Solutions, freeze-dried formulations, unit packages of freeze-dried formulations, injectable formulations, and methods for preparing injectable formulations.

Abstract

The present invention provides a TH-302 solution, a freeze-dried formulation, a unit package of the freeze-dried formulation, an injectable formulation, and a method for preparing the injectable formulation. The solution for preparing a freeze-dried formulation with a high drug loading capacity contains TH-302, water, tert-butyl alcohol, and mannitol. Water and tert-butyl alcohol are used as a mixed solvent, and the volume percentage of tert-butyl alcohol in the solution is (30±3)%, or the mass percentage is (24±2.4)%, or the content of tert-butyl alcohol in the solution is (235.5±23.55) mg / mL. The content of TH-302 in the solution is (8.16±0.82) mg / g or (8.00±0.80) mg / mL. Mannitol is used as an excipient, and the mass percentage of mannitol in the solution is (7.14±0.71)%, or the content of mannitol in the solution is (70±7) mg / mL. The pH of the solution is 4-9. The freeze-dried formulation contains TH-302 and mannitol. The drug loading dose of TH-302 in the freeze-dried formulation is (8.00 ± 0.80) mg / cm³. 3 The mass percentage of TH-302 in the freeze-dried formulation is (10.23±1.02)%. The mannitol content in the freeze-dried formulation is (70±7) mg / cm³. 3 Alternatively, the mannitol content in the freeze-dried formulation is the percentage remainder of (10.23±1.02)% by mass of TH-302.

Description

Technical Field

[0001] The present invention relates to the development of a freeze-dried preparation containing TH-302 and belongs to the technical field of pharmaceutical preparations.

Background Art

[0002] TH-302, that is, a prodrug of 2-nitroimidazole, was designed and synthesized in 2006 by researchers of Threshold Pharmaceuticals Inc. and is a selective hypoxia-activated DNA alkylating agent having high cytotoxicity (International Publication No. 2007002931, Phosphoramidate alkylator prodrugs). TH-302 can be converted into dibromo-isophosphoramide chloromethine having alkylating agent activity in the hypoxic region of a tumor or by activation with an acid, but is almost inactive under conditions of normal oxygen pressure or normal pH.

[0003] Researchers of Threshold Pharmaceuticals Inc. designed and developed preliminary freeze-dried preparations and injection preparations in 2007 (International Publication No. 2008083101, Phosphoramidate alkylator prodrugs for the treatment of cancer) and administered them in a Phase I clinical trial.

[0004] A solution (20 mL) of TH-302 (100 mg) and sucrose (1 g) was added to a freeze-dry vial, freeze-dried, and a freeze-dried unit dosage of TH-302 having a drug loading capacity of less than 5 mg / cm 3 was produced. For administration to humans, this unit dosage was dissolved in a 5% glucose injection and an appropriate amount of this solution was administered to patients.

[0005] A pharmaceutically acceptable liquid formulation containing 5% TH-302 was prepared by dissolving TH-302 in anhydrous ethanol. As used herein, a 5% TH-302 solution contains 5 g of TH-302 in 100 mL of solvent (e.g., ethanol).

[0006] In the Phase I clinical trial of TH-302, freeze-dried formulations were used in the follow-up dosing schedule for human patients. The freeze-dried TH-302 formulation for injection was prepared in 100 mL glass vials with a drug loading volume of 100 mg / 100 mL and stored at a controlled temperature of 2–8°C. For use, 250 mL of 5% glucose was injected into the vial containing the freeze-dried formulation, and the drug was administered intravenously via an infusion pump within 30 minutes.

[0007] Researchers at Threshold Pharmaceuticals designed and developed the injectable formulation in 2009 (International Publication No. 2010048330, Treatment of cancer using hypoxia activated prodrugs). Liquid formulations containing TH-302 in a concentration of 50 mg / mL to approximately 300 mg / mL, a nonionic surfactant (such as Tween 80), and ethanol as a carrier may further contain dimethylacetamide.

[0008] Clinical trials began in 2007 and have continued to the present, covering a wide range of solid tumors and hematological malignancies. The treatment regimens are also diverse, with various doses of TH-302 being used alone or in combination with other cancer treatments. Currently, there are 27 clinical trials in the United States using TH-302 as a treatment for various cancers and tumors (NCT02402062, NCT02020226, NCT02076230, NCT01381822, NCT02093962, NCT01440088, NCT02255110, NCT02342379, NCT01864538, NCT01149915, NCT02433639, NCT00 (0743379, NCT01485042, NCT01721941, NCT02047500, NCT00742963, NCT01497444, NCT00495144, NCT01746979, NCT01144455, NCT01403610, NCT01522872, NCT01833546, NCT02598687, NCT03098160, NCT02496832, and NCT02712567). These clinical trials have shown TH-302 to be a broad-spectrum anticancer drug candidate.

[0009] Based on the results of the aforementioned Phase I / II clinical trial, researchers at Threshold Pharmaceuticals found that the effective dose of TH-302 for treating various indications is as follows (International Publication No. 2012135757, Methods for treating cancer). The daily dose administered intravenously is 120 mg / m². 2 ~460 mg / m² 2 And, The weekly dose administered intravenously is 480 mg / m². 2 ~about 670mg / m 2 , or for example, 575 mg / m² 2 That is the case.

[0010] According to the above effective dose, for an average person (height 175cm, weight 75kg), the corresponding equivalent body surface area (BSA) (m²) is...2 ) = ([height (cm) × weight (kg)] / 3600) 1 / 2 = 1.90, so the corresponding dose is 228 - 1273 mg! In this case, when using a freeze-dried preparation with a drug loading capacity of less than 5 mg / cm 3 If a freeze-dried preparation with a drug loading capacity of less than 5 mg / cm is used (20 mL of an aqueous solution containing 100 mg of TH-302 and 1 g of sucrose is added to a 50 mL freeze-dry vial and freeze-dried to produce a freeze-dried unit dosage of TH-302, the drug loading capacity is less than 5 mg / cm 3 less), at least 3 vials are required, and when using a higher dose, 13 vials are required. This is inconvenient for clinical use and also involves an excessively high drug treatment cost for patients.

[0011] For the above reasons, in most of the subsequent Phase II / III clinical trials, researchers at Threshold Pharmaceuticals have used concentrated injections (International Publication No. 2015013448, Treatment of pancreatic cancer with a combination of a hypoxia-activated prodrug and a taxane).

[0012] TH-302 (concentrate for administration) for use in clinical trials is a sterile liquid formulation of TH-302. It is formulated using 70% anhydrous ethanol, 25% dimethylacetamide, and 5% polysorbate 80. The sterile liquid formulation is supplied by the provider in a 10 mL glass vial with a rubber stopper and flip-off seal. The TH-302 drug is a colorless to pale yellow clear solution and is essentially free of visible particulate matter. Each single-use vial contains a nominal filling volume of 6.5 mL of the TH-302 drug (corresponding to 100 mg / mL) when the nominal total amount of TH-302 is 650 mg, and is clearly labeled. The label includes the lot number, route of administration, required storage conditions, provider's name, and any appropriate cautionary statements required by applicable regulations. Dilution is required before administration according to pharmacy manuals.

[0013] The TH-302 drug is diluted before each administration with a commercially available 5% glucose aqueous solution to a total volume of 500 mL (1000 mL if the total dose is ≥1000 mg) to obtain the desired final concentration. Each dose of TH-302 is prepared using a 5% glucose aqueous solution (DEHP-free) that does not contain di(2-ethylhexyl)phthalate (DEHP) and administered by intravenous infusion using a DEHP-free intravenous administration device.

[0014] Clearly, in clinical trials, using a concentrated injectable formulation with a drug loading capacity of 100 mg / mL is more effective than using a drug loading capacity of 5 mg / cm³. 3 It is more convenient than using a freeze-dried formulation of less than 100 mL gauge. In the former case, one 10 mL gauge vial is sufficient to meet the drug therapy requirements of most patients, whereas in the latter case, 13 vials of the commonly used 100 mL gauge freeze-dried formulation are needed to meet the drug therapy requirements of most patients! However, researchers at Threshold Pharmaceuticals found that the aforementioned concentrated injectable formulation with a drug loading dose of 100 mg / mL (i.e., a concentrated injectable formulation of TH-302 prepared with 70% anhydrous ethanol, 25% dimethylacetamide, and 5% polysorbate 80) contained a large amount of dimethylacetamide. This adjuvant, which can increase drug solubility and improve the stability of the injectable formulation, is prone to causing allergic reactions after injection into the human body (International Publication No. 2015013448, Treatment of pancreatic cancer with a combination of a hypoxia-activated prodrug and a taxane).

[0015] Administration reactions to TH-302 (primarily induced by dimethylacetamide) have been observed. These reactions are characterized by lip swelling and urticaria in response to steroid and antihistamine treatment. It is recommended to include steroids such as dexamethasone (or equivalent) in the pre-administration antiemetic regimen. Symptoms and signs of hypersensitivity include fever, muscle pain, headache, rash, itching, urticaria, angioedema, chest discomfort, dyspnea, cough, cyanosis, and hypotension. Depending on the nature and severity of the above reactions, if discontinuation of treatment is necessary, it should be determined whether the reaction may be an immunoglobulin E-mediated process. If symptoms such as upper airway obstruction or hypotension suggestive of anaphylaxis or an anaphylactic-like reaction occur, researchers should consider treatment with antihistamines (e.g., diphenhydramine 25-50 mg orally, intramuscularly, or slowly intravenously, or equivalent) and low-dose steroids (e.g., hydrocortisone, 100 mg intravenously, or equivalent) as needed. If the event is clearly anaphylaxis, epinephrine (1 / 1000, 0.3-0.5 mL subcutaneously, or equivalent) should also be considered, as with standard treatment. In cases of bronchospasm, inhaled β-agonists should be considered. Allergic reactions may also be treated with antihistamines and low-dose steroids, depending on the severity. Responses to TH-302 administration should be evaluated and treated in a similar manner. For all responses to TH-302, researchers should consult with a medical monitor to determine appropriate course of action for future treatment.

[0016] The high-concentration TH-302 injection has resolved the issue of low drug loading doses in freeze-dried formulations. However, the use of the aforementioned adjuvants, which may cause adverse reactions, could lead to related adverse reactions in clinical trials, potentially increasing the risk of drug treatment for patients.

[0017] Freeze-dried formulations developed to date by dissolving TH-302 in a solution of water and sucrose and then freeze-drying them do not contain other adjuvants. However, because the drug content of the solution before freeze-drying is too low, it is impossible to obtain freeze-dried formulations with a high drug loading capacity that can meet the requirements for use in clinical trials and subsequent commercial production or sales.

[0018] In order to actually solve the aforementioned problems, it is still necessary for engineers in this field to develop freeze-dried formulations of TH-302 with high drug loading capacity, as well as solutions for such freeze-dried formulations. [Overview of the Initiative]

[0019] As a result of numerous experiments and continuous optimization, the inventors of this invention have proposed the preparation of a new high-concentration drug solution for producing freeze-dried formulations containing TH-302 and other similar drugs, as well as related freeze-dried formulations and methods thereof.

[0020] To facilitate understanding of the essence of this invention, the inventors' research process is briefly described below.

[0021] Screening for appropriate combinations of solvent and adjuvant to achieve high concentration [ka] To prepare the (TH-302) solution, the inventors initially considered modifying a concentrated injectable formulation of TH-302 with a drug load of 100 mg / mL developed by Threshold Pharmaceuticals, which was formulated using 70% anhydrous ethanol, 25% dimethylacetamide, and 5% polysorbate 80.

[0022] When anhydrous ethanol and water were used directly as solvents and sucrose was added, it was found that the resulting solution could not be freeze-dried, sometimes yielding only viscous oil, and the drug content of the solution only increased to a limited extent, failing to meet the requirements. Therefore, the researchers abandoned the simple ethanol + water scheme.

[0023] When using anhydrous ethanol and water directly as solvents, and adding sucrose or other excipients, and adjusting the type and amount of solubilizers added (such as PEG, Tween, and Span), it was found that solubility could be increased by adding small amounts of solubilizer, but the increase was very limited. Therefore, it is not possible to meet the requirements for high drug loading volumes.

[0024] The researchers also studied the solubility of TH-302 in various conventional single buffer solutions, considering that pH value can affect solubility. It was found that the pH value of the above solutions had little effect on the solubility of TH-302. Adjusting the pH value does not improve the solubility of TH-302 in aqueous solutions. Table 1 below shows the solubility data of TH-302 in aqueous solutions with various pH values ​​and various additives.

[0025] [Table 1]

[0026] Note: The ratios in the expressions "1% ethanol aqueous solution," "20% ethanol aqueous solution," "1% N,N-dimethylacetamide aqueous solution," "1% polyethylene glycol aqueous solution," and "1% Tween 80 aqueous solution" are all volume-based ratios.

[0027] Through numerous experiments, the inventors confirmed that, according to the threshold, simply using ethanol, aqueous solutions of various pH values, or ethanol + water as solvents is insufficient to develop a highly solubility solution that meets the requirements for freeze-drying a TH-302 freeze-dried formulation with a high drug loading capacity.

[0028] After numerous trials, the inventors found that the solubility of TH-302 is low in water or tert-butanol alone. However, surprisingly, the inventors found that the solubility of TH-302 is significantly increased in a mixed solvent of water and tert-butanol, and that the solubility is quite dependent on the mixing ratio of the solvents. Table 2 below shows the solubility data of TH-302 in various mixed solvents of tert-butanol + water or tert-butanol + ethanol.

[0029] [Table 2]

[0030] Note: The ratios of mixed solvents in Table 2 refer to volume ratios, and TH-302 is commercially purchased.

[0031] Tert-butanol is a colorless crystalline substance that is easily supercooled and becomes liquid in the presence of a small amount of water. It has a camphor-like odor and is hygroscopic. Its Chinese names include 2-methyl-2-propanol, tert-butanol, and trimethylmethanol. Since tert-butanol has a melting point of 25.7°C, it is a colorless, transparent liquid or colorless crystal at room temperature.

[0032] Generally speaking, tert-butanol has the following properties: 1. High freezing point. Pure tert-butanol may crystallize at room temperature (25°C), and after mixing with water, it can be frozen at several degrees below freezing. Both pure tert-butanol and mixtures of tert-butanol and water can be completely frozen in existing freeze-dryers. 2. Tert-butanol has a relatively high vapor pressure. A high vapor pressure is advantageous for sublimation, resulting in a shorter freeze-drying time. 3. Tert-butanol can be mixed with water in any ratio. This is extremely important because it can increase the solubility of some lipid-soluble drugs in water. For some drugs that are unstable in aqueous solutions, adding an appropriate amount of tert-butanol can suppress drug degradation and improve their stability. 4. Tert-butanol is easily freeze-dried and has a low residual content in formulations. During freeze-drying, most of the tert-butanol can sublimate in a single drying step, resulting in a very low residual content in formulations. 5. Tert-butanol can alter the crystallization mode of a solute and accelerate sublimation by forming needle-shaped crystals on its own during freezing. When a small amount of tert-butanol is added to water, the resulting tert-butanol-water cosolvent can alter the crystallization state of water, and the needle-shaped crystals formed during freezing have a large surface area. Sublimation of the ice crystals leaves behind tubular channels, which greatly reduce the flow resistance to water vapor and significantly increase the sublimation rate. Therefore, tert-butanol can be used to accelerate mass transfer during the freeze-drying process.

[0033] From the above-mentioned properties, tert-butanol + water can be used as a solvent to prepare a solution containing a high concentration of the compound of formula I, and tert-butanol itself is suitable for use as an adjuvant for freeze-drying due to its inherent properties.

[0034] From the results for the tert-butanol-water mixed solvent shown in Table 2, it can be inferred that TH-302 has relatively low solubility in aqueous solutions, and that the solubility of this active pharmaceutical ingredient increases as the concentration of tert-butanol in the tert-butanol aqueous solution increases, reaching the highest solubility in a 70% tert-butanol aqueous solution (V / V), and that its solubility decreases further as the concentration of tert-butanol in the tert-butanol aqueous solution increases.

[0035] In view of this, the present inventors proposed a scheme to obtain a solution having a high concentration of TH-302 by using tert-butanol + water as the solvent according to the present invention and adding appropriate (one or more) excipients. Furthermore, they developed a freeze-dried formulation of TH-302 with a high drug loading capacity, or an analogue based on this formulation.

[0036] The inventors further conducted experiments to investigate the effect of solvents containing tert-butanol in various volume ratios on the solubility of TH-302. The obtained additional solubility data are shown in Table 3 below.

[0037] [Table 3]

[0038] Note: The ratios of the mixed solvents in Table 3 refer to mass ratios, and TH-302 was synthesized by the applicants in small quantities.

[0039] Based on the above preliminary experiments, the present invention provides the following high-concentration solutions containing TH-302 or its analogues.

[0040] The present invention also relates to a solution comprising the compound of the following formula I, water, and tert-butanol, [ka] In the formula, R is independently selected from H, -CH3, and -CH2CH3, and X is independently selected from Br, Cl, OMs, and OTs. Water and tert-butanol are used as the mixed solvent. The content of the compound of formula I in the aforementioned solution is 1 mg / mL or more and 500 mg / mL or less. Provide the solution.

[0041] Furthermore, the present invention also relates to a solution for preparing a freeze-dried formulation with a high drug loading capacity, wherein the solution comprises a compound of the following formula I, water, tert-butanol, and an excipient. [ka] In the formula, R is independently selected from H, -CH3, and -CH2CH3, and X is independently selected from Br, Cl, OMs, and OTs. Water and tert-butanol are used as the mixed solvent. The content of the compound of formula I in the aforementioned solution is 5 mg / mL or more and 500 mg / mL or less. Provide the solution.

[0042] Preferably, the content of the compound of formula I in the solution is 5 mg / mL or more and 160 mg / mL or less. Preferably, the content of the compound of formula I in the solution is 8 mg / mL or more and 50 mg / mL or less. Preferably, the content of the compound of formula I in the solution is 8 mg / mL or more and 25 mg / mL or less. More preferably, the content of the compound of formula I in the solution is 8 mg / mL or more and 15 mg / mL or less. More preferably, the content of the compound of formula I in the solution is 8 mg / mL or more and 10 mg / mL or less.

[0043] Since water and tert-butanol can be miscible in any ratio, in order to improve the solubility of TH-302 and other similar compounds, the volume percentage of tert-butanol in the solution is 1-99%, preferably 5-95%, more preferably 30-60%, or The tert-butanol content in the above solution is 7.85 to 777.15 mg / mL, preferably 39.25 to 745.75 mg / mL, and more preferably 235.5 to 471 mg / mL.

[0044] Here, a volume percentage of tert-butanol from 1% to 99% of the solution corresponds to a tert-butanol content of 7.85 to 777.15 mg / mL in the solution. Specifically, when the volume percentage of tert-butanol in the solution is 1%, the tert-butanol content in the solution is 7.85 mg / mL. The conversion factor is the density of tert-butanol. Here, the density of the tert-butanol used by the applicant is 0.785 g / mL. In practice, the density of tert-butanol products from various manufacturers varies at various temperatures and is generally between 0.775 and 0.786 g / mL.

[0045] The solution for preparing a freeze-dried formulation with a high drug loading capacity according to the present invention comprises at least one excipient.

[0046] The term "pharmaceutically acceptable excipient" refers to any additive or carrier that may contribute to the stability of the active pharmaceutical ingredient in a formulation. During the preparation process of freeze-dried formulations, it is essential to add excipients or freeze-drying protectants to the solution used for freeze-drying.

[0047] Some drug solutions can be freeze-dried well in a vacuum, while others may disintegrate or melt into an oily substance immediately after freeze-drying. To obtain a stable freeze-dried formulation by successfully freeze-drying a specific drug solution, it is necessary to add several excipients that do not react with the drug. These excipients themselves do not sublimate during the sublimation stage of the freeze-drying process; instead, they are directly freeze-dried to form a framework, thus playing a role in giving shape. The drug can directly adsorb into or fill the gaps in the framework. Alternatively, the excipients can improve the solubility and stability of the freeze-dried product, or allow the freeze-dried product to have an aesthetic shape. Liquid formulations require the addition of several additional substances. These additional substances are collectively called "freeze-drying protectants" and may also be called fillers, bulking agents, excipients, buffers, bases, or frameworks. In general, freeze-drying protectants must be chemically inert to the drug solution.

[0048] Freeze-drying protective agents can be classified as follows, according to their chemical properties. Preparations: Skim milk, gelatin, proteins and their hydrolysates, peptides, yeast, broth, dextrin, methylcellulose, serum, and peptone, etc. Salts: Sodium thiosulfate, calcium lactate, monosodium glutamate, sodium chloride, potassium chloride, sodium sulfate, ammonium acetate, and ammonium chloride, etc. Sugars: Sucrose, lactose, maltose, glucose, raffinose, fructose, and hexose, etc. Alcohols: Sorbitol, ethanol, glycerin, mannitol, inositol, and xylitol, etc. Acids: Citric acid, phosphoric acid, tartaric acid, amino acids, and ethylenediaminetetraacetic acid (EDTA), etc. Bases: such as sodium hydroxide and sodium bicarbonate. Polymers: Dextran, polyethylene glycol, polysorbate, PVP, and poloxamer, etc. Others: Vitamin C, Vitamin E, Vitamin K, and thiourea, etc.

[0049] The polymers mentioned above have a wide range of degrees of polymerization (molecular weight). Polysorbate and polyethylene glycol are discussed as examples.

[0050] Polysorbates may have an average molecular weight in the range of about 500 g / mol to about 1900 g / mol, preferably about 800 g / mol to about 1600 g / mol, and more preferably about 1000 g / mol to about 1400 g / mol. Non-limiting examples of polysorbates include polysorbate-20, polysorbate-21, polysorbate-40, polysorbate-60, polysorbate-61, polysorbate-65, polysorbate-81, polysorbate-85, and polysorbate-120. Preferred polysorbates include polysorbate-20, polysorbate-80, and mixtures thereof.

[0051] Polyethylene glycol (PEG) may have an average molecular weight in the range of about 200 g / mol to about 600 g / mol, preferably about 200 g / mol to about 500 g / mol, and more preferably about 200 g / mol to about 400 g / mol. Non-limiting examples of PEG include PEG200, PEG300, PEG400, PEG540, and PEG600.

[0052] Poloxamer is HO(C2H4O) a (C3H6O) b (C2H4O) cPoloxamer has the general formula H, where a and c are in the range of 2 to 130, and b is in the range of 15 to 67. Poloxamer is a block copolymer of polyoxyethylene polyoxypropylene ether containing 81.8 ± 1.9% polyoxyethylene. There are various trademarks for poloxamer, including poloxamer 182, poloxamer 184, poloxamer 188, and poloxamer 407, which correspond to polymers of various molecular weights, for example, poloxamer 188 has a molecular weight of 7680 to 9510.

[0053] Freeze-drying protective agents perform many functions, which can be summarized as follows:

[0054] Microorganisms such as bacteria and viruses need to grow and reproduce in specific culture media. However, because microorganisms are difficult to isolate from these media, they can be effectively freeze-dried. Examples of media include broth, skim milk, and protein.

[0055] Some freeze-dried formulations have very low concentrations and small amounts of dried material. During freeze-drying, the dried components are carried away by the sublimation gas stream. To increase the drug concentration and dried material content so that the freeze-dried product can form relatively ideal aggregates, fillers must be added to maintain the solid content within a specific range. Examples of these fillers or excipients include sucrose, lactose, inositol, skim milk, hydrolyzed proteins, dextran, sorbitol, and polyvinylpyrrolidone (PVP).

[0056] Some biologically active substances are particularly fragile and can be damaged by physical or chemical factors during freeze-drying. Therefore, it is necessary to add several protective agents (e.g., dimethyl sulfoxide, glycerol, dextran, sugars, and PVP) to mitigate damage during freeze-drying.

[0057] By adding certain substances (e.g., mannitol, glycine, dextran, xylitol, and PVP), the decomposition temperature of the product can be increased, facilitating freeze-drying.

[0058] To accelerate freeze-drying and raise the eutectic point by changing the pH of the freeze-drying preparation, sodium bicarbonate, sodium hydroxide, etc., may be added.

[0059] To improve the storage stability of the product, raise the storage temperature, and extend the storage period, several antioxidants such as vitamin C, vitamin E, amino acids, sodium thiosulfate, thiourea, lecithin, and hydrolyzed proteins may be added.

[0060] By adding specific substances (e.g., amino acids, vitamin K, vitamin C, thiourea, sulfites, and sodium aspartate), free radicals can be removed and the stability of freeze-dried products can be improved.

[0061] This demonstrates that a single compound can perform multiple roles as a freeze-drying protective agent (excipient).

[0062] Due to the properties of the drug of the present invention, the freeze-drying protective agent (excipient) is selected from sugars, polyols, polyvinylpyrrolidone, proteins, poloxamers, or combinations thereof.

[0063] The sugars are selected from sucrose, dextran, cyclodextrin, maltodextrin, trehalose, lactose, maltose, and glucose.

[0064] Polyols are selected from the group consisting of glycerin, sorbitol, mannitol, inositol, ethylene glycol, polyethylene glycol (PEG), polysorbate, and adonitol.

[0065] The protein is selected from albumin, preferably bovine serum albumin and human albumin.

[0066] Poloxamer is selected from poloxamer 182, poloxamer 184, poloxamer 188, and poloxamer 407.

[0067] Preferably, the excipient is selected from PVP K12, sucrose, mannitol, albumin, or a combination thereof.

[0068] Generally speaking, excipients of the same type can be combined, and excipients that do not react with each other or with the drug can be combined. This combination should satisfy the requirements for drug compatibility.

[0069] In particular, the excipient is selected from sucrose and mannitol, and the content of sucrose or mannitol in the solution is in the range of 20 to 300 mg / mL, preferably in the range of 40 to 100 mg / mL, more preferably in the range of 60 to 80 mg / mL, and even more preferably in the range of 60 to 70 mg / mL.

[0070] Generally, only one of sucrose or mannitol is used, but in some special cases, a mixture of sucrose and mannitol may be used. In a preferred embodiment of the present invention, only mannitol or sucrose is used.

[0071] Similarly, the mass ratio of TH-302 to (one or more) excipients in a solution for preparing a freeze-dried formulation with a high drug loading capacity is in the range of 1:(0.5 to 20), preferably in the range of 1:(1 to 15), more preferably in the range of 1:(2 to 12.5), and even more preferably in the range of 1:(5 to 10).

[0072] The mass ratio of TH-302 and other similar compounds to the excipients represents the drug load ratio of the freeze-drying solution. Depending on the freeze-drying state of the excipients (lyophilization protectants, fillers, or skeletons), it will be understood that the freeze-dried product will have a skeleton obtained from (one or more) excipients, and that (one or more) drugs will be adsorbed or loaded onto that skeleton. Therefore, the drug load ratio of the drug solution before freeze-drying, i.e., the mass ratio of TH-302 to (one or more) excipients, is an important indicator.

[0073] An appropriate drug loading ratio means that, after freeze-drying the freeze-dried formulation, the drug is uniformly adsorbed to the framework and well distributed in the gaps and pores of the framework, so that the freeze-dried formulation can be rapidly and well dissolved during subsequent reconstitution (using 5% dextrose injection, physiological saline, etc.) and prepared for injection.

[0074] A "pharmaceutically acceptable buffer" refers to a weak acid or weak base used to maintain the pH value of the solution at a nearly constant level and to improve the stability of the active pharmaceutical ingredient in the solution.

[0075] The solution for preparing a freeze-dried formulation with a high drug loading capacity according to the present invention may also contain at least one buffer, which is selected from the group consisting of citrate buffer, borate buffer, lithium lactate, sodium lactate, potassium lactate, calcium lactate, lithium phosphate, sodium phosphate, potassium phosphate, calcium phosphate, lithium maleate, sodium maleate, potassium maleate, calcium maleate, lithium tartrate, sodium tartrate, potassium tartrate, calcium tartrate, lithium succinate, sodium succinate, potassium succinate, calcium succinate, lithium acetate, sodium acetate, potassium acetate, calcium acetate, or mixtures thereof.

[0076] Preferably, the buffer used in the solution for preparing the freeze-dried formulation with a high drug loading capacity according to the present invention is at least one citrate buffer. Non-limiting examples of suitable citrate buffers include lithium citrate monohydrate, sodium citrate monohydrate, potassium citrate monohydrate, calcium citrate monohydrate, lithium citrate dihydrate, sodium citrate dihydrate, potassium citrate dihydrate, calcium citrate dihydrate, lithium citrate trihydrate, sodium citrate trihydrate, potassium citrate trihydrate, calcium citrate trihydrate, lithium citrate tetrahydrate, sodium citrate trihydrate Examples include ammonium tetrahydrate, potassium citrate tetrahydrate, calcium citrate tetrahydrate, lithium citrate pentahydrate, sodium citrate pentahydrate, potassium citrate pentahydrate, calcium citrate pentahydrate, lithium citrate hexahydrate, sodium citrate hexahydrate, potassium citrate hexahydrate, calcium citrate hexahydrate, lithium citrate heptahydrate, sodium citrate heptahydrate, potassium citrate heptahydrate, and calcium citrate heptahydrate.

[0077] The solution for preparing a freeze-dried formulation with a high drug loading capacity according to the present invention may also contain at least one pH adjuster.

[0078] The pH adjusting agent of the present invention refers to a buffering substance or buffering solution used to appropriately adjust the pH that changes with an acid or alkali. Examples of pH adjusting agents include, but are not limited to, hydrochloric acid, sodium hydroxide, triethanolamine, phosphoric acid, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, or phosphoric acid, citric acid, lactic acid, tartaric acid, succinic acid, fumaric acid, malic acid, sodium bicarbonate, sodium carbonate, or mixtures thereof. The amount of pH adjusting agent added is such that the pH value of the solution is in the range of 4 to 9, preferably in the range of 6 to 8.

[0079] Since TH-302 and its analogues are typically acidic (the compound preparation process involves an acidic environment and slight hydrolysis, and the active pharmaceutical ingredient prepared or purchased is acidic), the pH adjuster is preferably selected from sodium hydroxide, triethanolamine, sodium bicarbonate, and bases such as sodium carbonate or alkali salts.

[0080] Naturally, the pH of TH-302 and its analogues may be alkaline due to other factors. In this case, it is necessary to add an acid or acidic salt (such as ammonium sulfate and ammonium chloride) to adjust the pH value.

[0081] The compound of formula I below is [ka] Selected from among, more preferably TH-302.

[0082] The specific synthesis method of the compound of formula I and the corresponding spectral data are disclosed in International Publication No. 2007002931 (corresponding to Chinese Patent Application Publication No. 101501054), which is incorporated herein by reference in its entirety.

[0083] The physiological and chemical properties and biological activities related to the compound of formula I and the three specific compounds mentioned above are described in the patents owned by Threshold Pharmaceuticals, Inc. (International Publication Nos. 2016011195, 2004087075, 2007002931, 2008151253, 2009018163, 2009033165, 2010048330, 2012142520, 2008083101, 2020007106, 2020118251, 2014169035, 2013116385, 2019173799, and 2016 No. 081547, No. 2014062856, No. 2015069489, No. 2012006032, No. 2018026606, No. 201004 No. 8330, No. 2015171647, No. 2013096687, No. 2013126539, No. 2013096684, No. 20120092 See also Nos. 88, 2012145684, 2016014390, 2019055786, 2012135757, 2015013448, 2016011328, 2013177633, 2016011195, and 2015051921, etc. These are incorporated herein by reference in their entirety.

[0084] The three preferred compounds described above have the same or similar physiological and chemical properties as TH-302.

[0085] The present invention relates to a solution for preparing a freeze-dried formulation with a high drug loading capacity, wherein the solution is The compound of formula I-1 below, water, tert-butanol, and sucrose, or The compound of formula I-1 below, water, tert-butanol, and mannitol, Includes, [ka] Water and tert-butanol are used as the mixed solvent. The volume content of tert-butanol in the solution is 30%, 40%, or 60%, or the corresponding volume content of tert-butanol in the solution is 235.5, 314, or 471 mg / mL. The content of the compound of formula I-1 in the aforementioned solution is 6, 7, 7.5, 8, 8.5, 10, 12.5, 15, 20, or 25 mg / mL. Sucrose or mannitol is used as an excipient, and the content of sucrose or mannitol in the solution is 40, 50, 60, 70, 75, 80, 90, or 100 mg / mL. Provide the solution.

[0086] The present invention relates to a solution for preparing a freeze-dried formulation with a high drug loading capacity, wherein the solution is A compound of formula I-1 below, water, tert-butanol, sucrose, and a pH adjuster, or The compound of formula I-1 below, water, tert-butanol, mannitol, and a pH adjuster. Includes, [ka] Water and tert-butanol are used as the mixed solvent. The volume content of tert-butanol in the solution is 30%, 40%, or 60%, or the volume content of tert-butanol in the solution is 235.5, 314, or 471 mg / mL. The content of the compound of formula I-1 in the aforementioned solution is 6, 7, 7.5, 8, 8.5, 10, 12.5, 15, 20, or 25 mg / mL. Sucrose or mannitol is used as an excipient, and the content of sucrose or mannitol in the solution is 40, 50, 60, 70, 75, 80, 90, or 100 mg / mL. The amount of pH adjusting agent present is such that the pH value of the solution is in the range of 4 to 9, preferably 6 to 8. Provide the solution.

[0087] The present invention relates to a solution for preparing a freeze-dried formulation with a high drug loading capacity, wherein the solution is The compound of formula I-1 below, water, tert-butanol, sucrose, and sodium bicarbonate, or The compound of formula I-1 below, water, tert-butanol, mannitol, and sodium bicarbonate. It consists of, [ka] Water and tert-butanol are used as the mixed solvent. The volume content of tert-butanol in the solution is 30%, 40%, or 60%, or the volume content of tert-butanol in the solution is 235.5, 314, or 471 mg / mL. The content of the compound of formula I-1 in the aforementioned solution is 6, 7, 7.5, 8, 8.5, 10, 12.5, 15, 20, or 25 mg / mL. Sucrose or mannitol is used as an excipient, and the content of sucrose or mannitol in the solution is 40, 50, 60, 70, 75, 80, 90, or 100 mg / mL. The amount of sodium bicarbonate present is such that the pH value of the solution is in the range of 4 to 9, preferably 6 to 8. Provide the solution.

[0088] In the solution for preparing the freeze-dried formulation with a high drug loading capacity as described above, the mass ratio of the compound to sucrose or mannitol is 1:2, 1:3, 1:3.5, 1:4, 1:5, 1:5.333, 1:5.6, 1:6, 1:1.67, 1:7, 1:8, 1:8.235, 1:8.75, or 1:9.375.

[0089] Naturally, buffers, pH adjusters, or other auxiliary materials for freeze-dried formulations may be added according to the above description and depending on the properties of the specific compound of Formula I.

[0090] Clearly, if other substances (environmental substances) are detected due to contact with containers, tubes, and equipment during solution preparation, or due to contamination thereof, those substances do not belong to the classification of auxiliary materials as described above. Similarly, the raw materials for tert-butanol, sucrose / mannitol, and the compounds of formula I inevitably contain impurities or other substances (environmental substances) present in the environment. These impurities or environmental substances also do not belong to the aforementioned classification of auxiliary materials.

[0091] All other substances (environmental substances) detected as a result of contact with or contamination of containers, tubes, and instruments, as well as impurities or other substances (environmental substances) inevitably present in the raw materials of tert-butanol, sucrose / mannitol, and compounds of formula I, should be within legal limits or in accordance with the corresponding product quality standards (pharmaceutical grade, medical grade, or equivalent quality standards).

[0092] Therefore, the expression "...consists of..." above refers to substances that are intentionally or artificially added during preparation (these substances are essential to the composition and can be detected by analytical testing equipment). In addition to these substances, no other substances are intentionally or artificially added. However, trace amounts of impurities, environmental substances, etc., are inevitably present.

[0093] The above describes the components (formulation formulation) of a solution for preparing a freeze-dried formulation with a high drug loading capacity. The following briefly explains how to use this solution.

[0094] The solutions provided above are for use only as intermediate semi-finished products for preparing freeze-dried formulations with high drug loading capacity and are not for use in clinical formulations. Generally speaking, the solutions are prepared immediately for use in the manufacturing facility. That is, the solutions are prepared in liquid formulation containers, directly filled into freeze-dried vials, and then sent in batches to freeze-drying manufacturing equipment for freeze-drying.

[0095] Therefore, solutions for preparing freeze-dried formulations with high drug loading capacity should be ensured to be stable after preparation and during the manufacturing and waiting processes for freeze-drying in a lyophilization facility, and should be stable at room temperature for at least 8 hours, preferably 24 hours, and even more so for 72 or 120 hours.

[0096] This is because the solution, after preparation, needs to be filtered, filled, and then sent to a freeze-dryer for freeze-drying. Filtration and filling are usually completed within 8 to 12 hours and are generally performed at room temperature. In contrast, the subsequent freeze-drying is carried out at low temperatures, and it takes 20 to 60 hours in a large freeze-dryer or freeze-drying system to cool the large volume of drug solution filled in vials to the set low temperature (-20°C to -55°C). Therefore, the drug solution for freeze-dried formulations should be stable at room temperature for a certain period of time. Experimental results have confirmed that the drug solution for freeze-dried formulations of TH-302 provided by the present invention has adequate stability.

[0097] The present invention also provides the use of a solution for preparing freeze-dried formulations with high drug loading capacity. The solution is suitable for use as a freeze-dried formulation solution and is used for the preparation of freeze-dried formulations by the freeze-drying process.

[0098] Based on the above-described solution for preparing freeze-dried formulations with high drug loading capacity, the present invention can provide freeze-dried formulations with high drug loading capacity.

[0099] Freeze-dried formulations are prepared by a freeze-drying process using the above-mentioned solution for preparing freeze-dried formulations with a high drug loading capacity as the freeze-drying formulation solution.

[0100] The freeze-dried formulation contains the compound of formula I below, an excipient, and a residual solvent component. [ka] In the formula, R is independently selected from H, -CH3, and -CH2CH3, and X is independently selected from Br, Cl, OMs, and OTs. The drug loading capacity of the compound of formula I in the aforementioned freeze-dried formulation is 5 mg / cm³. 3 Super 555.55mg / cm 3 The following, or, The drug loading capacity of the compound of formula I in the aforementioned freeze-dried formulation is 4.55 mg / cm³. 3 More than 500mg / cm 3 Less than, or The mass percentage of the compound of formula I in the freeze-dried formulation is 4.39% or more and less than 66.66%. The residual solvent components are water and tert-butanol.

[0101] The present invention also relates to a freeze-dried formulation comprising a compound of the following formula I, an excipient, a residual solvent component, and a pH adjuster, [ka] In the formula, R is independently selected from H, -CH3, and -CH2CH3, and X is independently selected from Br, Cl, OMs, and OTs. The drug loading capacity of the compound of formula I in the aforementioned freeze-dried formulation is 5 mg / cm³. 3 Super 555.55mg / cm 3 The following, or, The drug loading capacity of the compound of formula I in the aforementioned freeze-dried formulation is 4.55 mg / cm³. 3 More than 500mg / cm 3 Less than, or The mass percentage of the compound of formula I in the freeze-dried formulation is 4.39% or more and less than 66.66%. The residual solvent components are water and tert-butanol. We provide freeze-dried formulations.

[0102] The present invention also relates to a freeze-dried formulation comprising a compound of the following formula I and an excipient, [ka] In the formula, R is independently selected from H, -CH3, and -CH2CH3, and X is independently selected from Br, Cl, OMs, and OTs. The drug loading capacity of the compound of formula I in the aforementioned freeze-dried formulation is 5 mg / cm³. 3 Super 555.55mg / cm 3 The following, or, The drug loading capacity of the compound of formula I in the aforementioned freeze-dried formulation is 4.55 mg / cm³. 3 More than 500mg / cm 3 Less than, or The mass percentage of the compound of formula I in the freeze-dried formulation is 4.39% or more and less than 66.66%. We provide freeze-dried formulations.

[0103] Here, the drug loading capacity of the compound of formula I in the freeze-dried formulation is 5 mg / cm³. 3 Super 555.55mg / cm 3 The following conditions must be met, and the drug loading capacity of the compound of formula I in the freeze-dried formulation must be 4.55 mg / cm³. 3 More than 500mg / cm 3 Being less than represents two different situations.

[0104] As a result of extensive experiments, the applicants found that, when using a maximum filling volume of 25 mL in a 50 mL vial, for various freeze-drying processes to freeze-dry the above-mentioned freeze-dried formulation solution with a high drug loading capacity to obtain a freeze-dried formulation, there is a change of less than 10% between the volume of the pre-freeze-dried solution and the external volume of the freeze-dried formulation solid. The volume may increase by 10% due to the expansion of the freeze-dried cake after freeze-drying, or decrease by 10% due to the collapse of the powder cake. Based on the content of compound I in the freeze-dried formulation solution with a high drug loading capacity before freeze-drying being between 5 mg / mL and 500 mg / mL, and the maximum change being 10%, the volume can be calculated to be 1.1 times the original volume due to expansion, or 90% of the original volume due to collapse. Taking the example that the volume of the collapsed powder cake after freeze-drying is 90% of the original volume, the maximum drug loading capacity of the powder cake in this case can be calculated as 5 / 0.9 = 5.55 to 500 / 0.9 = 555.55. Depending on the process, there may be situations where the volume does not change. In this case, the drug loading capacity is in the range of 5 to 500. Combining the above two ranges, we get 5 to 555.55. That is, the drug loading capacity of the compound of formula I in a freeze-dried formulation is 5 mg / cm³. 3 Super 555.55mg / cm 3 The following applies:

[0105] Similarly, if the volume increases due to the expansion of the freeze-dried cake, the drug loading volume of the compound of formula I in the freeze-dried formulation becomes 4.55 mg / cm³. 3 More than 500mg / cm 3 It is less than.

[0106] The drug loading capacity of the freeze-dried formulation provided by this invention refers to the amount of compound of formula I as the active pharmaceutical ingredient contained per unit volume. Here, volume refers to the total external volume of the drug, including the internal void of the drug, in a unit packaged kit. For example, 20 mL of freeze-dried solution in a 100 mL freeze-dried vial contains 500 mg of drug. As a result of freeze-drying a preliminary freeze-dried solution, 20 mL of the solution is frozen, resulting in a loosely porous freeze-dried formulation. If the bottom area of ​​the internal space of the freeze-dried vial is measured as s and the height of the freeze-dried formulation in the freeze-dried vial is measured as h, then the total external volume of this unit packaged freeze-dried formulation is sh. Since 500 mg of the active pharmaceutical ingredient is loaded, its drug loading capacity is (500 / sh) mg / cm³ 3 Generally, the value of sh is approximately 20 cm. 3 The volume after freeze-drying will be 20 cm³ due to expansion. 3 It is possible that the volume after freeze-drying will be 20 cm³ due to decay. 3 It may be less than.

[0107] When determining the drug loading capacity of a freeze-dried formulation packaged in a specific unit package, first, the total external volume sh is measured and calculated as described above, and then all freeze-dried formulations packaged in the unit package are directly dissolved and the mass M of the drug contained therein is measured. Thus, the drug loading capacity (per unit volume) of the freeze-dried formulation of the present invention is given by = M / sh.

[0108] Here, the mass percentage of the compound of formula I in the freeze-dried formulation is 4.39% or more and less than 66.66%. This conversion value is calculated based on the content of the active pharmaceutical ingredient (API) and the excipients, as well as the residual tert-butanol and residual water in the freeze-dried formulation.

[0109] The mass content of compound I in a freeze-dried formulation refers to the percentage of the drug relative to the total mass of the freeze-dried formulation. This can be measured and calculated according to the following procedure.

[0110] When measuring the mass content of a drug in a freeze-dried formulation packaged in a specific unit package, first, the mass M1 of the freeze-dried formulation in the unit package is weighed, and then all of the freeze-dried formulation in the unit package is directly dissolved, and the mass M of the drug contained therein is measured. If the total mass of the empty vial and packaging cap after dissolution, washing, oven drying, and weighing is M2, then the total mass of the freeze-dried formulation is M1-M2, and therefore the mass content of the compound of formula I in the freeze-dried formulation according to the present invention is M / (M1-M2).

[0111] In the freeze-dried formulation described above, the compound of formula I is [ka] Selected from the following, preferably TH-302.

[0112] The excipients are selected from sugars, polyols, polyvinylpyrrolidone, proteins, poloxamers, or combinations thereof. The sugars are selected from sucrose, dextran, cyclodextrin, maltodextrin, trehalose, lactose, maltose, and glucose. Polyols are selected from the group consisting of glycerin, sorbitol, mannitol, inositol, ethylene glycol, polyethylene glycol, polysorbate, and adonitol. The protein is selected from albumin, preferably bovine serum albumin and human albumin. Poloxamer is selected from poloxamer 182, poloxamer 184, poloxamer 188, and poloxamer 407.

[0113] After freeze-drying the aforementioned solution, the solvents, water and tert-butanol, are sublimated. Therefore, the freeze-dried formulation contains only the remaining drug and excipients.

[0114] Clearly, it is impossible to completely remove water and tert-butanol during the freeze-drying sublimation process, so residues of water and tert-butanol are inevitably present. In fact, the amount of water and tert-butanol residue is an important quality indicator of freeze-dried formulations. The lower the residue, the better the quality and stability of the freeze-dried formulation, and the less likely patients are to experience adverse reactions after administering such a formulation. Residues can be minimized by adjusting the freeze-drying process. However, it is impossible to completely avoid residues.

[0115] Nevertheless, by extending the freeze-drying time and increasing the drying temperature, it is technically possible to reduce the water and tert-butanol content to below the detection limit, while still complying with product quality requirements, regardless of cost. In this case, the freeze-dried formulation can be considered to contain little to no water and tert-butanol.

[0116] Considering manufacturing costs and storage stability, the appropriate residual content of water and tert-butanol is as follows: The residual water content is 6% or less by mass, preferably 2% or less, more preferably 1% or less, and even more preferably 0.5% or less. The residual tert-butanol content is 1.75% or less by mass, preferably 1% or less, and even more preferably 0.5% or less.

[0117] Naturally, buffers, pH adjusters, or other adjuvants for freeze-drying formulations can also be added in accordance with the above description and in combination with the properties of the specific compounds of Formula I. Correspondingly, it is also detected that freeze-dried formulations may contain buffers, pH adjusters, or other adjuvants for freeze-drying.

[0118] Clearly, if other substances (environmental substances) that cannot be removed by sublimation are detected due to contact with containers, tubes, and equipment during the preparation of the solution or freeze-dried formulation, or due to contamination thereof, those substances do not belong to the classification of auxiliary materials as described above. Similarly, the raw materials for tert-butanol, sucrose, and the compounds of formula I inevitably contain impurities or other substances (environmental substances) present in the environment, which cannot be removed by sublimation. These impurities or environmental substances also do not belong to the aforementioned classification of auxiliary materials.

[0119] All other substances (environmental substances) that cannot be removed by sublimation and are detected as a result of contact with or contamination of containers, tubes, and instruments, or impurities or other substances (environmental substances) that cannot be removed by sublimation and are inevitably present in the raw materials of tert-butanol, sucrose, and the compound of formula I, should be within legal limits or in accordance with the corresponding product quality standards (pharmaceutical grade, medical grade, or equivalent quality standards).

[0120] "Containing the compound of formula I and excipients" means that the freeze-dried formulation is detectable to contain the compound of formula I, excipients, and the aforementioned residual environmental substances, in addition to unavoidable residual water and residual tert-butanol. The freeze-dried formulation may also contain other adjuvants.

[0121] The mass ratio of the compound of formula I to the excipient is in the range of 1:(0.5 to 20), preferably in the range of 1:(1 to 15), more preferably in the range of 1:(2 to 12.5), and even more preferably in the range of 1:(5 to 10).

[0122] The drug loading dose of the compound of formula I in freeze-dried formulations is 5.55 mg / cm³. 3 More than 177.77mg / cm 3 Preferably, 8.88 mg / cm³ 3 More than 55.55mg / cm 3More preferably, 8.88 mg / cm³ 3 More than 27.77mg / cm 3 More preferably, 8.88 mg / cm³ 3 More than 16.66mg / cm 3 More preferably, 8.88 mg / cm³ 3 More than 11.11mg / cm 3 The following, or, The drug loading dose of the compound of formula I in freeze-dried formulations is 4.55 mg / cm³. 3 More than 145.45mg / cm 3 Preferably, 7.27 mg / cm³ 3 More than 45.45mg / cm 3 More preferably, 7.27 mg / cm³ 3 More than 22.73mg / cm 3 More preferably, 7.27 mg / cm³ 3 More than 13.64mg / cm 3 More preferably, 7.27 mg / cm³ 3 More than 9.09mg / cm 3 The following applies:

[0123] Regarding the two situations described above, the former refers to a situation where the volume decreases due to decay after freeze-drying, while the latter refers to a situation where the volume increases due to expansion.

[0124] The present invention also relates to a freeze-dried formulation, the freeze-dried formulation essentially consisting of a compound of the following formula I-1, an excipient, residual water, and residual tert-butanol. [ka] The drug loading dose of the compound in the freeze-dried formulation is 6.66, 7.77, 8.33, 8.88, 9.44, 11.11, 13.88, 16.66, 22.22, or 27.77 mg / cm³. 3The drug loading dose of compound I-1 in the freeze-dried formulation is 5.45, 6.36, 6.82, 7.27, 7.73, 9.09, 11.36, 13.64, 18.18, or 22.73 mg / cm³. 3 And, The excipients are sucrose or mannitol. The mass ratio of the compound to the excipient is 1:2, 1:3, 1:3.5, 1:4, 1:5, 1:5.333, 1:5.6, 1:6, 1:7, 1:8, 1:8, 1:8.235, 1:8.75, or 1:9.375. The residual water content is 6% by mass or less, preferably 2% by mass or less, more preferably 1% by mass or less, and even more preferably 0.5% by mass or less. The content of residual tert-butanol is 1.75% by mass or less, preferably 1% by mass or less, and more preferably 0.5% by mass or less. We provide freeze-dried formulations.

[0125] The present invention also relates to a freeze-dried formulation, the freeze-dried formulation essentially comprising a compound of the following formula I-1, an excipient, residual water, residual tert-butanol, and a pH adjuster. [ka] The drug loading dose of the compound in the freeze-dried formulation is 6.66, 7.77, 8.33, 8.88, 9.44, 11.11, 13.88, 16.66, 22.22, or 27.77 mg / cm³. 3 The drug loading dose of compound I-1 in the freeze-dried formulation is 5.45, 6.36, 6.82, 7.27, 7.73, 9.09, 11.36, 13.64, 18.18, or 22.73 mg / cm³. 3 And, The excipients are sucrose or mannitol. The mass ratio of the compound to the excipient is 1:2, 1:3, 1:3.5, 1:4, 1:5, 1:5.333, 1:5.6, 1:6, 1:1.67, 1:7, 1:8, 1:8, 1:8.235, 1:8.75, or 1:9.375. The residual water content is 6% by mass or less, preferably 2% by mass or less, more preferably 1% by mass or less, and even more preferably 0.5% by mass or less. The content of residual tert-butanol is 1.75% by mass or less, preferably 1% by mass or less, and more preferably 0.5% by mass or less. The pH adjusting agent is sodium bicarbonate, at a concentration of 0.01 to 0.10 mg / cm³. 3 It exists in the amount of, We provide freeze-dried formulations.

[0126] The present invention also relates to a freeze-dried formulation, the freeze-dried formulation essentially consisting of a compound of the following formula and an excipient, [ka] The drug loading dose of the compound of formula I-1 in the freeze-dried formulation is 6.66, 7.77, 8.33, 8.88, 9.44, 11.11, 13.88, 16.66, 22.22, or 27.77 mg / cm³. 3 The drug loading dose of compound I-1 of formula I-1 in the freeze-dried formulation is 5.45, 6.36, 6.82, 7.27, 7.73, 9.09, 11.36, 13.64, 18.18, or 22.73 mg / cm³. 3 And, The excipients are sucrose or mannitol. The mass ratio of the compound of formula I-1 to the excipient is 1:2, 1:3, 1:3.5, 1:4, 1:5, 1:5.333, 1:5.6, 1:6, 1:1.67, 1:7, 1:8, 1:8, 1:8.235, 1:8.75, or 1:9.375. We provide freeze-dried formulations.

[0127] Clearly, if other substances (environmental substances) that cannot be removed by sublimation are detected due to contact with containers, tubes, and equipment during the preparation of the solution or freeze-dried formulation, or due to contamination thereof, those substances do not belong to the classification of auxiliary materials as described above. Similarly, the raw materials for tert-butanol, sucrose, and the compounds of formula I inevitably contain impurities or other substances (environmental substances) present in the environment, which cannot be removed by sublimation. These impurities or environmental substances also do not belong to the aforementioned classification of auxiliary materials.

[0128] All other substances (environmental substances) that cannot be removed by sublimation and are detected as a result of contact with or contamination of containers, tubes, and instruments, as well as impurities or other substances (environmental substances) that cannot be removed by sublimation and are inevitably present in the raw materials of tert-butanol, sucrose, and the compounds of formula I, should be within legal limits or in accordance with the corresponding product quality standards (pharmaceutical grade, medical grade, or equivalent quality standards).

[0129] "Containing the compound of formula I and excipients" means that the freeze-dried formulation is detectable to contain the compound of formula I, excipients, and the aforementioned residual environmental substances, in addition to unavoidable residual water and residual tert-butanol. The freeze-dried formulation may also contain other adjuvants.

[0130] "Essentially consisting of the compound of Formula I and the excipients" means that the freeze-dried formulation is detectable to contain only the compound of Formula I, the excipients, and the aforementioned residual environmental substances, in addition to the unavoidable residual water and residual tert-butanol. Apart from such components, the freeze-dried formulation contains no other substances.

[0131] The present invention provides a freeze-dried formulation prepared by a freeze-drying process using the above-described solution for preparing a freeze-dried formulation with a high drug loading capacity.

[0132] Pharmaceutical preparations and their freeze-dried powders can be stored in containers commonly used in the pharmaceutical field, such as plastic or glass containers, for example, standard USPI-type borosilicate glass containers. For example, the container used may be a syringe or a vial.

[0133] Based on the dosages of the compound of Formula I used in animal model experiments and clinical trials described in the background art section of this application, it is clear that the dosage of the compound administered each time may vary depending on the species (humans and other animals), the indication, or the patient. The dosage can range from a minimum of a few milligrams to a maximum of tens of thousands of milligrams. It is most preferable that the dosage for a particular administration can be met by a unit dose package or by a combination of multiple unit dose packages. Therefore, from the above perspective, we recommend appropriate dosages of freeze-dried formulations per formulation unit package for various species or indications.

[0134] The present invention also relates to a formulation unit package containing the freeze-dried formulation, The freeze-dried formulation is contained in a sealed container with a capacity of 1000 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of compound of formula I is 4,000 to 16,500 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 500 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of compound of formula I is 2,000 to 8,000 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 250 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of compound of formula I is 1,000 to 4,000 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 100 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of compound of formula I is 400 to 2,000 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 50 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of compound of formula I is 50 to 800 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 30 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of compound of formula I is 150 to 600 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 25 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of compound of formula I is 125 to 500 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 20 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of compound of formula I is 100 to 400 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 18 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of compound of formula I is 90 to 360 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 15 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of compound of formula I is 75 to 300 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 10 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of compound of formula I is 50 to 200 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 8 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of compound of formula I is 40 to 160 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 7 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of compound of formula I is 35 to 140 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 5 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of compound of formula I is 25 to 100 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 3 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of compound of formula I is 15 to 60 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 2 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of compound of formula I is 10 to 40 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 1.5 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of compound of formula I is 7.5 to 30 mg. We provide individual formulation packages.

[0135] These low doses are suitable for young humans or animals of comparable size, such as pigs, rats, and dogs, or other small, lightweight animals. The indications are not limited.

[0136] The freeze-dried formulations provided by the present invention are recommended to be administered by intravenous infusion. Therefore, the drug solution needs to be reconstituted. Generally, physiological saline (0.9%) or glucose injection (5%) is selected for reconstitution. For this purpose, the present invention provides an injectable formulation for intravenous injection containing TH-302, wherein the solvent is water, and the solute comprises the active pharmaceutical ingredient TH-302, an isotonic modifier, mannitol or sucrose, tert-butanol, and sodium bicarbonate, the isotonic modifier being selected from glucose and sodium chloride.

[0137] The present invention also relates to a method for preparing a solution of a freeze-dried formulation with a high drug loading capacity, wherein the preparation method is as follows: Procedure 1: Weigh the specified amount of the active pharmaceutical ingredient TH-302, add it to the tert-butanol aqueous solution, and stir until clear to obtain the first solution. Step 2: Dissolve the specified amount of mannitol or sucrose in an appropriate amount of water and stir until clear to obtain the second solution. Step 3: Mix the first solution and the second solution, then add the remaining specified amount of tert-butanol, then add an appropriate amount of water to the specified volume, and then add the specified amount of sodium bicarbonate, and mix uniformly while stirring. Includes, In the above procedure, The content of the active pharmaceutical ingredient TH-302 in the solution of the freeze-dried formulation with a high drug loading capacity is greater than 5 mg / mL and less than or equal to 500 mg / mL. The volume percentage of tert-butanol in a solution of a freeze-dried formulation with a high drug loading capacity is in the range of 1 to 99%, or the tert-butanol content in the solution is in the range of 7.85 to 777.15 mg / mL. The sucrose or mannitol content in the solution of freeze-dried formulations with a high drug loading capacity is in the range of 20-300 mg / mL. In freeze-dried formulations with a high drug loading capacity, the mass ratio of TH-302 to sucrose or mannitol in the solution is in the range of 1:(0.5~20). The volume ratio of tert-butanol in an aqueous solution of tert-butanol is in the range of 30-90%. The sodium bicarbonate content in the solution of freeze-dried formulations with a high drug loading capacity is in the range of 0.01 to 0.10 mg / mL. A method for preparation is provided.

[0138] The present invention provides, in this specification, a process for preparing the freeze-dried formulation.

[0139] The preparation method for this freeze-dried formulation, which has a high drug loading capacity, includes the following steps.

[0140] Pre-freeze-drying: The above drug solution is placed in a freeze-drying system and pre-freeze-dried. This pre-freeze-drying procedure includes maintaining the temperature at 0°C for a certain period of time and maintaining the temperature at -20°C to -55°C for a certain period of time.

[0141] Primary drying: After preliminary freeze-drying, the temperature is raised to a range of -10 to 10°C, and the drying is performed by maintaining this temperature for a certain period of time while keeping the material under vacuum.

[0142] Secondary drying: After primary drying, the temperature is raised to a range of 20-40°C, and the temperature is maintained for a certain period of time while a vacuum is maintained to perform drying.

[0143] As can be seen from the above explanation, freeze-dried formulations are obtained by directly filling the solution into freeze-dried bottles (vials) and freeze-drying them directly in a freeze-drying apparatus. Therefore, there is no sub-packaging process. Thus, the unit package corresponds to the corresponding freeze-dried bottle package. The packaging gauge is closely related to the gauge of the freeze-dried vial.

[0144] In particular, the numbers described in this application may have an error margin of ±10%. That is, -10% and +10% of the described numbers should be considered to be within the range of the numbers recorded in this application. In other words, if this application describes, for example, "content greater than 5 mg / mL but 500 mg / mL or less," then an actually measured content range of greater than 4.5 mg / mL and 550 mg / mL or less should naturally be considered equivalent to the above range.

[0145] Specifically, a value of (8.00 ± 0.80) corresponds to a range of 7.20 to 8.80. It should also be understood that if the actually measured content is between 6.48 and 9.68, it is equivalent to the aforementioned range.

[0146] Based on initial exploratory experiments, the applicant devised the following recommended scheme. Based on several considerations, the applicant used the following recommended scheme when manufacturing freeze-dried formulations in batches. The freeze-dried formulations manufactured as final products are expected to meet the requirements of subsequent clinical trials, be used as drug candidates in subsequent large-scale clinical trials, and, furthermore, be approved for future commercialization, manufacture, or sale.

[0147] The present invention relates to a solution for preparing a freeze-dried formulation with a high drug loading capacity, wherein the solution comprises a compound of the following formula (I-1), water, tert-butanol, and mannitol. [ka] Water and tert-butanol are used as a mixed solvent, wherein the volume percentage of tert-butanol in the solution is (30±3)%, or the mass percentage is (24±2.4)%, or the tert-butanol content in the solution is (235.5±23.55) mg / mL. The content of the compound of formula (I-1) in the aforementioned solution is (8.16±0.82) mg / g or (8.00±0.80) mg / mL. Mannitol is an excipient, and the mass percentage of mannitol in the solution is (7.14±0.71)%, or the mannitol content in the solution is (70±7) mg / mL. The pH value of the aforementioned solution is in the range of 4 to 9. Provide a solution Due to the inherent properties of TH-302 (compound of formula (I-1)) and the conditions in individual manufacturing batches, the pH value of the solution containing compound (I-1), water, tert-butanol, and mannitol is likely to be outside the range of 4-9 at certain temperatures or under certain environmental conditions. Therefore, it is necessary to incorporate a pH additive to adjust the pH value. Suitable pH adjusters include those commonly used in pharmaceutical solutions, such as Na2CO3, NaHCO3, K2CO3, and KHCO3. A preferred pH adjuster is NaHCO3. Therefore, the solution may further contain sodium bicarbonate as a pH adjuster.

[0148] Furthermore, the pH value of the solution is in the range of 6 to 8.

[0149] The present invention also relates to a freeze-dried formulation comprising a compound of the following formula (I-1) and mannitol, [ka] The drug loading capacity of the compound of formula (I-1) in the aforementioned freeze-dried formulation is (8.00 ± 0.80) mg / cm³. 3 The mass percentage of the compound of formula (I-1) in the freeze-dried formulation is (10.23±1.02)%, The mannitol content in the aforementioned freeze-dried preparation is (70±7) mg / cm³. 3 The mannitol content in the freeze-dried formulation is the percentage remainder of (10.23±1.02)% by mass of the compound of formula (I-1). We provide freeze-dried formulations.

[0150] After conducting numerous experiments, the inventors found that by adjusting the freeze-drying process, the volume of the freeze-dried solid obtained after freeze-drying could be essentially the same. That is, no significant collapse (resulting in a decrease in volume) or expansion (resulting in an increase in volume) of the vial wall was observed in the freeze-dried formulation. Therefore, when the water and tert-butanol used as solvents were removed by evaporation during the freeze-drying process, only TH-302 and mannitol remained in the freeze-dried formulation. In this case, the drug loading volume of the compound of formula (I-1) in the freeze-dried formulation was 8.00 mg / cm³. 3 The corresponding mass percentage is 10.23%. The mannitol content in this freeze-dried preparation is 70 mg / cm³. 3 Therefore, considering that the total amount of components added in various filling processes can differ by 10%, the above figures may vary within a ±10% margin of error.

[0151] The present invention also relates to a freeze-dried formulation comprising a compound of the following formula (I-1), mannitol, and a pH adjuster, [ka] The drug loading capacity of the compound of formula (I-1) in the aforementioned freeze-dried formulation is (8.00 ± 0.80) mg / cm³. 3 The mass percentage of the compound of formula (I-1) in the freeze-dried formulation is (10.23±1.02)%, The mannitol content in the aforementioned freeze-dried preparation is (70±7) mg / cm³. 3 The mannitol content in the freeze-dried formulation is the percentage remainder of (10.23±1.02)% by mass of the compound of formula (I-1). We provide freeze-dried formulations.

[0152] The present invention also relates to a freeze-dried formulation comprising a compound of the following formula (I-1), mannitol, a residual solvent component, and a pH adjuster, [ka] The drug loading capacity of the compound of formula (I-1) in the aforementioned freeze-dried formulation is (8.00 ± 0.80) mg / cm³. 3 The mass percentage of the compound of formula (I-1) in the freeze-dried formulation is (10.23±1.02)%, The mannitol content in the aforementioned freeze-dried preparation is (70±7) mg / cm³. 3 And, The residual solvent components are water and tert-butanol. We provide freeze-dried formulations.

[0153] Furthermore, this freeze-dried formulation is dissolved in water for injection to create an aqueous solution with a concentration of 5.0 mg / mL and a pH value in the range of 4.0 to 7.0.

[0154] The recommended measurement methods are described below.

[0155] The constituent components were accurately weighed at room temperature. The preparation was dissolved in sterile water for injection to obtain a solution containing 5.0 mg / mL of TH302 per 1 mL in a predetermined volume. Using a pH meter, the pH values ​​of at least two sample solutions were measured in parallel, and the average pH value was calculated.

[0156] The pH adjuster is sodium bicarbonate, and its content is 0.01 to 0.10 mg / cm³. 3 It is within the range of [the specified range].

[0157] In a preferred embodiment, the residual water content is 6% or less by mass, preferably 2% or less, more preferably 1% or less, and even more preferably 0.5% or less. The residual tert-butanol content is 1,795 ppm or less by mass, preferably 1,000 ppm or less, and more preferably 500 ppm or less.

[0158] Tert-butanol is used as a pharmaceutically acceptable adjuvant in the manufacture of TH-302 for injection. After freeze-drying, a small amount of residual tert-butanol may remain in the formulation. Tert-butanol has low toxicity in humans. To comply with pharmaceutical safety and quality requirements, the amount of residual tert-butanol in TH-302 for injection should be controlled within certain limits. As defined in the ICH HARMONISED GUIDELINE FOR RESIDUAL SOLVENTS Q3C(R8), the permitted daily exposure (PDE) for tert-butanol is 35 mg / day. Following this guideline, the maximum daily dose of TH-302 is estimated to be 1.5 g. If calculated strictly as 2 g, the corresponding maximum daily injectable dose is 2 g. If TH-302 is contained in 0.2 g / vial (50 mL vial), 10 vials will be required. If the mass of freeze-dried cake per vial is 1.95g (1.75g ​​mannitol + 0.20g active pharmaceutical ingredient TH-302), the total mass of 10 vials of freeze-dried cake is 19.5g, so the residual tert-butanol content is 35mg / 19.5g (0.1795% by mass, or 1795ppm) or less.

[0159] Considering the constant adjustments to formulations during the screening process, the individual measurement methods for tert-butanol, the expected variations in the loading volume of each vial filled in each process, and ease of calculation, the concentration limit for tert-butanol may be calculated based on the nominal amount of TH-302, and the residual tert-butanol content relative to the nominal mass percentage of the active pharmaceutical ingredient TH-302 can be determined to be 35 mg / 2.0 g = 1.75% or less.

[0160] Taking a freeze-dried formulation containing 200 mg of the active pharmaceutical ingredient (API) TH-302 as an example, the residual tert-butanol content can be calculated to be 1,795 ppm or less by mass, preferably 1,000 ppm or less, and more preferably 500 ppm or less, which is completely equivalent to the following value in percentage terms: the residual tert-butanol content is 1.75% or less of the nominal amount of TH-302 by mass, preferably 1% or less, and more preferably 0.5% or less.

[0161] Furthermore, the mass of the freeze-dried formulation per unit volume is (79.2 ± 7.9) mg / cm³. 3 That is the case.

[0162] Mass per unit volume is similar to density. The freeze-dried formulation is in the form of a porous cake in which the active pharmaceutical ingredient TH-302, mannitol, and sodium bicarbonate (the solvent of residual water and tert-butanol) are completely mixed to form a cellular structure with cavities formed within it. The magnitude of the mass per unit volume is a comprehensive indicator related to the drug loading capacity, mannitol content, and solvent of residual water and tert-butanol of the freeze-dried formulation. Studies have shown that homogeneous freeze-dried formulations have good rapid dissolution properties, making them convenient for subsequent combination use. The porosity of the freeze-dried formulation and its degree of porosity can be qualitatively examined at the microscopic level by direct observation or microscopic observation, or it can be characterized at the macroscopic level by the mass per unit volume of the freeze-dried formulation as described above.

[0163] As confirmed by the experiment, the mass per unit volume of the aforementioned freeze-dried formulation is (79.2 ± 7.9) mg / cm³. 3 In this case, the degree of porosity is appropriate, the drug loading capacity can meet the requirements, and the freeze-drying process can be easily carried out in large-scale production.

[0164] Clearly, this freeze-dried formulation can be prepared by freeze-drying the aforementioned solution for preparing a freeze-dried formulation with a high drug loading capacity.

[0165] The present invention also provides a formulation unit package containing the freeze-dried formulation, having the following features: The freeze-dried formulation is contained in a sealed container with a capacity of 1,000 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of compound of formula (I-1) is in the range of 1,600 to 5,333 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 500 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of compound of formula (I-1) is in the range of 800 to 2,666 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 250 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of compound of formula (I-1) is in the range of 400 to 1,333 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 100 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of compound of formula (I-1) is in the range of 160 to 533 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 50 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of compound of formula (I-1) is in the range of 80 to 266 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 30 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of compound of formula (I-1) is in the range of 48 to 160 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 25 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of compound of formula (I-1) is in the range of 40 to 133 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 20 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of compound I is in the range of 32 to 107 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 18 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of compound of formula (I-1) is in the range of 29 to 96 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 15 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of compound of formula (I-1) is in the range of 24 to 79 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 10 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of compound of formula (I-1) is in the range of 16 to 53 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 8 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of compound of formula (I-1) is in the range of 13 to 42 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 7 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of compound of formula (I-1) is in the range of 11 to 40 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 5 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of compound of formula (I-1) is in the range of 8 to 27 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 3 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of compound of formula (I-1) is in the range of 5 to 16 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 2 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of compound of formula (I-1) is in the range of 3 to 11 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 1.5 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of compound of formula (I-1) is in the range of 2 to 8 mg. We provide individual formulation packages.

[0166] The content of the aforementioned compound is 8.0 mg / cm³. 3 It is calculated based on this, but it is a nominal quantity, and the actual content may be within ±10% of the nominal quantity.

[0167] The present invention also provides an injectable preparation for intravenous injection, wherein the solvent is water, and the solute comprises the active pharmaceutical ingredient TH-302, mannitol, tert-butanol, sodium bicarbonate, and an isotonic modifier, wherein the content of the active pharmaceutical ingredient TH-302 is in the range of 0.1 to 4.0 mg / mL, the osmotic pressure of the injectable preparation is in the range of 260 to 320 mOsmol / kg, and the pH value is in the range of 4.0 to 9.0.

[0168] Similarly, the osmotic pressure of intravenous injection solutions needs to be adjusted. A commonly used injection solution comprises 5% by mass of glucose and 0.9% by mass of sodium chloride. Therefore, the solute may also contain an isotonic modifier selected from glucose and sodium chloride.

[0169] Furthermore, the present invention also provides a method for preparing the injectable agent, the following procedure, namely: Allow the freeze-dried preparation to stand at room temperature until it returns to room temperature. The freeze-dried preparation, returned to room temperature, is formulated into an aqueous solution for injection containing the active pharmaceutical ingredient TH-302 in an amount of (5±0.5) mg / mL using water for injection. An appropriate amount of the aforementioned aqueous solution for injection is injected into a 0.9% physiological saline injection or a 5% glucose injection for dilution to obtain an injectable preparation containing the active pharmaceutical ingredient TH-302 in an amount ranging from 0.1 to 4.0 mg / mL. The present invention provides a method for preparing an injectable drug, including [the specified element].

[0170] The present invention also relates to a method for preparing the injectable preparation, the method being printed in the instructions attached to the freeze-dried preparation unit package to provide specific instructions for preparing the injectable preparation for immediate use in a clinical setting, the prepared injectable preparation to be used within 8 hours, and the preparation method is as follows: Leave the freeze-dried preparation described above at room temperature for 30 to 120 minutes until it returns to room temperature. Use a syringe to take an appropriate volume of water for injection. Insert the syringe into the vial with the slanted end of the needle facing upwards and the tip of the needle at an angle of approximately 60° to the stopper of the vial, then inject along the inner wall of the vial, and after injection is complete, shake the vial containing the drug for at least 20 seconds to ensure that all freeze-dried blocks / powder are completely dissolved and uniformly mixed, and allow the vial to stand until no more air bubbles are observed to obtain an injectable aqueous solution containing the active pharmaceutical ingredient TH-302 in an amount of (5±0.5) mg / mL. A calculated amount of solution is taken from a container containing 0.9% physiological saline injection or 5% glucose injection, and an equal volume of the aqueous solution for injection is injected into the same container containing 0.9% physiological saline injection or 5% glucose injection for dilution, thereby obtaining an injectable preparation containing the active pharmaceutical ingredient TH-302 in an amount ranging from 0.1 to 4.0 mg / mL. The present invention provides a method for preparing an injectable drug, including [the specified element].

[0171] Furthermore, if the above solution contains a suspension mixture after being allowed to stand until no more bubbles are observed, the solution should be shaken for 2 minutes at a temperature in the range of 30-35°C until it becomes clear. [Brief explanation of the drawing]

[0172] [Figure 1] The solubility curves of TH-302 in tert-butanol aqueous solutions with various tert-butanol mass ratios are shown. [Figure 2] The 5-day stability curves of freeze-dried formulations prepared with 11 different excipients are shown. These curves, identified by the circle on the far right, represent the freeze-dried formulations prepared with PEG2000, P188, SBECD, mannitol, DSPE-MPEG2000, fructose, trehalose, PVPK12, sucrose, maltose, and lactose, respectively, from top to bottom. [Figure 3] The 10-day stability curves of freeze-dried formulations prepared with sucrose and mannitol as excipients are shown. In the figure, the top four curves represent freeze-dried formulations prepared with mannitol, and the bottom four curves represent freeze-dried formulations prepared with sucrose. [Figure 4] The stability curves for freeze-dried samples prepared with 100 mg / ml sucrose and 80 mg / ml mannitol as excipients are shown at high temperature (40°C) and room temperature (25°C). The horizontal axis represents days, and the vertical axis represents purity as a percentage, as measured by HPLC. [Figure 5] A photograph of a drug solution sample is shown. The drug solution sample crystallized at the bottom of the bottle containing the sample at an environment of 2-8°C. The label is covered with a mosaic. [Figure 6] The image shows photographs of four batches of freeze-dried formulations. From left to right, the photographs show freeze-dried formulations from batches 01, 02, 03, and 04, respectively. The labels are covered with a mosaic effect. [Figure 7]Comparison photos are shown of four batches of freeze-dried formulation samples using 40 ml of 5% glucose solution in the reconstitution experiment after adding the solution, and those samples after standing. The left photo shows the samples after adding the solution, and the right photo shows the samples after standing. In each photo, from left to right, there are batches 01, 02, 03, and 04. The labels are covered with a mosaic. [Figure 8] Comparison photos are shown of four batches of freeze-dried formulation samples using 50 ml of 5% glucose solution in the reconstitution experiment after adding the solution, and those samples after standing. The left photo shows the samples after adding the solution, and the right photo shows the samples after standing. In each photo, from left to right, there are batches 01, 02, 03, and 04. The labels are covered with a mosaic. [Figure 9] Sample photos of seven batches of freeze-dried formulations are shown. From left to right, there are batches 01 to 07. The labels are covered with a mosaic. [Figure 10] A schematic diagram of the arrangement of vials on the plate of a laboratory freeze dryer is shown. [Figure 11] A photo of a freeze-dried formulation sample is shown. [Figure 12] A photo of a sample with "neck lapping" in a freeze-dried formulation is shown. The labels are covered with a mosaic. [Figure 13] A schematic diagram of the angle at which the needle of a syringe penetrates the rubber stopper of a vial in the reconstitution of a freeze-dried formulation is shown. [Figure 14] A schematic diagram of the injection of an injectate along the inner wall of a vial after the needle of a syringe has penetrated the rubber stopper of the vial in the reconstitution of a freeze-dried formulation is shown.

Modes for Carrying Out the Invention

[0173] The present invention will be described below with specific examples. Those skilled in the art will understand that these examples are only used to explain the present invention and do not limit its scope in any way.

[0174] "Patient" and "individual" are used interchangeably and refer to a mammal that requires treatment for cancer. Generally, the patient is a human. Generally, the patient is a human diagnosed with cancer. In certain embodiments, "patient" or "individual" may refer to a non-human mammal used in the screening, characterization, and evaluation of drugs and therapies, such as non-human primates, dogs, cats, rabbits, pigs, mice, or rats.

[0175] "Prodrug" refers to a compound that is converted, after administration or application, by metabolism or other means, into a biologically active or more active compound (or drug) with respect to at least one property. A prodrug is chemically modified to be less active or inactive compared to the drug, and the chemical modification is such that after the prodrug is administered, the corresponding drug is generated from the prodrug by a metabolic process or other biological process. Prodrugs may have altered metabolic stability or transport properties, fewer side effects, or lower toxicity, or improved flavor, compared to the active drug. Prodrugs can be synthesized using reactants other than the corresponding drug.

[0176] "Treatment" or "treatment of a patient" means administering, using, or applying to a patient a therapeutically effective amount of a drug related to the present invention.

[0177] "Administering", "applying", or "using" a drug to a patient refers to direct administration or application, and the drug may be administered or applied to the patient by a medical professional, or may be self-administered or self-applied, and / or refers to indirect administration or application that may be the act of prescribing the drug. For example, a physician who instructs a patient to self-administer or apply a drug, or provides a prescription for a drug to the patient, is administering or applying the drug to the patient.

[0178] The "therapeutic dose" of a drug refers to the amount of drug that, when administered or applied to a cancer patient, is expected to produce an intended therapeutic effect, such as reduction, remission, mitigation, or disappearance of one or more symptoms of the patient's cancer. Therapeutic effects do not necessarily occur with a single dose, but may only occur after a series of doses have been administered. Therefore, a therapeutic dose of a drug may be administered or applied in one or more doses.

[0179] "Treatment" of a medical condition or patient means taking measures to obtain a beneficial or desired outcome (including clinical outcomes). For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, the alleviation or improvement of one or more symptoms of cancer, a reduction in the severity of the disease, a delay or slowing of disease progression, improvement, alleviation or stabilization of the condition, or other beneficial outcomes. Treatment of cancer may, in some cases, result in a partial response or a state of stability.

[0180] "Tumor cells" refers to tumor cells of any suitable species, such as animals of the Muridae, Canidae, Felidae, Equidae, or mammals such as humans.

[0181] The foregoing description relating to embodiments of the present invention is not intended to limit the invention. Those skilled in the art can make various modifications and changes in accordance with the present invention, and any modifications and changes that do not depart from the spirit of the invention shall be included in the claims appended to the present invention.

[0182] General Information Description Unless otherwise specified, the measurement or testing methods, equipment, and other information for each of the following experiments are as follows:

[0183] The water content was measured using the Karl Fischer KF method with a Mettler V10S Karl Fischer moisture meter.

[0184] The residual solvent tert-butanol content was measured using gas chromatography (GC). The instrument used was an Agilent 8860 gas chromatograph equipped with a 7696A automated headspace sampler and a flame ionization detector (FID). The chromatographic column was a capillary column packed with DB-624. The GC test parameters were as follows: carrier gas N2, inlet temperature 150°C, detector temperature 200°C, splitting ratio 20:1, temperature rise program: starting temperature 60°C, holding temperature for 5 minutes, raising temperature to 240°C at a rate of 30°C / min, then running at 60°C for 5 minutes, and headspace balancing at 85°C for 20 minutes.

[0185] The content and concentration of TH-302 were measured using high-performance liquid chromatography (HPLC). The instrument used was a Thermo Vanquish high-performance liquid chromatograph. The chromatographic column used was a YMC Pack AQ C18 4.6 mm × 250 mm, 5 μm. For the detection method, please refer to the HPLC method described in the patent, International Publication No. 2008083101, held by Threshold Pharmaceuticals (for details, please refer to the ethanol formulation of TH302 in Example 2).

[0186] Unless otherwise specified, the symbol " / " in the table means that it was not detected.

[0187] Unless otherwise specified, for the sake of calculation convenience, the index value for residual solvent (tert-butanol) in the test items for freeze-dried formulations refers to the mass percentage of residual tert-butanol content relative to the nominal amount of API in the freeze-dried formulation.

[0188] 1. Solubility experiment of TH-302 Solubility was measured using the saturated solution method. Specifically, solid TH-302 was directly added to the solvent until insoluble substances or turbidity appeared. After clarification over a certain period, the solution was directly filtered. To determine the concentration of TH-302, the clarified filtrate was directly collected. The obtained concentration accurately represents the solubility of TH-302 in this solvent system. For specific solubility data, please refer to Tables 1, 2, and 3.

[0189] For the method of measuring the concentration (mg / ml) of TH-302, please refer to the HPLC method described in the patent, International Publication No. 2008083101, held by Threshold Pharmaceuticals (for details, please refer to the ethanol formulation of TH302 in Example 2). An external standard method was used for quantification.

[0190] If the concentration is too high, the corresponding clear solution should be diluted first to a level where the concentration can be accurately measured quantitatively using HPLC.

[0191] When the mass ratio of tert-butanol in the data shown in Table 3 is plotted on the X-axis and the solubility of TH-302 in a tert-butanol-water mixed solvent on the Y-axis, the relationship curve shown in Figure 1 was obtained. This active pharmaceutical drug has low solubility in aqueous solutions. As the concentration of tert-butanol in the tert-butanol aqueous solution increased, the solubility of this active pharmaceutical component continued to increase. The solubility of this active pharmaceutical drug reached its maximum in a 70% tert-butanol aqueous solution (V / V), and then decreased as the concentration of tert-butanol in the tert-butanol aqueous solution further increased.

[0192] 2. Experiments to select the appropriate type of excipient (freeze-drying protective agent) for use in freeze-dried formulations. To explore the practical feasibility of using sugars, polyols, nonionic polymer surfactants, and other excipients commonly used for freeze-drying TH-302 drugs, the following excipients were selected for freeze-drying experiments, and their stability was investigated.

[0193] As saccharides, sucrose, lactose, maltose, fructose, and trehalose were selected.

[0194] As polyols, mannitol, glycerol, and sorbitol were selected.

[0195] As nonionic polymeric surfactants, PVPK12 (polyvinylpyrrolidone with a molecular weight of 5500), PEG2000 (polyethylene glycol 2000), and P188 (poloxamer, a block copolymer of polyoxyethylene polyoxypropylene ether, product number: 188) were selected.

[0196] Others: SBECD (sulfobutyl ether-β-cyclodextrin), and DSPE-MPEG2000 (phosphatidylethanolamine pegol).

[0197] According to Table 4 below, a drug solution for lyophilization was formulated.

[0198]

Table 4

[0199] ※The solvent used in the experimental group with PEG2000 and P188 as protective agents was 40% TBA / water.

[0200] The TH-302 active pharmaceutical ingredient and the lyophilization protectant were weighed according to the above formulation prescription, a specified amount of tert-butanol solution was added, and they were uniformly mixed until completely dissolved. Then, they were sub-packaged (into 5-ml vials, with 1 ml of the drug solution put into each vial, 8 vials for each formulation prescription), and rapidly freeze-dried in a small high-speed freeze dryer. The sub-packaged formulations were pre-freeze-dried in an ultra-low temperature refrigerator at -80°C for about 2.5 hours, and then put into the freeze dryer for about 10 - 90 hours (-30°C, absolute pressure 0.1 mbar) to obtain the freeze-dried formulations.

[0201] Experimental results Freeze-dried powder state: In all solvent groups except those using water as the solvent, a good white freeze-dried powder cake was obtained. However, when the freeze-dryer was heated to 0°C, an oily substance appeared in the glycerol group.

[0202] Reconstitution status: Reconstitution was successful for all groups except the sorbitol group (some groups dissolved after shaking).

[0203] High-temperature accelerated stability experiments were performed on the remaining groups of sucrose, mannitol, lactose, maltose, fructose, PVPK12, trehalose, DSPE-MPEG2000, SBECD, PEG2000, and P188.

[0204] After removing the freeze-dried formulation from the freeze-dryer, one vial was withdrawn at 0°C for HPLC testing of purity, water content, and residual solvent content of the active pharmaceutical ingredient. The vial was then placed in a 40°C incubator. HPLC purity was tested on days 3 and 5, respectively. The results are shown in Table 5 below.

[0205] [Table 5]

[0206] Using the data in Table 5, the curve shown in Figure 2 was obtained by plotting the number of days on the X-axis and the purity determined by HPLC on the Y-axis.

[0207] Conclusion of the experiment Generally speaking, the selection of a lyophilized protective agent for injection should take into account the following factors: good stability, suitability for use in conventional methods, and non-impact on efficacy, drug metabolism and pharmacokinetics (DMPK), and toxicity. Analysis of the curves in Figure 2 reveals that PEG2000 is used in a non-conventional manner, and that P188 / SBECD / DSPE-MPEG2000 may affect efficacy, DMPK, and toxic effects. Generally speaking, mannitol is the ideal excipient, with sucrose also showing potential (optimizing its amount and lyophilization conditions may improve stability and reduce tert-butanol residue).

[0208] To further evaluate the stability of mannitol and sucrose, samples of mannitol and sucrose were stored for 10 days, and then tested for purity by HPLC. The results were plotted on a curve, as shown in Figure 3.

[0209] By analyzing the curves in Figure 3, it can be seen that under the preliminary formulation conditions shown in Table 4, the stability of the formulation using mannitol is better than that of the formulation using sucrose.

[0210] 3. Experiments to investigate the effects of various freeze-drying protective agents (100 mg / ml sucrose or 80 mg / ml mannitol) on the stability of freeze-dried powder and the stability of reconstituted solutions. Based on their experience and the use of similar freeze-dried drug formulations, the inventors initially decided to use 100 mg / ml sucrose and 80 mg / ml mannitol as excipients and investigated the stability of the prepared freeze-dried formulation of TH-302 and the stability of the reconstituted solution.

[0211] 3.1 Experiments on the storage stability of freeze-dried formulations at room temperature and accelerated storage stability at high temperatures. Drug solutions for lyophilization were formulated according to Table 6 below. For each group, 40 vials were prepared at 1 mL / vial, resulting in a total of 40 mL of drug solution for each formulation.

[0212] [Table 6]

[0213] Drug solution preparation process 1. First, the specified amount of freeze-dried protective agent was dissolved in the specified amount of water and allowed to dissolve completely. 2. A specified amount of tert-butanol was added to the lyophilized protective agent aqueous solution and mixed uniformly. Then, a specified amount of API was added and stirred until the API was completely dissolved. 3. The pH of the drug solution formulated with sucrose was measured to be 4.24, and the pH was adjusted to 6.43 with 10 μL of NaHCO3 injection. Further adjustment to 7.13 was performed by adding another 10 μL of the injection.

[0214] The pH of the drug solution formulated with mannitol was measured to be 4.62, and the pH was adjusted to 7.31 with 12 μL of NaHCO3 injection.

[0215] The obtained solution was filtered using polyvinylidene fluoride (PVDF) and subpackaged in 1 mL / vial portions.

[0216] Using the same procedure as described above for "high-speed freeze-drying," the relevant parameters were adjusted to obtain a freeze-dried formulation.

[0217] After freeze-drying, the vial was sealed under vacuum and fitted with a crimp cap. A freeze-dried formulation was obtained. The residual solvent and residual water in the freeze-dried formulation were measured.

[0218] Freeze-dried formulation samples were placed in an incubator at 25°C or 40°C, and their purity was measured by HPLC on the corresponding day. The results are shown in Table 7 below.

[0219] [Table 7]

[0220] The stability curves for 100 mg / mL sucrose and 80 mg / mL mannitol were plotted using the purity obtained by HPLC (Table 7) on the Y-axis and time on the X-axis, and are shown in Figure 4.

[0221] By analyzing and comparing the data shown in Table 7 and Figure 4, it can be concluded that the stability of freeze-dried formulations prepared using drug solutions with mannitol as an excipient is better at 25°C or 40°C than that of formulations prepared using sucrose.

[0222] 3.2 Experiments on the stability and formulation characteristics of reconstituted freeze-dried formulations. One vial of freeze-dried powder was removed and dissolved in a 5% by mass aqueous glucose solution (D5W) to obtain a solution containing approximately 5 mg of API per 1 mL. The resulting solution was then left to stand at room temperature. The color and clarity of the solution were determined at 0, 6, and 24 hours, respectively. The results showed that in all cases, a colorless, transparent, and clear solution was observed.

[0223] The solution was diluted with a 30% acetonitrile aqueous solution for 0, 6, and 24 hours, respectively, to obtain a solution containing approximately 1 mg of API per 1 mL, and its purity was measured by HPLC. The results are shown in Table 8 below.

[0224] [Table 8]

[0225] One vial of freeze-dried powder was removed and dissolved in D5W to obtain a solution containing approximately 5 mg of API per 1 mL. The pH and osmotic pressure of the obtained solution were measured. The results are shown in Table 9 below. During the measurement, the pH and osmotic pressure of 5% glucose injection were also measured.

[0226] [Table 9]

[0227] The comparison revealed no significant difference in the stability and formulation characteristics of the reconstituted freeze-dried formulations between the formulation using 100 mg / mL sucrose and the formulation using 80 mg / mL mannitol. Both formulations can meet the requirements.

[0228] By comprehensively examining storage stability experiments of freeze-dried formulations at room temperature and under high-temperature acceleration, and experiments on the stability and formulation characteristics of reconstituted freeze-dried formulations, it was found that under existing rapid freeze-drying process conditions, freeze-dried formulation samples prepared using a drug solution containing mannitol as an excipient showed better stability at 25°C and 40°C than formulations using sucrose. However, the stability and formulation characteristics of those reconstituted using 5% glucose injection were only slightly different.

[0229] 4. Experiment to investigate the effects of pH and API concentration of the intermediate drug solution on sample reconstitution. Drug solutions for lyophilization were formulated according to Table 10 below.

[0230] [Table 10]

[0231] The density of tert-butanol is ρ = 0.785 g / ml. Therefore, the 40% volume ratio of tert-butanol in 200 ml of drug solution corresponds to a volume of 80 ml of tert-butanol and a mass of 62.80 g of tert-butanol.

[0232] Drug solution preparation process 1. First, the specified amount of freeze-dried protective agent was dissolved in approximately 80% of the specified amount of water while stirring until completely dissolved. 2. Add the specified amount of API to tert-butanol and the remaining water, and dissolve while stirring. 3. The solutions obtained from the two processes described above were uniformly mixed. 4. The mixed solution was filtered. The pH of the solutions for batches 01 and 02 was not adjusted, but the pH of the solutions for batches 03 and 04 was adjusted to 7.0. 5. Filling: Fill batch 01 with 14 mL, batch 02 with 10.5 mL, batch 03 with 14 mL, and batch 04 with 10.5 mL. The gauge of the filled vials is 50 mL.

[0233] One vial from each of batches 01, 02, 03, and 04 was placed in a medical-grade refrigerator (2-8°C) and the crystallization phenomenon was observed. As shown in Figure 5 below, it was found that they all exhibited different degrees of crystallinity. The crystallization supernatant and the reconstituted crystal solution (reconstituted with 5% glucose solution) from batch 01 were collected and their contents were measured. The results are shown in Table 11 below.

[0234] [Table 11]

[0235] The above-described filled formulation was freeze-dried in a vacuum freeze-dryer. The freeze-drying conditions, optimized through multiple trials, were used and are shown in Table 12 below. After freeze-drying, the vial was sealed under vacuum, and then a crimp cap was attached. The freeze-dried formulation shown in Figure 6 was obtained.

[0236] [Table 12]

[0237] The above freeze-dried formulations were reconstituted with 40 ml and 50 ml of 5% glucose solution, respectively. The results are shown in Table 13 below.

[0238] [Table 13]

[0239] Figure 7 shows a photograph of the formulation reconstituted with 40 mL of 5% glucose solution. For batch 3 (API concentration 15 mg / mL, pH adjusted to 7.13), there were no deposits on the vial walls, but several insoluble particles were present at the bottom of the vial. After standing for a certain period, the solution became clear. This indicates that excessively high API concentrations can be detrimental to reconstitution after freeze-dried formulations.

[0240] Figure 8 shows a photograph of the formulation reconstituted with 50 mL of 5% glucose solution. For batch 3 (API concentration adjusted to 15 mg / mL, pH to 7.03), there were no deposits on the vial wall or insoluble particles at the bottom of the vial, and the solution was clear. This indicates that increasing the volume of the reconstitution solution can improve the reconstituted freeze-dried formulation.

[0241] Conclusion of the experiment By comparing experimental results, the inventors found that although the solubility of API (TH-302) in a tert-butanol-water mixed solvent system can be 160 mg / mL or higher, the concentration of API should be kept within an appropriate range for the following reasons. Firstly, if the concentration of the API is excessively high, crystallization and stratification of the drug solution may occur during the cooling procedure of the freeze-drying process, thereby affecting the freeze-drying process. Secondly, excessively high drug concentrations can affect the reconstitution process and potentially lead to reconstitution failure.

[0242] Therefore, there is an appropriate range for API concentration. Through extensive experimentation, the inventors have found that a range of 5 to 160 mg / mL is preferable, or even better, that an API concentration of 8 to 50 mg / mL results in a stable drug solution and good quality of the subsequent freeze-dried formulation.

[0243] 5. Experiment to investigate the effects of API concentration, lyophilization protective agent concentration, and tert-butanol concentration on the residual solvent of freeze-dried powder. The effects of API concentration, lyophilization protective agent concentration, and tert-butanol concentration on the residual solvent of freeze-dried powder were investigated, and a blank freeze-dried powder formulation was used as a control.

[0244] Drug solutions for lyophilization were prepared according to Table 14 below. Each batch consisted of 10 vials, 1 mL per vial, for a total of 10 mL.

[0245] [Table 14]

[0246] Drug solution preparation process 1. First, the specified amount of freeze-dried protective agent was dissolved in the specified amount of water and allowed to dissolve completely. 2. For batches 01, 02, 03, and 04, the specified amount of tert-butanol was added to the lyophilized protective agent aqueous solution and mixed uniformly. Then, the specified amount of API was added and stirred until the API was completely dissolved.

[0247] For batch 05, tert-butanol was not added. The specified amount of API was added and stirred until the API was completely dissolved.

[0248] For batches 06 and 07, the specified amount of tert-butanol was added to the lyophilized protective agent aqueous solution and mixed uniformly. These were used as blank controls without the addition of API.

[0249] 3. In this experiment, the solution was subpackaged into 1 mL / vial without adjusting the pH.

[0250] The aforementioned subpackaged formulation was pre-freeze-dried in a -70°C refrigerator for 3 hours, and then dried in a freeze-dryer for 62 hours. After freeze-drying, the vial was sealed under vacuum, and then a crimp cap was attached. The freeze-dried formulation shown in Figure 9 was obtained.

[0251] The formulation was then infused with D5W for reconstitution. Reconstitution was easy for all batches 01-07, and the solution was clear.

[0252] Freeze-dried formulation samples were placed in a 40°C incubator, and their purity was measured by HPLC on the corresponding day. The results are shown in Table 15 below.

[0253] [Table 15]

[0254] The content of tert-butanol (2 vials) and water (1 vial) in the sample on day 0 was also measured. The results are shown in Tables 16 and 17 below.

[0255] [Table 16]

[0256] Note: TBA was not added to batch 05. "01 Middle Layer" and "01 Lower Layer" represent freeze-dried formulation samples obtained from the same batch by freeze-drying drug solutions placed on different plates (middle layer and lower layer) in a freeze-dryer.

[0257] [Table 17]

[0258] Conclusion of the experiment In samples prepared using a small freeze-dryer, the TBA residue levels in each vial varied, with the TBA content in formulations using sucrose being higher than that of formulations using mannitol.

[0259] Under 40°C conditions, the amount of the related substance increased significantly in formulations using sucrose, but only slightly in formulations using mannitol.

[0260] API concentration is not strongly related to the residual solvent content in freeze-dried formulations. Similarly, lyophilization protective agent concentration is not strongly related to the residual solvent content in freeze-dried formulations.

[0261] API concentration, lyophilization protective agent concentration, and tert-butanol concentration all affect the residual solvent content in freeze-dried formulations. In comparison, freeze-dried formulations obtained using mannitol as a lyophilization excipient (lyophilization protective agent) have lower residual solvent levels, and in high-temperature accelerated stability experiments, formulations formulated with mannitol are more stable than those formulated with sucrose.

[0262] 6. Experiments to investigate the effects of various API and mannitol concentrations, various proportions of tert-butanol, and various packing volumes on sample reconstruction. 6.1 Investigation of the effects of various pH values ​​and formulation (40% tert-butanol + 60 mg / ml mannitol + 10 mg / mL API) on sample reconstitution A 600 mL drug solution was prepared according to Table 18.

[0263] [Table 18]

[0264] The density of tert-butanol is ρ = 0.785 g / mL. Therefore, a volume of 240 mL corresponds to 188.4 g.

[0265] Drug solution preparation process 1. A specified amount of tert-butanol was added to the lyophilized protective agent aqueous solution while stirring and mixed uniformly. Then, a specified amount of API was added and dissolved while stirring. 2. Filtered using a sterile filter cup. Batch 01: 300 ml, no pH adjustment. Batch 02: pH 4.58 of 7.07 The solution was adjusted accordingly. Simultaneously, 6 ml of the solution was taken from each batch, stored in a refrigerator (2-8°C), and the crystallization phenomenon was observed. 3. Filling 200mg / vial: Batch 01, 21 Fill in mL 6 Obtain the vial, and for batch 02, 21 Fill in mL 6 I obtained a vial. 150mg / vial: Batch 01, 16 Fill in mL 6 Obtain the vial, and for batch 02, 16 Fill in mL 6 I obtained a vial. 100mg / vial: Batch 01, 10.5 Fill in mL 6 Obtain the vial, and for batch 02, 10.5 Fill in mL 6 I obtained a vial.

[0266] 6.2 Investigation of the effects of various pH values ​​and the packing volume of the formulation (30% tert-butanol + 70 mg / mL mannitol + 12.5 mg / mL API) on sample reconstitution. 600 ml of drug solution was prepared according to Table 19.

[0267] [Table 19]

[0268] Since the density of tert-butanol is ρ = 0.785 g / mL, the mass corresponding to 180 mL of tert-butanol is 141.3 g.

[0269] Drug solution preparation process 1. First, the specified amount of freeze-dried protective agent was dissolved in approximately the specified amount of water, stirring at 500 rpm until completely dissolved. 2. A specified amount of tert-butanol was added to the lyophilized protective agent aqueous solution and mixed uniformly. Then, a specified amount of API was added and dissolved while stirring. 3. Filtered using a sterile filter cup. Batch 03: 300 mL, no pH adjustment. Batch 04: pH 4.61 of 7.01 The solution was adjusted accordingly. Simultaneously, 6 mL of the solution was taken from each batch, stored in a refrigerator (2-8°C), and the crystallization phenomenon was observed. 4. Filling 200mg / vial: Batch 03, 17 Fill in mL 8 Obtain the vial, and for batch 04, 17 Fill in mL 8 I obtained a vial. 150mg / vial: Batch 03, 13 Fill in mL 7 Obtain the vial, and for batch 04, 13 Fill in mL 7 I obtained a vial. 100mg / vial: Batch 03, 8.5 Fill in mL 8 Obtain the vial, and for batch 04, 8.5 Fill in mL 8 I obtained a vial.

[0270] 6.3 Pre-freeze drying and freeze drying The aforementioned subpackaged formulations were freeze-dried in a vacuum freeze-dryer. Freeze-drying was performed using the parameters listed in Table 12. After freeze-drying, the vials were sealed under vacuum and then fitted with crimp caps. Freeze-dried formulations were obtained.

[0271] 6.4 Experimental Results (1) Crystallization phenomenon in intermediate drug solution Six mL of the solution was taken from batches 01 and 02 and stored in a refrigerator (2-8°C) for a set period of time (2 hours). As a result, the solution was clear and no crystallization was observed.

[0272] Six mL of the solution was taken from batches 03 and 04 and stored in a refrigerator (2-8°C) for a set period of time (2 hours). Crystallization was observed as a result.

[0273] (2) Sample reconstruction All of the freeze-dried preparations were white, cake-like solids.

[0274] The solvents used to reconstruct the samples and the phenomena observed after reconstruction are shown in Table 20 below.

[0275] [Table 20]

[0276] Atmospheric pressure refers to the conditions under which purified water is injected into a vial containing a freeze-dried preparation for reconstitution, with the vial's aluminum cap and rubber stopper open, allowing the vial to communicate with the external environment. If atmospheric pressure is not specified, it refers to the conditions under which purified water is directly injected into the vial containing the freeze-dried preparation via a syringe for reconstitution (in this case, the pressure inside the vial is lower than the external atmospheric pressure).

[0277] Samples from batch 04 (8.5 mL, 13 mL, and 17 mL) were collected after being stored at 40°C for 10 days and then reconstituted. The results are shown in Table 21 below.

[0278] [Table 21]

[0279] Brief summary of the experiment: Storing samples from batch 04 (8.5 mL, 13 mL, and 17 mL) at 40°C for 10 days did not affect reconstitution. The phenomena observed in the reconstituted samples were not significantly different from those observed in the sample on day 0.

[0280] Conclusion of the experiment Generally speaking, when the volume of a container is fixed, filling a freeze-dried drug solution with a smaller volume within a certain range makes it easier to reconstitute the resulting freeze-dried formulation.

[0281] Regarding whether the pH value of the drug solution should be adjusted, relatively speaking, freeze-dried formulations obtained by adjusting the pH value to approximately 7 exhibit better reconstitution performance.

[0282] Specific comparisons reveal that freeze-dried formulations obtained with lower API concentrations and lower mannitol content were easier to reconstitute. This suggests that simply increasing the API concentration and mannitol dosage to increase the drug loading capacity of freeze-dried formulations does not make reconstitution easier. To obtain a freeze-dried drug solution that is easy to reconstitute and has a higher drug loading capacity, appropriate API concentrations and mannitol content must be selected.

[0283] 7. Experiment to investigate the effect of pH on sample stability The effect of pH on sample stability was investigated.

[0284] The drug solution was prepared according to the plan shown in Table 22 below. A total volume of 6500 mL was prepared. 25 mL was filled into each vial, for a total of 260 vials.

[0285] [Table 22]

[0286] The density of tert-butanol is ρ = 0.785 g / mL, so the mass of 1950 ml of tert-butanol is 1530.75 g. The purity of the API is 99.26%.

[0287] Preparation of freeze-dried formulations 1. Preparation of the solution (1) Preparation of 70% tert-butanol aqueous solution: tert-butanol 350.81 g and water 150.42 The solution was obtained by weighing the amount in grams. (2) Preliminary dissolution: A specified amount of API was weighed and added to a 70% tert-butanol aqueous solution under stirring conditions, and dissolved while stirring. The resulting solution was recorded as Solution A. (3) Under stirring conditions, first, the specified amount of mannitol was dissolved in 70% of the remaining water. The resulting solution was stirred until clear and recorded as Solution B. (4) Solution A and Solution B were mixed and stirred uniformly. Then, the remaining water and the remaining tert-butanol were added to the mixed solution after rinsing. The pH of the resulting intermediate drug solution was 5.63 That was the case. (5) The drug solution was divided into three equal parts. Drug solution-01: The pH was measured after storage at room temperature for 24 hours without pH adjustment. 4.77 That was the case. Drug Solution-02: Sodium Bicarbonate 200 When μl was added and the pH was measured, 6.60 (The pH was between 6.0 and 6.5.) After storing at room temperature for 24 hours, the pH was measured again. 5.20 That was the case. Drug Solution-03: Sodium Bicarbonate 1000 When μl was added and the pH was measured, 7.20 (The pH was 7.0). After storing at room temperature for 24 hours, the pH was measured again. 6.90 The intermediate drug solution at 0 hours and the drug solution after 24 hours of storage at room temperature were collected, and their contents and related substances were measured.

[0288] 2. Filtration The above solution was filtered using a 250 mL sterile filter cup.

[0289] 3. Filling For drug solution batch-01, 24.5 Fill with g 78 A vial was obtained and recorded as batch 01. Regarding drug solution batch-02, 24.5 Fill with g 78 The vial was obtained and recorded as batch 02. Regarding drug solution batch 03, 24.5 Fill with g 78 The vial was obtained and recorded as batch 03.

[0290] 4. Half-stopper After packaging, the vials were partially capped and placed in a freeze-dryer for freeze-drying.

[0291] 5. Pre-freeze-drying and freeze-drying The subpackaged formulations described above were freeze-dried in a vacuum freeze-dryer. The arrangement of the vials in this freeze-dryer model is shown in Figure 10. Freeze-drying was performed using the freeze-drying parameters listed in Table 12. After freeze-drying, the vials were capped under vacuum and fitted with crimp caps. Freeze-dried formulations were obtained. The vials were manually capped under high vacuum at a plate temperature of 5.0°C before removal.

[0292] Experimental results The test results for the drug solution are shown in Table 23.

[0293] [Table 23]

[0294] Condition of the freeze-dried formulation sample: White, cake-like solid. A specific sample is shown in Figure 11. As shown in Figure 12, powder was adhering to the bottleneck of the sample bottle coming from the back box in the middle of the freeze-dryer. This may have been caused by improper handling during packaging.

[0295] (3) Measurement of reconstituted samples Freeze-dried formulations were collected and reconstituted from various batches and locations. The results are shown in Table 24 below.

[0296] [Table 24]

[0297] Note: The reconstitution solution for batch 02 (blank powder) was as clear as water. The reconstitution solutions for batches 01, 02, and 03 were slightly cloudier than water.

[0298] To further investigate the reconstitution effect using physiological saline, a commonly used intravenous injection solvent, the preparation was reconstituted with purified water to an API concentration of approximately 5 mg / mL, and then further diluted with physiological saline to 0.5 mg / mL (5 mL was withdrawn and added to 50 mL of sodium chloride injection, resulting in 0.5 mg / mL). A clear solution was obtained, and then the pH value and osmotic pressure data were measured. The results are shown in Table 25 below.

[0299] [Table 25]

[0300] After reconstitution with physiological saline, samples were taken at various time points, and the purity of the API in the solution was measured by HPLC. The results are shown in Table 26 below.

[0301] [Table 26]

[0302] (4) Solvent residue and stability test of the sample Freeze-dried formulation samples were placed in incubators at 40°C, 25°C, and 2–8°C, and their purity was measured by HPLC on the corresponding days. The results are shown in Table 27 below.

[0303] [Table 27]

[0304] Testing of residual solvents The tert-butanol content (2 vials) and water content (2 vials) of samples from batches 01, 02, and 03 on day 0 were measured. Samples were taken from the middle section of the freeze-drying machine plate. The results are shown in Table 28 below.

[0305] [Table 28]

[0306] 8. Examples of preparations after lyophilization of freeze-dried formulations using solutions with various tert-butanol concentrations, various excipients (sucrose or mannitol), various excipient amounts, various drug contents, and various pH values. Three main indicators of quality for freeze-dried formulations are reconstitution, stability, and residual solvent. From the above experiments, it was found that influencing factors include the solvent (volume ratio of tert-butanol in the tert-butanol-water mixed solvent), API concentration, type and amount of excipient (mannitol or sucrose), and the pH value of the drug solution. Taking these into consideration, the inventors conducted several multifactor experiments. The results are shown in Table 28 below.

[0307] [Table 29]

[0308] [Table 30]

[0309] [Table 31]

[0310] [Table 32]

[0311] [Table 33]

[0312] [Table 34]

[0313] Note: TBA stands for tert-butanol. Mannitol is mannitol. Sucrose is sucrose. The API is the active pharmaceutical ingredient TH-302. PBS stands for Phosphate Buffered Physiological Saline, and it has a stable pH value of approximately 7.4. In the formulation, "30% TBA / 70 Mannitol / 10 API-pH7.0" means that the volume ratio of tert-butanol is 30%, mannitol is 70 mg / mL, API is 10 mg / mL, and the pH is adjusted to 7.0 by adding NaHCO3. The preparation method was the same as described in the examples above. "40% TBA / 60 Mannitol / 10 API" means that the volume ratio of tert-butanol is 40%, mannitol is 60 mg / mL, API is 10 mg / mL, and the pH is not adjusted (weakly acidic), and the same applies to the other components.

[0314] Except for batches with a supply volume of 1 mL, 50 mL vials were used.

[0315] Conclusion of the experiment 1. Based on the solubility data of TH-302 in a tert-butanol-water mixed solvent system combined with the experiments described above, and the exploratory experimental results of 85 groups shown in Table 28 above, it can be estimated or confirmed that the compound can exist stably in solution when its content is in the range of 5 to 500 mg / mL. Although it may be necessary to adjust the freeze-drying parameters, for example, by pre-freeze-drying at -80°C, significantly lowering the absolute pressure, increasing the drying time, and lowering the drying temperature, it has been experimentally demonstrated that freeze-drying is possible when the content is greater than 8 mg / mL and less than or equal to 200 mg / mL. Preferably, when the content of the compound of formula I in the above solution is between 8 mg / mL and 25 mg / mL, the freeze-dried formulation has better stability and is easier to reconstitute. The content of the compound of formula I in the above solution is between 8 mg / mL and 15 mg / mL. Such a content range means that the freeze-drying conditions are relatively mild and the freeze-drying cycle is shorter. The fact that the compound of formula I in the solution is between 8 mg / mL and 10 mg / mL further means that this freeze-dried formulation is commercially viable for large-scale production under commercially appropriate freeze-drying cycles and low-temperature conditions. An inappropriate API content could lead to vial breakage or powder atomization in large freeze-dryers used in large-scale commercial production due to uneven heating temperatures, resulting in a yield of less than 90% of the acceptable level for commercial production.

[0316] 2. Based on the solubility data of TH-302 in a tert-butanol-water mixed solvent system combined with the above experiments, and the results of the 85 exploratory experiments shown in Table 28 above, it can be estimated or confirmed that the prepared TH-302 solution is stable and clear when the volume percentage of tert-butanol in the solution is 1-99%, or when the tert-butanol content in the solution is 7.85-777.15 mg / mL (under the condition that the density of tert-butanol is 0.785 g / mL). It may be necessary to adjust the freeze-drying parameters, for example, by pre-freezing at -80°C, lowering the absolute pressure during freeze-drying, increasing the drying time, or lowering the drying temperature. However, it has been experimentally demonstrated that freeze-drying is possible when the volume ratio is 5-95%, or when the tert-butanol content in the solution is 39.25-745.75 mg / mL (under the condition that the density of tert-butanol is 0.785 g / mL). Preferably, when the volume ratio of tert-butanol is 30-60%, or the tert-butanol content in the solution is 235.5-471 mg / mL (under the condition that the density of tert-butanol is 0.785 g / mL), the freeze-dried formulation has better stability and is easier to reconstitute.

[0317] In fact, in a tert-butanol-water mixed solvent system, an excessively high tert-butanol content affects the subsequent solubility of mannitol / sucrose, and the amount of mannitol / sucrose, which functions as an excipient, directly impacts the quality of the subsequent freeze-dried formulation. If the amount of mannitol / sucrose is too low, the drug solution may not be able to uniformly load the excipient backbone after freeze-drying, or furthermore, the freeze-dried powder cake may collapse during the drying stage of the freeze-drying process, or freeze-drying may fail. Therefore, the tert-butanol content in the mixed solvent cannot be inappropriately increased solely for the purpose of increasing the solubility of TH-302.

[0318] 3. Based on the solubility data of TH-302 in a tert-butanol-water mixed solvent system combined with the above experiments, and the results of the 85 exploratory experiments shown in Table 28, it can be seen that the freeze-dried formulation can be obtained when mannitol, PEG2000, P188, SBECD, DSPE-MPEG2000, sucrose, or other similar substances are used as excipients. However, after comparing the results, and considering factors other than those of the freeze-dried formulation itself, such as whether it can be conventionally used and commercially available, and whether it does not affect efficacy, DMPK, and toxicity, it is more appropriate to use sucrose and mannitol. Freeze-dried formulations prepared using such excipients have adequate stability in solution (lasting at least 24 hours at room temperature), the freeze-dried formulations are stable, can be immediately reconstituted, and it is easy to obtain products that meet the requirements for excipients for injectable formulations.

[0319] 4. In fact, as stated in item 2 above, a higher content of mannitol / sucrose as an excipient does not necessarily mean better efficacy. That is, a higher amount of excipient allows the freeze-dried skeleton to be loaded with more drug, which is beneficial for lyophilization production and improved stability of the freeze-dried formulation, but this stability is affected by the solubility in the tert-butanol-water mixed solvent system. If the amount of mannitol / sucrose is excessively high, it may not be able to dissolve completely in the mixed solvent, or the solution may become unstable, potentially leading to precipitation during the pre-lyophilization or cooling stage. Therefore, the amount of mannitol / sucrose used as an excipient in the freeze-dried solution should also be within an appropriate range to meet the above requirements.

[0320] The sucrose or mannitol content in the solution is 20–300 mg / mL. Within this range, the above requirements can be met, namely that the drug solution is stable and less prone to precipitation during the pre-freeze-drying or cooling stage of the freeze-drying process. A content of 40–100 mg / mL is more preferable, allowing for the acquisition of well-stabilized reconstituted solutions and freeze-dried formulations. A content of 60–70 mg / mL is even more preferable. Such a content range means that the freeze-drying conditions are relatively mild and the freeze-drying cycle conforms to commercial production practices (less than 10 days, i.e., 240 hours per production cycle).

[0321] 5. As stated in item 2 above, if the amount of mannitol / sucrose is too low, the drug solution may not be able to be uniformly loaded into the excipient skeleton after freeze-drying, or furthermore, the freeze-dried powder cake may collapse during the drying stage of the freeze-drying process, or freeze-drying may fail. Conversely, if the amount of mannitol / sucrose is too high, although it is possible to produce a freeze-dried formulation with good appearance and stability, it means that the amount of drug TH-302 in the freeze-dried formulation per unit package may be too low to meet the requirements for commercial sale and commercial use. Therefore, an appropriate ratio of mannitol / sucrose to the active drug is essential. In this solution, the mass ratio of the compound of formula I to the excipient is in the range of 1:(0.5~20). Such a ratio can satisfy the requirements stated in items 1~4 above with respect to the other components, and can enable the prepared freeze-dried formulation to have good appearance and stability. Preferably, the above mass ratio is in the range of 1:(2 to 12.5), and within this range, a better freeze-dried formulation that is easily reconstituted can be obtained. More preferably, when the above mass ratio is in the range of 1:(5 to 10), the prepared freeze-dried formulation has excellent properties. That is, the freeze-dried formulation has good stability, is easy to reconstitute, the freeze-drying conditions are mild, and the freeze-drying cycle is short.

[0322] 6. Experimental phenomena demonstrated that the pH value of a drug solution for freeze-drying directly affects the stability of the drug solution, the level of complexity of its reconstitution, and the stability of the corresponding freeze-dried formulation. Experiments showed that when no pH adjuster (base or basic salt) was added to the drug solution, the drug solution became acidic, resulting in poor stability of the acidic drug solution and insufficient reconstitution and low stability of the corresponding freeze-dried formulation. Adding a pH adjuster (base or basic salt) significantly improved these aspects.

[0323] 7. It should be noted that the filling volume of the drug solution in the vial can also affect the appearance and reconstitution of the final freeze-dried powder cake (the powder is either in a sprayed state or adhered to the vial's bottleneck) due to several factors. Therefore, the ratio of the filling volume of the drug solution in the vial to the volume of the vial must be appropriate. Based on the experimental data and actual production practices described above, it is appropriate that the filling volume of the freeze-dried drug solution be 1 / 3 to 1 / 2 of the volume of the sealed container, and the volume of the corresponding freeze-dried formulation be 1 / 3 to 1 / 2 of the volume of the sealed container.

[0324] 9. Saturation solubility of the drug in D5W solution (under strong shaking conditions) Approximately 60 mg of the TH-302 sample was accurately weighed and placed in a clear vial. 6 mL of 5% glucose aqueous solution (D5W) was added. The resulting solution was vigorously shaken for 2 minutes to obtain a solubility-preservation solution. The solubility-preservation solution was observed to contain an insoluble substance. Therefore, the solubility-preservation solution was filtered, and it became clear. The filtered filtrate was left at room temperature for 24 hours and remained clear. After sampling and appropriate dilution, the solution content was measured by HPLC and found to be 7.25 mg / mL. This value was precisely the saturation solubility of the drug in the D5W solution.

[0325] Thus, it can be seen that the concentration of TH-302 in TH-302-containing injectable preparations for intravenous administration should be within the range of 0 to 7.25 mg / mL. In fact, considering the isotonic and osmotic properties of glucose contained in intravenous injectable preparations, further research is needed to determine appropriate infusion formulations for intravenous drip infusion.

[0326] Statistical results of height measurements for pre-freeze-drying and post-freeze-drying of drug solutions in 10.50 mL vials (calculation of drug loading volume and drug mass percentage). The drug solution for Experiment 7 was filled into vials. 25 mL of the drug solution was filled into 50 mL vials, and the liquid level after filling was measured. The average height was 26.5 mm. After freeze-drying, the height of the freeze-dried powder cake in vials at various plate positions in the freeze-dryer was measured, and the volume difference compared to the 25 mL before filling was roughly calculated.

[0327] In multi-batch experiments, it was found that the volume before freeze-drying increased in some batches, decreased in others, and remained almost unchanged in some batches. By marking the liquid level before and after freeze-drying and statistically analyzing the volume change, it was found that the maximum increase or decrease in volume was 10%. In other words, the volume change was within ±10%.

[0328] When the volume of freeze-dried powder cake decreases by 10% after freeze-drying, If the API content of the drug solution is between 5 mg / mL and 500 mg / mL, the drug loading capacity of the freeze-dried powder cake after lyophilization is 5 / 0.9 to 500 / 0.9, i.e., 5.55 to 555.55 mg / cm³. 3 The following results were obtained using the same method. When the compound content of formula I in the drug solution is in the range of 5 to 160 mg / mL, the drug loading capacity of the freeze-dried powder cake is 5.55 to 177.77 mg / cm³. 3 That is the case. When the compound content of formula I in the drug solution is in the range of 8 to 50 mg / mL, the drug loading capacity of the freeze-dried powder cake is 8.88 to 55.55 mg / cm³. 3 That is the case. When the compound content of formula I in the drug solution is in the range of 8 to 25 mg / mL, the drug loading capacity of the freeze-dried powder cake is 8.88 to 27.77 mg / cm³. 3 That is the case. When the compound content of formula I in the drug solution is in the range of 8 to 15 mg / mL, the drug loading capacity of the freeze-dried powder cake is 8.88 to 16.66 mg / cm³. 3 That is the case. If the compound content of formula I in the drug solution is in the range of 8 to 10 mg / mL, the drug loading capacity of the freeze-dried powder cake is 8.88 to 11.11 mg / cm³. 3 That is the case. If the drug solution contains 6, 7, 7.5, 8, 8.5, 10, 12.5, 15, 20, or 25 mg / mL, the corresponding drug loading volumes for freeze-dried powder cakes are 6.66, 7.77, 8.33, 8.88, 9.44, 11.11, 13.88, 16.66, 22.22, or 27.77 mg / cm³. 3 That is the case.

[0329] When the volume of freeze-dried powder cake increases by 10% after freeze-drying, If the API content of the drug solution is between 5 mg / mL and 500 mg / mL, the drug loading capacity of the freeze-dried powder cake after lyophilization is 5 / 1.1 to 500 / 1.1, i.e., 4.55 to 454.55 mg / cm³. 3 The following results were obtained using the same method. When the compound content of formula I in a drug solution is in the range of 5 to 160 mg / mL, the drug loading capacity of the freeze-dried powder cake is 4.55 to 145.45 mg / cm³. 3 That is the case. When the compound content of formula I in the drug solution is in the range of 8 to 50 mg / mL, the drug loading capacity of the freeze-dried powder cake is 7.27 to 45.45 mg / cm³. 3 That is the case. When the content of the compound of formula I in the drug solution is in the range of 8 - 25 mg / mL, the drug loading capacity of the freeze-dried powder cake is 7.27 - 22.73 mg / cm 3 . When the content of the compound of formula I in the drug solution is in the range of 8 - 15 mg / mL, the drug loading capacity of the freeze-dried powder cake is 7.27 - 13.64 mg / cm 3 . When the content of the compound of formula I in the drug solution is in the range of 8 - 10 mg / mL, the drug loading capacity of the freeze-dried powder cake is 7.27 - 9.09 mg / cm 3 . Assuming that the content of the drug solution is 6, 7, 7.5, 8, 8.5, 10, 12.5, 15, 20 or 25 mg / mL, the corresponding drug loading capacities of the freeze-dried powder cake are 5.45, 6.36, 6.82, 7.27, 7.73, 9.09, 11.36, 13.64, 18.18, or 22.73 mg / cm 3 .

[0330] The preparation obtained by freeze-drying the drug solution contains API drug, excipient, as well as residual tert-butanol solvent and residual water. Since the mass ratio of the API drug to the excipient in the freeze-dried preparation prepared by the applicants is in the range of 1:(0.5 - 20), the maximum allowable tert-butanol content is 1.75% by mass, and the water content is 6% by mass. Therefore, the lower limit based on the mass percentage of the API drug in the freeze-dried preparation is (1 / (20 + 1))*(1 - 6% - 1.75%) = 4.76%*92.25% = 4.39%, and the upper limit is (1 / (0.5 + 1))*(1 - 0% - 0%) = 66.66%. That is, the content of the API drug in the freeze-dried preparation is not less than 4.39% and less than 66.66%.

[0331] Based on initial exploratory experiments and several considerations, the applicant used the following recommended scheme for batch production of the freeze-dried formulation. The freeze-dried formulation produced as the final product is expected to meet the requirements for subsequent clinical trials, be used as a candidate drug in subsequent large-scale clinical trials, and be approved for future commercialization, manufacture, or sale.

[0332] 11. Experiments on the preparation, stability, and reconstitution of small batch freeze-dried formulations. 11.1 Based on "7. Experiments to investigate the effect of pH on sample stability" and the relevant preparation methods in the formulations shown in Table 22, freeze-dried formulations were prepared using the following laboratory simulation and in-line manufacturing processes.

[0333] 1. Preparation of the solution (1) Preliminary dissolution of the active pharmaceutical ingredient: A portion of tert-butanol and sterile water for injection were weighed, stirred, and mixed uniformly to prepare a 70% (w / w) tert-butanol solution. Next, a specified amount of the active pharmaceutical ingredient was weighed and added to the 70% (w / w) tert-butanol solution, and stirred until the active pharmaceutical ingredient was completely dissolved (confirmed visually). (2) Approximately 70% (w / w) of the total volume of water for injection was added to the liquid preparation tank, and then the specified amount of mannitol was added and stirred to dissolve it. (3) The remaining tert-butanol in the total prescribed amount was added to the liquid preparation tank, stirred, and mixed uniformly. (4) The pre-dissolved active pharmaceutical ingredient was added to the liquid preparation tank, and then sterile water for injection was added to the total volume of the preparation. The solution was stirred and mixed uniformly. (5) A 5% (by mass) sodium bicarbonate solution was added to adjust the pH value to a range of 4.0 to 9.0.

[0334] 2. Filtration The above solution was filtered using a sterile filter.

[0335] 3. Filling A 50 mL penicillin bottle was used for filling. The theoretical filling volume is 25 mL per vial (density 0.98 g / mL, recorded as 24.5 g per vial). Since the filling volume is controlled within a tolerance of ±2%, the acceptable actual filling volume is (25.0 ± 0.5) mL per vial, and the mass is controlled within the range of (24.5 ± 0.49) g per vial.

[0336] 4. Half-stopper After filling, the vials were partially capped and placed in a freeze-dryer for freeze-drying.

[0337] 5.Lyophilization The aforementioned subpackaged formulations were freeze-dried in a vacuum freeze-dryer. Freeze-drying was performed using the adjusted freeze-drying parameters listed in Table 12. After freeze-drying, the vials were capped under vacuum and fitted with crimp caps. Freeze-dried formulations were obtained. The vials were manually capped under high vacuum with a plate temperature of 5.0°C before removal.

[0338] The above liquid preparation and freeze-drying processes ultimately yielded a total of 174 vials of freeze-dried formulation, each containing 0.20 g of active pharmaceutical ingredient (i.e., each vial of the freeze-dried formulation is labeled as containing 0.20 g of TH-302). The average filling weight is 1.98 g per vial (i.e., the average mass of the powder cake in this batch of freeze-dried formulations is 1.98 g, and the total volume is 25 cm³). 3 Each freeze-dried powder cake contains 1.75g ​​of mannitol, 0.20g of the active pharmaceutical ingredient TH-302, and other components.

[0339] The sampling and test results of the intermediate drug solution (i.e., the drug solution after pH adjustment) from the above batch are shown in Table 29 below.

[0340] [Table 35]

[0341] The intermediate drug solution contains water (used as a solvent) and tert-butanol, as well as mannitol as an excipient. Since mannitol is relatively stable, its detection is not performed during quality control.

[0342] The test results of the freeze-dried formulations sampled from the above batch are shown in Table 30 below.

[0343] [Table 36]

[0344] pH test method: One vial of this product was removed, dissolved in 38 mL of sterile water for injection, and a solution containing 5.0 mg of TH-302 per mL was prepared. The pH was determined using a pH meter. Two samples were measured in parallel, and the average value was calculated.

[0345] The tert-butanol content was tested by gas chromatography as specified in the pharmacopoeia. One vial of the freeze-dried formulation was removed and allowed to return completely to room temperature. While maintaining a vacuum, approximately 30 mL of water was added using a disposable syringe, and the solution was shaken to dissolve. The cap was removed (note: to avoid loss of solution, the action of opening the rubber stopper should be gentle and slow), and the solution was transferred to a 50 mL volumetric flask. The inner surfaces of the sample vial and rubber stopper were washed with water at least three times each, the washings were added to the volumetric flask, diluted with water to the marked volume, and shaken uniformly. Two samples were prepared in parallel, and both samples were tested by gas chromatography, and finally the average value was calculated. The tert-butanol entry refers to the mass percentage of tert-butanol relative to a nominal amount of 200 mg of the active pharmaceutical ingredient, calculated based on a nominal amount of 200 mg of TH-302.

[0346] The water content was determined by the Karl Fischer method. To ensure uniformity and representativeness of the sampling, the following procedure was employed: One vial of the freeze-dried formulation was removed and allowed to return to room temperature. Any water adhering to the surface of the vial was wiped off, and the cap was removed to release the pressure inside the vial. The stopper was opened, and the sample was stirred with a spoon to prevent the sample from adhering to the inner wall and bottom of the vial. The stopper was covered, the vial was placed horizontally, rotated for more than 5 cycles, and then inverted up and down more than 5 times. The stopper was opened, the sample was stirred with a spoon for more than 5 cycles, and the stopper was covered (the mouth of the vial was sealed with Parafilm if necessary). The sample was ready for use. Approximately 0.20 g of the freeze-dried formulation sample was precisely weighed (in reality, the actual mass was less than 0.20 g because it was not possible to completely transfer the freeze-dried powder on the inner wall of the vial to the titration cell of the moisture meter, so precise weighing was performed by the mass difference method), and added to the titration cell. The sample was first stirred for 2 minutes, and then titrated. Alternatively, USP <921> The percentage of water by mass was determined by referring to the law.

[0347] The total amount of impurities refers to the total amount of impurities in the active pharmaceutical ingredient other than TH-302 (excluding the excipients tert-butanol and mannitol). For the TH-302 active pharmaceutical ingredient, the total amount of impurities was determined using the HPLC purity test method described above.

[0348] Following the same procedure, another batch of the freeze-dried formulation (batch number 1202206002) was prepared in a freeze-drying tray with a total mass of 529.54 g. The above product was heat-sealed using a double-layer low-density polyethylene (PE) self-sealing bag as the inner layer and a single-layer 300 mm unprinted composite film (meeting relevant requirements) as the outer layer.

[0349] Following the same procedure, another batch of the freeze-dried formulation (batch number F00106301) was prepared, totaling 600 vials with an average filling amount of 1.97 g per vial, and used for stability studies.

[0350] 11.2 Study of the stability of freeze-dried formulations stored under proposed long-term storage conditions (-20℃±5℃) and accelerated storage conditions (25℃±5℃) The purpose of this study was to investigate the time-dependent fluctuation patterns of test indicators related to the TH-302 freeze-dried formulation under proposed long-term storage conditions (-20°C ± 5°C) and high-temperature accelerated conditions (25°C ± 5°C) in order to establish standards for the quality stability and storage period (shelf life) of this freeze-dried formulation.

[0351] The samples from the two batches mentioned above (1202206002 and 1202206001) were stored in a light-shielded environment at -20±5°C for long-term stability testing. Batch F00106301 underwent both long-term stability testing and accelerated stability testing (stored in a light-shielded environment at 25°C±5°C). The specific test results are shown in Table 31 below.

[0352] [Table 37]

[0353] Identification: Check whether the retention time of the HPLC chromatogram of the test sample matches the retention time of the reference standard solution.

[0354] Values ​​for the content test items: The first value represents the percentage of the actual TH-302 content relative to the nominal amount (the indicated TH-302 content is 0.20g), and the second value represents the mass percentage of TH-302 (calculated based on an average filling amount of 1.98g) as 9.8%.

[0355] Based on the long-term data above, under long-term storage conditions (-20°C ± 5°C), the percentage of total impurities changed gradually. After 3 months and 6 months of storage, there were no significant changes in impurities, pH value, moisture content, or content. Furthermore, data from another batch (F00106301, prepared using TH-302 active pharmaceutical ingredient at a higher purity) showed that the aforementioned indicators did not show significant changes even when stored under accelerated conditions (25 ± 5°C). Under these accelerated conditions, it was inferred that these results would be similar for samples stored for more than 18 months at -20°C ± 5°C.

[0356] 11.3 Study on the freeze-thaw cycle stability of freeze-dried formulations The freeze-thaw test consisted of a cycle of placing the sample in the dark for two days (-20°C ± 5°C) followed by another two days (25°C ± 5°C). The HPLC purity of TH-302 in the freeze-dried sample was determined.

[0357] [Table 38]

[0358] The API data represents the HPLC purity of the TH-302 active pharmaceutical ingredient itself, which was used in the preparation of two batches of freeze-dried formulations.

[0359] The data in the lower column for day 0 (1202206001) represents the purity of the freeze-dried formulation from batch 1202206001 when tested on day 0.

[0360] Based on the long-term data described above, when each freeze-dried formulation stored under long-term storage conditions (-20°C ± 5°C) is removed and placed in a room temperature environment (25°C ± 5°C), the change in the purity of the TH-302 active pharmaceutical ingredient in each freeze-dried formulation remains minimal even after repeating the above procedure three times. This demonstrates that the freeze-dried formulation has good stability against sudden temperature shocks, providing experimental evidence for its stability during actual transport and delivery where it may be exposed to room temperature for relatively long periods.

[0361] 11.4 Stability experiment of freeze-dried formulations using 0.9% NaCl and 5% D5W The freeze-dried formulation for injection was removed from storage at -20±5°C and allowed to return completely to room temperature. 38 mL of sterile water for injection was drawn into a disposable sterile syringe (50 mL). With the beveled end of the needle tube facing upwards and the needle tip at an angle of approximately 60° to the stopper, the needle was inserted into the vial to avoid generating fragments from needle puncture (see Figure 1), and the water was injected in a circular motion along the inner wall of the vial (Figure 2). After injection, the vial was shaken vigorously for at least 20 seconds to ensure that all of the freeze-dried cake / powder was completely dissolved and uniformly mixed, yielding approximately 40 mL of solution containing approximately 5.0 mg of TH-302 per mL. If the freeze-dried cake / powder does not completely dissolve after shaking vigorously for 20 seconds, it can be shaken in a 30-35°C water bath until completely dissolved.

[0362] The above 5.0 mg / mL solution, diluted with sterile water for injection, was prepared into 5 mg / mL compatible solutions with sterile water for injection, 4 mg / mL compatible solutions with 5% glucose injection, 0.5 mg / mL compatible solutions with 5% glucose injection, 4 mg / mL compatible solutions with 0.9% sodium chloride injection, and 0.5 mg / mL compatible solutions with 0.9% sodium chloride injection, and each was tested. The test items included the characteristics of the solution, osmotic pressure, pH value, color of the solution, content, and HPLC percentage of impurities (total impurity amount). The results are shown in Table 33 below.

[0363] [Table 39]

[0364] [Table 40]

[0365] Based on the conformity test results described above, each conformity solution meets specific requirements in terms of test items, including content, related substances, pH value, osmotic pressure, solution color, and characteristics. This demonstrates that the aforementioned freeze-dried formulations, when reconstituted with clinically commonly used sterile water for injection, 5% glucose injection, and 0.9% sodium chloride injection, can all meet the requirements for intravenous injection if they are compatible with these solutions.

[0366] 12. Experiments on the preparation, stability, and reconstitution compatibility of large batch freeze-dried formulations. Based on the freeze-dried formulation preparation experiments described in Section 11 above, larger-scale freeze-dried formulation preparations were carried out using batches F00106301 and F00115101, respectively. Stability tests under high-temperature accelerated conditions (25°C ± 5°C) and freeze-thaw tests were performed. The results are shown in Table 34 below.

[0367] [Table 41]

[0368] The results in Table 34 show that the quality of the freeze-dried formulation prepared after scale-up remained largely unchanged, and each indicator met the required standards.

[0369] A Good Manufacturing Practice (GMP) batch of this API active pharmaceutical ingredient, using provisional quality standards, was manufactured under GMP conditions. This batch will be considered for use in subsequent clinical trials. High-temperature accelerated stability testing and conformity testing were performed on batch 296220901, and the specific experimental data are shown in Tables 35 and 36 below.

[0370] [Table 42]

[0371] [Table 43]

[0372] A 5-day light exposure* represents a vial containing the freeze-dried formulation that is not packaged, while a 5-day light exposure* represents the normal case where it is packaged. 1 represents a loose white cake, and 2 represents a loose pale yellow cake. A value of 3 indicates that the color is lighter than standard color solution No. 0.5, and a value of 4 indicates that the color is between standard color solution No. 0.5 and No. 1. Insoluble particle 1 represents the number of particles with a particle size of ≥ 10 μm, and insoluble particle 2 represents the number of particles with a particle size of ≥ 25 μm.

[0373] [Table 44]

[0374] [Table 45]

[0375] The experimental data described above demonstrates that the stability and reconstitution compatibility of freeze-dried formulations prepared on a large scale from the above freeze-dried formulation formula meet the required standards. Furthermore, the results of accelerated testing suggest that this freeze-dried formulation can be stored stably for 18 months at -20°C ± 5°C in the proposed light-shielded environment.

Claims

1. A solution for preparing a freeze-dried formulation with a high drug loading capacity, wherein the solution comprises a compound of the following formula (I-1), water, tert-butanol, and mannitol. 【Chemistry 1】 Water and tert-butanol are used as a mixed solvent, wherein the volume percentage of tert-butanol in the solution is (30 ± 3)%, or the mass percentage is (24 ± 2.4)%, or the tert-butanol content in the solution is (235.5 ± 23.55) mg / mL. The content of the compound of formula (I-1) in the solution is (8.16 ± 0.82) mg / g or (8.00 ± 0.80) mg / mL. Mannitol is an excipient, and the mass percentage of mannitol in the solution is (7.14 ± 0.71)%, or the mannitol content in the solution is (70 ± 7) mg / mL. The pH value of the aforementioned solution is in the range of 4 to 9. solution.

2. The solution according to claim 1, wherein the solution further comprises sodium bicarbonate as a pH adjusting agent.

3. The solution according to claim 1, wherein the pH value of the solution is in the range of 6 to 8.

4. A freeze-dried formulation comprising the compound of the following formula (I-1) and mannitol, 【Chemistry 2】 The drug loading capacity of the compound of formula (I-1) in the freeze-dried formulation is (8.00 ± 0.80) mg / cm³. ３ The mass percentage of the compound of formula (I-1) in the freeze-dried formulation is (10.23 ± 1.02)%, The mannitol content in the aforementioned freeze-dried preparation is (70 ± 7) mg / cm³. ３ or, the mannitol content in the freeze-dried formulation is the percentage remainder of the mass percentage (10.23 ± 1.02)% of the compound of formula (I-1). Freeze-dried formulation.

5. A freeze-dried formulation comprising the compound of the following formula (I-1), mannitol, and a pH adjuster, 【Transformation 3】 The drug loading capacity of the compound of formula (I-1) in the freeze-dried formulation is (8.00 ± 0.80) mg / cm³. ３ The mass percentage of the compound of formula (I-1) in the freeze-dried formulation is (10.23 ± 1.02)%, The mannitol content in the aforementioned freeze-dried preparation is (70 ± 7) mg / cm³. ３ or, the mannitol content in the freeze-dried formulation is the percentage remainder of the mass percentage (10.23 ± 1.02)% of the compound of formula (I-1). Freeze-dried formulation.

6. A freeze-dried formulation comprising the compound of the following formula (I-1), mannitol, residual solvent components, and a pH adjuster, 【Chemistry 4】 The drug loading capacity of the compound of formula (I-1) in the freeze-dried formulation is (8.00 ± 0.80) mg / cm³. ３ The mass percentage of the compound of formula (I-1) in the freeze-dried formulation is (10.23 ± 1.02)%, The mannitol content in the aforementioned freeze-dried preparation is (70 ± 7) mg / cm³. ３ And, The residual solvent components are water and tert-butanol. Freeze-dried formulation.

7. When dissolved in water for injection, an aqueous solution with a concentration of 5.0 mg / mL and a pH value in the range of 4.0 to 7.0 is obtained. A freeze-dried formulation according to any one of claims 4 to 6.

8. The pH adjusting agent is sodium bicarbonate, and its content is 0.01 to 0.10 mg / cm³. ３ A freeze-dried formulation according to claim 5 or 6, which is within the range of [the specified range].

9. The residual water content is 6% or less by mass, preferably 2% or less, more preferably 1% or less, and even more preferably 0.5% or less. The residual tert-butanol content is 1,795 ppm or less by mass, preferably 1,000 ppm or less, and more preferably 500 ppm or less. The freeze-dried formulation according to claim 6.

10. The mass of the freeze-dried formulation per unit volume is (79.2 ± 7.9) mg / cm³. ３ The freeze-dried formulation according to any one of claims 4 to 6.

11. A freeze-dried formulation according to any one of claims 4 to 6, prepared by freeze-drying the solution according to claim 1.

12. A formulation unit package containing a freeze-dried formulation according to any one of claims 4 to 11, having the following features: The freeze-dried formulation is contained in a sealed container with a capacity of 1,000 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of the compound of formula (I-1) is in the range of 1,600 to 5,333 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 500 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of the compound of formula (I-1) is in the range of 800 to 2,666 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 250 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of the compound of formula (I-1) is in the range of 400 to 1,333 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 100 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of the compound of formula (I-1) is in the range of 160 to 533 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 50 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of the compound of formula (I-1) is in the range of 80 to 266 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 30 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of the compound of formula (I-1) is in the range of 48 to 160 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 25 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of the compound of formula (I-1) is in the range of 40 to 133 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 20 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of the compound of formula (I-1) is in the range of 32 to 107 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 18 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of the compound of formula (I-1) is in the range of 29 to 96 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 15 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of the compound of formula (I-1) is in the range of 24 to 79 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 10 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of the compound of formula (I-1) is in the range of 16 to 53 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 8 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of the compound of formula (I-1) is in the range of 13 to 42 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 7 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of the compound of formula (I-1) is in the range of 11 to 40 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 5 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of the compound of formula (I-1) is in the range of 8 to 27 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 3 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of the compound of formula (I-1) is in the range of 5 to 16 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 2 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of the compound of formula (I-1) is in the range of 3 to 11 mg, or The freeze-dried formulation is contained in a sealed container with a capacity of 1.5 mL, the volume of the freeze-dried formulation is 1 / 5 to 2 / 3, preferably 1 / 3 to 1 / 2, of the volume of the sealed container, and the amount of the compound of formula (I-1) is in the range of 2 to 8 mg. Pharmaceutical unit packaging.

13. An injectable preparation for intravenous injection, wherein the solvent is water, and the solute comprises the active pharmaceutical ingredient TH-302, mannitol, tert-butanol, sodium bicarbonate, and an isotonic modifier, wherein the content of the active pharmaceutical ingredient TH-302 is 0.1 to 4.0 mg / mL, the osmotic pressure of the injectable preparation is 260 to 320 mOsmol / kg, and the pH value is in the range of 4.0 to 9.

0.

14. The injectable preparation according to claim 13, wherein the isotonic adjusting agent is selected from glucose and sodium chloride.

15. The following procedure, that is, The freeze-dried preparation according to any one of claims 4 to 12 is left to stand at room temperature until it returns to room temperature. The freeze-dried preparation, returned to room temperature, is formulated into an aqueous solution for injection containing the active pharmaceutical ingredient TH-302 in an amount of (5 ± 0.5) mg / mL using water for injection. An appropriate amount of the aforementioned aqueous solution for injection is injected into a 0.9% physiological saline injection or a 5% glucose injection for dilution to obtain an injectable preparation containing the active pharmaceutical ingredient TH-302 in an amount ranging from 0.1 to 4.0 mg / mL. A method for preparing an injectable agent according to claim 14, comprising:

16. A method for preparing an injectable preparation according to claim 14, wherein the preparation method is printed in an instruction manual attached to the formulation unit package of the freeze-dried preparation in order to provide specific instructions for preparing the injectable preparation for immediate use in a clinical setting, the prepared injectable preparation should be used within 8 hours, and the method is the following procedure, namely: The freeze-dried preparation according to any one of claims 4 to 12 is left to stand at room temperature for 30 to 120 minutes until it returns to room temperature. Use a syringe to take an appropriate volume of water for injection. Insert the syringe into the vial with the slanted end of the needle facing upwards and the tip of the needle at an angle of approximately 60° to the stopper, then inject along the inner wall of the vial, and after injection is complete, shake the vial containing the drug for at least 20 seconds to ensure that all freeze-dried blocks / powder are completely dissolved and uniformly mixed, and allow the vial to stand until no more air bubbles are observed to obtain an injectable aqueous solution containing the active pharmaceutical ingredient TH-302 in an amount of (5 ± 0.5) mg / mL. A calculated amount of solution is taken from a container containing 0.9% physiological saline injection or 5% glucose injection, and an equal volume of the aqueous solution for injection is injected into the same container containing 0.9% physiological saline injection or 5% glucose injection for dilution, thereby obtaining an injectable preparation containing the active pharmaceutical ingredient TH-302 in an amount ranging from 0.1 to 4.0 mg / mL. A preparation method including the following.

17. The preparation method according to claim 16, wherein if the solution contains a suspension mixture after being allowed to stand until no more bubbles are observed, the solution should be shaken for 2 minutes at a temperature in the range of 30 to 35°C until it becomes clear.

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