (10% FCS was added, and concentrations of 5 or 10 μM, respectively) The drug (ie, I7457 or I7557) was tested and then continued for approximately 24 hours. The drug is washed off and the cells are treated with radiation. Electron beam energy of about 6 MeV was delivered by a linear accelerator (Clinac 1800, Varian Associates, Inc., Palo Alto, CA, USA) and applied about 0, 0.5, 1, at a rate of about 2.4 Gy per minute. 2 or 4 Gy (Gray) dose. At each irradiation, another RP-60C free chamber (CAPINTEL, Inc., Ramsey, NY, USA) that was also irradiated was used as a control to ensure that the electron energy reached equilibrium. After irradiation, the cells were returned to the incubation room and cultured for 10-14 days, then stained with 3% crystal violet and the number of cell populations formed was calculated ( 50 cells are 1 cell population).

Example 1 I7457 and I7557 inhibit the growth of drug-resistant CML K562R cells

The human CML cell line K562R resistant to imatinib was obtained by induction using the procedure described in the "Materials and Methods" section above. The growth inhibition activities of I7457 and I7557 were evaluated by cell survival (MTT) and cell cycle analysis, as well as immuno dot blotting and Liu staining, respectively.

Figures 1 and 2 show the effect of I7457 and I7557 on cell viability against imatinib CML cells, respectively. The results shown in the figure show that both I7457 and I7557 are effective in reducing the number of cells, and this effect is dose-dependent; when using 20 μM of the test compound, the number of drug-resistant CML cells can be reduced to at least 65 of the control cells. %.

Cell cycle analysis further showed that at least 60% of CML cells were arrested in the G ₂ /M phase, and the population of cells in the sub-G1 (sub-G1) population increased with increasing dose of I7457 or I7557 (see Table 1).

In addition, compared to the control cells (Fig. 3A), CML cells treated with 20 μM of I7457 (Fig. 3B) or I7557 (Fig. 3B) began to round, giving the appearance a balloon shape, which means CML cells are undergoing apoptosis and mitotic catastrophe. The above-mentioned mitotic events were further confirmed by staining analysis of α- and β-tubulin by drug-treated CML cells (data not shown) and immunoblot analysis. Figure 4 is a graph showing the content of β-tubulin in drug-treated CML cells measured by immuno-ink dot analysis. It can be seen from the figure that compared with the control cells, treatment with I7457 and I7557 It can reduce the content of β-tubulin in cells.

I7457 or I7557-induced mitotic inactivation is further explored by measuring the expression of mitotic inactivation complex proteins and their upstream regulatory proteins; the above mitotic inactive complex proteins such as Cdc2 kinase and cyclin ( Cyclin) B1; and upstream regulatory proteins such as Plk-1, Chk-1 and Chk-2. The results showed that compared with the control group, there was a higher expression of cyclin B1 and phosphorylated Plk-1 in CML cells treated with I7457 and I7557 (Figs. 5 and 6); The expression of phosphorylated Chk-1, phosphorylated Chk-2 and phosphorylated cdc2 kinase was lower in C7 cells treated with I7457 and I7557 (Figs. 7, 8, and 9).

This example also explores the amount of expression of another key mitotic protein-phosphorylated histone H3. Histone H3 is only phosphorylated during cell mitosis, so if the cells are arrested during mitosis, the protein content will increase. As expected, among the drug-resistant CML cells treated with I7457 and I7557, the amount of phosphorylated histone H3 was much higher than that of the control cells (Fig. 10). A mitotic inhibitor known to interfere with microtubule formation in mitotic cells, nocodazole (100 nM), was used as a positive control. In summary, the results of this example show that mitosis is inhibited in drug-resistant CML cells treated with I7457 and I7557.

Example 2 I7457 and I7557 inhibit the growth of human pancreatic cancer-like epithelial cell line PANC-1

The effects of the compounds of the present invention, I7457 and I7557, on PANC-1 cells were investigated by a method similar to that of Example 1 above, using cell viability assay, cell cycle analysis, and immunization of cell cycle-associated proteins. Ink dot analysis and appearance analysis.

The results of the cell viability assay showed that I7457 and I7557 were able to effectively reduce the number of cancer cells by at least 50% at a concentration of 20 μM compared to the control cells (data not shown). Cell cycle analysis showed that at least 50% of the cells were arrested in the G ₂ /M phase after only 24 hours of treatment with I7457 and I7557, and the number of cells in the sub-G1 population increased with increasing dose of I7457 or I7557 ( See Table 2). As for the protein associated with the cell cycle, the amount of expression was similar to that described above for drug-resistant CML cells (data not shown).

Example 3 I7457 and I7557 enhance the sensitivity of human pancreatic cancer cells to radiation treatment

In the present example, the inventors explored the effects of the compounds of the present invention, I7457 and I7557, on the susceptibility of the pancreatic cancer cell lines Panc-1 and BxPc-3 to radiation treatment.

To this end, the pancreatic cancer cells are first treated with radiation according to the procedures disclosed in the "Materials and Methods" section of this article, and then the remaining cancer cells are continued to be cultured and observed by clonogenic assay. The ability of cells to subsequently form cell communities. Cell population analysis is a test in the field of cancer research that is commonly used to test drugs or radiation treatment, and to continue the ability of cancer cells themselves to continue unrestricted, by estimating the number of colonies formed by these cancer cells. Each cell population must contain at least 50 cells. The test results are shown in Tables 3 and 4 below.

The results showed that, regardless of the Panc-1 or BxPc-3 cell line, the two compounds disclosed in the present invention, I7457 and I7557, were effective at enhancing the sensitivity of these cancer cell lines to radiation treatment at a concentration of 10 μM. It also enhances the effect of radiation treatment on killing cancer cells. Thus, the two compounds of the invention, I7457 and I7557, have the potential to be subsequently developed as anticancer drug adjuvants.

Example 4 I7457 and I7557 inhibit the growth of human non-small cell lung cancer cell line SEG-1

The effects of the compounds of the present invention, I7457 and I7557, on SEG-1 cells were investigated by a method similar to that of Example 1 above, using cell viability analysis, cell cycle analysis, and immunostaining of cell cycle-related proteins. Point analysis and appearance analysis.

The results of the cell viability assay showed that I7457 and I7557 were able to effectively reduce the number of cancer cells by at least 70% at a concentration of 20 μM compared to control cells (data not shown). Cell cycle analysis showed that at least 45% of cells were arrested in the G ₂ /M phase after only 24 hours of treatment with I7457 and I7557, and the number of cells in the sub-G1 population increased with increasing dose of I7457 or I7557 ( The data is not shown). As for the protein associated with the cell cycle, the amount of expression was similar to that described above for drug-resistant CML cells (data not shown).

Example 5 I7457 and I7557 inhibit the growth of human esophageal cancer cell lines 81T/VGH and BE-3

Using the method similar to the above Example 1, the compounds I7457 and I7557 of the present invention were probed into the human esophageal squamous cell carcinoma line 81T/VGH and the human esophagus by cell viability analysis. The effect of the growth ability of the human esophageal adenocarcinoma line BE-3 was shown in Tables 5 and 6 below.

For 81T/VGH cells, cell viability analysis showed that after treatment with I7457 and I7557 at 20 nM for 48 hours, both compounds were able to effectively reduce the number of cancer cells by at least 50% compared to control cells (Table 5). ). Similar results were also observed in the cell viability assay of BE-3 cells, and I7457 and I7557 effectively inhibited the growth of approximately 50% of cancer cells at a concentration of about 20 nM (Table 6).

It will be understood that the above-described embodiments and examples are merely illustrative, and that those skilled in the art can align various modifications. The description, examples, and materials set forth above are intended to be illustrative of the present invention and are illustrative of the invention. The present disclosure has been disclosed in the above embodiments, but it is not intended to limit the disclosure, and any person skilled in the art can make various changes and refinements without departing from the spirit and scope of the disclosure. The scope of protection of the disclosure is subject to the definition of the scope of the patent application.

The above and other objects, features, advantages and embodiments of the present disclosure will become more apparent and understood.

1 depicts the effect of I7457 on cell viability of CML K562R cells against imatinib, in accordance with an embodiment of the present invention;

Figure 2 is a graph showing the effect of I7557 on cell viability of CML K562R cells against imatinib according to an embodiment of the present invention;

Fig. 3 is a photograph showing CML K562R cells resistant to imatinib treated by Liu staining according to an embodiment of the present invention, wherein Fig. 3A is a control group cell, and Fig. 3B is a 20 μM I7457 treatment. Cells, and Figure 3C shows cells treated with 20 μM of I7557;

Figure 4 is a graph showing the content of β-tubulin in CML K562R cells treated with I7457 and I7557 against imatinib according to an embodiment of the present invention;

Figure 5 is a graph showing the content of cyclin B1 in CML K562R cells against imatinib treated by I7457 and I7557, respectively, according to an embodiment of the present invention;

Figure 6 is a graph showing the content of p-Plk-1 in CML K562R cells treated with I7457 and I7557 against imatinib according to an embodiment of the present invention;

Figure 7 is a graph showing the content of p-Chk-1 in CML K562R cells treated with I7457 and I7557 against imatinib according to an embodiment of the present invention;

Figure 8 is a graph showing the content of p-Chk-2 in CML K562R cells treated with I7457 and I7557 against imatinib according to an embodiment of the present invention;

Figure 9 is a graph showing the content of p-Cdc2 kinase in CML K562R cells against imatinib treated with I7457 and I7557, respectively, according to an embodiment of the present invention;

Figure 0 is a graph showing the content of phosphorylated histone H3 in imatinib-resistant CML K562R cells treated with I7457 and I7557, respectively, according to an embodiment of the present invention.

Claims (16)

6-methylsulfinylhexyl isothiocyanate for use in the manufacture of a medicament for the treatment of cancer selected from the group consisting of pancreatic cancer, chronic myeloid leukemia, non-small cell lung cancer and the esophagus A group of cancers. The use of claim 1, wherein the chronic myeloid leukemia is resistant to imatinib. The use of claim 1, wherein the cancer is pancreatic cancer. The use of claim 3, wherein the pancreatic cancer is treated with radiation in a subsequent treatment. A method for delaying growth of a majority of cancer cells, comprising administering an effective amount of 6-methylsulfinylhexyl isothiocyanate as claimed in claim 1 or a pharmaceutically acceptable salt thereof, wherein the cancer is pancreas Dirty cancer, chronic myeloid leukemia, non-small cell lung cancer or esophageal cancer. The method of claim 5, wherein the chronic myeloid leukemia is resistant to imatinib. The method of claim 5, wherein the cancer is pancreatic cancer. The method of claim 7, further comprising administering the effective amount of 6-methylsulfinylhexyl isothiocyanate or a pharmaceutically acceptable salt thereof as claimed in claim 1 Dirty cancer is treated with radiation. The use of 6-methylsulfonylhexyl isothiocyanate for the manufacture of a medicament for the treatment of cancer selected from the group consisting of pancreatic cancer, chronic myeloid leukemia, non-small cell lung cancer and esophageal cancer The group formed. The use of claim 9, wherein the chronic myeloid leukemia is resistant to imatinib. The use of claim 9, wherein the cancer is pancreatic cancer. The use of claim 11, wherein the pancreatic cancer is treated with radiation in a subsequent treatment. A method for delaying the growth of a majority of cancer cells comprising administering an effective amount of 6-methylsulfonylhexyl isothiocyanate of claim 9 or a pharmaceutically acceptable salt thereof, wherein the cancer is a pancreas Cancer, chronic myeloid leukemia, non-small cell lung cancer or esophageal cancer. The method of claim 13, wherein the chronic myeloid leukemia is resistant to imatinib. The method of claim 13, wherein the cancer is pancreatic cancer. The method of claim 15, further comprising administering the effective amount of 6-methylsulfonylhexyl isothiocyanate or a pharmaceutically acceptable salt thereof as claimed in claim 1 to the pancreas The cancer is treated with radiation.