KR102086827B1 - Ulipristal acetate For Treatment Of Leiomyosarcoma - Google Patents

Abstract

The present invention includes 17α-acetoxy-11β- (4-N, N-dimethylamino-phenyl) -19-norpregna-4,9-diene-3,20-dione or a metabolite thereof The present invention relates to woolly pristal acetate for treating uterine myoma.

Description

 Ullipristal Acetate for treatment of uterine sarcoma {Ulipristal acetate For Treatment Of Leiomyosarcoma}

FIELD OF THE INVENTION The present invention relates to ulipristal acetate for the treatment of uterine myoma, and more particularly, to the invention of uterine myoma sarcoma treatment for ulipristal acetate used as an emergency contraceptive pill or uterine myoma.

Cancer is a group of diseases that can ultimately invade and destroy surrounding normal tissues or organs, and can metastasize any organ of an individual from its primary path to create new growth sites, which can take the individual's life away.

Cancer is largely divided into carcinoma and non-epithelial malignant tumors (sarcoma) that occur in epithelial cells, depending on the tissue cell pathologically caused by the primary pathology.

The carcinoma is further subdivided into squamous cell carcinoma, adenocarcinoma, basal cell carcinoma, melanoma, and the like, and the sarcoma may be subdivided into fibrosarcoma, osteosarcoma, angiosarcoma, lymphadenopathy, leukemia, and myoma.

Uterine sarcoma is a malignant tumor that occurs in the muscles or connective tissue of the uterus. The uterus divides the lower part into the cervix and the upper part into the body. Uterine sarcomas occur more in the body than the cervix, and uterine myoma and uterine sarcoma, which are benign tumors of the uterine body, are difficult to distinguish. In particular, the rapid growth of post-menopausal myoma may be a typical symptom of myoma.

Treatment of uterine sarcoma includes surgery, radiation therapy, chemotherapy, etc. like general cancer, but there are difficulties such as side effects and long recovery time due to surgery or radiation therapy. In the past, alternative treatments using emergency contraceptives and uterine fibroids have become of interest.

In order to solve the problems of the prior art, to provide a therapeutic drug for uterine myoma sarcoma as a new use of the existing emergency contraceptive pill and ulcer free acetate, uterine fibroid therapy.

In order to solve the above problems, the present invention provides the following Chemical Formula 1, α-acetoxy-11β- (4-N, N-dimethylamino-phenyl) -19-norpregna-4,9-diene-3,20 Provided are Ulifrystal Acetate for treatment of uterine myoma comprising dione or a metabolite thereof.

[Formula 1]

In the present invention, the metabolite is monodemethylated CDB-2914 (CDB-3877), dedemethylated CDB-2914 (CDB-3963), 17 alpha-hydroxy CDB-2914 (CDB- 3236) or an aromatic A-ring derivative of CDB-2914 (CDB-4183).

The present invention, Ulifrystal Acetate (UPA) induces the death of uterine myoma sarcoma cells and at the same time induces the cancer cells to enter the resting state has the effect of maintaining the growth of the cancer cells is effective in the treatment of uterine sarcoma.

Figure 1 is a graph of the killing effect of the uterine myoma cell line (SK-UT-1B) for 24 hours according to the concentration of woolly pristal acetate.
Figure 2 is a graph of the killing effect of the uterine myoma cell line (SK-UT-1B) for 48 hours according to the concentration of woolly phthalate acetate.
Figure 3 is a graph of the killing effect of the uterine myoma cell line (SK-UT-1B) for 72 hours according to the concentration of woolly pristal acetate.
FIG. 4A shows the cell cycle (A) by flow cytometry (FACSCalibur Flow Cytometer, Becton Dickinson, San Jose, Calif., USA) of uterine myoma cell line (SK-UT-1B) according to the concentration of ulipristal acetate for 24 hours. Sub_G1), (B) is a graph of G0 / G1 change, and FIG. 4B shows the contents of Sub-G1 and G0 / G1 of the uterine myoma cell line (SK-UT-1B).
FIG. 5A shows the cell cycle (A) by flow cytometry (FACSCalibur Flow Cytometer, Becton Dickinson, San Jose, Calif., USA) of uterine myoma cell line (SK-UT-1B) according to the concentration of ulipristal acetate for 48 hours. Sub_G1), (B) is a graph of G0 / G1 change, and FIG. 5B shows the contents of Sub-G1 and G0 / G1 of the uterine myoma cell line (SK-UT-1B).
FIG. 6A shows the cell cycle (A) by flow cytometry (FACSCalibur Flow Cytometer, Becton Dickinson, San Jose, Calif., USA) of uterine myoma cell line (SK-UT-1B) according to the concentration of ulipristal acetate for 72 hours. Sub_G1), (B) is a graph of G0 / G1 changes, and FIG. 6B shows the contents of Sub-G1 and G0 / G1 of the uterine myoma cell line (SK-UT-1B).

Hereinafter, a preferred embodiment of the present invention will be described in detail. First, in describing the present invention, a detailed description of related known functions or configurations will be omitted so as not to obscure the subject matter of the present invention.

As used herein, the terms 'about', 'substantially', and the like, are used at, or in close proximity to, numerical values when manufacturing and material tolerances inherent in the meanings indicated are intended to aid the understanding of the invention. Accurate or absolute figures are used to assist in the prevention of unfair use by unscrupulous infringers.

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

UPA, already known as CDB-2914, is used in the present invention as 17α-acetoxy-11β- [4-N, N-dimethylamino-phenyl) -19-norpregna-4,9-diene-3,20 Dione, having the general formula (I):

[Formula 1]

Woollypristal acetate (UPA) has been approved for emergency contraception (trade name EllaOne®), and for the treatment of uterine fibroids (trade name Esmya®). Various other potential clinical applications have been proposed in Chabbert-Buffet et al, Human Reproduction, 2005, 11 (3): 293-307.

Also a metabolite of woolly phthalate acetate (UPA) such as monodemethylated CDB-2914 (CDB-3877) (formula 2) dimethylated CDB-2914 (CDB-3963) (Formula 3) 17 alpha-hydroxy CDB-2914 (CDB-3236) (Formula 4); Aromatic A-ring derivatives of CDB-2914 (CDB-4183) (Formula 5) are included. Preferably the metabolite is monodemethylated CDB-2914 (CDB-3877) (Formula 2)

         [Formula 2]

        [Formula 3]

       [Formula 4]

      [Formula 5]

Woollypristal acetate (UPA) or a metabolite thereof may be administered via a variety of routes, eg, orally, intravenously, or transdermally. Preferred routes of administration are oral routes. Injection into the tumor site is also possible. Other routes of administration, including intravaginal or intrauterine routes, are included. Particularly useful are devices that allow sustained release of UPA or its metabolites, especially intravaginal or intrauterine devices. Subcutaneous implants may also be considered further.

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

Capsules are solid dosage forms which preferably use hard or soft gelatin shells as containers for a mixture of active and inactive ingredients. The process of producing and manufacturing hard gelatin and soft elastic capsules is well known to those skilled in the art.

Compressed tablets may include any excipients, which are diluents that increase the bulk of the active ingredient, thus allowing the production of compressed tablets of practical size. There is also a need for a binder, which is an agent that imparts the nature of aggregation to the material in powder form. Starch, gelatin, sugars such as lactose or dextrose, and natural and synthetic gums are used. Disintegrants are necessary for tablets to facilitate the breakdown of the tablets. Disintegrants include starch, clays, celluloses, algins, gums and crosslinked polymers. Finally, small amounts of materials known as lubricants and lubricants are included in the tablets, which prevents the tablet material from adhering to the surface during the manufacturing process and improves the flow properties of the powdered material during manufacture. Colloidal silicon dioxide is most commonly used as a lubricant, and compounds such as talc or stearic acid are most commonly used as lubricants. The production and manufacturing process of compressed tablets is well known to those skilled in the art.

Hereinafter, the present invention will be described in more detail through the following experimental methods and experimental results. However, the present invention is not limited by this experimental method.

Experimental Method

1. Cell death effect experiment Annexin  V- FITC / PI apoptosis  assay)

Experimental method was inoculated with uterine sarcoma cell line (SK-UT-1B) at 1.0 x 10 ⁶ / 100mm cell culture dish density and adapted for 2 days with 2% FBS-DMEM medium (37 ° C, 5% CO ₂ concentration). ). After dissolving Ulpriprise Acetate (UPA) in DMSO, treated with cell lines (SK-UT-1B) at 5, 10, 20, 40, and 60 μM concentrations, and incubated for 24 hours, 48 hours, and 72 hours, The experiment was carried out.

Apoptosis effect of the cells (SK-UT-1B) was measured using a FITC Annexin V + PI (Propidium Iodide) detection kit (BD Biosciences, San Diego, CA, USA) to determine the degree of apoptosis. After mixing the cells with 100μl binding solution, 5μl FITC-Annexin V and 5μl propidium iodide (PI) were added and reacted at room temperature for 15 minutes in the dark. Annexin V-FITC and PI fluorescence measurements were measured using a flow cytometer (FACSCalibur Flow Cytometer, Becton Dickinson, San Jose, Calif., USA). , 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 Daily adaptation was performed. After dissolving Ulpristal Acetate (UPA) in DMSO, the cells were treated at 5, 10, 20, 40, and 60 μM concentrations, and then cultured for 24 hours, 48 hours, and 72 hours, and harvested at a predetermined time.

Cells were washed with PBS at 24, 48 and 72 hours, respectively, and treated with 0.05% Trypsin-0.53 mM EDTA, and then cells were harvested and washed twice with cold phosphate buffer (PBS, Phosphate Buffered Saline pH 7.4) and 70%. Fixed with ethanol at -20 ° C overnight. The fixed cells were washed twice with cold phosphate buffer solution and treated with RNase A (10mg / mL) for 30 minutes in a 37 ℃ constant temperature water bath. Finally, 50mg / of Propidium Iodide (Sigma-Aldrich St. Louis, MO) Cells were stained by treatment with mL. Cell cycle measurements were measured using a flow cytometer (FACSCalibur Flow Cytometer, Becton Dickinson, San Jose, Calif., USA), and analysis was performed using CellQuest pro version 6.0 software (Becton Dickinson, Franklin Lakes, NJ, USA). The DNA content of the step was analyzed.

* Experiment result

1. Confirmation of apoptosis marker protein expression

1, 2, and 3 are graphs of the killing effect of the uterine myoma cell line (SK-UT-1B) for 24, 48, and 72 hours according to the concentration of woolly freetal acetate (UPA), Annexin V-FITC / PI apoptosis assay method shows that apoptosis marker protein expression is increasing.

When apoptosis begins, the cell membrane phospholipid, phosphatidyl serine (PS), moves out of the cell membrane and is exposed out of the cell. Annexin V is a calcium dependent phospholipid binding protein that attaches to negatively charged phospholipids such as PS. Therefore, cells undergoing apoptosis can be identified by Annexin V and propidium iodide (PI) staining patterns. Initial cell death was detected using a flow cytometer through a simple staining procedure using Annexin V kit (BD Pharmingen, San Diego, Calif., USA). Normally living cells are not labeled with Annexin V and PI, cells with early cell death process are labeled with Annexin V and not with PI. Cells of late cell death process can be distinguished by being labeled with both Annexin V and PI.

1 to 3 can be seen that the expression of apoptosis marker protein also increases with the increase in the concentration of woolly pristal acetate (UPA), it can be seen that the degree of apoptosis also increases.

In particular, uterine sarcoma cell line (SK-UT-1B) shows more than 60% cell death at 40 and 60 μM concentration of woolly freetal acetate (UPA) for 48 and 72 hours.

2. Confirm cell cycle changes

Figure 4a. 5a, 6a are flow cytometry (FACSCalibur Flow Cytometer, Becton Dickinson, San Jose, Calif., USA) of uterine myoma cell line (SK-UT-1B) according to the concentration of woolly free acetate (UPA) for 24, 48 and 72 hours. (A) Cell cycle (Sub_G1), (B) is a graph of the G0 / G1 change by, Figure 4b. 5b and 6b are graphs showing the content of Sub-G1 and G0 / G1 of the uterine myoma cell line (SK-UT-1B) for 24, 48 and 72 hours.

In the cell cycle, G1 phase is a DNA synthesis preparatory phase to determine whether to start the next cycle or enter the resting phase, Sub-G1 phase is a marker of cell death with less DNA than normal cells. G0 / G1 is used as an indicator of cell cycle blocking, or proliferation.

Typically, the cell death effect of an anticancer agent or the prerequisite effect of inhibiting the growth of cancer cells is the position of cells in the life cycle, in which the cells actually divide the cell cycle. Should be observed as a whole.

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

In some cell death mechanisms, Sub-G1 and GO / G1 may be increased at the same time, and this phenomenon is thought to simultaneously express the effects of cancer cell death and growth inhibition.

On the other hand, other mechanisms of apoptosis inhibit the growth of cancer cells as much as possible (confirmed by the increase of G0 / G1). If the limit is reached, G0 / G1 may decrease and may rapidly move toward Sub_G1.

Eventually, the killing effect of the cells is the same, but the only difference in whether the sub-G1 and G0 / G1 is expressed at the same time or sequentially in the cell cycle changes.

In general, if sub-G1 increases statistically, it is certain that apoptosis effect occurs. In addition, if G0 / G1 increases together, some may commit suicide and some may inhibit growth.

Alternatively, there is a section in which Sub-G1 increases and G0 / G1 begins to decrease, indicating that sub-G1 increases relatively when the limit is reached.

In the present experiment, the percentage of cells in sub-G1 phase was gradually increased according to the concentration of Ulpriprital Acetate (UPA) in comparison with the control group by treatment with Ulpriprisal Acetate (UPA) in the myoma sarcoma cell line (SK-UT-1B). It can be seen that the increase. However, during the initial 24 hours, the maximum effect occurs when the concentration of the Ulifrystal acetate (UPA) is 40μM, but when observed for 48, 72 hours, Figure 4b. As can be seen in 5b and 6b, the percentage of cells in the sub-G1 phase at 20,40,60 μM of Ulpriprisyl Acetate (UPA) concentration is 60-80%.

In addition, the ratio of G0 / G1 was decreased by Ullipristal Acetate (UPA) treatment in uterine myoma sarcoma cell line (SK-UT-1B). Growth inhibition is stronger than apoptosis, but if the treated farmhouse exceeds 20μM, it can be seen that the cell death stage sub-G1 is increased beyond the limitation of cell growth inhibition by G0 / G1.

More specifically 4b. As can be seen from 5b and 6b, the maximum effect of apoptosis occurs at 60% or more of the cell percentage when the concentration of Ulifrystal Acetate (UPA) is 40 μM during the first 24 hours, but the Ulifrystal Acetate (UPA) for 48 and 72 hours. Even at concentrations of 20, 40 and 60 μM, all show an effect of more than 60% of the cell percentage.

3. Conclusion

In the cell death experiment, it was confirmed that the effect of cell death in uterine myoma sarcoma cell line (SK-UT-1B) increased as the concentration of Ulpripristal Acetate (UPA) increased, and cell cycle analysis entered the resting period (G0 / G1). It can be seen that the number of cells to increase first and then enter Sub_G1 to cause apoptosis effect.

The present invention described above is not limited to the above-described embodiments and the accompanying drawings, and various substitutions, modifications, and alterations are possible within the scope without departing from the technical spirit of the present invention. It will be evident to those who have knowledge of.

Claims (2)

A pharmaceutical composition for treating uterine sarcoma comprising 17α-acetoxy-11β- (4-N, N-dimethylamino-phenyl) -19-norpregna-4,9-diene-3,20-dione Composition.

[Formula 1]
Monodemethylated CDB-, a metabolite of 17α-acetoxy-11β- (4-N, N-dimethylamino-phenyl) -19-norpregna-4,9-diene-3,20-dione 2914 (CDB-3877) (Formula 2), dimethylated CDB-2914 (CDB-3963) (Formula 3), 17 alpha-hydroxy CDB-2914 (CDB-3236) (Formula 4) or CDB- A pharmaceutical composition for treating uterine myoma sarcoma, comprising any one of the aromatic A-ring derivatives of 2914 (CDB-4183) (Formula 5).

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

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