CN112089710B

CN112089710B - Application of 4-hydroxyisoleucine in preparation of antitumor drugs

Info

Publication number: CN112089710B
Application number: CN202010789738.6A
Authority: CN
Inventors: 李国栋; 袁保梅; 祁园明; 杨永会; 杨佳丽; 吴亚红
Original assignee: Zhengzhou University
Current assignee: Zhengzhou University
Priority date: 2020-08-07
Filing date: 2020-08-07
Publication date: 2022-04-01
Anticipated expiration: 2040-08-07
Also published as: WO2022028615A1; CN112089710A

Abstract

The invention relates to application of 4-hydroxyisoleucine (4-HIL) in preparing a medicament for preventing and/or treating tumors. The 4-HIL can block the periodic process of tumor cells, induce the apoptosis of the tumor cells, inhibit the growth of tumors and reduce the volume of the tumors, and has obvious antitumor activity.

Description

Application of 4-hydroxyisoleucine in preparation of antitumor drugs

Technical Field

The application relates to the technical field of medicines, in particular to application of 4-hydroxyisoleucine (4-HIL) in preventing and/or treating tumors and application of a combination of the 4-hydroxyisoleucine (4-HIL) and chemotherapeutic drugs in preventing and/or treating tumors.

Background

Cancer is currently the leading cause of harm to human health. Based on the data available and the aging characteristics of the population, experts predict that 2025 years will have a global cancer with up to about 58572 new cases per year. Traditional treatment methods for tumors include surgical treatment, chemotherapy, and radiation therapy. However, the operation treatment is only suitable for the early tumor treatment, and radiotherapy and chemotherapy both have considerable toxic and side effects, which bring great pain to patients. The existing chemotherapy drugs and the like have the problems of easy drug resistance, easy relapse, great toxic and side effects and the like in the later treatment period. Therefore, the development of new drugs for specifically treating malignant tumors with low side effects has been a desire of anti-tumor researchers.

The drug treatment plays an important role in three major therapies of malignant tumors, and the search of high-efficiency and low-toxicity anti-tumor active ingredients from plants becomes a hot spot of common attention of domestic and foreign scholars. According to incomplete statistics, the antitumor preparation derived from the botanical drugs accounts for 32.25 percent of the total antitumor drugs. The traditional Chinese medicine in China has a long history, which can be traced back to thousands of years ago. The traditional Chinese medicine has the advantages of unique efficacy, relatively high side effect and the like, and more scholars at home and abroad begin to pay attention to the research on the effect and the action mechanism of the traditional Chinese medicine and chemical components thereof on treating the tumor in recent years. At present, the anti-tumor activity screening of more than 3000 Chinese herbal medicines in 28 families is carried out in China, and the effective rate is about more than 200. A large number of research works prove that the hit rate of screening the anti-tumor drugs from natural animal and plant drugs is much higher than that of the synthetic drugs, so that the screening of the natural anti-tumor drugs is a significant work and is valued by people. Paclitaxel, camptothecin, vincristine and the like are widely accepted as the first choice drugs for resisting tumors. Therefore, the research and development of new antitumor drugs from natural drugs has wide clinical application prospect.

4-hydroxyisoleucine is a non-protein amino acid present in Trigonella species, and is predominantly present in the seed of Trigonella foenum graecum, accounting for approximately 80% of the total free amino acid content of the seed. Fenugreek (Trigonella foenum-graecum) is an annual herb plant of the genus Trigonella of the family Leguminosae, widely distributed in Asia, Europe, and Africa. Fenugreek seeds are traditional Chinese medicinal materials, and the main two pharmacological actions are diabetes and cholesterol reduction. In recent years, (2S, 3R, 4S) -4-hydroxyisoleucine isolated from fenugreek seeds has been reported in the literature to promote insulin secretion, and this promotion also explains the anti-diabetic activity of fenugreek seeds. At present, no report on the antitumor activity of 4-hydroxyisoleucine exists in the prior art.

Disclosure of Invention

The invention discovers the activity of the 4-HIL in the aspect of anti-tumor in a breakthrough way, and provides the application of the active ingredients of the traditional Chinese medicine in the aspect of preparing anti-tumor medicines.

Specifically, the invention provides an application of 1, 4-hydroxyisoleucine (4-HIL) or pharmaceutically acceptable salts thereof, analogues thereof and metabolites thereof in preparing a medicament for preventing and/or treating tumors.

Further wherein the tumor is a solid tumor, preferably a 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, renal cancer, cholangiocarcinoma, gastric cancer, liver cancer, more preferably a breast cancer, colorectal cancer, melanoma, and head and neck squamous cell carcinoma.

Further, the 4-HIL blocks tumor cell cycle progression, induces tumor cell apoptosis, inhibits tumor growth, and reduces tumor volume.

Further, the half maximal Inhibitory Concentration (IC) of said 4-HIL on tumor cells₅₀) Less than 5 mM.

Further, the medicament also comprises a second active ingredient, the second active ingredient being a chemotherapeutic agent.

Preferably, the chemotherapeutic is selected from the group consisting of gefitinib, erlotinib, afatinib, doxorubicin, pemetrexed, cisplatin, paclitaxel, gemcitabine, vinorelbine, irinotecan, avastin, 5-fluorouracil, methotrexate, oxaliplatin.

More preferably, the chemotherapeutic agent is doxorubicin.

Further, the combination of 4-HIL and chemotherapeutic agent exhibits a synergistic effect in inhibiting tumor cell growth.

Further, the concentration of 4-HIL in the drug is 0.5-10mM, preferably 4mM, and the concentration of the chemotherapeutic agent is 1-5. mu.M, preferably 2.5. mu.M.

In one embodiment, the medicament of the invention is administered by oral, parenteral, or topical administration.

Specifically, the dosage forms of the medicine are tablets, granules, capsules, powder, pills, oral liquid, powder injection for injection, transdermal patches, gels and ointments.

Drawings

FIG. 14-HIL shows in vitro proliferation inhibitory effects on tumor cell lines 4T1, B16OVA, CT26, MCF-7, KYSE450, RKO, HT29 and HepG 2.

FIG. 24 mM 4-HIL in combination with 2.5. mu.M of the chemotherapeutic drug doxorubicin showed proliferation inhibitory effect on the mouse breast cancer 4T1 cell line.

FIG. 30 mM and 5mM of 4-HIL are shown to have an effect on the cell cycle progression of the mouse breast cancer 4T1 cell line, as well as cell cycle distribution.

FIG. 44-Effect of HIL on apoptosis of mouse breast cancer 4T1 cell line, 4-HIL promotes apoptosis of 4-T1 tumor cells in a dose-dependent manner, and the effect of different concentrations of 4-HIL on the apoptosis rate of 4T1 cells.

FIG. 550 mg/kg and 150mg/kg doses of 4-HIL are shown to inhibit the growth of subcutaneous CT26 tumor, 5A tumor volume as a function of drug concentration and time, 5B tumor weight during administration, and 5C tumor weight statistics.

FIG. is a graph comparing the growth inhibition of 4-HIL at 650 mg/kg and 150mg/kg doses on established subcutaneous colon carcinoma CT26 tumors in wild type mice.

FIG. 7150 mg/kg dose of 4-HIL shows the growth inhibitory effect on the colon cancer CT26 tumor established subcutaneously in nude mice, and the tumor volume in nude mice.

FIG. 8200 mg/kg of 4-HIL shows the growth inhibitory effect on tumors of subcutaneous melanoma B16OVA established in wild type mice, FIG. 8A is a graph showing the change in tumor volume with the administration concentration and time, and FIG. 8B is a graph showing the change in mouse body weight during the administration.

FIG. 9200 mg/kg of 4-HIL shows growth inhibition effect on tumor of breast cancer MCF-7 cell line established in the fourth breast pad of nude mice, FIG. 9A is a graph showing tumor volume according to administration concentration and time and FIG. 9B is a graph showing weight change of mice during administration.

Detailed Description

In a first aspect of the present invention, the present invention provides an application of 1, 4-hydroxyisoleucine (4-HIL) or a pharmaceutically acceptable salt thereof, an analog thereof, a metabolite thereof in the preparation of a medicament for preventing and/or treating tumors.

Wherein the tumor is 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, gastric cancer, liver cancer, more preferably breast cancer, colorectal cancer and melanoma.

Further, the 4-HIL blocks cell cycle progression, induces apoptosis, inhibits tumor growth, and reduces tumor volume.

Further, the half Inhibitory Concentration (IC) of said 4-HIL on cancer cells₅₀) Less than 5 mM.

The chemotherapeutic is selected from the group consisting of gefitinib, erlotinib, afatinib, doxorubicin, pemetrexed, cisplatin, paclitaxel, gemcitabine, vinorelbine, irinotecan, avastin, 5-fluorouracil, methotrexate, oxaliplatin.

Preferably, the chemotherapeutic agent is doxorubicin.

Further, the combination of 4-HIL and chemotherapeutic agent exhibits a synergistic effect in inhibiting cancer cell growth.

In a second aspect of the invention, the invention provides the use of a combination of 4-HIL or a pharmaceutically acceptable salt thereof, an analogue thereof, a metabolite thereof and a chemotherapeutic agent in the manufacture of a medicament for the prevention and/or treatment of tumours.

Wherein the tumor is a solid tumor, preferably a 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, gastric cancer and liver cancer, more preferably breast cancer, colorectal cancer, melanoma, head and neck squamous cell carcinoma.

Preferably, the chemotherapeutic agent is doxorubicin.

A third aspect of the invention provides a pharmaceutical composition comprising 4-HIL or a pharmaceutically acceptable salt thereof, an analogue, a metabolite thereof and a pharmaceutically acceptable carrier.

In a fourth aspect, the invention provides a pharmaceutical composition comprising 4-HIL or a pharmaceutically acceptable salt thereof, an analogue, metabolite or chemotherapeutic agent thereof, and a pharmaceutically acceptable carrier.

Preferably, the chemotherapeutic agent is doxorubicin.

The pharmaceutical compositions of the present invention may be administered orally, parenterally, or topically.

In the present invention, 4-HIL or a pharmaceutically acceptable salt thereof, or an analog or metabolite thereof ("active compound") can be formulated into a pharmaceutical composition or formulation, which can be administered by any suitable route, including, but not limited to, oral or non-oral administration. In one example of the invention, a composition or formulation containing 4-HIL or a pharmaceutically acceptable salt thereof, or an analog or metabolite thereof, is administered via the oral route, which may be in solid or liquid form. Solid compositions or formulations include tablets, pills, capsules, dispersible powders, granules, and the like. Oral compositions also include mouth washes, which are sticky to the mouth, and sublingual tablets. The capsule includes hard capsule and soft capsule. In such solid compositions or preparations for oral use, one or more of the active compounds may be mixed with or accompanied by diluents, binders, disintegrants, lubricants, stabilizers, solubilizers and subsequently formulated into preparations in a conventional manner. When desired, such preparations may be coated with a coating agent, or it may be coated in two or more coats. In another aspect, liquid compositions for oral administration include pharmaceutically acceptable aqueous solutions, suspensions, emulsions, syrups, elixirs, and the like. In such compositions, one or more of the active compounds may be dissolved, suspended, or emulsified in a conventional diluent (e.g., purified water, ethanol, or mixtures thereof, etc.). In addition to such diluents, the compositions can also contain wetting agents, suspending agents, emulsifying agents, sweetening agents, flavoring agents, perfuming agents, preservatives, buffering agents, and the like.

Pharmaceutical compositions for parenteral administration include solutions, suspensions, emulsions, and solid injectable compositions, which are dissolved or suspended in a solvent immediately before use. Injections may be prepared by dissolving, suspending, or emulsifying one or more active ingredients in a diluent. Examples of the diluent are distilled water for injection, physiological saline, vegetable oil, alcohol, and a combination thereof. In addition, the injection may contain stabilizers, solubilizers, suspending agents, emulsifiers, buffers, preservatives and the like. The final formulation step of the injection is sterilized or prepared by aseptic procedures. The pharmaceutical compositions of the present invention may also be formulated as sterile solid preparations, for example, by lyophilization, and may be used after sterilization, or dissolved in sterile water for injection or other sterile diluents immediately prior to use.

Advantageous effects

Experiments show that the 4-HIL can inhibit the proliferation of various murine and human tumor cell lines such as colon cancer, colorectal cancer, breast cancer, melanoma, esophageal squamous cell carcinoma, liver cancer and the like, influence the cell cycle process and induce cell apoptosis. The combination of 4-HIL and the chemotherapeutic drug doxorubicin has a significant synergistic inhibitory effect on the proliferation of 4T1 cells. In an in vivo animal experiment, the 4-HIL can obviously inhibit the growth rate of the xenografted colon cancer cell line CT26 in the skin of wild mice and nude mice. 4-HIL also inhibited the subcutaneous growth rate of the xenografted melanoma cell line B16OVA in wild-type mice and the subcutaneous growth rate of the xenografted breast cancer cell line MCF-7 in wild-type mice. The proliferation inhibition effect of the 4-HIL on various tumor cells is proved through in vitro cell experiments and in vivo animal experiments, and the 4-HIL has no obvious toxic or side effect and has wide antitumor activity.

Detailed Description

Example 1

The experimental method comprises the following steps: MTT method detects the inhibition effect of 4-HIL on the in vitro growth of tumor cell lines 4T1, B16OVA, CT26, MCF-7, KYSE450, RKO, HT29 and HepG 2.

Inoculating 2000 tumor cell strains CT26, B16OVA, 4T1, RKO, KYSE450 and HepG2 in each hole to a 96-hole plate, wherein the volume of each hole is 200 uL/hole, inoculating 5000 tumor cell strains MCF-7 and HT29 in the logarithmic growth phase to the 96-hole plate, wherein the volume of each hole is 200 uL/hole, removing an old culture medium after culturing for 24 hours, adding a fresh serum-free culture medium for starvation treatment for six hours, respectively adding 4-HIL with different concentrations after the starvation treatment is finished, and finally placing in a culture box for culturing for 24 hours, 48 hours and 72 hours respectively; adding MTT 20 uL/hole in dark 4h before detection by a microplate reader, placing in an incubator for incubation for 4h, discarding the culture solution, adding 150uLDMSO in each hole in dark, and shaking by a shaking table for 10min to fully dissolve the crystals; at 490nm, the absorbance was measured and the cell viability was calculated.

The statistical method comprises the following steps: the comparison between groups was performed by t-test. The results are expressed as mean ± standard deviation (means ± SD).

Cell proliferation inhibition (%) (1-experimental OD/control OD) × 100%.

The experimental results are as follows: the results are shown in FIG. 1, 4-HIL has obvious inhibition effect on the proliferation of different tumor cell lines, and the inhibition effect of 4-HIL on the proliferation of tumor cell lines is gradually enhanced along with the increase of the concentration of 4-HIL and the prolonging of the action time.^*p<0.05，^**p<0.01，^***p<0.001。

The cell proliferation inhibition results are shown in table 1.

TABLE 1 inhibitory effect of 4-HIL on tumor cell line proliferation at various concentrations (%)

As is clear from the results in Table 1, 4mM 4-HIL showed an inhibitory effect of 50% or more on most tumor cell lines, and its IC was found to be₅₀About 4 mM. Relative to other tumor cell lines, 4-HIL had no significant inhibitory effect on HepG2, its IC₅₀The value is 20mM or more.

Example 2

Proliferation inhibition of 4T1 cell line by combined application of 4-HIL and chemotherapeutic drug adriamycin

Proliferation inhibition of 4T1 cells by 2.5. mu.M DOX, a combination of 0.5mM, 1mM, 2mM and 4mM 4-HIL and 2.5. mu.M DOX was determined using the MTT method.

The experimental procedure was as in example 1.

The statistical method and the cell proliferation inhibition rate were the same as those in example 1.

The experimental results are as follows: the combination of 4-HIL and DOX has obviously improved growth inhibition effect on 4T1, has very significant difference compared with that of DOX with 2.5 mu M alone, after 72 hours of action, the proliferation inhibition rate of DOX with 2.5 mu M on 4T1 cells is 53.81%, the combination of DOX with 4-HIL with 0.5mM, 1mM, 2mM and 4mM obviously enhances the proliferation inhibition effect of DOX, and the proliferation inhibition effect is enhanced along with the increase of the concentration of 4-HIL and the prolonging of the action time, and the combination of the two has synergistic inhibition effect on 4T1 tumor cell lines. The results of cell growth inhibition are shown in fig. 2 and table 2.

TABLE 2 proliferation inhibitory Effect of different concentrations of 4-HIL in combination with DOX on 4T1 cell line

Compared to 2.5 μ M DOX alone,^*p<0.05，^**p<0.01，^***p<0.001。

example 3: 4-HIL blocks cell cycle progression

The breast cancer cell line 4T1 in logarithmic growth phase was added at 2X 10⁴The cells/well are uniformly placed in a 6-well plate, and 3 cells are arrangedAn aperture; adding 1640 culture medium diluted 4-HIL (0 mM and 5 mM) into each well respectively to make the total volume of each well be 2mL, and continuously culturing for 8 hours; collecting cells, processing by using a cell cycle kit, and detecting by using a flow cytometry detector.

The statistical method comprises the following steps: the comparison between groups was performed by t-test. The results are expressed as mean ± standard deviation (means ± SD),^*p<0.05，^**p<0.01，^***p<0.001。

the results of the experiment are shown in FIG. 3. 4-HIL acts on 4T1 cells for 48 hours, G1 phase cells are remarkably increased, G2 and S phase cells are remarkably reduced, 4-HIL blocks 4T1 cells at G1 phase, the progress of 4T1 cell cycle is influenced, and the proliferation of 4T1 cells is inhibited.

Example 4: 4-HIL-induced apoptosis

The breast cancer cell line 4T1 in logarithmic growth phase was added at 2X 10⁴Uniformly placing each cell/hole in a 6-hole plate, and arranging 3 multiple holes; adding 1640 diluted 4-HIL (0 mM, 4mM, 6mM and 8 mM) culture medium into each well respectively to make the total volume of each well be 2mL, and continuously culturing for 48 hours; and collecting cells, treating by using an apoptosis kit, and detecting by using a flow cytometry detector.

the results of the experiment are shown in FIG. 4. From the results of FIG. 4, it can be seen that the apoptosis rate was significantly increased with the increase in the concentration of 4-HIL, indicating that 4-HIL can promote apoptosis.

Example 5: 4-HIL in vivo antitumor Activity

Laboratory animal

BALB/C, C57BL/6 female mice, 6 weeks old, SPF grade, purchased from Biotech, Inc., of Viton, Beijing.

The experimental animals are fed aseptically in the whole process and are free to eat drinking water. The cage, the feed, the padding and the drinking water are sterilized and disinfected at high pressure, and the feeding environment meets the requirements of environmental facilities of medical experimental animals.

Wild type CT26 tumor model: carrying out tumor loading: collecting good-growth-state CT26 colorectal cancer cells, and adjusting the cell concentration to 1 × 10⁶cells/mL, 200. mu.L (2X 10. mu.L) were inoculated subcutaneously in the right back of each BABL/c mouse⁵cells); from day 8, the volume of the mice grows to about 40-80mm³The mice were weighed using an electronic balance every other day, the mice tumors were measured using a digital vernier caliper, and the change in tumor volume of the mice was calculated and recorded according to the formula V1/2 a (length) b (width) c (height). The experiment was divided into Control group, 4-HIL 50mg/kg/d group and 4-HIL 150mg/kg/d group, and after 14 days of continuous administration, mice were sacrificed for subsequent experiments. The statistical method comprises the following steps: the comparison between groups was performed by t-test. The results are expressed as mean ± standard deviation (means ± SD),^*p<0.05，^**p<0.01，^***p<0.001。

the results of the experiment are shown in fig. 5 and 6. The results of FIGS. 5A, 5C and 6 show that after 14 days of administration, the 4-HIL 50mg/kg/d group and the 150mg/kg/d group were significantly different (p <0.001) compared to the Control group, and that the 4-HIL 50mg/kg/d group and the 150mg/kg/d group were also significantly different (p < 0.05). The results of FIG. 5B show that 50mg/kg/d and 150mg/kg/d of 4-HIL have little effect on the body weight of mice, and have no statistical difference from the Control group, which indicates that 4-HIL is safe and effective, and does not affect the proliferation of normal cells while exerting an anti-tumor effect.

Nude mouse CT26 tumor model: carrying out tumor loading: collecting good-growth-state CT26 colorectal cancer cells, and adjusting the cell concentration to 1 × 10⁶cells/mL, 200. mu.L (2X 10. mu.L) were inoculated subcutaneously in the right back of each BABL/c nude mouse⁵cells); from day 6 of tumor bearing, the volume of the mice grows to about 40-80mm³The mice were weighed using an electronic balance every other day, the mice tumors were measured using a digital vernier caliper, and the change in tumor volume of the mice was calculated and recorded according to the formula V1/2 a (length) b (width) c (height). The experiment was divided into two Control groups and 4-HIL 150mg/kg/d group, and after 14 days of continuous administration, mice were sacrificed for subsequent experiments. The statistical method comprises the following steps: the comparison between groups was performed by t-test. The results are expressed as mean ± standard deviation (means ± SD),^*p<0.05，^**p<0.01，^***p<0.001。

the results of the experiment are shown in FIG. 7. The results in FIG. 7 show that 14 days after administration, 150mg/kg of 4-HIL significantly decreased tumor volume and was significantly different between the two groups (p <0.05) in the BALB/c nude mouse tumor model.

B16OVA tumor model: carrying out tumor loading: collecting B16-OVA melanoma cells with good growth state, and adjusting cell concentration to 2.5 × 10⁶cells/mL, 200. mu.L (5X 10. mu.L) were inoculated subcutaneously in the right back of each BABL/c mouse⁵cells); from the 9 th day of tumor bearing, the volume of the mice grows to about 30-70mm³The sigmoid grouping was started and the drug was administered, and the mouse body weight was measured every other day using an electronic balance, the mouse tumor was measured with a digital vernier caliper, and the change in the mouse tumor volume was calculated and recorded according to the formula V of 1/2 × a (length) × b (width) × c (height). The experiment was divided into two Control groups and 4-HIL200mg/kg/d group, and after 14 days of continuous administration, mice were sacrificed for subsequent experiments. The statistical method comprises the following steps: the comparison between groups was performed by t-test. The results are expressed as mean ± standard deviation (means ± SD),^*p<0.05，^**p<0.01，^***p<0.001。

the results of the experiment are shown in FIG. 8. The results in FIG. 8A show that 14 days after administration of 150mg/kg of 4-HIL in the B16OVA tumor model showed a significant reduction in tumor volume compared to the Control group, with a very significant difference between the two groups (p < 0.01). The results in FIG. 8B show that 200mg/kg of 4-HIL had no significant effect on the body weight of the mice, and there was no statistical difference between the two compared to the Control group.

MCF-7 tumor model: carrying out tumor loading: collecting MCF-7 breast cancer cells with good growth state, and adjusting cell concentration to 2 × 10⁷cells/mL, inoculated at 200. mu.L (2X 106cells) per BALB/c nude mouse by injection at the fourth breast pad; from the 9 th day of tumor bearing, the volume of the mice grows to about 30-70mm³The mice are weighed every other day by using an electronic balance, the tumors of the mice are measured by a digital vernier caliper, and the volume change of the tumors of the mice is calculated and recorded according to the formula V which is 1/2 multiplied by a (length) multiplied by b (width) multiplied by c (height); the experiment was divided into two Control groups and 4-HIL200mg/kg/d group, and after 14 days of continuous administration, mice were sacrificed for subsequent experimentsAnd (6) testing. The statistical method comprises the following steps: the comparison between groups was performed by t-test. The results are expressed as mean ± standard deviation (means ± SD),^*p<0.05，^**p<0.01，^***p<0.001。

the results of the experiment are shown in FIG. 9.

The results in FIG. 9A show that 14 days after administration, 150mg/kg of 4-HIL significantly decreased tumor volume compared to the Control group in the MCF-7 tumor model, with a very significant difference between the two groups (p < 0.01). The results in FIG. 9B show that 200mg/kg of 4-HIL had no significant effect on the body weight of the mice, and there was no statistical difference between the two compared to the Control group.

The foregoing describes preferred embodiments of the present invention, but is not intended to limit the invention thereto. Modifications and variations of the embodiments disclosed herein may be made by those skilled in the art without departing from the scope and spirit of the invention.

Claims

1. Use of a pharmaceutical composition consisting of 4-hydroxyisoleucine (4-HIL) and doxorubicin in the manufacture of a medicament for the treatment of a tumor, wherein said tumor is breast cancer 4T1 cells, the combination of 4-HIL and doxorubicin exhibits a synergistic effect in inhibiting the growth of tumor cells, the concentration of 4-HIL in said medicament is 0.5-4mM and the concentration of doxorubicin is 2.5. mu.M.