KR20190085456A - Ulipristal Acetate For Treatment Of Colnon cancer - Google Patents

Abstract

The present invention relates to ulipristal acetate for treating colorectal cancer, comprising 17α-acetoxy-11β-(4-N,N-dimethylamino-phenyl)-19-norpregna-4,9-diene-3,20-dione, chemical formula 1, or a metabolite thereof.

Description

  ≪ Desc / Clms Page number 2 > Ullium phosphate for treatment of colorectal cancer.

The present invention relates to a method for the treatment of colorectal cancer. The present invention relates to a method for the treatment of colorectal cancer.

Cancer is a group of diseases that can eventually take away the life of an individual by infiltrating and destroying surrounding normal tissues or organs, and transferring any organ of the individual from the primary lesion to a new growth site.

Cancer is generally classified into carcinoma, malignant tumor originating from epithelial cells, and sarcoma, malignant tumor originating from non-epithelial cells, depending on the histological cell originating from the primary lesion.

The carcinoma is subdivided into squamous cell carcinoma, adenocarcinoma, basal cell carcinoma, melanoma, etc., and the sarcoma is divided into fibroid, osteosarcoma, angiosarcoma, lymph node disease, leukemia, and muscular carcinoma.

According to data from the Central Cancer Registry, 224,177 cancer cases occurred in Korea in 2012, among which 28,988 cases of men and women were combined, accounting for 12.9% of the total, which is the third most common cancer. Therefore, in the country, men and women over the age of 50 are required to have a fecal occult blood test once a year.

Colon cancer is a malignant tumor composed of cancer cells in the large intestine. It causes symptoms such as change in bowel habits, diarrhea, constipation, stool, abdominal pain, abdominal distension, fatigue, anorexia or indigestion.

Causes of colorectal cancer can be divided into genetic or environmental factors. Among the environmental factors, changes in Western eating habits (excessive fat intake, fiber and calcium deficiency) and lack of exercise were found to be the most common causes.

For the treatment of colorectal cancer, the treatment method is decided according to the degree of tissue penetration of cancer cells. In primary cancer, most cases are resection, chemotherapy or radiation therapy. However, as a side effect of surgery, postoperative complications such as pulmonary complication, anastomotic leakage, hemorrhage or intestinal obstruction may occur.

Side effects of chemotherapy include leukocytosis, thrombocytopenia, hair loss, nausea (nausea, nausea), vomiting, and fatigue. Side effects of radiation therapy include pelvic pain, changes in bowel habits, dysuria, anal pain, diarrhea Or hair loss is known.

In addition, colorectal cancer recurs in about 20-50% of cases even after radical resection. Local recurrence, distant metastasis, and recurrence with local recurrence and distant metastasis appear. Generally, a large number of recurrences accompanied by local recurrence and distant metastasis are present. In the case of metastasis, it is classified as the most advanced stage of colorectal cancer, and its prognosis is poor.

Therefore, it is necessary to develop new therapeutic potentials for the prevention and treatment of metastatic cancer, which is difficult to perform and has poor prognosis, while complementing current treatments with many side effects.

In order to solve the problems of the prior art, there is provided a therapeutic agent for colorectal cancer as a novel use of Ulifurstal Acetate which is a conventional contraceptive pill and a treatment agent for uterine myoma.

In order to solve the above-mentioned problems, the present invention provides a process for preparing 17α-acetoxy-11β- (4-N, N-dimethylamino-phenyl) -19-norpregna-4,9- -Dione or a metabolite thereof, for the treatment of colorectal cancer.

        [Chemical Formula 1]

The present invention also relates to the use of the above metabolites in monodermethylated CDB-2914 (CDB-3877), didemethylated CDB-2914 (CDB-3963), 17 alpha -hydroxy CDB- 3236) or an aromatic A-ring derivative of CDB-2914 (CDB-4183).

The present invention, Ulyspora pratale acetate (UPA), induces the death of colorectal cancer cells and induces the entry of cancer cells into a resting state, thereby maintaining the growth of cancer cells in a quiescent state, which is effective in the treatment of colon cancer.

FIG. 1 is a graph showing the killing effect of the colon cancer cell line (HT29) for 24 hours depending on the concentration of urolifestalacetate.
2 is a graph showing the killing effect of the colon cancer cell line (HT29) for 48 hours depending on the concentration of urolifastalacetate.
FIG. 3 is a graph showing the killing effect of the colon cancer cell line (HT29) for 72 hours depending on the concentration of wilipristal acetate.
4 is a graph showing the killing effect of a colon cancer cell line (HT116) for 24 hours depending on the concentration of urolifastalacetate.
5 is a graph showing the killing effect of the colon cancer cell line (HT116) for 48 hours depending on the concentration of urolifastalacetate.
FIG. 6 is a graph showing the killing effect of the colon cancer cell line (HT116) for 72 hours depending on the concentration of wilf pristine acetate.
FIG. 7A is a graph showing the results of (A) cell cycle (Sub_G1), (FACSCalibur Flow Cytometer, Becton Dickinson, San Jose, CA, USA) of colorectal cancer cell line (HT29) B) G0 / G1 change, and FIG. 7B shows the content of Sub-G1 and G0 / G1 of the colon cancer cell line (HT29).
FIG. 8A shows the cell cycle (A) cell cycle (Sub_G1), (B) cell cycle (B) by a colon cancer cell line (HT29) flow cytometer (Becton Dickinson, San Jose, CA, USA) ), And FIG. 8B shows the contents of Sub-G1 and G0 / G1 of the colon cancer cell line (HT29).
FIG. 9A shows the cell cycle (A) cell cycle (Sub_G1), (B) cell cycle by a colon cancer cell line (HT29) flow cytometer (Becton Dickinson, San Jose, CA, USA) FIG. 9B is a graph showing changes in G0 / G1, and FIG. 9B shows the content of Sub-G1 and G0 / G1 in the colon cancer cell line (HT29).
FIG. 10A is a graph showing the results of (A) cell cycle (Sub_G1), (B) cell cycle (B) by a colorectal cancer cell line (HT29) flow cytometer (Becton Dickinson, San Jose, CA, USA) FIG. 10B is a graph showing changes in G0 / G1, and FIG. 10B shows the content of Sub-G1 and G0 / G1 in the colon cancer cell line (HT116).
FIG. 11A shows the cell cycle (A) cell cycle (Sub_G1), (B) cell cycle (FACSCalibur Flow Cytometer, Becton Dickinson, San Jose, CA, USA) ), And FIG. 11B shows the contents of Sub-G1 and G0 / G1 of the colon cancer cell line (HT116).
Figure 12a shows the cell cycle (A) cell cycle (Sub_G1), (B) cell cycle (B) by a colon cancer cell line (HT29) flow cytometer (Becton Dickinson, San Jose, CA, USA) ), And FIG. 12B shows the contents of Sub-G1 and G0 / G1 of the colon cancer cell line (HT116).

Hereinafter, preferred embodiments of the present invention will be described in detail. In the following description of the present invention, a detailed description of known functions and configurations incorporated herein will be omitted so as to avoid obscuring the subject matter of the present invention.

As used herein, the terms " about, " " substantially, " " etc. ", when used to refer to a manufacturing or material tolerance inherent in the stated sense, Accurate or absolute numbers are used to help prevent unauthorized exploitation by unauthorized intruders of the referenced disclosure.

Uriferustal acetate (UPA) is a progesterone receptor modulator that effectively binds to and inhibits progesterone receptors in progesterone target tissues

UPA, which is already known as CDB-2914, is present in the present invention as 17α-acetoxy-11β- [4-N, N-dimethylamino-phenyl) -19-norpregna-4,9- -Dione, < / RTI >

         [Chemical Formula 1]

Monodemethylated CDB-2914 (CDB-3877) (Formula 2), which is also a metabolite of wooly pristeacetate (UPA); Didemethylated CDB-2914 (CDB-3963) (Formula 3); 17 alpha-hydroxy CDB-2914 (CDB-3236) (Formula 4); An aromatic A-ring derivative of CDB-2914 (CDB-4183) (Formula 5). The preferred metabolite is mono-demethylated CDB-2914 (CDB-3877) (Formula 2)

         (2)

         (3)

         [Chemical Formula 4]

          [Chemical Formula 5]

The woolly pristare acetate (UPA) or metabolite thereof may be administered via various routes, for example, orally, intravenously, or transdermally. The preferred route of administration is oral. Injection into the tumor site is also possible. Vaginal or intrauterine routes, and the like. Devices that enable sustained release of UPA or its metabolites, particularly vaginal or intrauterine devices, are particularly useful. Subcutaneous implants may also be considered.

Oral solid dosage forms are preferably compressed tablets, or capsules, which may be coated or uncoated.

Capsules are solid dosage forms, preferably rigid or soft gelatin shells, for use as a container for a mixture of active and inactive ingredients. The production and manufacture of hard gelatine and soft elastic capsules is well known to those skilled in the art.

Compressed tablets may contain any excipient, which is a diluent that increases the bulk of the active ingredient, thereby making it possible to produce compressible tablets of practical size. A binder is also needed, which is an agent that imparts the property of agglomeration to a powdery material. Starch, gelatin, saccharides such as lactose or dextrose, and natural and synthetic gums are used. Disintegration is required for tablets to promote disintegration of tablets. Disintegrants include starch, clay, cellulose, algin, gum, and cross-linked polymers. Finally, small amounts of materials known as lubricants and lubricants are included in the tablet, which prevents the refining material from adhering to the surface during manufacture and improves the flow properties of the powder material during manufacture. Colloidal silicon dioxide is the most commonly used lubricant and compounds such as talc or stearic acid are most commonly used as lubricants. The production and manufacture of compressed tablets is well known to those skilled in the art.

Hereinafter, the present invention will be described in more detail with reference to experimental methods and experimental results. However, the present invention is not limited by these experimental methods.

<Experimental Method>

1. Experiments on cell killing effect Annexin  V- FITC / PI apoptosis  assay)

Experimental method colon cancer cell lines (HCT116 and HT29) to ^{1.0 x 10 6 / 100mm cell culture} dish at a density, which was inoculated 2 days adaptation to 2% FBS-DMEM culture medium (37 ℃, 5% CO ₂ concentration). Ulipristal acetate was dissolved in DMSO and cultured at 5, 10, 20, 40, and 60 μM concentrations for 24, 48, and 72 hours, respectively.

Cell apoptosis was assessed using FITC Annexin V + PI (Propidium Iodide) detection kit (BD Biosciences, San Diego, Calif., USA). After mixing the cells with 100 μl of the binding solution, 5 μl of FITC-Annexin V and 5 μl of propidium iodide (PI) were added and allowed to react in the dark for 15 minutes at room temperature. Annexin V-FITC and PI fluorescence measurements were performed using a flow cytometer (FACSCalibur Flow Cytometer, Becton Dickinson, San Jose, Calif., USA) and analyzed with CellQuest pro version 6.0 software (Becton Dickinson, Franklin Lakes, NJ , USA)

2. Cell cycle analysis

For cell cycle analysis by flow cytometry (FACSCalibur Flow Cytometer), 1.0 x at 10 ⁶ / 100mm cell culture dish density of the cells inoculated with the 2% FBS-DMEM culture medium in the 37 ℃, an incubator of 5% CO ₂ concentration 2 Day adaptation. Ulysine stearate (UPA) was dissolved in DMSO and cultured at 5, 10, 20, 40, and 60 μM for 24, 48, and 72 hours, respectively.

  Cells were washed with PBS (0.05% Trypsin - 0.53 mM EDTA) at 24, 48 and 72 hours, washed twice with cold PBS (Phosphate Buffered Saline pH 7.4) Lt; RTI ID = 0.0 &gt; -20 C &lt; / RTI &gt; The fixed cells were washed twice with cold phosphate buffer solution and then treated with RNase A (10 mg / mL) for 30 minutes in a constant temperature water bath at 37 ° C. Finally, Propidium Iodide (Sigma-Aldrich St. Louis, MO) To stain the cells. Cell cycle measurements were performed using a flow cytometer (FACSCalibur Flow Cytometer, Becton Dickinson, San Jose, CA, USA) and analyzed using CellQuest pro version 6.0 software (Becton Dickinson, Franklin Lakes, NJ, USA) The DNA content of the step was analyzed.

* Experiment result

1. Identification of increased expression of apoptotic markers

1, 2, and 3 are graphs showing the killing effect of the colon cancer cell line (HT29) for 24, 48, and 72 hours according to the concentration of urolifastalacetate (UPA) (HT116) for 24, 48, and 72 hours depending on the concentration of UPA, and the expression of apoptotic markers is increased by Annexin V-FITC / PI apoptosis assay .

When apoptosis begins, phosphatidylserine (PS), a cell membrane phospholipid, moves out of the cell membrane and is exposed outside the cell. Annexin V is a calcium-dependent phospholipid binding protein that attaches to negatively charged phospholipids such as PS. Therefore, cells that undergo apoptosis can be identified through the Annexin V and propidium iodide (PI) staining patterns. Early cell death was detected using a flow cytometer using a simple staining procedure using the Annexin V kit. Normally live cells are not labeled with Annexin V and PI, whereas cells in early apoptosis are labeled with Annexin V and not with PI. Cells of the late apoptotic process can be distinguished as being labeled in both Annexin V and PI.

FIGS. 1 to 3 and FIGS. 4 to 6 show that expression of apoptotic marker protein is increased as the concentration of urolifastalacetate (UPA) increases in colon cancer cell lines (HT29) and (HT116) Cell death is also increased.

In particular, the colon cancer cell line (HT116) shows 40% ~ 50% cell death of the colon cancer cell line (HT116) at a concentration of 60 μM of urolifastrate acetate (UPA) for 72 hours

2. Identification of cell cycle changes

7a. 8a and 9a were measured by using a flow cytometer (FACSCalibur Flow Cytometer, Becton Dickinson, San Jose, CA, USA) of a colorectal cancer cell line (HT29) according to the concentration of urolifrise acetate (UPA) for 24, ) Cell cycle (Sub_G1), (B) G0 / G1 change, and Fig. 7b. 8b and 9b are graphs showing the contents of Sub-G1 and G0 / G1 of colon cancer cell line (HT29) for 24, 48 and 72 hours.

10A. 11a and 12a were measured by using a flow cytometer (FACSCalibur Flow Cytometer, Becton Dickinson, San Jose, CA, USA) for colorectal cancer cell lines (HT116) according to the concentration of urolifrise acetate (UPA) for 24, ) Cell cycle (Sub_G1), (B) G0 / G1 change, and Fig. 10b. 11b and 12b are graphs showing the contents of Sub-G1 and G0 / G1 of colorectal cancer cell lines (HT116) for 24, 48 and 72 hours.

The G1 group in the cell cycle is a step of starting the next cycle as a DNA synthesis preparation unit, or determining whether it enters the dormant period, and the Sub-G1 group is an index factor of apoptosis with a DNA level lower than that of normal cells. G0 / G1 blocks cell cycle. That is, it is used as a growth inhibition index.

In general, the cytotoxic effect of anticancer drugs or the total effect of inhibiting the growth of cancer cells is considered to be a potency in the life cycle in which the cell actually divides the cell cycle, In the first place.

Therefore, the killing effect of the cells can be confirmed by apoptosis assay, or by the increase of Sub-G1 in the cell cycle change.

Some of the mechanisms of apoptosis may be increased simultaneously with Sub-G1 and GO / G1, and this phenomenon appears to be the simultaneous expression of the effects of cancer cell death and growth inhibition.

On the other hand, when the mechanism of other cell deaths reaches the limit of inhibiting the growth of cancer cells (confirmed by an increase of G0 / G1), the G0 / G1 decreases and the sub_G1 increases sharply.

As a result, the cell killing effect is the same, but there is only difference in the expression of Sub-G1 and G0 / G1 at the same time or sequentially in the cell cycle change.

In general, if a statistically significant increase in Sub-G1 is evident, apoptosis will occur, and if G0 / G1 is increased together, suicide will be partial and growth will be partially inhibited.

Or that Sub-G1 increases and G0 / G1 begins to decrease indicates that cell growth is inhibited, and sub-G1 is relatively increased when the limit is reached.

In this experiment, the percentage of cells in the sub-G1 stage was significantly lowered in the colorectal cancer cell lines (HT29) and (HT116) according to the concentration of urolifristalacetate (UPA) . Overall, the higher the treatment concentration, the greater the percentage of cells in the sub-G1 phase.

More specifically, in colorectal cancer cell line (HT29), 24, 48, and 72 hours of application, the maximum effect occurs when the urolifastalacetate (UPA) concentration is 60 μM, and the second highest effect occurs when the dose is 40 μM.

In colorectal cancer cell line (HT116), the maximal effect occurs when the urolithiasitic acetate (UPA) concentration is 40 μM at 48 and 72 hours, and the second highest effect occurs at 60 μM. but. And the highest effect is obtained at 60 μM for 24 hours.

The inhibitory effect of G0 / G1 ratio on cell proliferation is similar to that of G0 / G1 ratio in the colon cancer cell line (HT29) G0 / G1 ratio decreased with increasing sub-G1 ratio in 48 hour and 72 hour application, while sub-G1 ratio and G0 / G1 ratio tended to increase simultaneously in early 24 hours in colorectal cancer cell line (HT116) The number of cells entering the dormancy (G0 / G1) increases firstly, and then the cell enters the Sub_G1, resulting in apoptosis.

3. Conclusion

In the cell death experiment, it was confirmed that the degree of apoptosis was increased in colorectal cancer cells as the concentration of urolifastalacetate (UPA) was increased. In the cell cycle analysis, the number of cells entering the dormant period and the number of apoptotic cells Or in succession. However, experimentally, it has been shown that colon cancer cells reach self-destruction when the concentration of urolifrise acetate (UPA) is applied for more than 24 hours.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the inventions. It will be clear to those who have knowledge of.

Claims (2)

A method for the treatment and / or prophylaxis of colorectal cancer, including 17α-acetoxy-11β- (4-N, N-dimethylamino-phenyl) -19-norpregna-4,9-diene-3,20- Therapeutic wooly pristine acetate.

[Chemical Formula 1]
The method according to claim 1,
The metabolite may be monodermethylated CDB-2914 (CDB-3877), didemethylated CDB-2914 (CDB-3963), 17 alpha-hydroxy CDB- Characterized in that it is an aromatic A-ring derivative of CDB-2914 (CDB-3236) (Formula 4) or CDB-2914 (CDB-4183) (Formula 5).

(2)

(3)

[Chemical Formula 4]

[Chemical Formula 5]

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