Solution, freeze-drying preparation, freeze-drying preparation unit package, injection and injection preparation method

Abstract

The invention provides a TH-302 solution, a lyophilized preparation, a lyophilized preparation unit package, an injection and a preparation method of the injection. The solution for preparing the freeze-drying preparation with the high drug loading capacity contains TH-302, water, tert-butyl alcohol and mannitol, the water and the tert-butyl alcohol serve as a mixed solvent, the volume percent of the tert-butyl alcohol relative to the solution is (30 + / -3)% or the mass percent of the tert-butyl alcohol relative to the solution is (24 + / -2.4)%, or the content of the 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; the mannitol is an excipient, the mass percent of the mannitol in the solution is (7.14 + / -0.71)%, or the content of the mannitol in the solution is (70 + / -7) mg / mL, and the pH value of the solution is 4-9. The freeze-dried preparation contains TH-302 and mannitol, the drug loading capacity of TH-302 in the freeze-dried preparation is (8.00 + / -0.80) mg / cm < 3 >, or the mass percent of TH-302 in the freeze-dried preparation is (10.23 + / -1.02)%, and the content of mannitol in the freeze-dried preparation is (70 + / -7) mg / cm < 3 >, or the content of mannitol in the freeze-dried preparation is the balance of the mass percent of TH-302 (10.23 + / -1.02)%.

Description

Solutions, lyophilized preparations, unit packages of lyophilized preparations, injections, and methods for preparing injections Technical Field

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

[0002] TH-302 is a 2-nitroimidazole prodrug designed and synthesized by researchers at Threshold Pharmaceuticals in 2006 (WO2007002931A2, Phosphoramidate alkylator prodrugs). It is a highly cytotoxic, selective, hypoxia-activated DNA alkylating agent. It can be activated in hypoxic tumor areas or by acid to convert to dibromoisophosphoramide mustard, an alkylating agent. However, it is virtually inactive under normoxia or normal pH conditions.

[0003] In 2007, researchers at Threshold Pharmaceuticals (WO2008083101A1, Phosphoramidate alkylate or prodrugs for the treatment of cancer) designed and developed preliminary freeze-dried formulations and injection formulations, 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 lyophilization vials and lyophilized to produce lyophilized unit dosage forms of TH-302 with a drug loading of less than 5 mg / cm 3 For human administration, the unit dosage form is dissolved in 5% dextrose injection and an appropriate amount of this solution is administered to the patient;

[0005] TH-302 is dissolved in anhydrous ethanol to produce a 5% TH-302 pharmaceutically acceptable liquid formulation. As used herein, a 5% solution of TH-302 contains 5 g of TH-302 in 100 mL of a solvent (e.g., ethanol);

[0006] The subsequent Phase I clinical trial dosing regimen for TH-302 in human patients used a lyophilized formulation. The TH-302 lyophilized formulation for injection was prepared in a 100 mL glass vial with a drug loading of 100 mg / 100 mL and stored under controlled conditions at 2-8°C. When used, the formulation was injected into 250 mL of 5% glucose injection in the lyophilized formulation bottle and administered intravenously within 30 minutes via an infusion pump.

[0007] Researchers at Threshold Pharmaceuticals designed and developed an injectable formulation in 2009 (WO2010048330A1, Treatment of cancer using hypoxia activated prodrugs):

[0008] Liquid preparation, containing 50 mg / mL to about 300 mg / mL of TH-302, a nonionic surfactant (such as Tween 80), a carrier ethanol, and may also contain dimethylacetamide.

[0009] Clinical trials have been ongoing since 2007, covering a variety of solid tumors and blood cancers, and the medication regimens are also diverse: different doses of TH-302 are used alone or in combination with other cancer treatment drugs. Currently, there are 27 clinical trials registered in the United States for TH-302 as a therapeutic drug for the treatment of various cancers and tumors (NCT02402062, NCT02020226, NCT02076230, NCT01381822, NCT02093962, NCT01440088, NCT02255110, NCT02342379, NCT01864538, NCT01149915, NCT02433639, NCT00743379, NCT01485042, NCT01721941, NCT02047500, NCT00742963, NCT01497444, NCT00495144, NCT01746979, NCT01144455, NCT01403610, NCT01522872, NCT01833546, NCT02598687, NCT03098160, NCT02496832, NCT02712567) These clinical trials have shown that TH-302 is a broad-spectrum anti-cancer drug candidate.

[0010] After the aforementioned Phase I / II clinical trials, Threshold discovered that the effective doses of TH-302 for treating various indications are as follows (WO2012135757A2, Methods for treating cancer):

[0011] 120 mg / m 2 Up to 460 mg / m 2 The daily dose is administered intravenously;

[0012] 480 mg / m 2 Up to approximately 670 mg / m 2 Or for example 575 mg / m 2 The weekly dose is administered intravenously.

[0013] According to the above effective dose, for an average person (175 cm tall, 75 kg in weight), the corresponding equivalent body surface area (BSA) is 2 ) = ([Height (cm) × Weight (kg)] / 3600) 1 / 2 =1.90, the corresponding dose is 228-1273 mg! In this case, if the drug loading is less than 5 mg / cm 3 A lyophilized formulation (20 mL of an aqueous solution containing 100 mg of TH-302 and 1 g of sucrose is added to a 50 mL lyophilization vial and lyophilized to produce a lyophilized unit dosage form of TH-302 with a drug loading of less than 5 mg / cm 3 ) requires at least 3 bottles, and if it is a larger dose, 13 bottles are needed. This situation is not convenient for clinical use, and the cost of medication for patients is too high.

[0014] For this reason, in most subsequent Phase II / III clinical trials, Threshold used a concentrated injection (WO2015013448A1, Treatment of pancreatic cancer with a combination of a hypoxia-activated protein drug and a taxane):

[0015] TH-302 (concentrate for administration solution) used in clinical trials is a sterile liquid formulation of TH-302. TH-302 is formulated with 70% anhydrous ethanol, 25% dimethylacetamide, and 5% polysorbate 80. It is provided by the sponsor in 10 mL glass vials with rubber stoppers and flip-off closures. The TH-302 drug product is a clear, colorless to pale yellow solution that is essentially free of visible particulates. For a nominal total amount of 650 mg of TH-302, each single-use vial contains a nominal fill volume of 6.5 mL of TH-302 drug product (equivalent to 100 mg / mL) and is clearly labeled, disclosing the batch number, route of administration, required storage conditions, the sponsor's name, and appropriate precautionary labeling as required by applicable regulations. Dilution is required according to the pharmacy manual prior to administration.

[0016] Prior to administration, dilute to a total volume of 500 mL (1000 mL for a total dose of ≥1000 mg) with a commercially available 5% dextrose aqueous solution to achieve the desired final concentration. Each dose of TH-302 was prepared with a 5% dextrose aqueous solution that was free of di(2-ethylhexyl) phthalate (DEHP) and administered intravenously using a DEHP-free intravenous infusion device.

[0017] Obviously, the use of concentrated injections with a drug loading of 100 mg / mL in clinical practice is more effective than using concentrated injections with a drug loading of less than 5 mg / cm 3 The lyophilized preparations are convenient: the former's 10mL specification vial can meet the medication requirements of most patients, while the latter requires 13 bottles of commonly used 100mL specification lyophilized preparation vials to meet the medication requirements of most patients!

[0018] However, Threshold discovered that the concentrated injection with a drug loading of 100 mg / mL (TH-302 injection prepared with 70% anhydrous ethanol, 25% dimethylacetamide, and 5% polysorbate 80) contained a large amount of dimethylacetamide. This excipient, which increases drug solubility and improves injection stability, can easily cause allergic reactions after infusion into the human body (WO2015013448A1, Treatment of pancreatic cancer with a combination of a hypoxia-activated prodrug and a taxane):

[0019] Reactions to the administration of TH-302 (primarily caused by dimethylacetamide) have been observed. These reactions are characterized by lip swelling and urticaria, which respond to treatment with steroids and antihistamines. It is recommended to include steroids such as dexamethasone (or equivalent) in the antiemetic regimen before administration. Symptoms and signs of hypersensitivity reactions include fever, myalgia, headache, rash, itching, urticaria, angioedema, chest discomfort, dyspnea, cough, cyanosis and hypotension. If the nature and severity of the reaction require termination of treatment, it should be determined whether the reaction may or may not be an immunoglobulin E-mediated process. If there are symptoms suggestive of an allergic or anaphylactic reaction, such as upper airway obstruction or hypotension, researchers should consider treatment with antihistamines (e.g., diphenhydramine 25-50 mg orally, intramuscularly or slowly iv, or equivalent) and low-dose steroids (e.g., hydrocortisone, 100 mg iv, or equivalent), as appropriate. If the event is clearly anaphylactic, epinephrine (1 / 1000, 0.3-0.5 mL subcutaneously, or equivalent) should be considered along with standard treatment. In the case of bronchospasm, inhaled beta-agonists should be considered. Antihistamines and low-dose steroids can also be used to treat idiosyncratic reactions, depending on their severity. Reactions to the administration of TH-302 should be evaluated and treated in a similar manner. For all reactions to TH-302, researchers should consult with medical monitors to determine the appropriate course of action for future treatment.

[0020] The high-concentration concentrated TH-302 injection solves the problem of low drug loading in lyophilized preparations. However, due to the use of the aforementioned excipients that may cause adverse reactions, related adverse reactions may occur in clinical trials, increasing the risk of medication for patients.

[0021] The currently developed freeze-dried preparation uses water and sucrose solution to dissolve TH-302 for freeze-drying and does not contain other excipients. However, since the drug content of the solution before freeze-drying is too low, it is impossible to obtain a high-drug-load freeze-dried preparation that is suitable for clinical use and subsequent commercial production and sales.

[0022] To resolve the above practical contradictions, relevant technical personnel need to develop TH-302 freeze-dried preparations with high drug loading and solutions for freeze-dried preparations.

[0023] Summary of the Invention

[0024] After many experiments and continuous optimization, the inventors of this application have proposed a new solution formulation with high drug concentration for producing lyophilized preparations of TH-302 and other similar drugs, as well as related lyophilized preparations and methods.

[0025] In order to facilitate understanding of the essence of the present invention, the following briefly describes the inventor's exploration process.

[0026] In order to screen the solvent combination and excipient combination to prepare high concentration The inventors first thought of improving the concentrated injection of TH-302 (i.e., TH-302) developed by Threshold with a drug loading of 100 mg / mL: a concentrated injection of TH-302 prepared with 70% anhydrous ethanol, 25% dimethylacetamide, and 5% polysorbate 80 was prepared:

[0027] Directly using anhydrous ethanol and water as solvents and adding sucrose resulted in either inability to freeze-dry and only a viscous oily substance being obtained; or the drug content of the solution was limited and did not meet the requirements. Therefore, the simple ethanol + water solution was abandoned.

[0028] Directly using anhydrous ethanol and water as solvents, and adding sucrose or other excipients, and adjusting the type and amount of solubilizers (PEG, Tween, Span, etc.), it was found that adding a small amount of them improved the solubility but was very limited and could not meet the requirements of high drug loading;

[0029] Considering that pH may affect solubility, the solubility of TH-302 in various single conventional buffer solutions was also studied. It was found that the pH value of the solution had little effect on the solubility of TH-302, and adjusting the pH value did not increase the solubility of the aqueous solution. The solubility data of TH-302 in aqueous solutions with different pH values ​​and different additives are shown in Table 1 below.

[0030] Table 1: Solubility data of TH-302 in aqueous solutions with different pH values ​​and different additives

[0031] Note: 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 by volume ratio.

[0032] Multiple experimental attempts confirmed that using Threshold's single ethanol, aqueous solutions of different pH values, and ethanol + water as solvents could not develop a high-solubility solution that met the requirements for freeze-drying to prepare a high-drug-loaded TH-302 freeze-dried preparation.

[0033] After many attempts, the inventors found that TH-302 has low solubility in water alone and in tert-butanol alone. However, they unexpectedly discovered that the solubility of TH-302 is greatly improved in a mixed solvent of the two, and the solubility is highly dependent on the mixing ratio of the two. The solubility data of TH-302 in different mixed solvents of tert-butanol + water and tert-butanol + ethanol are shown in Table 2 below.

[0034] Table 2: Solubility data of TH-302 in different tert-butyl alcohol + water and tert-butyl alcohol + ethanol mixed solvents

[0035] Note: The ratio of mixed solvents in Table 2 is by volume, and TH-302 was purchased commercially.

[0036] Tert-Butanol occurs as colorless crystals that are easily supercooled and become 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. Its melting point is 25.7°C, so it occurs as a colorless, transparent liquid or colorless crystals at room temperature.

[0037] In general, its characteristics are as follows:

[0038] 1. High freezing point. Pure tert-butyl alcohol may crystallize at room temperature (25°C), and can be frozen at several degrees below zero after mixing with water. It can be completely frozen in existing freeze dryers.

[0039] 2. Tert-butyl alcohol has a high vapor pressure, which is conducive to sublimation and saves freeze-drying time.

[0040] 3. Tert-butyl alcohol and water can be mixed in any proportion. This is extremely important. It can increase the solubility of some fat-soluble drugs in water. At the same time, for some drugs that are unstable in aqueous solution, adding an appropriate amount of tert-butyl alcohol can inhibit the decomposition of the drug and enhance the stability of the drug.

[0041] 4. Tert-Butanol is easy to freeze-dry and the residual amount in the preparation is low. During the freeze-drying process, most of the tert-Butanol can be sublimed in the primary drying stage, and the residual amount in the preparation is very low.

[0042] 5. Tert-butanol itself forms needle-shaped crystals during freezing, which can change the crystallization pattern of the solute and facilitate sublimation. When a small amount of tert-butanol is added to water to form a tert-butanol-water co-solvent, the crystallization state of the water can be changed, forming needle-shaped crystals with a large surface area during the freezing process. At the same time, after the ice crystals sublime, they leave behind tubular channels, greatly reducing the flow resistance of water vapor and significantly increasing the sublimation rate. Therefore, tert-butanol can be used to accelerate the mass transfer process during the freeze-drying process.

[0043] In summary, it can be seen that tert-butanol + water as a solvent can not only prepare a high-concentration solution of the compound of Formula I, but also the properties of tert-butanol itself are also suitable as a lyophilization auxiliary material.

[0044] Based on the tert-butanol-water mixed solvent results in Table 2, it can be inferred that TH-302 has a low solubility in aqueous solution; as the concentration of tert-butanol in the tert-butanol aqueous solution increases, the solubility of the raw material increases continuously; it is inferred that the solubility is highest in a 70% tert-butanol aqueous solution (V / V), and then as the concentration of tert-butanol in the tert-butanol aqueous solution increases, the solubility decreases.

[0045] Therefore, the inventors proposed the solution of the present invention in which tert-butanol + water is used as a solvent and appropriate excipients are added to obtain a high-concentration solution, and based on this prescription, developed a freeze-dried preparation of TH-302 and similar compounds with a high drug loading.

[0046] Further solubility experiments were conducted on TH-302 using different volume ratios of tert-butyl alcohol solvents, and the following further solubility data were obtained, as shown in Table 3 below.

[0047] Table 3: Solubility data of TH-302 in tert-butanol-water mixed solvents with different tert-butanol mass ratios

[0048] Note: The ratio of the mixed solvents in Table 3 is by mass, and TH-302 was synthesized by the applicant in small quantities.

[0049] Based on the above preliminary experiments, the present invention provides the following high-concentration solution containing a TH-302-like compound.

[0050] A solution containing a compound of the following formula I as well as water and tert-butanol:

[0051] wherein R is independently selected from H, -CH3, -CH2CH3, and X is independently selected from Br, Cl, OMs, and OTs.

[0052] Water and tert-butyl alcohol were used as mixed solvents.

[0053] The content of the compound of formula I in the solution is greater than or equal to 1 mg / mL and less than or equal to 500 mg / mL.

[0054] Furthermore, a solution for preparing a high drug loading lyophilized preparation is provided, comprising a compound of the following formula I, water, tert-butanol, and an excipient:

[0055] wherein R is independently selected from H, -CH3, -CH2CH3, and X is independently selected from Br, Cl, OMs, and OTs.

[0056] Water and tert-butyl alcohol were used as mixed solvents.

[0057] The content of the compound of formula I in the solution is greater than or equal to 5 mg / mL and less than or equal to 500 mg / mL,

[0058] Preferably, the content of the compound of formula I in the solution is greater than or equal to 5 mg / mL and less than or equal to 160 mg / mL,

[0059] Preferably, the content of the compound of formula I in the solution is greater than or equal to 8 mg / mL and less than or equal to 50 mg / mL,

[0060] Preferably, the content of the compound of formula I in the solution is greater than or equal to 8 mg / mL and less than or equal to 25 mg / mL;

[0061] More preferably, the content of the compound of formula I in the solution is greater than or equal to 8 mg / mL and less than or equal to 15 mg / mL,

[0062] Further preferably, the content of the compound of formula I in the solution is greater than or equal to 8 mg / mL and less than or equal to 10 mg / mL.

[0063] Since water and tert-butanol are miscible in any ratio, in order to improve the solubility of TH-302 and similar compounds, the volume percentage of tert-butanol relative to the solution is selected to be 1%-99%, preferably 5%-95%, and more preferably 30%-60%;

[0064] or

[0065] The content of tert-butanol in the solution is 7.85-777.15 mg / mL, preferably 39.25-745.75 mg / mL, and more preferably 235.5-471 mg / mL.

[0066] Here, the volume percentage of tert-butanol relative to the solution is 1%-99% and the tert-butanol content in the solution is 7.85-777.15 mg / mL. The volume percentage of tert-butanol relative to the solution is 1%, which corresponds to a tert-butanol content of 7.85 mg / mL in the solution. The conversion factor is the density of tert-butanol. The density of tert-butanol used by the applicant here is 0.785 g / mL. In fact, the density of products from different manufacturers at different temperatures is different, generally between 0.775-0.786 g / mL.

[0067] The solution for preparing the high-drug-loaded lyophilized preparation of the present invention contains at least one excipient.

[0068] Pharmaceutically acceptable excipients refer to additives or carriers that can contribute to the stability of the API in the formulation. In the preparation of lyophilized formulations, it is necessary to add excipients or lyoprotectants to the solution used for lyophilization.

[0069] Some drug solutions can be successfully freeze-dried in a vacuum, while others quickly collapse or melt into an oily substance after lyophilization. To successfully freeze-dry certain drug solutions and produce stable lyophilized formulations, it is necessary to add excipients that do not react with the drug. These excipients themselves will not sublime during the sublimation stage of the lyophilization process, but will be directly lyophilized into a skeleton, which acts to impart a specific shape, while the drug can be directly adsorbed or fill the voids of the skeleton. Alternatively, to improve the solubility and stability of the lyophilized product or to give it an aesthetically pleasing shape, additional substances must be added to the liquid formulation. These are collectively referred to as lyoprotectants, sometimes also called fillers, excipients, buffers, bases, or skeletons. Generally speaking, lyoprotectants must be chemically inert to the drug solution.

[0070] According to the chemical composition of freeze-drying protective agents, they can be divided into the following categories:

[0071] Complex: skim milk, gelatin, protein and hydrolysate, polypeptide, yeast, broth, dextrin, methyl cellulose, serum, peptone, etc.

[0072] Salts: sodium thiosulfate, calcium lactate, sodium glutamate, sodium chloride, potassium chloride, sodium sulfate, ammonium acetate, ammonium chloride, etc.

[0073] Sugars: sucrose, lactose, maltose, glucose, raffinose, fructose, hexose, etc.

[0074] Alcohols: sorbitol, ethanol, glycerol, mannitol, inositol, xylitol, etc.

[0075] Acids: citric acid, phosphoric acid, tartaric acid, amino acids, ethylenediaminotetraacetic acid (EDTA), etc.

[0076] Alkali: sodium hydroxide, sodium bicarbonate, etc.

[0077] Polymers: dextran, polyethylene glycol, polysorbate, PVP, poloxamer, etc.

[0078] Others: Vitamin C, Vitamin E, Vitamin K, Thiourea, etc.

[0079] The degree of polymerization (molecular weight) of the above polymers is wide. Take polysorbate and polyethylene glycol as examples.

[0080] The molecular weight average of polysorbate can be about 1900g / mol at about 500g / mol, preferably about 1600g / mol at about 800g / mol, more preferably in the scope of about 1400g / mol at about 1000g / mol.The non-limiting example of polysorbate comprises: polysorbate-20, polysorbate-21, polysorbate-40, polysorbate-60, polysorbate-61, polysorbate-65, polysorbate-81, polysorbate-85 and polysorbate-120.Preferred polysorbate comprises polysorbate-20, polysorbate-80 and their mixture.

[0081] The average molecular weight of polyethylene glycol (PEG) is in the range of about 200 g / mol to about 600 g / mol, preferably in the range of about 200 g / mol to about 500 g / mol, more preferably in the range of about 200 g / mol to about 400 g / mol. Non-limiting examples of PEG include: PEG200, PEG300, PEG400, PEG540 and PEG600.

[0082] Poloxamer, whose general formula is HO(C2H4O) a (C3H6O) b (C2H4O) c H, where a and c are 2-130, and b is 15-67. It contains 81.8±1.9% polyoxyethylene and is a polyoxyethylene polyoxypropylene ether block copolymer. It has different designations: Poloxamer 182, Poloxamer 184, Poloxamer 188, and Poloxamer 407, corresponding to polymers of different molecular weights. For example, Poloxamer 188 has a molecular weight of 7680-9510.

[0083] There are many functions of freeze-drying protectants, which are summarized as follows:

[0084] Microorganisms such as bacteria and viruses need to grow and reproduce in specific culture media, but these culture media are often difficult to separate from the microorganisms. They can be successfully freeze-dried in these culture media, such as broth, skim milk, and protein.

[0085] Some freeze-dried preparations have very low concentrations and minimal dry matter content. During freeze-drying, the dried portion is carried away by the sublimating airflow. To increase the concentration, increase the dry matter content, and form a more ideal agglomerate after freeze-drying, fillers are added to keep the solid matter concentration within a certain range. These fillers or excipients include sucrose, lactose, inositol, skim milk, hydrolyzed protein, dextran, sorbitol, and polyvinylpyrrolidone (PVP).

[0086] Some bioactive substances are particularly fragile and may be damaged due to physical or chemical reasons during freezing and drying. Therefore, it is necessary to add some protective agents to reduce damage during freezing and drying. Examples include dimethyl sulfoxide, glycerol, dextran, sugars, and PVP.

[0087] Adding certain substances can increase the disintegration temperature of the product and make it easier to freeze-dry. They are mannitol, glycine, dextran, xylitol and PVP.

[0088] In order to change the pH of the freeze-dried preparation, thereby increasing the eutectic point and facilitating freeze-drying, they are sodium bicarbonate, sodium hydroxide, etc.

[0089] In order to improve the storage stability of the product, increase the storage temperature and prolong the storage time, some antioxidants such as vitamin C, vitamin E, amino acids, sodium thiosulfate, thiourea, lecithin and hydrolyzed protein are added.

[0090] Adding certain substances can eliminate free radicals and increase the stability of freeze-dried products. They are amino acids, vitamin K, vitamin C, thiourea, sulfite compounds, sodium aspartate, etc.

[0091] It can be found that the same compound can play a variety of roles as a lyoprotectant (excipient).

[0092] According to the properties of the drug of the present invention, the lyoprotectant (excipient) is selected from sugars, polyols, polyvinyl pyrrolidones, proteins, poloxamers or a combination thereof.

[0093] The carbohydrate is selected from sucrose, dextran, cyclodextrin, maltodextrin, trehalose, lactose, maltose, and glucose;

[0094] The polyols are selected from glycerol, sorbitol, mannitol, inositol, ethylene glycol, polyethylene glycol (PEG), polysorbate, and adonitol;

[0095] The protein is selected from albumin, preferably bovine serum albumin and human albumin;

[0096] The poloxamer is selected from poloxamer 182, poloxamer 184, poloxamer 188, and poloxamer 407.

[0097] Preferably, the excipient is selected from PVP K12, sucrose, mannitol, albumin or a combination thereof.

[0098] Generally speaking, excipients of the same type can be combined, and excipients that do not react with each other and with drugs can be combined. The combination should meet the requirements of drug compatibility.

[0099] Particularly, the excipient is selected from sucrose and mannitol, and the content of sucrose and mannitol in the solution is 20-300 mg / mL, preferably 40-100 mg / mL, more preferably 60-80 mg / mL, and further preferably 60-70 mg / mL.

[0100] Sucrose or mannitol is generally used alone, and can be mixed in special circumstances. The preferred solution of the present application is to use only mannitol or only sucrose.

[0101] Similarly, the mass ratio of TH-302 to excipient in the solution for preparing the high drug loading lyophilized preparation of the present invention is 1 (0.5-20), preferably 1: (1-15), more preferably 1: (2-12.5), and further preferably 1: (5-10).

[0102] The mass ratio of TH-302 and similar compounds to excipients is the drug loading ratio of the lyophilized solution. Based on the lyophilized state of the aforementioned excipients (lyoprotectants, fillers, and matrix), the excipients serve as the matrix upon which the drug is adsorbed or loaded. Therefore, the drug loading ratio of the drug solution before lyophilization—that is, the mass ratio of TH-302 to excipients—is a crucial indicator.

[0103] A suitable drug loading ratio means that after freeze-drying, the drug and the skeleton are evenly adsorbed, and the drug is well distributed on the surface of the skeleton's voids and pores. In this way, during the subsequent reconstitution process (5% glucose injection, normal saline, etc.), the freeze-dried preparation can be well and quickly dissolved for injection.

[0104] A pharmaceutically acceptable buffer is a weak acid or base that allows the pH of a solution to be maintained at a nearly constant level, and is used to enhance the stability of the drug substance in solution.

[0105] The solution for preparing a high drug load lyophilized formulation of the present invention may further contain at least one buffer selected from 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 a mixture thereof.

[0106] Preferably, the buffer used in the solution for preparing the high drug load lyophilized formulation 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 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, calcium citrate heptahydrate.

[0107] The solution for preparing the high drug loading lyophilized preparation of the present invention may further contain at least one pH regulator.

[0108] The pH adjuster of the present invention refers to a buffer substance or buffer used to appropriately adjust the pH of a solution that changes with acid or base. The pH adjuster includes, but is not limited to, hydrochloric acid, sodium hydroxide, triethanolamine, phosphoric acid, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, phosphoric acid, citric acid, lactic acid, tartaric acid, succinic acid, fumaric acid, malic acid, sodium bicarbonate, sodium carbonate, or mixtures thereof. The pH adjuster is added in an amount such that the solution has a pH of 4-9, preferably 6-8.

[0109] Because TH-302 and its analogs are generally acidic, the compound preparation process involves an acidic environment and a trace amount of hydrolysis, so the prepared or purchased API may be acidic. Therefore, the pH adjuster is preferably selected from bases such as sodium hydroxide, triethanolamine, sodium bicarbonate, sodium carbonate, or alkaline salts.

[0110] Of course, it is also possible that the pH of TH-302 and its analogs is alkaline due to other reasons. In this case, it is necessary to add acid or acidic salt (such as ammonium sulfate, ammonium chloride, etc.) to adjust it.

[0111] The compound of formula I is selected from

[0112] TH-302 is more preferred.

[0113] The specific synthesis method and corresponding spectral data of the compound of formula I are disclosed in WO2007002931 (corresponding Chinese publication CN101501054A), which is incorporated herein in its entirety.

[0114] The relevant physicochemical properties and biological activities of the compound of formula I and these three specific compounds can be found in the patents applied for by Threshold (such as WO2016011195A2, WO2004087075A1, WO2007002931A1, WO2008151253A2, WO2009018163A1, WO2009033165A2, WO2010 048330A2, WO2012142520A1, WO2008083101A2, WO2020007106A1, WO2020118251A1, WO2 014169035A1, WO2013116385A1, WO2019173799A2, WO2016081547A1, WO2014062856A1, W O2015069489A1, WO2012006032A2, WO2018026606A2, WO2010048330A2, WO2015171647A 1. WO2013096687A1, WO2013126539A2, WO2013096684A2, WO2012009288A2, WO201214568 4A2, WO2016014390A2, WO2019055786A2, WO2012135757A2, WO2015013448A2, WO2016011328A2, WO2013177633A2, WO2016011195A2, WO2015051921A2), the present invention hereby introduces all the above-mentioned related application text information.

[0115] The three preferred compounds have the same or similar physical and chemical properties as TH-302.

[0116] The present invention provides a solution for preparing a high drug loading lyophilized preparation, comprising

[0117] The compound of the following formula I-1, water, tert-butanol and sucrose;

[0118] or

[0119] The compound of formula I-1 below, as well as water, tert-butyl alcohol and mannitol;

[0120] Water and tert-butyl alcohol were used as mixed solvents.

[0121] The volume content of tert-butanol relative to the solution is 30%, 40%, 60% or the corresponding content of tert-butanol in the solution is 235.5, 314, 471 mg / mL,

[0122] The content of the compound of formula I-1 in the solution is 6, 7, 7.5, 8, 8.5, 10, 12.5, 15, 20, 25 mg / mL,

[0123] Sucrose or mannitol is used as an excipient, and its content in the solution is 40, 50, 60, 70, 75, 80, 90, or 100 mg / mL.

[0124] The present invention provides a solution for preparing a high drug loading lyophilized preparation, comprising

[0125] Compound I-1, water, tert-butyl alcohol, sucrose, and a pH regulator are provided below;

[0126] or

[0127] Compound I-1, water, tert-butyl alcohol, mannitol, and a pH regulator:

[0128] Water and tert-butyl alcohol were used as mixed solvents.

[0129] The volume content of tert-butanol relative to the solution is 30%, 40%, 60% or the content of tert-butanol in the solution is 235.5, 314, 471 mg / mL,

[0130] The content of the compound of formula I-1 in the solution is 6, 7, 7.5, 8, 8.5, 10, 12.5, 15, 20, 25 mg / mL,

[0131] Sucrose or mannitol is used as an excipient, and the content in the solution is 40, 50, 60, 70, 75, 80, 90, 100 mg / mL.

[0132] The content of the pH regulator is such that the pH value of the solution is 4-9, preferably 6-8.

[0133] The present invention provides a solution for preparing a high drug loading lyophilized preparation, comprising

[0134] The compound of the following formula I-1, water, tert-butyl alcohol, sucrose and sodium bicarbonate;

[0135] or

[0136] The compound of the following formula I-1, water, tert-butyl alcohol, mannitol and sodium bicarbonate;

[0137] Water and tert-butyl alcohol were used as mixed solvents.

[0138] The volume content of tert-butanol relative to the solution is 30%, 40%, 60% or the content of tert-butanol in the solution is 235.5, 314, 471 mg / mL,

[0139] The content of the compound of formula I-1 in the solution is 6, 7, 7.5, 8, 8.5, 10, 12.5, 15, 20, 25 mg / mL,

[0140] Sucrose or mannitol is used as an excipient, and the content in the solution is 40, 50, 60, 70, 75, 80, 90, 100 mg / mL.

[0141] The content of sodium bicarbonate is such that the pH value is 4-9, preferably 6-8.

[0142] The above solution for preparing a high drug loading lyophilized preparation, wherein:

[0143] The mass ratio of the compound to sucrose or mannitol in the solution 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, and 1:9.375.

[0144] Of course, according to the above description and in combination with the properties of the specific compound of Formula I, a buffer, a pH adjuster or other auxiliary materials of the lyophilized preparation can also be added.

[0145] Obviously, if other substances (environmental substances) are detected during the solution preparation process due to contact or contamination of containers, pipes, or tools, this is not limited. Similarly, the raw materials of tert-butyl alcohol, sucrose / mannitol, and the compound of Formula I will inevitably be contaminated with impurities or other substances (environmental substances) in the environment, and these impurities or environmental substances are also not limited.

[0146] All raw materials of the compound of Formula I, which are inevitably contaminated with impurities or other substances (environmental substances) detected due to contact or contamination of containers, pipes, and tools, or tert-butyl alcohol and sucrose / mannitol, or the content of other substances (environmental substances) in the environment should be within the legal limits or meet the quality standards of the corresponding products (pharmaceutical grade, medical grade or equivalent quality standards).

[0147] Therefore, the above-mentioned "consisting of xxx" refers to substances that are intentionally and artificially added during preparation (these substances must be contained and can be detected by analytical testing instruments). Apart from these substances, no other intentionally and artificially added substances are contained. However, there will still inevitably be trace amounts of impurities, environmental substances, etc.

[0148] The above content describes the composition (prescription) of the solution for preparing the high drug loading lyophilized preparation. The following briefly describes its use.

[0149] The solutions provided above are intended only as intermediates for the preparation of high-drug-load lyophilized formulations and are not intended for use as prescriptions for clinical preparations. Generally, they are prepared on-site, i.e., directly filled into lyophilization vials after preparation in a dispensing container, and then shipped in batches to the lyophilization production facility for lyophilization.

[0150] Therefore, the solution for preparing high-drug-load lyophilized preparations should be stable during the production and waiting process from preparation to lyophilization in the lyophilization equipment, that is, it should have stability for at least 8 hours at room temperature, and better still, it should have stability for 24 hours, or even 72 hours or 120 hours.

[0151] This is because the solution needs to be filtered and filled after preparation before it can enter the freeze dryer for freeze drying. Filtration and filling are generally completed within 8-12 hours and are generally performed at room temperature. Although the subsequent freeze drying is low temperature, a large freeze dryer or freeze drying system takes 20-60 hours to cool a large amount of liquid medicine filled in a vial to a set low temperature (-20 to -55°C). Therefore, the liquid medicine of the freeze-dried preparation should be stable within a certain period of time and under room temperature regulation. After experiments, it has been verified that the liquid medicine of the TH-302 freeze-dried preparation provided by the present invention has suitable stability.

[0152] The invention relates to a use of a solution for preparing a lyophilized preparation with a high drug load, wherein the solution is used as a lyophilized preparation solution and is used to prepare a lyophilized preparation through a lyophilization process.

[0153] Based on the above solution for preparing a lyophilized preparation with high drug loading, the present invention can prepare a lyophilized preparation with high drug loading.

[0154] The lyophilized preparation is prepared by a lyophilization process using the above-mentioned solution for preparing the lyophilized preparation with high drug loading as the lyophilized preparation solution.

[0155] A lyophilized preparation comprising a compound of the following formula I, excipients, and residual solvent components:

[0156] wherein R is independently selected from H, -CH3, -CH2CH3, and X is independently selected from Br, Cl, OMs, and OTs.

[0157] The drug loading of the compound of formula I in the lyophilized formulation is greater than 5 mg / cm 3 Less than or equal to 555.55 mg / cm 3 ,or

[0158] The drug loading of the compound of formula I in the lyophilized formulation is greater than or equal to 4.55 mg / cm 3 And less than 500mg / cm 3 ,or

[0159] The mass percentage of the compound of formula I in the lyophilized preparation is greater than or equal to 4.39% and less than 66.66%,

[0160] The residual solvent components are water and tert-butanol.

[0161] A lyophilized preparation comprising a compound of the following formula I, excipients, residual solvent components, and a pH adjuster:

[0162] wherein R is independently selected from H, -CH3, -CH2CH3, and X is independently selected from Br, Cl, OMs, and OTs.

[0163] The drug loading of the compound of formula I in the lyophilized formulation is greater than 5 mg / cm 3 Less than or equal to 555.55 mg / cm 3 ,or

[0164] The drug loading of the compound of formula I in the lyophilized formulation is greater than or equal to 4.55 mg / cm 3 And less than 500mg / cm 3 ,or

[0165] The mass percentage of the compound of formula I in the lyophilized preparation is greater than or equal to 4.39% and less than 66.66%,

[0166] The residual solvent components are water and tert-butanol.

[0167] A lyophilized formulation comprising a compound of the following formula I and excipients:

[0168] wherein R is independently selected from H, -CH3, -CH2CH3, and X is independently selected from Br, Cl, OMs, and OTs.

[0169] The drug loading of the compound of formula I in the lyophilized formulation is greater than 5 mg / cm 3 Less than or equal to 555.55 mg / cm 3 ,or

[0170] The drug loading of the compound of formula I in the lyophilized formulation is greater than or equal to 4.55 mg / cm 3 And less than 500mg / cm 3 ,or

[0171] The mass percentage of the compound of formula I in the lyophilized preparation is greater than or equal to 4.39% and less than 66.66%.

[0172] The drug loading of the compound of formula I in the lyophilized formulation is greater than 5 mg / cm 3 Less than or equal to 555.55 mg / cm 3The drug loading of the compound of formula I in the lyophilized formulation is greater than or equal to 4.55 mg / cm 3 And less than 500mg / cm 3 These are two different situations.

[0173] After multiple experiments, the applicant discovered that, using different lyophilization processes, the volume of the high-drug-load lyophilized formulation solution lyophilized to obtain a lyophilized formulation in a 50mL vial with a maximum fill volume of 25mL, the volume of the solution before lyophilization and the apparent volume of the solid formulation after lyophilization varied by no more than 10%. This could be due to expansion of the powder cake after lyophilization, or a decrease of 10% due to collapse. Based on a pre-lyophilization concentration of the compound of Formula I greater than or equal to 5 mg / mL and less than or equal to 500 mg / mL, and assuming a maximum change of 10%, the calculation assumes that the volume increases by 1.1 times the original volume due to expansion and decreases by 90% due to collapse. Taking the case where the powder cake collapses to 90% of its original volume after freeze-drying as an example, the maximum drug loading capacity of the powder cake at this time is 5 / 0.9 = 5.55 to 500 / 0.9 = 555.55. Due to different processes, the volume may not change. In this case, the drug loading range is 5 to 500. The union of the two ranges is 5 to 555.55, which means that the drug loading capacity of the compound of formula I in the freeze-dried preparation is greater than 5 mg / cm 3 Less than or equal to 555.55 mg / cm 3 .

[0174] Similarly, when the freeze-dried powder cake expands and its volume increases, the drug loading of the compound of formula I in the freeze-dried preparation is greater than or equal to 4.55 mg / cm 3 And less than 500mg / cm 3 .

[0175] The drug loading of the lyophilized preparation provided by the present invention refers to the amount of the raw material drug of the compound of formula I contained in the unit volume. The volume here refers to the total apparent volume of the drug in the unit packaging kit, which includes the internal space of the drug. For example, a 100mL freeze-dried bottle contains 20mL of freeze-dried solution, and 20mL of freeze-dried solution contains 500mg of drug. After the freeze-drying preparation solution undergoes the freeze-drying process, the 20mL solution is frozen into a loose and porous freeze-dried preparation. By measuring the bottom area of ​​the inner space of the freeze-dried bottle as s, the height of the freeze-dried preparation in the freeze-dried bottle as h, then the total apparent volume of the freeze-dried preparation of this bottle of unit packaging is sh, which contains 500mg of raw material drug, and its drug loading is (500 / sh)mg / cm 3 Generally, the value of sh is around 20cm. 3 About, the volume may expand to more than 20cm after freeze-drying 3, may collapse after freeze-drying and have a volume less than 20 cm 3 .

[0176] When measuring the drug loading of a unit package of a lyophilized preparation, the aforementioned apparent total volume sh is first measured and calculated. Then, the entire unit package of the lyophilized preparation is directly dissolved and the mass M of the drug contained therein is detected. The drug loading (per unit volume) of the lyophilized preparation of the present invention is then calculated as M / sh.

[0177] Here, the mass percentage of the compound of formula I in the lyophilized preparation is greater than or equal to 4.39% and less than 66.66%, which can be calculated based on the API content, excipients, and residual tert-butanol and water content in the lyophilized preparation.

[0178] The mass content of the compound of formula I in a lyophilized formulation refers to the percentage of the drug in the total mass of the lyophilized formulation. It can be measured and calculated as follows:

[0179] When measuring the drug mass content of a unit package of a lyophilized preparation, first weigh the mass M1 of the unit package of the lyophilized preparation, then directly dissolve the entire unit package of the lyophilized preparation and detect the mass M of the drug contained therein. After dissolution, clean, dry, and weigh the total mass of the empty bottle and packaging cap as M2. The total mass of the lyophilized preparation is M1-M2, and the mass content of the compound of formula I in the lyophilized preparation of the present invention = M / (M1-M2).

[0180] In the above-mentioned lyophilized preparation, the compound of formula I is selected from

[0181] Preferred is TH-302.

[0182] The excipient is a sugar, a polyol, a polyvinyl pyrrolidone, a protein, a poloxamer or a combination thereof,

[0183] The sugar is selected from sucrose, dextran, cyclodextrin, maltodextrin, trehalose, lactose, maltose, glucose,

[0184] The polyols are selected from glycerol, sorbitol, mannitol, inositol, ethylene glycol, polyethylene glycol, polysorbate, and aconitol.

[0185] The protein is selected from albumin, preferably bovine serum albumin, human albumin,

[0186] The poloxamer is selected from poloxamer 182, poloxamer 184, poloxamer 188, and poloxamer 407.

[0187] After the solution is freeze-dried, the water and tert-butyl alcohol used as solvents will sublime, so what remains in the freeze-dried preparation are the drug and excipients.

[0188] Obviously, the sublimation process during freeze-drying cannot completely remove water and tert-butyl alcohol, so residual water and tert-butyl alcohol are unavoidable. In fact, the amount of residual water and tert-butyl alcohol is an important quality indicator for freeze-dried preparations: the lower the residual amount, the better the quality of the freeze-dried preparation, the greater its stability, and the fewer adverse reactions patients will experience after use. Reducing residual water and tert-butyl alcohol can be solved by adjusting the freeze-drying process, but residual water and tert-butyl alcohol cannot be completely eliminated.

[0189] However, by extending the freeze-drying time and increasing the drying temperature, the content of water and tert-butanol can be technically reduced to below the detection limit without considering the cost and meeting the product quality requirements. In this case, it can be considered that the freeze-dried preparation contains almost no water and tert-butanol.

[0190] Taking into account the production cost and storage stability, the residual amount of water and tert-butanol has an appropriate value: the residual water content is less than or equal to 6% by mass, preferably less than or equal to 2%, more preferably less than or equal to 1%, and further preferably less than or equal to 0.5%.

[0191] The residual tert-butanol content is less than or equal to 1.75% by mass, preferably less than or equal to 1%, and more preferably less than or equal to 0.5%.

[0192] Of course, according to the above description and in combination with the properties of the specific compound of Formula I, a buffer, a pH adjuster or other lyophilized preparation excipients can be added to the solution. In this way, the lyophilized preparation after lyophilization will also be detected to contain a buffer, a pH adjuster or other lyophilized preparation excipients.

[0193] Obviously, if other substances that cannot be sublimed (environmental substances) are detected during the preparation of solutions or lyophilized preparations due to contact or contamination of containers, pipes, or tools, this is not limited. Similarly, the raw materials of tert-butyl alcohol, sucrose, and the compound of Formula I will inevitably be contaminated with impurities or other substances that cannot be sublimed (environmental substances) in the environment, and these impurities or environmental substances are also not limited.

[0194] All raw materials of the compound of Formula I, which are detected to contain other non-sublimable substances (environmental substances) due to contact or contamination of containers, pipes, and tools, tert-butyl alcohol and sucrose, are inevitably contaminated with impurities or other non-sublimable substances (environmental substances) in the environment. The content range of these substances should be within the legal limit or meet the quality standards of the corresponding products (pharmaceutical grade, medical grade or equivalent quality standards).

[0195] The compound of formula I and the excipients mentioned below refer to the lyophilized preparations that can detect the compound of formula I, the excipients and the above-mentioned residual environmental substances, except for unavoidable and residual water and tert-butanol. In addition, other excipients may also be contained.

[0196] The mass ratio of Compound I to the excipient is 1:(0.5-20), preferably 1:(1-15), more preferably 1:(2-12.5), and further preferably 1:(5-10).

[0197] The drug loading of the compound of formula I in the lyophilized formulation is greater than or equal to 5.55 mg / cm 3 Less than or equal to 177.77 mg / cm 3 , preferably greater than or equal to 8.88 mg / cm 3 Less than or equal to 55.55 mg / cm 3 , more preferably greater than or equal to 8.88 mg / cm 3 Less than or equal to 27.77 mg / cm 3 , more preferably greater than or equal to 8.88 mg / cm 3 Less than or equal to 16.66 mg / cm 3 , and further preferably greater than or equal to 8.88 mg / cm 3 Less than or equal to 11.11 mg / cm 3 ;

[0198] or

[0199] The drug loading of the compound of formula I in the lyophilized formulation is greater than or equal to 4.55 mg / cm 3 Less than or equal to 145.45 mg / cm 3 , preferably greater than or equal to 7.27 mg / cm 3 Less than or equal to 45.45 mg / cm 3 , more preferably greater than or equal to 7.27 mg / cm 3 Less than or equal to 22.73 mg / cm 3 , more preferably greater than or equal to 7.27 mg / cm 3 Less than or equal to 13.64 mg / cm 3 , and further preferably greater than or equal to 7.27 mg / cm 3 Less than or equal to 9.09 mg / cm 3 .

[0200] There are two situations here. The former is the situation where the volume collapses and becomes smaller after freeze-drying, and the latter is the situation where the volume expands and becomes larger.

[0201] A lyophilized preparation consisting essentially of the compound of the following formula I-1, excipients, residual water, and residual tert-butyl alcohol:

[0202] The drug loading of the compound in the lyophilized formulation was 6.66, 7.77, 8.33, 8.88, 9.44, 11.11, 13.88, 16.66, 22.22, and 27.77 mg / cm 3 , or the drug loading of compound I-1 in the lyophilized formulation was 5.45, 6.36, 6.82, 7.27, 7.73, 9.09, 11.36, 13.64, 18.18, 22.73 mg / cm 3 ;

[0203] The excipients are sucrose or mannitol;

[0204] 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, 1:8.235, 1:8.75, 1:9.375;

[0205] The residual water content is less than or equal to 6% by mass, preferably less than or equal to 2%, more preferably less than or equal to 1%, and further preferably less than or equal to 0.5% by mass;

[0206] The residual tert-butanol content is less than or equal to 1.75% by mass, preferably less than or equal to 1%, and more preferably less than or equal to 0.5%.

[0207] A lyophilized preparation is essentially composed of the compound of the following formula I-1, excipients, residual water, residual tert-butyl alcohol, and a pH adjuster:

[0208] The drug loading of the compound in the lyophilized formulation was 6.66, 7.77, 8.33, 8.88, 9.44, 11.11, 13.88, 16.66, 22.22, and 27.77 mg / cm 3 , or the drug loading of compound I-1 in the lyophilized formulation was 5.45, 6.36, 6.82, 7.27, 7.73, 9.09, 11.36, 13.64, 18.18, 22.73 mg / cm 3 ;

[0209] The excipients are sucrose or mannitol;

[0210] 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, 1:8.235, 1:8.75, 1:9.375;

[0211] The residual water content is less than or equal to 6% by mass, preferably less than or equal to 2%, more preferably less than or equal to 1%, and further preferably less than or equal to 0.5% by mass;

[0212] The residual tert-butanol content is less than or equal to 1.75% by mass, preferably less than or equal to 1%, and more preferably less than or equal to 0.5% by mass;

[0213] The pH regulator is sodium bicarbonate, with a content of 0.01-0.10 mg / cm 3 .

[0214] The lyophilized preparation is essentially composed of the following compound and excipients:

[0215] The drug loading of compound I-1 in the lyophilized formulation was 6.66, 7.77, 8.33, 8.88, 9.44, 11.11, 13.88, 16.66, 22.22, and 27.77 mg / cm 3 , or the drug loading of compound I-1 in the lyophilized formulation was 5.45, 6.36, 6.82, 7.27, 7.73, 9.09, 11.36, 13.64, 18.18, 22.73 mg / cm 3 ;

[0216] The excipients are sucrose or mannitol;

[0217] The mass ratio of compound 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, 1:8.235, 1:8.75, and 1:9.375.

[0218] Obviously, if other substances that cannot be sublimed (environmental substances) are detected during the preparation of solutions or lyophilized preparations due to contact or contamination of containers, pipes, or tools, this is not limited. Similarly, the raw materials of tert-butyl alcohol, sucrose, and the compound of Formula I will inevitably be contaminated with impurities or other substances that cannot be sublimed (environmental substances) in the environment, and these impurities or environmental substances are also not limited.

[0219] All raw materials of the compound of Formula I, which are detected to contain other non-sublimable substances (environmental substances) due to contact or contamination of containers, pipes, and tools, tert-butyl alcohol and sucrose, are inevitably contaminated with impurities or other non-sublimable substances (environmental substances) in the environment. The content range of these substances should be within the legal limit or meet the quality standards of the corresponding products (pharmaceutical grade, medical grade or equivalent quality standards).

[0220] The compound and excipients of the following formula I refer to a freeze-dried preparation in which, except for unavoidable, residual water and tert-butanol, the compound, excipients and the aforementioned residual environmental substances can be detected. In addition, other excipients may also be contained.

[0221] Essentially consisting of the compound of formula I and excipients means that, except for unavoidable, residual water and tert-butanol, the lyophilized preparation can only be detected to contain the compound of formula I, excipients and the above-mentioned residual environmental substances, and does not contain any other substances.

[0222] The present invention provides a freeze-dried preparation, which is prepared by a freeze-drying process using the above series of solutions for preparing freeze-dried preparations with high drug loading.

[0223] The pharmaceutical preparation and its freeze-dried powder can be stored in a container commonly used in the pharmaceutical field, which can include: a plastic container or a glass container, such as a standard USPI type borosilicate glass container. For example, the container used can be a syringe or a vial.

[0224] According to the animal model experiments and clinical trial dosages of the compound of Formula I in the background art, different species (humans and other animals), different indications, and different patients may require different dosages per administration, ranging from a minimum of a few milligrams to a maximum of tens of thousands of milligrams. It is best that one unit dose package or a combination of multiple unit dose packages can meet the requirements of a certain dosage administration. Therefore, based on the above, we provide recommended unit packaging specifications for lyophilized preparations corresponding to different species and different indications.

[0225] The present invention also provides a preparation unit package containing the above-mentioned freeze-dried preparation:

[0226] The lyophilized preparation is contained in a sealed container with a volume of 1000 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I is 4000-16500 mg; or

[0227] The lyophilized preparation is contained in a sealed container with a volume of 500 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I is 2000-8000 mg; or

[0228] The lyophilized preparation is contained in a sealed container with a volume of 250 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I is 1000-4000 mg; or

[0229] The lyophilized preparation is contained in a sealed container with a volume of 100 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I is 400-2000 mg; or

[0230] The lyophilized preparation is contained in a sealed container with a volume of 50 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I is 50-800 mg; or

[0231] The lyophilized preparation is contained in a sealed container with a volume of 30 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I is 150-600 mg; or

[0232] The lyophilized preparation is contained in a sealed container with a volume of 25 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I is 125-500 mg; or

[0233] The lyophilized preparation is contained in a sealed container with a volume of 20 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I is 100-400 mg; or

[0234] The lyophilized preparation is contained in a sealed container with a volume of 18 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I is 90-360 mg; or

[0235] The lyophilized preparation is contained in a sealed container with a volume of 15 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I is 75-300 mg; or

[0236] The lyophilized preparation is contained in a sealed container with a volume of 10 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I is 50-200 mg; or

[0237] The lyophilized preparation is contained in a sealed container with a volume of 8 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I is 40-160 mg; or

[0238] The lyophilized preparation is contained in a sealed container with a volume of 7 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I is 35-140 mg; or

[0239] The lyophilized preparation is contained in a sealed container with a volume of 5 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I is 25-100 mg; or

[0240] The lyophilized preparation is contained in a sealed container with a volume of 3 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I is 15-60 mg; or

[0241] The lyophilized preparation is contained in a sealed container with a volume of 2 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I is 10-40 mg; or

[0242] The lyophilized preparation is contained in a sealed container with a volume of 1.5 mL. The volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and contains 7.5-30 mg of the compound of formula I.

[0243] These small doses are suitable for minors among humans or animals of comparable size, such as pigs, rats, dogs and other small animals in size and weight, and the indications are not limited.

[0244] The lyophilized preparation provided by the present invention is recommended to be administered by intravenous infusion, so the drug solution needs to be reconstituted. Normal saline (0.9%) or glucose injection (5%) is generally used for reconstitution. For this purpose, an intravenous injection solution containing TH-302 is provided, wherein the solvent is water, and the solutes include TH-302 raw material, an isotonicity adjusting agent, mannitol or sucrose, tert-butanol and sodium bicarbonate. The isotonicity adjusting agent is selected from glucose and sodium chloride.

[0245] The present invention also provides a method for preparing a solution for preparing a high drug loading lyophilized preparation, comprising the following steps:

[0246] Operation 1: Weigh the prescribed amount of TH-302 API, add it to the tert-butyl alcohol aqueous solution, and stir until clear to obtain the first solution;

[0247] Step 2: Dissolve the prescribed amount of mannitol or sucrose in an appropriate amount of water and stir until clear to obtain the second solution;

[0248] Operation 3: Mix the first solution and the second solution and finally add the remaining amount of tert-butyl alcohol, then add appropriate amount of water to the predetermined volume, add the prescribed amount of sodium bicarbonate, stir and mix evenly,

[0249] in,

[0250] The content of TH-302 API in the solution of the high drug loading lyophilized preparation is greater than 5 mg / mL and less than or equal to 500 mg / mL,

[0251] The volume percentage of tert-butanol relative to the high drug loading lyophilized preparation is 1%-99%, or the content of tert-butanol in the solution is 7.85-777.15 mg / mL,

[0252] The content of sucrose or mannitol in the solution of the high drug loading lyophilized preparation is 20-300 mg / mL,

[0253] The mass ratio of TH-302 to sucrose or mannitol in the solution of the high drug loading lyophilized preparation is 1:(0.5-20),

[0254] The volume ratio of tert-butanol in the tert-butanol aqueous solution is 30-90%,

[0255] The content of sodium bicarbonate in the solution of the high drug loading lyophilized preparation is 0.01-0.10 mg / mL.

[0256] The present invention provides a preparation process for a lyophilized preparation.

[0257] The preparation method of a high drug loading lyophilized preparation comprises the following steps:

[0258] Pre-freezing: placing the above-mentioned drug solution in a freeze-drying system for pre-freezing, the pre-freezing process includes keeping it at 0°C for a period of time and keeping it at -20 to -55°C for a period of time;

[0259] Once dried, after pre-freezing, the temperature is raised to -10 to 10°C and kept warm for a period of time, and vacuum drying is maintained;

[0260] Secondary drying: After the primary drying is completed, the temperature is raised to 20 to 40°C and kept warm for a period of time, and vacuum drying is maintained.

[0261] From the above description, it can be seen that the freeze-dried preparation is obtained by directly filling the solution into the freeze-dried bottle (cillin bottle) and directly freeze-drying it through the freeze-drying equipment. Therefore, there is no subpackaging link, that is, the unit packaging is the corresponding freeze-dried bottle packaging, and the packaging specifications are closely related to the specifications of the freeze-dried bottle.

[0262] It should be noted that all numbers appearing in this application have an error of ±10%, that is, numbers within -10% and +10% should be considered to be within the range of the numbers described in this application. For example, if the words "content is greater than 5 mg / mL and less than or equal to 500 mg / mL" appear in this application, then if the actual tested content is greater than 4.5 mg / mL and less than or equal to 550 mg / mL, it should naturally be determined to be equivalent to the above range.

[0263] In particular, for the case of (8.00±0.80), the corresponding range is 7.20-8.80. Therefore, if the actual test result shows that the content is greater than or equal to 6.48 and less than or equal to 9.68, it should naturally be determined to be equivalent to the above range.

[0264] Based on the preliminary exploratory experiments, the applicant proposed the following recommended scheme: Based on certain considerations, the applicant has used the following recommended scheme to carry out relevant batch production of freeze-dried preparations. The finished freeze-dried preparations produced meet the requirements of subsequent clinical trials and are expected to become candidate drugs for subsequent large-scale clinical trials and even for commercial production in the future.

[0265] A solution for preparing a high-drug-load lyophilized formulation contains a compound of the following formula I-1, water, tert-butyl alcohol, and mannitol:

[0266] Water and tert-butanol are used as a mixed solvent, the volume percentage of tert-butanol relative to 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;

[0267] 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;

[0268] Mannitol is an excipient, and its mass percentage in the solution is (7.14±0.71)% or the mannitol content in the solution is (70±7) mg / mL,

[0269] The solution has a pH of 4-9.

[0270] Due to the properties of TH-302 (Compound I-1) and the production batch, the pH of the solution containing Compound I-1, water, tert-butanol, and mannitol may not be between 4 and 9 under specific temperature and environment. Therefore, a pH additive needs to be added for adjustment. Recommended pH adjusters are Na2CO3, NaHCO3, K2CO3, KHCO3, etc. commonly used in medicine, preferably NaHCO3. Therefore, the above solution also contains sodium bicarbonate as a pH adjuster.

[0271] Furthermore, the pH value of the solution is 6-8.

[0272] A lyophilized preparation containing the compound of the following formula I-1 and mannitol:

[0273] The drug loading of the compound of formula I-1 in the freeze-dried preparation was (8.00±0.80) mg / cm 3 Or the mass percentage of the compound of formula I-1 in the lyophilized preparation is (10.23±1.02)%,

[0274] The content of mannitol in the freeze-dried preparation is (70±7) mg / cm 3 , or the content of mannitol in the lyophilized preparation is the remainder of (10.23±1.02)% by mass of the compound of formula I-1.

[0275] After experimentation, it was found that the volume of the freeze-dried solid obtained after freeze-drying by adjusting the freeze-drying process using the above solution remained essentially unchanged: no significant collapse (resulting in a smaller volume) or spray bottle (resulting in an increased volume) was observed in the freeze-dried preparation. Therefore, as the water and tert-butyl alcohol used as solvents were evaporated during the freeze-drying process, only TH-302 and mannitol remained in the freeze-dried preparation. Therefore, the drug loading of the compound of formula I-1 in the freeze-dried preparation was 8.00 mg / cm 3 , the corresponding mass percentage is 10.23%; the content of mannitol in the freeze-dried preparation is 70 mg / cm 3 Taking into account the 10% difference in filling volume during the filling process, the above values ​​have a variation range of ±10%.

[0276] A lyophilized preparation containing the compound of the following formula I-1, mannitol, and a pH adjuster:

[0277] The drug loading of the compound of formula I-1 in the freeze-dried preparation was (8.00±0.80) mg / cm 3 Or the mass percentage of the compound of formula I-1 in the lyophilized preparation is (10.23±1.02)%,

[0278] The content of mannitol in the freeze-dried preparation is (70±7) mg / cm 3 , or the content of mannitol in the lyophilized preparation is the remainder of (10.23±1.02)% by mass of the compound of formula I-1.

[0279] A lyophilized preparation comprising a compound of the following formula I-1, mannitol, residual solvent components, and a pH adjuster:

[0280] The drug loading of the compound of formula I-1 in the freeze-dried preparation was (8.00±0.80) mg / cm3 Or the mass percentage of the compound of formula I-1 in the lyophilized preparation is (10.23±1.02)%,

[0281] The content of mannitol in the freeze-dried preparation is (70±7) mg / cm 3 ,

[0282] The residual solvent components are water and tert-butanol.

[0283] Furthermore, the lyophilized preparation is dissolved in water for injection to form a 5.0 mg / mL aqueous solution with a pH value of 4.0-7.0.

[0284] The recommended testing methods are as follows:

[0285] Accurately weigh at room temperature, add water for injection to dissolve and dilute to a solution containing 5.0 mg of TH-302 per 1 mL. Use a pH meter to measure, and measure at least two samples in parallel to obtain the average value.

[0286] The pH regulator is sodium bicarbonate, and its content is 0.01-0.10 mg / cm 3 .

[0287] Preferably, the residual water content is less than or equal to 6% by mass, preferably less than or equal to 2%, more preferably less than or equal to 1%, and further preferably less than or equal to 0.5%.

[0288] The residual tert-butanol content is less than or equal to 1795 ppm by mass, preferably 1000 ppm, and more preferably less than or equal to 500 ppm.

[0289] Tert-butyl alcohol, a pharmaceutical excipient used in the production of TH-302 for injection, may leave a small amount of residue after lyophilization. Tert-butyl alcohol has low toxicity to humans. To ensure drug safety and quality, limits should be established to control the residual tert-butyl alcohol in TH-302 for injection. According to the ICHO3C(R8) document, the PDE (permitted daily exposure) for tert-butyl alcohol is 35 mg / day. Based on literature estimates, the maximum daily dose of TH-302 is 1.5 g. Strictly calculating this as 2 g, the corresponding maximum daily dose for injection is 2 g. Assuming a TH-302 content of 0.2 g per bottle (50 mL vial), 10 bottles are required. Each bottle of lyophilized powder contains 1.95 g (1.75 g mannitol and 0.20 g of the API TH-302). Therefore, the total mass of the 10 bottles of lyophilized powder is 19.5 g. Therefore, the residual tert-butyl alcohol content is less than or equal to 35 mg / 19.5 g (mass percentage = 0.1795%), or 1795 ppm.

[0290] Based on the continuous adjustment of the prescription during the prescription screening process, the operability and calculation convenience of the tert-butanol detection method, and considering that the differences in the process may lead to changes in the amount per bottle, the limit of tert-butanol can also be calculated according to the labeled amount of TH-302. The residual tert-butanol content relative to the labeled mass percentage of the raw material TH-302 is less than or equal to 35mg / 2.0g = 1.75%.

[0291] According to the conversion, taking a lyophilized preparation containing 200 mg of TH-302 API as an example, the above-mentioned residual tert-butanol content is less than or equal to 1795 ppm by mass, preferably 1000 ppm, and more preferably less than or equal to 500 ppm, which is completely equivalent to: the residual tert-butanol content is less than or equal to 1.75% by mass of the labeled amount, preferably less than or equal to 1%, and more preferably less than or equal to 0.5%.

[0292] Furthermore, the unit volume mass of the freeze-dried preparation was (79.2±7.9) mg / cm 3 .

[0293] Mass per unit volume is similar to density. The lyophilized preparation is a porous cake in which the TH-302 API is fully fused with mannitol and sodium bicarbonate (residual solvent water and tert-butanol) to form a honeycomb structure, with cavities formed within the honeycomb. The size of the mass per unit volume is a comprehensive indicator that is related to the drug loading, mannitol content, residual solvent water, and tert-butanol of the lyophilized preparation. Studies have shown that uniform and high-quality lyophilized preparations have good rapid dissolution characteristics, which facilitates subsequent compatibility. Whether the lyophilized preparation is loose and porous, and the degree of looseness and porosity, can be qualitatively studied at the microscopic level through direct observation or microscopic observation, while at the macroscopic level, it can be characterized by the mass per unit volume of the above-mentioned lyophilized preparations.

[0294] The experimental results show that the unit volume mass of the freeze-dried preparation is (79.2±7.9) mg / cm 3 It is relatively suitable: the degree of porosity is appropriate, the drug loading capacity also meets the requirements, and the freeze-drying process is relatively easy to implement in large-scale production.

[0295] Obviously, the above-mentioned lyophilized preparation can be prepared by freeze-drying the above-mentioned solution for preparing the lyophilized preparation with high drug loading.

[0296] The preparation unit packaging containing the above-mentioned freeze-dried preparation has the following characteristics:

[0297] The lyophilized preparation is contained in a sealed container with a volume of 1000 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I-1 is 1600-5333 mg; or

[0298] The lyophilized preparation is contained in a sealed container with a volume of 500 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I-1 is 800-2666 mg; or

[0299] The lyophilized preparation is contained in a sealed container with a volume of 250 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I-1 is 400-1333 mg; or

[0300] The lyophilized preparation is contained in a sealed container with a volume of 100 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I-1 is 160-533 mg; or

[0301] The lyophilized preparation is contained in a sealed container with a volume of 50 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I-1 is 80-266 mg; or

[0302] The lyophilized preparation is contained in a sealed container with a volume of 30 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I-1 is 48-160 mg; or

[0303] The lyophilized preparation is contained in a sealed container with a volume of 25 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I-1 is 40-133 mg; or

[0304] The lyophilized preparation is contained in a sealed container with a volume of 20 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I-1 is 32-107 mg; or

[0305] The lyophilized preparation is contained in a sealed container with a volume of 18 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I-1 is 29-96 mg; or

[0306] The lyophilized preparation is contained in a sealed container with a volume of 15 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I-1 is 24-79 mg; or

[0307] The lyophilized preparation is contained in a sealed container with a volume of 10 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I-1 is 16-53 mg; or

[0308] The lyophilized preparation is contained in a sealed container with a volume of 8 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I-1 is 13-42 mg; or

[0309] The lyophilized preparation is contained in a sealed container with a volume of 7 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I-1 is 11-40 mg; or

[0310] The lyophilized preparation is contained in a sealed container with a volume of 5 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I-1 is 8-27 mg; or

[0311] The lyophilized preparation is contained in a sealed container with a volume of 3 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I-1 is 5-16 mg; or

[0312] The lyophilized preparation is contained in a sealed container with a volume of 2 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I-1 is 3-11 mg; or

[0313] The lyophilized preparation is contained in a sealed container with a volume of 1.5 mL. The volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and contains 2-8 mg of the compound of formula I-1.

[0314] The above compound content is based on 8.0 mg / cm 3 The calculated value is the labeled amount, and the actual content is within the range of ±10% of the labeled amount.

[0315] An injection for intravenous injection, wherein the solvent is water, and the solutes include TH-302 raw material drug, mannitol, tert-butyl alcohol, sodium bicarbonate, and an isotonicity adjusting agent, wherein the content of TH-302 raw material drug is 0.1-4.0 mg / mL, the osmotic pressure of the injection is 260-320 mOsmol / kg, and the pH value is 4.0-9.0.

[0316] Similarly, as a solution for intravenous injection, the osmotic pressure needs to be adjusted. Commonly used injections are 5% by mass glucose injection and 0.9% by mass sodium chloride injection. Therefore, the solute may also include an isotonicity adjusting agent selected from glucose and sodium chloride.

[0317] Furthermore, a method for preparing the above-mentioned injection is also provided, comprising the following steps:

[0318] The above-mentioned lyophilized preparation is placed at room temperature until it returns to room temperature;

[0319] The lyophilized preparation returned to room temperature was prepared into an aqueous solution for injection with a TH-302 API content of (5±0.5) mg / mL using water for injection;

[0320] An appropriate amount of the above aqueous solution for injection is injected into 0.9% saline injection or 5% glucose injection to dilute the solution to a content of TH-302 raw material drug of 0.1-4.0 mg / mL.

[0321] The preparation method of the above-mentioned injection is printed in the instructions attached to the unit packaging of the lyophilized preparation, which is used to guide clinical preparation and immediate use. The prepared injection should be injected within 8 hours and includes the following steps:

[0322] The lyophilized preparation according to any one of claims 4 to 12 is placed at room temperature for 30 to 120 minutes until it returns to room temperature;

[0323] Use a syringe to draw an appropriate volume of water for injection. Insert the syringe with the bevel facing upward and the needle at an angle of approximately 60° to the stopper, then inject along the inner wall of the bottle. After injection, shake the vial for at least 20 seconds to ensure that all lyophilized blocks / powders are completely dissolved and mixed evenly. Let it stand to eliminate bubbles, and obtain an aqueous solution for injection with a TH-302 API content of (5±0.5) mg / mL.

[0324] A calculated amount of solution is extracted from a container containing 0.9% saline injection or 5% glucose injection, and an amount of the above-mentioned aqueous solution for injection equal to the extracted amount is injected into the container containing 0.9% saline injection or 5% glucose injection to dilute the TH-302 raw material to a content of 0.1-4.0 mg / mL.

[0325] In addition, if there is suspended matter in the solution after standing to eliminate bubbles, it should be shaken at 30-35°C for 2 minutes until it becomes clear. BRIEF DESCRIPTION OF THE DRAWINGS

[0326] Figure 1 shows the solubility curves of TH-302 in aqueous tert-butyl alcohol solutions at different mass ratios;

[0327] Figure 2 shows the stability curves of lyophilized formulations prepared with 11 excipients over a 5-day period, distinguished by the rightmost circle, which from top to bottom are PEG2000, P188, SBECD, mannitol, DSPE-MPEG2000, fructose, trehalose, PVPK12, sucrose, maltose, and lactose;

[0328] FIG3 shows the stability curves of the lyophilized formulations prepared with sucrose and mannitol as excipients over a period of 10 days. The four upper curves in the figure are curves for the mannitol formulation, and the four lower curves are curves for the sucrose formulation;

[0329] FIG4 is a stability curve of a freeze-dried sample prepared with 100 mg / mL sucrose and 80 mg / mL mannitol as excipients at a high temperature of 40°C and a room temperature of 25°C, with the horizontal axis representing days and the vertical axis representing the percentage purity by HPLC;

[0330] Figure 5 shows a photo of the bottom of a liquid sample crystallized at 2-8°C, with the label coded and covered.

[0331] Figure 6 shows samples of four batches of freeze-dried preparations, from left to right: batches 01, 02, 03, and 04, with the labels coded.

[0332] Figure 7 shows comparative photographs of four batches of freeze-dried preparation samples after addition of solution and after standing in a 40 mL 5% glucose solution reconstitution experiment. The left picture shows the photo after addition of solution, and the right picture shows the photo after addition of solution and standing. From left to right in each photo are batches 01, 02, 03, and 04, and the labels have been coded;

[0333] Figure 8 shows comparative photographs of four batches of freeze-dried preparation samples after addition of solution and after standing in a 50 mL 5% glucose solution reconstitution experiment. The left picture shows the photo after addition of solution, and the right picture shows the photo after addition of solution and standing. From left to right in each photo are batches 01, 02, 03, and 04, and the labels have been coded.

[0334] Figure 9 shows samples of seven batches of freeze-dried preparations, from left to right, batches 01 to 07, with the labels already coded;

[0335] FIG10 is a schematic diagram of the placement of vials on the shelf of the experimental freeze dryer;

[0336] Figure 11 is a photo of a freeze-dried preparation sample;

[0337] Figure 12 shows a photo of a freeze-dried preparation with "neck wrapping"; the label has been coded and covered.

[0338] FIG13 is a schematic diagram showing the angle at which a syringe needle penetrates a vial stopper during reconstitution of a lyophilized preparation;

[0339] Figure 14 is a schematic diagram of the syringe needle piercing the stopper of the vial and injecting the injection solution along the inner wall of the vial when the lyophilized preparation is reconstituted. DETAILED DESCRIPTION

[0340] The present invention is described below with reference to specific examples. Those skilled in the art will appreciate that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0341] "Patient" and "subject" are used interchangeably to refer to a mammal in need of cancer treatment. Typically, the patient is a human. Typically, the patient is a human diagnosed with cancer. In certain embodiments, a "patient" or "subject" may refer to a non-human mammal, such as a non-human primate, dog, cat, rabbit, pig, mouse, or rat, used to screen, characterize, and evaluate drugs and therapies.

[0342] "Prodrug" refers to a compound that, after administration or administration, is metabolized or otherwise converted to a biologically active or more active compound (or drug) with respect to at least one property. A prodrug is chemically modified relative to a drug in a manner that renders it less active or inactive relative to the drug, but the chemical modification allows for the production of the corresponding drug by metabolism or other biological processes following administration of the prodrug. A prodrug may have altered metabolic stability or delivery characteristics, fewer side effects or lower toxicity, or an improved flavor relative to the active drug. Prodrugs may be synthesized using reactants other than the corresponding drug.

[0343] "Treatment" or "treating a patient" refers to administering, using or applying to a patient a therapeutically effective amount of a drug related to the present invention.

[0344] "Administering" or "applying" a drug to a patient refers to direct administration or administration (which may be administered or administered to a patient by a medical professional or may be self-administered or administered) and / or indirect administration or administration, which may be the act of prescribing a drug. For example, a physician who instructs a patient to self-administer or administer a drug and / or provides a prescription for a drug to a patient is administering or administering a drug to a patient.

[0345] A "therapeutically effective amount" of a drug refers to an amount of the drug that, when administered or used to a patient suffering from cancer, will have the desired therapeutic effect (e.g., alleviation, amelioration, remission, or elimination of the clinical manifestations of one or more cancers in the patient). The therapeutic effect does not necessarily occur by administering or applying a single dose and may only occur after administering or applying a series of doses. Thus, a therapeutically effective amount can be administered or applied in one or more doses.

[0346] "Treatment" of a condition or patient refers to taking steps to obtain beneficial or desired results (including clinical results). For the purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms of cancer; reduction in the extent of the disease; delay or slowing of disease progression; improvement, remission, or stabilization of the disease state; or other beneficial results. In some instances, treatment of cancer may result in a partial response or stabilization of the disease.

[0347] "Tumor cell" refers to a tumor cell of any appropriate species (eg, mammalian, such as murine, canine, feline, equine, or human).

[0348] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention. As long as they do not depart from the spirit of the present invention, they should fall within the scope of the claims attached to the present invention.

[0349] General Information

[0350] Unless otherwise specified in the following experiments, the detection or examination methods, instruments, etc. are as follows:

[0351] The moisture content was determined using the Karl Fischer (KF) method using a Mettler V10S Karl Fischer titrator.

[0352] The residual solvent, tert-butyl alcohol, was determined using gas chromatography (GC) using an Agilent 8860 gas chromatograph equipped with a 7696A automatic headspace sampler and an FID detector. The chromatographic column was a capillary column packed with DB-624. GC parameters included nitrogen as carrier gas, an inlet temperature of 150°C, a detector temperature of 200°C, a split ratio of 20:1, and a heating program of 60°C for 5 minutes, followed by a temperature increase of 30°C / min to 240°C, followed by a 5-minute hold at 60°C, and a headspace equilibration at 85°C for 20 minutes.

[0353] The content and concentration of TH-302 were determined using high-performance liquid chromatography (HPLC) using a Thermo Vanquish HPLC instrument and a YMC pack AQ C18 4.6 mm × 250 mm, 5 μm column. The detection method was based on the HPLC method described in patent application WO2008083101A1 filed by Threshold (specifically, Example 2: An Ethanol Formulation of TH302).

[0354] Unless otherwise specified, “ / ” in the table means not tested.

[0355] Unless otherwise specified, for the convenience of calculation, the residual solvent (tert-butanol) index values ​​in the freeze-dried preparation test items all represent the residual tert-butanol content as a mass percentage relative to the API labeled amount of the freeze-dried preparation.

[0356] 1. TH-302 solubility test

[0357] The solubility was determined experimentally using the saturated solution method, where solid TH-302 was directly added to the solvent until insoluble matter or turbidity appeared. After a period of clarification, the solution was directly filtered and the clarified filtrate was used to directly test the concentration of TH-302. The obtained concentration was the solubility of TH-302 in the solvent system. Specific solubility data are shown in Tables 1, 2, and 3.

[0358] The concentration (mg / mL) of TH-302 was measured according to the HPLC method in patent WO2008083101A1 applied by Threshold Corporation (specifically, Example 2. An Ethanol Formulation of TH302). The external standard method was used for quantification.

[0359] If the concentration is too high, the corresponding clear solution should be diluted to a level that can be accurately quantitatively detected by HPLC.

[0360] Using the mass ratio of tert-butanol in the data of Table 3 as the X-axis and the solubility of TH-302 in the tert-butanol-water mixed solvent as the Y-axis, the relationship curve in Figure 1 is obtained: the solubility of the raw material drug in the aqueous solution is low; as the concentration of tert-butanol in the tert-butanol aqueous solution increases, the solubility of the raw material drug continues to increase; the solubility of the raw material drug is highest in 70% tert-butanol aqueous solution (w / w), and then as the concentration of tert-butanol in the tert-butanol aqueous solution increases, the solubility decreases.

[0361] 2. Screening experiment of excipients (lyoprotectants) in freeze-dried preparations

[0362] In order to explore the feasibility of commonly used sugars, polyols, non-ionic polymer surfactants, etc. as freeze-drying excipients for TH-302 drugs, the following excipients were selected for freeze-drying experiments and stability tests.

[0363] Sugar selection: sucrose, lactose, maltose, fructose, trehalose

[0364] Polyol selection: mannitol, glycerol, sorbitol

[0365] Non-ionic polymer surfactants include: PVPK12 (polyvinyl pyrrolidone with a molecular weight of 5500), PEG2000 (polyethylene glycol 2000), and P188 (Poloxamer is a polyoxyethylene polyoxypropylene ether block copolymer with a brand name of 188).

[0366] Others: SBECD (sulfobutyl ether-β-cyclodextrin), DSPE-MPEG2000 (phosphatidylethanolamine)

[0367] Prepare the lyophilized solution according to Table 4 below.

[0368] Table 4: Preparation scheme of lyophilized drug solution prepared with different lyophilization excipients

[0369] API: TH-302, TBA: tert-butyl alcohol, water: water.

[0370] ※The solvent used in the PEG2000 and P188 protective agent experimental groups was 40% TBA / water.

[0371] According to the above prescription, the TH-302 raw material and lyoprotectant were weighed, the prescribed amount of tert-butanol solution was added, and the mixture was mixed evenly until completely dissolved. The mixture was then aliquoted (using 5 mL vials, each filled with 1 mL of the drug solution, and 8 vials for each prescription) and rapidly freeze-dried in a small rapid freeze dryer: the aliquoted preparation was pre-frozen in a -80°C ultra-low temperature refrigerator for approximately 2.5 h, and then freeze-dried in a freeze dryer for approximately 10-90 h (-30°C, absolute pressure of 0.1 mbar) to obtain a freeze-dried preparation.

[0372] Experimental results

[0373] Freeze-dried powder: Except for water as the solvent, all other solvent groups successfully produced white freeze-dried powder cakes. However, the glycerol group produced an oily substance when the freeze dryer was heated to 0°C.

[0374] Redissolution status: Except for the sorbitol group, all other groups were successfully re-dissolved (partially dissolved after shaking).

[0375] The remaining sucrose, mannitol, lactose, maltose, fructose, PVPK12, trehalose, DSPE-MPEG2000, SBECD, PEG2000, and P188 formulation groups were subjected to high-temperature accelerated stability experiments.

[0376] After the freeze-dried preparation was taken out of the freeze dryer, one bottle was taken out at 0°C to test the HPLC purity of the raw material and the water and residual solvent contents. It was then placed in a 40°C thermostat and the HPLC purity was tested on the 3rd and 5th days respectively. The results are shown in Table 5 below.

[0377] Table 5: Accelerated stability test data at 40°C for lyophilized formulations with different excipients.

[0378] ND: Not Detected

[0379] The data in Table 5 were plotted with days as the X-axis and HPLC purity as the Y-axis to obtain the curve shown in FIG2 .

[0380] Experimental Conclusion

[0381] Generally speaking, the selection of a lyoprotectant for injectable formulations requires a balance of stability, routine use, and no impact on efficacy, DMPK, or toxicology. Analyzing the curves in Figure 2, PEG2000 is not routinely used, while P188 / SBECD / DSPE-MPEG2000 may impact efficacy, DMPK, and toxicology. Overall, mannitol is ideal, and sucrose also has potential for development (optimizing dosage and lyophilization conditions may improve stability and reduce residual tert-butyl alcohol).

[0382] In order to further evaluate the stability of mannitol and sucrose, the mannitol and sucrose samples were subjected to HPLC purity tests after being placed for 10 days, and the results were plotted as a curve, as shown in Figure 3.

[0383] Analysis of Figure 3 shows that under the preliminary formulation conditions in Table 4, the stability of the mannitol formulation is better than that of the sucrose formulation.

[0384] 3. Experimental study on the effects of different lyophilization protectants (100 mg / mL sucrose, 80 mg / mL mannitol) on the stability of lyophilized powder and reconstituted solution

[0385] Based on experience and similar lyophilized drug preparations, it was preliminarily determined to use 100 mg / mL sucrose and 80 mg / mL mannitol as excipients to investigate the stability of the prepared TH-302 lyophilized preparation and the stability of the solution after reconstitution.

[0386] 3.1 Accelerated storage stability test of freeze-dried preparations at room temperature and high temperature

[0387] Prepare the lyophilized solution according to Table 6 below. Prepare 40 bottles of each, 1 mL / bottle, for a total of 40 mL of solution per prescription.

[0388] Table 6: Preparation of lyophilized solution with 100 mg / mL sucrose and 80 mg / mL mannitol as excipients

[0389] Liquid preparation process:

[0390] 1. First dissolve the prescribed amount of lyophilized protective agent in the prescribed amount of water until completely dissolved.

[0391] 2. Add the prescribed amount of tert-butyl alcohol to the lyophilization protectant aqueous solution and mix well. Then add the prescribed amount of API and stir until completely dissolved.

[0392] 3. If the pH of the sucrose solution is 4.24, adjust the pH to 6.43 with 10 μL of NaHCO3 injection. Then add another 10 μL to adjust the pH to 7.13.

[0393] The pH of the mannitol solution was measured to be 4.62, and 12 μL of NaHCO3 injection was used to adjust the pH to 7.31.

[0394] Filter through PVDF and dispense into 1 mL / bottle.

[0395] A similar operation to the aforementioned "rapid freeze drying" was used and relevant parameters were adjusted to obtain a freeze-dried preparation.

[0396] After lyophilization, the tubes were vacuum-pressed and capped to obtain the lyophilized preparation. The lyophilized preparations were then tested for residual solvent and moisture.

[0397] The freeze-dried preparation samples were placed in thermostats at 25°C and 40°C, and the HPLC purity was tested on the corresponding days. The results are shown in Table 7 below.

[0398] Table 7: Stability test data of lyophilized samples prepared with 100 mg / mL sucrose and 80 mg / mL mannitol as excipients at high temperature 40°C and room temperature 25°C

[0399] NA: Not detected

[0400] A stability curve of 100 mg / mL sucrose and 80 mg / mL mannitol was prepared using the HPLC purity in Table 7 as the Y coordinate and time as the X coordinate, as shown in FIG4 .

[0401] From the analysis and comparison of Table 7 and Figure 4, it can be seen that the stability of the freeze-dried preparation samples prepared by using mannitol as the excipient at 25°C and 40°C is better than that of the sucrose preparation.

[0402] 3.2 Reconstitution and stability test of freeze-dried preparations

[0403] Take one tube of lyophilized powder and dissolve it in D5W (5% by mass glucose in water) to form a solution containing approximately 5 mg of API per 1 mL. The solution was placed at room temperature and the color and clarity of the solution were measured at 0, 6, and 24 hours. The results were all colorless, transparent, and clear solutions.

[0404] At 0, 6, and 24 hours, the solution was diluted with 30% acetonitrile aqueous solution to a solution containing approximately 1 mg of API per mL and the HPLC purity was tested. The results are shown in Table 8 below.

[0405] Table 8: Stability test data of lyophilized samples prepared with 100 mg / mL sucrose and 80 mg / mL mannitol as excipients after reconstitution at room temperature (25°C)

[0406] Each vial of lyophilized powder was dissolved in D5W to form a solution containing approximately 5 mg of API per 1 mL. The pH and osmotic pressure of the solution were measured. The results are shown in Table 9 below. The pH and osmotic pressure of the 5% glucose injection were also measured.

[0407] Table 9: pH and osmotic pressure data of lyophilized samples prepared with 100 mg / mL sucrose and 80 mg / mL mannitol as excipients after reconstitution with D5W at room temperature (25°C)

[0408] By comparison, it was found that in terms of the stability of the reconstitution of the lyophilized preparations, there was little difference between the 100 mg / mL sucrose and 80 mg / mL mannitol formulations, and both met the requirements.

[0409] Based on the room temperature and high temperature accelerated storage stability tests of freeze-dried preparations and the reconstitution compatibility stability tests of freeze-dried preparations, relatively speaking, under the existing rapid freeze-drying process conditions, the stability of freeze-dried preparation samples prepared with mannitol as the excipient at 25°C and 40°C were better than those of sucrose prescriptions, while the reconstitution compatibility stability of 5% glucose injection was not much different.

[0410] 4. Experiment to investigate the effect of pH of drug intermediates and API concentration on sample re-dissolution

[0411] Prepare the lyophilized solution according to Table 10 below.

[0412] Table 10: Lyophilized solution preparation scheme to investigate the effects of API concentration and pH on sample reconstitution

[0413] The density of tert-butanol is ρ = 0.785 g / mL. A 40% volume ratio corresponds to 80 mL of tert-butanol in 200 mL of the drug solution, and the corresponding mass of tert-butanol is 62.80 g.

[0414] Liquid preparation process:

[0415] 1. Dissolve the prescribed amount of lyophilized protective agent in about 80% of the prescribed amount of water under stirring until completely dissolved;

[0416] 2. Add the prescribed amount of API to tert-butyl alcohol and the remaining water and stir to dissolve;

[0417] 3. Mix the solutions in steps 1 and 2 above evenly;

[0418] 4. Filtration: No pH adjustment was made for batches 1 and 2, and pH was adjusted to 7.0 for batches 3 and 4;

[0419] 5. Filling: Batch 1 is filled with 14 mL; Batch 2 is filled with 10.5 mL; Batch 3 is filled with 14 mL; Batch 4 is filled with 10.5 mL. The filled vials are 50 mL in size.

[0420] One bottle from each of batches 01, 02, 03, and 04 was placed in a medical refrigerator (2-8°C) and observed for crystallization: varying degrees of crystallization were observed, as shown in Figure 5 below. The supernatant from batch 01 and its reconstitution solution (reconstituted with 5% glucose) were collected and assayed for content, with the results shown in Table 11 below.

[0421] Table 11: Reconstitution of intermediate solution 01 after storage at 2-8°C, content of supernatant after storage and HPLC purity

[0422] The above-mentioned subpackaged preparations were freeze-dried in a vacuum freeze dryer. The freeze-drying conditions were optimized multiple times and are shown in Table 12 below. After freeze-drying, the capsules were vacuum-pressed and capped. The resulting freeze-dried preparations are shown in Figure 6.

[0423] Table 12: Freeze-drying conditions after multiple optimizations

[0424] The above lyophilized preparation was reconstituted with 40 mL and 50 mL of 5% glucose. The results are shown in Table 13 below.

[0425] Table 13: Visual observation after reconstitution of 40 mL and 50 mL 5% glucose

[0426] Figure 7 shows a photograph of reconstituted 40 mL of 5% glucose. Batch 3 (15 mg / mL API concentration, pH adjusted to 7.13) showed no adherent matter on the bottle wall and a few insoluble particles at the bottom of the bottle. After standing for a period of time, the solution became clear, indicating that excessively high API concentration may be detrimental to subsequent reconstitution of the lyophilized preparation.

[0427] A photograph of the reconstituted 50 mL of 5% glucose is shown in FIG8 . In Batch 3 (15 mg / mL API concentration, pH adjusted to 7.03), there is no adherence to the bottle wall, no insoluble particles at the bottom of the bottle, and the solution is clear, indicating that increasing the amount of reconstitution solution improves the reconstitution of the lyophilized preparation.

[0428] Experimental Conclusion

[0429] Through comparative experimental results, it was found that although the solubility of API (TH-302) in the tert-butyl alcohol-water mixed solvent system can be as high as 160 mg / mL or more, due to:

[0430] First, too high a concentration during the freeze-drying process will cause the drug solution to crystallize and stratify, affecting the freeze-drying process;

[0431] Second, excessively high drug concentration will affect reconstitution and lead to reconstitution failure.

[0432] Therefore, there is an appropriate range for API concentration. After many experiments, we found that 5-160 mg / mL is more appropriate, or further using 8-50 mg / mL to obtain a stable drug solution and better quality of subsequent freeze-dried preparations.

[0433] 5. Experiment to investigate the effects of API concentration, lyophilization protectant concentration, and tert-butyl alcohol concentration on the residual solvent of lyophilized powder

[0434] The effects of API concentration, lyoprotectant concentration, and tert-butanol concentration on the residual solvent in the lyophilized powder were investigated, and blank lyophilized powder was prepared as a control.

[0435] Prepare the lyophilized solution according to Table 14 below. Each batch has 10 bottles, 1 mL / bottle, a total of 10 mL

[0436] Table 14: Lyophilized drug solution preparation scheme to investigate the effects of API concentration, lyoprotectant concentration, and tert-butyl alcohol concentration on residual solvent in lyophilized powder

[0437] Liquid preparation process:

[0438] 1. First dissolve the prescribed amount of lyophilized protective agent in the prescribed amount of water until completely dissolved.

[0439] 2. For batches 01, 02, 03, and 04, add the prescribed amount of tert-butyl alcohol to the lyophilization protectant aqueous solution and mix thoroughly. Then add the prescribed amount of API and stir until completely dissolved.

[0440] For batch 05 of prescription, tert-butyl alcohol was not added, and the prescribed amount of API was added and stirred to dissolve it completely.

[0441] For batches 06 and 07, add the prescribed amount of tert-butyl alcohol to the lyophilization protectant aqueous solution and mix thoroughly. No API was added to serve as a blank control.

[0442] 3. The pH was not adjusted in this experiment and the solution was dispensed into 1 mL bottles.

[0443] The aliquoted preparation was pre-frozen in a -70°C freezer for 3 hours and then dried in a freeze dryer for 62 hours. After freeze drying, the capsules were plugged and capped. The resulting freeze-dried preparation is shown in Figure 9.

[0444] The preparations were reconstituted by injecting into D5W. Batches 01 to 07 were easily reconstituted and the solutions were clear.

[0445] The freeze-dried preparation samples were placed in a 40°C thermostat and tested for HPLC purity on corresponding days. The results are shown in Table 15 below.

[0446] Table 15: HPLC purity data (%) of 5 batches of freeze-dried samples at high temperature of 40°C

[0447] At the same time, the tert-butyl alcohol (2 bottles) and water (1 bottle) contents of the 0-day samples were measured, and the results are shown in Tables 16 and 17 below.

[0448] Table 16: Residual tert-butyl alcohol content data of 7 batches of freeze-dried samples measured at 0 days

[0449] Note: TBA is not added to 05. 01 middle and 01 bottom represent freeze-dried preparation samples obtained by freeze-drying the same batch of drug solution on different plates (middle and bottom) of the freeze dryer.

[0450] Table 17: Residual water content data of 7 batches of freeze-dried samples measured at 0 days

[0451] Experimental Conclusion

[0452] In the samples prepared by the small freeze dryer, the TBA residue in each bottle of sample was uneven, and the TBA residue in the sucrose formulation was higher than that in the mannitol formulation.

[0453] At 40℃, the related substances in the sucrose formulation increased significantly, while the related substances in the mannitol formulation increased slightly.

[0454] The API concentration has little relationship with the residual solvent content in the freeze-dried preparation, and the lyoprotectant concentration has little relationship with the residual solvent content in the freeze-dried preparation.

[0455] The API concentration, lyoprotectant concentration, and tert-butanol concentration all affect the residual solvent content in the freeze-dried preparation: in comparison, the solvent residue in the freeze-dried preparation obtained by using mannitol as a freeze-drying excipient (lyoprotectant) is lower, and in the high-temperature accelerated stability experiment, the stability of the mannitol formulation is better than that of the sucrose formulation.

[0456] 6. Experimental study on the effects of different API concentrations and mannitol concentrations, different proportions of tert-butyl alcohol, and different filling volumes on sample reconstitution

[0457] 6.1 Investigating the Effects of Different pH and Filling Volumes on Sample Reconstitution in the Formula (40% tert-Butanol + 60 mg / mL Mannitol + 10 mg / mL API)

[0458] Prepare 600 mL of the solution according to Table 18.

[0459] Table 18: Effects of different pH and filling volumes on sample reconstitution for the formulation (40% tert-butyl alcohol + 60 mg / mL mannitol + 10 mg / mL API)

[0460] The density of tert-butanol ρ = 0.785 g / mL, so 240 mL corresponds to 188.4 g.

[0461] Liquid preparation process:

[0462] 1. Add the prescribed amount of tert-butyl alcohol to the lyophilization protectant aqueous solution while stirring and mix well; then add the prescribed amount of API and stir to dissolve;

[0463] 2. Filter through a sterile filter cup. Batch 01: 300 mL, no pH adjustment; Batch 02: pH 4.58, adjust pH to 7.07; simultaneously, take 6 mL of each solution and place it in a refrigerator (2-8°C) to observe crystallization.

[0464] 3. Filling:

[0465] 200mg / bottle: Batch 01 is filled with 21mL, resulting in 6 bottles; Batch 02 is filled with 21mL, resulting in 6 bottles;

[0466] 150mg / bottle: Batch 01 is filled with 16mL, resulting in 6 bottles; Batch 02 is filled with 16mL, resulting in 6 bottles;

[0467] 100mg / bottle: Batch 01 is filled with 10.5mL, resulting in 6 bottles; Batch 02 is filled with 10.5mL, resulting in 6 bottles.

[0468] 6.2 Investigating the Effects of Different pH and Filling Volumes on Sample Reconstitution in the Formulation (30% tert-Butanol + 70 mg / mL Mannitol + 12.5 mg / mL API)

[0469] Prepare 600 mL of the solution according to Table 19.

[0470] Table 19: Effects of different pH and filling volumes on sample reconstitution for the formulation (30% tert-butyl alcohol + 70 mg / mL mannitol + 12.5 mg / mL API)

[0471] The density of tert-butanol is ρ = 0.785 g / mL, so the mass corresponding to 180 mL is 141.3 g.

[0472] Liquid preparation process:

[0473] 1. Dissolve the prescribed amount of lyophilized protective agent in approximately the prescribed amount of water under stirring at 500 rpm until completely dissolved.

[0474] 2. Add the prescribed amount of tert-butyl alcohol to the lyophilization protectant aqueous solution and mix well; then add the prescribed amount of API and stir to dissolve;

[0475] 3. Filter through a sterile filter cup. Batch 03: 300 mL, no pH adjustment; Batch 04: pH 4.61, adjust pH to 7.01; simultaneously, take 6 mL of each solution and place it in a refrigerator (2-8°C) to observe crystallization.

[0476] 4. Filling:

[0477] 200mg / bottle: Batch 03 is filled with 17mL, resulting in 8 bottles; Batch 04 is filled with 17mL, resulting in 8 bottles;

[0478] 150mg / bottle: Batch 03 is filled with 13mL, resulting in 7 bottles; Batch 04 is filled with 13mL, resulting in 7 bottles;

[0479] 100mg / bottle: Batch 03 is filled with 8.5mL, resulting in 8 bottles; Batch 04 is filled with 8.5mL, resulting in 8 bottles;

[0480] 6.3 Prefreezing and freeze-drying

[0481] The above-mentioned subpackaged preparations were freeze-dried in a vacuum freeze dryer using the parameters shown in Table 12. After freeze-drying, the jars were vacuum-sealed and capped to obtain the freeze-dried preparations.

[0482] 6.4 Experimental Results

[0483] (1) Crystallization of drug solution intermediates

[0484] 6 mL of the solution from batches 01 and 02 was placed in a refrigerator (2-8°C) for a period of time (2 h) and then became clear without any crystallization;

[0485] 6 mL of the solutions from batches 03 and 04 were placed in a refrigerator (2-8°C) for a period of time (2 h) and then crystallized.

[0486] (2) Sample reconstitution

[0487] All freeze-dried preparations are white solid blocks.

[0488] The solvents used for sample reconstitution and the reconstitution phenomena are shown in Table 20.

[0489] Table 20: Experimental results on the effects of different API concentrations and mannitol concentrations, different proportions of tert-butyl alcohol, and different filling volumes on sample reconstitution

[0490] Normal pressure means that the aluminum cap and rubber stopper of the vial containing the lyophilized preparation are opened to the outside world and purified water is injected for reconstitution; no normal pressure is marked means that purified water is directly injected into the vial containing the lyophilized preparation through a syringe (at this time, the air pressure in the vial is less than the external atmospheric pressure) for reconstitution.

[0491] Sample 04 (8.5 mL, 13 mL, 17 mL) was placed at 40°C for 10 days, sampled, and redissolved. The results are shown in Table 21 below.

[0492] Table 21: Reconstitution results of 4 batches of samples with different filling volumes after storage at 40°C for 10 days

[0493] Experimental summary: The re-dissolution of sample 04 (8.5mL, 13mL, 17mL) was not affected by placing it at 40℃ for 10 days, and the re-dissolution phenomenon was not much different from that of the sample on day 0.

[0494] Experimental conclusion:

[0495] In general, for a container with a fixed volume, within a certain range, the reconstitution of the lyophilized preparation obtained by filling the lyophilized liquid with a smaller volume will be easier;

[0496] Regarding whether the pH value of the drug solution should be adjusted, relatively speaking, the lyophilized preparation obtained by adjusting the pH value to around 7 has a better reconstitution condition.

[0497] Through specific comparison, it can be found that the lyophilized preparations obtained with lower API concentration and lower mannitol content are easier to reconstitute. This suggests that simply increasing the API concentration and mannitol dosage to increase the drug loading of the lyophilized preparation will lead to difficulty in reconstitution. It is necessary to select appropriate API concentration and mannitol content to obtain a lyophilized solution with a higher drug loading and easy reconstitution.

[0498] 7. Experiment to investigate the effect of pH on sample stability

[0499] Investigating the effect of pH on sample stability

[0500] Prepare the solution according to Table 22 below. Prepare a total of 6500 mL and fill each bottle with 25 mL, for a total of 260 bottles.

[0501] Table 22: Solution preparation scheme for investigating the effect of pH on sample stability

[0502] 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 API purity is 99.26%.

[0503] Preparation process of freeze-dried preparation.

[0504] 1. Liquid preparation

[0505] ① Preparation of 70% tert-butanol aqueous solution: weigh 350.81g of tert-butanol and 150.42g of water.

[0506] ② Predissolution: Weigh the prescribed amount of API, add the API into 70% tert-butyl alcohol aqueous solution under stirring, stir and dissolve, and record it as solution A.

[0507] ③ Under stirring conditions, first dissolve the prescribed amount of mannitol in 70% of the remaining water, stir until clear, and record it as solution B.

[0508] ④ Mix solutions A and B and stir evenly. Rinse with the remaining water and tert-butyl alcohol and add the mixture; the pH of the intermediate solution is 5.63.

[0509] ⑤ Divide the liquid medicine into 3 equal parts,

[0510] Solution-01: No pH adjustment; pH value was measured at 4.77 after being placed at room temperature for 24 hours.

[0511] Solution 02: Add 200 μl of sodium bicarbonate and measure pH 6.60 (pH range 6.0-6.5). Place at room temperature for 24 hours and measure pH 5.20.

[0512] Solution-03: Add 1000 μl of sodium bicarbonate and measure pH 7.20 (pH is 7.0). Place at room temperature for 24 hours and measure pH 6.90. Take the intermediate of the 0-hour solution and the solution placed at room temperature for 24 hours and test the content and related substances.

[0513] 2. Filter

[0514] Filter using a 250 mL sterile filter cup.

[0515] 3. Filling

[0516] The drug solution - Batch 01 is filled with 24.5 g, and 78 bottles are obtained, which is Batch 01.

[0517] The drug solution - Batch 02 is filled with 24.5 g, and 78 bottles are obtained, which is Batch 02.

[0518] The drug solution - Batch 03 was filled with 24.5 g, resulting in 78 bottles, which is Batch 03.

[0519] 4. Half-cut

[0520] After filling, half of the stopper is added and the bottle is placed in a freeze dryer for freeze drying.

[0521] 5. Pre-freezing and freeze-drying

[0522] The above-mentioned packaged preparations were freeze-dried in a vacuum freeze dryer. The order of vials arranged in this freeze dryer is shown in Figure 10. Freeze-drying was performed using the freeze-drying parameters in Table 12. After freeze-drying, the vials were vacuum-sealed and capped. This resulted in the freeze-dried preparation. Shelf temperature before discharge was 5.0°C, and the vials were manually sealed under high vacuum.

[0523] Experimental results

[0524] The results of the drug solution test are shown in Table 23.

[0525] Table 23: pH data of different batches of drug solution after pH adjustment and storage for different time periods

[0526] The sample state of the freeze-dried preparation is white solid block. The specific sample is shown in Figure 11. The bottleneck of the sample in the middle and rear boxes of the freeze dryer is surrounded by powder as shown in Figure 12. This may be caused by improper operation during filling.

[0527] (3) Sample re-dissolution test

[0528] Freeze-dried preparations from different batches and locations were sampled for reconstitution, and the results are shown in Table 24 below.

[0529] Table 24: Reconstitution test data of freeze-dried preparations at different shelf locations from different batches after freeze-drying of different pH solutions

[0530] Note: The solution of batch 02 (blank powder) after reconstitution is as clear as water; the solutions of batches 01, 02 and 03 after reconstitution are slightly more turbid than water.

[0531] To further investigate the reconstitution effect with the commonly used intravenous infusion solution - normal saline: After reconstitution with purified water to an API concentration of approximately 5 mg / mL, it was diluted again with normal saline to 0.5 mg / mL (5 mL was drawn and added to 50 mL of sodium chloride injection, 0.5 mg / mL): clear solutions were obtained, and then the pH and osmotic pressure data were tested. The results are shown in Table 25 below.

[0532] Table 25: Reconstitution test data of freeze-dried preparations at different shelf locations from different batches after freeze-drying of different pH solutions

[0533] Samples were taken at different time periods to test the HPLC purity of the API in the solution reconstituted with saline. The results are shown in Table 26 below.

[0534] Table 26: HPLC purity data of API in solutions reconstituted with saline at different concentrations and time periods

[0535] (4) Sample solvent residue and stability test

[0536] The freeze-dried preparation samples were placed in thermostats at 40°C, 25°C and 2-8°C, and the HPLC purity was tested at the corresponding time. The results are shown in Table 27 below.

[0537] Table 27: Lyophilized preparation samples were placed in thermostats at 40°C, 25°C, and 2-8°C, and HPLC purity data were tested at the corresponding time.

[0538] Residual solvent detection.

[0539] The tert-butyl alcohol (2 bottles) and water (2 bottles) contents of batches 01, 02, and 03 on day 0 were measured respectively. Note that samples were taken from the middle of the freeze dryer shelf. The results are shown in Table 28 below.

[0540] Table 28: Residual solvent content of lyophilized preparations prepared from three batches of drug solutions with different pH values

[0541] 8. Preparation Examples of Solutions with Different Tert-Butanol Concentrations, Different Excipients (Sucrose, Mannitol), Different Excipient Doses, Different Drug Contents, and Different pH Values ​​after Lyophilization

[0542] Since the quality indicators of lyophilized preparations include three main evaluation indicators such as reconstitution, stability, and residual solvent, and the above experiments show that the influencing factors include solvent (volume ratio of tert-butanol in tert-butanol-water mixed solvent), API concentration, excipient type (mannitol, sucrose) and dosage, pH value of the drug solution, etc., multiple multi-factor experiments were conducted, and the results are shown in Table 28 below.

[0543] Table 28: Data of freeze-dried formulation examples of different tert-butyl alcohol volume ratios, API concentrations, excipient types and amounts, and pH values ​​of the drug solution

[0544] Remark:

[0545] TBA is tert-butyl alcohol.

[0546] Mannitol is mannitol.

[0547] Sucrose is sucrose.

[0548] The API is TH-302 raw material drug.

[0549] PBS is phosphate-buffered saline, and its pH value is stable at around 7.4.

[0550] In the prescription, 30% TBA / 70Mannitol / 10API-pH7.0 means 30% by volume of tert-butanol, 70 mg / mL of mannitol, and 10 mg / mL of API, and finally NaHCO3 is added to adjust the pH to 7.0, and the preparation method is the same as the above embodiment; 40% TBA / 60Mannitol / 10API means 40% by volume of tert-butanol, 60 mg / mL of mannitol, and 10 mg / mL of API, without pH adjustment (weakly acidic), and so on.

[0551] Except for the batch with a filling volume of 1 mL, which uses a 5 mL volume vial, the rest use a 50 mL volume vial.

[0552] Experimental Conclusion

[0553] 1. Combining the solubility data of TH-302 in a tert-butyl alcohol-water mixed solvent system with the above experiments and the results of the 85 exploratory experiments in Table 28 above, it can be inferred or verified that the compound can be stably present in solution at a content of 5-500 mg / mL. Experiments have also shown that lyophilization is possible at a content greater than 8 mg / mL but less than or equal to 200 mg / mL. However, adjustment of lyophilization parameters may be required, such as pre-freezing at -80°C, lower absolute pressure during lyophilization, longer drying time, and lower drying temperature. Preferably, when the content of the compound of Formula I in solution is greater than or equal to 8 mg / mL and less than or equal to 25 mg / mL, the lyophilized formulation is more stable and easier to reconstitute. The content of the compound of formula I in the solution is greater than or equal to 8 mg / mL and less than or equal to 15 mg / mL. Such a content range will mean that the lyophilization conditions are relatively mild and the lyophilization cycle is shorter. The content of the compound of formula I in the solution is greater than or equal to 8 mg / mL and less than or equal to 10 mg / mL, which further means that the lyophilized preparation has commercially suitable lyophilization cycles and lyophilization low temperature conditions and is valuable for commercial large-scale production. Inappropriate API content will result in large-scale lyophilizers used in commercial large-scale production, resulting in broken bottles and dusting due to uneven temperature, resulting in a yield lower than 90% of commercial production acceptance.

[0554] 2. The solubility data of TH-302 in the tert-butanol-water mixed solvent system, combined with the above experiments and the results of the 85 exploratory experiments in Table 28 above, can be inferred or verified that the TH-302 solution prepared when the volume percentage of tert-butanol relative to the solution is 1%-99% or 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) is stable and clear. Experiments have also shown that lyophilization can be performed when the volume ratio is 5%-95% or 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). However, the lyophilization parameters may need to be adjusted, such as pre-freezing at -80°C, lower absolute pressure during lyophilization, longer drying time, and lower drying temperature. Preferably, 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 preparation has better stability and is easier to reconstitute.

[0555] In fact, in the tert-butanol-water mixed solvent system, too high a tert-butanol content will affect the subsequent dissolution of mannitol / sucrose. The amount of mannitol / sucrose used as excipients will directly affect the quality of the subsequent freeze-dried preparation: too low a mannitol / sucrose content will result in the drug solution being unable to be evenly loaded on the excipient skeleton after freeze-drying, and may even cause the freeze-dried powder cake to collapse or freeze-drying failure during the drying stage of the freeze-drying process. Therefore, the tert-butanol content in the mixed solvent cannot be blindly increased to increase the solubility of TH-302.

[0556] 3. According to the solubility data of TH-302 in the tert-butyl alcohol-water mixed solvent system and in combination with the above experiments and the results of the 85 exploratory experiments in Table 28 above, it can be seen that lyophilized preparations can be obtained using mannitol, PEG2000, P188, SBECD, DSPE-MPEG2000, sucrose or similar substances as excipients. However, after comparing and considering the reasons other than the lyophilized preparation itself, such as whether it is routinely used and easily commercially available, whether it affects the efficacy, DMPK and toxicology, etc., it is more appropriate to use sucrose and mannitol. The lyophilized preparations prepared therefrom have suitable solution stability (at least 24 hours at room temperature), lyophilized preparation stability, resolubility and are easy to obtain products that meet the requirements of injection preparation excipients.

[0557] 4. In fact, as stated in Article 2, as an excipient, the higher the content of mannitol / sucrose, the better: although a larger amount can serve as a freeze-dried skeleton to load more drugs, and is beneficial to freeze-dried production and improves the stability of the freeze-dried preparation, it is affected by its solubility in the tert-butyl alcohol-water mixed solvent. Excessive mannitol / sucrose dosage may make the mixed solvent unable to completely dissolve or the solution unstable, resulting in precipitation during the pre-freezing stage or cooling stage of freeze-drying. Therefore, the amount of mannitol / sucrose as an excipient in the freeze-dried liquid also has an appropriate range to meet the above requirements.

[0558] The content of sucrose and mannitol in the solution is 20-300 mg / mL. This range can meet the above requirements: the drug solution is stable and is not prone to precipitation during the pre-freezing stage or the cooling stage of freeze-drying. A further preferred range is 40-100 mg / mL, which can produce a reconstituted solution and a freeze-dried preparation with good stability. A more preferred range is 60-70 mg / mL, as this content range will mean that the freeze-drying conditions are relatively mild and the freeze-drying cycle is consistent with commercial production practices (no more than 10 days, i.e., a 240-hour production cycle).

[0559] 5. As described in item 2 above, a too low mannitol / sucrose ratio will result in uneven loading of the drug solution onto the excipient matrix after lyophilization, and may even lead to collapse of the lyophilized powder cake or lyophilization failure during the drying phase of the lyophilization process. While a too high mannitol / sucrose content can produce a lyophilized formulation with better appearance and stability, it means that the content of the Chinese drug TH-302 per unit of lyophilized formulation is too low, which does not meet commercial sales and usage requirements. Therefore, an appropriate mannitol / sucrose to drug ratio is essential. The mass ratio of the compound of Formula I to the excipient in the solution is 1:(0.5-20). This ratio meets the various other ingredient requirements in items 1-4 above and also ensures that the prepared lyophilized formulation has better appearance and stability. A ratio of 1:(2-12.5) is preferred for a lyophilized formulation that is easier to reconstitute, and a more preferred ratio of 1:(5-10) results in a lyophilized formulation with excellent properties: good stability, easy reconstitution, mild lyophilization conditions, and a short lyophilization cycle.

[0560] 6. Experimental phenomena show that the pH value of the freeze-dried drug solution will directly affect the stability of the drug solution, the difficulty of reconstitution of the freeze-dried preparation, and the stability of the freeze-dried preparation. Experiments have shown that if the pH regulator (alkali or alkaline salt) is not added to the drug solution, the drug solution is acidic, and the acidic drug solution has poor stability, the freeze-dried preparation is not easily reconstituted, and the stability of the freeze-dried preparation is poor; after adding the pH regulator (alkali or alkaline salt), these situations are significantly improved.

[0561] 7. It is worth pointing out that the filling volume of the drug solution in the vial also affects the appearance of the final lyophilized powder cake (dusting or adhering to the bottleneck of the vial) and reconstitution through various factors. Therefore, the ratio of the volume of the drug solution filled in the vial to the volume of the vial must be appropriate. Based on the above experimental data and combined with actual production, the filling volume of the lyophilized drug solution is 1 / 3 to 1 / 2 of the volume of the closed container, and the corresponding volume of the lyophilized preparation is 1 / 3 to 1 / 2 of the volume of the closed container, which is more appropriate.

[0562] 9. Saturated solubility of drug in D5W solution (strong shaking conditions)

[0563] Approximately 60 mg of TH-302 sample was accurately weighed and placed in a transparent vial. 6 mL of 5% glucose injection (D5W) was added and vigorously shaken for 2 minutes to prepare a solubility stock solution. The solubility stock solution was observed to contain insoluble matter. The solubility stock solution was filtered, leaving it clear. The filtered filtrate remained clear after standing at room temperature for 24 hours. A sample was taken and appropriately diluted. HPLC analysis revealed a solution content of 7.25 mg / mL, which was the saturated solubility of the drug in D5W solution.

[0564] It can be seen from this that the concentration of the intravenous injection solution containing TH-302 should be within the range of 0-7.25 mg / mL. In fact, considering the isotonicity and osmotic pressure of glucose contained in the intravenous injection solution, the infusion formula for intravenous drip needs further research and determination.

[0565] 10. Statistical results of height measurement of drug solution in 50 mL vials before and after lyophilization (calculation of drug loading and drug mass percentage)

[0566] Using the drug solution from Experiment 7, we filled 25 mL of the drug solution into 50 mL vials and measured the liquid level after filling, which averaged 26.5 mm. After lyophilization, we measured the height of the freeze-dried powder cake in the vials at different shelf positions in the freeze dryer and used this to roughly calculate the volume difference relative to the 25 mL before filling.

[0567] In the multiple batch experiments conducted, it was found that the volume of some batches increased before and after freeze-drying, some batches decreased, and some batches remained almost unchanged. After marking the liquid level before and after freeze-drying and statistically analyzing the volume changes, it was found that the maximum increase or decrease in volume was 10%, that is, the volume change was within ±10%.

[0568] In the case where the volume of the freeze-dried powder decreases by 10% before and after freeze-drying:

[0569] When the API content of the drug solution is greater than or equal to 5 mg / mL and less than or equal to 500 mg / mL, the drug loading of the freeze-dried powder after lyophilization is 5 / 0.9 to 500 / 0.9, that is, 5.55-555.55 mg / cm 3 , the same calculation method:

[0570] The drug content of the compound of formula I in the solution is 5-160 mg / mL, and the drug loading of the powder after freeze-drying is 5.55-177.77 mg / cm 3 ;

[0571] The drug loading of the powder after freeze-drying of the drug solution containing the compound of formula I in the range of 8-50 mg / mL is 8.88-55.55 mg / cm 3 ;

[0572] The drug loading of the powder after freeze-drying of the drug solution containing the compound of formula I in the range of 8-25 mg / mL is 8.88-27.77 mg / cm 3 ;

[0573] The drug content of the compound of formula I in the solution is 8-15 mg / mL, and the drug loading of the powder after freeze-drying is 8.88-16.66 mg / cm 3 ;

[0574] The drug loading of the powder after freeze-drying of the drug solution containing the compound of formula I in the range of 8-10 mg / mL is 8.88-11.11 mg / cm 3 .

[0575] If the drug content of the liquid is 6, 7, 7.5, 8, 8.5, 10, 12.5, 15, 20, 25 mg / mL, the corresponding drug loading of the freeze-dried powder is 6.66, 7.77, 8.33, 8.88, 9.44, 11.11, 13.88, 16.66, 22.22, 27.77 mg / cm 3 .

[0576] In the case where the volume of the freeze-dried powder increases by 10% before and after freeze-drying:

[0577] When the API content of the drug solution is greater than or equal to 5 mg / mL and less than or equal to 500 mg / mL, the drug loading of the freeze-dried powder after lyophilization is 5 / 1.1 to 500 / 1.1, that is, 4.55-454.55 mg / cm 3 , the same calculation method:

[0578] The drug content of the compound of formula I in the solution is 5-160 mg / mL, and the drug loading of the powder after freeze-drying is 4.55-145.45 mg / cm 3 ;

[0579] The drug content of the compound of formula I in the solution is 8-50 mg / mL, and the drug loading of the powder after freeze-drying is 7.27-45.45 mg / cm 3 ;

[0580] The drug content of the compound of formula I in the solution is 8-25 mg / mL, and the drug loading of the powder after freeze-drying is 7.27-22.73 mg / cm 3 ;

[0581] The drug content of the compound of formula I in the solution is 8-15 mg / mL, and the drug loading of the powder after freeze-drying is 7.27-13.64 mg / cm 3 ;

[0582] The drug content of the compound of formula I in the solution is 8-10 mg / mL, and the drug loading of the powder after freeze-drying is 7.27-9.09 mg / cm 3 .

[0583] If the drug content of the liquid is 6, 7, 7.5, 8, 8.5, 10, 12.5, 15, 20, 25 mg / mL, the corresponding drug loading of the freeze-dried powder is 5.45, 6.36, 6.82, 7.27, 7.73, 9.09, 11.36, 13.64, 18.18, 22.73 mg / cm3 .

[0584] Since the lyophilized drug solution contains API drugs, excipients, and residual tert-butanol solvent and water, and the mass ratio of the API drugs and excipients in the lyophilized preparation prepared by the applicant is 1:(0.5-20), and the maximum allowable tert-butanol content is 1.75% by mass, and the water content is 6% by mass, then the lower limit of the mass percentage of the API drug in the lyophilized 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 API drug in the lyophilized preparation is greater than or equal to 4.39% and less than 66.66%.

[0585] Based on the preliminary exploratory experiments and taking certain considerations into account, the applicant has used the following recommended scheme to carry out relevant batch production of freeze-dried preparations. The finished freeze-dried preparations produced meet the requirements of subsequent clinical trials and are expected to become candidate drugs for subsequent large-scale clinical trials and even be commercially marketed in the future.

[0586] 11. Preparation, stability and reconstitution experiments of small batches of freeze-dried preparations

[0587] 11.1 Prepare the freeze-dried preparation according to the following laboratory simulation and production process, referring to the preparations in "7. Experimental Study on the Effect of pH on Sample Stability" and the formulation in Table 22.

[0588] 1. Liquid preparation

[0589] ① Predissolve the API: Weigh a portion of tert-butyl alcohol and water for injection, stir and mix thoroughly to prepare a 70% (w / w) tert-butyl alcohol solution. Then weigh the prescribed amount of API and add it to the 70% (w / w) tert-butyl alcohol solution, stirring until visually dissolved.

[0590] ② Add about 70% (w / w) of the total amount of water for injection into the liquid preparation tank, add the prescribed amount of mannitol, and stir to dissolve.

[0591] ③ Add the remaining tert-butyl alcohol in the total amount of the prescription into the liquid preparation tank, start stirring and mix evenly.

[0592] ④ Add the pre-dissolved API to the preparation tank and add water for injection to the total preparation volume. Turn on the agitator and mix thoroughly.

[0593] ⑤ Add sodium bicarbonate solution to adjust the pH value to 4.0-9.0. The mass percentage of sodium bicarbonate solution is 5%.

[0594] 2. Filter using a sterilizing filter.

[0595] 3. Filling

[0596] Use 50mL vials for filling, with a theoretical filling volume of 25mL / vial (density of 0.98g / mL is recorded as 24.5g / vial). If the filling volume is controlled within ±2%, the acceptable actual filling volume is (25.0±0.5)mL / vial, and the quality is controlled at (24.5±0.49)g / vial.

[0597] 4. Half-cut-in

[0598] After filling, half of the stopper is added and the bottle is placed in a freeze dryer for freeze drying.

[0599] 5. Freeze-drying

[0600] The above-packaged preparations were freeze-dried in a vacuum freeze dryer using the freeze-drying parameters in Table 12 after adjustment. After freeze-drying, the tubes were vacuum-sealed and capped. This yielded the freeze-dried preparations. Shelf temperature before discharge was 5.0°C, and the tubes were manually sealed under high vacuum.

[0601] According to the above process, the drug solution was prepared and lyophilized to finally obtain 174 bottles of freeze-dried preparations, with a specification of 0.20g / bottle (i.e., the bottle of freeze-dried preparation contains 0.20g of TH-302 raw material drug), and an average filling amount of 1.98g / bottle (i.e., the average weight of the powder cake of this batch of freeze-dried preparations is 1.98g, 25cm 3 The freeze-dried powder contains 1.75g ​​mannitol, 0.20g TH-302 raw material and others).

[0602] The sampling and testing results of the intermediate solution of the above batch, i.e., the solution after pH adjustment, are shown in Table 29 below.

[0603] Table 29: Test results of intermediate solution of batch 1202206001

[0604] In addition to water and tert-butanol as solvents, the intermediate drug solution also contains the excipient mannitol. Due to the relatively stable properties of mannitol, mannitol is not tested in quality control.

[0605] The results of the sampling and testing of the above batches of freeze-dried preparations are shown in Table 30 below.

[0606] Table 30: Test results of freeze-dried preparations in batch 1202206001

[0607] pH test method: Take 1 bottle of this product, add 38mL of water for injection to dissolve it, and make a solution containing 5.0mg of TH-302 per 1mL. Use a pH meter to measure it. Measure two samples in parallel and take the average value.

[0608] The tert-butanol content test is carried out using the gas chromatography method specified in the pharmacopoeia. Take 1 bottle of freeze-dried preparation, fully rewarm it to room temperature, add about 30mL of water with a disposable syringe while maintaining the vacuum degree, shake well to dissolve, open the lid (note that the action of opening the stopper should be gentle and slow to avoid solution loss), transfer the solution to a 50mL volumetric flask, wash the test bottle and the inner surface of the stopper with water at least 3 times each, add the washing solution to the volumetric flask, and then dilute with water to the scale and shake well. Prepare 2 copies in parallel, use gas chromatography to detect two samples, and finally take the average value. The tert-butanol project refers to the mass percentage of tert-butanol relative to the labeled amount of 200mg of raw materials calculated according to the labeled amount of TH-302 200mg.

[0609] The moisture content test is determined using the Karl Fischer method. To ensure sampling uniformity and representativeness, the following operations are used for sampling: Take 1 bottle of freeze-dried preparation, return to room temperature, wipe off the water attached to the surface of the bottle, open the lid, release the pressure in the bottle, open the stopper, stir with a spoon so that the sample does not adhere to the inner wall and bottom of the bottle, cover the bottle with the stopper, turn the bottle horizontally and rotate it for no less than 5 times, turn the bottle upside down for no less than 5 times, open the stopper, stir the sample with a spoon for no less than 5 times, cover the bottle with the stopper (seal the bottle mouth with sealing film if necessary), and set aside. Accurately weigh about 0.20g of freeze-dried preparation sample (actually, since it is impossible to completely transfer the freeze-dried powder on the inner wall of the bottle to the titration cell of the moisture meter, the actual mass is less than 0.20g, and it is accurately weighed by the mass difference method) and add it to the titration cell. Stir in advance for 2 minutes before starting titration. Or refer to USP <921> The water mass percentage was determined by this method.

[0610] Total impurities refer to the total amount of impurities other than TH-302 in the API excluding the excipients tert-butyl alcohol and mannitol, and are determined using the above-mentioned TH-302 API HPLC purity test method.

[0611] Following the same procedure, another batch of lyophilized preparation (batch number 1202206002) totaling 529.54 g was prepared in lyophilization trays and heat-sealed using a double-layer low-density polyethylene (PE) ziplock bag as the inner layer and a single-layer 300 mm unprinted composite film (in compliance with relevant requirements) as the outer layer.

[0612] The same process was used to prepare another batch of lyophilized preparations (batch number F00106301) totaling 600 bottles with an average filling volume of 1.97 g / bottle for stability study.

[0613] 11.2 Stability Study of Lyophilized Formulations under Proposed Long-Term Storage Conditions (-20°C ± 5°C) and Accelerated Storage Conditions (25 ± 5°C)

[0614] The changes of relevant test indicators of TH-302 freeze-dried preparations over time under the proposed long-term storage conditions (-20℃±5℃) and high-temperature accelerated conditions (25±5℃) were investigated to provide a basis for the quality stability and storage time (shelf life) of the freeze-dried preparations.

[0615] The above two batches of samples (1202206002 and 1202206001) were stored in a -20±5℃ dark environment for long-term stability experiments, and the F00106301 batch was subjected to long-term stability experiments and accelerated stability experiments in a 25±5℃ dark environment. The specific experimental results are shown in Table 31 below.

[0616] Table 31: Quality index test data of three batches of lyophilized preparations at different time periods of long-term and accelerated storage

[0617] Identify whether the retention time of HPLC spectrum is consistent with that of reference solution.

[0618] The numerical values ​​of the content test items are: the first value is the percentage of the amount of TH-302 contained to the labeled amount (the amount of TH-302 contained is 0.20g); the second value is the mass percentage of TH-302 contained, which is 9.8% (based on the average loading amount of 1.98g).

[0619] According to the above long-term data, the percentage of total impurity content changes slowly under long-term storage conditions (-20℃±5℃), and the changes in impurities, pH, moisture and content are not obvious after 3 months and 6 months of storage; according to the data of another batch of F00106301 using higher purity TH-302 raw materials, it can be seen that under accelerated conditions (25±5℃), the above indicators still do not change significantly. It is further speculated based on the accelerated conditions that these results are equivalent to the results of storage at -20℃±5℃ for more than 18 months.

[0620] 11.3 Freeze-thaw cycle stability study of lyophilized preparations

[0621] The freeze-thaw test was performed by placing the test sample in a dark place (-20℃±5℃) for 2 days and (25±5℃) for 2 days as one cycle, and the HPLC purity of TH-302 in the freeze-dried sample was tested.

[0622] Table 32: HPLC purity test data (%) of the raw material TH-302 and two batches of lyophilized preparations prepared after three freeze-thaw cycles

[0623] The data of the API project indicates the HPLC test purity of the TH-302 raw material itself used to prepare these two batches of lyophilized preparations.

[0624] The data under 1202206001-0 day represents the purity of the lyophilized formulation of batch 1202206001 when tested on day 0.

[0625] According to the above long-term data, even after being taken out of long-term storage conditions (-20℃±5℃) and placed in a room temperature environment (25±5℃), the purity of the raw material TH-302 in the lyophilized preparation changed very little after three operations. This proves that the lyophilized preparation has good stability against sudden temperature shock and provides experimental evidence for the stability of the lyophilized preparation during actual transportation and transportation of the lyophilized preparation during long-term exposure to room temperature and transportation.

[0626] 11.4 Stability test of freeze-dried preparation (0.9% NaCl, 5% D5W)

[0627] Remove the lyophilized preparation for injection from -20°C ± 5°C and allow it to fully return to room temperature. Use a 50mL disposable sterile syringe to draw 38mL of sterile water for injection. Insert the needle with the bevel facing up and the tip of the needle at an angle of approximately 60° to the vial stopper (see Figure 1) to avoid needle puncture and debris. Inject in a circular motion along the inner wall of the vial (see Figure 2). After injection, shake the vial vigorously for at least 20 seconds to ensure that all lyophilized particles / powder are completely dissolved and evenly mixed. This yields approximately 40mL of solution, containing approximately 5.0mg of TH-302 per 1mL. If the lyophilized particles / powder do not completely dissolve after 20 seconds of vigorous shaking, shake in a 30°C to 35°C water bath until completely dissolved.

[0628] The 5.0 mg / mL solution diluted with the above-mentioned water for injection was used to prepare 5 mg / mL sterile water for injection solution, 4 mg / mL 5% glucose injection solution, 0.5 mg / mL 5% glucose injection solution, 4 mg / mL 0.9% sodium chloride injection solution, and 0.5 mg / mL 0.9% sodium chloride injection solution, and tested respectively. The test items included solution properties, osmotic pressure, pH, solution color, content, and impurity HPLC percentage (total impurities). The results are shown in Table 33.

[0629] Table 33: Test results of different compatible solutions

[0630] According to the above compatibility test results, the content, related substances, pH, osmotic pressure, solution color, properties and other test items of each compatibility solution are in compliance with the regulations, which shows that the above-mentioned lyophilized preparations can be reconstituted and combined with the commonly used clinical water for injection, 5% glucose injection, and 0.9% sodium chloride injection to meet the requirements of intravenous injection.

[0631] 12. Preparation, stability and reconstitution compatibility experiments of large batches of freeze-dried preparations

[0632] Based on the freeze-dried preparation experiment in Part 11 above, a larger-scale freeze-dried preparation was prepared. The batch numbers were F00106301 and F00115101, and stability tests and freeze-thaw experiments were carried out under high-temperature accelerated conditions (25±5°C). The results are shown in Table 34.

[0633] Table 34: Results of high temperature accelerated storage and multiple freeze-thaw tests of two batches (F00106301, F00115101) of lyophilized formulations

[0634] The results in Table 34 show that the quality of the freeze-dried preparation prepared after scale-up has hardly changed, and all indicators are qualified.

[0635] For this purpose, a GMP batch was produced under GMP conditions using API raw materials with provisional quality standards. This batch will be considered for use in subsequent clinical trials. High-temperature accelerated stability tests and compounding experiments were conducted on this batch 296220901. The specific experimental data are shown in Tables 35 and 36 below.

[0636] Table 35: Results of high temperature accelerated storage and multiple freeze-thaw experiments on GMP batch 296220901 lyophilized formulation

[0637] Exposure to light for 5 days* indicates that the vial containing the lyophilized preparation is not packaged, while exposure to light for 5 days represents the normal situation in which the vial is packaged.

[0638] 1 indicates white loose lumps; 2 indicates light yellow loose lumps.

[0639] 3 means the color is lighter than the No. 0.5 standard colorimetric solution; 4 means the color is between the No. 0.5 and No. 1 standard colorimetric solutions.

[0640] Insoluble particles 1 indicates the number of particles with a diameter ≥ 10 μm; insoluble particles 2 indicates the number of particles with a diameter ≥ 25 μm.

[0641] Table 36: Stability test results of the reconstituted solution of GMP batch 296220901 lyophilized preparation

[0642] The above experimental data prove that the stability and reconstitution compatibility of the freeze-dried preparation obtained after large-scale preparation of the above freeze-dried preparation formula are qualified. In addition, based on the results of the accelerated experiment, it can be inferred that the freeze-dried preparation can be stably stored for 18 months in the proposed light-proof environment of -20±5℃.

Claims

1. A solution for preparing a high drug loading lyophilized preparation, comprising a compound of the following formula I-1, water, tert-butyl alcohol and mannitol: Water and tert-butanol are used as a mixed solvent, the volume percentage of tert-butanol relative to the solution is (30±3)% or the mass percentage is (24±2.4)%, or the content of tert-butanol 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 its mass percentage in the solution is (7.14±0.71) % or the mannitol content in the solution is (70±7) mg / mL, The solution has a pH of 4-9.

2. The solution according to claim 1, further comprising sodium bicarbonate as a pH adjuster.

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

4. A lyophilized preparation comprising a compound of the following formula I-1 and mannitol: The drug loading of the compound of formula I-1 in the freeze-dried preparation was (8.00±0.80) mg / cm 3 Or the mass percentage of the compound of formula I-1 in the lyophilized preparation is (10.23±1.02)%, The content of mannitol in the freeze-dried preparation is (70±7) mg / cm 3 , or the content of mannitol in the lyophilized preparation is the remainder of (10.23±1.02)% of the mass percentage of the compound of formula I-1.

5. A lyophilized preparation comprising a compound of the following formula I-1, mannitol, and a pH regulator: The drug loading of the compound of formula I-1 in the freeze-dried preparation was (8.00±0.80) mg / cm 3 Or the mass percentage of the compound of formula I-1 in the lyophilized preparation is (10.23±1.02)%, The content of mannitol in the freeze-dried preparation is (70±7) mg / cm 3 , or the content of mannitol in the lyophilized preparation is the remainder of (10.23±1.02)% of the mass percentage of the compound of formula I-1.

6. A lyophilized preparation comprising a compound of the following formula I-1, mannitol, residual solvent components, and a pH adjuster: The drug loading of the compound of formula I-1 in the freeze-dried preparation was (8.00±0.80) mg / cm 3 Or the mass percentage of the compound of formula I-1 in the lyophilized preparation is (10.23±1.02)%, The content of mannitol in the freeze-dried preparation is (70±7) mg / cm 3 , The residual solvent components are water and tert-butyl alcohol.

7. The lyophilized preparation according to any one of claims 4 to 6, The lyophilized preparation is dissolved in water for injection into a 5.0 mg / mL aqueous solution with a pH value of 4.0-7.

0.

8. The freeze-dried preparation according to claim 5 or 6, in, The pH regulator is sodium bicarbonate, and its content is 0.01-0.10 mg / cm 3 .

9. The lyophilized preparation according to claim 6, in, The residual water content is less than or equal to 6% by mass, preferably less than or equal to 2%, more preferably less than or equal to 1%, and further preferably less than or equal to 0.5%. The residual tert-butyl alcohol content is less than or equal to 1795 ppm by mass, preferably 1000 ppm, and more preferably less than or equal to 500 ppm.

10. The lyophilized preparation according to any one of claims 4 to 6, in, The unit volume mass of the freeze-dried preparation is (79.2±7.9) mg / cm 3 .

11. The lyophilized preparation according to any one of claims 4 to 6, which is prepared by freeze-drying the solution according to claim 1.

12. A preparation unit package containing the freeze-dried preparation according to any one of claims 4 to 11, having the following characteristics: The lyophilized preparation is contained in a sealed container with a volume of 1000 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I-1 is 1600-5333 mg; or The lyophilized preparation is contained in a sealed container with a volume of 500 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I-1 is 800-2666 mg; or The lyophilized preparation is contained in a sealed container with a volume of 250 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I-1 is 400-1333 mg; or The lyophilized preparation is contained in a sealed container with a volume of 100 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I-1 is 160-533 mg; or The lyophilized preparation is contained in a sealed container with a volume of 50 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I-1 is 80-266 mg; or The lyophilized preparation is contained in a sealed container with a volume of 30 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I-1 is 48-160 mg; or The lyophilized preparation is contained in a sealed container with a volume of 25 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I-1 is 40-133 mg; or The lyophilized preparation is contained in a sealed container with a volume of 20 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I-1 is 32-107 mg; or The lyophilized preparation is contained in a sealed container with a volume of 18 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I-1 is 29-96 mg; or The lyophilized preparation is contained in a sealed container with a volume of 15 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I-1 is 24-79 mg; or The lyophilized preparation is contained in a sealed container with a volume of 10 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I-1 is 16-53 mg; or The lyophilized preparation is contained in a sealed container with a volume of 8 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I-1 is 13-42 mg; or The lyophilized preparation is contained in a sealed container with a volume of 7 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I-1 is 11-40 mg; or The lyophilized preparation is contained in a sealed container with a volume of 5 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I-1 is 8-27 mg; or The lyophilized preparation is contained in a sealed container with a volume of 3 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I-1 is 5-16 mg; or The lyophilized preparation is contained in a sealed container with a volume of 2 mL, the volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I-1 is 3-11 mg; or The lyophilized preparation is contained in a sealed container with a volume of 1.5 mL. The volume of the lyophilized preparation is 1 / 5 to 2 / 3 of the volume of the sealed container, preferably 1 / 3 to 1 / 2, and the content of the compound of formula I-1 is 2-8 mg.

13. An injection for intravenous injection, wherein the solvent is water, and the solutes include TH-302 raw material drug, mannitol, tert-butyl alcohol, sodium bicarbonate, and an isotonicity adjusting agent. in, The content of TH-302 raw material is 0.1-4.0 mg / mL, the osmotic pressure of the injection is 260-320 mOsmol / kg, and the pH value is 4.0-9.

0. The injection according to claim 13 , wherein the isotonicity adjusting agent is selected from glucose and sodium chloride.

15. The method for preparing the injection according to claim 14, comprising the following steps: Placing the lyophilized preparation according to any one of claims 4 to 12 at room temperature until it returns to room temperature; The above-mentioned lyophilized preparation restored to room temperature is prepared into an aqueous solution for injection with a TH-302 raw material drug content of (5±0.5) mg / mL using water for injection; An appropriate amount of the above aqueous solution for injection is injected into 0.9% saline injection or 5% glucose injection to dilute the solution to a content of TH-302 raw material drug of 0.1-4.0 mg / mL.

16. The method for preparing the injection of claim 14, which is printed in the instructions attached to the unit package of the freeze-dried preparation, is used to guide the preparation and use of the preparation after clinical preparation, and the prepared injection should be injected within 8 hours, comprising the following operations: The lyophilized preparation according to any one of claims 4 to 12 is placed at room temperature for 30 to 120 minutes until it returns to room temperature; Use a syringe to draw an appropriate volume of water for injection, insert the needle with the bevel of the needle upward and the needle tip at about 60 degrees to the bottle stopper, and inject along the inner wall of the bottle. After the injection is completed, shake the vial for at least 20 seconds to ensure that all lyophilized blocks / powders are completely dissolved and mixed evenly, and let it stand to eliminate bubbles to obtain an aqueous solution for injection with a TH-302 API content of (5±0.5) mg / mL; Draw a calculated amount of solution from a container containing 0.9% saline injection or 5% glucose injection, and inject the same amount of the above-mentioned aqueous solution for injection as the drawn amount into the container containing 0.9% saline injection or 5% glucose injection to dilute the TH-302 raw material drug content to 0.1-4.0 mg / mL.

17. The preparation method according to claim 16, if there is a suspended substance in the solution after standing to eliminate bubbles, it should be shaken at 30-35°C for 2 minutes until it becomes clear.

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