A pharmaceutical composition for preventing and treating lung cancer, a preparation method thereof, and use thereof

Abstract

The present application relates to a kind of drug composition for preventing and treating lung cancer, composition is by MDM2 inhibitor and CDK9 inhibitor according to molar ratio (1-10):1 It is composed, wherein the MDM2 inhibitor is selected from Idasanutlin, Nutlin-3, Nutlin-3a, Nutlin-3b, MX69, NVP-CGM097, MI-773 (SAR405838), RG-7112, HDM201 (Siremadlin), YH239-EE, NSC207895, Serdemetan (JNJ-26854165) Any one or combination thereof, the CDK9 inhibitor is selected from NVP-2, AZD-4573, P276-00, CDKI-73, LY2857785, BS-194, PHA767491, roscovitine, CYC065, CR8 Any one or combination thereof.The present application scientifically configures and synergistically utilizes the anticancer drug of different action mechanisms, selectively causes tumor cell DNA damage, induces tumor cell apoptosis, and effectively inhibits tumor cell growth.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a pharmaceutical composition for preventing and treating lung cancer, a preparation method thereof, and an application thereof. Background Art

[0002] Lung cancer, a malignant tumor originating from the bronchial mucosa or glands of the lungs, is one of the fastest-growing malignant tumors in terms of incidence and mortality. Lung cancer ranks first in both incidence and mortality among malignant tumors in men and second in women.

[0003] The p53 gene is a tumor suppressor gene. Its product, p53 protein, promotes apoptosis in cancer cells, preventing carcinogenesis and tumor growth, and helps cells repair genetic defects. In approximately 50% of human cancer cells, p53 is inactivated due to mutations. Interactions with other proteins also contribute to the loss of p53's normal biological functions.

[0004] MDM2 (murine double mimute 2) is the most important inhibitor of p53. The design and development of novel anti-cancer drugs targeting MDM2-p53 is a hot topic and a key focus in global oncology drug research and development. Idasanutlin is a potent MDM2 inhibitor that selectively binds to the p53 site on the surface of MDM2, preventing MDM2 from binding to p53 and thus degrading p53. It exhibits strong inhibitory activity against wild-type p53-carrying cancer cells, including human osteosarcoma (SJSA-1), human colon cancer cells (HCT-116), and human colon adenocarcinoma cells (RKO). Potential indications include chronic lymphocytic leukemia, acute lymphocytic leukemia, acute myeloid leukemia, multiple myeloma, melanoma, neuroblastoma, and mantle cell lymphoma. Idasanutlin also exhibits potent inhibitory activity against p53-carrying non-small cell lung cancer cell lines and pediatric neuroblastoma.

[0005]

[0006] CDK9 is a serine kinase that plays a major role in the regulation of transcriptional elongation without affecting the cell cycle process. CDK9 inhibitors block the phosphorylation of the C-terminal region of RNA Poly-II by the positive transcription elongation factor P-TEFb (positive transcription elongation factor b) by degrading and inhibiting CDK9, inhibiting transcription, rapidly reducing intracellular mRNA levels, and causing tumor cell apoptosis, making it a hot topic in the development of anti-tumor drugs. NVP-2 is an effective and selective ATP-competitive cyclin-dependent kinase 9 (CDK9) probe that can inhibit CDK9 / CycT activity (IC 50 0.514nM). NVP-2 inhibits CDK1 / CycB (IC 50 NVP-2 has inhibitory effects on several kinases, including MDM2 (0.584 μM), CDK2 / CycA (0.706 μM), and CDK16 / CycY (0.605 μM). NVP-2 can induce apoptosis. However, there are no reports on the combined use of MDM2 and NVP-2 inhibitors for the treatment of lung cancer. Summary of the Invention

[0007] The object of the present invention is to provide a pharmaceutical composition for preventing and treating lung cancer, which composition is composed of an MDM2 inhibitor and a CDK9 inhibitor in a molar ratio of (1-10):1, wherein the MDM2 inhibitor is selected from any one of Idasanutlin, Nutlin-3, Nutlin-3a, Nutlin-3b, MX69, NVP-CGM097, MI-773 (SAR405838), RG-7112, HDM201 (Siremadlin), YH239-EE, NSC207895, and Serdemetan (JNJ-26854165), or a combination thereof, and the CDK9 inhibitor is selected from any one of NVP-2, AZD-4573, P276-00, CDKI-73, LY2857785, BS-194, PHA767491, roscovitine, CYC065, and CR8, or a combination thereof.

[0008] In a preferred technical solution of the present invention, the composition is composed of an MDM2 inhibitor and a CDK9 inhibitor in a molar ratio of 1-5:1, preferably 2-4:1.

[0009] In a preferred technical solution of the present invention, the composition consists of Idasanutlin and NVP-2 in a molar ratio of (1-10):1, preferably 1-5:1.

[0010] In a preferred technical solution of the present invention, the molar ratio of Idasanutlin to NVP-2 in the composition is selected from any one of 2.4:1, 10:3.3, 1:1, 3.3:0.43, 3.3:1.4, 10:1.4, 10:4.3, 1.4:1, 4.3:1, 4.3:3, and 13:3.

[0011] In a preferred technical solution of the present invention, the MDM2 inhibitor and the CDK9 inhibitor are administered simultaneously or sequentially.

[0012] In a preferred technical solution of the present invention, the pharmaceutical composition contains an MDM2 inhibitor and a CDK9 inhibitor and a pharmaceutically acceptable carrier.

[0013] In the preferred technical solution of the present invention, the amount or type of the pharmaceutically acceptable carrier in the pharmaceutical composition is determined according to factors such as the physicochemical properties and content of the active ingredient in the composition, the type of preparation, the dissolution and bioavailability of the preparation, and the like.

[0014] In a preferred technical solution of the present invention, the administration method of the drug or pharmaceutical composition is selected from any one of oral administration, injection administration, mucosal administration, and skin administration.

[0015] In the preferred technical solution of the present invention, the pharmaceutical composition of the present invention can be in various dosage forms well known in the art and can be prepared using conventional formulation techniques in the art. The formulation suitable for the present invention is selected from any one of oral formulations, injections, and external preparations.

[0016] In the preferred technical solution of the present invention, the oral preparation is selected from any one of oral liquid preparations, tablets, capsules, granules, syrups, powders, dews, effervescents, suspensions, pills, dripping pills, mixtures, pastes, emulsions, and teas.

[0017] In a preferred technical solution of the present invention, the external preparation is selected from any one of a gel, ointment, patch, cream, unguent, liniment, lotion, suppository, smear, gel, ointment, aerosol, dry powder inhaler, spray, and atomizer.

[0018] In a preferred technical solution of the present invention, the injection is selected from any one of a solution injection, an emulsion injection, a suspension injection, a sterile powder for injection, and a large infusion.

[0019] The pharmaceutically acceptable carriers described herein are commonly used excipients or auxiliary materials well known in the art for preparing the desired formulations, including but not limited to fillers (also known as diluents), lubricants (also known as glidants or anti-adherents), dispersants, wetting agents, binders, disintegrants, pH adjusters, osmotic pressure regulators, pore formers, solubilizers, antioxidants, antibacterial agents, analgesics, suspending agents, emulsifiers, co-emulsifiers, lyoprotectants, flavoring agents, and fragrance agents. Binders include, for example, syrup, gum arabic, gelatin, sorbitol, tragacanth gum, cellulose or its derivatives, gelatin slurry, starch slurry, polyvinyl pyrrolidone, and the like. Preferably, the cellulose derivative is selected from microcrystalline cellulose, sodium carboxymethyl cellulose, ethyl cellulose, methyl cellulose, hydroxypropyl cellulose, and hydroxypropyl methyl cellulose, and preferably, the starch derivative is selected from sodium carboxymethyl starch, sodium starch glycolate, pregelatinized starch, modified starch, hydroxypropyl starch, potato starch, and corn starch, or any one or a combination thereof. Fillers include, for example, lactose, powdered sugar, dextrin, starch or its derivatives, cellulose or its derivatives, inorganic calcium salts, sorbitol, and mannitol. Preferably, the inorganic calcium salt is selected from calcium chloride, calcium sulfate, calcium phosphate, calcium hydrogen phosphate, and precipitated calcium carbonate. Preferably, the cellulose derivative is selected from microcrystalline cellulose, sodium carboxymethyl cellulose, ethyl cellulose, and hydroxypropyl methyl cellulose. Preferably, the starch derivative is selected from sodium carboxymethyl starch, sodium starch glycolate, pregelatinized starch, modified starch, hydroxypropyl starch, potato starch, and corn starch, or a combination thereof. Lubricants include, for example, micropowdered silica gel, magnesium stearate, talc, colloidal silicon dioxide, aluminum hydroxide, boric acid, hydrogenated vegetable oil, polyethylene glycol, and sodium lauryl sulfate. Disintegrants include, for example, starch or its derivatives, cross-linked polyvinyl pyrrolidone, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, and cross-linked sodium carboxymethyl cellulose. Preferably, the starch derivative is selected from sodium carboxymethyl starch, sodium starch glycolate, pregelatinized starch, modified starch, hydroxypropyl starch, potato starch, and corn starch, or a combination thereof. Wetting agents, such as sodium lauryl sulfate, polysorbate (Tween), water, or alcohol. Antioxidants, such as sodium sulfite, sodium bisulfite, sodium metabisulfite, sodium thiosulfate, vitamin C, vitamin E, butylated phenol, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate (PG), tert-butylhydroquinone (TBHQ), etc. Antibacterial agents (fungicides), such as phenol, cresol, chlorobutanol, benzyl alcohol, etc. Analgesics, such as chlorobutanol, benzyl alcohol, lidocaine, procaine, etc. Suspending agents, such as microcrystalline cellulose, sodium carboxymethylcellulose, ethylcellulose, methylcellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, syrup, glycerin, gelatin, gum arabic, tragacanth gum, sodium (potassium) alginate, pectin, etc. Acid-base regulators (pH regulators), such as hydrochloric acid, citric acid, sodium (potassium) hydroxide, sodium (potassium) citrate, sodium (potassium) citrate, sodium (potassium) malate, sodium (potassium) dihydrogen phosphate, disodium (potassium) hydrogen phosphate, etc.Osmotic pressure regulators, such as sodium (potassium) chloride and glucose. Emulsifiers, such as sodium stearate, potassium stearate, triethanolamine stearate, magnesium stearate, calcium stearate, sodium lauryl sulfate, sodium dodecylbenzenesulfonate, polysorbate (Tween), Span, PEG-100, benzyl alcohol, polyvinyl alcohol, gum tragacanth, gum arabic, Pluronic F-68, lecithin, and soy lecithin. Co-emulsifiers, such as n-butanol, ethylene glycol, ethanol, propylene glycol, glycerol, and polyglycerol esters. Lyoprotectants, such as sucrose, lactose, galactose, glucose, trehalose, mannitol, and sorbitol. Solubilizers, such as Tween-80, Pluronic F-68, PEG-100, benzyl alcohol, bile salts, deoxycholates, glycerol, propylene glycol, and polyethylene glycol. Flavoring agents, such as honey, syrup, essences, and sweeteners.

[0020] In a preferred technical solution of the present invention, the pharmaceutical composition of the present invention is prepared with a pharmaceutically acceptable sustained-release preparation carrier or a controlled-release preparation carrier according to the preparation method of sustained-release preparation or controlled-release preparation well known in the art, such as adding a blocker coating or making a matrix-type preparation, or after microencapsulating the drug of the present invention, it is made into a sustained-release preparation or a controlled-release preparation.

[0021] The sustained-release preparation carrier or controlled-release preparation carrier of the present invention includes but is not limited to oily admixtures, hydrophilic colloids, water-insoluble, enteric-soluble, biodegradable inhibitors, etc., wherein the oily admixture is selected from glyceryl monostearate, hydrogenated castor oil, mineral oil, polysiloxane or dimethylsiloxane, etc.; the hydrophilic colloid is selected from methylcellulose (MC), sodium carboxymethylcellulose (CMC-Na), hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), hydroxypropyl methylcellulose (HMPC), polyvinyl pyrrolidone (PVP), gum arabic, tragacanth or carbopol, polyvinyl alcohol (PVA), pectin, alginate, chitosan, xanthan gum, guar gum, guar gum, gelatin, agar, Any one or a combination of galactomannan; the water-insoluble retarder is selected from any one or a combination of ethyl cellulose (EC), cellulose acetate (CA), polyethylene, polypropylene, polysiloxane, ethylene vinyl acetate copolymer (EVA), polymethyl methacrylate; the enteric retarder is selected from cellulose acetate phthalate (CAP), hydroxypropyl methylcellulose phthalate (HMPCP), polyvinyl alcohol phthalate (PVAP), hydroxypropyl methylcellulose acetate succinate (HPMCAS) acrylic resin, etc.; the biodegradable retarder is selected from waxes, fatty acids and their fats, fatty alcohols, etc., such as carnauba wax, stearic acid, glyceryl monostearate, stearyl alcohol, cetyl alcohol, etc.

[0022] Another object of the present invention is to provide use of the pharmaceutical composition of the present invention in preparing a drug for preventing and treating lung cancer.

[0023] In a preferred technical solution of the present invention, the lung cancer is selected from any one of non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, small cell lung cancer, lung adenosquamous carcinoma, and large cell neuroendocrine carcinoma, or a combination thereof.

[0024] Unless otherwise specified, when the present invention refers to the percentage between liquids, the percentage is volume / volume percentage; otherwise, the percentage is weight / weight percentage.

[0025] Compared with the prior art, the present invention has the following beneficial technical effects:

[0026] 1. The present invention scientifically combines and synergistically utilizes anticancer drugs with different mechanisms of action to selectively trigger DNA damage in tumor cells, induce tumor cell apoptosis, safely and effectively inhibit tumor cell growth, reduce the dosage of each drug and its side effects, reduce treatment costs and drug costs, and improve the safety and effectiveness of drugs.

[0027] 2. The preparation method of the present invention has the advantages of simple operation, high yield, better cost, wide applicability to the population, and suitability for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The in vitro inhibition of lung cancer cell growth by the composition of the present invention was studied, including (a) dose-response curves of single drug or combination treatment for 72 hours; (b) Western immunoblot analysis;

[0029] Figure 2 Study on the in vivo inhibitory effect of the composition of the present invention on lung cancer cell growth, including: (a) treatment regimen for mice bearing A549 xenograft tumors and treated with a vector; (b) tumor weights in each group; (c) weight changes in mice in each group; and (d) protein immunoblotting results for each tumor group. DETAILED DESCRIPTION

[0030] The present invention will be described below with reference to Examples, but the present invention is not limited to these Examples.

[0031] The experimental materials used in the examples were all commercially available.

[0032] A549 cells (non-small cell lung cancer cells) were obtained from the American Type Culture Collection (ATCC, VA, USA).

[0033] Test Example 1 Study on the inhibitory effect of the pharmaceutical composition of the present invention on the growth of lung cancer cells

[0034] Idasanutlin and NVP-2 were dissolved in DMSO and added to RPMI-1640 medium to prepare 10 mM drug stock solutions. The drug stock solutions were further diluted with RPMI-1640 medium to prepare drug test solutions at concentrations of 0.00014 μM, 0.00043 μM, 0.0014 μM, 0.0043 μM, 0.013 μM, 0.04 μM, 0.13 μM, 0.4 μM, and 1.2 μM.

[0035] Test method:

[0036] ① A549 cells in the logarithmic growth phase were seeded in a 96-well plate with 3000-4000 cells per well, and the culture plate was pre-cultured in an incubator for 24 hours (at 37° C., 5% CO 2 ).

[0037] ② Replace the culture medium in the well plate and add 100 μl of drug test solution of corresponding concentration to the culture plate, see Table 1 for details.

[0038] ③ Incubate the culture plate in an incubator for 72 hours, add 10 μl of CCK-8 solution to each well, and then incubate the culture plate in an incubator for another 2 hours.

[0039] ④ Measure the absorbance at 450 nm using a CCK8 kit enzyme reader.

[0040] Inhibition%=100-(Signal cmpd -Signal Ave_PC ) / (Signal Ave_VC -Signal Ave_PC )×100.

[0041] See the results Figure 1 (a), (b), (c).

[0042] Table 1

[0043]

[0044] Experimental Example 2 Animal Experimental Study on the Inhibition of Lung Cancer Cells by the Pharmaceutical Composition of the Present Invention

[0045] Idasanutlin was dissolved in DMSO, and water was added to prepare a 1 mg / ml solution; NPV-2 was dissolved in DMSO, and water was added to prepare a 2 mg / ml solution.

[0046] Human lung cancer A549 cells (from ATCC) were routinely cultured in RPMI-1640 medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator. After three passages in vitro, when the cells grew to more than 80% and the fusion rate reached the required amount, the cells were digested and collected. The cells were washed with PBS and counted, and the cell concentration was adjusted to approximately 5×10 7 / mL, place it in a 4mL centrifuge tube and keep it on ice for later use.

[0047] Twenty-eight 6-week-old female nude mice weighing 16-18 g were obtained from Vital River Laboratory Animal Technology (Beijing, China) and housed in a specific pathogen-free animal facility. Human lung cancer A549 tumor cells were inoculated subcutaneously. The mice were placed in a lateral position. The axilla of the forelimb was disinfected with 75% alcohol. A 1 mL syringe was used to draw up 100 μL of the cell suspension, i.e., 5 × 10 6 cells / mouse / 100μL.

[0048] Tumor volume calculation: volume = (longest diameter × shortest diameter2) / 2.

[0049] When the tumor grows to 100 mm 3 Afterwards, the animals were randomly divided into groups of 7 each and fed according to different drug administration forms, namely:

[0050] Model group: Oral administration of the same volume of vehicle (10% DMSO + 90% distilled water) every day;

[0051] Test group 1: Idasanutlin solution was administered orally at a dose of 10 mg / kg (mouse body weight) per day;

[0052] Test group 2: NVP-2 solution was administered orally at a dose of 20 mg / kg (mouse body weight) per day;

[0053] Test group 3: 10 mg / kg (mouse body weight) of Idasanutlin solution and 20 mg / kg (mouse body weight) of NVP-2 solution were orally administered every day.

[0054] Administration was performed by gavage once daily for 30 consecutive days. The first day of administration was defined as experimental day 1. Tumors were measured every three days using a vernier caliper to measure the long diameter (a) and short diameter (b) of the tumors, and changes in tumor volume were recorded. At the end of the experiment, the mice were dissected and the tumors were weighed.

[0055] Data were entered and analyzed using GraphPad Prism 6. Data are expressed as mean ± SEM (standard error of mean) and analyzed using one-way ANOVA. P ≤ 0.05 was considered statistically significant.

[0056] The activity of drug inhibitors was evaluated based on tumor growth inhibition (TGI): TGI (%) = (Vc-Vt) / (Vc-V0)*100, where Vc and Vt correspond to the median volumes of the control and experimental groups, respectively, and V0 is the median volume of the control group at the beginning of the study.

[0057] The tumor was terminated when its maximum diameter was approximately 1.5 cm. The mice were anesthetized before euthanasia to minimize pain, after which the tumors were removed and weighed. The results were analyzed using one-way analysis of variance (ANOVA) using SPSS 13.0 or GraphPad Prism 8.0. Data are reported as mean ± SD, with P < 0.05 as the threshold for significance. Figure 2 .

[0058] Test groups 1 and 2 resulted in moderate tumor growth inhibition (TGI) % values ​​of 33.0% and 47.2%, respectively, in A549 xenograft tumors, while test group 3 significantly reduced tumor size with a TGI % value of 89.2%. The composition of the present invention significantly inhibited tumor growth.

[0059] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention. As long as they do not depart from the spirit of the present invention, they should fall within the scope of protection of the claims of the present invention.

Claims

1. A pharmaceutical composition for preventing and treating lung cancer, comprising an MDM2 inhibitor and a CDK9 inhibitor in a molar ratio of (1-10):1, wherein: The MDM2 inhibitor is Idasanutlin, and the CDK9 inhibitor is NVP-2.

2. The pharmaceutical composition according to claim 1, wherein the composition is composed of Idasanutlin and NVP-2 in a molar ratio of (1-5):

1.

3. The pharmaceutical composition according to claim 1, wherein the molar ratio of Idasanutlin to NVP-2 in the composition is selected from any one of 2.4:1, 10:3.3, 1:1, 3.3:0.43, 3.3:1.4, 10:1.4, 10:4.3, 1.4:1, 4.3:1, 4.3:3, and 13:

3.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the MDM2 inhibitor and the CDK9 inhibitor are administered simultaneously or sequentially. 5 . The pharmaceutical composition according to claim 1 , comprising an MDM2 inhibitor and a CDK9 inhibitor and a pharmaceutically acceptable carrier.

6. The pharmaceutical composition according to any one of claims 1 to 3, wherein the administration method of the pharmaceutical composition is selected from any one of oral administration, injection, mucosal administration, and skin administration.

7. The pharmaceutical composition according to any one of claims 1 to 3, wherein the pharmaceutical composition is in various dosage forms well known in the art and is prepared using conventional formulation techniques in the art, and is preferably selected from any one of an oral formulation, an injection, and a topical formulation. The pharmaceutical composition according to claim 7 , wherein the oral preparation is an oral liquid preparation.

9. The pharmaceutical composition according to claim 7, wherein the oral preparation is selected from any one of tablets, capsules, granules, syrups, powders, dews, effervescents, suspensions, pills, and mixtures.

10. The pharmaceutical composition according to claim 7, wherein the external preparation is selected from any one of a gel, an ointment, a cream, a liniment, a lotion, a suppository, a dry powder inhaler, and an atomizer.

11. The pharmaceutical composition according to claim 7, wherein the injection is selected from any one of solution injection, emulsion injection, suspension injection, and sterile powder for injection.

12. The pharmaceutical composition according to claim 5, wherein the pharmaceutically acceptable carrier is a commonly used excipient or auxiliary material well known in the art for preparing the desired preparation, including fillers, lubricants, dispersants, wetting agents, binders, disintegrants, pH regulators, osmotic pressure regulators, pore formers, solubilizers, antioxidants, suspending agents, emulsifiers, co-emulsifiers, lyoprotectants, flavoring agents, and fragrance agents.

13. The pharmaceutical composition according to claim 5, which is prepared by combining the pharmaceutical composition with a pharmaceutically acceptable sustained-release preparation carrier or a controlled-release preparation carrier according to a method for preparing sustained-release preparations or controlled-release preparations well known in the art to prepare a sustained-release preparation or a controlled-release preparation.

14. Use of the pharmaceutical composition according to any one of claims 1 to 13 in preparing a medicament for preventing and treating lung cancer.

15. The use according to claim 14, wherein the lung cancer is non-small cell lung cancer. The use according to claim 14 , wherein the lung cancer is lung adenocarcinoma.

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