WO2020000704A1

WO2020000704A1 - Use of ampk inhibitor, compound c, in drug for treating tumors

Info

Publication number: WO2020000704A1
Application number: PCT/CN2018/106620
Authority: WO
Inventors: 万晓春; 黄诗然; 章桂忠
Original assignee: 中国科学院深圳先进技术研究院
Priority date: 2018-06-29
Filing date: 2018-09-20
Publication date: 2020-01-02
Also published as: CN110652515A

Abstract

Disclosed is the new use of an AMPK inhibitor, compound C. Specifically, disclosed is the use of the compound C inhibitor in the preparation of a drug for treating tumors. Compound C can play the role of fighting against hematologic tumors through the inhibition of the activity of tumor cells and induction of the apoptosis of tumor cells. The new use of the AMPK inhibitor, compound C, of the present invention provides a new drug for treating hematologic tumors in a clinical setting.

Description

Application of AMPK inhibitor Compound C in tumor medicine

Technical field

The invention belongs to the technical field of medicine, and specifically relates to the application of an AMPK inhibitor Compound C in the treatment of tumor drugs.

Background technique

Tumor (neoplasm) is a non-hereditary genetic disease. Genes have been changed in normal cells under the action of tumorigenic factors, losing normal regulation of their growth, resulting in abnormal proliferation. Tumor cells have three distinct basic characteristics: immortality, migration, and loss of contact inhibition. The core idea of tumor treatment is to kill and clear tumor cells.

At present, one of the mechanisms of tumor treatments commonly used in clinical practice, such as chemotherapy, radiotherapy, hormone therapy, and some biological therapies, is to induce tumor cells to undergo apoptosis. For example, apoptosis induced by radiotherapy is mainly mediated by Fas, and some cell cycle related proteins and Bcl-2 and Bax also play a key role in this process. Chemotherapy is to induce tumor cells to undergo apoptosis through some chemical drugs to achieve the purpose of suppressing tumor treatment. Drugs such as doxorubicin and 5-fluorouracil can effectively cause cell DNA damage, resulting in cell G1 phase arrest to complete repair or enter apoptosis. Paclitaxel and colchicine, which act on microtubules, mediate apoptosis through the Bcl-2 family. At present, chemotherapy is still one of the main strategies for cancer treatment at this stage and in the future. Large toxic side effects and the development of drug resistance are the main obstacles facing tumor chemotherapy.

Therefore, no matter whether it is radiotherapy or chemotherapy, its induction of apoptosis of tumor cells is lacking in targeting. While killing tumor cells, it also harvests the life of normal cells at the same time, with great toxic and side effects. In addition, tumor recurrence and drug resistance often occur after radiochemotherapy, which also severely limits the clinical treatment effect. At present, tumor treatment is ineffective, and the five-year survival rate of patients is very low, and new therapies with high efficiency and low toxicity are urgently needed.

Compound C (Dorsomorphin) is a potent and reversible selective AMPK inhibitor, competing for its ATP binding site. Ki was 109 nM in the cell-free assay. Studies have shown that Compound C selectively inhibits AMPK activity, while other structurally-related kinases include ZAPK, SYK (Spleen Tyrosine Kinase), PKCθ (Protein Kinase, C-θ), PKA (Protein Kininase A), and JAK3 (Janus and Kinase 3) No significant inhibitory effect. Therefore, at present in the art, Compound C is often used as a specific AMPK inhibitor, and other new uses of Compound C have not been found, such as its use for treating tumors.

technical problem

The purpose of the present invention is to overcome the above-mentioned shortcomings of the prior art and provide an application method of Compound C, an AMPK inhibitor, to solve the current compound C is only used for specific AMPK inhibitors and the side effects of current methods and drugs for treating cancer Large, prone to technical problems such as drug resistance.

Another object of the present invention is to provide a medicament for treating tumors, so as to solve the technical problems of toxic and side effects of existing medicaments for treating tumours, prone to drug resistance, and susceptible to recurrence of tumours.

Technical solutions

In order to achieve the above-mentioned object of the present invention, in one aspect of the present invention, an application method of the AMPK inhibitor Compound C is provided. Specifically, the application of the AMPK inhibitor Compound C in the preparation of a medicine for treating tumors.

In another aspect of the present invention, a medicament for treating tumors is provided. The medicine for treating a tumor includes an effective dose of an active ingredient for treating a tumor, and the active ingredient includes an AMPK inhibitor Compound C.

Compared with the prior art, the application of the compound AMP, an AMPK inhibitor of the present invention, in the preparation of drugs for treating tumors has the following beneficial effects:

Beneficial effect

It has been experimentally proven that the AMPK inhibitor Compound C has a relatively strong effect of inhibiting tumor cell activity and inducing apoptosis of tumor cells, and exerts an anti-hematological tumor effect. Therefore, after preparing the tumor drug by using the AMPK inhibitor Compound C as an active ingredient, the tumor drug can be endowed with a significant effect on inhibiting tumor cell activity and inducing tumor cell apoptosis.

The medicine for treating tumors of the present invention uses the AMPK inhibitor Compound C as an active ingredient. Therefore, the medicine for treating tumors can effectively inhibit the activity of tumor cells and induce and promote tumor cell apoptosis to achieve relief and reduction of malignant tumors on the human body. Harm, relieve clinical symptoms, improve quality of life, and prolong life.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be further described below with reference to the accompanying drawings and embodiments. In the drawings:

FIG. 1 is a graph showing the effect of Compound C and Rapamycin reagents of different concentrations on the activity of Jurkat lymphoma cells in Example 11 of the present invention; FIG.

2 is a graph showing the effect of Compound C and Rapamycin reagents on K562 lymphoma cell activity in different concentrations in Example 11 of the present invention;

FIG. 3 is a graph showing the effect of Compound C and control reagent (DMSO) on the apoptosis of Jurkat lymphoma cells in Example 12 of the present invention; FIG.

FIG. 4 is a graph showing the effect of Compound C and control reagent (DMSO) on K562 lymphoma cell apoptosis in Example 12 of the present invention.

Embodiments of the invention

In order to make the technical problems, technical solutions, and beneficial effects of the present invention clearer and clearer, the following further describes the present invention in detail with reference to the embodiments and the attached tables. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

Explanation of proper names:

Tumor (tumour, neoplasm): refers to a new organism (neogrowth) formed by the body's local tissue proliferation under the action of various tumorigenic factors, because this new organism is mostly a mass-shaped protrusion, also called neoplasm (neoplasm).

Compound C (Dorsomorphin): A potent, reversible, and selective AMPK inhibitor with Ki of 109 nM in cell-free assays. Its molecular structure is as follows:

AMPK (Adenosine Monophosphate-activated Protein Kinase): It is a key sensor of cell energy. The protein kinase can maintain the balance of ATP generation and consumption of eukaryotic cells by sensing the energy state of the cell, that is, energy homeostasis. Its activation helps to correct metabolic disorders and bring the cell metabolism to physiological balance, thus becoming a treatment of metabolic disorders. And possible targets for cancer. Many studies have shown that the activation of AMPK can stimulate the body to produce ATP and provide energy for physiological activities; at the same time, the activation of AMPK can strongly inhibit the synthesis of proteins and fats to reduce the consumption of ATP. Among them, the inhibition of AMPK activity by Dorsomorphin almost completely inhibited the proteolytic effect of autophagy in HT-29 cells.

In one aspect, the embodiment of the present invention provides an application method of the AMPK inhibitor Compound C. Specifically, the application of the AMPK inhibitor Compound C in the preparation of a medicine for treating tumors. According to relevant experiments, it is known that the AMPK inhibitor Compound C is an active ingredient, which can effectively inhibit tumor cell activity and induce and promote tumor cell apoptosis. Specifically, it is as follows in Example 1 and the experimental results of the effects of Compound C at different concentrations on the activity of blood tumor cells. Therefore, the compound AMPK inhibitor Compound C can be used as an effective anti-tumor component to inhibit tumor cell activity and induce and promote tumor cell apoptosis, to alleviate and reduce the harm of malignant tumors to the human body, and to relieve clinical symptoms. , Improve the quality of life and extend the clinical effect of life.

In one embodiment, the AMPK inhibitor Compound C can inhibit tumor cell activity and promote tumor cell apoptosis. The tumor can be a blood tumor, that is, the AMPK inhibitor Compound C can be used in the preparation of a drug for treating blood tumors. . In a specific embodiment, the hematological tumor includes at least one of lymphoma, myeloid leukemia, and myeloma. Wherein, the lymphoma includes T / B lymphoma.

Of course, in addition to the AMPK inhibitor Compound C, which can inhibit the activity of blood tumor cells and promote the apoptosis of blood tumor cells, it can also be other types of tumors.

In addition, when the AMPK inhibitor Compound C is used to prepare a tumor therapy drug, a corresponding dosage form can be prepared according to different modes of administration. Then, the compound AMPK inhibitor Compound C can be compounded with components acceptable to the drug in the corresponding dosage form, such as excipients, etc., and can also be compounded with other anti-tumor active ingredients to form an anti-tumor composite active ingredient.

In addition, according to the molecular structural formula of Compound C, appropriate modification of its molecular body to introduce other groups, such as substitution and modification of carbon atom hydrogen, is also within the scope of the present disclosure.

On the other hand, based on the application of the AMPK inhibitor Compound C described above, the embodiment of the present invention also provides a medicine for treating tumors. The medicine for treating tumors includes an effective dose of active ingredients for treating tumors, and of course, further pharmaceutically acceptable auxiliary ingredients may be further used. The effective dose refers to an effective amount for treating a tumor, and refers to an amount of a compound of the present invention sufficient to show benefit or clinical significance to an individual. Those skilled in the art will understand that the actual amount or dose administered and the duration of administration will depend on the nature and severity of the disease being prevented or treated, the age and general condition of the subject being prevented or treated, and the Medicine way and so on.

Wherein, the active ingredient includes Compound C, an AMPK inhibitor; of course, as mentioned above, the active ingredient may also include other active ingredients that are effective against tumors. The effect component may be a component that can improve the antitumor effect of the AMPK inhibitor Compound C after being compounded with the AMPK inhibitor Compound C. In an embodiment, in an embodiment, the content of the AMPK inhibitor Compound C in the medicine for treating tumors is 5-20 μM.

The auxiliary component may be a related auxiliary material of a corresponding dosage form prepared according to the drug administration method of treating a tumor. In one embodiment, the excipient is a pharmaceutically acceptable carrier of the AMPK inhibitor Compound C. In a specific embodiment, the carrier includes at least one of an antibody capable of coupling to an AMPK inhibitor Compound C, a targeted recombinant protein, a nanoparticle, and an exosome. By using the carrier, the stability of the AMPK inhibitor Compound C can be improved, and the effect of targeted drug delivery in vivo can be improved, and the purpose of targeted killing tumor cells can be achieved.

Therefore, the medicine for treating tumors uses the compound AMPK inhibitor Compound C described above as an active ingredient. Therefore, the medicine for treating tumors can effectively inhibit tumor cell activity and induce and promote tumor cell apoptosis to achieve remission and reduction of malignancy. The harm of tumors to the human body, alleviates clinical symptoms, improves quality of life, and prolongs life.

In addition, the medicine for treating tumors can be prepared according to a conventional medicine preparation method. For example, when the medicine for treating tumors contains an antibody capable of coupling to an AMPK inhibitor Compound C, the antibody can be coupled to AMPK to inhibit the conventional method. Agent Compound C.

In combination with specific examples, the application of Compound C in the preparation of tumor drugs is further described in detail.

1. Examples of effects of Compound C on tumor cell activity and apoptosis

Example 11

MTS detection of different concentrations of Compound C on blood tumor cell viability

S11: Cell pretreatment

1) Resuspend the two lymphoma cells (Jurkat, K562) to 5 × 10 ⁴ cells / mL, respectively, and inoculate them into 96-well plates;

2) Add Compound C to a 96-well plate and set concentration gradients of 0 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, and 20 μM. After 48 h of culture, check the cell survival status;

S12: MTS detects cell viability

1) Mix 2mL MTS and 100μL PMS gently;

2) Add 20 μLMTS mixed solution to each well, mix thoroughly, put it in a carbon dioxide cell incubator, and incubate for 3 hours in the dark;

3) Detect the wavelengths at OD490nm and OD630nm with a microplate reader;

4) Using the OD value at Compound C concentration of 0 μM as a control, analyze the cell survival rate under each concentration gradient.

Using Rapamycin reagent as a control, the same comparative example was performed according to the method of Example 11. The results of experiments showed that the effects of Compound C and Rapamycin reagent on the activity of Jurkat lymphoma cells are shown in Figure 1, and the effects of Compound C and Rapamycin reagent on the activity of K562 lymphoma cells are shown in Figure 2. It can be seen from Figures 1 and 2 that Compound C has a significant effect on inhibiting the activity of blood tumor cells, and that Compound C has a stronger ability to inhibit the activity of tumor cells than Rapamycin at the same dose.

Example 12

Effect of Compound C on Apoptosis of Tumor Cells by Flow Cytometry

S11: Cell pretreatment

1) Lymphoma cells (Jurkat, K562) were resuspended to 3 × 10 ⁵ cells / mL and seeded in 12-well plates; 2) Apoptosis was detected after 48 h incubation with Compound C;

S12: Analysis of apoptosis by flow cytometry

1) Collect the cell suspension;

2) Centrifuge the cell suspension at 800g for 5min at 4 ° C, discard the supernatant, and collect the cells in the pellet;

3) Wash the cells twice with pre-chilled PBS, and resuspend the cells with 100 μL 1 × AnnexinV Binding Buffer;

4) Add 5 μL of Annexin V-FITC and 5 μL of PI staining solution to each sample, mix gently, and react at room temperature for 15 minutes in the dark, and pay attention to setting single staining and blank control. Follow-up flow compensation needs to be adjusted;

5) After incubation, add 400 μL of pre-chilled 1 × Annexin VBinding Buffer to each sample and mix gently; test within 1h;

6) Flow cytometry detection of apoptosis: Annexin V-FITC single positive cells are early apoptotic cells, Annexin V-FITC / PI double positive cells are late apoptotic cells.

Using the DMSO reagent as a control group (Ctrl), the same comparative example was performed according to the method of Example 12. After experimental inspection, it was found that the effect of Compound C and the control group (Ctrl) on the apoptosis of Jurkat lymphoma cells is shown in Figure 3, and the effect of Compound C and the control group (Ctrl) on the apoptosis of K562 lymphoma cells is shown in Figure 4. It can be seen from Figures 1 and 2 that Compound C has a significant apoptotic effect on Jurkat and K562.

The embodiments described above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention shall be included in the present invention. Within the scope of the invention.

Claims

Application of AMPK inhibitor Compound C in the preparation of drugs for treating tumors.
The application according to claim 1, wherein the medicine for treating tumors is a medicine for treating blood tumors.
The application according to claim 2, wherein the blood tumor comprises at least one of T / B lymphoma, myeloid leukemia, and myeloma.
A medicine for treating tumors, comprising an effective dose of an active ingredient for treating tumors, characterized in that the active ingredient comprises the AMPK inhibitor Compound C according to any one of claims 1-3.
The medicament according to claim 4, wherein the content of the AMPK inhibitor Compound C is 5-20 μM.
The medicament according to claim 4 or 5, further comprising a pharmaceutically acceptable carrier of the compound AMPK inhibitor Compound C.
The medicament according to claim 6, further comprising a pharmaceutically acceptable carrier of Compound C, an AMPK inhibitor. The carrier includes at least one of an antibody coupled to an AMPK inhibitor Compound C, a targeted recombinant protein, a nanoparticle, and an exosome.