WO2022028615A1 - Method for treating tumor - Google Patents

Abstract

Uses of 4-hydroxyisoleucine in preventing and/or treating a tumor. The uses comprise single-drug administration, combined use with a second active ingredient, or radiotherapy. Provided in the present invention is a novel tumor fighting method.

Description

Methods of treating tumors

Background of the Invention

Tumor treatments include surgery, chemotherapy, and radiotherapy. However, surgical treatment is only suitable for early tumor treatment, and radiotherapy has toxic and side effects that cannot be ignored.

The general structure of 4-hydroxyisoleucine is

, these amino acids and their analogs have been reported to be anti-diabetic, especially (2S, 3R, 4S)-4-hydroxyisoleucine (4-HIL) with the following structural formula

.

SUMMARY OF THE INVENTION

The invention discloses that 4-hydroxyisoleucine can block the process of tumor cell cycle, induce tumor cell apoptosis, inhibit tumor growth and reduce tumor volume. The median inhibitory concentration (IC ₅₀ ) of 4-hydroxyisoleucine on tumor cells can be less than 5 mM.

The invention also discloses that the combination of 4-hydroxyisoleucine and the second active ingredient or radiotherapy can resist tumor. The second active ingredient can be a chemotherapeutic agent, and the combination of 4-hydroxyisoleucine and the chemotherapeutic agent can exhibit a synergistic effect in inhibiting tumor cell growth.

Accordingly, the present invention provides a method of preventing and/or treating a tumor comprising administering to a subject, the medicament comprising 4-hydroxyisoleucine and a pharmaceutically acceptable alternative form thereof, the pharmaceutically acceptable alternative form Included are active analogs, lactone forms, salts, prodrugs or active metabolites of the amino acid. The prevention of tumors according to the present invention may not include: inhibiting the proliferation of cell lines related to the risk of developing cancer.

Description of drawings

Figure 1 4-HIL inhibits the proliferation of tumor cell lines 4T1, B16OVA, CT26, MCF-7, KYSE450, RKO, HT29 and HepG2 in vitro.

Figure 2 The inhibitory effect of 4-HIL at 4 mM in combination with doxorubicin (DOX) at 2.5 μM on mouse breast cancer 4T1 cell line.

Figure 3 The effect of 4-HIL on cell cycle progression of mouse breast cancer 4T1 cell line.

Figure 4 4-HIL promotes apoptosis of 4-T1 tumor cells.

Figure 5 Inhibitory effect of 4-HIL on CT26 tumor in wild-type mice at doses of 50 mg/kg and 150 mg/kg, 5A is the graph of the change in tumor volume with drug concentration and time, 5B is the graph of the body weight of mice during administration, 5C is the graph of tumor weight summary graph.

Figure 6 Photographs of tumor growth changes at 50 mg/kg and 150 mg/kg doses of 4-HIL.

Figure 7 The growth inhibitory effect of 4-HIL at a dose of 150 mg/kg on CT26 tumors established in nude mice: tumor volume in nude mice.

Figure 8. The growth inhibitory effect of 200 mg/kg 4-HIL on the established wild-type mouse melanoma B16OVA tumor. Figure 8A is a graph of the change in tumor volume with administration concentration and time, and Figure 8B is a graph of the change in mouse body weight during administration.

Figure 9. The growth inhibitory effect of 200 mg/kg 4-HIL on the tumor of breast cancer MCF-7 cell line established in the fourth breast pad of nude mice. Graph of changes in mouse body weight during the period.

Figure 10 shows the tumor volume growth curve under radiotherapy.

Detailed description of the invention

In a first aspect, the present invention provides a pharmaceutical composition comprising a medicament and a pharmaceutically acceptable carrier thereof, the medicament comprising 4-HIL or a pharmaceutically acceptable alternative form thereof, the pharmaceutically acceptable alternative form comprising A pharmaceutically acceptable salt, active analog, lactone form, prodrug or metabolite. The carrier may include diluents, binders, disintegrants, lubricants, solubilizers.

Further, the medicament further comprises a second active ingredient, and the second active ingredient is a chemotherapeutic agent. The chemotherapeutic agent may be selected from gefitinib, erlotinib, afatinib, doxorubicin, pemetrexed, cisplatin, paclitaxel, gemcitabine, vinorelbine, irinotecan, bevacizumab , 5-fluorouracil, methotrexate, oxaliplatin group. Preferably, the chemotherapeutic agent is doxorubicin. The active ingredients may be present in physical isolation or in admixture.

In the second aspect, the present invention correspondingly provides a method for preventing and/or treating a tumor, comprising administering the pharmaceutical composition described in the first aspect to a subject, and the tumor can be a solid tumor, preferably breast cancer, colorectal cancer, melanoma, esophageal cancer, lung cancer, head and neck squamous cell carcinoma, pancreatic cancer, thyroid cancer, cervical cancer, ovarian cancer, endometrial cancer, kidney cancer, bile duct cancer, and gastric cancer.

4-HIL or its pharmaceutically acceptable alternative form (eg, its pharmaceutically acceptable salt), when used in combination with one or more second active ingredients, the active ingredients are administered synchronously or asynchronously, and the administration of each active ingredient The route and dosage form of the drug may also be different. Such routes of administration include oral, parenteral or topical administration. The pharmaceutical dosage forms include solid or liquid forms, and the solid forms include tablets, granules, capsules, and pills, and the liquid forms include oral liquids and injections, and the injections include injection powders or injections.

Further, the method also includes combined use with radiotherapy.

detailed description

Unless otherwise specified, the experimental methods and English abbreviations used in this embodiment follow conventional practices, such as:

Statistical methods: t-test was used for comparison between groups; experimental results were expressed as mean ± standard deviation (means ± SD). The meaning of the significant mark * involved in each chart of the present invention: *p<0.05, **p<0.01, ***P<0.001. Mouse tumor volume calculation formula: V=1/2*a(length)*b(width)*c(height).

 

Example 1

Experimental methods: MTT assay was used to detect the inhibitory effect of 4-HIL on the growth of tumor cell lines 4T1, B16OVA, CT26, MCF-7, KYSE450, RKO, HT29 and HepG2.

The tumor cell lines in the logarithmic growth phase were inoculated into 96-well plates at 200uL/well, of which CT26, B16OVA, 4T1, RKO, KYSE450, HepG2 were 2000 cells per well, MCF-7 and HT29 were 5000 cells per well, and cultured for 24h Then, the old medium was removed and fresh serum-free medium was added for starvation treatment for six hours. After the starvation treatment, different concentrations of 4-HIL were added, and finally placed in the incubator for 24h, 48h, and 72h respectively; For the first 4 hours, add MTT 20uL/well in the dark, incubate for 4 hours in the incubator, discard the culture medium, add 150uL DMSO to each well in the dark, shake to fully dissolve the crystals; at 490nm wavelength, measure the absorbance value, and count the cells survival rate, etc.

Cell viability (%) = sample well OD value * 100/control well OD value;

Cell proliferation inhibition rate (%)=(1-OD of experimental group/OD of control group)*100%.

Experimental results:

As shown in Figure 1, 4-HIL has obvious inhibitory effect on the proliferation of various tumor cell lines, and with the increase of 4-HIL concentration and the prolongation of action time, the inhibitory effect of 4-HIL on the proliferation of tumor cell lines is gradually enhanced. Some cell proliferation inhibition rates are shown in Table 1.

Table 1 Proliferation inhibition rate (%) of tumor cell lines by some concentrations of 4-HIL

cell line/concentration	0.5mM	1mM	2mM	4mM
4T1	7.75±2.89	31.43±4.43	33.33±5.36	63.91±2.74
CT26	17.91±5.73	15.20±5.14	19.10±6.04	52.90±1.03
B16-OVA	25.36±10.58	38.04±4.10	46.05±6.61	55.90±4.23
MCF-7	35.63±3.43	41.68±3.88	32.31±1.72	53.84±2.78
HT29	23.29±15.01	28.54±13.87	35.19±11.54	50.32±9.58
KYSE450	4.71±7.77	6.28±4.45	37.14±13.45	57.08±3.57
RKO	19.93±2.43	20.04±12.65	25.40±2.22	47.09±5.54
HepG2	ND *	ND	ND	ND

*ND indicates that this concentration is not described here, and the IC ₅₀ value of 4-HIL for HepG2 is above 20 mM.

Example 2

Inhibitory effect of 4-HIL combined with doxorubicin on the proliferation of 4T1 cell line

The MTT method was used to measure the inhibitory effect of the combination of 0.5mM, 1mM, 2mM, 4mM 4-HIL and 2.5μM DOX on the proliferation of 4T1 cells, and a single-drug control group was set up according to the practice of control experiments.

The experimental method and the formula of cell proliferation inhibition rate are the same as those in Example 1.

Experimental results: The combination of 4-HIL and DOX significantly improved the growth inhibitory effect of 4T1, and with the increase of 4-HIL concentration and prolonged action time, the proliferation inhibitory effect was also enhanced. synergistic inhibition. The results of cell proliferation inhibition are shown in Fig. 2 and Table 2.

Table 2 The effect of 2.5μM DOX combined with different concentrations of 4-HIL

4-HIL concentration	Proliferation inhibition rate of combined use of DOX (%)
0	53.81±3.20
0.5mM	71.33±1.98 *
1mM	79.59±4.56 *
2mM	84.13±5.93 **
4mM	99.68±7.37 ***

Example 3: 4-HIL arrests cell cycle progression

The breast cancer cell line 4T1 in the logarithmic growth phase was placed in a 6-well plate at 2×10 ⁴ cells/well, and 3 duplicate wells were set up; 0mM and 5mM 4T1 diluted in 1640 medium were added to each well. -HIL, the total volume of each well was 2 mL, and cultured for 8 hours; the cells were collected, processed with a cell cycle kit, and detected by flow cytometry.

Experimental results: 4-HIL acted on 4T1 cells for 48 hours, the cells in G1 phase were significantly increased, and the cells in G2 and S phases were significantly decreased. 4-HIL blocked 4T1 cells in G1 phase. The cell cycle distribution is shown in Figure 3.

Example 4: 4-HIL induces apoptosis

The breast cancer cell line 4T1 in the logarithmic growth phase was placed in a 6-well plate at 2×10 ⁴ cells/well, and 3 duplicate wells were set up; 0mM, 4mM and 6mM diluted in 1640 medium were added to each well respectively. , 8 mM 4-HIL, the total volume of each well was 2 mL, and cultured for 48 hours; cells were collected, treated with apoptosis kit, and detected by flow cytometry.

The experimental results are shown in Figure 4.

Example 5: In vivo antitumor activity of 4-HIL

Experimental animals:

6-week-old BALB/c, C57BL/6 female mice, SPF grade.

Wild-type CT26 tumor model:

Tumor-bearing: CT26 colorectal cancer cells, 200 μL (2×10 ⁵ cells) were subcutaneously injected into the right back of each BABL/c mouse;

From the 8th day of tumor bearing, the mice were grouped into S-shaped groups and administered when the tumor volume reached about 40-80 mm ³ , the weight of the mice was weighed every other day, and the tumor size of the mice was measured, and the tumor volume of the mice was calculated and recorded according to the aforementioned formula. Variety. The experiment was divided into Control group, 4-HIL 50 mg/kg/d group and 4-HIL 150 mg/kg/d group. After continuous administration for 14 days, the mice were sacrificed. The experimental results are shown in Figure 5 and Figure 6.

 

Nude mouse CT26 tumor model:

Tumor bearing: CT26 colorectal cancer cells, 200 μL (2×10 ⁵ cells) were subcutaneously injected into the right back of each BABL/c nude mouse;

From the 6th day of tumor bearing, when the tumor volume of mice grows to about ^40-80mm , the mice were grouped into S-shaped groups and administered, and the weight of the mice was weighed and the tumor size of the mice was measured every other day, and the tumors of the mice were calculated and recorded according to the aforementioned formula. Volume change. The experiment was divided into two groups, the Control group and the 4-HIL 150 mg/kg/d group. After continuous administration for 14 days, the mice were sacrificed.

The experimental results are shown in Figure 7.

 

B16OVA tumor model:

Tumor bearing: B16-OVA melanoma cells, 200 μL (5×10 ⁵ cells) were subcutaneously injected into the right back of each BABL/c mouse;

From the 9th day of tumor bearing, when the tumor volume of mice grows to about ^30-70mm , the mice are divided into S-shaped groups and administered. The weight of mice is weighed and the tumor size of mice is measured every other day. The tumor of mice is calculated and recorded according to the aforementioned formula. Volume change. The experiment was divided into two groups, the Control group and the 4-HIL 200 mg/kg/d group. After continuous administration for 14 days, the mice were sacrificed.

The experimental results are shown in Figure 8.

 

MCF-7 tumor model:

Tumor-bearing: MCF-7 breast cancer cells, 200 μL (2×10 ⁶ cells) were injected into the fourth breast pad of each BALB/c nude mouse;

From the 9th day of tumor bearing, when the tumor volume of mice grows to about ^30-70mm , the mice are divided into S-shaped groups and administered. The weight of mice is weighed and the tumor size of mice is measured every other day. The tumor of mice is calculated and recorded according to the aforementioned formula. Volume change; the experiment was divided into two groups: Control group and 4-HIL 200 mg/kg/d group. After continuous administration for 14 days, the mice were sacrificed.

The experimental results are shown in Figure 9.

 

Combination radiation therapy for colon cancer:  

Tumor-bearing: CT26 colon cancer cells, 200 μL (2×10 ⁵ cells) were injected into the fourth mammary fat pad of each BABL/c mouse;

From the 6th day of tumor bearing, the weight of the mice was weighed, and the tumor size of the mice was measured. According to the formula V=1/2 × a (length) × b (width) × c (height), the changes in the tumor volume of the mice were calculated and recorded; On the 10th day (the tumor volume of mice grows to about 40-60mm ³ ), the mice were randomly divided into 4 groups: Control group-saline group, 4-HIL group (administered dose 50mg/kg/d), X-GY Group (10Gy), "4HIL+X-GY" group (50mg/kg/day+10Gy irradiation), the animals in each group were irradiated with a dose of 10Gy, and then administered by intraperitoneal injection every day for 10 consecutive days ; 24 hours after the end of administration, the mice were sacrificed, and various indicators were detected.

The experimental results are shown in Figure 10.

Claims (9)

1. Use of 4-hydroxyisoleucine, its lactone form or its pharmaceutically acceptable salt in the preparation of a medicament for preventing and/or treating tumors, the structure of said 4-hydroxyisoleucine is

   

   .
2. The use of claim 1, wherein the use comprises arresting tumor cell cycle progression, inducing tumor cell apoptosis, inhibiting tumor growth and/or reducing tumor volume.
3. The application according to claim 1, wherein the three-dimensional structure of the 4-hydroxyisoleucine is

   

   .
4. The application according to claim 1, wherein the tumor is a solid tumor, preferably selected from breast cancer, colorectal cancer, melanoma, esophageal cancer, lung cancer, head and neck squamous cell carcinoma, pancreatic cancer, thyroid cancer, The group consisting of cervical, ovarian, endometrial, renal, bile duct, and gastric cancers.
5. The use according to any one of claims 1-4, wherein the 4-hydroxyisoleucine is used in combination with a second active ingredient or radiotherapy, and the second active ingredient is a chemotherapeutic agent.
6. The use according to claim 5, wherein the chemotherapeutic agent is selected from the group consisting of gefitinib, erlotinib, afatinib, doxorubicin, pemetrexed, cisplatin, paclitaxel, gemcitabine, vinorelbine The group consisting of pyridoxine, irinotecan, bevacizumab, 5-fluorouracil, methotrexate and oxaliplatin, preferably doxorubicin.
7. The use according to any one of claims 1-6, wherein the use comprises administration by oral, parenteral and/or topical administration.
8. A pharmaceutical composition comprising the 4-hydroxyisoleucine in admixture or physical separation and a second active ingredient, the second active ingredient being a chemotherapeutic agent.
9. The pharmaceutical composition according to claim 8, wherein the chemotherapeutic agent is selected from the group consisting of gefitinib, erlotinib, afatinib, doxorubicin, pemetrexed, cisplatin, paclitaxel, gemcitabine, The group consisting of vinorelbine, irinotecan, Avastin, 5-fluorouracil, methotrexate, and oxaliplatin.