阅读说明：本技术 紫檀芪在制备top1酶抑制剂中的应用 (Application of pterostilbene in preparation of TOP1 enzyme inhibitor ) 是由 陈西敬 张愈甜 李莹 孙长城 刘金峰 于 2020-06-28 设计创作，主要内容包括：本发明涉及紫檀芪的一种新的用途,具体公开了紫檀芪在制备DNA拓扑异构酶I(topoisomerase I,TOP1)抑制剂中的应用,还公开了紫檀芪在制备用于具有TOP1介导的DNA复制损伤特征的肿瘤疾病的药物中的应用,所述肿瘤包括但不限于非小细胞肺癌、乳腺癌、肝癌、卵巢癌、回盲肠癌、结肠癌或大肠癌。紫檀芪能够有效抑制TOP1酶的酶活,抑制其对超螺旋DNA的解旋能力,且对TOP1酶的抑制效果与喜树碱持平。此外,紫檀芪能够有效抑制多种肿瘤细胞的增殖,具有广谱抗肿瘤效果；还能在细胞内下调TOP1酶蛋白的浓度,对体外非增值型细胞无细胞毒性,安全性较高。(The invention relates to a new application of pterostilbene, and particularly discloses an application of pterostilbene in preparation of a DNA topoisomerase I (TOP 1) inhibitor, and also discloses an application of pterostilbene in preparation of a medicine for treating tumor diseases with TOP1 mediated DNA replication damage characteristics, wherein the tumors include but are not limited to non-small cell lung cancer, breast cancer, liver cancer, ovarian cancer, ileocecum cancer, colon cancer or colorectal cancer. The pterostilbene can effectively inhibit the enzyme activity of TOP1 enzyme, inhibit the unwinding capability of the pterostilbene on supercoiled DNA, and has the inhibiting effect on TOP1 enzyme which is equivalent to that of camptothecin. In addition, pterostilbene can effectively inhibit the proliferation of various tumor cells and has a broad-spectrum anti-tumor effect; can also reduce the concentration of TOP1 enzyme protein in cells, has no cytotoxicity to non-proliferation cells in vitro, and has high safety.)

1. Application of pterostilbene in preparation of DNA topoisomerase I (TOP 1) inhibitor is provided.

2. Use of pterostilbene in the manufacture of a medicament for use in a neoplastic disease characterized by TOP 1-mediated DNA replication damage.

3. The use of claim 2, wherein the tumor includes but is not limited to: non-small cell lung cancer, breast cancer, liver cancer, ovarian cancer, ileocecal cancer, colon cancer or carcinoma of large intestine.

4. Application of pterostilbene in preparing medicine for inhibiting proliferation of A549, MCF-7, Hep G2, A2780, HCT-116, HCT-8, SW480, LOVO, COLO205 and/or CL187 tumor cells.

5. The use according to any one of claims 1to 4, wherein the pterostilbene is present at a concentration of 0.25 to 500 μ M; preferably 0.25, 0.5, 2.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 300, 400, 500 μ M.

6. The use according to any one of claims 1to 4, wherein pterostilbene is formulated as follows: fully dissolving pterostilbene by adopting a proper amount of solvent to prepare mother liquor; and then diluting the mother liquor by the solvent according to gradient to prepare pterostilbene solutions with different concentrations.

7. The use of claim 6, the solvent including but not limited to: purified water, physiological saline, and glucose water; ethanol, propylene glycol, glycerol, polyethylene glycol 200(400, 600), butanol and benzyl alcohol; dimethylformamide, dimethylacetamide; glycerol triacetate, ethyl acetate, ethyl oleate, benzyl benzoate, and isopropyl myristate; peanut oil, corn oil, sesame oil; dimethyl sulfoxide; cyclodextrin, hydroxypropyl-beta-cyclodextrin and/or sulfobutyl-beta-cyclodextrin.

8. The use according to any one of claims 1to 4, wherein pterostilbene is present in the form of a pharmaceutical composition comprising pterostilbene and any pharmaceutically acceptable carrier.

9. The use according to claim 8, wherein the pharmaceutical composition is a solid, semi-solid or liquid formulation.

Technical Field

The invention belongs to the field of medical application, particularly relates to a new application of pterostilbene, and further relates to an application of pterostilbene in preparation of a TOP1 enzyme inhibitor.

Background

Cancer is a disease threatening human health, and causes death second to heart disease, and has a tendency to exceed the mortality rate of heart disease, thus causing a heavy economic and mental burden to society and families. International agency for research on cancer (IARC) states that 1810 million new cancer pathologies are predicted worldwide in 2018, and approximately 960 million people die of cancer. In China, there are approximately 280.4 million cancer cases and 229.6 million cancer death cases, and the incidence and mortality of cancer are in a significantly rising trend compared to the previous year's assessment report. The existing conventional tumor treatment method is surgical drug-adding treatment, but most of the anti-tumor drugs have the defects of large toxic and side effects, weak targeting property, easy generation of drug resistance and the like.

DNA topoisomerase I (TOP 1) is an enzyme universally existing in mammalian cells, is involved in many important cellular processes such as DNA replication, transcription and chromatin remodeling, can regulate the topological structure of DNA, and unwinds the positive and negative supercoils of DNA, and is the only enzyme found so far that can form DNA 3' -tyrosyl phosphodiester bond. TOP1 has been proved to be an effective target for treating tumor, and camptothecin and its derivatives are TOP1 enzyme inhibitors, which have wide application in antitumor clinical treatment. However, camptothecin and its derivatives have poor water solubility and most of them have high cytotoxicity, so that their clinical application is limited. Therefore, the research and development of new low-toxicity and high-targeting antitumor drugs are of great significance.

In the process of searching for antitumor drugs, compounds of natural sources attract attention of scientists. Pterostilbene was originally derived from sandalwood and is thus named, and is later found and extracted in large quantities in berries such as blueberries, grapes, and the like. Pterostilbene is a dimethylated derivative of resveratrol, and compared with resveratrol, pterostilbene has more excellent oral absorption utilization rate. Pterostilbene has anticancer, antiinflammatory, antioxidant and analgesic effects. At present, a great deal of research shows that pterostilbene has a good effect in the treatment and the protection of skin diseases, particularly in the fields of oxidation resistance and the like. At present, a patent of application of pterostilbene in preparation of an anti-cervical cancer medicament (CN201010249037.X) exists in China, and the patent publication shows that the pterostilbene has low acute toxicity and can be used for preparing the anti-cervical cancer medicament. However, no report related to pterostilbene in preparation of TOP1 enzyme inhibitor is seen at home and abroad at present.

Disclosure of Invention

In order to solve the technical problems, the invention provides an application of pterostilbene in preparation of a TOP1 enzyme inhibitor, and discloses that pterostilbene can inhibit activity of TOP1 enzyme and tumor diseases with TOP 1-mediated DNA replication damage characteristics.

The pterostilbene has the molecular formula as follows: c₁₆H₁₆O₃Molecular weight: 256.30, the structural formula is as follows:

in one embodiment, the invention provides the use of pterostilbene in the preparation of a TOP1 enzyme inhibitor.

In one embodiment, the invention also provides the use of pterostilbene in the preparation of a medicament for a neoplastic disease characterized by TOP 1-mediated DNA replication damage.

In one embodiment, the tumor includes, but is not limited to, non-small cell lung cancer, breast cancer, liver cancer, ovarian cancer, ileocecal cancer, colon cancer, or large intestine cancer. Further, pterostilbene can inhibit proliferation of A549, MCF-7, Hep G2, A2780, HCT-116, HCT-8, SW480, LOVO, COLO205, and/or CL187 tumor cells.

In one embodiment, the concentration of pterostilbene in the inhibitor is 0.25-500 μ M. Further, the concentration of pterostilbene is 0.25, 0.5, 2.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 300, 400, 500 μ M.

In one embodiment, the inhibitor is formulated using methods conventional in the art, including: fully dissolving pterostilbene by adopting a proper amount of solvent to prepare mother liquor; and then diluting the mother liquor to the concentration according to a gradient by using the solvent. Further, the solvents include, but are not limited to: purified water, physiological saline, aqueous glucose, and other aqueous solvents; alcohols and polyols such as ethanol, propylene glycol, glycerol, polyethylene glycol 200(400, 600), butanol, benzyl alcohol, and the like; amides such as dimethylformamide, dimethylacetamide and the like; esters such as glycerol triacetate, ethyl acetate, ethyl oleate, benzyl benzoate, isopropyl myristate, and the like; vegetable oils such as peanut oil, corn oil, sesame oil, etc.; sulfoxides such as dimethyl sulfoxide, etc.; cyclodextrins, such as hydroxypropyl-beta-cyclodextrin and sulfobutyl-beta-cyclodextrin, and the like.

In one embodiment, the use is where pterostilbene is present in a pharmaceutical composition comprising pterostilbene and any pharmaceutically acceptable carrier. The carrier means any carrier which is substantially free of long term or permanent deleterious effects when administered, and includes the meanings of "pharmaceutically acceptable medium, stabilizer, diluent, additive, adjuvant or vehicle". The carrier is typically mixed with the active compound or may be used to dilute or encapsulate the active compound and may be a solid, semi-solid or liquid formulation. It will be appreciated that the active ingredient should be soluble or be released as a suspension in the desired carrier or diluent. Any variety of pharmaceutically acceptable carriers can be used, including but not limited to aqueous media such as water, saline, glycine, hyaluronic acid, and the like; solid carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, sodium saccharine, cellulose, glucose, sucrose, and magnesium carbonate; a solvent; dispersing the reagent; coating; antibacterial and antifungal agents; isotonic and absorption delaying agents; or any other inactive ingredient. The choice of a pharmaceutically acceptable carrier may depend on the mode of administration. Except insofar as some pharmaceutically acceptable carriers are incompatible with the active ingredient, use thereof in pharmaceutically acceptable compositions is contemplated. Non-limiting examples of specific uses of such pharmaceutical carriers are found in pharmaceutical dosage forms and drug delivery systems (Howard. Anseletal, eds., Lippincott Williams & Wilkinspublysepers, 7the d.1999); redmon: science and practice of medicine ((alfonsor. gennarrated., Lippincott, Williams & Wilkins, 20the d.2000); goodman & gilman "pharmacology basis for treatment" (joel g. hardman, eds., McGraw-hill professional, 10the d.2001)); handbook of pharmaceutical excipients (Raymond. Roweeet al., APhA publications, 4the edition 2003). These protocols are conventional procedures and any modifications are intended to be included within the scope of those skilled in the art and derived from the teachings herein.

Further, the pharmaceutical composition may optionally include, but is not limited to, other pharmaceutically acceptable ingredients (or pharmaceutical ingredients) including, but not limited to, buffers, preservatives, tonicity adjusting agents, salts, antioxidants, tonicity adjusting agents, physiological substances, pharmacological substances, fillers, emulsifiers, wetting agents, sweeteners or flavoring agents, and the like. Various buffers and methods of adjusting pH may be used in the preparation of the pharmaceutical compositions disclosed herein, provided that the preparation resulting therefrom is pharmaceutically acceptable. Such buffers include, but are not limited to, acetate buffers, citrate buffers, phosphate buffers, neutral buffered saline, phosphate buffered saline, and borate buffers. It is understood that acids or bases can be used to adjust the pH of the composition as desired. Pharmaceutically acceptable antioxidants include, but are not limited to, sodium metabisulfite, sodium thiosulfate, acetylcysteine, butyl hydroxyanisole and di-tert-butyl p-cresol. Useful preservatives include, but are not limited to, benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric acetate, phenylmercuric nitrate, stable oxychloro compositions, and chelating agents, such as DTPA or DTPA-bisamide, calcium DTPA, and CaNaDTPA-bisamide. Tonicity adjusting agents useful in the pharmaceutical composition include, but are not limited to, salts (e.g., sodium chloride, potassium chloride), mannitol or glycerol and other pharmaceutically acceptable tonicity adjusting agents. The pharmaceutical compositions may be provided as salts and may be formed with a variety of acids, including but not limited to hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid, and the like. The salts are more soluble in aqueous or other protic solvents than in the corresponding free base form. It is to be understood that the pharmaceutical composition may include the agent and other substances known in the pharmaceutical art.

Further, the pharmaceutical composition can be prepared into a solid preparation. Solid state formulations suitable for enteral or parenteral administration include, but are not limited to, capsules, tablets, pills, lozenges, troches, and powders and granules suitable for inhalation or reconstitution into sterile injectable solutions or dispersions. The therapeutic compounds or compositions disclosed herein for such administration can be prepared according to any method known in the art for the manufacture of pharmaceutical compositions. In this solid dosage form, the therapeutic compound may be mixed with: (a) at least one inert common excipient (or carrier), for example, sodium citrate or dicalcium phosphate or (b) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, isomalt, and silicic acid, (c) binders, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, (d) humectants, for example, glycerol, (e) disintegrating agents, for example, agar-agar, calcium carbonate, corn starch, potato starch, tapioca starch, alginic acid, certain complex silicates, and sodium carbonate, (f) solution retarding agents, for example, paraffin, (g) absorption accelerators, for example, quaternary ammonium compounds, (h) wetting agents, for example, cetyl alcohol and glycerol monostearate, (i) adsorbents, for example, kaolin and bentonite, (j) lubricants, for example, talc, stearic acid, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate or mixtures thereof, and (k) a buffering agent. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. In solid formulations, the therapeutically effective amount of pterostilbene may typically be between about 0.0001% (w/w) and about 60% (w/w), between about 0.001% (w/w) and about 40.0% (w/w), or between about 0.01% (w/w) and about 20.0% (w/w).

Further, the pharmaceutical composition can be prepared into a semi-solid preparation. Semisolid formulations suitable for topical administration include, without limitation, ointments, creams, salves, and gels. The therapeutic compounds or compositions disclosed herein for such administration can be prepared according to any method known in the art for the manufacture of pharmaceutical compositions. In semi-solid formulations, the therapeutically effective amount of pterostilbene may typically be between about 0.0001% (w/v) and about 60% (w/v), between about 0.001% (w/v) and about 40.0% (w/v), or between about 0.01% (w/v) and about 20.0% (w/v).

Further, the pharmaceutical composition can be prepared into a liquid preparation. Liquid preparations suitable for enteral or parenteral administration include, without limitation, solutions, syrups, elixirs, dispersions, emulsions, and suspensions including, without limitation, those for intravenous administration. The therapeutic compounds or compositions disclosed herein for such administration may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions. In this liquid dosage form, the pterostilbene may be mixed with: (a) suitable aqueous and nonaqueous carriers, (b) diluents, (c) solvents, for example, water, ethanol, propylene glycol, polyethylene glycol, glycerol, vegetable oils (such as rapeseed oil and olive oil), and injectable organic esters (such as ethyl oleate); and/or a flow agent (e.g., a surfactant or a coating agent such as lecithin). In the case of dispersions and suspensions, the flowability can also be controlled by maintaining a specific particle size. In liquid formulations, the therapeutically effective amount of pterostilbene may typically be between about 0.0001% (w/v) and about 60% (w/v), between about 0.001% (w/v) and about 40.0% (w/v), or between about 0.01% (w/v) and about 20.0% (w/v).

The invention has the beneficial effects that:

the invention provides application of pterostilbene in preparation of TOP1 enzyme inhibitors. Experiments show that pterostilbene can effectively inhibit the enzyme activity of TOP1 enzyme, inhibit the unwinding capability of the pterostilbene on supercoiled DNA, and has the same inhibition effect on TOP1 enzyme as camptothecin. In addition, pterostilbene can effectively inhibit the proliferation of various tumor cells and has a broad-spectrum anti-tumor effect; can also reduce the concentration of TOP1 enzyme protein in cells, has no cytotoxicity to non-proliferation cells in vitro, and has high safety.

Drawings

Figure 1 shows that pterostilbene inhibits the activity of TOP1 enzyme in vitro.

FIG. 2 is a graph of the inhibition of pterostilbene on the proliferation of various tumor cells.

Fig. 3 is a graph of pterostilbene inhibition of TOP1 enzyme protein content in CL187 cells.

Fig. 4 shows the inhibition of intracellular TOP1 enzyme gene expression by pterostilbene.

FIG. 5 is a graph showing the effect of pterostilbene on proliferating normal cells in vitro.

FIG. 6 is a graph of the effect of pterostilbene on non-proliferating normal cells in vitro.

Detailed Description

The technical solutions of the present invention are further illustrated by the following specific embodiments, but it is easily understood by those skilled in the art that the specific material ratios, process conditions and results thereof described in the examples are only for illustrating the present invention, and should not also limit the present invention described in detail in the claims.