CD73 inhibitor pharmaceutical composition, preparation method therefor, and use thereof

Abstract

Provided are a CD73 pharmaceutical composition, a preparation method therefor, and use thereof. The pharmaceutical composition comprises an active ingredient, a pH buffering agent, and water for injection. The active ingredient is a free acid of a compound represented by formula (I) having the following structure or a trisodium salt thereof, and the content thereof is 0.01-50 mg / ml. The pharmaceutical composition is simple in composition and convenient to use, and the stability of the pharmaceutical composition can meet the requirements of industrial production of pharmaceutical preparations and meet the requirements of clinical research and commercialization of drugs.

Description

A CD73 inhibitor pharmaceutical composition, its preparation method and application Technical Field

[0001] The present invention belongs to the field of drug synthesis, and specifically relates to a CD73 inhibitor pharmaceutical composition, a preparation method and application thereof. Technical Background

[0002] CD73, also known as extracellular 5'-nucleotidase (Ecto-5'-nucleotidase, eNT), is a 70kDa protein molecule. Under normal circumstances, it is expressed on vascular endothelial cells and some blood cells. It is anchored to the cell membrane surface through glycosylphosphatidylinositol (GPI) and regulates the metabolism of adenosine triphosphate (ATP) together with CD39. Among them, CD39 (also known as extracellular nucleoside diphosphate hydrolase-NTPDase1) can catalyze ATP to generate adenosine monophosphate (AMP), producing only a small amount of adenosine diphosphate (ADP). The main function of CD73 is to catalyze the conversion of extracellular nucleotides (such as 5'AMP) into their corresponding nucleosides (such as adenosine).

[0003] Nucleosides, particularly adenosine, produced by CD73 are considered endogenous regulators of a wide range of physiological functions. Adenosine regulates the cardiovascular system, central nervous system, respiratory system, kidneys, adipocytes, platelets, and the immune system. Within the immune system, extracellular adenosine can act on a wide variety of immune cells and mediate anti-inflammatory responses. In many tissues, adenosine also promotes fibrosis.

[0004] CD73 expression has been found in many tumor cell types, including leukemia, bladder cancer, glioma, glioblastoma, ovarian cancer, melanoma, prostate cancer, thyroid cancer, esophageal cancer, and breast cancer. Furthermore, CD73 expression has been found on the surface of immunosuppressive cells, including regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs). High CD73 expression has also been found to be associated with angiogenesis, invasion, chemotherapy resistance, metastasis, and shorter survival in various tumors, including breast cancer and melanoma.

[0005] Mechanistic studies have shown that malignant tumor cells release large amounts of ATP under chemotherapy and other stressors, which is rapidly converted into adenosine and accumulates in the tumor microenvironment. The release of extracellular ATP due to cell death or intracellular stress activates the immune response, but the ATP metabolite adenosine has immunosuppressive activity. A key point is that adenosine in the tumor inhibits infiltrating effector T lymphocytes by activating adenosine receptors (such as A2A), thereby promoting tumor development. Therefore, the accumulation of extracellular adenosine in tumor tissue is an important mechanism for tumor immune escape.

[0006] Reducing CD73 expression with interfering RNA or overexpressing CD73 in tumor cells can modulate tumor growth and migration. CD73 knockout mice are less susceptible to organ transplant rejection and spontaneous tumor development. Genetic deletion of the A2A receptor gene can induce T cell-dependent tumor rejection. In mouse models, treatment with an antibody that binds to mouse CD73 inhibits breast tumor growth and migration.

[0007] Therefore, targeting CD73 represents a potential therapeutic strategy that can enhance the efficacy of anti-tumor therapy and provide a new therapeutic strategy for limiting the further development of tumors. At the same time, targeting CD73 can also be used to treat other diseases mediated by adenosine, such as enhancing immune responses, enhancing immune effects, enhancing inflammatory responses, and treating neurological disorders, neurodegenerative diseases, and central nervous system diseases, such as depression, Parkinson's disease, sleep disorders, fibrosis, and other immune-inflammatory diseases.

[0008] Shanghai Abbisko Therapeutics Co., Ltd. has discovered a small molecule compound (WO2018113584A1) with CD73 inhibitory effects during its long-term research. Representative compounds are as follows:

[0009] The Chinese name is: (((((2R,3S,4R,5R)-5-(6-chloro-4-(((R)-6-fluoro-2,3-dihydro-1H-inden-1-yl)amino)-1H-pyrazolo[3,4-b]pyridin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphino)methyl)phosphonic acid (compound of formula (I)). The compound has a strong inhibitory effect on CD73 enzymatic and cytological activity and can be used to treat tumors, immune-related diseases and metabolic diseases mediated at least in part by CD73, such as prostate cancer, colorectal cancer, and prostate cancer. Colon cancer, rectal cancer, pancreatic cancer, stomach cancer, endometrial cancer, cervical cancer, brain cancer, liver cancer, bladder cancer, ovarian cancer, testicular cancer, head cancer, neck cancer, skin cancer (including melanoma and basal cell carcinoma), mesothelial lining cancer, white blood cell cancer (including lymphoma and leukemia), esophageal cancer, breast cancer, muscle cancer, connective tissue cancer, lung cancer (including small cell lung cancer and non-small cell lung cancer), adrenal cancer, thyroid cancer, kidney cancer, bone cancer, brain tumor, glioblastoma, mesothelioma, renal cell carcinoma, sarcoma (including Kaposi's sarcoma), choriocarcinoma, epidermal basal cell carcinoma, testicular seminoma, etc.

[0010] During the drug development process, the applicant developed a pharmaceutical preparation using the trisodium salt of the compound of formula (I). The structure of the trisodium salt of the compound of formula (I) is as follows:

[0011] The molecular formula is: C 21 H 21 ClFN4Na3O9P2, molecular weight is 658.78, the trisodium salt of the compound of formula (I) is usually in an amorphous form and has certain hygroscopicity. TGA shows a weight loss of about 4.6% from room temperature to 150°C. The trisodium salt of the compound of formula (I) shows very good solubility in water (>100 mg / mL) and has good chemical stability after being placed in a buffer solution with a pH value of 6 to 9 for 2 weeks, but some degradation will occur after being placed in a buffer solution with a pH value of 3 to 4 for 2 weeks. In addition, during the further development of clinical preparations, it was found that the preparation had poor stability and was prone to visible foreign matter, making it unsuitable for the development of injectable drugs. Therefore, there is an urgent need to optimize the formulation and preparation process, and further develop a pharmaceutical preparation whose stability can meet the requirements of industrial production to meet the needs of clinical drug research and market launch. Summary of the Invention

[0012] The object of the present invention is to provide a CD73 pharmaceutical composition whose stability can meet the requirements of industrial production.

[0013] In a first aspect, the present invention provides a CD73 pharmaceutical composition comprising an active ingredient, a pH buffer and water for injection; wherein the active ingredient is the free acid of the compound of formula (I) or its trisodium salt, and the content is 0.01 to 50 mg / ml.

[0014] In the present invention, the active ingredient, the free acid of the compound of formula (I) or its trisodium salt, can exist in its free form in the solution. In the free acid solution, it exists in its ionic form, for example, anions such as -1-valent anion, -2-valent anion, -3-valent anion, -5-valent anion of the compound of formula (I), which release 1, 2 or 3 Na in its trisodium salt solution. + ions, correspondingly freeing the anions of the compounds of formula (I).

[0015] As a preferred solution, the active ingredient is the trisodium salt of the compound of formula (I), and the content is 0.01 to 30.0 mg / ml.

[0016] As a preferred solution, the active ingredient is the trisodium salt of the compound of formula (I), and the content is 0.01 to 20.0 mg / ml.

[0017] As a preferred solution, the pH value of the pharmaceutical composition is 3.0-10.0.

[0018] As a further preferred embodiment, the pH value of the pharmaceutical composition is 5.0-9.0.

[0019] As a further preferred embodiment, the pH value of the pharmaceutical composition is 5.0-8.0 or 6.0-9.0.

[0020] As a preferred embodiment, the pH buffer is selected from citrate buffer, phosphate buffer, carbonate buffer and histidine buffer.

[0021] As a further preferred embodiment, the pH buffer solvent is a citrate buffer.

[0022] As a preferred embodiment, the pharmaceutical composition further comprises an osmotic pressure regulator, and the osmotic pressure regulator is selected from one or more of sodium chloride, mannitol, sucrose and glycerol.

[0023] As a preferred solution, the content of the osmotic pressure regulator is 0.1% to 10% of the total weight of the pharmaceutical composition.

[0024] As a preferred solution, the content of the osmotic pressure regulator is 0.5-10% of the total weight of the pharmaceutical composition;

[0025] As a preferred solution, the content of the osmotic pressure regulator is 1 to 10% of the total weight of the pharmaceutical composition;

[0026] As a preferred solution, the content of the osmotic pressure regulator is 5-10% of the total weight of the pharmaceutical composition. As a preferred solution, the content of the active ingredient is 0.01-2.0 mg / ml, and the pH value of the pharmaceutical composition is 3.0-9.0.

[0027] As a further preferred embodiment, the content of the active ingredient is 0.01 to 1.0 mg / ml, and the pH value of the pharmaceutical composition is 5.0 to 8.0.

[0028] As a further preferred solution, the content of the active ingredient is 0.1-0.5 mg / ml, and the pH value of the pharmaceutical composition is 5.0-8.0.

[0029] As a preferred solution, the content of the active ingredient is 0.01 to 30.0 mg / ml, and the pH value of the pharmaceutical composition is 3.0 to 10.0; the pharmaceutical composition further includes a pharmaceutically acceptable stabilizer.

[0030] As a further preferred embodiment, the content of the active ingredient is 0.01 to 25.0 mg / ml, and the pH value of the pharmaceutical composition is 5.0 to 9.0; the pharmaceutical composition further includes a pharmaceutically acceptable stabilizer, and the pharmaceutically acceptable stabilizer is selected from one or more of a cosolvent, a surfactant, a cyclodextrin and a chelating agent.

[0031] As a further preferred embodiment, the content of the active ingredient is 0.01 to 20.0 mg / ml, and the pH value of the pharmaceutical composition is 6.0 to 9.0; the pharmaceutical composition further includes a pharmaceutically acceptable stabilizer, and the pharmaceutically acceptable stabilizer is selected from one or more of a cosolvent, a surfactant, a cyclodextrin and a chelating agent; the cosolvent is selected from one or more of propylene glycol, N,N-dimethylacetamide, polyethylene glycol 400 and ethanol; the surfactant is selected from one or more of polysorbate 80, polysorbate 20, polyoxyethylene castor oil and polyethylene glycol-15 hydroxystearate; the cyclodextrin is one or more of hydroxypropyl-β-cyclodextrin or sulfobutyl ether β-cyclodextrin; and the chelating agent is disodium edetate.

[0032] As a further preferred embodiment, the pharmaceutically acceptable stabilizer is polysorbate 20 and / or disodium edetate; the content of polysorbate 20 is 0.001% to 10% of the total weight of the pharmaceutical composition; the content of disodium edetate is 0.001% to 1% of the total weight of the pharmaceutical composition.

[0033] As a further preferred embodiment, the pharmaceutically acceptable stabilizer is polysorbate 20 and / or disodium edetate; the content of polysorbate 20 is 0.01-2% of the total weight of the pharmaceutical composition; the content of disodium edetate is 0.01-0.5% of the total weight of the pharmaceutical composition.

[0034] As a further preferred embodiment, the pharmaceutically acceptable stabilizer is polysorbate 20 and / or disodium edetate; the content of polysorbate 20 is 0.1-2% of the total weight of the pharmaceutical composition; the content of disodium edetate is 0.01-0.2% of the total weight of the pharmaceutical composition. As a further preferred embodiment, the pharmaceutical composition comprises an active ingredient, the content of the active ingredient is 0.01-0.5 mg / ml, the content of the osmotic pressure regulator is 0.1%-10% of the total weight of the pharmaceutical composition, and the pH value of the pharmaceutical composition is 5.0-8.0; or,

[0035] The content of the active ingredient is 0.1 to 20 mg / ml, and the pH value of the pharmaceutical composition is 6.0 to 9.0; the pharmaceutically acceptable stabilizer is polysorbate 20 and / or disodium edetate; the content of polysorbate 20 is 0.01% to 2% of the total weight of the pharmaceutical composition; the content of disodium edetate is 0.01% to 0.5% of the total weight of the pharmaceutical composition; and the content of the osmotic pressure regulator is 0.1% to 10% of the total weight of the pharmaceutical composition.

[0036] As a further preferred embodiment, the pharmaceutical composition comprises an active ingredient, the content of the active ingredient is 0.5 to 20 mg / ml, the pH value of the pharmaceutical composition is 6.0 to 8.5, the pharmaceutically acceptable stabilizer is polysorbate 20 and / or disodium edetate; the content of the polysorbate 20 is 0.1% to 2% of the total weight of the pharmaceutical composition; the content of the disodium edetate is 0.01 to 0.2% of the total weight of the pharmaceutical composition, and the content of the osmotic pressure regulator is 5% to 10% of the total weight of the pharmaceutical composition.

[0037] As a preferred embodiment, the pharmaceutical composition comprises an active ingredient, and the content of the active ingredient is 0.01 mg / ml.

[0038] As a preferred solution, the pharmaceutical composition comprises an active ingredient, and the content of the active ingredient is 0.5 mg / ml.

[0039] As a preferred embodiment, the pharmaceutical composition comprises an active ingredient, and the content of the active ingredient is 0.8 mg / ml.

[0040] As a preferred solution, the pharmaceutical composition comprises an active ingredient, and the content of the active ingredient is 1.0 mg / ml.

[0041] As a preferred embodiment, the pharmaceutical composition comprises an active ingredient, and the content of the active ingredient is 4.0 mg / ml.

[0042] As a preferred embodiment, the pharmaceutical composition comprises an active ingredient, and the content of the active ingredient is 5.0 mg / ml.

[0043] As a preferred embodiment, the pharmaceutical composition comprises an active ingredient, and the content of the active ingredient is 10.0 mg / ml.

[0044] As a preferred embodiment, the pharmaceutical composition comprises an active ingredient, and the content of the active ingredient is 20.0 mg / ml.

[0045] As a preferred solution, the pH value of the pharmaceutical composition is 5.0.

[0046] As a preferred solution, the pH value of the pharmaceutical composition is 6.0.

[0047] As a preferred solution, the pH value of the pharmaceutical composition is 6.5.

[0048] As a preferred solution, the pH value of the pharmaceutical composition is 7.0.

[0049] As a preferred solution, the pH value of the pharmaceutical composition is 7.5.

[0050] As a preferred solution, the pH value of the pharmaceutical composition is 8.0.

[0051] As a preferred solution, the pH value of the pharmaceutical composition is 8.5.

[0052] As a preferred solution, the pH value of the pharmaceutical composition is 9.0.

[0053] A second aspect of the present invention provides a lyophilized powder of CD73 drug, wherein the lyophilized powder is obtained by lyophilizing the aforementioned pharmaceutical composition.

[0054] A third aspect of the present invention provides a method for preparing a CD73 pharmaceutical composition, comprising the following steps:

[0055] Step 1: dissolving the active ingredient, pH buffer, and optional osmotic pressure regulator in water for injection to prepare a mixed solution;

[0056] Step 2: Adjust the pH value of the mixed solution to 3-10; optionally,

[0057] Step 3: Add water for injection to the target content of active ingredient, pH buffer, and osmotic pressure regulator, and filter to obtain a pharmaceutical composition;

[0058] The pH buffer is selected from citrate buffer, phosphate buffer, carbonate buffer and histidine buffer;

[0059] The osmotic pressure regulator is selected from one or more of sodium chloride, mannitol, sucrose and glycerol.

[0060] As a preferred solution, the pH buffer solvent is a citrate buffer.

[0061] As a preferred embodiment, the method comprises the following steps: when the content of the active ingredient in step 1 is 0.01 to 1.0 mg / ml, in step 2, adjusting the pH value of the pharmaceutical composition to 5.0 to 8.0; or,

[0062] When the content of the active ingredient in step 1 is 0.01-20.0 mg / ml,

[0063] Step 1 comprises dissolving a pharmaceutically acceptable stabilizer in water for injection, wherein the pharmaceutically acceptable stabilizer is selected from one or more of a cosolvent, a surfactant, a cyclodextrin and a chelating agent. In step 2, the pH value of the pharmaceutical composition is adjusted to 6.0 to 9.0.

[0064] A fourth aspect of the present invention provides a use of the aforementioned pharmaceutical composition in the preparation of a drug for treating tumors, immune-related diseases and disorders, and metabolic diseases that are at least partially mediated by CD73.

[0065] As a preferred embodiment, the tumor is selected from prostate cancer, colon cancer, rectal cancer, pancreatic cancer, gastric cancer, endometrial cancer, cervical cancer, brain cancer, liver cancer, bladder cancer, ovarian cancer, testicular cancer, head cancer, neck cancer, skin cancer, mesothelial lining cancer, white blood cell cancer, esophageal cancer, breast cancer, muscle cancer, connective tissue cancer, lung cancer, adrenal cancer, thyroid cancer, kidney cancer, bone cancer, brain tumor, glioblastoma, mesothelioma, renal cell carcinoma, sarcoma, choriocarcinoma, epidermal basal cell carcinoma and testicular seminoma.

[0066] As a further preferred embodiment, the tumor is selected from the group consisting of skin cancer, colon cancer, pancreatic cancer, breast cancer, prostate cancer, lung cancer, leukemia cancer, brain tumor, ovarian cancer and sarcoma.

[0067] As a further preferred embodiment, the skin cancer is melanoma and basal cell carcinoma; the white blood cell cancer is lymphoma and leukemia; the lung cancer is small cell lung cancer and non-small cell carcinoma; and the sarcoma is Kaposi's sarcoma.

[0068] As a preferred embodiment, the immune-related diseases and disorders are selected from rheumatoid arthritis, renal failure, lupus erythematosus, asthma, psoriasis, ulcerative colitis, pancreatitis, allergies, fibrosis, anemia, fibromyalgia, Alzheimer's disease, congestive heart failure, stroke, aortic valve stenosis, arteriosclerosis, osteoporosis, Parkinson's disease, infection, Crohn's disease, ulcerative colitis, allergic contact dermatitis and eczema, systemic sclerosis and multiple sclerosis.

[0069] The fifth invention of the present invention provides the aforementioned pharmaceutical composition for use as a drug for treating tumors, autoimmune diseases and disorders and metabolic diseases that are at least partially mediated by CD73.

[0070] As a preferred embodiment, the tumor is selected from prostate cancer, colon cancer, rectal cancer, pancreatic cancer, gastric cancer, endometrial cancer, cervical cancer, brain cancer, liver cancer, bladder cancer, ovarian cancer, testicular cancer, head cancer, neck cancer, skin cancer, mesothelial lining cancer, white blood cell cancer, esophageal cancer, breast cancer, muscle cancer, connective tissue cancer, small cell lung cancer, adrenal cancer, thyroid cancer, kidney cancer, bone cancer, brain tumor, glioblastoma, mesothelioma, renal cell carcinoma, sarcoma, choriocarcinoma, epidermal basal cell carcinoma, cell carcinoma and testicular seminoma; the immune-related diseases and disorders are selected from rheumatoid arthritis, renal failure, lupus erythematosus, asthma, psoriasis, ulcerative colitis, pancreatitis, allergies, fibrosis, anemia, fibromyalgia, Alzheimer's disease, congestive heart failure, stroke, aortic valve stenosis, arteriosclerosis, osteoporosis, Parkinson's disease, infection, Crohn's disease, ulcerative colitis, allergic contact dermatitis and eczema, systemic sclerosis and multiple sclerosis.

[0071] The present invention also relates to a method for treating tumors, immune diseases and disorders, and metabolic diseases mediated at least in part by CD73, comprising administering a therapeutically effective amount of the aforementioned pharmaceutical composition to a patient in need thereof.

[0072] As a further preferred embodiment, the tumor is selected from prostate cancer, colon cancer, rectal cancer, pancreatic cancer, gastric cancer, endometrial cancer, cervical cancer, brain cancer, liver cancer, bladder cancer, ovarian cancer, testicular cancer, head cancer, neck cancer, skin cancer, mesothelial lining cancer, white blood cell cancer, esophageal cancer, breast cancer, muscle cancer, connective tissue cancer, small cell lung cancer, adrenal cancer, thyroid cancer, kidney cancer, bone cancer, brain tumor, glioblastoma, mesothelioma, renal cell carcinoma, sarcoma, choriocarcinoma, epidermal Basal cell carcinoma and testicular seminoma; the immune diseases and disorders are selected from rheumatoid arthritis, renal failure, lupus erythematosus, asthma, psoriasis, ulcerative colitis, pancreatitis, allergies, fibrosis, anemia, fibromyalgia, Alzheimer's disease, congestive heart failure, stroke, aortic stenosis, arteriosclerosis, osteoporosis, Parkinson's disease, infection, Crohn's disease, ulcerative colitis, allergic contact dermatitis and eczema, systemic sclerosis and multiple sclerosis.

[0073] As a further preferred embodiment, the skin cancer is melanoma and basal cell carcinoma; the white blood cell cancer is lymphoma or leukemia; the lung cancer is small cell lung cancer or non-small cell carcinoma; and the sarcoma is Kaposi's sarcoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 shows the X-ray powder diffraction pattern of the pentasodium salt of the compound of formula (I). The abscissa represents the 2θ value (degrees) and the ordinate represents the peak intensity.

[0075] Figure 2 shows the pentasodium salt of the compound of formula (I) 1 H-NMR spectrum. The horizontal axis represents the compound displacement (ppm), and the vertical axis represents the signal intensity.

[0076] Figure 3 shows the X-ray powder diffraction pattern of the trisodium salt of the compound of formula (I). The abscissa represents the 2θ value (degrees) and the ordinate represents the peak intensity.

[0077] Figure 4 is a mDSC spectrum of the trisodium salt of the compound of formula (I). The abscissa represents temperature (°C), the left ordinate represents reversing heat flow (w / g), and the right ordinate represents irreversing heat flow (w / g).

[0078] Figure 5 is a TGA spectrum of the trisodium salt of the compound of formula (I), wherein the abscissa represents temperature (°C) and the ordinate represents weight (%).

[0079] Figure 6 is a diagram of the trisodium salt of the compound of formula (I) 1H-NM spectrum. The horizontal axis represents the compound displacement (ppm), and the vertical axis represents the signal intensity. DETAILED DESCRIPTION

[0080] After in-depth research, the present invention found that the trisodium salt of the compound of formula (I) is the main cause of the stability problem of the injection. At the same time, the inventors of the present application found through extensive research that the trisodium salt of the compound of formula (I) can avoid the formation of visible foreign matter in the injection by adjusting the pH value at low content levels; or, at high content levels, by selecting different types of stabilizers and adjusting the pH value at the same time to control the stability of the injection, thereby avoiding the problem of visible foreign matter during production and storage. On this basis, the present invention provides a CD73 pharmaceutical composition and its preparation method and application. The pharmaceutical composition of the present invention has simple components, is easy to use, and has a stability that can meet the requirements of industrial production. It meets the needs of clinical research and marketing of drugs. The CD73 pharmaceutical composition provided by the present invention can be widely used in the preparation of treatments for tumors, immune-related diseases and metabolic diseases that are at least partially mediated by CD73, and is expected to accelerate the development of a new generation of CD73 inhibitor drugs. On this basis, the present invention was completed.

[0081] Detailed Description: Unless otherwise stated or specifically indicated, the following terms used in the specification and claims have the following meanings.

[0082] As used herein, the term "osmotic pressure regulator" refers to glucose, mannitol, xylitol, fructose, lactose, maltose, sucrose, trehalose, glycerol, glycine, histidine or arginine, and the like.

[0083] As used herein, the term "pharmaceutically acceptable stabilizer" refers to one or more of a cosolvent, a surfactant, a cyclodextrin, or a chelating agent.

[0084] The co-solvent may be selected from methanol, ethanol, isopropanol, propylene glycol, benzyl alcohol, glycerol, dimethyl sulfoxide, dimethylformamide, n-methylpyrrolidone, PEG200, PEG400 and mixtures thereof.

[0085] The surfactant may be selected from ionic surfactants or non-ionic surfactants, wherein the ionic surfactants include but are not limited to disodium ethylenediaminetetraacetic acid, lecithin or bile salts and mixtures thereof, and the non-ionic surfactants include but are not limited to monoglycerides, polyethylene glycol fatty alcohol ethers, sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene 12-hydroxystearate or poloxamer and mixtures thereof.

[0086] The surfactant is preferably selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, PEG3350, poloxamers, polyoxyethylene castor oil, polyethylene glycol-15 hydroxystearate, polyethylene glycol, polypropylene glycol and mixtures thereof.

[0087] The cyclodextrin may be selected from the group consisting of β-cyclodextrin, γ-cyclodextrin, sulfobutyl-ether-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, and randomly methylated β-cyclodextrin, and mixtures thereof.

[0088] The chelating agent can be selected from ethylenediaminetetraacetic acid (EDTA), salts of EDTA include dipotassium EDTA, disodium EDTA, calcium disodium EDTA, sodium EDTA, trisodium EDTA and potassium EDTA, diethylenetriaminepentaacetic acid 5 (DTPA), nitrilotriacetic acid (NTA), N-(2-acetylamino)-2-iminodiacetic acid (ADA), bis(aminoethyl) glycol ether, N,N,N',N'-tetraacetic acid (EGTA), a suitable salt of deferoxamine (DEF) is deferoxamine mesylate, trans-diaminocyclohexanetetraacetic acid (DCTA), ethylenediamine, propylenediamine, diethylenetriamine, triethylenetetramine (triethylenetetramine), 8-hydroxyquinolate. Where possible, the chelating agent used in the present invention can exist in the form of the free acid or free base or salt of the compound, or in the anhydrous, solvate or hydrated form of the compound or the corresponding salt.

[0089] The pH buffer used in the present invention is also called a buffer, which is named after the specific components used to prepare the pH buffer. The pH buffer refers to any buffer that can maintain the pH value of the preparation in the aqueous solution state between 3.0 and 10.0, such as citrate buffer, phosphate buffer, disodium hydrogen phosphate-citrate buffer, TRIS, glycyl-glycine, N-bicine, sodium dihydrogen phosphate buffer, disodium hydrogen phosphate buffer, sodium acetate buffer, sodium carbonate buffer, sodium phosphate buffer, histidine salt buffer, lysine salt buffer, arginine salt buffer, or a mixture thereof. The buffer is not specified, which can be Na + , K + NH4 + The name of the buffer solution and its preparation method can be found in Part IV of the Chinese Pharmacopoeia.

[0090] The carrier system configuration of the present invention is as follows: According to the carrier composition described in the examples, the required active ingredient (the free acid of the compound of formula (I) or its trisodium salt), osmotic pressure regulator, and stabilizer (cosolvent, surfactant, cyclodextrin or chelating agent) are weighed and quantitatively prepared in a specific buffer solution.

[0091] The term "compound of formula (I)" refers to a compound having the following structure:

[0092] The term "active ingredient" refers to the free acid of the compound of formula (I) or a pharmaceutically acceptable salt thereof; the active ingredient is preferably the free acid of the compound of formula (I) or a basic salt thereof, or an anhydrate, hydrate or solvate thereof.

[0093] As used herein, the organic solvent is selected from alcohols, chloroalkanes, ketones, ethers, cyclic ethers, esters, alkanes, cycloalkanes, benzenes, amides or sulfoxides, or mixtures thereof.

[0094] The organic solvent is selected from methanol, ethanol, n-propanol, isopropanol, dichloromethane, acetonitrile, acetone, 1,4-dioxane, tetrahydrofuran, 2-methyltetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide, ethyl acetate, isopropyl acetate, methyl tert-butyl ether or 2-methoxyethyl ether, or a mixture thereof, or an aqueous solution thereof.

[0095] Chemical bases used to obtain the basic salts are those that form non-toxic basic salts with the compounds of formula (I). Such non-toxic basic salts include, but are not limited to, those derived from such pharmacologically acceptable cations, such as basic salts derived from alkali metal cations (e.g., lithium, potassium, and sodium) and alkaline earth metal cations (e.g., calcium and magnesium); ammonium or water-soluble amine addition salts, such as N-methylglucamine (meglumine), tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, diethylamine, triethylamine, ethylamine, piperidine, lower alkanolammonium, lysine, arginine, morpholine, and tris(hydroxymethyl)aminomethane; and other basic salts of pharmaceutically acceptable organic amines.

[0096] (1) Reagents and excipients

[0097] (2) Experimental equipment

[0098] (3) Test method

[0099] 1) Purity and content determination method

[0100] According to the current edition of the Chinese Pharmacopoeia <0512> The purity and content were determined by HPLC.

[0101] 2) Osmotic pressure test method

[0102] According to the current edition of the Chinese Pharmacopoeia <0632> Osmolality measures the osmotic pressure of an injection solution.

[0103] 3) Appearance inspection method

[0104] Carefully observe the sample's color and properties using a visual inspection method. A clear, colorless liquid is considered a pass. Opalescence, visible foreign matter, or other signs are considered a failure. If a failure occurs, subsequent testing is terminated.

[0105] 4) Insoluble particle test method

[0106] According to the current edition of the Chinese Pharmacopoeia <0903> The insoluble particle test method is used to determine the insoluble particles in the injection.

[0107] 5) Density test method

[0108] According to the current edition of the Chinese Pharmacopoeia <0601> Oscillating density meter method.

[0109] 6) Viscosity test method

[0110] According to the current edition of the Chinese Pharmacopoeia <0633> Rotational viscometer method.

[0111] 7) X-ray powder diffraction test method (XRPD)

[0112] The crystal form of the sample was characterized by XRPD using a Bruker D8 Advance instrument. Specific instrument parameters are shown in the table below. X-ray path: Reflection mode. Detector: LYNXEYE_XE_T (1D mode). Aperture angle: Max. Radiation source: Cu / K-Alpha1. Primary beam path slits: Dual primary motorized 10.0mm slits, SollerMount, 2.5° axis. Secondary beam path slits: Detector OpticsMount, 2.5° slits, Dual secondary motorized 5.2mm slits. Scan mode: Continuous scan. Scan type: Dual beam path mode. Step size: 0.02°, Step time: 0.12 s / step. Scan range: 3° to 40°. Sample rotation speed: 15 rpm. Sample plate: Single crystal silicon wafer, flat plate.

[0113] 8) Melting point and melting enthalpy test method (DSC)

[0114] The experimental method for characterizing samples using differential scanning calorimetry (DSC) involves taking a small amount of sample powder and placing it in a press-fit aluminum pan that comes with the instrument. After loading the sample, the pan is press-fitted with the pan, and then the pan is inserted into the instrument for testing. The DSC analysis used in this patent uses a TA Discovery 2500 or Q2000 instrument. Scan parameters are set to use a nitrogen atmosphere, a heating rate of 10°C / min, and a temperature range of 30 to 250°C.

[0115] 9) Modulated Differential Scanning Calorimetry (mDSC)

[0116] Instrument: TA Discovery 2500; Sample: Tzero pan and Tzero sealed lid with 0.7 mm diameter hole; Pan temperature range: 0°C / 30°C to 250°C; Heating rate: 2°C / min; Nitrogen flow rate: 50 mL / min; Temperature adjustment range: + / -1°C / min; Sample size: approximately 0.5-2 mg.

[0117] 10) Thermogravimetric analysis (TGA)

[0118] Thermogravimetric analysis (TGA) characterization involves taking a small amount of sample powder and placing it in an aluminum pan supplied with the instrument. The TGA analysis method used in this patent utilizes a TA Discovery 5500 or Q5000 instrument. Scan parameters are set to a nitrogen atmosphere and a heating rate of 10°C / min. The termination temperature is set at 300°C or when the remaining sample weight is less than 80% of the initial value.

[0119] 11) Residual solvent test method ( 1 H-NMR)

[0120] The residual solvent was determined by 1H-NMR analysis using a Bruker Avance-AV 400M instrument with a 5 mm PABBO BB / 19F-1H / D Z-GRD Z108618 / 0406 probe, 8 scans, a temperature of 297.6 K, and a relaxation delay of 1 second.

[0121] 12) Ion content test method (IC)

[0122] The sodium ion content was tested using ion chromatography (IC) using a Metrohm 940 professional IC instrument.

[0123] Instrument: Metrohm 940professional IC; detector: conductivity detector; eluent (anion): 3.2mmol / LNa2CO3+1.0mmol / L NaHCO3; eluent (cation): 2.5mmol / L; MSA suppressor supplement: 0.5% H2SO4; chromatographic column: Anion A SUPP 5-150 or Cation Column C4-150; column temperature: 30℃; flow rate: 0.7mL / min (anion) or 0.9mL / min (cation); diluent: ACN:H2O (v:v=1:1).

[0124] The present invention is further described in detail and completely in the following examples, which are only used to illustrate specific embodiments of the present invention and should not be interpreted as limiting the scope of the present invention in any way.

[0125] Example 1

[0126] (1) Preparation method of free acid of compound of formula (I)

[0127] Step 1: Synthesis of (2R,3R,4R,5R)-2-(acetoxymethyl)-5-(4,6-dichloro-1H-pyrazolo[3,4-b]pyridin-1-yl)tetrahydrofuran-3,4-diyl diacetate

[0128] 4,6-Dichloro-1H-pyrazolo[3,4-b]pyridine (3.1 g, 16.49 mmol) was placed in hexamethyldisilazane (30 mL), and ammonium sulfate (22 mg, 0.16 mmol) was added. The reaction mixture was heated to 140°C and stirred for 3 hours. The hexamethyldisilazane was removed by rotary evaporation under reduced pressure, and the residue was dissolved in acetonitrile (60 mL). β-D-ribofuranose 1,2,3,5-tetraacetate (5.8 g, 18.14 mmol) was added. The reaction mixture was cooled to 0°C in an ice bath. Trimethylsilyl trifluoromethanesulfonate (5.5 g, 24.7 mmol) was added dropwise with stirring and cooling in an ice bath. The reaction mixture was slowly warmed to room temperature and stirred overnight. The acetonitrile was removed by rotary evaporation under reduced pressure below 30°C. The residue was diluted with ethyl acetate and washed with saturated sodium bicarbonate. The layers were separated, and the aqueous phase was extracted with ethyl acetate. The organic phases were combined and evaporated under reduced pressure. The residue was separated by column chromatography (ethyl acetate / petroleum ether = 15 / 85) to obtain (2R,3R,4R,5R)-2-(acetoxymethyl)-5-(4,6-dichloro-1H-pyrazolo[3,4-b]pyridin-1-yl)tetrahydrofuran-3,4-diyl diacetate (5.6 g, yield: 76%). MS m / z (ESI): 446.2, 448.2 [M+H] + .

[0129] Step 2: Synthesis of (2R,3R,4R,5R)-2-(acetoxymethyl)-5-(6-chloro-4-(((R)-6-fluoro-2,3-dihydro-1H-inden-1-yl)amino)-1H-pyrazolo[3,4-b]pyridin-1-yl)tetrahydrofuran-3,4-diyl diacetate

[0130] (2R,3R,4R,5R)-2-(Acetoxymethyl)-5-(4,6-dichloro-1H-pyrazolo[3,4-b]pyridin-1-yl)tetrahydrofuran-3,4-diyl diacetate (0.40 g, 0.90 mmol) was dissolved in N-methylpyrrolidone (2.0 mL), and (R)-6-fluoro-2,3-dihydro-1H-inden-1-amine hydrochloride (0.25 g, 1.35 mmol) and N,N-diisopropylethylamine (0.46 g, 3.60 mmol) were added. The reaction was stirred in a sealed tube at 100 ° C for 44.3 hours. After completion of the reaction, water was added for dilution, and the mixture was extracted with ethyl acetate. The organic phase was washed with water and concentrated. The crude product was separated by column chromatography (EA / PE = 4 / 6) to give (2R,3R,4R,5R)-2-(acetoxymethyl)-5-(6-chloro-4-(((R)-6-fluoro-2,3-dihydro-1H-inden-1-yl)amino)-1H-pyrazolo[3,4-b]pyridin-1-yl)tetrahydrofuran-3,4-diyl diacetate (0.44 g, yield 85%). MS m / z (ESI): 561.3 [M+H] + .

[0131] Step 3: Synthesis of (2R,3R,4S,5R)-2-(6-chloro-4-(((R)-6-fluoro-2,3-dihydro-1H-inden-1-yl)amino)-1H-pyrazolo[3,4-b]pyridin-1-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol

[0132] (2R,3R,4R,5R)-2-(Acetoxymethyl)-5-(6-chloro-4-(((R)-6-fluoro-2,3-dihydro-1H-inden-1-yl)amino)-1H-pyrazolo[3,4-b]pyridin-1-yl)tetrahydrofuran-3,4-diyl diacetate (0.44 g, 0.78 mmol) was dissolved in methanol (3.0 mL) and potassium carbonate (0.54 g, 3.90 mmol) was added and the reaction was stirred at room temperature for 1 hour. After the reaction was completed, methanol was concentrated under reduced pressure, and the crude product was separated by column chromatography (DCM / MEOH = 14 / 1) and concentrated to give (2R,3R,4S,5R)-2-(6-chloro-4-(((R)-6-fluoro-2,3-dihydro-1H-inden-1-yl)amino)-1H-pyrazolo[3,4-b]pyridin-1-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (0.27 g, yield 76%). MS m / z (ESI): 435.2 [M+H] + .

[0133] Step 4: Preparation of (((((2R,3S,4R,5R)-5-(6-chloro-4-(((R)-6-fluoro-2,3-dihydro-1H-inden-1-yl)amino)-1H-pyrazolo[3,4-b]pyridin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)hydroxyphosphino)methyl)phosphinocarboxylic acid

[0134] ((3aR,4R,6R,6aR)-6-(6-chloro-4-(((R)-6-fluoro-2,3-dihydro-1H-inden-1-yl)amino)-1H-pyrazolo[3,4-b]pyridin-1-yl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxazol-4-yl)methanol (0.27 g, 0.62 mmol) was dissolved in anhydrous trimethyl phosphate (3.0 mL), cooled to 0°C, and a solution of methylenebisphosphonic dichloride (0.62 g, 2.50 mmol) in trimethyl phosphate (2.0 mL) was slowly added dropwise. After the addition was complete, the reaction was continued at 0°C for 30 minutes. After completion, the reaction was quenched with water and stirred for 10 minutes. After that, the mixture was directly purified by reverse phase (H2O / ACN=4 / 1) to obtain the target compound (((((2R,3S,4R,5R)-5-(6-chloro-4-(((R)-6-fluoro-2,3-dihydro-1H-inden-1-yl)amino)-1H-pyrazolo[3,4-b]pyridin-1-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)hydroxyphosphino)methyl)phosphinocarboxylic acid (89.4 mg, yield 24%). MS m / z (ESI): 593.0 [M+H] + .

[0135] 1H NMR(400MHz,MeOH-d4)δ8.19(s,1H),7.28(dd,J=8.3,5.1Hz,1H),7.00(ddd,J=17.9,8.9,2.4Hz,2H),6.5 0(s,1H),6.33(d,J=3.7Hz,1H),5.32(t,J=7.3Hz,1H),4.71(dd,J=5.2,3.8Hz,1H),4.57(t,J=5.2Hz,1H) ,4.30(ddd,J=11.0,7.3,3.8Hz,1H),4.26–4.08(m,2H),3.05(ddd,J=16.1,8.7,3.9Hz,1H),2.93(dt,J=1 5.9,8.0Hz,1H),2.70(dtd,J=11.9,7.6,4.0Hz,2H),2.42(t,J=21.0Hz,2H),2.07(dq,J=12.9,8.0Hz,1H).

[0136] (2) Preparation method of pentasodium salt of compound of formula (I) (API:Na=1:5)

[0137] The free acid of the compound of formula (I) was dispersed in water, and an excess of aqueous sodium hydroxide was added. The mixture was stirred at room temperature until clear. Methanol was slowly added until solid precipitated. The mixture was stirred overnight at room temperature and the solid was collected. Ion chromatography confirmed that the molar ratio of API:Na was 1:5.

[0138] (3) Preparation method of trisodium salt of compound of formula (I) (API:Na=1:3)

[0139] Approximately 90 mg of the pentasodium salt of the compound of formula (I) was weighed and suspended in 0.6 mL of ethanol at 25°C for 2 days. The solid portion (suspension) was collected by filtration and dried at 22-25°C / 40-60% RH for approximately 2 hours to obtain 60 mg of the trisodium salt of the amorphous form of the compound of formula (I) as an off-white powder with a purity of 99.8% and a yield of 72%.

[0140] (4) Characterization of the properties of the pentasodium salt and trisodium salt of the compound of formula (I)

[0141] By HPLC, XRPD, DSC, TGA, 1 The relevant properties of the compound of formula (I) and its sodium salt obtained above were measured by H-NMR, IC, and KF as follows. The specific test methods are detailed in the definition:

[0142] Example 2

[0143] Weigh the required amount of the trisodium salt of the compound of formula (I) into a vial and add pH buffer to the target content according to the table below; adjust the solution to the target pH with HCl or NaOH and record the appearance; continue stirring at 25°C and 700 rpm and record the appearance after 24 hours; transfer the sample to a centrifuge tube and centrifuge, and analyze the pH value and content of the trisodium salt of the compound of formula (I) in the supernatant.

[0144] Table 2.1 Solubility of trisodium salt of compound of formula (I) in buffer solutions of different pH values ​​at 25°C

[0145] Experimental results:

[0146] The solubility study results of the trisodium salt of the compound of formula (I) in buffer solutions of different pH values ​​are shown in the table above. The results show that the trisodium salt of the compound of formula (I) exhibits good solubility (>97 mg / mL) in buffer solutions with a pH value range of 3 to 10.

[0147] Example 3

[0148] Weigh the required amount of the trisodium salt of the compound of formula (I) into a vial and add H2O to a 2 mg / mL stock solution. Dilute the stock solution to 1 mg / mL using the different pH buffers listed in the table below. If a clear solution is obtained, dispense the sample into vials, store the sample at 40°C, and test the purity and content of the trisodium salt of the compound of formula (I) at each time point.

[0149] Table 3.1 Stability of the trisodium salt of the compound of formula (I) in different pH buffers at 40°C

[0150] Experimental results:

[0151] The results of the stability study are shown in the table above, which show that the trisodium salt of the compound of formula (I) exhibits good chemical stability at pH 6-9, while at pH 3-4, some degradation occurs at 40°C over 14 days.

[0152] Example 4

[0153] Weigh the required amount of trisodium salt of the compound of formula (I) into a vial, add co-solvents to the target content of trisodium salt of the compound of formula (I) according to the table below, and record the appearance; maintain stirring at 25°C, and record the appearance after 24 hours; transfer the sample to a centrifuge tube and centrifuge, analyze the content of trisodium salt of the compound of formula (I) in the supernatant by HPLC, and characterize potential undissolved residues by XRPD.

[0154] Table 4.1 Solubility of trisodium salt of compound of formula (I) in cosolvents at 25°C

[0155] Experimental results:

[0156] The solubility results of the trisodium salt of the compound of formula (I) in co-solvents are shown in the table above. The results show that the trisodium salt of the compound of formula (I) exhibits good solubility in propylene glycol (up to 48 mg / mL), while it exhibits relatively low solubility (<5 mg / mL) in other co-solvents (N,N-dimethylacetamide, polyethylene glycol 400 and ethanol).

[0157] Example 5

[0158] Weigh the required amount of trisodium salt of the compound of formula (I) into a vial, and dissolve the trisodium salt of the compound of formula (I) in the carrier solution listed in the table below to 50 mg / mL; place the sample at 40°C and test the pH, osmotic pressure, content, and purity at each time point.

[0159] Table 5.1 Compatibility study results of the trisodium salt of the compound of formula (I) with different osmotic pressure regulators

[0160] Experimental results:

[0161] The compatibility study results of the trisodium salt of the compound of formula (I) with different osmotic pressure regulators are shown in the table above. The results show that the trisodium salt of the compound of formula (I) exhibits good stability with all osmotic pressure regulators (sodium chloride, mannitol, sucrose and glycerol).

[0162] Example 6

[0163] Weigh the required amount of the trisodium salt of the compound of formula (I) into a vial and add water to a 20 mg / mL stock solution. Dilute the stock solution to 0.5 mg / mL and 10 mg / mL using buffer solutions of varying pH values ​​and water. Filter the formulation and fill it into vials. Store the sample at 25°C and test the formulation for visible foreign matter at each time point.

[0164] Table 6.1

[0165] Experimental results:

[0166] The results of the effects of pH and concentration on visible foreign matter are shown in the table above. The results show that all solutions of the trisodium salt of the compound of Formula (I) exhibited visible foreign matter at a concentration of 10 mg / mL, while no foreign matter was visible at a concentration of 0.5 mg / mL at pH values ​​between 5.0 and 8.0. Therefore, lower concentrations help prevent the formation of visible foreign matter.

[0167] Example 7

[0168] The required amount of trisodium salt of the compound of formula (I) was weighed into a vial, and the trisodium salt of the compound of formula (I) was dissolved in the carrier solution shown in the table below to a target content of 10 mg / mL. The preparation was then filtered and filled into vials. The sample was placed at 25°C, and the preparation was tested for visible foreign matter at each time point.

[0169] Table 7.1

[0170] Experimental results:

[0171] The results of the effects of stabilizers on visible foreign matter are summarized in the table below. The experiments show that the presence of disodium EDTA and polysorbate 20 appears to help prevent the formation of visible foreign matter. These stabilizers will be used in subsequent stability studies.

[0172] Example 8

[0173] Weigh the required amount of the trisodium salt of the compound of formula (I) into a vial and dissolve the trisodium salt of the compound of formula (I) in the carrier solution listed below to a target concentration of 10 mg / mL. Then, filter the formulation and fill it into vials. Store the samples at 4°C, 25°C, and 40°C, and test the formulation for visible foreign matter at each time point.

[0174] Table 8.1

[0175] Table 8.2

[0176] Experimental results:

[0177] The results of a study on visible foreign matter in different buffer solutions of stabilizers are detailed in Tables 8.1 and 8.2. The results show that visible foreign matter was present in F1-F7 at various stages of the experiment. However, no foreign matter was observed in F8 (0.02% disodium EDTA dissolved in pH 9.0 carbonate buffer) and F9-F11 (0.5% / 1.0% / 0.5% polysorbate 20 dissolved in pH 6.0 citrate buffer), suggesting that the buffer solution may have contributed to this.

[0178] Therefore, based on the results of studies F1 to F8, the trisodium salt of the compound of formula (I) exhibits a better visible foreign matter effect in a buffer solution with a pH value in the range of 7 to 9.

[0179] Based on the research results of F8 and F9-F11, an appropriate ratio of polysorbate 20 and disodium edetate can be selected as stabilizers, and citrate buffer is recommended for subsequent formulation screening studies.

[0180] Example 9

[0181] The required amount of trisodium salt of the compound of formula (I) was weighed into a vial, and the trisodium salt of the compound of formula (I) was dissolved in the carriers listed in Table 9.1 below to prepare the target content. The pH was adjusted, and the preparation was filtered and filled into vials. The samples were placed at -20°C, 2-8°C, and 25°C. The preparation was tested for osmotic pressure, pH, content, purity, and visible foreign matter at each time point.

[0182] Table 9.1 Preparations with different formulations

[0183] Table 9.2 Summary of formulation screening results

[0184] Table 9.3 Summary of 1-week stability test results of formulations under different test conditions

[0185] Table 9.4 Summary of 2-week stability test results of formulations under different test conditions

[0186] Table 9.5 Summary of 4-week stability test results of formulations under different test conditions

[0187] Experimental results:

[0188] The stability results of the trisodium salt preparation screening of the compound of formula (I) are shown in Tables 9.1 to 9.5 above. The results show that C1 to C10 are stable in terms of pH, osmotic pressure, content, purity and insoluble particulate matter.

[0189] Formulation screening was performed based on visible foreign matter. A formulation containing disodium EDTA and the surfactant polysorbate 20 appeared to help prevent the formation of visible foreign matter. C8 was selected as the primary prototype for the following studies.

[0190] Example 10

[0191] The desired amount of the trisodium salt of the compound of formula (I) was weighed into a vial. The trisodium salt of the compound of formula (I) was dissolved in the carriers listed in the table below to the target concentration. The pH was adjusted. The formulation was then filtered and filled into vials. The samples were placed at 40°C and tested for content, purity, and visible foreign matter at each time point. The formulation was placed at -20°C for 1 day, then at 25°C for 2 hours. The formulation was then inspected for visible foreign matter for a third cycle.

[0192] Table 10.1

[0193] Experimental results:

[0194] The experimental results are summarized in the table above. The results show that the optimal content of the trisodium salt of the compound of formula (I) in the formulation is in the range of 0.5 mg / mL to 20 mg / mL.

[0195] Example 11

[0196] The required amount of trisodium salt of the compound of formula (I) was weighed into a glass bottle, and the trisodium salt of the compound of formula (I) was dissolved with the carriers listed in the table below to prepare a target content of 10 mg / mL. The sample was placed at 40°C, and the content and purity were tested at each time point.

[0197] Table 11.1

[0198] Experimental results:

[0199] Based on the pH range validation study, all formulations showed good chemical stability within the pH range of 6.0 to 8.5.

[0200] Example 12

[0201] Follow the steps below to prepare the formula in the table below.

[0202] 1) Weigh the 250 mL bottle and record the weight;

[0203] 2) Add 85 mL of water to the bottle;

[0204] 3) Weigh and add 485.383 mg of sodium citrate dehydrate to the solution. Add 67.17 mg of citric acid to the solution.

[0205] 4) Add 20 mg of disodium edetate and mix well;

[0206] 5) Add 500 mg of polysorbate 20 and mix well;

[0207] 6) Weigh 444.6 mg of the trisodium salt of the compound of formula (I) corresponding to 400 mg of the free acid into a 250 mL bottle;

[0208] 7) Weigh 7 g of sucrose into a 250 mL bottle and mix thoroughly;

[0209] 8) detecting the pH value in step 7;

[0210] 9) Adjust the pH to 7.50 ± 0.2 with 0.1 M NaOH or 0.1 M HCl if necessary, and record the volume of acid or base added;

[0211] 10) Weigh the vial after step 9 and add purified water to make the solution 100 mL;

[0212] 11) Testing the pH and osmotic pressure of the final solution;

[0213] 12) Filter through a 0.2 μm filter and place in a vial;

[0214] 13) Test the product's pH, appearance, osmotic pressure, content, viscosity, insoluble particles, density and purity;

[0215] 14) Test for visible foreign matter at 40°C / 75% RH, 25°C / 75% RH, and 2-8°C.

[0216] Table 12.1 Injection Preparation Composition

[0217] Table 12.2 Evaluation of properties of preparation products

[0218] Table 12.3 Appearance inspection results of preparations under different storage conditions

[0219] From the above, it can be seen that the properties of the 4 mg / mL preparation are stable, and no visible foreign matter appears after storage for 2 weeks under different conditions.

[0220] Example 13

[0221] The 4 mg / mL formulation obtained in Example 12 was diluted with 5% glucose solution and saline. The pH, appearance, and osmotic pressure were recorded at each time point. The results of the formulation dilution test are recorded in the table below.

[0222] Table 13.1 Dilution results of preparations

[0223] Experiments have shown that the 4 mg / mL preparation is stable within 24 hours after being diluted with 5% glucose solution and normal saline, ensuring the stability and safety of clinical use.

Claims

1. A CD73 pharmaceutical composition, characterized in that: The pharmaceutical composition comprises an active ingredient, a pH buffer and water for injection; wherein the active ingredient is the free acid of the compound of formula (I) or its trisodium salt, and the content is 0.01-50 mg / ml.

2. The pharmaceutical composition according to claim 1, characterized in that The active ingredient is trisodium salt of the compound of formula (I), and the content is 0.01-30.0 mg / ml; Preferably, the active ingredient is the trisodium salt of the compound of formula (I), and the content is 0.1 to 20.0 mg / ml.

3. The pharmaceutical composition according to claim 1, characterized in that The pH value of the pharmaceutical composition is 3.0 to 10.0; Preferably, the pH value of the pharmaceutical composition is 5.0 to 9.0; More preferably, the pH value of the pharmaceutical composition is 5.0-8.0 or 6.0-9.

0.

4. The pharmaceutical composition according to claim 1, characterized in that The pH buffer is selected from citrate buffer, phosphate buffer, carbonate buffer or histidine buffer; preferably, the pH buffer solvent is selected from citrate buffer.

5. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutical composition further comprises an osmotic pressure regulator, and the osmotic pressure regulator is selected from one or more of sodium chloride, mannitol, sucrose and glycerol.

6. The pharmaceutical composition according to claim 5, characterized in that The content of the osmotic pressure regulator is 0.1% to 10% of the total weight of the pharmaceutical composition; Preferably, the content of the osmotic pressure regulator is 0.5-10% of the total weight of the pharmaceutical composition; Preferably, the content of the osmotic pressure regulator is 1 to 10% of the total weight of the pharmaceutical composition; Preferably, the content of the osmotic pressure regulator is 5-10% of the total weight of the pharmaceutical composition.

7. The pharmaceutical composition according to claim 1, characterized in that The content of the active ingredient is 0.01-2.0 mg / ml, and the pH value of the pharmaceutical composition is 3.0-9.

0.

8. The pharmaceutical composition according to claim 7, characterized in that The content of the active ingredient is 0.01-1.0 mg / ml, and the pH value of the pharmaceutical composition is 5.0-8.

0.

9. The pharmaceutical composition according to claim 7, characterized in that The content of the active ingredient is 0.1-0.5 mg / ml, and the pH value of the pharmaceutical composition is 5.0-8.

0.

10. The pharmaceutical composition according to claim 1, characterized in that The content of the active ingredient is 0.01-30.0 mg / ml, and the pH value of the pharmaceutical composition is 3.0-10.0; the pharmaceutical composition further comprises a pharmaceutically acceptable stabilizer.

11. The pharmaceutical composition according to claim 10, characterized in that The content of the active ingredient is 0.01-25.0 mg / ml, and the pH value of the pharmaceutical composition is 5.0-9.0; the pharmaceutical composition further includes a pharmaceutically acceptable stabilizer, and the pharmaceutically acceptable stabilizer is selected from one or more of a cosolvent, a surfactant, a cyclodextrin and a chelating agent.

12. The pharmaceutical composition according to claim 10, characterized in that The content of the active ingredient is 0.01 to 20.0 mg / ml, and the pH value of the pharmaceutical composition is 6.0 to 9.0; the pharmaceutical composition further includes a pharmaceutically acceptable stabilizer, and the pharmaceutically acceptable stabilizer is selected from one or more of a cosolvent, a surfactant, a cyclodextrin and a chelating agent; the cosolvent is selected from one or more of propylene glycol, N,N-dimethylacetamide, polyethylene glycol 400 and ethanol; the surfactant is selected from one or more of polysorbate 80, polysorbate 20, polyoxyethylene castor oil and polyethylene glycol-15 hydroxystearate; the cyclodextrin is one or more of hydroxypropyl-β-cyclodextrin or sulfobutyl ether β-cyclodextrin; and the chelating agent is disodium ethylenediaminetetraacetic acid.

13. The pharmaceutical composition according to claim 12, characterized in that The pharmaceutically acceptable stabilizer is polysorbate 20 and / or disodium edetate; The content of polysorbate 20 is 0.001% to 10% of the total weight of the pharmaceutical composition; The content of disodium edetate is 0.001-1% of the total weight of the pharmaceutical composition; Preferably, the content of polysorbate 20 is 0.01-2% of the total weight of the pharmaceutical composition; The content of disodium edetate is 0.01-0.5% of the total weight of the pharmaceutical composition; Preferably, the content of polysorbate 20 is 0.1-2% of the total weight of the pharmaceutical composition; The content of disodium edetate is 0.01-0.2% of the total weight of the pharmaceutical composition.

14. The pharmaceutical composition according to claim 10, characterized in that The pharmaceutical composition comprises an active ingredient, the content of the active ingredient is 0.01-0.5 mg / ml, the content of the osmotic pressure regulator is 5%-10% of the total weight of the pharmaceutical composition, and the pH value of the pharmaceutical composition is 5.0-8.0; or, The content of the active ingredient is 0.1-20 mg / ml, and the pH value of the pharmaceutical composition is 6.0-9.0; the pharmaceutically acceptable stabilizer is polysorbate 20 and / or disodium edetate; the content of polysorbate 20 is 0.01%-2% of the total weight of the pharmaceutical composition; the content of disodium edetate is 0.01%-0.5% of the total weight of the pharmaceutical composition, and the content of the osmotic pressure regulator is 0.1%-10% of the total weight of the pharmaceutical composition; Preferably, the content of the active ingredient is 0.5-20 mg / ml, the pH value of the pharmaceutical composition is 6.0-8.5, and the pharmaceutically acceptable stabilizer is selected from polysorbate 20 and / or disodium edetate; the content of polysorbate 20 is 0.1%-2% of the total weight of the pharmaceutical composition; the content of disodium edetate is 0.01-0.2% of the total weight of the pharmaceutical composition, and the content of the osmotic pressure regulator is 5%-10% of the total weight of the pharmaceutical composition.

15. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutical composition comprises an active ingredient, and the content of the active ingredient is 0.01 mg / ml, 0.5 mg / ml, 0.8 mg / ml, 1.0 mg / ml, 4.0 mg / ml, 5.0 mg / ml, 10.0 mg / ml or 20.0 mg / ml.

16. The pharmaceutical composition according to claim 1, characterized in that The pH value of the pharmaceutical composition is 5.0, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 or 9.

0.

17. A CD73 drug lyophilized powder, characterized in that: The pharmaceutical composition according to claim 1 is freeze-dried to obtain a freeze-dried powder.

18. A method for preparing the CD73 pharmaceutical composition according to claim 1, comprising the following steps: Step 1: dissolving the active ingredient, pH buffer, and optional osmotic pressure regulator in water for injection to prepare a mixed solution; Step 2: adjusting the pH value of the mixed solution to 3-10; optionally, Step 3: Add water for injection to the target content of active ingredient, pH buffer, and osmotic pressure regulator, and filter to obtain a pharmaceutical composition; The pH buffer is selected from citrate buffer, phosphate buffer, carbonate buffer and histidine buffer; preferably, the pH buffer solvent is selected from citrate buffer; The osmotic pressure regulator is selected from one or more of sodium chloride, mannitol, sucrose and glycerol.

19. The preparation method according to claim 18, characterized in that: When the content of the active ingredient in step 1 is 0.01-1.0 mg / ml, the pH value of the pharmaceutical composition is adjusted to 5.0-8.0 in step 2; or, When the content of the active ingredient in step 1 is 0.01 to 20.0 mg / ml, Step 1 comprises dissolving a pharmaceutically acceptable stabilizer in water for injection, wherein the pharmaceutically acceptable stabilizer is selected from one or more of a co-solvent, a surfactant, a cyclodextrin and a chelating agent. In step 2, the pH value of the pharmaceutical composition is adjusted to 6.0 to 9.

0.

20. Use of the pharmaceutical composition of claim 1 in the preparation of a medicament for treating tumors, immune-related diseases and disorders or metabolic diseases at least partially mediated by CD73.

21. The use according to claim 20, characterized in that The tumor is selected from the group consisting of prostate cancer, colon cancer, rectal cancer, pancreatic cancer, gastric cancer, endometrial cancer, cervical cancer, brain cancer, liver cancer, bladder cancer, ovarian cancer, testicular cancer, head cancer, neck cancer, skin cancer, mesothelial lining cancer, white blood cell cancer, esophageal cancer, breast cancer, muscle cancer, connective tissue cancer, lung cancer, adrenal cancer, thyroid cancer, kidney cancer, bone cancer, brain tumor, glioblastoma, mesothelioma, renal cell carcinoma, sarcoma, choriocarcinoma, epidermal basal cell carcinoma and testicular seminoma; Preferably, the tumor is selected from the group consisting of skin cancer, colon cancer, pancreatic cancer, breast cancer, prostate cancer, lung cancer, leukemia cancer, brain tumor, ovarian cancer and sarcoma.

22. The use according to claim 20, characterized in that The immune-related diseases and disorders are selected from rheumatoid arthritis, renal failure, lupus erythematosus, asthma, psoriasis, ulcerative colitis, pancreatitis, allergies, fibrosis, anemia fibromyalgia, Alzheimer's disease, congestive heart failure, stroke, aortic stenosis, arteriosclerosis, osteoporosis, Parkinson's disease, infection, Crohn's disease, ulcerative colitis, allergic contact dermatitis and eczema, systemic sclerosis and multiple sclerosis.

23. The pharmaceutical composition of claim 1 for use as a medicament for treating tumors, autoimmune diseases and disorders or metabolic diseases mediated at least in part by CD73.

24. The pharmaceutical composition according to claim 23, characterized in that The tumor is selected from prostate cancer, colon cancer, rectal cancer, pancreatic cancer, gastric cancer, endometrial cancer, cervical cancer, brain cancer, liver cancer, bladder cancer, ovarian cancer, testicular cancer, head cancer, neck cancer, skin cancer, mesothelial lining cancer, white blood cell cancer, esophageal cancer, breast cancer, muscle cancer, connective tissue cancer, small cell lung cancer, adrenal cancer, thyroid cancer, kidney cancer, bone cancer, brain tumor, glioblastoma, mesothelioma, renal cell carcinoma, sarcoma, choriocarcinoma, epidermal basal cell carcinoma and Testicular seminoma; the immune-related diseases and disorders are selected from rheumatoid arthritis, renal failure, lupus erythematosus, asthma, psoriasis, ulcerative colitis, pancreatitis, allergies, fibrosis, anemia fibromyalgia, Alzheimer's disease, congestive heart failure, stroke, aortic valve stenosis, arteriosclerosis, osteoporosis, Parkinson's disease, infection, Crohn's disease, ulcerative colitis, allergic contact dermatitis and eczema, systemic sclerosis and multiple sclerosis.

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