USE OF 3-O-SULFAMATE-16,16-DIMETHYL-D-HOMOEQUILENIN TO TREAT ONCOLOGICAL DISEASES

Abstract

The invention relates to the field of medicine and to the chemical and pharmacological industry, and concerns agents for the treatment of cancer. 3-O-Sulfamate 16,16-dimethyl-D-homoequilenin is proposed as an anti-cancer agent in monotherapy and adjuvant therapy of oncological diseases such as hepatocellular carcinoma, gastric carcinoma, lung cancer, chronic myelogenous leukemia, and breast cancer including triple negative breast cancer. The new compound has not any uterotropic activity in animals and does not influence on endometrium.

Claims

1. (canceled)

2. A method of treating cancer, the method comprising administering to a patient in need thereof an effective amount of 3-O-sulfamate-16,16-dimethyl-D-homoequilenin of formula (3) ##STR00003##

3. The method of claim 2, wherein the cancer is hepatocellular carcinoma, gastric carcinoma, lung cancer, chronic myelogenous leukemia, or breast cancer.

4. The method of claim 2, wherein the patient is a female patient with an early stage of breast cancer.

5. The method of claim 2, wherein the patient is a female patient with an advanced form of breast cancer.

6. The method of claim 3, wherein the cancer is a triple negative form of breast cancer.

7. The method of claim 3, wherein 3-O-sulfamate-16,16-dimethyl-D-homoequilenin of formula (3) is administered as monotherapy or adjuvant therapy.

8. The method of claim 3, wherein the patient has been treated with tamoxifen.

9. The method of claim 3, wherein the patient is a woman in postmenopause.

10. The method of claim 3, wherein 3-O-sulfamate-16,16-dimethyl-D-homoequilenin of formula (3) is administered orally.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0094] The invention is illustrated by the following figures:

[0095] FIG. 1 depicts the staining of breast tumor with ERα antibodies.

[0096] Visualization of horseradish peroxidase+diaminobenzidine, magnification ×100 (A). A specific staining of breast duct cell nuclei and nuclei of single tumor cells (B), magnification ×400.

[0097] FIG. 2 depicts the average volume of tumors at the beginning of the experiment in mice receiving the CAF therapy.

[0098] FIG. 3 depicts the average total volume of tumors at the beginning of the experiment in mice receiving the CAF therapy.

[0099] FIG. 4 depicts the relative change in the average total volume of tumors established at the beginning of the experiment in mice receiving the CAF therapy.

[0100] FIG. 5 depicts the average volume of tumors in mice when assessing the antitumor activity of the medicament.

[0101] FIG. 6 depicts the average total volume of tumors in mice when assessing the antitumor activity of the medicament.

[0102] FIG. 7 depicts the relative change in the average total volume of tumors in mice when assessing the antitumor activity of the medicament.

EXAMPLES

[0103] The invention can be further illustrated by examples of carrying out thereof disclosed herein below.

Example 1

[0104] The study analysis was carried out on an passaged MCF-7 human cells culture culture (breast adenocarcinoma). Normal human dermal fibroblasts (HDF) of early passages were used as a negative control. The cells were cultivated in Carrel vials in DMEM/F12 medium (Biolot) with 1.0% of antibiotic-free fetal bovine embryonic serum (Biolot) added in the 5% CO.sub.2 atmosphere, at 37° C.

[0105] The cells were seeded on Carrel vials at 50×10.sup.4 cells per flask. To study the proliferative activity of the cells under conditions of sulfatase inhibition, 24 hours after seeding the culture medium was replaced with the medium containing sulfatase inhibitors to a final concentration of 50 μg/ml, and then these tumor cell lines were incubated for various periods of time (24 to 72 hrs). The inhibitor was dissolved in DMSO. The final concentration of DMSO in the culture medium did not exceed 0.5%. To exclude the cytotoxic effect of DMSO, a control sample comprising DMSO without sulfatase inhibitor was prepared. To exclude nonspecific detrimental effects of the compounds, normal human skin fibroblasts were used. Then the cells were detached with the versene-trypsin solution (Biolot), plated onto Carrel vials containing a fresh complete culture medium. The cells were counted when untreated control cells reached the maximum cell density per unit of surface area of a culture flask (monolayer), and their number was set as 100%. The proliferative activity of all of the studied cell cultures exposed to sulfatase inhibitors was determined in triplicate.

[0106] The study of the obtained sulfamate effects on the proliferation of passaged human MCF-7 cell culture (breast adenocarcinoma) has demonstrated that the steroid of formula (3) at a concentration of 20 μg/ml completely blocks the proliferation of tumor cells but it does not influence the growth of human skin fibroblasts having no estrogen receptors. The proliferation of tumor cells is inhibited to the same extent as under the action of Tamoxifen which has been used in clinical practice for more than 30 years. This is very important because sulfamates and tamoxifen have different mechanisms of action, and there is a potential for their use as a combination.

[0107] In the experiments on FVB mice, transgenic for HER-2/neu (ER−/PR−/HER2+) having breast tumours, the agent inhibits the proliferation of tumours more effectively than clinically used Tamoxifen, which suggests the potential of their use as a combination for the treatment of breast cancer.

[0108] In triple negative breast cancer MDA-MB-231 cell line, the compound inhibits cell proliferation by 83%, which is similar to etoposide, a chemotherapeutic medicament used in clinical practice.

[0109] The medicament was active against hepatocellular carcinoma SK-Hep-1 cells (IC.sub.50=53.4 μM), lung cancer A549 (IC.sub.50=29.6 μM), gastric adenocarcinoma SNU638 (IC.sub.50=22.8 μM), as well as against chronic myelogenous leukemia cells (K562).

Example 2

[0110] Control group. There were 10 mice, 10 tumors per group. At the beginning of the therapy V.sub.average=1.5±0.4 mm.sup.3. Without a specific treatment tumor sizes reached V.sub.average=233.2±86.5 mm.sup.3 by Day 33 after transplantation. The number of dead mice in this group before the end of the observation period was 2 out of 10.

[0111] Medicament group, 10 mg/kg. There were 7 mice, 7 tumors per group.

[0112] At the beginning of the therapy V.sub.average=1.0±0.4 mm.sup.3 without significant differences from the control group. On Day 7 after the beginning of the treatment, non-significant TGI=74.3% was recorded (V.sub.average=5.1±2.3 mm.sup.3 versus V.sub.average=20.0±6.0 mm.sup.3 in the control, p<0.220). On Day 10 after the beginning of the treatment, TGI=68.0% (V.sub.average=24.8±12.2 mm.sup.3 versus V.sub.average=77.3±32.0 mm.sup.3 in the control, p<0.181). On Day 14 after the beginning of the treatment, the maximum significant TGI of 94.3% was observed (V.sub.average=6.9±2.2 mm.sup.3 versus V.sub.average=120.6±44.4 mm.sup.3 in the control, p<0.03) which remained at approximately the same level and was 90.6% (V.sub.average=17.6±0.7 mm.sup.3 versus V.sub.average=186.4±65.0 mm.sup.3 in control, p<0.061) and 90.2% (V.sub.average=26.1±12.1 mm.sup.3 versus V.sub.average=266.5±91.3 mm.sup.3 in control, p<0.061) by Day 17 and Day 21, respectively. The number of dead mice in this group before the end of the observation period was 5 out of 7.

[0113] Tamoxifen group, 30 mg/kg. There were 7 mice, 7 tumors per group. At the beginning of therapy V.sub.average=0.9±0.2 mm.sup.3 without significant differences from the control group. On Day 7, TGI was 12.6% (V.sub.average=18.8±7.4 mm.sup.3 versus V.sub.average=20.0±6, mm.sup.3 in the control, p<0.962) and did not exceed these values until the end of the observation period. On Day 17 and Day 21 after the beginning of the treatment, negative TGI was observed with a maximum value on Day 21 TGI=−44.2 (V.sub.average=384.8±103.2 mm.sup.3 versus V.sub.average=266.5±91.3 mm.sup.3 in control, p<0.106). The number of dead mice in this group before the end of the observation period was 6 out of 7.

[0114] Tamoxifen group, 10 mg/kg. There were 7 mice, 7 tumors per group. At the beginning of therapy V.sub.average=0.7±0.1 mm.sup.3 without significant differences from the control group. On Day 7 after the beginning of the treatment, non-significant TGI=63.9% was recorded (V.sub.average=7.2±3.9 mm.sup.3 versus V.sub.average=20.2±6.0 mm.sup.3 in the control, p<0.364). On Day 10 after the beginning of the treatment, TGI=56.9% was recorded (V.sub.average=33.4±16.1 mm.sup.3 versus V.sub.average=77.3±32.0 mm.sup.3 in the control, p<0.315). Through Day 14 to 17 an increase in TGI to 60.6% was observed (V.sub.average=47.5±16.8 mm.sup.3 versus V.sub.average=120.6±44.4 mm.sup.3 in the control, p<0.193) and 68.9% (V.sub.average=58.0±18.2 mm.sup.3 versus V.sub.average=186.4±65.0 mm.sup.3 in the control, p<0.070), respectively. On Day 21 after the beginning of the treatment and until the end of the observation period, TGI was below biologically significant values (<50%). The number of dead mice in this group before the end of the observation period was 2 out of 7.

[0115] The final results of the measurements and statistical significance of their differences between the groups are shown in Tables 10 and 11.

TABLE-US-00010 TABLE 10 The average tumor volumes with SEM in mice in the groups (M ± m) Days from the beginning of the treatment Groups 0 3 7 10 14 17 21 24 Steroid, 1.0 ± 0.4 1.2 ± 0.4 5.1 + 2.3 24.8 ± 12.2 6.9 ± 2.2 17.6 ± 0.7 26.2 ± 12.1 32.0 ± 6.9 10 mg/kg Tamoxifen 0.9 ± 0.2 2.0 ± 0.4 18.8 ± 7.4  67.6 ± 21.1 117.1 ± 41.7  218.3 ± 63.6 384.8 ± 103.2 392.1 30 mg/kg Tamoxifen 0.7 ± 0.1 1.0 + 0.3 7.2 ± 3.9 33.4 ± 16.1 47.5 ± 16.8  58.0 ± 18.2 145.5 ± 70.9  211.1 ± 94.0 10 mg/kg Control 1.5 ± 0.4 2.2 + 0.5 20.0 ± 6.0  77.3 ± 32.0 120.6 + 44.4  186.4 ± 65.0 266.5 ± 91.3  233.2 ± 86.5

TABLE-US-00011 TABLE 11 Effects of the medicament and Tamoxifen on SKBR-3 growth inhibition over the entire observation period, TGI % Days from the beginning of the treatment Group 7 10 14 17 21 24 1 Medicament, 10 mg/kg 74.3 68.0 94.3 90.6 90.2 86.3 2 Tamoxifen, 30 mg/kg 6.2 12.6 2.9 −17.1 −44.4 −68.1 3 Tamoxifen, 10 mg/kg 63.9 56.9 60.6 68.9 45.4 9.5

Example 3

[0116] Control group. There were 12 mice, 12 tumors per group. At the beginning of the therapy V.sub.average=3.8±1.0 mm.sup.3. Without a specific treatment tumor sizes reached V.sub.average=299.1±100.5 mm.sup.3 by Day 30 after transplantation. The number of dead mice in this group before the end of the observation period was 6 out of 12.

[0117] Medicament group, 10 mg/kg. There were 10 mice, 10 tumors per group. At the beginning of therapy V.sub.average=1.6±0.4 mm.sup.3 without significant differences from the control group. On Day 7 after the beginning of the treatment, significant TGI=66.1% was recorded (V.sub.average=27.0±5.1 mm.sup.3 versus V.sub.average=79.6±14.2 mm.sup.3 in the control, p<0.002). On Day 10 after the beginning of the treatment, TGI=78.7% was observed (V.sub.average=21.6±4.9 mm.sup.3 versus V.sub.average=101.2±22.5 mm.sup.3 in the control, p<0.007). On Day 14 after the beginning of the treatment, the maximum non-significant TGI equal to 83.1% was observed (V.sub.average=27.6±2.6 mm.sup.3 versus V.sub.average=163.3±31.0 mm.sup.3 in control, p<0.06). On Day 17 after the beginning of the treatment, TGI decreased to 76.1% (V.sub.average=46.3±5.3 mm.sup.3 versus V.sub.average=193.6±39.6 mm.sup.3 in the control, p<0.04). The number of dead mice in this group before the end of the observation period was 10 out of 10.

[0118] Tamoxifen group, 30 mg/kg. There were 10 mice, 10 tumors per group. At the beginning of therapy V.sub.average=4.1±1.2 mm.sup.3 without significant differences from the control group. On Day 7 and until the end of the observation period, TGI was below a biologically significant threshold and did not exceed 44.5% (V.sub.average=44.1±8.8 mm.sup.3 versus V.sub.average=79.6±14.2 mm.sup.3 in the control, p<0.10). The number of dead mice in this group before the end of the observation period was 4 out of 10.

[0119] Tamoxifen group, 10 mg/kg. There were 10 mice, 10 tumors per group. At the beginning of therapy V.sub.average=2.3±0.8 mm.sup.3 without significant differences from the control group. In this group, no tumor growth inhibition was detected during the entire observation period. The number of dead mice in this group before the end of the observation period was 6 out of 9.

[0120] The results of the measurements and statistical significance of their differences between the groups are shown in Tables 12 and 13.

TABLE-US-00012 TABLE 12 The average tumor volumes with SEM in mice in the groups (M ± m) Days from the beginning of the treatment Groups 0 3 7 10 14 17 21 Medicament, 1.57 ± 0.35  5.65 ± 1.64 26.98 ± 5.12  21.59 ± 4.88 27.59 + 2.57 46.25 + 5.31 i-g. 10 Tamoxifen, 4.14 + 1.22 17.12 ± 4.12 44.13 ± 8.81   61.37 ± 12.03 119.53 ± 18.54 141.85 ± 35.07 199.54 ± 43.55 30 mg/kg (2) Tamoxifen, 2.33 ± 0.76 25.61 ± 5.56 79.17 ± 18.80 122.09 + 27.84 193.24 ± 54.10 237.32 ± 77.07 322.76 ± 88.25 10 mg/kg (3) Control (4) 3.84 ± 1.04 28.52 ± 6.27 79.57 ± 14.20 101.19 ± 22.51 163.25 ± 31.00 193.58 + 39.56  299.05 ± 100.50

TABLE-US-00013 TABLE 13 Effect of the medicament and Tamoxifen on TNBC_Kad growth inhibition over the entire observation period, TGI % Days from the beginning of the treatment Group 7 10 14 17 21 1 Medicament, 10 mg/kg 66.1 78.7 83.1 76.1 — 2 Tamoxifen, 30 mg/kg 44.5 39.4 26.8 26.7 33.3 3 Tamoxifen, 10 mg/kg 0.5 −20.7 −18.4 −22.6 −7.9

[0121] The carried out comparative studies of the antitumor activity of the medicament after multiple administrations to athymic immunodeficient Balb/c nude mice in two models of subcutaneous xenografts of human SKBR-3 and TNBC-Kad breast cancers which are different in phenotype.

[0122] A pilot experiment of the antitumor activity of the medicament in SKBR3 model in comparison with Tamoxifen (dosage form) has shown that the therapy with the medicament at a dose of 10 mg/kg was the most effective among all studied treatment regimens: on Day 10 after the beginning of the therapy, a significant antitumor effect was revealed, i.e. the maximum TGI=91.2% (V.sub.average=2.2±2.0 mm.sup.3 versus V.sub.average=24.9±12.5 mm.sup.3 in control, p<0.018) alongside with a relatively good tolerability of the medicament. Tamoxifen at 10 mg/kg dose was not effective: a biologically significant TGI value was not reached over the entire observation period. On the contrary, on Days 10 to 17 after the beginning of the treatment, the tumor growth stimulation was recorded.

[0123] A repeated experiment of the antitumor activity of the medicament in the same SKBR3 model in comparison with Tamoxifen (the reference drug) has shown that the medicament at 10 mg/kg dose has a high antitumor effect with maximum significant TGI on Day 14 (94.3%, p<0.03) and Day 24 (86.3%, p<0.04) that reproduced the result obtained in the first experiment.

[0124] An experiment of the antitumor activity of the medicament in TNBC_Kad model in comparison with Tamoxifen (the reference drug) has shown that the medicament at 10 mg/kg dose inhibits the tumor growth by 66.1 and 78.7% on Days 7 and 10 after the beginning of the treatment (the significant differences from the control groups and groups with tamoxifen 30 mg/kg being observed). The effect lasts for 10 days at approximately the same level. Tamoxifen at 30 mg/kg dose causes a weak non-significant inhibition of tumor growth by 44.5% on Day 7 which decreases to the end of observation. Tamoxifen at 10 mg/kg dose is not effective in this model.

INDUSTRIAL APPLICABILITY

[0125] The conducted experiments have shown that the claimed use of the compound is characterized by: [0126] the absence of uterotropic activity, [0127] a wide range of anti-cancer activities including activity against triple negative breast cancer, [0128] the absence of toxicity, [0129] the hypocholesterolemic activity and the absence of effects on the content of triglycerides in blood serum, [0130] the absence of negative effects on the endometrium.

[0131] The invention can be used for: [0132] the monotherapy and adjuvant therapy of early hormone-positive breast cancer in postmenopausal women; [0133] the monotherapy and adjuvant therapy of early stage of hormone-positive breast cancer in postmenopausal women having treated with tamoxifen for 2-3 years; [0134] the treatment of advanced breast cancer at late stages; [0135] the treatment of triple negative breast cancer; [0136] the monotherapy and adjuvant therapy of hepatocellular carcinoma; [0137] the monotherapy and adjuvant therapy of gastric carcinoma; [0138] the monotherapy and adjuvant therapy of lung cancer; [0139] the monotherapy and adjuvant therapy of chronic myelogenous leukemia.