Polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer micelle having osimertinib, etoposide, and docetaxel encapsulated therein and use thereof

Abstract

The present invention relates to a polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (PCL S-PAC-PEG S) micelle in which osimertinib, etoposide, and docetaxel are encapsulated, and used thereof. Specifically, the present invention relates to a pharmaceutical composition for preventing or treating cancer, wherein osimertinib, which is a target therapeutic agent, and etoposide and docetaxel, which are both anticancer chemotherapeutic drugs, are encapsulated in a Soluplus® polymer to form micelles, whereby anticancer activity can be effectively induced through an improvement in the solubility of the poorly soluble drug in water and a synergistic action between the drugs. The pharmaceutical composition for cancer treatment according to the present invention enables effective solubilization of osimertinib, etoposide, and docetaxel by encapsulating the poorly soluble drugs in micelles formed using the PCL-PVAc-PEG (Soluplus®) polymer, and thus can be advantageously used as an intravenous injection. With the advantage of avoiding first-pass metabolism in the liver, unlike oral dosage forms, intravenous injection can reduce unnecessary loss of drugs and also increase bioavailability compared to oral dosage forms, and thus is an effective administration method capable of expecting a high effect with the same amount of drugs. Therefore, development of such a formulation allows effective administration of osimertinib, etoposide, and docetaxel. In addition, the composition of the present invention can effectively treat non-small cell lung cancer having an EGFR-sensitive mutation while preventing the development of anticancer drug resistance, which can be caused by single drug administration, by using a targeted therapeutic agent and chemotherapy in combination. Furthermore, the composition of the present invention employs a biocompatible polymer as a material of the micelle and thus has an advantage of being safe for the human body. Moreover, by combining osimertinib, etoposide, and docetaxel at a specific molar ratio and encapsulating same in Soluplus® micelles, the composition of the present invention has excellent encapsulation efficiency of three kinds of drugs and has high monodispersibility having a consistent particle size of 50-60 nm. Thus, the composition can maintain consistent product quality and has excellent formulation stability.

Description

Polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer micelles loaded with osimertinib, etoposide, and docetaxel and their uses

[0001] The present invention relates to polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (PCL-PVAc-PEG) micelles encapsulating osimertinib, etoposide and docetaxel and their uses, and more particularly, to a micelle encapsulating osimertinib, a targeted therapeutic agent, and etoposide and docetaxel, anticancer chemotherapeutic drugs, in the form of Soluplus. ® ) relates to a pharmaceutical composition for preventing or treating cancer, which can improve the solubility of a poorly soluble drug in water by encapsulating it in a polymer to form micelles, and can effectively induce anticancer activity through synergistic effects between drugs.

[0002] Cancer remains a major global health problem, with a rising mortality rate compared to the past. Lung cancer, in particular, has long maintained one of the highest mortality rates, with the global death toll projected to reach 3 million by 2035. While early detection of lung cancer can improve survival rates through surgical resection, early symptoms are often minimal, making early diagnosis challenging. Furthermore, lung cancer easily metastasizes and has a high risk of recurrence, making surgery difficult and the prognosis poor if not detected early.

[0003] Lung cancer is primarily classified into small cell lung cancer and non-small cell lung cancer (NSCLC). NSCLC accounts for approximately 80-85% of lung cancer cases. Unlike Caucasians, mutations in the epidermal growth factor receptor (EGFR) gene are observed in 30-40% of Asian NSCLC patients, and specific anticancer drugs targeting these mutations are known to be highly effective. However, some EGFR mutations confer drug resistance to cancer cells, a problem that remains a significant challenge.

[0004] In clinical settings, targeted therapies are often used alone rather than in combination with other chemotherapeutic agents due to their lack of selectivity for target patients and the generally antagonistic interactions between targeted therapies and chemotherapy. However, previous studies have described some notable exceptions. The combination of gefitinib and docetaxel (DTX) demonstrated antagonism in cell lines resistant to EGFR-tyrosine kinase inhibitors (EGFR-TKIs), but synergistic effects in cell lines sensitive to EGFR-TKIs. Another study investigated the interactions of gefitinib with various chemotherapeutic agents in several cancer cell lines. In particular, the combination of gefitinib and cisplatin demonstrated antagonism in H1975 cells. However, synergistic effects were observed when gefitinib was combined with Taxol. Furthermore, the phase 3 FLAURA2 study reported that the combination of osimertinib (OSM), cisplatin, and pemetrexed reduced the risk of disease progression or death by 38% compared to monotherapy. These findings highlight the potential benefits of combination therapy.

[0005] Meanwhile, drugs used to treat non-small cell lung cancer generally require administration through solubilizers due to their low water solubility. However, Cremophor ®Commonly used solubilizing agents, such as EL, N,N-Dimethylacetamide (DMA), and ethanol (EtOH), pose a risk of various adverse effects, including vascular toxicity, skin and mucosal inflammation, and cell damage. To address these issues, biocompatible and biodegradable polymers have been proposed to reduce solubilizing agent toxicity and extend drug retention time in the body.

[0006] Against this backdrop, the inventors of the present invention sought to explore a drug combination that enhances the therapeutic efficacy of targeted therapy and chemotherapy in the effective treatment of lung cancer, which has a high incidence of recurrence and metastasis. As a result, the optimal molar ratio was established by combining the targeted therapy osimertinib with the chemotherapeutic agents etoposide and docetaxel, and the optimal polymer was identified that could maintain excellent encapsulation efficiency, high monodispersity, and a small size of 60 nm or less when encapsulating these drugs. In addition, the micelles loaded with osimertinib, etoposide, and docetaxel exhibited excellent formulation stability; excellent cytotoxicity against lung cancer cell lines (NCI-H1975-Luc) and tumor spheroids; slow and sustained drug release; and biocompatibility evaluation revealed a hemolysis rate of 0.51%, which is within a safe range where hemolysis does not occur, demonstrating excellent hemocompatibility. The present invention was completed by demonstrating that the NCI-H1975-Luc cell-transplanted nude mouse xenograft model exhibited excellent anticancer effects and that it could be usefully used in the treatment of lung cancer.

[0007] The purpose of the present invention is to effectively improve the solubility of a poorly soluble drug in water so that it can be used as an intravenous injection formulation (the injection formulation can reduce the disadvantages and side effects of oral administration); to prevent the occurrence of anticancer drug resistance that can be caused by single drug administration by using a combined targeted therapeutic agent and chemotherapy, and to effectively treat non-small cell lung cancer with EGFR sensitive mutations; and to use a biocompatible polymer (Soluplus) that is safe for the human body as a material for micelles. ® ) to overcome the problem of highly toxic organic solvents used to dissolve poorly soluble drugs in the past; and to provide a pharmaceutical composition for preventing or treating cancer, which can slowly and continuously release a drug by encapsulating the drug inside a micelle.

[0008] In order to achieve the above purpose of the present invention,

[0009] The present invention provides a pharmaceutical composition for preventing or treating cancer, comprising, as an active ingredient, polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (PCL-PVAc-PEG) micelles in which osimertinib, etoposide, and docetaxel are loaded.

[0010] In one embodiment of the present invention, osimertinib, etoposide, and docetaxel may be combined in a molar ratio of 4:6:7.

[0011] In one embodiment of the present invention, the micelle may have a particle size of 50 nm to 60 nm.

[0012] In one embodiment of the present invention, the pharmaceutical composition may be a preparation for intravenous injection.

[0013] In one embodiment of the present invention, the pharmaceutical composition may further include one or more components selected from the group consisting of amino acids, sugars, lipids, vitamins, electrolytes, pH adjusters, stabilizers, osmotic pressure adjusters, and solubilizers.

[0014] In one embodiment of the present invention, the cancer may be lung cancer.

[0015] The pharmaceutical composition for cancer treatment according to the present invention comprises a poorly soluble drug in PCL-PVAc-PEG (Soluplus) ® ) By encapsulating osimertinib, etoposide, and docetaxel in micelles using a polymer, they can be effectively solubilized, and thus can be usefully used as intravenous injections. Intravenous injection can avoid the hepatic first-pass effect compared to oral administration formulations, thereby reducing unnecessary drug loss, and can also increase bioavailability compared to oral administration formulations, making it an effective administration method that can expect a high effect with the same amount of drug. Therefore, the development of such formulations enables effective administration of osimertinib, etoposide, and docetaxel. In addition, the composition of the present invention can effectively treat non-small cell lung cancer with EGFR sensitivity mutations while preventing the occurrence of anticancer drug resistance that may be induced by single drug administration by using a targeted therapeutic agent and chemotherapy in combination. In addition, the composition of the present invention has the advantage of being safe for the human body by using a biocompatible polymer as a material for the micelles. In addition, the composition of the present invention has excellent encapsulation efficiency of the three drugs by combining osimertinib, etoposide, and docetaxel at a specific molar ratio and encapsulating them in a soluplus micelle, and has high monodispersity with a consistent particle size of 50 to 60 nm, thereby enabling maintenance of consistent product quality and having the advantage of excellent stability of the formulation.

[0016] Figure 1 is a schematic diagram briefly showing the manufacturing process of OSM / ETP / DTX-loaded soluplus micelles (OSM: osimertinib, ETP: etoposide, DTX: docetaxel).

[0017] Figure 2a shows the polymerization of various polymers (mPEO-b-PCL, Pluronic F127) with OSM / ETP / DTX. ® , Soluplus ® ) shows the particle distribution of micelles manufactured by applying (OSM: osimertinib, ETP: etoposide, DTX: docetaxel, Solu: Soluplus) ® ).

[0018] Figure 2b shows a transmission electron microscope (TEM) image of OSM / ETP / DTX-loaded Soluplus micelles (OSM: osimertinib, ETP: etoposide, DTX: docetaxel).

[0019] Figure 2c shows the results of confirming the change in particle size over time of OSM / ETP / DTX-loaded Soluplus micelles under various temperature conditions (4°C, 25°C, 37°C) (OSM: osimertinib, ETP: etoposide, DTX: docetaxel).

[0020] Figure 2d shows the results of examining the change in polydispersity index (PDI) over time of OSM / ETP / DTX-loaded Soluplus micelles under various temperature conditions (4°C, 25°C, 37°C) (OSM: osimertinib, ETP: etoposide, DTX: docetaxel).

[0021] Figure 3 shows the results of analyzing the cytotoxicity of single and combination formulations of osimertinib (OSM), etoposide (ETP), and docetaxel (DTX) in a lung cancer cell line (NCI-H1975-Luc) after short-term treatment (48 hours) (A: free DTX, B: DTX-loaded Soluplus micelles, C: free OSM / DTX, D: OSM / DTX-loaded Soluplus micelles, E: free ETP / DTX, F: ETP / DTX-loaded Soluplus micelles, G: free OSM / ETP / DTX, H: OSM / ETP / DTX-loaded Soluplus micelles).

[0022] Figure 4a is a morphological image of NCI-H1975-Luc tumor spheroids on days 0 and 35 of culture.

[0023] Figure 4b shows the luminescence intensity of cancer cells imaged using an imaging system (IVIS) after drug treatment with various agents in NCI-H1975-Luc tumor spheroids (OSM: osimertinib, ETP: etoposide, DTX: docetaxel).

[0024] Figure 4c shows the relative total flux over time after treatment of NCI-H1975-Luc tumor spheroids with free DTX and DTX-loaded Soluplus micelles (OSM: osimertinib, ETP: etoposide, DTX: docetaxel).

[0025] Figure 4d shows the relative total flux over time after treatment of NCI-H1975-Luc tumor spheroids with free OSM / DTX and OSM / DTX-loaded Soluplus micelles (OSM: osimertinib, DTX: docetaxel).

[0026] Figure 4e shows the relative total flux over time after treatment of free ETP / DTX and ETP / DTX-loaded Soluplus micelles in NCI-H1975-Luc tumor spheroids (ETP: etoposide, DTX: docetaxel).

[0027] Figure 4f shows the relative total flux over time after treatment of NCI-H1975-Luc tumor spheroids with free OSM / ETP / DTX and OSM / ETP / DTX-loaded Soluplus micelles (OSM: osimertinib, ETP: etoposide, DTX: docetaxel).

[0028] Figure 5 shows the drug release patterns of (A) OSM, (B) ETP, and (C) DTX in OSM / ETP / DTX solution and OSM / ETP / DTX soluplus micelle (OSM: osimertinib, ETP: etoposide, DTX: docetaxel).

[0029] Figures 6a to 6e show the results of examining the change in body weight of ICR mice after administering OSM / ETP / DTX solution and OSM / ETP / DTX Soluplus micelles at various doses three times at one-week intervals (6a: 2 / 3 / 5 mg / kg, 6b: 4 / 6 / 10 mg / kg, 6c: 6 / 9 / 15 mg / kg, 6d: 8 / 12 / 20 mg / kg, 6e: 10 / 15 / 25 mg / kg).

[0030] Figure 7 shows the results of the biocompatibility evaluation of the OSM / ETP / DTX solution and OSM / ETP / DTX Soluplus micelle (OSM: osimertinib, ETP: etoposide, DTX: docetaxel).

[0031] Figure 8 shows the results of the anticancer efficacy evaluation according to the administration of OSM / ETP / DTX solution and OSM / ETP / DTX Soluplus micelle to a nude mouse xenograft model transplanted with NCI-H1975-Luc cells (8A: schedule for the anticancer efficacy evaluation, 8B: IVIS imaging of the luminescence intensity of cancer cells over time after drug administration, 8C: relative total flux over time after drug administration, 8D: volume of collected tumors according to the administration group).

[0032] The present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising as active ingredients a polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (PCL-PVAc-PEG) micelle encapsulating an osimertinib compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof; an etoposide compound represented by the following chemical formula 2 or a pharmaceutically acceptable salt thereof; and docetaxel represented by the following chemical formula 3.

[0033] <Chemical Formula 1>

[0034]

[0035] <Chemical Formula 2>

[0036]

[0037] <Chemical Formula 3>

[0038]

[0039] The compound osimertinib is a targeted anticancer drug used to treat non-small cell lung cancer. It selectively inhibits EGFR tyrosine kinase activity, thereby suppressing cancer cell proliferation. It is effective even in patients resistant to existing treatments. It is used to treat non-small cell lung cancer patients with EGFR gene mutations or those positive for the EGFR T790M mutation.

[0040] Etoposide is a drug capable of forming a four-dimensional complex structure. It binds to DNA and topoisomerase II, preventing DNA strand recombination and inducing damage. This drug is used to treat glioblastoma, lung cancer, sarcoma, non-lymphocytic leukemia, and lymphoma, but is known to cause bone marrow suppression as a side effect.

[0041] The above docetaxel compound is a taxane-based drug used to treat various types of solid tumors such as breast cancer, head and neck cancer, gastric cancer, prostate cancer, and non-small cell lung cancer. Docetaxel is known to induce apoptosis by inhibiting microtubule disassembly and preventing microtubule aggregation, and is also known to block apoptosis by inducing phosphorylation of the oncoprotein bcl-2.

[0042] The osimertinib, etoposide and docetaxel compounds represented by the above chemical formulas 1 to 3 may be commercially available, or may be directly synthesized and used using a conventionally known synthetic method.

[0043] The osimertinib, etoposide and docetaxel compounds represented by the above chemical formulas 1 and 2 can be prepared as pharmaceutically acceptable salts and solvates according to methods conventional in the art.

[0044] Pharmaceutically acceptable salts are useful acid addition salts formed with pharmaceutically acceptable free acids. Acid addition salts are prepared by conventional methods, for example, by dissolving the compound in an excess of an aqueous acid solution and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone, or acetonitrile. Equimolar amounts of the compound and an acid or alcohol (e.g., glycol monomethyl ether) in water can be heated, and the mixture can then be evaporated to dryness, or the precipitated salt can be filtered off with suction.

[0045] At this time, organic acids and inorganic acids can be used as free acids, and inorganic acids such as hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, and tartaric acid can be used, and organic acids such as methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, and hydroiodic acid can be used.

[0046] Additionally, pharmaceutically acceptable metal salts can be prepared using bases. Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving a compound in an excess of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering out the undissolved compound salt, and evaporating and drying the filtrate. Among the metal salts, sodium, potassium, or calcium salts are particularly pharmaceutically suitable. Furthermore, the corresponding silver salts are obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

[0047] Pharmaceutically acceptable salts of the osimertinib, etoposide and docetaxel compounds having structures of the above chemical formulae 1 to 3 of the present invention include acidic or basic salts that may be present in the compounds having the structures of the chemical formulae, unless otherwise indicated. For example, pharmaceutically acceptable salts include sodium, calcium and potassium salts of hydroxyl groups, and other pharmaceutically acceptable salts of amino groups include hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p-toluenesulfonate (tosylate) salts, and can be prepared through a salt preparation method or process known in the art.

[0048] In the present invention, the polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (PCL-PVAc-PEG) micelle is Soluplus, a polymer of polyethylene glycol (PEG), a hydrophilic substance, and polyvinyl caprolactam-polyvinyl acetate (PCL-PVAc), a hydrophobic component. ® It is composed of a combination of and acts as a carrier that can contain drugs or a vehicle that can deliver drugs.

[0049] Above, Soluplus ® Although it is a single molecule, when this substance dissolves in water and exceeds a certain concentration (critical micelle concentration, CMC), it forms spherical micelles. When a micelle is formed, a hydrophilic shell (shall) and a hydrophobic core are formed. The hydrophilic shell is made of PEG, the hydrophilic part of the polymer, and the hydrophobic core is made of PCL-PVAc, the hydrophobic part of the polymer.

[0050] The drug delivery composition of the present invention is solubilized in such a way that the poorly soluble compounds osimertinib, etoposide, and docetaxel are hardly present in the aqueous layer or hydrophilic portion of the micelle, but are distributed in the hydrophobic portion, i.e., the nucleus, thereby being encapsulated within the micelle.

[0051] The present invention utilizes a lyophilization method to prepare micelles. Briefly, tert-butanol is added to osimertinib, etoposide, docetaxel, and an amphiphilic polymer, and the mixture is completely dissolved using ultrasonication. Distilled water is then added, the solution is gently mixed using ultrasonication, and then rapidly cooled in a -70°C freezer for approximately 1 hour before being lyophilized. The lyophilized sample is reconstituted in distilled water, centrifuged, and the supernatant filtered, thereby preparing the micelles of the present invention.

[0052] In one specific embodiment of the present invention, osimertinib, etoposide and docetaxel may be combined in a molar ratio of 4:6:7.

[0053] In the following examples, the combination index (CI) was evaluated when a drug mixture of osimertinib, etoposide, and docetaxel in a molar ratio of 4:6:7 was treated with NCI-H1975-Luc non-small cell lung cancer. As a result, the CI value was 0.17 at the molar ratio, confirming a very strong synergistic effect.

[0054] The micelle of the present invention may have an average particle diameter of 50 nm to 60 nm.

[0055] For reference, nano-drug delivery systems, such as micelles, accumulate in tumors primarily through enhanced permeability and retention. While the gaps between endothelial cells in normal tissues are approximately 6–7 nm, the neovascularization of tumor tissues is incomplete, resulting in wider gaps of approximately 200 nm. Furthermore, tumor tissues lack mature lymphatic tissues, making them unable to remove foreign substances within the tissue. Consequently, nanoparticles released from the bloodstream are retained within the tumor tissue. This therapeutic effect achieved by utilizing drug delivery systems that exploit differences in tumor blood vessel walls is called the enhanced permeability and retention (EPR) effect, and this system can be used to infiltrate or retain drugs in tumors. Nanoparticles smaller than 200 nm can penetrate neovascularization in tumor tissues without entering normal tissues. In addition, when the particle size of the micelle is less than 100 nm, it can avoid phagocytosis and absorption by the reticuloendothelial system (RES), thereby extending the systemic circulation. Therefore, when the particle size of the micelle is sufficiently small to avoid phagocytosis and absorption by the reticuloendothelial system, it can remain in the body for a long time, thereby increasing the bioavailability of the drug to be delivered. Therefore, the micelle of the present invention has a sufficiently small particle size to avoid phagocytosis and absorption by the reticuloendothelial system, thereby allowing it to remain in the body for a long time, thereby increasing the bioavailability of the drug to be delivered (osimertinib, etoposide, and docetaxel).

[0056] The pharmaceutical composition according to the present invention may include a pharmaceutically acceptable carrier in addition to the active ingredient. At this time, the pharmaceutically acceptable carrier is one commonly used in formulations, and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above ingredients, the composition may further include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

[0057] The pharmaceutical composition of the present invention may be in the form of granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable liquids and sustained-release preparations of active compounds, and preferably, liquids for intravenous injection.

[0058] The active ingredients of the pharmaceutical composition of the present invention, osimertinib, etoposide, and docetaxel, can be developed into an intravenous injection using water by dissolving them in the aqueous layer. Intravenous injection can avoid the hepatic first-pass effect compared to oral administration formulations, thereby reducing unnecessary drug loss. In addition, it can increase bioavailability compared to oral administration formulations, making it an effective administration method that can expect high efficacy with the same amount of drug. Therefore, the development of such a formulation enables the effective administration of osimertinib, etoposide, and docetaxel compounds.

[0059] If the pharmaceutical composition of the present invention is an intravenous formulation, it may include pharmaceutical ingredients typically included in intravenous formulation compositions. For example, the intravenous formulation composition may include one or more ingredients selected from the group consisting of amino acids, sugars, lipids, vitamins, electrolytes, pH adjusters, stabilizers, osmotic pressure adjusters, and solubilizers. The specific compositions of amino acid injection solutions, sugar injection solutions, or lipid injection solutions are well known in the art.

[0060] The pharmaceutical composition of the present invention can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method, and the dosage varies depending on the patient's condition and weight, the degree of disease, the drug form, the route of administration, and the time of administration, but can be appropriately selected by those skilled in the art.

[0061] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level may be determined based on factors including the type and severity of the patient's disease, the activity and sensitivity of the drug to the drug, the time of administration, the route of administration and excretion rate, the duration of treatment, concurrently used drugs, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. It is important to take all of the above factors into consideration and administer an amount that achieves the maximum effect with the minimum amount without causing side effects, and this can be easily determined by those skilled in the art.

[0062] Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, condition, weight, absorption rate, inactivation rate and excretion rate of the active ingredient in the body, type of disease, and concomitantly administered drugs.

[0063] Examples of types of cancer that can exhibit the therapeutic effect of the pharmaceutical composition of the present invention include lung cancer, and preferably non-small cell lung cancer with an EGFR sensitive mutation.

[0064] The present invention also provides a method for treating cancer, comprising administering to a subject a pharmaceutical composition comprising polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (PCL-PVAc-PEG) micelles loaded with therapeutically effective amounts of osimertinib, etoposide, and docetaxel.

[0065] As used herein, the term "subject" refers to any animal, including humans, that has or has developed the cancer disease of the present invention. Lung cancer can be treated by administering the pharmaceutical composition of the present invention to a subject.

[0066] Hereinafter, the present invention will be described in more detail through examples. These examples are intended to more specifically illustrate the present invention, and the scope of the present invention is not limited to these examples.

[0067]

[0068] <Example>

[0069]

[0070] 1. Materials and Methods

[0071]

[0072] ingredient

[0073] Soluplus ®, polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (PCL-PVAc-PEG) was obtained from BASF (Ludwigshafen, Rhineland-Palatinate, Germany). mPEG-b-PCL (MW: 2,000:2,000 Da) was purchased from PolySciTech® (West Lafayette, IN, USA). Pluronic® F127, dimethyl sulfoxide (DMSO), formalin, DMA, Cremophor EL, MTT, and tert-butyl alcohol were purchased from Sigma-Aldrich (St. Louis, MO, USA). D-luciferin potassium salt was purchased from GoldBio (St. Louis, MO, USA). Matrigel, ultra-low attachment (ULA) 24-well plates, ULA 100-pi plates, 96-well plates (transparent, black), and 100-pi plates were purchased from Corning, Inc. (Corning, NY, USA). Etoposide (hereinafter abbreviated as 'ETP') was purchased from TCI Chemicals (Chuo-ku, Tokyo, Japan). Osimertinib (hereinafter abbreviated as 'OSM') and docetaxel (hereinafter abbreviated as 'DTX') were purchased from LC Laboratories® (Woburn, MA, USA). Acetonitrile (ACN), distilled water (DW), and methanol were purchased from Honeywell Burdick & Jackson (Muskegon, MI, USA). Phosphate-buffered saline (PBS; pH 7.4) and Spectra / Por® Dialytic Membrane Biotech CE Tubing (MWCO: 20 kD) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Sterile DW was purchased from JW Pharmaceutical Corporation (Gwacheon, Gyeonggi-do, Korea). 10×PBS (pH 7.4) was purchased from Tech & Innovation (Chuncheon, Gangwon-do, Korea), and formic acid was purchased from Dongyang Chemical Industry (Jung-gu, Seoul, Korea).

[0074]

[0075] cell culture

[0076] Non-small cell lung cancer cell line NCI-H1975-Luc cells (JCRB cell band, Osaka, Japan) were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution. Cells were cultured at 37°C under 5% CO2 conditions. When NCI-H1975-Luc cells overgrew, the T75 flask with attached cells was washed with Dulbecco's phosphate-buffered saline (DPBS) and the cells were detached using trypsin. The cells were then transferred to another T75 flask and cultured in RPMI 1640 medium. All materials required for cell culture were purchased from Corning, Inc.

[0077]

[0078] High-performance liquid chromatography (HPLC) analysis

[0079] The concentrations of OSM, ETP, and DTX in the samples were measured using a Waters high-performance liquid chromatography (HPLC) system (Milford, Massachusetts, USA) equipped with a 2695 separation module and a 2996 photodiode array. A Fortis C18 chromatography column (5 μm, 4.6 × 250 mm; Fortis Technologies Ltd., Cheshire, UK) was used and maintained at 30°C during the analysis. All samples were eluted using an isocratic mode with a mobile phase consisting of acetonitrile / water (70:30, v / v). The sample injection volume was 10 μL, and the flow rate was 1.0 mL / min in the mobile phase. Osimertinib, etoposide, and docetaxel were detected at wavelengths of 271, 284, and 229 nm, respectively. The retention times of OSM, ETP, and DTX were 1.84, 2.66, and 4.79 min, respectively. The concentration of each drug was quantified using a calibration curve generated from the peak area.

[0080]

[0081] Combination index (CI) analysis

[0082] To assess drug-drug interactions, a combination index (CI) analysis was performed. CI values ​​were calculated using the equation (Equation 1).

[0083] (Formula 1)

[0084] Combination index (CI) = (D)1 / (D x )1+(D)2 / (D x )2+(D)3 / (D x )3

[0085] Here, (D)1, (D)2, (D)3 are the IC of each combination drug considering the molar fraction. 50 Represents the value, (D x )1, (D x )2, (D x )3 is the IC of individual drugs50 It represents the value. A CI value >1 indicates an antagonistic effect, a CI = 1 indicates an additive effect, and a CI value <1 indicates a synergistic effect. Depending on the CI, the synergistic effect can be further classified as follows: CI <0.1 indicates very strong synergy, 0.1-0.3 indicates strong synergy, 0.3-0.7 indicates synergy, 0.7-0.85 indicates moderate synergy, 0.85-0.9 indicates weak synergy, and 0.9-1.1 indicates almost additive effect.

[0086]

[0087] Preparation of OSM / ETP / DTX solutions

[0088] To prepare an OSM / ETP / DTX solution, a solvent was prepared by combining Cremophor EL®, DMA (dimethylacetamide), EtOH, and PBS in a ratio of 1:1:1:2 (v / v / v / v). The OSM / ETP / DTX solution was prepared by adding OSM, ETP, and DTX to the solvent at concentrations of 2 mg / mL, 3 mg / mL, and 5 mg / mL, respectively.

[0089]

[0090] Manufacturing of OSM free drugs, OSM / DTX free drugs, ETP / DTX free drugs, and OSM / ETP / DTX free drugs

[0091] OSM free drug was prepared by dissolving 2 mg of OSM in 100 μL DMSO and then diluting 1000-fold with RPMI. In the same manner as OSM free drug, OSM / DTX free drug was prepared by dissolving 2 mg of OSM and 5 mg of DTX, ETP / DTX free drug was prepared by dissolving 3 mg of ETP and 5 mg of DTX, and OSM / ETP / DTX free drug was prepared by dissolving 2 mg of OSM, 3 mg of ETP, and 5 mg of DTX in 100 μL DMSO and then diluting 1000-fold with RPMI medium.

[0092]

[0093] Preparation of OSM / ETP / DTX-loaded micelles

[0094] OSM / ETP / DTX-loaded micelles were prepared using a lyophilization method. Considering the pharmacodynamic capacity, 2 mg of OSM, 3 mg of ETP, 5 mg of DTX, and 100 mg of amphiphilic polymer were used. Next, 1 mL of tert-butanol was added at 60°C, and the mixture was completely dissolved using sonication. Next, 1 mL of DW was added at 60°C, and the solution was gently mixed using sonication to maintain clarity. The sample was then rapidly cooled in a -70°C freezer (Deep Freezer DF 8517; IlShinBioBase, Dongducheon, Korea) for approximately 1 h. The cooled sample was placed in a freeze dryer (Advantage Pro; SP Scientific, Warminster, PA, USA) and dried for 24 h. After drying, the sample was reconstituted in 1 mL of DW and centrifuged at 16,600 × g for 5 min at 4°C (Hanil Science, Gimpo, Korea). The supernatant was filtered through a 0.2 μm filter (Corning Inc.) to obtain micelles loaded with OSM / ETP / DTX.

[0095]

[0096] Physicochemical properties of micelles

[0097] The physicochemical properties of micelles were evaluated using a Litesizer 500 instrument (Anton Paar, Graz, Austria) and HPLC. The sizing angle was automatically selected between side scattering (90°) and back scattering (175°). The encapsulation efficiency (EE, %) and drug loading capacity (DL, %) of the micelles were quantified using HPLC based on the formulas (Eq. 2) and (Eq. 3).

[0098] (Formula 2)

[0099] DL% = (Weight of drug in micelle / Total weight of supplied drug and polymer) × 100

[0100] (Formula 3)

[0101] EE% = (Weight of drug in micelles / Weight of drug supplied) × 100

[0102] The results of each sample analysis are presented as the mean ± standard deviation (SD) of three individual experiments.

[0103]

[0104] Imaging of micelles using transmission electron microscopy

[0105] Images of micelles were obtained using a transmission electron microscope (TEM) (JEM-2100 Plus, JEOL, Tokyo, Japan). In the sample preparation procedure for TEM measurements, micelle suspensions diluted 5-fold with purified water (DW) were dropped onto 200-mesh formvar-coated copper grids and dried in a drying oven at 60°C for 72 h. Finally, micelles loaded with osimertinib / etoposide / docetaxel were measured using a TEM operating at 200 kV.

[0106]

[0107] Stability assessment

[0108] The stability test of micelles was performed for two weeks at 4°C (refrigerated), 25°C (room temperature), and 37°C (incubator). Twenty-μL ​​samples were collected at intervals of 0, 1, 2, 5, 7, 14, 17, 20, 24, 27, 30, and 60 days and diluted 50-fold with distilled water. The average particle size and polydispersity index (PDI) were measured using DLS. The stability of micelles stored at 25°C for five months was analyzed. Data from three independent experiments were plotted using SigmaPlot software (version 10.0, Systat Software, San Jose, CA, USA).

[0109]

[0110] In vitro cytotoxicity evaluation

[0111] Cytotoxicity is a biological marker that detects drug-induced cell death. In this experiment, cytotoxicity was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

[0112] In detail, NCI-H1975-Luc cells were seeded at 5 × 10 in a clear 96-well plate. 3 Cells were seeded at a density of 10 cells / well and cultured at 5% CO2 and 37°C for 24 h. The culture medium was removed, and drug treatment was performed using free DTX, free OSM / DTX, free ETP / DTX, free OSM / ETP / DTX, DTX-loaded Soluplus micelles, ETP / DTX-loaded Soluplus micelles, and OSM / ETP / DTX-loaded Soluplus micelles. The free drug was dissolved in DMSO and diluted more than 1000-fold with RPMI 1640 medium, while the micelles were diluted more than 10-fold. After 48 h of drug exposure, the drug was removed, and each well was treated with 100 μL of MTT reagent (0.5 mg / mL) and cultured for 4 h. The MTT reagent was replaced with 100 μL of DMSO. The plate was shaken at 200 rpm for 10 min on an orbital shaker (NB-101S; N-BIOTEK, Bucheon, Korea). Cell viability was assessed by measuring absorbance at 540 nm using a microplate reader (Spectra Max ID3; Molecular Devices, San Jose, CA, USA). This experiment was performed six times independently, and the results were analyzed using GraphPad Prism version 8 (GraphPad Software, La Jolla, CA, USA).

[0113]

[0114] Manufacturing of NCI-H1975-Luc tumor spheroids

[0115] NCI-H1975-Luc tumor spheroids were created by mixing Matrigel with cancer cells to promote 3D cell culture and maintain natural cell morphology. First, 10 μL of Matrigel was dispensed into a ULA 100-pi plate, followed by 4 μL of cancer cell suspension (1.2 × 10 5 (cells / μL) was infiltrated into Matrigel. Then, 25 mL of RPMI 1640 medium was added and culture was maintained at 37°C, 5% CO2. After 24 h, the tumor spheroids were continued to be cultured and shaken at 50 rpm in an orbital shaker.

[0116]

[0117] Cytotoxicity evaluation using tumor spheroids

[0118] A stock solution of D-luciferin (30 mg / mL) prepared in DW was diluted 200-fold in RPMI 1640 medium. Then, 100 μL of the diluted D-luciferin solution was added to each well of a black 96-well plate, followed by the addition of NCI-H1975-Luc tumor spheroids cultured for 5 weeks. The inventive images were quantified using IVIS (VISQUE InVivo Smart-LF; VIEWORKS, Anyang, Korea). Then, the drug was administered to each well at a concentration of 10 μM for each experimental group of free DTX, free OSM / DTX, free ETP / DTX, free OSM / ETP / DTX, DTX-loaded Soluplus micelles, ETP / DTX-loaded Soluplus micelles, and OSM / ETP / DTX-loaded Soluplus micelles. The medium was replaced on days 1 and 2, and luminescence imaging was performed on days 2 and 7 to measure the luminescence status of the cells. This experiment was performed five times independently, and the results were analyzed using GraphPad Prism version 8.

[0119]

[0120] In Vitro Drug Release Evaluation

[0121] The in vitro drug release behavior from micelles was evaluated over 168 hours by dialysis using phosphate buffered saline (PBS, pH 7.4) as the release medium.

[0122] Specifically, the OSM / ETP / DTX solution and the OSM / ETP / DTX-loaded Soluplus micelle samples were each inserted into a dialysis membrane bag (MWCO 20 kDa). The dialysis membrane bag was tied and placed in 2.0 L of PBS release medium on a hot plate stirrer at 37°C. The PBS release medium was maintained at 37°C and continuously stirred using a magnetic bar at 200 rpm, and replaced with fresh medium at 8, 24, 72, 120, and 168 h. At each sampling time point (0, 2, 4, 6, 8, 24, 48, 72, 120, 168 h), 20 μL of each sample was collected, diluted 10-fold with acetonitrile, and then analyzed for drug concentration using HPLC. This experiment was performed three times independently, and the results were graphed using Sigma Plot version 10.0.

[0123]

[0124] In vitro blood compatibility evaluation

[0125] All animal procedures were approved by the Animal Ethics Committee of Chungbuk National University (Approval Number: CBNUA-2153-23-01, Approval Date: July 17, 2023). The hemolytic effects of OSM / ETP / DTX solutions (2 / 3 / 5 mg / kg) and OSM / ETP / DTX-loaded Soluplus micelles (2 / 3 / 5 mg / kg) were evaluated using male Sprague-Dawley rats (Orient Bio, Seongnam, Korea). Blood was extracted from rats via the retroorbital plexus, centrifuged at 2,000 rpm for 10 min to separate erythrocytes, and the erythrocytes were diluted 10-fold in PBS. 200 μL of the diluted erythrocyte suspension was added to each 800 μL sample and incubated at 37°C and 50 rpm for 2 h. Subsequently, the supernatant was collected by centrifugation at 2500 rpm for 5 minutes, and the absorbance was measured at 540 nm using a microplate reader. The hemolysis rate was calculated using the formula (Equation 4).

[0126] (Formula 4)

[0127] Hemolysis rate (%) = [(sample absorbance - negative control absorbance) / (positive control absorbance - negative control absorbance)] × 100

[0128] The negative control group used PBS, and the positive control group used sterile distilled water. Samples were prepared according to the ratio of the administered dose to the total blood volume of the rats. This experiment was performed three times independently, and the results were plotted using Sigma Plot version 10.0.

[0129]

[0130] In Vivo Toxicity Evaluation

[0131] Before evaluating the anticancer effect, a toxicity assessment was designed to determine the appropriate dose and administration period. Toxicity was evaluated using 6-week-old male ICR outbred mice (Orient Bio). Mice were divided into control (wild type) and various groups receiving OSM / ETP / DTX solutions or OSM / ETP / DTX-loaded Soluplus micelles at various concentrations (2 / 3 / 5, 4 / 6 / 10, 6 / 9 / 15, 8 / 12 / 20, 10 / 15 / 25 mg / kg). Mice were weighed at designated intervals (days 0, 2, 5, 7, 9, 12, 14, 16, 19, and 21), and drugs were administered via the tail vein on days 1, 8, and 15. Euthanasia was defined as a 20% weight loss compared to the pre-treatment body weight, a rapid 10-15% weight loss, or abnormal behavior. When these symptoms were observed, euthanasia was performed using CO2.

[0132]

[0133] Evaluation of anticancer efficacy using a nude mouse xenograft model

[0134] Six-week-old male BALB / c nude mice were obtained from Orient Bio. NCI-H1975-Luc cell pellets were suspended in DPBS and 7 × 10 4cells / μL. Subsequently, a mixture of 100 μL of Matrigel and 100 μL of cell suspension was injected subcutaneously into mice. Two weeks after tumor cell implantation, the mice were divided into control and treatment groups, including OSM / ETP / DTX solution (2 / 3 / 5 mg / kg) and OSM / ETP / DTX-loaded Soluplus micelles (2 / 3 / 5 mg / kg). Bioluminescence intensity was measured using IVIS in randomly selected mice with saturation signals, and drugs were administered on days 0, 7, and 14. D-luciferin solution was prepared to obtain bioluminescence images before and after drug treatment. D-luciferin potassium salt (15 mg) was dissolved in 1 mL of DPBS and filtered through a 0.2 μm syringe filter. Filtered D-luciferin solution was injected intraperitoneally at 10 mg / kg on days 0, 3, 7, 10, 14, 17, and 21, and bioluminescence images were obtained using an IVIS system 15 minutes later. The acquired images were CleVue TM Bioluminescence signal intensity was measured and quantified using software (VIEWORKS). Tumor Growth Inhibition (TGI) (%) was calculated using the formula (Equation 5). At the end of the experiment, mice were euthanized using CO2, and tumor volume was calculated using the chemical formula (Equation 6).

[0135] (Formula 5)

[0136] TGI (%) = [(B - A) / B] × 100

[0137] Here, A represents the relative total flux of the treatment group after drug administration. B represents the average bioluminescence signal intensity of the control group (before drug administration). This calculation allowed for the evaluation of the TGI ratio after drug treatment.

[0138] Meanwhile, the total flux is the area value and the fluorescence intensity value [unit: radiance (p / sec / cm2 / sr)] is the product of [ / sr].

[0139] (Formula 6)

[0140] Tumor volume (mm) 3 ) = (major axis × minor axis) 2 ) / 2

[0141]

[0142] H&E staining

[0143] After evaluating the anticancer efficacy, H&E staining was performed to analyze the histological structure. Tissues from the liver, spleen, heart, lung, kidney, and tumor were collected and fixed in formalin. The tissues were dehydrated and paraffin blocks were prepared. Tissue sections obtained from the paraffin blocks were mounted on slides and stained with H&E. Images of the tissue sections were acquired using a slide scanner (Pannoramic SCAN II, 3DHISTECH, Budapest, Hungary) and viewed using CaseViewer 2.4 software (3DHISTECH).

[0144]

[0145] Statistical analysis

[0146] All data were analyzed using t-tests in GraphPad Prism version 8.0. A p-value < 0.05 was considered statistically significant.

[0147]

[0148] 2. Results

[0149]

[0150] Evaluation of the synergistic interaction between OSM, ETP, and DTX

[0151] To develop a formulation that can effectively treat cancer cells by combining drugs used in actual clinical practice, we used OSM, ETP, and DTX to confirm the synergistic effect among the drugs. When the drug mixture of OSM, ETP, and DTX in a molar ratio of 4:6:7 was treated with NCI-H1975-Luc non-small cell lung cancer, the combination index (CI) was evaluated. A CI less than 1 indicates synergism, and the CI of this drug combination was 0.17, confirming a very strong synergistic effect.

[0152]

[0153] IC of single drugs of OSM, ETP, and DTX and their combination drugs 50 Values ​​and combination indices (n=6) Drug (molar ratio) OSM / ETP / DTX (4:6:7) OSMETPDTXIC 50 (μM)OSM0.020.73--ETP0.03-25.1-DTX0.03--0.22Combination index (CI)0.17---OSM: Osimertinib, ETP: Etoposide, DTX: Docetaxel

[0154]

[0155] Physicochemical characteristics of OSM / ETP / DTX-loaded micelles

[0156] To find the polymer that best encapsulates OSM / ETP / DTX, mPEG-b-PCL and Pluronic ⓡ F-127, Soluplus ⓡ Various physicochemical characteristics were compared and analyzed using the polymer. The manufactured micelles used 100 mg of polymer, 2 mg of osimertinib, 3 mg of etoposide, and 5 mg of docetaxel.

[0157] The results are detailed in Table 2 and Figure 2 below.

[0158]

[0159] Physicochemical properties of OSM / ETP / DTX-loaded micelles according to polymer type (n=3)Polymer typeOSM encapsulation efficiency (%)ETP encapsulation efficiency (%)DTX encapsulation efficiency (%)OSM drug loading rate (%)ETP drug loading rate (%)DTX drug loading rate (%)Particle size (nm)Polydispersity index (PDI)Zeta potential (mV)mPEG-b-PCL80.5±0.4978.8±0.4873.8±1.081.58±0.012.30±0.013.51±0.0525.5±0.930.25±0.0110.7±5.60Pluronic® F-12772.0±1.9073.1±1.7961.0±1.461.41± 0.042.13± 0.042.13± 0.05988± 13600.31± 0.0813.0± 3.20Soluplus®70.6± 1.9569.6± 1.6664.8± 2.111.38± 0.042.03± 0.053.09± 0.1052.3± 0.630.12± 0.0221.9± 4.30OSM: Osimertinib, ETP: Etoposide, DTX: Docetaxel

[0160]

[0161] All of the manufactured micelles exhibited excellent water solubility, which facilitates clinical drug administration. Analysis of the physicochemical properties of the manufactured micelles revealed that mPEG-b-PCL micelles exhibited superior encapsulation efficiency compared to other polymeric micelles. However, since particle uniformity was considered a key factor in the present invention, Soluplus, which has high monodispersity, was selected over mPEG-b-PCL, which has excellent encapsulation efficiency. ⓡ was selected as the final formulation (see Figure 2a). Higher particle uniformity improves reproducibility, maintains consistent product quality, and enhances stability. Excellent formulation stability offers several benefits, including enhanced patient trust, improved storage convenience, improved safety, sustained efficacy, and consistent dosing. These benefits enable patients to use the drug more safely and effectively.

[0162] The morphology of the selected OSM / ETP / DTX-loaded Soluplus micelles was observed by transmission electron microscopy and confirmed to be spherical (see Fig. 2b). DLS and TEM analyses confirmed that the particle size of the OSM / ETP / DTX-loaded Soluplus micelles was approximately 50 nm. These results indicate that micelles with a particle size of less than 100 nm have very low absorption by the reticuloendothelial system and can accumulate in tumors by enhancing the EPR (Permeability and Retention) effect.

[0163] For reference, the gaps between endothelial cells in normal tissues are approximately 6–7 nm, whereas the neovascularization of tumor tissues is incomplete, with gaps as wide as 200 nm. Therefore, nanoparticles smaller than 200 nm can penetrate neovascularization of tumor tissues without entering normal tissues. Furthermore, tumor tissues lack mature lymphatic tissues, making them unable to remove foreign substances within the tissue. Consequently, nanoparticles released from the bloodstream are retained within the tumor tissues. This therapeutic effect achieved by utilizing a drug delivery system that exploits the differences in tumor blood vessel walls is called the enhanced permeability and retention (EPR) effect, and this system can be used to penetrate or retain drugs in cancer. Furthermore, micelle particle sizes smaller than 100 nm can avoid phagocytosis and uptake by the reticuloendothelial system (RES), thereby extending systemic circulation. Therefore, if the particle size of the micelle is sufficiently small to avoid phagocytosis and absorption by the reticuloendothelial system, it can remain in the body for a long time, thereby increasing the bioavailability of the drug to be delivered.

[0164]

[0165] Stability Evaluation of OSM / ETP / DTX-Loaded Solublus Micelles

[0166] The stability of OSM / ETP / DTX-loaded Soluplus micelles was evaluated by measuring the average particle size and polydispersity index (PDI) at various temperature conditions (4, 25, and 37°C) over a period of 2 months (see Figures 2c and 2d). The average particle size was stable at both 4°C and 25°C and remained below 60 nm for 60 days. However, at 37°C, the average particle size remained below 60 nm for the first 30 days, but then increased continuously to 72 nm after 60 days. Furthermore, micelles stored at 25°C for 150 days maintained a particle size of 60 nm (see Figures 2e and 2f).

[0167] The long-term stability of OSM / ETP / DTX-loaded Soluplus micelles was demonstrated through the above experiments. A notable aspect is the ability of Soluplus micelles to consistently maintain a small size of less than 60 nm, suggesting their potential for effective treatment against cancer cells. First, particle sizes in the 10-60 nm range are known to exhibit higher cellular uptake compared to particles of other sizes, and the micelles of the present invention satisfy this requirement. Second, micelles can accumulate in cancer cells over a long period of time through the EPR effect. These properties hold great potential for targeted therapeutic strategies that leverage the permeability of cancer cells.

[0168]

[0169] In vitro cytotoxicity evaluation

[0170] To evaluate the cytotoxicity of the OSM / ETP / DTX-loaded soluplus micelles used in the present invention, two methods were used: MTT assay and tumor spheroid assay using NCI-H1975-Luc cells.

[0171] Both methods were performed based on DTX, which has a strong cell killing effect and a high molar ratio, and the physicochemical properties of the Soluplus micelle formulation used for cytotoxicity evaluation are detailed in Table 3 below.

[0172] First, IC of free DTX, DTX-loaded soluplus micelles, free OSM / DTX, OSM / DTX-loaded soluplus micelles, free ETP / DTX, ETP / DTX-loaded soluplus micelles, free OSM / ETP / DTX, and OSM / ETP / DTX-loaded soluplus micelles using MTT assay. 50 The values ​​are shown in Fig. 3. In NCI-H1975-Luc cells, the IC of free DTX and DTX-loaded Soluplus micelles 50 There was a statistically significant difference in the values ​​(p < 0.05), and no significant difference was observed between the free and Solublus micelles of other identical drugs.

[0173]

[0174] Physicochemical properties of Soluplus micelle formulations used in cytotoxicity evaluation (Soluplus ⓡ100 mg fixed, n=3) Formulation OSM encapsulation efficiency (%) ETP encapsulation efficiency (%) DTX encapsulation efficiency (%) OSM drug loading rate (%) ETP drug loading rate (%) DTX drug loading rate (%) Particle size (nm) Polydispersity index (PDI) Zeta potential (mV) Vehicle------60.8± 0.820.09± 0.03-5.10± 0.30 DTX micelles (DTX, 5 mg)--76.7± 3.34--3.65± 0.1655.6± 0.370.06± 0.03-5.50± 1.90 OSM / DTX micelles (OSM, 2 mg; DTX, 5 mg)76.5± 6.02-72.6± 6.711.50± 0.12-3.46± 0.3250.6± 0.740.10± 0.0222.1± 3.70ETP / DTX micelles (ETP, 3 mg; DTX, 5 mg)-78.1± 4.1376.2± 4.74-2.28± 0.123.63± 0.2357.6± 1.180.07± 0.03-7.10± 2.00OSM / ETP / DTX micelles (OSM, 2 mg; ETP, 3 mg; DTX, 5 mg)71.2± 2.5971.0± 2.0671.5± 2.431.40± 0.052.07± 0.063.41± 0.1252.8± 0.410.13± 0.0623.4± 3.00OSM: Osimertinib, ETP: Etoposide, DTX: Docetaxel

[0175]

[0176] Another cytotoxicity assessment was performed using tumor spheroids cultured for 5 weeks (see Fig. 4a). The tumor spheroids were spherical in shape, and the luminescence intensity of cancer cells was imaged using an in vivo imaging system (IVIS) after treatment with various formulations containing 10 μM of the total drug, respectively (see Fig. 4b). On day 7, there was no statistically significant difference in the relative total flux between the same drug treatment groups (see Figs. 4c-4f). However, compared to the control group, free OSM / DTX, free OSM / ETP / DTX, OSM / DTX-loaded Soluplus micelles, and OSM / ETP / DTX-loaded Soluplus micelles all showed statistically significant differences (p < 0.0001). In addition, free ETP / DTX showed a statistically significant difference (p < 0.01) compared to the control group.

[0177] Cytotoxicity assessments using MTT and tumor spheroids yielded discrepant results, likely due to differences in the tumor environment. The MTT assay was conducted on 2D (two-dimensional) cancer cells, while the tumor spheroid assay used 3D (three-dimensional) cancer cells. 3D cancer cells better reflect the histological, functional, and microenvironmental characteristics of in vivo human tumor tissues than 2D cells. Furthermore, differences in drug treatment methods, which produce different results, appear to be a major factor in the discrepancies. While the MTT assay was conducted at a constant drug concentration, the tumor spheroid assay removed the drug 24 hours after treatment and replaced it with fresh medium, mimicking the excretion process in vivo. Although this method is not a perfect biomimetic, it provides an opportunity to more realistically assess the apoptotic effects of drugs absorbed by cancer cells.

[0178] In the cytotoxicity evaluation using tumor spheroids, the OSM / DTX and OSM / ETP / DTX treatment groups showed similar results in terms of efficacy. However, considering that the total drug concentration in all groups was 10 μM, the amount of osimertinib was significantly reduced in the OSM / ETP / DTX treatment group compared to the OSM / DTX treatment group. Considering the molar ratio of OSM, ETP, and DTX, the amount of osimertinib in the OSM / DTX treatment was approximately 1.55 times higher than that in the OSM / ETP / DTX treatment. Therefore, considering the high cost of OSM, the OSM / ETP / DTX formulation was judged to be advantageous in terms of cost-effectiveness.

[0179]

[0180] In Vitro Drug Release Assay

[0181] The drug release patterns of the OSM / ETP / DTX solution and the OSM / ETP / DTX-loaded Soluplus micelles are detailed in Fig. 5. The OSM / ETP / DTX-loaded Soluplus micelles released 44.5% of OSM, 51.1% of ETP, and 54.2% of DTX over 7 days. For the OSM / ETP / DTX solution, OSM exceeded 51.9% after 8 hours, and a total of 87.1% was released over 7 days. ETP exceeded 56.5% after 6 hours, and 99.3% was released over 7 days. DTX was released 53.3% after 8 hours, and 91.7% on day 7. A statistically significant difference was observed between the same drugs in the solution and micelles on day 7. The parameter values ​​for drug release are shown in Table 4 below.

[0182]

[0183] Rate constant (k) and release index (n) for each drug release of OSM / ETP / DTX solution and OSM / ETP / DTX-loaded Soluplus micelles. Formulation Rate constant (k) Release index (n) OSM / ETP / DTX solution OSM 3 2.3 ± 0.96 0.19 ± 0.01 ETP 4 2.0 ± 2.39 0.17 ± 0.01 DTX 3 2.4 ± 0.96 0.20 ± 0.01 OSM / ETP / DTX Soluplus ⓡ MicellesOSM10.3 ± 0.840.28 ± 0.02ETP10.8 ± 0.970.31 ± 0.02DTX10.7 ± 0.840.31 ± 0.02OSM: Osimertinib, ETP: Etoposide, DTX: Docetaxel

[0184]

[0185] In the OSM / ETP / DTX solution, the rate constant (k) of ETP was higher than those of OSM and DTX. However, in the OSM / ETP / DTX-loaded Soluplus micelles, the k values ​​of OSM, ETP, and DTX were similar. This indicates that Soluplus micelles can release all drugs continuously at similar rates, unlike solutions where drugs are released at different rates. Both the solution and Soluplus micelles followed Fick's law of diffusion, and the higher release index (n) in Soluplus micelles than in solutions indicates more complex interactions. This result suggests that drug release may be slower due to drug-polymer interactions and polymer degradation.

[0186]

[0187] In Vivo Toxicity Evaluation

[0188] The results of the toxicity evaluation using ICR mice are shown in Figure 6. After administering three doses at one-week intervals, the body weight changes and survival rates of the mice were observed. There were no cases of weight loss greater than 20% compared to before administration, rapid weight loss of 10-15%, or abnormal behavior. However, one mouse died on day 8 in the OSM / ETP / DTX solution (10 / 15 / 25 mg / kg) administration group. No cases of weight loss or death as mentioned above were observed in the other test groups. When comparing the body weight gain rate after 21 days with the control group, there was no statistically significant difference in the OSM / ETP / DTX-loaded Soluplus micelle (2 / 3 / 5 mg / kg) administration group, but significant differences were observed in the other groups. The groups with statistically significant differences are shown in Figure 6. OSM / ETP / DTX-loaded Soluplus micelles (2 / 3 / 5 mg / kg) did not exhibit abnormal behavior or rapid weight loss, satisfying the toxicity guidelines suggested by Charmaine et al. Based on these findings, OSM / ETP / DTX Soluplus micelles (2 / 3 / 5 mg / kg) were selected as the final formulation.

[0189]

[0190] In vitro blood compatibility evaluation

[0191] Prior to the evaluation of anticancer efficacy, a biohemorrhage compatibility evaluation was conducted according to the administered doses of 2 / 3 / 5 mg / kg. As a result, as shown in Fig. 7, the OSM / ETP / DTX-loaded Soluplus micelles showed a hemolysis rate of 0.51%, whereas the OSM / ETP / DTX solution showed a hemolysis rate of 4.72%, confirming a statistically significant difference between the two groups (p < 0.001). Generally, nanoparticles suitable for biomedical applications are known to be hemocompatible when the hemolysis rate is less than 5%. The hemolysis rate of 0.51% of the OSM / ETP / DTX-loaded Soluplus micelles satisfies this criterion and is evaluated to exhibit excellent hemocompatibility as it falls within a safe range where almost no hemolysis occurs.

[0192]

[0193] Evaluation of anticancer efficacy using a nude mouse xenograft model

[0194] Using a nude mouse xenograft model transplanted with NCI-H1975-Luc (Luciferase-labeled human NCI-H1975 cells), the anticancer effect was evaluated according to the schedule specified in Figure 8A.

[0195] The intensity changes of the luminescence signal were recorded as images at 7-day intervals using IVIS (see Fig. 8B), and the change rate of total flux over 3 weeks based on the total flux on day 0 was graphically represented (see Fig. 8C). Comparative analysis of the relative total flux on day 21 showed that there was no statistically significant difference between the model group and the OSM / ETP / DTX solution (2 / 3 / 5 mg / kg) group, but the OSM / ETP / DTX-loaded Soluplus micelles (2 / 3 / 5 mg / kg) group showed a significant difference (p < 0.001). In addition, there was a statistically significant difference between the OSM / ETP / DTX solution group and the OSM / ETP / DTX-loaded Soluplus micelles group (p < 0.05). On the third day after the first administration, the relative total flux of the OSM / ETP / DTX solution group was 75.1%, whereas the OSM / ETP / DTX-loaded Soluplus micelle group showed 80.1%. However, on the seventh day, the relative total flux of the OSM / ETP / DTX solution group increased significantly to 176% compared to that on the third day, whereas the relative total flux of the OSM / ETP / DTX-loaded Soluplus micelle group decreased to 60.5%, which was lower than that on the third day. The relative total flux of the OSM / ETP / DTX-loaded Soluplus micelle continuously decreased over time, while the relative total flux of the OSM / ETP / DTX solution repeatedly increased and decreased over time. Specifically, the relative total flux of the OSM / ETP / DTX solution initially decreased and then tended to increase every 7 days.

[0196] In cytotoxicity experiments using tumor spheroids, the OSM / ETP / DTX solution group and the OSM / ETP / DTX-loaded Soluplus micelle group showed similar results. However, the results in animal experiments are thought to be different due to differences in drug release patterns. While the solution rapidly decomposed in the body, the micelles did not decompose and were released slowly, resulting in a sustained anticancer effect.

[0197] Finally, when comparing the tumor growth inhibition rate (TGI, %), the OSM / ETP / DTX solution group showed a TGI of 54.6%, and the OSM / ETP / DTX-loaded Soluplus micelle group showed a TGI of 96.5%. On the 14th day, one mouse administered the OSM / ETP / DTX solution died with a body weight loss of more than 20%, but the remaining mice did not show a body weight loss of more than 20% compared to the first day, a rapid weight loss of 10-15%, or death. After the experiment, all mice were euthanized, and the tumor volumes were collected and compared. Statistically significant differences were observed in all experimental groups compared to the model group (see Figure 8D). Statistically significant differences were also observed between the OSM / ETP / DTX solution and the OSM / ETP / DTX-loaded Soluplus micelle (p < 0.0001).

[0198]

[0199] Relative total flux (% of day) by group according to period Group Period 0 days 3 days 7 days 10 days 14 days 17 days 21 days Model (untreated group) 100 196.19±42.38 267.55±81.05 221.54±106.63 251.83±81.9 1302.06±114.14 325.44±167.07 OSM / ETP / DTX solution administration group 100 75.13±25.45 175.87±49.67 157.78±44.65 185.74±66.58 110.14±19.59 147.79±120.61 OSM / ETP / DTX Soluplus ⓡMicelline administration group 100 80.12 ± 13.3 260.51 ± 18.3 145.37 ± 17.6 730.59 ± 16.6 416.15 ± 9.3 311.30 ± 8.19 OSM: Osimertinib, ETP: Etoposide, DTX: Docetaxel

[0200]

[0201] Comparison of tumor volumes collected by group Group tumor volume (mm) 3 ) Model (untreated group) 791.01±418.71 OSM / ETP / DTX solution administration group 147.52±60.15 OSM / ETP / DTX Soluplus ⓡ Micelles administered group 35.39±13.92 OSM: osimertinib, ETP: etoposide, DTX: docetaxel

[0202]

[0203] The present invention has been described above, focusing on preferred embodiments thereof. Those skilled in the art will appreciate that the present invention can be implemented in modified forms without departing from its essential characteristics. Therefore, the disclosed embodiments should be considered illustrative rather than limiting. The scope of the present invention is set forth in the claims, not the foregoing description, and all differences within the scope equivalent thereto should be construed as being encompassed by the present invention.

[0204]

[0205] This invention was made possible with the support of the following national research and development project.

[0206] [National Research and Development Project Supporting This Invention]

[0207] [Project ID] 1711189389

[0208] [Assignment Number] 2022R1C1C1007107

[0209] [Ministry Name] Ministry of Science and ICT

[0210] [Name of Project Management (Specialist) Institution] National Research Foundation of Korea

[0211] [Research Project Name] Individual Basic Research (Ministry of Science and ICT)

[0212] [Research Project Name] Dual-Release Formulation of pH-Sensitive Micelle and Temperature-Sensitive Gel for Combination Drug Delivery to Peritoneal Metastatic Ovarian Cancer

[0213] [Project Performing Organization Name] Chungbuk National University

[0214] [Research Period] March 1, 2023 - February 29, 2024

[0215]

[0216] [National Research and Development Project Supporting This Invention]

[0217] [Project ID] 1345370811

[0218] [Project Number] 2021RIS-001

[0219] [Ministry Name] Ministry of Education (P13)

[0220] [Name of Project Management (Specialist) Institution] National Research Foundation of Korea

[0221] [Research Project Name] Local Government-University Cooperation-Based Regional Innovation Project (086123605005223002301400)

[0222] [Research Project Name] Local Government-University Cooperation-Based Regional Innovation Project (Chungbuk Regional Innovation Platform)

[0223] [Project Implementation Organization Name] (Chungbuk Regional Innovation Platform) Chungbuk National University

[0224] Research Period: April 1, 2023 - February 29, 2024

Claims

1. A pharmaceutical composition for preventing or treating cancer, comprising, as an active ingredient, polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (PCL-PVAc-PEG) micelles encapsulating osimertinib, etoposide, and docetaxel.

2. In paragraph 1, A pharmaceutical composition for preventing or treating cancer, characterized in that the above osimertinib, etoposide, and docetaxel are combined in a molar ratio of 4:6:

7.

3. In paragraph 1, A pharmaceutical composition for preventing or treating cancer, characterized in that the micelle has a particle size of 50 nm to 60 nm.

4. In paragraph 1, A pharmaceutical composition for preventing or treating cancer, characterized in that the pharmaceutical composition is a preparation for intravenous injection.

5. In paragraph 1, A pharmaceutical composition for preventing or treating cancer, characterized in that the pharmaceutical composition further comprises at least one component selected from the group consisting of amino acids, sugars, lipids, vitamins, electrolytes, pH adjusters, stabilizers, osmotic pressure adjusters, and solubilizers.

6. In any one of paragraphs 1 to 5, A pharmaceutical composition for preventing or treating cancer, characterized in that the cancer is lung cancer.

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