Mitochondrial targeting copper ion carrier anti-cancer drug as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological pharmacy, and particularly relates to construction and application of a mitochondrial targeting copper ion carrier anti-cancer drug. The anti-cancer drug is prepared from copper salt and pyruvate, and the preparation method comprises the following steps: dissolving the copper salt in an organic solvent to obtain a copper salt solution with a certain concentration; dissolving pyruvate in an organic solvent to obtain a pyruvate solution with a certain concentration; mixing and stirring the copper salt solution and the pyruvate solution according to a certain molar ratio; and finally, centrifuging the solution, taking a supernatant solution, and freeze-drying to prepare copper pyruvate solid powder. According to the invention, a new anticancer drug copper pyruvate is prepared by coordination of trace element copper ions necessary for a human body and micromolecular pyruvate in a coordination bond manner. The polypeptide has a good mitochondrial targeting effect, kills tumor cells through copper death, and can effectively inhibit tumor volume increase of tumor-bearing mice. The preparation method is low in preparation cost, can be used in the fields of cancer treatment and the like, and has a good medical prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of biopharmaceuticals and relates to a mitochondrial targeted copper ion carrier anticancer drug and a preparation method and application thereof. Background Art

[0002] Copper death is a new cancer treatment method. Excessive intracellular copper ions stimulate the lipoylation aggregation process of mitochondrial-related proteins, promote the degradation of iron-sulfur cluster proteins, lead to protein toxicity stress, and ultimately induce cancer cell death. Currently, the main commercially available mitochondrial-targeted copper ion carrier anticancer drug is ilisimol. However, ilisimol is expensive and its targeting effect is not obvious.

[0003] Pyruvate is an α-keto acid that plays an important role in biochemical metabolic pathways. Pyruvate is an important intermediate in sugar metabolism in all biological cells and the mutual conversion of various substances in the body. Because the molecule contains activated ketone and carboxyl groups, it is widely used as a basic raw material in various fields such as chemistry, pharmaceuticals, food, agriculture and environmental protection. It can be prepared by chemical synthesis and biotechnology. Pyruvate is mainly involved in the metabolism of sugars, fats, etc. in the human body, and is also one of the intermediates of carbohydrate metabolism. Pyruvate plays an important role in processes such as glycolysis: it can be produced from phosphoenolpyruvate during the glycolysis of cytoplasmic glucose. Each mole of glucose provides the cell with 2 moles of pyruvate, 2 moles of ATP and 2 moles of NADH in this process. + H + Pyruvate can continue to enter the Krebs cycle for further oxidation and decomposition. 2 moles of pyruvate obtained from 1 mole of glucose can be decomposed into 6 moles of CO2 and 8 moles of NADH in the mitochondria. + H + , 2 moles of FADH2 and 2 moles of GTP. In anaerobic respiration, pyruvate can be converted into lactic acid or ethanol. The metabolites of pyruvate are excreted in the urine.

[0004] In summary, in view of the advantages of pyruvate, such as good biocompatibility, strong mitochondrial targeting and low price, the preparation of copper pyruvate to provide a mitochondrial targeted copper ion carrier anticancer drug is of great significance for cancer treatment applications. Summary of the invention

[0005] In view of the deficiencies of the prior art and actual needs, the present invention provides a mitochondrial targeted copper ion carrier anticancer drug and a preparation method and application thereof, wherein the mitochondrial targeted copper ion carrier anticancer drug is copper pyruvate. The mitochondrial targeted copper ion carrier anticancer drug has a simple preparation method, low price, good biosafety, strong mitochondrial targeting and tumor cell killing effect, and can be used in medical fields such as anticancer treatment.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a mitochondrial targeted copper ion carrier anticancer drug, wherein the mitochondrial targeted copper ion carrier anticancer drug is copper pyruvate.

[0008] In a second aspect, the present invention provides a method for preparing the mitochondrial targeted copper ion carrier anticancer drug according to the first aspect, the preparation method comprising the following steps:

[0009] (1) dissolving a copper salt in an organic solvent to obtain a copper salt solution of a certain concentration;

[0010] (2) dissolving pyruvate in an organic solvent to obtain a pyruvate solution of a certain concentration;

[0011] (3) mixing the copper salt solution and the pyruvate solution in a certain molar ratio and stirring;

[0012] (4) Centrifuging the above solution, taking the supernatant solution, and freeze-drying it to prepare copper pyruvate solid powder.

[0013] Preferably, the copper salt solution in step (1) has a mass concentration of 10-16 mg·mL -1 .

[0014] Preferably, the mass concentration of the pyruvate solution in step (2) is 6.5-13 mg·mL -1 .

[0015] Preferably, the organic solvent in steps (1) and (2) includes but is not limited to dimethyl sulfoxide, N,N-dimethylformamide, and chloroform.

[0016] Preferably, in step (3), the molar ratio of the copper salt to the pyruvate is 1:(2-4).

[0017] Preferably, the stirring time in step (3) is 0.5-24 h, the stirring rate is 300-700 r / min, and the stirring temperature is 20-37 °C.

[0018] Preferably, the centrifugation rate in step (4) is 5000-10000 rpm, and the centrifugation time is 5 min-2 h.

[0019] Preferably, the freeze-drying time in step (4) is 12 to 48 hours.

[0020] Compared with the prior art, the present invention has the following technical effects:

[0021] (1) The present invention combines copper ions, which are essential trace elements for the human body, with small molecule pyruvate through coordination bonds to prepare a new anticancer drug copper pyruvate;

[0022] (2) The copper pyruvate prepared by the present invention has a good mitochondrial targeting effect and kills tumor cells through copper death, which can effectively inhibit the growth of tumor volume in tumor-bearing mice;

[0023] (3) The preparation cost of the present invention is low, and it can be used in the fields of anti-cancer treatment and has good medical prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is the chemical structural formula of copper pyruvate;

[0025] Figure 2 It is a solid powder diagram of copper pyruvate;

[0026] Figure 3 This is the XPS spectrum of copper pyruvate;

[0027] Figure 4 This is the result diagram of copper pyruvate mitochondrial targeting;

[0028] Figure 5 This is a graph showing the therapeutic effect of copper pyruvate on triple-negative breast cancer in animals;

[0029] Figure 6 This is a graph showing the biosafety results of copper pyruvate. DETAILED DESCRIPTION

[0030] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0031] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0032] Example 1

[0033] This embodiment prepares a mitochondrial targeted copper ion carrier anticancer drug, and the preparation method includes the following steps:

[0034] (1) Dissolve cupric chloride dihydrate in dimethyl sulfoxide to a concentration of 10 mg mL -1 ;

[0035] (2) Dissolve sodium pyruvate in dimethyl sulfoxide to a concentration of 13 mg mL -1 ;

[0036] (3) Mix the copper chloride and sodium pyruvate solutions in a molar ratio of 1:2 and stir at 500 r / min at 20°C for 8 h;

[0037] (4) The solution in step (3) was centrifuged at 10,000 rpm for 10 min, the supernatant solution was taken, and freeze-dried for 48 h to obtain copper pyruvate solid powder.

[0038] Example 2

[0039] This embodiment prepares a mitochondrial targeted copper ion carrier anticancer drug, and the preparation method includes the following steps:

[0040] (1) Dissolve cupric chloride dihydrate in N,N-dimethylformamide to a concentration of 10 mg mL -1 ;

[0041] (2) Dissolve sodium pyruvate in N,N-dimethylformamide to a concentration of 13 mg mL -1 ;

[0042] (3) Mix the copper chloride and sodium pyruvate solutions in a molar ratio of 1:2 and stir at 400 rpm for 8 h at 20 °C.

[0043] (4) The solution in step (3) was centrifuged at 10,000 rpm for 10 min, the supernatant solution was taken, and freeze-dried for 48 h to obtain copper pyruvate solid powder.

[0044] Example 3

[0045] This embodiment prepares a mitochondrial targeted copper ion carrier anticancer drug, and the preparation method includes the following steps:

[0046] (1) Dissolve cupric chloride dihydrate in chloroform to a concentration of 10 mg mL -1 ;

[0047] (2) Dissolve sodium pyruvate in chloroform to a concentration of 13 mg mL -1 ;

[0048] (3) Mix the copper chloride and sodium pyruvate solutions in a molar ratio of 1:3 and stir at 500 r / min at 25°C for 12 h;

[0049] (4) The solution in step (3) was centrifuged at 10,000 rpm for 10 min, the supernatant solution was taken, and freeze-dried for 36 h to obtain copper pyruvate solid powder.

[0050] Example 4

[0051] This embodiment prepares a mitochondrial targeted copper ion carrier anticancer drug, and the preparation method includes the following steps:

[0052] (1) Dissolve copper sulfate pentahydrate in dimethyl sulfoxide to a concentration of 16 mg mL -1 ;

[0053] (2) Dissolve sodium pyruvate in dimethyl sulfoxide to a concentration of 6.5 mg mL -1 ;

[0054] (3) Mix the copper chloride and sodium pyruvate solutions in a molar ratio of 1:2 and stir at 37°C and 300 r / min for 8 h;

[0055] (4) The solution in step (3) was centrifuged at 8000 rpm for 30 min, the supernatant solution was taken, and freeze-dried for 24 h to obtain copper pyruvate solid powder.

[0056] Example 5

[0057] This embodiment prepares a mitochondrial targeted copper ion carrier anticancer drug, and the preparation method includes the following steps:

[0058] (1) Dissolve copper sulfate pentahydrate in N,N-dimethylformamide at a concentration of 16 mg mL -1 ;

[0059] (2) Dissolve sodium pyruvate in N,N-dimethylformamide to a concentration of 6.5 mg mL -1 ;

[0060] (3) Mix the copper chloride and sodium pyruvate solutions in a molar ratio of 1:3 and stir at 700 rpm at 30°C for 24 h;

[0061] (4) The solution in step (3) was centrifuged at 5000 rpm for 1 h, the supernatant solution was taken, and freeze-dried for 24 h to obtain copper pyruvate solid powder.

[0062] Example 6

[0063] This embodiment prepares a mitochondrial targeted copper ion carrier anticancer drug, and the preparation method includes the following steps:

[0064] (1) Dissolve copper sulfate pentahydrate in chloroform to a concentration of 12 mg mL -1 ;

[0065] (2) Dissolve sodium pyruvate in chloroform to a concentration of 10 mg mL -1 ;

[0066] (3) Mix the copper chloride and sodium pyruvate solutions in a molar ratio of 1:4 and stir at 500 r / min at 20°C for 12 h;

[0067] (4) The solution in step (3) was centrifuged at 10,000 rpm for 10 min, the supernatant solution was taken, and freeze-dried for 48 h to obtain copper pyruvate solid powder.

[0068] Experimental Example 1 Detection of mitochondrial targeting of copper pyruvate

[0069] This test example studies the mitochondrial targeting effect of Example 1 on 4T1 mouse breast cancer cells.

[0070] 1.0 × 10 cells were cultured per well in a 6-well plate. 5 4T1 cells were added with 1 mL of culture medium (1640 + 10% fetal bovine serum + 1% double antibody) and cultured for 24 h before replacement with 1 mL of fresh culture medium containing copper pyruvate, copper chloride dihydrate or an equal volume of PBS (10 μg mL -1 , Cu). Continue to culture for 24 h, collect cells, separate mitochondria, cytoplasm and other parts of cells using mitochondrial separation kit, determine protein content of each component using BCA kit, and determine copper content of each component by atomic absorption spectrometer. Calculate copper concentration of each cell component. The experimental results are shown in Figure 4 As shown, the copper pyruvate prepared in Example 1 has a good tumor cell mitochondrial targeting effect and significantly increases the mitochondrial copper concentration of 4T1 cells.

[0071] Experimental Example 2 Testing the Effect of Copper Pyruvate on the Treatment of Triple-Negative Breast Cancer in Animals

[0072] This test example studies the mitochondrial targeting effect of Example 1 on 4T1 mouse breast cancer cells.

[0073] Balb / c mice (7 weeks old, female) were used in the experiment and were raised in a specific pathogen-free (SPF) animal environment. Each mouse was injected subcutaneously at the back of the right hind leg with 2×10 6 4T1 cells were used to establish a triple-negative breast cancer model. The tumor-bearing mice were randomly divided into two groups: Group 1, injected with PBS (200 μL); Group 2, injected with copper pyruvate (200 μL, 4 μg mL -1 , Cu), and the tumor volumes of the mice were monitored every two days.

[0074] The experimental results are as follows Figure 5 As shown, the copper pyruvate prepared in Example 1 can effectively inhibit the growth of tumor volume in mice.

[0075] Test Example 3 Testing the biological safety of copper pyruvate

[0076] This test example studies the mitochondrial targeting effect of Example 1 on 4T1 mouse breast cancer cells.

[0077] To examine the biocompatibility, mice were euthanized 30 days after different treatments, and heart, liver, spleen, lung, and kidney tissues were obtained for H&E staining.

[0078] The experimental results are as follows Figure 6 As shown, the copper pyruvate prepared in Example 1 has good biocompatibility.

[0079] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A mitochondrial targeted copper ion carrier anticancer drug, characterized in that Prepared from copper salts and pyruvate.

2. The mitochondrial targeted copper ion carrier anticancer drug according to claim 1, characterized in that: The copper salt is selected from copper chloride, copper bromide and copper sulfate.

3. The mitochondrial targeted copper ion carrier anticancer drug according to claim 1, characterized in that: The pyruvate is selected from sodium pyruvate and potassium pyruvate.

4. The method for preparing the mitochondrial targeted copper ion carrier anticancer drug according to claim 1, characterized in that: The following steps are involved: (1) dissolving a copper salt in an organic solvent to obtain a copper salt solution of a certain concentration; (2) dissolving pyruvate in an organic solvent to obtain a pyruvate solution of a certain concentration; (3) mixing the copper salt solution and the pyruvate solution in a certain molar ratio and stirring; (4) Centrifuging the above solution, taking the supernatant solution, and freeze-drying it to prepare copper pyruvate solid powder.

5. The method for preparing the mitochondrial targeted copper ion carrier anticancer drug according to claim 4, characterized in that: The mass concentration of the copper salt solution in step (1) is 10-16 mg·mL -1 .

6. The method for preparing the mitochondrial targeted copper ion carrier anticancer drug according to claim 4, characterized in that: The mass concentration of the pyruvate solution in step (2) is 6.5-13 mg·mL -1 .

7. The method for preparing the mitochondrial targeted copper ion carrier anticancer drug according to claim 4, characterized in that: The organic solvent in steps (1) and (2) includes but is not limited to dimethyl sulfoxide, N,N-dimethylformamide, and chloroform.

8. The method for preparing the mitochondrial targeted copper ion carrier anticancer drug according to claim 4, characterized in that: The molar ratio of the copper salt to the pyruvate in step (3) is 1:(2-4).

9. The method for preparing the mitochondrial targeted copper ion carrier anticancer drug according to claim 4, characterized in that: The stirring time in step (3) is 0.5-24 h, the stirring rate is 300-700 r / min, and the stirring temperature is 20-37 °C.

10. The method for preparing the mitochondrial targeted copper ion carrier anticancer drug according to claim 4, characterized in that: In step (4), the centrifugal speed is 5000-10000 rpm, the centrifugal time is 5 min-2 h, and the freeze-drying time is 12-48 h.

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