CN116421590B - Application of chlorhexidine diacetate in preparing medicine for preventing or/and treating liver cancer - Google Patents

Abstract

The application relates to a new application of chlorhexidine diacetate, in particular to an application of chlorhexidine diacetate in preparing medicines for preventing or/and treating liver cancer. Further, the liver cancer cell is SNU449. Compared with the prior art, the application has the following beneficial effects: the chlorhexidine diacetate is applied to the preparation of the medicine for preventing or/and treating liver cancer for the first time. The inventor discovers that the chlorhexidine diacetate can specifically inhibit the proliferation of liver cancer cells, and is hopeful to be a novel drug for overcoming liver cancer by inhibiting the RNA helicase DDX 17.

Description

Application of chlorhexidine diacetate in the preparation of medicines for preventing or/and treating liver cancer

technical field

The application relates to the technical field of medicines, in particular to the use of chlorhexidine diacetate in the preparation of medicines for preventing or/and treating liver cancer.

Background technique

Hepatocellular carcinoma (HCC) is referred to as liver cancer for short. Liver cancer refers to malignant tumors that occur in the liver, including primary liver cancer and metastatic liver cancer. People usually refer to primary liver cancer when they refer to liver cancer. Primary liver cancer is one of the most common malignant tumors in clinical practice. According to the latest statistics, there are about 600,000 new patients with liver cancer every year in the world, ranking fifth among malignant tumors, and its mortality rate ranks second among all tumors. According to cell type, primary liver cancer can be divided into hepatocellular carcinoma, cholangiocellular carcinoma and mixed liver carcinoma. According to the shape of the tumor, it can be divided into nodular type, massive type and diffuse type.

There are various treatment methods for liver cancer, including surgery and targeted therapy, but at present, surgical treatment represented by hepatectomy is still the first choice for the treatment of liver cancer. At present, the treatment drugs for liver cancer include sorafenib, lenvatinib, donafenib, bevacizumab combined with atezolizumab, regorafenib, apatinib, cabozantinib, etc. . The treatment of conventional chemotherapy drugs (such as doxorubicin, fluorouracil, cisplatin, α-interferon, etc.) not only has serious side effects, but also usually cannot significantly alleviate the disease or prolong life, and the curative effect of most other drugs is unsatisfactory. In the molecular targeted therapy of liver cancer, sorafenib is currently the standard treatment for advanced hepatocellular carcinoma. The tyrosine kinase inhibitor sorafenib, which has been proven to prolong the survival of patients with liver cancer, has also been found to be effective. Obvious side effects. In the studies of other new targeted drugs that emerged after sorafenib, the overall survival rate of patients with the primary treatment endpoint was not better than that of sorafenib. The reason is that hepatocellular carcinoma does not have clear driver genes or biomarkers like lung cancer, which makes it very difficult to develop new drug targets for human liver cancer. In addition to immune checkpoint inhibitors, various inhibitors for various cancer targets, such as various growth factor receptors, C-MET and other inhibitors are in progress, but the overall treatment of liver cancer is still lack of effective There is an urgent need to develop more effective anticancer drugs clinically.

Chlorhexidine diacetate belongs to the biguanide family, the molecular formula is C ₂₆ H ₃₈ Cl ₂ N ₁₀ O ₄ , and the molecular weight is 625.55. It is often used as a broad-spectrum antibacterial and disinfectant. The corresponding mechanism of action involves disturbing the cell membrane. It has a strong bactericidal effect on Gram-positive bacteria, negative bacteria and fungi, and is also effective on Pseudomonas aeruginosa. It is often used for skin disinfection, wound washing, and surgical instrument disinfection. At present, there is no research on the use of chlorhexidine diacetate on liver cancer cells.

Contents of the invention

Based on this, the application provides a new application of chlorhexidine diacetate, specifically the application of chlorhexidine diacetate in the preparation of drugs for the prevention or/and treatment of liver cancer, chlorhexidine diacetate can effectively inhibit liver cancer cells, It has obvious curative effect on liver cancer.

The technical scheme of the application includes:

Use of chlorhexidine diacetate in preparing medicines for preventing or/and treating liver cancer.

In one embodiment, the treatment of liver cancer includes: inhibiting the proliferation of liver cancer cells.

In one embodiment, inhibiting the proliferation of liver cancer cells includes inhibiting the expression of DDX17 protein.

In one of the embodiments, the liver cancer cell is the liver cancer cell line SNU449.

In one of the embodiments, the medicine comprises chlorhexidine diacetate and pharmaceutically acceptable auxiliary materials.

In one of the embodiments, the dosage form of the medicine is a tablet.

In one of the embodiments, the dosage form of the medicine is a capsule.

In one embodiment, the dosage form of the drug is oral liquid, oral granule or oral powder.

In one of the embodiments, the dosage form of the drug is injection.

In one of the embodiments, the injection is freeze-dried powder or emulsion for injection.

Compared with the traditional technology, the application of chlorhexidine diacetate of the present application in the preparation of drugs for preventing or/and treating liver cancer includes the following beneficial effects:

This study found for the first time that chlorhexidine diacetate can be used as a chemotherapy drug, which can significantly inhibit the malignant growth, plate colony formation ability and DNA synthesis ability of liver cancer cells (such as SNU449). It was also found that chlorhexidine diacetate can significantly inhibit the expression of DDX17 protein with the increase of drug concentration, and chlorhexidine diacetate is expected to become a new generation of drugs targeting DDX17 to inhibit the malignant proliferation of liver cancer cells.

Description of drawings

In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the accompanying drawings that need to be used in the description of the specific embodiments or prior art. Obviously, the accompanying drawings in the following description The figures show some implementations of the present application, and those skilled in the art can obtain other figures based on these figures without any creative effort.

Fig. 1 is the schematic diagram of the structural formula of the chlorhexidine diacetate of an embodiment;

Fig. 2 is the experimental result figure that the MTS experiment of an embodiment detects chlorhexidine diacetate to the viability of liver cancer cell SNU449 cell;

Fig. 3 is the experimental result figure of chlorhexidine diacetate to the colony formation of liver cancer cell SNU449 in the plate colony formation experiment of an embodiment;

Fig. 4 is the experimental result figure that EDU experiment detects chlorhexidine diacetate to the synthetic ability of liver cancer cell SNU449 DNA in an embodiment;

Fig. 5 is the experimental result diagram of using surface plasmon resonance technology to measure the interaction affinity between chlorhexidine diacetate and DDX17 protein in an embodiment;

Fig. 6 is a Western blot Western blot diagram comparing the expression levels of DDX17 in the control group treated with DMSO and treated with different concentrations of chlorhexidine diacetate in one embodiment.

Detailed ways

In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the relevant drawings. Preferred embodiments of the application are shown in the accompanying drawings. However, the present application can be embodied in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the application more thorough and comprehensive.

In addition, the terms "first" and "second" are used for descriptive purposes only, and cannot be interpreted as indicating or implying relative importance or implicitly specifying the quantity of indicated technical features. Thus, the features defined as "first" and "second" may explicitly or implicitly include at least one of these features. In the description of the present application, "plurality" means at least two, such as two, three, etc., unless otherwise specifically defined. In the description of the present application, "several" means at least one, such as one, two, etc., unless otherwise specifically defined.

When a numerical range is disclosed herein, such range is considered continuous and includes the minimum and maximum values of that range and every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum of the range is included. Furthermore, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise indicated, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this application belongs. The terms used herein in the specification of the application are only for the purpose of describing specific embodiments, and are not intended to limit the application. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

"Pharmaceutically acceptable" refers to those ligands, materials, compositions and/or dosage forms which, within the scope of sound medical judgment, are suitable for administration to a patient and commensurate with a reasonable benefit/risk ratio.

"Pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. As used herein, the language "pharmaceutically acceptable carrier" includes buffers, sterile water for injection, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorbing agents compatible with pharmaceutical administration Retardants and the like. Each carrier must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Suitable examples include, but are not limited to: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch, potato starch, and substituted or unsubstituted beta-cyclodextrins; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyhydric Alcohols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) Esters, such as ethyl oleate and ethyl laurate; (13) Agar; (14) Buffers, such as magnesium hydroxide and hydrogen (15) Alginic acid; (16) Pyrogen-free water; (17) Isotonic saline; (18) Ringer's solution; (19) Ethanol; (20) Phosphate buffered saline; and (21) Drug formulation Other non-toxic compatible substances used in the product.

Application method

The dosage form and administration method of the compound of the present application or its pharmaceutical composition are not particularly limited.

Representative modes of administration include, but are not limited to, oral, intratumoral, rectal, parenteral (intravenous, intramuscular, or subcutaneous) injection, and topical administration.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is admixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with (a) fillers or extenders, for example, Starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders such as hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, For example, glycerol; (d) disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow agents, such as paraffin; (f) Absorption accelerators such as quaternary ammonium compounds; (g) wetting agents such as cetyl alcohol and glyceryl monostearate; (h) adsorbents such as kaolin; and (i) lubricants such as talc, hard Calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 ,3-Butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances. Besides such inert diluents, the compositions can also contain adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. Suspensions, for example, may contain suspending agents, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar or mixtures of these substances.

Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous or non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols, and suitable mixtures thereof.

Dosage forms for topical administration include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers, or propellants that may be required if necessary.

The application provides a use of chlorhexidine diacetate in the preparation of medicines for preventing or/and treating liver cancer.

Optionally, treating liver cancer includes: inhibiting the proliferation of liver cancer cells.

Further optionally, inhibiting the proliferation of liver cancer cells includes inhibiting the expression of DDX17 protein.

Optionally, the liver cancer cells are the liver cancer cell line SNU449.

Optionally, the medicine comprises chlorhexidine diacetate and pharmaceutically acceptable auxiliary materials.

It can be understood that, depending on the dosage form of the drug, the type and amount of the selected excipients are also different. Chlorhexidine diacetate can be combined with appropriate types of excipients to prepare drugs in different forms (such as liquid, semi-solid, solid), and correspondingly form different dosage forms.

Optionally, the dosage form of the medicine is a tablet.

It can be understood that, in this application, a tablet is a tablet-like or special-shaped tablet-shaped solid preparation that is uniformly mixed with a drug and an excipient, and is mainly taken for oral administration. There are also buccal tablets, sublingual tablets, and buccal patches. film etc. In order to increase the stability, mask the bad smell of the drug, improve the appearance of the tablet, etc., the tablet can be coated.

In one specific example, the tablet is a coated tablet.

Optionally, the dosage form of the medicine is a capsule.

It can be understood that, in this application, capsules refer to medicines or solid preparations filled with suitable auxiliary materials in hollow hard capsules or sealed in soft capsules, which can be divided into hard capsules, soft capsules (capsules) ), mainly for oral use, is also a solid dosage form widely used in clinical practice.

Optionally, the dosage form of the drug is oral liquid, oral granule or oral powder.

In one of the specific examples, the dosage form of the drug is oral granules.

It can be understood that, in this application, oral granules refer to dry granular preparations with a certain particle size prepared by mixing raw materials and suitable excipients, which are commonly used oral solid dosage forms. Unless otherwise specified, the sum of coarse particles larger than No. 1 sieve and fine particles smaller than No. 5 sieve in granules shall not exceed 15%. Granules can be swallowed directly or washed into water for drinking.

Optionally, the dosage form of the drug is injection.

It can be understood that, in this application, injections refer to sterile solutions (including emulsions and suspensions) made of drugs for injection into the body and sterile powders for making solutions or suspensions before use. or concentrated solution.

In one specific example, the injection is freeze-dried powder for injection.

In one specific example, the injection is an emulsion for injection.

Above, the examples of the present application are only examples of the types of "dosage forms". In addition to the several dosage forms of "liquid" and "injection", chlorhexidine diacetate can also be prepared into other suitable and pharmaceutically acceptable dosage forms.

Further description below in conjunction with specific examples, the raw materials involved in the following specific examples, if no special instructions, all can be derived from commercially available, as the chlorhexidine diacetate described in the embodiment of the application is commercially available, so The instruments used, unless otherwise specified, are commercially available, and the processes involved, unless otherwise specified, are routinely selected by those skilled in the art.

The following are specific examples.

Example 1

(1) Prepare cells

Human liver cancer cells SNU449 were purchased from Guangzhou Geneo Biological Company;

Human liver cancer cell SNU449 cell culture method: Prepare mixed medium, including DMEM medium + 10% fetal bovine serum (FBS) + 1% penicillin/streptomycin double antibody) (P/S), add the cells to the prepared mixture The culture medium was then cultured in a 37°C, 5% CO ₂ cell incubator. After 48-72 hours, the cell fullness of the culture dish was observed, and subculture was carried out when the cells grew to 90%-100%.

(2) Effect of Chlorhexidine diacetate on the proliferation of liver cancer cells SNU449

Chlorhexidine diacetate (15 μM) was used to treat liver cancer cells SNU449, and then the growth of liver cancer cells at four time points of 0h, 24h, 48h, and 72h were detected by MTS assay.

Combined with the results in Figure 2, it was shown that after SNU449 was treated with 15 μM chlorhexidine diacetate, compared with the control group treated with dimethyl sulfoxide (DMSO), after 72 hours of culture, chlorhexidine diacetate could significantly reduce the growth (P<0.05).

(3) Effect of chlorhexidine diacetate on colony formation ability of liver cancer cells SNU449 plate

Liver cancer cells SNU449 were inoculated in six wells, the number of cells was 0.1k~0.3k cells/well, cultured for 10 days according to the method of conventional culture of liver cancer cells, and then treated with 15μM chlorhexidine diacetate for 2~3 days to remove After the culture medium, wash with 1×PBS, add 1ml of methanol to fix the cells for 30 minutes, stain with 0.1% crystal violet, remove the crystal violet after 60 minutes, wash with 1×PBS, take pictures and count after drying, the control group is also treated as above, But no chlorhexidine diacetate was added.

Combined with the results shown in Figure 3, compared with the DMSO-treated control group, treatment with chlorhexidine diacetate can significantly reduce the plate colony formation ability of liver cancer cell SNU449 (P<0.05).

(4) Effect of chlorhexidine diacetate on the DNA synthesis ability of liver cancer cells SNU449

Liver cancer cells SNU449 were inoculated in six wells containing glass slides, and treated by adding (15 μM) chlorhexidine diacetate for 2-3 days the next day, using the EDU kit (Beyond, C0085S), and under a fluorescent microscope To observe the effect of chlorhexidine diacetate on the DNA synthesis ability of liver cancer cells SNU449.

Combined with the results shown in Figure 4, compared with the control group without chlorhexidine diacetate, treatment with chlorhexidine diacetate can significantly reduce the DNA synthesis ability of liver cancer cells SNU449 (P<0.05).

(5) Chlorhexidine diacetate has interaction affinity with DDX17 protein

RNA helicase DDX17 is DEAD-box17 (DDX17) is one of the important members of the RNA helicase DDX family, and its helicase core region contains 9 conserved motifs (Q, I, Ia, Ib, II-VI) . DDX17, a DEAD box protein characterized by the conserved motif Asp-Glu-Ala-Asp (DEAD), is involved in many cellular processes with altered RNA secondary structure, such as translation initiation, nuclear and mitochondrial splicing, and ribosome and spliceosome assembly. This gene encodes the DEAD box protein, an ATPase activated by various RNAs but not dsDNA, which can participate in almost all processes related to RNA metabolism, from transcription to translation. Play an important role in the growth of cancer. Studies have found that the expression of DDX17 is significantly increased in a variety of tumors, including liver cancer, lung cancer, breast cancer, and prostate cancer. In addition, multiple studies have shown that DDX17 can promote malignant proliferation, migration and invasion of liver cancer.

Using surface plasmon resonance (SPR) to establish a DDX17 protein-FDA listed compound interaction assay model, by dividing 1729 compounds in the FDA listed drug library into 36 groups of mixtures at about 50 compounds/group, and performing SPR at a single concentration point (10 μM ) screening, 4 groups of compounds were selected according to the descending order of RU value. Then, a total of 200 compounds from the 4 groups were sequenced by single-concentration (10 μM) SPR one by one, and sorted according to the descending order of RU value. Finally, the top-ranked compounds were set to multiple concentrations for SPR testing to obtain affinity data KD and kinetic numbers Kd and ka. Compounds with higher binding affinity to DDX17 were screened based on binding mode analysis. The DDX17 protein-compound interaction assay model was established using surface plasmon resonance (SPR).

The results in Figure 5 show that chlorhexidine diacetate has a high interaction affinity with DDX17 protein.

(6) Chlorhexidine diacetate inhibits the expression level of RNA helicase DDX17

Chlorhexidine diacetate was used to treat liver cancer cells SNU449, and the effect of chlorhexidine diacetate at different concentrations on the expression level of DDX17 protein was detected by WB experiment. The specific operation process was as follows:

Liver cancer cells SNU449 were inoculated in six wells, treated with 3.75 μM, 7.5 μM and 15 μM chlorhexidine diacetate for 2 to 3 days on the next day, and then the DDX17 protein was lysed, the concentration was measured, and SDS polyacrylamide gel was prepared. Gel (SDS-PAGE), electrophoresis, membrane transfer and blocking, blocking and incubation with primary antibody incubation, secondary antibody incubation, and imaging.

The results in Figure 6 show that compared with the control group treated with DMSO, with the increase of the drug concentration of chlorhexidine diacetate, chlorhexidine diacetate inhibits the gradual enhancement of the expression level of DDX17. The inhibitory effect is the strongest. According to the analysis of the above results, it can be seen that chlorhexidine diacetate can be used as a small molecule compound inhibitor of DDX17 protein, and can inhibit the malignant proliferation of liver cancer cells.

The technical features of the above-mentioned embodiments can be combined arbitrarily. To make the description concise, all possible combinations of the technical features in the above-mentioned embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, should be considered as within the scope of this specification.

The above-mentioned embodiments only represent several implementation modes of the present application, and the description thereof is relatively specific and detailed, but it should not be construed as limiting the scope of the patent for the invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the scope of protection of the patent application should be based on the appended claims.

Claims (10)

1. The application of chlorhexidine diacetate in preparing medicine for treating liver cancer is provided.

2. Use of chlorhexidine diacetate according to claim 1 for the manufacture of a medicament for the treatment of liver cancer comprising: inhibit proliferation of liver cancer cells.

3. The use of chlorhexidine diacetate according to claim 2 for the manufacture of a medicament for the treatment of liver cancer, wherein inhibiting proliferation of liver cancer cells comprises inhibiting expression of DDX17 protein.

4. The use of chlorhexidine diacetate according to claim 3 for the preparation of a medicament for the treatment of liver cancer, wherein the liver cancer cell is the liver cancer cell line SNU449.

5. Use of chlorhexidine diacetate according to any of claims 1 to 4 for the manufacture of a medicament for the treatment of liver cancer, characterized in that the medicament comprises chlorhexidine diacetate and pharmaceutically acceptable excipients.

6. The use of chlorhexidine diacetate in the manufacture of a medicament for the treatment of liver cancer according to claim 5 wherein the dosage form of the medicament is a tablet.

7. The use of chlorhexidine diacetate in the manufacture of a medicament for treating liver cancer according to claim 5, wherein the dosage form of the medicament is a capsule.

8. The use of chlorhexidine diacetate in the preparation of a medicament for treating liver cancer according to claim 5, wherein the dosage form of the medicament is an oral liquid, an oral granule or an oral powder.

9. The use of chlorhexidine diacetate in the preparation of a medicament for treating liver cancer according to claim 5, wherein the dosage form of the medicament is an injection.

10. The use of chlorhexidine diacetate in the preparation of a medicament for treating liver cancer according to claim 9, wherein the injection is a lyophilized powder for injection or an emulsion for injection.