Pharmaceutical composition containing elemene and its preparation method and use

Abstract

This invention relates to a pharmaceutical composition comprising elemol, a protein carrier, and an oil for injection, exhibiting good safety and stability.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical preparations, and more specifically, to pharmaceutical compositions containing elemol, methods for their preparation, and uses. Background Technology

[0002] Elemene is a volatile oily compound extracted from plants such as Curcuma Wenyujin YHChen et C.ling and Cymbopoqon citratus ((DC.)) Stapt. This compound is a pale yellow or yellow clear liquid with a pungent anise odor. Currently, elemene used clinically is a mixture containing α-elemene, β-elemene, δ-elemene, and γ-elemene, and is widely used for malignant pleural effusion, lung cancer, gastrointestinal tumors, and other superficial tumors. Because currently marketed elemene and its formulations contain excipients such as castor oil polyoxyethylene esters, which are highly irritating, most patients experience severe phlebitis, fever, local pain, allergic reactions, or mild gastrointestinal reactions after taking the drug. In actual clinical use, due to the high incidence and even higher recurrence rate of phlebitis, the intravenous infusion rate must be strictly controlled, resulting in a long infusion time.

[0003] Elemene is almost insoluble in water but highly soluble in lipids, especially in organic solvents such as petroleum ether and diethyl ether. Its molecular formula is C63-C62 ... 15 H 24 It is composed solely of hydrocarbons. Due to its unique chemical structure and physicochemical properties, its formulation as a pharmaceutical preparation presents significant challenges and obstacles. To increase the solubility of elemol, excipients such as castor oil polyoxyethylene are added to marketed formulations during the preparation of injectable solutions, leading to severe hemolysis and high irritation. Furthermore, although the market availability of paclitaxel albumin nanoparticles offers a new pharmaceutical formulation for addressing clinical side effects, the chemical structure and physicochemical properties of elemol, which is oily at room temperature, make its preparation into albumin formulations extremely difficult. Therefore, no relevant research or development has been conducted to date. Summary of the Invention

[0004] This application provides a pharmaceutical composition comprising elemol, injectable oil, and a protein carrier, which can effectively reduce one or more side effects when the pharmaceutical composition is administered to the human body.

[0005] In one or more embodiments of this application, the protein carrier used in the pharmaceutical composition comprises a protein, and any suitable protein may be used. Examples of suitable proteins include, but are not limited to, albumin, immunoglobulins, including but not limited to IgA, lipoproteins, apolipoprotein B, α-acid glycoprotein, β-2-macroglobulin, thyroglobulin, transferrin, fibronectin, factor VII, factor VIII, factor IX, factor X, and analogs. The protein carrier may be of natural origin or synthesized synthetically. In some embodiments, the protein carrier is a non-blood protein, such as casein, α-lactalbumin, and β-lactoglobulin. In some embodiments, the protein carrier comprises albumin, such as human serum albumin (HSA), bovine serum albumin, etc. Human serum albumin is a highly soluble globulin, Mr65K, composed of 585 amino acids. HSA is the most abundant protein in plasma and constitutes 70-80% of the colloid osmotic pressure of human plasma.

[0006] A pharmaceutical composition comprising β-elemene, an oil for injection, and a protein carrier, wherein the weight ratio of β-elemene to the oil for injection is 1:1 to 1:3; the weight ratio of β-elemene to the protein carrier is 1:0.5 to 1:3; and the most preferred weight ratio of β-elemene:oil for injection:protein carrier is 1:3:2.7.

[0007] The preparation process of the pharmaceutical composition of the present invention contains an organic solvent, preferably selected from one or more of chloroform, dichloromethane, tert-butanol, isopropanol, ethyl acetate, ethanol, tetrahydrofuran, dioxane, acetonitrile, acetone, dimethyl sulfoxide, dimethylformamide, and methylpyrrolidone. More preferably, the organic solvent is a mixture of dichloromethane and ethanol. More preferably, the volume ratio of ethanol to dichloromethane is 1:1-8, and even more preferably, the volume ratio of ethanol to dichloromethane is 3:7.

[0008] The preparation process of the pharmaceutical composition of the present invention includes the following steps:

[0009] (1) Weigh out the prescribed amounts of β-elemene and soybean oil, add an appropriate amount of organic solvent and vortex mix to obtain the oil phase;

[0010] (2) Take 20% human serum albumin, add an appropriate amount of ultrapure water to dilute it, and obtain an aqueous solution of human serum albumin of appropriate concentration as the aqueous phase;

[0011] (3) The oil phase is slowly added drop by drop to the aqueous phase, and the two phases are uniformly mixed by ultrasonication to form a primary emulsion; the obtained primary emulsion is homogenized by a high-pressure homogenizer.

[0012] (4) The homogenized emulsion is subjected to a rotary evaporator to remove organic solvents, and then filtered through a microporous membrane to remove bacteria, thus obtaining β-elemene albumin nanoparticles.

[0013] In step (3), the pressure of high-pressure homogenization is 700-1000 bar, preferably 1000 bar; the number of homogenizations is 10-25 times, preferably 10-15 times, and most preferably 15 times.

[0014] The pharmaceutical composition of the present invention further comprises a lyophilization protectant, wherein the lyophilization protectant is selected from one or more of glucose, sucrose, maltose, lactose, mannose, trehalose, glycine, and dextran, and more preferably sucrose is the lyophilization protectant. Even more preferably, the weight ratio of the lyophilization protectant to the volume of the solution of the pharmaceutical composition is 1:100-5:100 g / mL, preferably 3:100 g / mL.

[0015] This invention also provides the use of the pharmaceutical composition in the preparation of a medicament for the prevention or treatment of cancer, preferably the cancer including adrenocortical carcinoma, myeloid metaplasia of unknown cause, AIDS-related cancer, anal cancer, appendiceal cancer, astrocytoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, glioma, ependymoma, oligodendroglioma, meningioma, craniopharyngioma, hemangioblastoma, medulloblastoma, neuroectodermal tumor, visual pathway and hypothalamic glioma and malignant glioma, breast cancer, bronchial adenoma, carcinoid tumor, central nervous system lymphoma, cervical cancer, colon cancer, colorectal cancer, chronic... Myeloproliferative disorders, endometrial cancer, ependymoma, esophageal cancer, Ewing's family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, germ cell carcinomas, gestational trophoblastic tumors, head and neck cancer, liver cancer, laryngeal cancer, leukemia, lip and oral cavity cancer, lung cancer, lymphoma, medulloblastoma, melanoma, mesothelioma, metastatic squamous cell carcinoma of the neck, multiple endocrine neoplasia syndrome, myelodysplastic syndrome, myelodysplastic / myeloproliferative disorders, nasal cavity and sinus cancer, nasopharyngeal carcinoma, neuroblastoma, neuroendocrine carcinoma, oropharyngeal cancer, brain tumors, bone metastases, gastric cancer, colorectal cancer, esophageal cancer, ovarian cancer, pancreas Cancer, breast cancer, skin cancer, parathyroid cancer, penile cancer, peritoneal cancer, pharyngeal cancer, pheochromocytoma, pineal cell carcinoma and supratentorial primitive neuroectodermal tumor, pituitary adenoma, pleural pulmonary blastoma, lymphoma, primary central nervous system lymphoma, pulmonary lymphangioleiomyomatosis, rectal cancer, kidney cancer, renal pelvis and ureter cancer, rhabdomyosarcoma, salivary gland cancer, skin cancer, small intestine cancer, squamous cell carcinoma, testicular cancer, pharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, or vaginal cancer; preferably, the cancers are lung cancer, liver cancer, esophageal cancer, nasopharyngeal carcinoma, brain tumors, bone metastases, gastric cancer, intestinal cancer, uterine cancer, cervical cancer, germ cell cancer. Cancer, endometrial cancer, gestational trophoblastic tumor, breast cancer, skin cancer, lymphoma, leukemia, or malignant melanoma; more preferably, the cancer is a brain tumor; even more preferably, the brain tumor is a glioma, brainstem glioma, cerebellum or cerebrum astrocytoma, malignant glioma, ependymoma, oligodendroglioma, meningioma, craniopharyngioma, hemangioblastoma, medulloblastoma, visual pathway and hypothalamic glioma or malignant glioma; even more preferably, the cerebellum or cerebrum astrocytoma is a fibrous astrocytoma or diffuse astrocytoma or anaplastic (malignant) astrocytoma.

[0016] The pharmaceutical composition described in this invention is preferably used for the prevention or treatment of lung cancer or glioma.

[0017] The present invention further provides a method of administration of the pharmaceutical composition, such as via enteral, inhalation, intraperitoneal, intrabladder, intramuscular, intravenous, intratracheal, subcutaneous, intraocular, intrathecal, transdermal, rectal, or vaginal administration, with intravenous administration being preferred.

[0018] The present invention further provides a sealed container for holding the pharmaceutical composition, preferably a unit-dose container or a multi-dose container, preferably a liquid composition or a dry solid composition, preferably a lyophilized pharmaceutical composition, preferably a sterile pharmaceutical composition, and preferably a pre-filled syringe.

[0019] In one or more embodiments of this application, the nanoformulations or nanoparticles described herein may be present in a dried formulation (such as a lyophilized composition) or suspended in a biocompatible medium. Suitable biocompatible media include, but are not limited to, water, aqueous buffered media, saline, buffered saline, optionally buffered amino acid solutions, optionally buffered protein solutions, optionally buffered sugar solutions, optionally buffered vitamin solutions, optionally buffered synthetic polymer solutions, lipid-containing emulsions, etc.

[0020] Unless otherwise clearly stated, “individual” as used herein refers to mammals, including but not limited to primates, humans, cattle, horses, felines, canines, or rodents.

[0021] As used herein, “treatment” is a method of obtaining beneficial or desired results, including clinical outcomes. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, any one or more of the following: reduction of one or more symptoms caused by a disease, alleviation of disease severity, stabilization of disease (e.g., prevention or delay of disease progression), prevention or delay of disease spread (e.g., metastasis), prevention or delay of disease onset or recurrence, delay or slowing of disease progression, improvement of disease status, provision of disease relief (whether partial or complete), reduction of the dosage of one or more other medications necessary for treating the disease, delay of disease progression, increased quality of life, and / or prolonged survival. In some embodiments, the composition reduces the severity of one or more cancer-related symptoms by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% compared to corresponding symptoms in the same subjects prior to treatment or compared to corresponding symptoms in other subjects who did not receive the composition. “Treatment” also includes alleviation of the pathological consequences of cancer. The methods of this invention are contemplated to include any one or more of these therapeutic aspects.

[0022] As used herein, "pharmaceutically acceptable" or "pharmacologically compatible" means a substance that is not biologically or otherwise undesirable, for example, that can be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a harmful manner with any other components contained in the composition. Pharmaceutically acceptable carriers or excipients preferably meet standards for toxicological and manufacturing testing.

[0023] In this document, references to "approximately" a value or parameter include (and describe) the implementation of that value or parameter itself. For example, a description of "approximately X" includes a description of "X".

[0024] The pharmaceutical compositions of the present invention have various suitable formulations. The formulations and methods described below are merely exemplary and not limiting.

[0025] Orally administered formulations may be made from (a) liquid solutions, such as an effective amount of the active ingredient dissolved in a diluent such as water, saline, or orange juice; (b) capsules, sachets, or tablets, each containing a predetermined amount of the active ingredient in solid or granular form; (c) suspensions in suitable liquids; and (d) suitable emulsions. Tablet forms may include one or more of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, gum arabic, gelatin, colloidal silica, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffers, wetting agents, preservatives, flavoring agents, and pharmacologically compatible excipients. Lozenge forms may contain the active ingredient in flavorings, typically sucrose and gum arabic or tragacanth, and lozenges containing the active ingredient in an inert matrix, such as gelatin and glycerin, or sucrose and gum arabic, emulsions, gels, etc., which, in addition to the active ingredient, contain excipients known in the art.

[0026] Parenteral formulations include aqueous and non-aqueous, isotonic sterile injectable solutions, which may contain antioxidants, buffers, bacteriostatic agents, and solutes that make the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that may include suspending agents, solubilizers, thickeners, stabilizers, and preservatives. The formulations may be present in single-dose or multi-dose sealed containers, such as ampoules and vials, and may be stored immediately before use under lyophilized (freeze-dried) conditions, requiring only the addition of a sterile liquid excipient, such as water, for injection. Temporary injectable solutions and suspensions can be prepared from sterile powders, granules, and tablets of the aforementioned types.

[0027] Aerosols comprising the pharmaceutical compositions of the present invention, said pharmaceutical compositions comprising aqueous and non-aqueous, isotonic sterile solutions, which may contain antioxidants, buffers, antibacterial agents and solutes, and aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickeners, stabilizers and preservatives, which, alone or in combination with other suitable components, can be formulated into aerosol formulations for administration by inhalation. These aerosol formulations can be placed in pressurized, acceptable propellants, such as difluoromethane, propane, nitrogen, etc. They can also be formulated into non-pressurized pharmaceutical preparations, for example in nebulizers or sprayers.

[0028] Other suitable formulations are also possible; for example, suppositories can be prepared using various bases, such as emulsified bases or water-soluble bases. Vaginal formulations can be presented as vaginal suppositories, tampons, creams, gels, pastes, foams, or sprays, containing, in addition to the active ingredient, a suitable carrier known in the art.

[0029] Instruction manual illustrations

[0030] Figure 1 The values ​​represent changes in tumor volume (A), tumor weight (B), tumor growth inhibition (C), and body weight (D) in the lung cancer model of Example 5, where **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

[0031] Figure 2 The survival time of different drug groups in the glioma model of Example 6 is shown. Detailed Implementation

[0032] The following embodiments are further illustrative of the present invention and are not intended to limit the scope of the invention. The present invention is further described in detail below with reference to the embodiments; however, those skilled in the art should understand that the present invention is not limited to these embodiments and the preparation methods used. Furthermore, those skilled in the art can make equivalent substitutions, combinations, improvements, or modifications to the present invention based on the description thereof, but all such substitutions and modifications will be included within the scope of the present invention.

[0033] Example 1

[0034] The sample preparation process is as follows:

[0035] (1) Weigh out the prescribed amount of β-elemene and soybean oil, add an appropriate amount of organic solvent and vortex mix to obtain the oil phase.

[0036] (2) Take 20% human serum albumin, add an appropriate amount of ultrapure water to dilute it, and obtain an aqueous solution of human serum albumin of appropriate concentration as the aqueous phase.

[0037] (3) The oil phase is slowly added drop by drop to the water phase, and the two phases are mixed evenly by ultrasonic action to form a primary emulsion; the obtained primary emulsion is homogenized by a high-pressure homogenizer.

[0038] (4) The homogenized emulsion is subjected to a rotary evaporator to remove organic solvents, and then filtered through a microporous membrane to remove bacteria, thus obtaining β-elemene albumin nanoparticles.

[0039] 1.1 Selection of Organic Solvents

[0040] The sample was prepared according to the sample preparation process, including 0.25 g of β-elemene raw material, 0.75 g of soybean oil, 2.5 mL of organic solvent, and 3.375 mL of 20% human serum albumin solution, to prepare a 3% human serum albumin aqueous solution as the aqueous phase. The specific test results are shown in Table 1.

[0041] Table 1. Effects of Organic Solvents

[0042]

[0043] 1.2 Selection of human serum albumin dosage

[0044] The sample was prepared according to the sample preparation process, including 0.25 g of β-elemene raw material, 0.75 g of soybean oil, and 2.5 mL of organic solvent (a mixed solution of dichloromethane and ethanol (7:3, v / v)). The effects of human serum albumin concentrations of 1% (equivalent to 0.225 g albumin), 2% (equivalent to 0.45 g albumin), and 3% (equivalent to 0.675 g albumin) on the formulation were investigated, and the results are shown in Table 2.

[0045] Table 2 Effect of albumin dosage

[0046]

[0047] 1.3 Selection of soybean oil dosage

[0048] The sample was prepared according to the sample preparation process, including 0.25 g of β-elemene raw material, 2.5 mL of organic solvent (a mixed solution of dichloromethane and ethanol (7:3, v / v)), and 3.375 mL of 20% human serum albumin solution, to prepare a 3% human serum albumin aqueous solution as the aqueous phase. The specific test results are shown in Table 3.

[0049] Table 3. Effect of soybean oil usage

[0050]

[0051] 1.4 Investigation of Homogeneous Pressure

[0052] Table 4 Prescription Dosage

[0053]

[0054] The dosages in Table 4 were prepared according to the sample preparation method. In step (3), after the initial emulsion was formed by ultrasonication with a probe, the prepared initial emulsion was transferred to a high-pressure homogenizer for homogenization. After cycling at 300-400 bar for 4-5 times, the homogenization was continued for 10 times at 500 bar, 700 bar, and 1000 bar to investigate the effect of homogenization pressure on the formulation. The results are shown in Table 5. The nanoparticle size decreased with increasing homogenization pressure. The nanoparticles with the smallest size and stable placement were obtained when the homogenization pressure was 1000 bar.

[0055] Table 5. Effect of Homogeneous Pressure

[0056]

[0057] 1.5 Examination of the number of homogenization cycles

[0058] The dosages in Table 4 were prepared according to the sample preparation method. In step (3), after the initial emulsion was formed by ultrasonication with a probe, the prepared initial emulsion was transferred to a high-pressure homogenizer for homogenization. After cycling at 300-400 bar for 4-5 times, the homogenization was carried out at a pressure of 1000 bar for 5, 10, 15, 20, and 25 times respectively to investigate the effect of the number of homogenization cycles on the formulation. The results are shown in Table 6. As the number of homogenization cycles gradually increased, the particle size of the prepared nanoparticles decreased with the increase of the number of homogenization cycles.

[0059] Table 6. Effect of Homogenization Times

[0060]

[0061] Example 2

[0062] (1) Weigh 0.25g of β-elemene raw material and 0.75g of soybean oil and dissolve them in 2.5mL of dichloromethane / ethanol (7:3) mixed solvent. Vortex mix to obtain the oil phase.

[0063] (2) Take 3.375 mL of 20% human serum albumin solution and add an appropriate amount of ultrapure water to dilute it to obtain a 3% human serum albumin aqueous solution, which is the aqueous phase.

[0064] (3) The oil phase is slowly added drop by drop to the aqueous phase. Under the action of ultrasound, the oil and water phases are mixed evenly to form a primary emulsion. Then, the mixture is homogenized by a high-pressure homogenizer. After cycling at 300-400 bar for 4-5 times, the pressure is increased to 1000 bar and the cycle is continued for 15 times. After homogenization, the organic solvent is removed by a rotary evaporator and the mixture is filtered to remove bacteria, thus obtaining the β-elemene albumin nanoparticle solution.

[0065] Example 3

[0066] 3.1 Main Instruments

[0067] CPA225D electronic balance (Sartorius GmbH, Germany);

[0068] Scientz-IID Cell Disruptor (Ningbo SCIENTZ Company);

[0069] N-1300 rotary evaporator (Shanghai EYELA Company);

[0070] Zetasizer Nano ZS90 laser particle size analyzer (Malvern);

[0071] AH-NANO high-pressure homogenizer (ATS Corporation);

[0072] UPH-II-10T Ultrapure Water System (Chengdu ULUPURE Company);

[0073] 3K-15 Tabletop High-Speed ​​Centrifuge (SIGMA);

[0074] Ultimate 3000 high-performance liquid chromatograph (Thermo Fisher);

[0075] H-600 transmission electron microscope (HITACHI).

[0076] 3.2 Medicines and Reagents

[0077] The sample (β-ELE-AN) prepared in Example 2;

[0078] Acetonitrile (chromatographic grade);

[0079] Ultrapure water (homemade);

[0080] 3.3 Particle size distribution and zeta potential

[0081] Take 0.1 ml of β-ELE-AN, dilute it with ultrapure water about 40 times, and then use a Malvern particle size analyzer to measure the particle size and potential. The average particle size of the sample was 84.9 ± 4.0 nm, the PDI was 0.18 ± 0.02, and the Zeta potential was -23.5 ± 4.0 mV.

[0082] 3.4 Storage stability assessment

[0083] β-ELE-AN nanoparticles were placed at 4℃ and 25℃, and their particle size and PDI were measured on days 1, 3, 5, and 7 to investigate their stability under these conditions. The results showed that no obvious aggregation or precipitation was observed after 7 days of placement at 4℃ and 25℃, and the particle size did not increase or decrease significantly.

[0084] Example 4

[0085] 4.1 Main Instruments

[0086] Ultimate 3000 high-performance liquid chromatograph (Thermo Fisher);

[0087] 1101378091E Refrigerated Dryer (LABCONCO, USA).

[0088] 4.2 Medicines and Reagents

[0089] The sample (β-ELE-AN) prepared in Example 2;

[0090] Mannitol and lactose (Tianjin Jindong Tianzheng Fine Chemical Reagent Factory);

[0091] Glucose, sucrose (Chengdu CHRON CHEMICALS);

[0092] 4.3 Screening of freeze-drying protectants

[0093] Freshly prepared β-ELE-AN was dispensed into 2 ml vials. Appropriate amounts of mannitol, lactose, glucose, and sucrose were added to the nanoparticle solution, and the solutions were shaken to ensure complete dissolution. After pre-freezing at -40°C overnight, the solutions were transferred to a freeze dryer for freeze-drying. The freeze-drying procedure is shown in Table 7. After freeze-drying, the appearance and color of the freeze-dried samples were observed. Each sample was reconstituted with 2 ml of ultrapure water, and its dispersibility was observed. The particle size and PDI were also measured. The results are shown in Table 8.

[0094] Table 7. Freeze-drying process

[0095]

[0096] Table 8. Effect of freeze-drying protectants

[0097]

[0098] Example 5

[0099] 5.1 Main Instruments

[0100] CPA225D electronic balance (Sartorius GmbH, Germany);

[0101] ZH-Bluestar Brain Stereotype Instrument (Anhui Zhenghua Biological Instrument Equipment Co., Ltd.)

[0102] 5.2 Main Drugs and Reagents

[0103] β-ELE-AN (homemade);

[0104] Commercially available β-elemene injection (CSPC Grand Pharmaceutical Co., Ltd.) (β-ELE-INJ);

[0105] Physiological saline for injection (Sichuan Kelun Pharmaceutical Co., Ltd.);

[0106] DMEM incomplete culture medium (Hyclone, USA);

[0107] Fetal bovine serum (Gibco, USA);

[0108] Trypsin (Solarbio, China), etc.

[0109] 5.3 Cell lines used in the experiment

[0110] Healthy male C57BL / 6 mice, weighing 20-22g; and healthy male Kunming mice, weighing 20-22g, were purchased from Chengdu Dashuo Biotechnology Co., Ltd. They were fed standard feed at a temperature of 25±2℃ and a humidity of 50±10%, with free access to food and water.

[0111] The C6 glioma cell line and the Lewis lung cancer cell line were both purchased from the Shanghai Institute of Cell Biology.

[0112] 5.4 Establishment of the C57BL / 6 Lewis lung cancer mouse model

[0113] Lewis lung cancer cells, required for establishing the animal model, were cultured under the following conditions: When the cells reached approximately 90% confluence, the culture medium was discarded, PBS buffer was added, and the cells were washed once. After discarding the PBS solution, 2 mL of trypsin digestion solution was added, and the cells were gently shaken to ensure the cell surface was completely wetted. The culture dish was then placed in an incubator for 2 minutes, after which the digestion solution was discarded, and 2 mL of complete culture medium was added to terminate the digestion. The cells were gently dispersed into a single-cell suspension using a pipette. The resulting cell suspension was passaged in separate dishes at a 1:3 ratio. Once the cells reached 90% confluence again, the above steps were repeated.

[0114] On the day of the experiment, healthy LLC cells were collected, the culture medium was discarded, and the cells were washed once with PBS buffer. 2 mL of trypsin solution was added to the dish, and the cells were digested in a cell culture incubator for 2 minutes. The trypsin was then discarded, and 2 mL of complete culture medium was added to stop the digestion. The bottom of the dish was repeatedly agitated with a pipette to form a single-cell suspension. The cell suspension was collected and centrifuged at 2000 rpm for 3 minutes. The supernatant was discarded, and the cell pellet was resuspended in PBS buffer. The cell concentration was adjusted to 1 × 10⁻⁶ cells / mL. 6 Cells / 100μL. Male C57BL / 6 mice aged 6–8 weeks were used. The hair on the right side of the abdomen was removed, and the area was disinfected by wiping with an alcohol swab. 100μL of LLC cell suspension, pre-adjusted to a suitable concentration, was subcutaneously injected using a 1ml syringe, minimizing leakage of the LLC cell suspension during the process. The appearance of small tumor protrusions at the injection site indicates successful modeling.

[0115] 5.5 Administration Method

[0116] On day 12 after LLC cell inoculation, mice that successfully modeled the tumor were randomly divided into three groups of six each. On days 12, 14, 16, and 18, mice were injected intravenously with saline, β-ELE-INJ, and β-ELE-AN, respectively, at a dose of 60 mg / kg. The long and short diameters of the tumor were measured every other day, and the mice's weight was recorded. The tumor size was calculated using the formula: TumorVolume(mm) 3 ) = 1 / 2 × Width 2 ×Length. All mice were sacrificed on day 24 post-inoculation. Tumor tissue was removed, washed, dried, photographed, and weighed. The tumor inhibition rate was calculated using the formula: TIR% = (W... control –W treated ) / W control ×100%. The tumor group was then fixed in 4% paraformaldehyde solution overnight, and then replaced with fresh fixative for 48 hours before sectioning.

[0117] 5.6 Examination of tumor volume

[0118] Figure 1 Tumor size and body weight changes were observed in each group of mice. Tumor volume results showed: saline group > β-ELE-INJ group > β-ELE-AN group. On day 24, at a dose of 60 mg / kg, the average tumor volume in the saline group reached 1135.99 mm. 3 The tumor volume in the commercially available injection group was 452.83 mm. 3 The mean tumor volume in the β-ELE-AN group was 198.05 mm. 3 Compared with the saline group, both the β-ELE-AN and β-ELE-INJ groups showed significant tumor growth inhibition. Furthermore, at the same dosage, the average tumor volume in the β-ELE-AN group was significantly smaller than that in the commercially available injection group, being 2.29 times larger. The tumor tissue was harvested and photographed on day 24. Figure 1 As shown, the average tumor weight in the physiological group was 0.8776g, and the average tumor weight in the β-ELE-AN group was 0.2063g, with a tumor inhibition rate of 76%. The average tumor weight in the β-ELE-INJ group was 0.4698g, which was significantly greater than that in the β-ELE-AN group.

[0119] Example 6

[0120] 6.1 Establishment of a mouse model bearing C6 glioma

[0121] This experiment used C6 cells to establish a glioma model. The cell culture method was as follows: When the cells grew to about 90%, the culture medium was discarded, PBS buffer was added, and the cells were washed once. After discarding the PBS solution, 2 mL of trypsin digestion solution was added, and the cells were gently shaken to completely wet the cell surface. The culture dish was then placed in an incubator for 2 minutes, after which the digestion solution was discarded, and 2 mL of complete culture medium was added to stop the digestion. The cells were gently dispersed into a single-cell suspension by pipetting. The resulting cell suspension was passaged in a 1:3 ratio. When the cells grew to 90% again, the above operation was repeated.

[0122] On the day of the experiment, C6 cells in good growth condition were collected, the culture medium was discarded, and the cells were washed once with PBS buffer. 2 mL of trypsin was added to the dish, and the cells were digested in a 37°C incubator for 1 min. The trypsin was then discarded, and 2 mL of complete culture medium was added to stop the digestion. The bottom of the dish was repeatedly agitated with a pipette to form a single-cell suspension. The cell suspension was collected, centrifuged at 2000 rpm for 3 min, the supernatant was discarded, and the cell pellet was resuspended in PBS buffer. The cell concentration was adjusted to 1 × 10⁻⁶ cells / mL. 6 5μL / piece.

[0123] Healthy male Kunming mice were anesthetized by intraperitoneal injection of 4% chloral hydrate. The hair on the top of the head was removed, and the area was disinfected with alcohol-soaked cotton. A small incision was made along the sagittal midline of the head using sterilized ophthalmic scissors. The tissue membrane was quickly removed and the skull bone separated using a cotton swab soaked in 10% hydrogen peroxide. Residual hydrogen peroxide solution was then wiped away with a cotton swab soaked in PBS buffer. The mouse head was fixed to a stereotaxic apparatus, positioned 1.8 mm to the right and 0.6 mm posterior to the anterior fontanelle. At this location, a small hole was drilled using a 2 mL syringe needle, taking care not to drill too deeply to avoid bleeding. 5 μL of C6 cells were aspirated using a micro-injection needle and injected perpendicularly into the hole, advancing the needle 4 mm and withdrawing it 1 mm. The cell suspension was slowly injected into the brain. The needle remained in the hole for 5 minutes before being removed to prevent cell fluid leakage. Finally, the wound was sutured with sutures and the wound surface was smeared with a cotton swab soaked in antibiotics to prevent infection.

[0124] 6.2 Dosing regimen

[0125] Eight days after establishing the C6 glioma-bearing Kunming mouse model, mice were randomly divided into three groups of ten mice each. On days 8, 10, 12, and 14 after successful modeling, mice were intravenously injected with saline, β-ELE-INJ, and β-ELE-AN, respectively, with β-ELE at a dose of 40 mg / kg. The number of deaths and the number of days were recorded promptly. Graphpad software was used to calculate the survival rate, median survival time, and other data for each group of tumor-bearing mice, and survival curves were plotted.

[0126] 6.3 Survival period assessment

[0127] Orthotopic C6 gliomas cannot record changes in tumor volume and size. A survival study was used to verify the in vivo efficacy of β-ELE-AN against gliomas in animals. The survival study results are as follows: Figure 2 As shown, the experimental results indicate that mice bearing C6 gliomas administered β-ELE-AN exhibited longer survival. The median survival time was 15 days in the physiological group, 25 days in the β-ELE-INJ group, and 42 days in the β-ELE-AN group. After 60 days, all mice in the physiological group died, 30% of the mice in the β-ELE-INJ group survived, and 50% of the mice in the β-ELE-AN group still survived. The experimental results demonstrate that β-ELE-AN can effectively prolong the survival time of mice bearing C6 gliomas.

Claims

1. A pharmaceutical composition comprising β-elemene, an oil for injection, and a protein carrier, wherein: The oil used for injection is soybean oil; The protein carrier is human serum albumin; The weight ratio of β-elemene:injectable oil:protein carrier is 1:3:2.7; The pharmaceutical composition contains an organic solvent during its preparation. The organic solvent is selected from dichloromethane, ethyl acetate, or a mixture of dichloromethane and ethanol, wherein the volume ratio of ethanol to dichloromethane is 3:

7.

2. The pharmaceutical composition according to claim 1, wherein the preparation process comprises the following steps: Step (1) Weigh the prescribed amount of β-elemene and soybean oil, add an appropriate amount of organic solvent and vortex mix to obtain the oil phase; Step (2) Take 20% human serum albumin, add an appropriate amount of ultrapure water to dilute it, and obtain an aqueous solution of human serum albumin of appropriate concentration as the aqueous phase; Step (3) The oil phase is slowly added drop by drop to the aqueous phase, and the two phases are uniformly mixed by ultrasonic action to form a primary emulsion; the obtained primary emulsion is homogenized by a high-pressure homogenizer. After homogenization in step (4), the organic solvent was removed by rotary evaporator and then sterilized by microporous filter membrane to obtain β-elemene albumin nanoparticles.

3. The pharmaceutical composition according to claim 2, characterized in that... In step (3), the pressure of high-pressure homogenization is 700-1000 bar.

4. The pharmaceutical composition according to claim 3, characterized in that... In step (3), the pressure of high-pressure homogenization is 1000 bar.

5. The pharmaceutical composition according to claim 2, characterized in that... In step (3), the high-pressure homogenization is performed 10-25 times.

6. The pharmaceutical composition according to claim 5, characterized in that... In step (3), the high-pressure homogenization is performed 10-15 times.

7. The pharmaceutical composition according to claim 6, characterized in that... In step (3), the high-pressure homogenization is performed 15 times.

8. The pharmaceutical composition according to claim 1, further comprising a lyophilization protectant, wherein the lyophilization protectant is selected from one or more of glucose, sucrose, maltose, lactose, mannose, trehalose, glycine, and dextran.

9. The pharmaceutical composition according to claim 8, wherein the lyophilization protectant is sucrose.

10. The pharmaceutical composition according to claim 8, wherein the weight ratio of the lyophilization protectant to the volume of the solution of the pharmaceutical composition is 1 g / 100 ml to 5 g / 100 ml.

11. The pharmaceutical composition according to claim 10, wherein the weight ratio of the lyophilization protectant to the volume of the solution of the pharmaceutical composition is 3 g / 100 mL.

12. Use of the pharmaceutical composition of any one of claims 1-11 in the preparation of a medicament for treating cancer, wherein the cancer is selected from bladder cancer, bone cancer, glioma, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, gallbladder cancer, liver cancer, laryngeal cancer, leukemia, oral cancer, lung cancer, lymphoma, melanoma, mesothelioma, nasopharyngeal carcinoma, brain tumor, bone metastasis, gastric cancer, ovarian cancer, pancreatic cancer, skin cancer, pituitary adenoma, rectal cancer, kidney cancer, and thyroid cancer.

13. The use according to claim 12, characterized in that, The cancer in question is glioma.

14. A sealed container comprising the pharmaceutical composition according to any one of claims 1-11.

15. The sealed container according to claim 14, characterized in that, The sealed container is a single-dose container or a multi-dose container.

16. The sealed container according to claim 14, characterized in that, The pharmaceutical composition is a liquid composition or a dry solid composition.

17. The sealed container according to claim 14, characterized in that, The pharmaceutical composition is lyophilized.

18. The sealed container according to claim 14, characterized in that, The pharmaceutical composition is sterile.

19. The sealed container according to claim 14, characterized in that, The sealed container is a pre-filled syringe.

Images