WO2023025291A1 - 冻干制剂溶液及冻干制剂、方法和用途 - Google Patents

冻干制剂溶液及冻干制剂、方法和用途 Download PDF

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WO2023025291A1
WO2023025291A1 PCT/CN2022/115176 CN2022115176W WO2023025291A1 WO 2023025291 A1 WO2023025291 A1 WO 2023025291A1 CN 2022115176 W CN2022115176 W CN 2022115176W WO 2023025291 A1 WO2023025291 A1 WO 2023025291A1
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freeze
compound
equal
solution
volume
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PCT/CN2022/115176
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English (en)
French (fr)
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于继兵
段建新
蔡晓宏
房启龙
申广彬
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深圳艾欣达伟医药科技有限公司
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Priority to CN202280046255.4A priority Critical patent/CN117651548A/zh
Priority to EP22860637.2A priority patent/EP4393480A1/en
Priority to KR1020247009526A priority patent/KR20240047452A/ko
Publication of WO2023025291A1 publication Critical patent/WO2023025291A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention relates to the development of a freeze-dried preparation of TH-302, belonging to the technical field of pharmaceutical preparations.
  • TH-302 is a prodrug of 2-nitroimidazoles. It is a selective hypoxia-activated DNA alkylating agent with high cytotoxicity designed and synthesized by researchers from Threshold Pharmaceuticals in 2006 (WO2007002931A2, Phosphoramidate alkylator prodrugs). It can be converted into dibromoifosfamide mustard with alkylating agent activity in the hypoxic area of the tumor or when it is activated by acid, but it has almost no activity under normoxic or normal pH conditions.
  • a solution (20 mL) of TH-302 (100 mg) and sucrose (1 g) was added to a lyophilized vial and lyophilized to produce a lyophilized unit dosage form of TH-302 with a drug loading of less than 5 mg/cm 3 .
  • the unit dosage form is dissolved in 5% dextrose injection, and an appropriate amount of this solution is administered to the patient;
  • TH-302 was dissolved in absolute ethanol to produce a pharmaceutically acceptable liquid formulation of 5% TH-302.
  • a 5% solution of TH-302 contains 5 g TH-302 in 100 mL of solvent (e.g., ethanol);
  • TH-302 uses lyophilized preparations, and TH-302 lyophilized preparations for injection are prepared in 100mL glass vials with a drug loading of 100mg/100ml and controlled at 2-8°C. Store under conditions, inject 250mL of 5% glucose injection into a freeze-dried preparation bottle when used, and infuse intravenously within 30 minutes through an infusion pump.
  • Threshold pharmaceutical company researchers designed and developed the injection formula in 2009 (WO2010048330A1, Treatment of cancer using hypoxia activated prodrugs):
  • the liquid preparation contains 50mg/ml to about 300mg/ml of TH-302, a nonionic surfactant (such as Tween 80), a carrier ethanol, and may also contain dimethylacetamide.
  • Threshold Company found that the effective dose of TH-302 for treating various indications is (WO2012135757A2, Methods for treating cancer):
  • Weekly doses of 480 mg/m 2 to about 670 mg/m 2 or, for example, 575 mg/m 2 are administered intravenously.
  • a lyophilized preparation with a drug loading of less than 5 mg/ cm3 (20 mL of an aqueous solution containing 100 mg of TH-302 and 1 g of sucrose is added to a 50-ml lyophilized vial and lyophilized to produce a lyophilized unit of TH-302
  • Dosage form, drug load less than 5mg/cm 3 requires at least 3 bottles, and if it is a larger dose, 13 bottles are required, which is not convenient for clinical use, and the drug cost for patients is too high.
  • Threshold used concentrated injections (WO2015013448A1, Treatment of pancreatic cancer with a combination of a hypoxia-activated prodrug and a taxane) in most of the subsequent phase II/III clinical trials:
  • TH-302 (Concentrate for Administration Solution) for clinical trials is a sterile liquid formulation of TH-302.
  • TH-302 was formulated with 70% absolute ethanol, 25% dimethylacetamide and 5% polysorbate 80. It is supplied by the sponsor in a 10 mL glass vial with a rubber stopper and flip-off closure.
  • TH-302 drug product is a clear, colorless to pale yellow solution substantially free of visible particles.
  • each single-use vial contains TH-302 drug product in a nominal fill volume of 6.5 mL (equivalent to 100 mg/mL) and is clearly labeled as The batch number, route of administration, required storage conditions, name of the sponsor and appropriate precautionary labels required by applicable regulations are disclosed. It needs to be diluted according to the pharmacy manual before administration.
  • TH-302 Dilute to a total volume of 500 mL (1000 mL for a total dose > 1000 mg) with commercially available 5% dextrose in water before administration to obtain the desired final concentration.
  • Each dose of TH-302 was prepared with 5% dextrose in water without bis(2-ethylhexyl) phthalate (DEHP-free) and administered intravenously using a DEHP-free IV administration set.
  • TH-302 mainly caused by dimethylacetamide
  • TH-302 mainly caused by dimethylacetamide
  • a steroid such as dexamethasone (or equivalent) in the antiemetic regimen prior to administration is recommended.
  • Symptoms and signs of hypersensitivity reactions include fever, myalgia, headache, rash, pruritus, urticaria, angioedema, chest discomfort, dyspnea, cough, cyanosis, and hypotension. If the nature and severity of the reaction warrant discontinuation of therapy, it should be determined that the reaction may or may not be an immunoglobulin E-mediated process.
  • antihistamines e.g., diphenhydramine 25-50 mg orally, intramuscularly or slowly i.v., or e.g. effector
  • low-dose steroid e.g., hydrocortisone, 100 mg i.v., or equivalent
  • epinephrine epinephrine (1/1000, 0.3-0.5 mL subcutaneously, or equivalent
  • inhaled beta-agonists should be considered.
  • idiosyncratic reactions can also be treated with antihistamines and low-dose steroids.
  • Response to administration of TH-302 should be evaluated and treated in a similar manner.
  • investigators should consult with medical supervisors to determine the appropriate course of action for future treatment.
  • the high-concentration TH-302 injection solves the problem of low drug loading in freeze-dried preparations, but due to the use of the above-mentioned excipients that may cause adverse reactions, related adverse reactions will occur in clinical trials, increasing the risk of medication for patients;
  • the currently developed freeze-dried preparation uses water and sucrose solution to dissolve TH-302 for freeze-drying, and does not contain other excipients.
  • the drug content of the solution before freeze-drying is too low, it cannot be used in clinical medicine and subsequent commercial production and sales. high drug loading lyophilized formulation.
  • the inventors of the present application have proposed a new high drug concentration solution prescription for producing TH-302 and other similar drug freeze-dried preparations and related freeze-dried preparations and methods after many experiments and continuous optimization.
  • Table 1 Solubility data of TH-302 in different pH aqueous solutions and different additive aqueous solutions
  • 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 volume ratios.
  • solubility of TH-302 in a single water is small, and the solubility in a single tert-butanol is also small, but unexpectedly found that the solubility of TH-302 in a mixed solvent of the two is greatly improved. And the solubility has a great relationship with the mixing ratio of the two.
  • Table 2 The solubility data of different tert-butanol + water, tert-butanol + ethanol mixed solvents for TH-302 are shown in Table 2 below.
  • Tert-butanol is a colorless crystal, easily supercooled, and becomes a liquid in the presence of a small amount of water. It has a camphor-like smell and is hygroscopic. Chinese name 2-methyl-2-propanol, tert-butanol, trimethylmethanol, etc. The melting point is 25.7°C, so it is a colorless transparent liquid or colorless crystal at room temperature.
  • tert-butanol may crystallize at room temperature (25°C), and after mixing with water, it can also freeze at a few degrees below zero, and it can be completely frozen in existing lyophilizers.
  • the vapor pressure of tert-butanol is relatively high. High vapor pressure facilitates sublimation and saves freeze-drying time.
  • Tert-butanol and water can be mixed in any proportion. This is extremely important, as it can increase the solubility of some fat-soluble drugs in water. At the same time, for some unstable drugs in aqueous solution, adding an appropriate amount of tert-butanol can inhibit the decomposition of the drug and enhance the stability of the drug.
  • Tert-butanol is easy to freeze-dry and has low residue in the preparation. During the freeze-drying process, most of the tert-butanol can be sublimated in the primary drying stage, and the residual amount in the preparation is very low.
  • Tert-butanol itself forms needle-like crystals during freezing, which can change the crystallization mode of the solute and facilitate sublimation.
  • a small amount of tert-butanol is added to water to form a tert-butanol-water co-solvent, the crystallization state of water can be changed, and needle-like crystals are formed during the freezing process, with a large surface area, and at the same time, tubular channels are left after the ice crystal sublimates. , so that the water vapor flow resistance is greatly reduced, and the sublimation rate is significantly increased, so tert-butanol can be used to accelerate the mass transfer process in the freeze-drying process.
  • 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 suitable as a freeze-drying auxiliary material.
  • TH-302 has low solubility in aqueous solution; along with the increase of tert-butanol concentration in tert-butanol aqueous solution, the solubility of raw material drug is constantly increasing; The solubility is highest in butanol aqueous solution (V/V), and then decreases with the increase of tert-butanol concentration in tert-butanol aqueous solution.
  • tert-butanol+water of the present invention can be used as a solvent and adding an appropriate excipient can obtain a high-concentration solution, and based on this prescription, develop TH-302 and its analogous compounds with high drug loading Freeze-dried formulations.
  • the present invention provides the following high-concentration solutions containing TH-302 similar compounds.
  • each R is independently selected from H, -CH 3 , -CH 2 CH 3
  • each X is independently selected from Br, Cl, OMs, OTs,
  • 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.
  • a solution for preparing a high-loaded lyophilized preparation which contains the compound of the following formula I, water, tert-butanol, and excipients:
  • each R is independently selected from H, -CH 3 , -CH 2 CH 3
  • each X is independently selected from Br, Cl, OMs, OTs,
  • 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,
  • 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,
  • 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,
  • 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;
  • 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,
  • 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.
  • the selected tert-butanol is 1%-99% relative to the volume percentage of the solution, preferably 5%-95%, More preferably 30%-60%;
  • the content of tert-butanol in the solution is 7.85-777.15 mg/ml, preferably 39.25-745.75 mg/ml, more preferably 235.5-471 mg/ml.
  • tert-butanol relative to the volume percentage of the solution is 1%-99% and the content of tert-butanol in the solution is 7.85-777.15mg/ml is a corresponding relationship, and the volume percentage of tert-butanol relative to the solution is 1%.
  • the content of tert-butanol in the corresponding solution is 7.85mg/ml
  • the conversion factor is the density of tert-butanol.
  • the density of tert-butanol used by the applicant is 0.785g/ml. In fact, the density of products from different manufacturers at different temperatures They are all different, generally between 0.775-0.786g/ml.
  • the solution used in the present invention for preparing the freeze-dried formulation with high drug loading contains at least one excipient.
  • a pharmaceutically acceptable excipient refers to an additive or carrier that can contribute to the stability of the drug substance in the preparation. During the preparation of the lyophilized preparation, the excipient is added to the solution used for lyophilization or lyophilized. Dry protectant is a must.
  • Some drug solutions can be successfully lyophilized in vacuum, while others collapse or melt into oils quickly after lyophilization.
  • some excipients that do not react with the drug need to be added. These excipients themselves will not be sublimated in the sublimation stage of the lyophilization process, but can be directly absorbed Freeze-drying becomes a skeleton, which plays the role of giving shape, and the drug can be directly adsorbed or filled in the void of the skeleton; or improve the solubility and stability of the freeze-dried product, or make the freeze-dried product have a beautiful shape, etc.
  • lyoprotectants sometimes also called fillers, fillers, excipients, buffers, bases, frameworks, etc.
  • the lyoprotectant must be chemically inert to the drug solution.
  • Salts sodium thiosulfate, calcium lactate, sodium glutamate, sodium chloride, potassium chloride, sodium sulfate, ammonium acetate, ammonium chloride, etc.
  • Sugars sucrose, lactose, maltose, glucose, raffinose, fructose, hexose, etc.
  • Alcohols sorbitol, ethanol, glycerin, mannitol, inositol, xylitol, etc.
  • Acids citric acid, phosphoric acid, tartaric acid, amino acids, ethylenediaminetetraacetic acid (EDTA), etc.
  • Alkalis sodium hydroxide, sodium bicarbonate, etc.
  • Polymers dextran, polyethylene glycol, polysorbate, PVP, poloxamer, etc.
  • the degree of polymerization (molecular weight) of the above-mentioned polymers is wide. Take polysorbate and polyethylene glycol as examples.
  • the average molecular weight of the polysorbate may range from about 500 g/mol to about 1900 g/mol, preferably from about 800 g/mol to about 1600 g/mol, more preferably from about 1000 g/mol to about 1400 g/mol.
  • Non-limiting examples of polysorbates include: polysorbate-20, polysorbate-21, polysorbate-40, polysorbate-60, polysorbate-61, polysorbate -65, polysorbate-81, polysorbate-85 and polysorbate-120.
  • Preferred polysorbates include polysorbate-20, polysorbate-80, and mixtures thereof.
  • 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.
  • PEGs include: PEG200, PEG300, PEG400, PEG540, and PEG600.
  • Poloxamers whose general formula is HO(C 2 H 4 O) a (C 3 H 6 O) b (C 2 H 4 O) c H, where a and c are 2-130, and b is 15-67 .
  • the polyoxyethylene content is 81.8 ⁇ 1.9%.
  • It is polyoxyethylene polyoxypropylene ether block copolymer, which has different grades: Poloxamer 182, Poloxamer 184, Poloxamer 188, Poloxamer Poloxamer 407, which corresponds to polymers of different molecular weights, such as Poloxamer 188, has a molecular weight of 7680-9510.
  • Lyoprotectants have many functions, which are summarized as follows:
  • Microorganisms such as bacteria and viruses need to grow and reproduce in specific culture media, but these culture media and microorganisms are often difficult to separate, and they can be successfully freeze-dried in these culture media. Examples include broth, skim milk, and protein.
  • freeze-dried preparations have a very low concentration and very little dry matter content, and the dried part will be taken away by the sublimation airflow during freeze-drying.
  • filling materials are sucrose, lactose, inositol, skim milk, hydrolyzed protein, dextran, sorbitol, polyvinylpyrrolidone (PVP) and the like.
  • Some biologically active substances are particularly fragile and will be harmed due to physical or chemical reasons during freezing and drying, so some protective agents need to be added to reduce damage during freezing and drying.
  • Some biologically active substances are particularly fragile and will be harmed due to physical or chemical reasons during freezing and drying, so some protective agents need to be added to reduce damage during freezing and drying.
  • Adding certain substances can increase the disintegration temperature of the product, making the product easy to freeze-dry. They are mannitol, glycine, dextran, xylitol and PVP etc.
  • antioxidants such as vitamin C, vitamin E, amino acids, sodium thiosulfate, thiourea, lecithin, and hydrolyzed protein.
  • Adding certain substances can eliminate free radicals and increase the stability of freeze-dried products. They are amino acids, vitamin K, vitamin C, thiourea, sulfites, sodium aspartate, etc.
  • the lyoprotectant is selected from sugars, polyalcohols, polyvinylpyrrolidones, proteins, poloxamers or combinations thereof.
  • the sugar is selected from sucrose, dextran, cyclodextrin, maltodextrin, trehalose, lactose, maltose, glucose;
  • Polyhydric alcohols are selected from glycerin, sorbitol, mannitol, inositol, ethylene glycol, polyethylene glycol (PEG), polysorbate, side calendula alcohol;
  • Proteins are selected from albumin, preferably bovine serum albumin and human albumin;
  • the poloxamer is selected from poloxamer 182, poloxamer 184, poloxamer 188, poloxamer 407.
  • the excipient is selected from PVP K12, sucrose, mannitol, albumin or a combination thereof.
  • excipients of the same type can be combined, and excipients that do not react with each other and the drug can be combined, and the combination should meet the compatibility requirements of the drug.
  • 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 , more preferably 60-70 mg/ml.
  • sucrose or mannitol are generally used, and they can be used in combination under special circumstances.
  • the preferred scheme of the present application is to use only mannitol or only sucrose.
  • the mass ratio of TH-302 and excipients in the solution used to prepare high-loaded lyophilized preparations in the present invention is 1: (0.5-20), preferably 1: (1-15), more preferably 1:(2-12.5), more preferably 1:(5-10).
  • the mass ratio of TH-302 and other similar compounds to excipients is the drug-loading ratio of the solution for lyophilization.
  • the lyophilized body after lyophilization uses the excipient as the framework, and the drug will be adsorbed or loaded on the framework, so the freeze-dried
  • the drug-loading ratio of the prodrug solution that is, the mass ratio of TH-302 to the excipient, is an important indicator.
  • Appropriate drug-loading ratio means that after lyophilization of the lyophilized solution, the drug and the matrix are evenly adsorbed, and the drug will be better distributed on the surface of the voids and pores of the matrix. In this way, in the subsequent reconstitution (5% glucose injection, During the process of normal saline, etc.), the lyophilized preparation can be well and quickly dissolved for injection administration.
  • a pharmaceutically acceptable buffer refers to a weak acid or base that allows the pH of the solution to be maintained at a nearly constant level, and is used to enhance the stability of the drug substance in solution.
  • the solution used in the preparation of the high-loaded lyophilized preparation of the present invention may also 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 mixtures thereof.
  • 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
  • the buffer used in the solution used to prepare the high drug loading lyophilized formulation is at least one citrate buffer.
  • suitable citrate buffers include: lithium citrate monohydrate, sodium citrate monohydrate, potassium citrate monohydrate, calcium citrate monohydrate, lithium citrate dihydrate, lemon Sodium Citrate Dihydrate, Potassium Citrate Dihydrate, Calcium Citrate Dihydrate, Lithium Citrate Trihydrate, Sodium Citrate Trihydrate, Potassium Citrate Trihydrate, Calcium Citrate Trihydrate, Citric Acid Lithium 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,
  • the solution used for preparing the freeze-dried preparation with high drug loading may further contain at least one pH regulator.
  • the pH adjuster of the present invention refers to the buffer substance or buffer used for proper adjustment of the pH changed according to acid or alkali
  • the pH adjuster includes but not limited to hydrochloric acid, sodium hydroxide, triethanolamine, phosphoric acid, dihydrogen phosphate Sodium, dipotassium phosphate, monosodium phosphate, monopotassium phosphate or phosphoric acid, citric acid, lactic acid, tartaric acid, succinic acid, fumaric acid, malic acid, sodium bicarbonate, sodium carbonate or mixtures thereof.
  • the added content of the pH regulator makes the pH value of the solution 4-9, preferably 6-8.
  • the compound preparation process contains an acidic environment, and there is a small amount of hydrolysis at the same time, so the prepared or purchased raw materials will appear acidic.
  • the pH adjuster is preferably selected from alkalis such as sodium hydroxide, triethanolamine, sodium bicarbonate, sodium carbonate, or salts with alkalinity.
  • TH-302 or I-1 More preferred is TH-302.
  • the invention provides a solution for preparing a high-loaded lyophilized preparation, which contains
  • 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, 471mg/ml,
  • the content of the compound of formula I-1 in solution is 6,7,7.5,8,8.5,10,12.5,15,20,25mg/ml,
  • 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.
  • the invention provides a solution for preparing a high-loaded lyophilized preparation, which contains
  • 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, 471mg/ml,
  • 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, 25mg/ml,
  • Sucrose or mannitol are used as excipients, and the content in the solution is 40, 50, 60, 70, 75, 80, 90, 100mg/ml,
  • the content of the pH regulator makes the pH of the solution 4-9, preferably 6-8,
  • the present invention provides a solution for preparing a high drug loading lyophilized preparation, comprising
  • 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, 471mg/ml,
  • 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, 25mg/ml,
  • Sucrose or mannitol are used as excipients, and the content in the solution is 40, 50, 60, 70, 75, 80, 90, 100mg/ml,
  • the content of sodium bicarbonate is such that the pH value is 4-9, preferably 6-8.
  • 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, 1:9.375.
  • buffers, pH regulators or other adjuvants for lyophilized preparations can also be added according to the above description and in combination with the properties of the specific compound of formula I.
  • tert-butanol, sucrose/mannitol, and the raw materials of the compound of formula I must be doped with impurities or other environmental substances (environmental substances), and these impurities or environmental substances are also not limited here.
  • the raw material that tert-butanol and sucrose/mannitol, sodium bicarbonate, the compound of formula I have must be doped with impurity or other environment, these impurity or environmental substance, also Not limited to this.
  • the above-mentioned “consisting of xxx” refers to substances added intentionally and artificially during preparation (these substances must be contained and can be detected by analytical testing instruments), and no other intentionally and artificially added substances are contained except for these substances Substances, but there will still inevitably be trace amounts of impurities, environmental substances, etc.
  • the solution provided above is only used as an intermediate semi-finished product for the preparation of high-loaded lyophilized preparations, and cannot be used as a prescription for clinical preparations. Generally speaking, it is ready-to-use at the production site, that is, it is directly filled into a freeze-drying bottle after being prepared in a liquid preparation container, and then sent to the freeze-drying production equipment in batches for freeze-drying.
  • the solution for the preparation of high-loaded lyophilized preparations should be stable during the production and waiting process of lyophilization in lyophilization equipment after the preparation is completed, that is, it should have stability for at least 8 hours at room temperature, and more optimal , should have 24 hours or even 72 hours, 120 hours of stability.
  • the present invention can prepare freeze-dried preparations with high drug loading.
  • the freeze-dried preparation is prepared by using the above-mentioned solution for preparing the freeze-dried preparation with high drug loading as the freeze-dried preparation solution through a freeze-drying process.
  • Freeze-dried preparations containing the compound of the following formula I, excipients, and residual solvent components:
  • each R is independently selected from H, -CH 3 , -CH 2 CH 3
  • each X is independently selected from Br, Cl, OMs, OTs,
  • the drug loading amount of the compound of formula I in the freeze-dried preparation is greater than 5 mg/cm 3 and less than or equal to 555.55 mg/cm 3 , or
  • the drug loading amount 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 500 mg/cm 3 , or
  • 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%
  • the remaining solvent components are water and tert-butanol.
  • Freeze-dried preparations containing the compound of the following formula I and excipients, residual solvent components, and pH regulators:
  • each R is independently selected from H, -CH 3 , -CH 2 CH 3
  • each X is independently selected from Br, Cl, OMs, OTs,
  • the drug loading amount of the compound of formula I in the freeze-dried preparation is greater than 5 mg/cm 3 and less than or equal to 555.55 mg/cm 3 , or
  • the drug loading amount 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 500 mg/cm 3 , or
  • 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%
  • the remaining solvent components are water and tert-butanol.
  • a lyophilized formulation comprising a compound of the following formula I and excipients:
  • each R is independently selected from H, -CH 3 , -CH 2 CH 3
  • each X is independently selected from Br, Cl, OMs, OTs,
  • the drug loading amount of the compound of formula I in the freeze-dried preparation is greater than 5 mg/cm 3 and less than or equal to 555.55 mg/cm 3 , or
  • the drug loading amount 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 500 mg/cm 3 , or
  • the mass percentage of the compound of formula I in the freeze-dried preparation is greater than or equal to 4.39% and less than 66.66%.
  • the drug loading of the compound of formula I in the lyophilized preparation is greater than 5 mg/cm 3 and less than or equal to 555.55 mg/cm 3
  • the drug loading of the compound of formula I in the lyophilized preparation is greater than or equal to 4.55 mg/cm 3 and less than 500 mg/cm 3
  • the volume may increase by 10% due to the expansion of the powder cake after freeze-drying, or the volume may decrease by 10% due to the collapse of the powder cake.
  • the calculation is based on a maximum change of 10%, that is, the expansion becomes larger
  • the volume is made 1.1 times of the original volume, and the collapse becomes smaller so that the volume is 90% of the original volume.
  • the drug loading range is 5 to 500, and the two ranges are combined to obtain 5 to 555.55, that is, the drug loading of the compound of formula I in the freeze-dried preparation is greater than 5 mg/cm 3 and less than or equal to 555.55 mg/cm 3 .
  • the drug loading of the compound of formula I in the lyophilized preparation is greater than or equal to 4.55 mg/cm 3 but less than 500 mg/cm 3 .
  • the drug loading of the lyophilized preparation refers to the amount of the bulk drug of the compound of formula I contained in a unit volume, where the volume refers to the total external volume of the drug in the unit packaging kit, including the internal void of the drug.
  • a 100ml lyophilized vial contains 20ml of lyophilized solution, and 20ml of lyophilized solution contains 500mg of drug. After the preparation solution before freeze-drying goes through the freeze-drying process, 20ml of the solution is frozen into a loose and porous freeze-dried preparation.
  • the height of the freeze-dried preparation in the freeze-dried bottle is h
  • the total apparent volume of the freeze-dried preparation in this unit package is sh, and it contains 500mg of raw material drug, so its drug loading is (500/sh) mg/cm 3 , and generally the value of sh will be 20cm 3 Left or right, the volume may expand after lyophilization to be larger than 20cm 3 , or it may collapse after lyophilization and the volume may be less than 20cm 3 .
  • 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%, and its conversion can be calculated according to the API content, excipients, residual tert-butanol and water content in the lyophilized preparation.
  • the mass content of the compound of formula I in the freeze-dried preparation refers to the percentage of the drug in the total mass of the freeze-dried preparation. It can be measured and calculated by the following operations:
  • the compound of formula I is selected from
  • the excipients are sugars, polyalcohols, polyvinylpyrrolidones, proteins, poloxamers or combinations thereof,
  • the sugar is selected from sucrose, dextran, cyclodextrin, maltodextrin, trehalose, lactose, maltose, glucose,
  • Polyhydric alcohols are selected from glycerin, sorbitol, mannitol, inositol, ethylene glycol, polyethylene glycol, polysorbate, side calendula alcohol,
  • Proteins are selected from albumin, preferably bovine serum albumin, human albumin,
  • the poloxamer is selected from poloxamer 182, poloxamer 184, poloxamer 188, poloxamer 407.
  • the freeze-dried preparation can be considered as It contains almost no water and tert-butanol.
  • 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 percentage, preferably less than or equal to 2%, more preferably less than or equal to 1% %, more preferably less than or equal to 0.5%,
  • the residual tert-butanol content is less than or equal to 1.75% by mass, preferably less than or equal to 1%, more preferably less than or equal to 0.5%.
  • Containing the compound of the following formula I and excipients means that in addition to the unavoidable residual water and tert-butanol, the lyophilized preparation can be detected to contain the compound of formula I, excipients and the above-mentioned residual environmental substances, in addition to In addition, it can also contain other auxiliary materials.
  • the mass ratio of compound I to the excipient is 1:(0.5-20), preferably 1:(1-15), more preferably 1:(2-12.5), further preferably 1:(5-10 ).
  • the drug loading of the compound of formula I in the lyophilized preparation is greater than or equal to 5.55 mg/cm 3 and less than or equal to 177.77 mg/cm 3 , preferably greater than or equal to 8.88 mg/cm 3 and less than or equal to 55.55 mg/cm 3 , more preferably It is greater than or equal to 8.88 mg/cm 3 and less than or equal to 27.77 mg/cm 3 , more preferably greater than or equal to 8.88 mg/cm 3 and less than or equal to 16.66 mg/cm 3 , still more preferably greater than or equal to 8.88 mg/cm 3 cm 3 and less than or equal to 11.11mg/cm 3 ;
  • the drug loading of the compound of formula I in the lyophilized preparation is greater than or equal to 4.55 mg/cm 3 and less than or equal to 145.45 mg/cm 3 , preferably greater than or equal to 7.27 mg/cm 3 and less than or equal to 45.45 mg/cm 3 , more preferably It is greater than or equal to 7.27 mg/cm 3 and less than or equal to 22.73 mg/cm 3 , more preferably greater than or equal to 7.27 mg/cm 3 and less than or equal to 13.64 mg/cm 3 , still more preferably greater than or equal to 7.27 mg/cm 3 cm 3 and less than or equal to 9.09mg/cm 3 .
  • the former is the situation that the volume collapses and becomes smaller after freeze-drying
  • the latter is the situation that the volume expands and becomes larger.
  • the freeze-dried preparation is essentially composed of the compound of the following formula I-1 and excipients, residual water and residual tert-butanol:
  • the drug loading amount of the compound in the lyophilized preparation is 6.66, 7.77, 8.33, 8.88, 9.44, 11.11, 13.88, 16.66, 22.22, 27.77 mg/cm 3
  • the drug loading amount of the compound I-1 in the lyophilized preparation is 5.45, 6.36, 6.82, 7.27, 7.73, 9.09, 11.36, 13.64, 18.18, 22.73mg/ cm3 ;
  • the excipient is sucrose or mannitol
  • the mass ratio of the compound to the excipient is 1:2, 1:3, 1:3.5, 1:4, 1:5, 1:5.333, 1:5.6, 1:6, 1:7, 1:8 , 1:8.235, 1:8.75, 1:9.375;
  • 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%, further preferably less than or equal to 0.5%;
  • the residual tert-butanol content is less than or equal to 1.75% by mass, preferably less than or equal to 1%, more preferably less than or equal to 0.5%.
  • the freeze-dried preparation is essentially composed of the compound of the following formula I-1 and excipients, residual water and residual tert-butanol, and a pH regulator:
  • the drug loading amount of the compound in the lyophilized preparation is 6.66, 7.77, 8.33, 8.88, 9.44, 11.11, 13.88, 16.66, 22.22, 27.77 mg/cm 3
  • the drug loading amount of the compound I-1 in the lyophilized preparation is 5.45, 6.36, 6.82, 7.27, 7.73, 9.09, 11.36, 13.64, 18.18, 22.73mg/ cm3 ;
  • the excipient is sucrose or mannitol
  • the mass ratio of the compound to the excipient is 1:2, 1:3, 1:3.5, 1:4, 1:5, 1:5.333, 1:5.6, 1:6, 1:1.67, 1:7 , 1:8, 1:8.235, 1:8.75, 1:9.375;
  • 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%, further preferably less than or equal to 0.5%;
  • the residual tert-butanol content is less than or equal to 1.75% by mass, preferably less than or equal to 1%, more preferably less than or equal to 0.5%;
  • the pH regulator is sodium bicarbonate, the content of which is 0.01-0.10 mg/cm 3 .
  • freeze-dried preparation essentially consists of the compound of the following formula and excipients:
  • the drug loading amount of compound I-1 in the freeze-dried preparation is 6.66, 7.77, 8.33, 8.88, 9.44, 11.11, 13.88, 16.66, 22.22, 27.77 mg/cm 3 , or the drug loading amount of compound I-1 in the freeze-dried preparation 5.45, 6.36, 6.82, 7.27, 7.73, 9.09, 11.36, 13.64, 18.18, 22.73 mg/cm 3 ;
  • the excipient is sucrose or mannitol
  • the mass ratio of compound I-1 to the excipients 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.
  • Containing the compound of the following formula I and excipients means that in addition to the unavoidable residual water and tert-butanol, the lyophilized preparation can detect the compound containing I, excipients and the above-mentioned residual environmental substances, in addition , may also contain other excipients.
  • the lyophilized preparation can only detect the compound containing I, excipients and the above-mentioned residual environmental substances, except Besides, it does not contain other substances.
  • the present invention provides a freeze-dried preparation, which is prepared through a freeze-drying process using the above-mentioned series of solutions for preparing freeze-dried preparations with high drug loading.
  • the pharmaceutical formulation and its freeze-dried powder can be stored in containers commonly used in the pharmaceutical field, which may include: plastic containers or glass containers, such as standard USPI type borosilicate glass containers.
  • the container used may be a syringe or a vial.
  • the present invention also provides a preparation unit package filled with the above-mentioned freeze-dried preparation:
  • the freeze-dried preparation is contained in a closed container with a volume of 1000ml, the volume of the freeze-dried preparation is 1/5 to 2/3 of the volume of the closed container, preferably 1/3 to 1/2, and the content of the compound of formula I is 4000-16500mg; or
  • the freeze-dried preparation is contained in a closed container with a volume of 500ml, the volume of the freeze-dried preparation is 1/5 to 2/3 of the volume of the closed container, preferably 1/3 to 1/2, and the content of the compound containing formula I 2000-8000mg; or
  • the freeze-dried preparation is contained in a closed container with a volume of 250ml, the volume of the freeze-dried preparation is 1/5 to 2/3 of the volume of the closed container, preferably 1/3 to 1/2, and the content of the compound containing formula I 1000-4000mg; or
  • the freeze-dried preparation is contained in a closed container with a volume of 100ml, the volume of the freeze-dried preparation is 1/5 to 2/3 of the volume of the closed container, preferably 1/3 to 1/2, and the content of the compound of formula I is 400-2000mg; or
  • the freeze-dried preparation is contained in a closed container with a volume of 50ml, the volume of the freeze-dried preparation is 1/5 to 2/3 of the volume of the closed container, preferably 1/3 to 1/2, and the content of the compound containing formula I 50-800mg; or
  • the freeze-dried preparation is contained in a closed container with a volume of 30ml, the volume of the freeze-dried preparation is 1/5 to 2/3 of the volume of the closed container, preferably 1/3 to 1/2, and the content of the compound containing formula I 150-600mg; or
  • the freeze-dried preparation is contained in a closed container with a volume of 25ml, the volume of the freeze-dried preparation is 1/5 to 2/3 of the volume of the closed container, preferably 1/3 to 1/2, and the content of the compound containing formula I 125-500mg; or
  • the freeze-dried preparation is contained in a closed container with a volume of 20ml, the volume of the freeze-dried preparation is 1/5 to 2/3 of the volume of the closed container, preferably 1/3 to 1/2, and the content of the compound of formula I is 100-400mg; or
  • the freeze-dried preparation is contained in a closed container with a volume of 18ml, and the volume of the freeze-dried preparation is 1/5 to 2/3 of the volume of the closed container, preferably 1/3 to 1/2, and contains the content of the compound of formula I 90-360mg; or
  • the freeze-dried preparation is contained in a closed container with a volume of 15ml, the volume of the freeze-dried preparation is 1/5 to 2/3 of the volume of the closed container, preferably 1/3 to 1/2, and the content of the compound of formula I is 75-300mg; or
  • the freeze-dried preparation is contained in a closed container with a volume of 10ml, the volume of the freeze-dried preparation is 1/5 to 2/3 of the volume of the closed container, preferably 1/3 to 1/2, and the content of the compound containing formula I 50-200mg; or
  • the freeze-dried preparation is contained in an airtight container with a volume of 8ml, and the volume of the freeze-dried preparation is 1/5 to 2/3 of the volume of the airtight container, preferably 1/3 to 1/2, containing the content of the compound of formula I 40-160mg; or
  • the freeze-dried preparation is contained in a closed container with a volume of 7ml, and the volume of the freeze-dried preparation is 1/5 to 2/3 of the volume of the closed container, preferably 1/3 to 1/2, containing the content of the compound of formula I 35-140mg; or
  • the freeze-dried preparation is contained in a closed container with a volume of 5ml, the volume of the freeze-dried preparation is 1/5 to 2/3 of the volume of the closed container, preferably 1/3 to 1/2, and the content of the compound containing formula I 25-100mg; or
  • the freeze-dried preparation is contained in a closed container with a volume of 3ml, the volume of the freeze-dried preparation is 1/5 to 2/3 of the volume of the closed container, preferably 1/3 to 1/2, and the content of the compound of formula I is 15-60mg; or
  • the freeze-dried preparation is contained in a closed container with a volume of 2ml, and the volume of the freeze-dried preparation is 1/5 to 2/3 of the volume of the closed container, preferably 1/3 to 1/2, containing the content of the compound of formula I 10-40mg; or
  • the freeze-dried preparation is contained in a closed container with a volume of 1.5ml, the volume of the freeze-dried preparation is 1/5 to 2/3 of the volume of the closed container, preferably 1/3 to 1/2, containing the compound of formula I
  • the content is 7.5-30mg.
  • small doses are suitable for minors in humans or animals of equivalent size, such as pigs, rats, dogs and other animals of small size and weight, and the indications are not limited.
  • the lyophilized preparation provided by the present invention is recommended to be administered by intravenous infusion, so it is necessary to reconstitute the medicinal solution, which generally uses physiological saline (0.9%) or glucose injection (5%).
  • a drug containing TH-302 Its solvent for intravenous injection is water, and the solute includes TH-302 crude drug, adjusting isotonicity reagent, mannitol or sucrose, tert-butanol and sodium bicarbonate, and described adjusting isotonicity reagent is selected from glucose, sodium chloride .
  • the present invention also provides a preparation method for preparing a solution of a high-loaded lyophilized preparation, comprising the following operations:
  • Operation 1 Weigh the raw material drug of TH-302 in the prescribed amount, add it to the aqueous solution of tert-butanol, and stir until it becomes clear to obtain the first solution;
  • Operation 2 Dissolve the prescribed amount of mannitol or sucrose in an appropriate amount of water, and stir until clear to obtain the second solution;
  • Operation 3 Mix the first solution and the second solution and finally add the remaining prescription amount of tert-butanol, then add an appropriate amount of water to set the volume to the predetermined volume, add the prescription amount of sodium bicarbonate, stir and mix evenly,
  • the content of the TH-302 bulk drug in the solution of the high-loaded lyophilized preparation is greater than 5 mg/ml and less than or equal to 500 mg/ml,
  • the volume percentage of tert-butanol relative to the high drug loading freeze-dried preparation is 1%-99%, or the content of tert-butanol in the solution is 7.85-777.15mg/ml,
  • sucrose or mannitol in the solution of the high drug loading freeze-dried preparation is 20-300 mg/ml
  • the mass ratio of TH-302 to sucrose or mannitol in the solution of the high-loaded lyophilized preparation is 1:(0.5-20),
  • the volume ratio of tert-butanol in the aqueous solution of tert-butanol is 30-90%
  • the content of sodium bicarbonate in the solution of the high drug loading freeze-dried preparation is 0.01-0.10 mg/ml.
  • the present invention herein provides a process for the preparation of lyophilized formulations.
  • a method for preparing a high-loading freeze-dried preparation comprising the following operations:
  • the above-mentioned liquid medicine is placed in a freeze-drying system for pre-freezing.
  • the pre-freezing process includes a period of time at 0°C and a period of time at -20 to -55°C;
  • One-time drying after pre-freezing is completed, the temperature is raised to -10 to 10°C and kept for a period of time, and the vacuum is maintained for drying;
  • the temperature is raised to 20 to 40°C and kept for a period of time, and the vacuum is maintained for drying.
  • freeze-dried preparation is obtained by directly filling the solution into the freeze-dried bottle (vial), and directly freeze-dried through the freeze-drying equipment, so there is no sub-packaging link, that is, the unit package is the corresponding freeze-dried bottle Packaging, the specifications of the packaging are closely related to the specifications of the freeze-drying bottle.
  • the numbers appearing in this application all have an error of ⁇ 10%, that is, -10% and +10% of the numbers should be regarded as within the range of numbers recorded in this application, that is, as appears in this application, "the content is greater than 5mg/ml but less than or equal to 500mg/ml", in fact, if the test results are greater than 4.5mg/ml but less than or equal to 550mg/ml, it should be taken for granted that it is equivalent to the above range.
  • Fig. 1 is the solubility curve of the tert-butanol aqueous solution of different mass ratios to TH-302;
  • Figure 2 shows the stability curves of lyophilized preparations prepared with 11 excipients within 5 days, which are distinguished by the circle on the far right. From top to bottom, they are PEG2000, P188, SBECD, mannitol, DSPE-MPEG2000, fructose, Trehalose, PVPK12, sucrose, maltose, lactose;
  • Fig. 3 is the stability curve of the lyophilized preparation prepared by sucrose and mannitol as excipients within 10 days, the 4 curves in the upper part of the figure are the curves of the mannitol prescription, and the 4 curves in the lower part are the curves of the sucrose prescription;
  • Fig. 4 is the stability curve of 100mg/ml sucrose, 80mg/ml mannitol prepared as excipient freeze-dried sample at high temperature 40 °C and room temperature 25 °C, the abscissa is the number of days, and the ordinate is the percentage purity of HPLC;
  • Figure 5 is a photo of the crystallization at the bottom of the liquid sample placed in a 2-°C environment, and the label has been coded and covered;
  • Figure 6 is a photo of samples of 4 batches of freeze-dried preparations, from left to right they are batches 01, 02, 03, and 04, and the labels have been coded and covered;
  • Figure 7 is a comparison photo of adding the solution and standing in the 40ml 5% glucose solution reconstitution experiment of 4 batches of freeze-dried preparation samples.
  • the picture on the left is the photo after adding the solution, and the right is the photo after adding the solution and standing. From left to right in the photo are batches 01, 02, 03, and 04, and the labels have been coded and covered;
  • Figure 8 is a comparison photo of adding the solution and standing in the 50ml 5% glucose solution reconstitution experiment of 4 batches of freeze-dried preparation samples.
  • the picture on the left is the photo after adding the solution, and the right is the photo after adding the solution and standing. From left to right in the photo are batches 01, 02, 03, and 04, and the labels have been coded and covered;
  • Figure 9 is a photo of samples of 7 batches of freeze-dried preparations, from left to right they are batches 01 to 07, and the labels have been coded and covered;
  • Fig. 10 is a schematic diagram of placing vials on the shelf of the experimental lyophilizer
  • Fig. 11 is the photo of freeze-dried preparation sample
  • Figure 12 is a photo of a sample with "neck wrapping" in the freeze-dried preparation, and the label has been coded and covered.
  • a patient and “individual” are used interchangeably and refer to a mammal in need of cancer treatment.
  • the patient is a human.
  • the patient is a human being diagnosed with cancer.
  • a "patient” or “individual” may refer to a non-human mammal, such as a non-human primate, dog, cat, rabbit, pig, mouse, for screening, characterization and evaluation of drugs and therapies or rats.
  • Prodrug refers to a compound (or drug) that, upon 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.
  • Prodrugs are chemically modified relative to drugs in such a way that they are less or inactive relative to the drug, but chemically modified such that the corresponding drug is produced by metabolism or other biological processes after administration of the prodrug.
  • Prodrugs may have altered metabolic stability or delivery characteristics, fewer side effects or toxicity, or improved flavor relative to the active drug.
  • Prodrugs can be synthesized using reactants other than the corresponding drug.
  • Treating or “treating a patient” refers to administering, using or administering to a patient a therapeutically effective amount of a drug relevant to the present invention.
  • administering or “administering” "using" a drug to a patient refers to direct administration or administration (which may be administered or administered to the patient by a medical professional or may be self-administered or administered) and/or indirect administration or administration, which may prescribe the drug the behavior of.
  • direct administration or administration which may be administered or administered to the patient by a medical professional or may be self-administered or administered
  • indirect administration or administration which may prescribe the drug the behavior of.
  • a physician who instructs a patient to self-administer or administer a drug and/or provides a prescription for the drug to the patient is administering or administering the drug to the patient.
  • a "therapeutically effective amount" of a drug means that when administered or administered or used to a patient with cancer, it will have a desired therapeutic effect (such as alleviation, improvement, remission or elimination of the clinical manifestations of one or more cancers in the patient) amount of drug.
  • a therapeutic effect does not necessarily occur through the administration or administration of one dose, and may only occur after the administration or administration of a series of doses. Thus, a therapeutically effective amount may be administered or administered in one or more doses.
  • Treatment of a condition or patient refers to steps taken to obtain beneficial or desired results (including clinical results).
  • beneficial or desired clinical outcomes include, but are not limited to, alleviation or amelioration of one or more symptoms of cancer; reduction in extent of disease; delay or slowing of disease progression; amelioration, remission or stabilization of disease state; or other beneficial outcomes.
  • treatment of cancer can result in a partial response or stable disease.
  • Tumor cell refers to a tumor cell of any appropriate species (eg, a mammal such as murine, canine, feline, equine or human).
  • the moisture content was determined by the Karl Fischer KF method, and the instrument used was a Mettler V10S Karl Fischer moisture meter.
  • the content of residual solvent tert-butanol was determined by gas chromatography GC.
  • the instrument used was Agilent 8860 gas chromatograph equipped with 7696A automatic headspace sampler and FID detector.
  • the chromatographic column was a capillary column filled with DB-624. ; GC test parameters are carrier gas as N 2 ; inlet temperature is 150°C; detector temperature is 200°C; split ratio is 20:1; Raise the temperature to 240°C, run at 60°C for 5 minutes; headspace balance at 85°C for 20 minutes;
  • the content and concentration of TH-302 are determined by high-performance liquid chromatography (HPLC).
  • HPLC high-performance liquid chromatography
  • the instrument used is Thermo Vanquish high-performance liquid chromatography.
  • the chromatographic column YMC pack AQ C18 4.6mm ⁇ 250mm, 5 ⁇ m.
  • the HPLC method specifically Example 2.An Ethanol Formulation of TH302).
  • Solubility is measured by the saturated solution method, that is, the solid TH-302 is directly put into the solvent until insoluble matter or turbidity appears, after a period of clarification, it is filtered directly, and the clear filtrate is taken to directly detect the concentration of TH-302.
  • the obtained concentration is is the TH-302 solubility of the solvent system, see Table 1, Table 2, and Table 3 for specific solubility data.
  • TH-302 concentration concentration (mg/ml)
  • HPLC method specifically Example 2.An Ethanol Formulation of TH302
  • WO2008083101A1 applied by Threshold Company
  • the external standard method is used for quantification.
  • the concentration is too high, the corresponding clear solution should be diluted to the extent that HPLC can accurately and quantitatively detect.
  • the mass ratio of tert-butanol in the data in Table 3 is used as the X-axis, and the solubility of tert-butanol-water mixed solvent to TH-302 is used as the Y-axis to obtain the relationship curve in Figure 1: the solubility of the bulk drug in the aqueous solution is low; With the increase of the concentration of tert-butanol in the tert-butanol aqueous solution, the solubility of the bulk drug increases; increases, the solubility decreases.
  • Nonionic polymer surfactants are selected: PVPK12 (polyvinylpyrrolidone with a molecular weight of 5500), PEG2000 (polyethylene glycol 2000), P188 (Poloxamer is polyoxyethylene polyoxypropylene ether block copolymer, The grade is 188);
  • SBECD sulfobutyl ether- ⁇ -cyclodextrin
  • DSPE-MPEG2000 cultured phosphatidylethanolamine
  • API TH-302
  • TBA tert-butanol
  • water water.
  • Freeze-dried powder state Except for water as the solvent, all other solvent groups successfully obtained white freeze-dried powder. However, the glycerol group turned into an oily substance when the temperature of the lyophilizer was raised to 0°C.
  • sucrose, mannitol, lactose, maltose, fructose, PVPK12, trehalose, DSPE-MPEG2000, SBECD, PEG2000, and P188 prescription groups were subjected to high-temperature accelerated stability tests.
  • Table 5 High temperature 40°C accelerated stability test data of lyophilized formulations with different excipients.
  • lyoprotectant for injection should take into account good stability, and routine use will not affect the efficacy, DMPK and toxicology. Analyzing the curve in Figure 2, PEG2000 is not routinely used, and P188/SBECD/DSPE-MPEG2000 may have an impact on drug efficacy, DMPK and toxicity. Generally speaking, mannitol is ideal, and sucrose also has development potential (optimized dosage and freeze-drying conditions Potentially improved stability and reduced tert-butanol residues).
  • the mannitol and sucrose samples were tested for purity by HPLC after being placed on the 10th day, and the results were plotted as a curve, as shown in FIG. 3 .
  • the measured pH of the sucrose prescription liquid is 4.24. Use 10 ⁇ L of NaHCO 3 injection to adjust the pH to 6.43. Add another 10 ⁇ L to adjust the pH to 7.13.
  • the measured pH of the liquid prescribed for mannitol was 4.62, and the pH was adjusted to 7.31 using 12 ⁇ L of NaHCO 3 injection.
  • the lyophilized preparation samples were placed in incubators at 25°C and 40°C, and the HPLC purity was tested on the corresponding days respectively. The results are shown in Table 7 below.
  • Table 7 Stability test data of freeze-dried samples prepared with 100mg/ml sucrose and 80mg/ml mannitol as excipients at high temperature 40°C and room temperature 25°C
  • HPLC purity in Table 7 is used as the Y coordinate, and the time is used as the X coordinate to make a stability curve of 100 mg/ml sucrose and 80 mg/ml mannitol, as shown in FIG. 4 .
  • Table 8 Stability test data of freeze-dried samples prepared with 100mg/ml sucrose and 80mg/ml mannitol as excipients after reconstitution at room temperature 25°C
  • Table 9 pH value and osmotic pressure data of freeze-dried samples prepared with 100mg/ml sucrose and 80mg/ml mannitol as excipients after reconstitution with D5W at room temperature 25°C
  • Table 10 Preparation scheme of lyophilized medicinal solution for investigating the influence of API concentration and pH on sample redissolution
  • Table 11 Content of 01 intermediate drug solution after storage at 2-8°C, content of supernatant after storage, and HPLC purity
  • the above subpackaged preparations are freeze-dried in a vacuum freeze dryer.
  • the freeze-drying conditions were optimized several times, as shown in Table 12 below. After freeze-drying, vacuum stopper and cover. A lyophilized formulation was obtained, as shown in Figure 6.
  • the above-mentioned freeze-dried preparation was reconstituted with 40ml and 50ml of 5% glucose, and the results are shown in Table 13 below.
  • the API concentration has an appropriate range. After many tests, it is found that 5-160mg/ml is more suitable, or the drug solution obtained by further using 8-50mg/ml is stable and the quality of the subsequent freeze-dried preparation is better.
  • Table 14 The preparation scheme of lyophilized medicinal solution to investigate the influence of API concentration, lyoprotectant concentration and tert-butanol concentration on the residual solvent of lyophilized powder
  • the preparation was injected into D5W for reconstitution, batches 01 to 07 were easy to reconstitute, and the solution was clear.
  • the lyophilized preparation samples were placed in a 40°C incubator, and the HPLC purity was tested on corresponding days, and the results are shown in Table 15 below.
  • Table 15 HPLC purity data (%) of 5 batches of freeze-dried samples at a high temperature of 40°C
  • Table 16 Residual tert-butanol content data of 7 batches of freeze-dried samples determined at 0 days
  • TBA was not added in 05.
  • 01 middle and 01 lower represent the freeze-dried preparation samples of the same batch of liquid medicine after being freeze-dried on different plates (middle layer and lower layer) of the freeze dryer.
  • Table 17 Residual water content data of 7 batches of freeze-dried samples measured at 0 days
  • the API concentration has little relationship with the residual solvent content in the lyophilized formulation, and the lyoprotectant concentration has little relationship with the residual solvent content in the lyophilized formulation.
  • API concentration, lyoprotectant concentration, and tert-butanol concentration all affect the residual solvent content in the preparation after lyophilization: in comparison, in the lyophilized preparation obtained by using mannitol as a lyophilization excipient (lyoprotectant), Solvent residues are low, and in the high temperature accelerated stability test, the stability of mannitol formulation is better than that of sucrose formulation.
  • 150mg/bottle Fill 16 mL in batch 01, get 6 bottles; fill 16 mL in batch 02, get 6 bottles;
  • the above subpackaged preparations are freeze-dried in a vacuum freeze dryer.
  • the parameters in Table 12 were used for freeze-drying, and after the freeze-drying was completed, the vacuum plug was pressed and the cap was crimped. A freeze-dried formulation was obtained.
  • the lyophilized preparations were all white blocky solids.
  • Atmospheric pressure means opening the aluminum cap and rubber stopper of the vial containing the lyophilized preparation to communicate with the outside world and then injecting purified water for reconstitution; normal pressure not marked means directly injecting purified water 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) to redissolve.
  • Table 21 Reconstitution results of 04 batches of samples with different filling volumes stored at 40°C for 10 days
  • the lyophilized preparation obtained with lower API concentration and lower mannitol content is easier to reconstitute, which suggests that simply increasing the API concentration and the amount of mannitol to increase the drug loading of the lyophilized preparation will lead to Reconstitution is difficult, and appropriate API concentration and mannitol content must be selected to obtain a lyophilized drug solution with higher drug loading and easy reconstitution.
  • API 2Pre-dissolution Weigh the prescribed amount of API, add API to 70% tert-butanol aqueous solution under stirring, stir and dissolve, and record it as A solution.
  • Medicinal Solution-01 No pH adjustment; left at room temperature for 24 hours to measure pH 4.77.
  • Drug solution-02 add 200 ⁇ l of sodium bicarbonate, measure pH 6.60 (pH is 6.0-6.5). Leave it at room temperature for 24 hours to measure pH 5.20.
  • Solution-03 add 1000 ⁇ l of sodium bicarbonate, measure pH 7.20 (pH is at 7.0). Place at room temperature for 24 hours to measure pH 6.90 , respectively take the 0-hour liquid intermediate and place it at room temperature for 24 hours to detect the content and related substances.
  • Liquid medicine-01 batch was filled with 24.5 g, and 78 bottles were obtained, which was batch 01.
  • Liquid medicine-03 batches were filled with 24.5 g, and 78 bottles were obtained, which was batch 03.
  • Table 23 pH value data of different batches of medicinal solutions stored for different periods of time after pH adjustment
  • Sample state of freeze-dried preparation white blocky solid, the specific sample is shown in Figure 11, and the bottle neck of the sample bottle in the middle layer of the freeze dryer is surrounded by powder, as shown in Figure 12, which may be caused by improper operation during filling.
  • Table 24 Reconstitution detection data of lyophilized preparations located in different positions on the shelf in different batches after lyophilization of different pH liquid medicines
  • Table 25 Reconstitution detection data of lyophilized preparations located in different positions on the shelf in different batches after lyophilization of different pH liquid medicines
  • Table 26 HPLC purity data of API in the solution reconstituted with normal saline by sampling at different concentrations and at different time periods
  • the lyophilized preparation samples were placed in incubators at 40°C, 25°C, and 2-8°C, and the HPLC purity was tested at corresponding times. The results are shown in Table 27 below.
  • Table 27 Samples of lyophilized preparations were placed in incubators at 40°C, 25°C and 2-8°C, and HPLC purity data were detected at corresponding times
  • the quality index of the lyophilized preparation includes three main inspection indexes such as reconstitution, stability, and residual solvent, and through the above experiments, it can be found that the influencing factors include solvent (volume ratio of tert-butanol in tert-butanol-water mixed solvent), API concentration, type of excipients (mannitol, sucrose) and dosage, pH value of medicinal solution, etc., therefore, many multi-factor experiments were carried out, and the results are shown in Table 28.
  • Table 28 Example data of freeze-dried preparations of medicinal liquids with different volume ratios of tert-butanol, API concentrations, types and dosages of excipients, and pH values of medicinal liquids
  • TBA is tert-butanol.
  • Mannitol is mannitol.
  • sucrose sucrose
  • API is TH-302 API.
  • PBS is phosphate-buffered saline with a stable pH of around 7.4.
  • 30% TBA/70Mannitol/10API-pH7.0 in the prescription means 30% tert-butanol by volume, 70mg/ml mannitol, 10mg/ml API, and finally add NaHCO3 to adjust the pH to 7.0.
  • the preparation method is as above Example: 40% TBA/60Mannitol/10API means 40% tert-butanol by volume, 60mg/ml mannitol, 10mg/ml API, no pH adjustment (weak acidity), and so on.
  • the stability of the freeze-dried preparation is better and the reconstitution is easier.
  • the content of the compound of formula I in the solution is greater than or equal to 8mg/ml and less than or equal to 15mg/ml, such a content range will mean that the freeze-drying conditions are relatively mild, the freeze-drying cycle is shorter, and the content of the compound of formula I in the solution is greater than Or equal to 8mg/ml and less than or equal to 10mg/ml will further imply that the lyophilized preparation has commercial mass production value under commercially suitable lyophilization cycle and lyophilization low temperature conditions: inappropriate API content will lead to commercial mass production
  • the large-scale lyophilizers used in the world are broken bottles and powder sprayed due to uneven temperature, so the yield is lower than 90% of the commercial production acceptance.
  • Drying parameters such as pre-freezing at -80°C, lower absolute air pressure during freeze-drying, longer drying time and lower drying temperature, etc.
  • the volume ratio of tert-butanol is 30%-60% or the content of tert-butanol in the solution is 235.5-471mg/ml (under the condition that the density of tert-butanol is 0.785g/ml), the freeze-dried preparation is more stable Well, reconstitution is easier.
  • the excipients used are mannitol, PEG2000, P188, SBECD, DSPE- Freeze-dried preparations can be obtained from MPEG2000, sucrose or similar substances, but after comparison, the reasons for the freeze-dried preparation itself, such as whether it is routinely used and easy to buy commercially, whether it will affect the efficacy, DMPK and toxicity etc., considering that it is more appropriate to use sucrose and mannitol, the lyophilized preparations prepared by them have suitable solution stability (at least 24 hours at room temperature), lyophilized preparation stability, reconstitution and are easy to obtain products that meet the requirements of injection preparation excipients .
  • the content of sucrose and mannitol in the solution is 20-300mg/ml, such a range can meet the above requirements: the liquid medicine is stable, and it is not easy to precipitate during the pre-freezing stage or cooling stage of lyophilization.
  • the further preferred range is 40-100 mg/ml, which can obtain a reconstituted solution and a freeze-dried formulation with good stability. More preferably 60-70mg/ml, such a content range will mean that the freeze-drying conditions are relatively mild, and the freeze-drying cycle is in line with commercial production practice (not more than 10 days, that is, a production cycle of 240 hours).
  • too low mannitol/sucrose will lead to the inability to uniformly load the drug solution on the excipient skeleton after lyophilization, and even cause the freeze-dried powder cake to collapse or freeze-dry during the drying stage of the freeze-drying process Fail; and although too high mannitol/sucrose content can obtain the lyophilized preparation of better appearance and stability, but mean the content of the lyophilized preparation Chinese medicine TH-302 of unit packaging unit is too low, does not meet commercial sale and Use requirements, therefore a suitable ratio of mannitol/sucrose to drug is necessary.
  • the mass ratio of the compound of formula I in the solution to the excipient is 1: (0.5-20), such a ratio can meet the requirements of various other ingredients in the above items 1-4, and can make the prepared lyophilized preparation Have better outward appearance and stability; Preferably be 1:(2-12.5) can obtain the lyophilized preparation that better reconstitution is easy, more preferably 1:(5-10) prepared lyophilized preparation will have excellent Properties:
  • the freeze-dried preparation has good stability, easy reconstitution, mild freeze-drying conditions, and short freeze-drying cycle.
  • the filling volume of the liquid medicine in the vial also affects the appearance of the final freeze-dried powder cake (spraying powder or adhering to the bottleneck of the vial) and reconstitution through various factors, so the vial
  • the ratio of the volume of the filled liquid medicine to the volume of the vial must be appropriate.
  • the filling volume of the freeze-dried liquid medicine is 1/3 to 1/2 of the volume of the airtight container, and the corresponding freeze-dried
  • the volume of the preparation is preferably 1/3 to 1/2 of the volume of the closed container.
  • TH-302 sample was accurately weighed, placed in a transparent vial, added 6 ml of 5% glucose injection (D5W), and shaken vigorously for 2 minutes, as a solubility stock solution. Observation finds that the solubility stock solution has insoluble matter, the solubility stock solution is filtered and becomes clear, the filtered filtrate is placed at room temperature for 24 hours, the filtrate is still clear, the sample is properly diluted and detected by HPLC and the content of the solution is 7.25mg/ ml, this value is the saturation solubility of the drug in the D5W solution.
  • D5W 5% glucose injection
  • the concentration of the intravenous injection containing TH-302 should be within 0-7.25mg/ml.
  • the infusion formula during intravenous infusion Further research is needed to confirm.
  • the content of the compound of formula I in the solution is 5.55-177.77 mg/cm 3 in the drug solution of the drug solution after lyophilization;
  • the content of the compound of formula I in the solution is 8.88-55.55mg/cm 3 in the drug solution of the drug solution after lyophilization;
  • the content of the compound of formula I in the solution is 8.88-27.77 mg/cm 3 in the drug solution of the drug solution after lyophilization;
  • the content of the compound of formula I in the solution is 8.88-16.66 mg/cm 3 in the drug solution of the drug solution after lyophilization;
  • the drug solution with the compound of formula I in the solution at a content of 8-10 mg/ml has a powder cake drug loading of 8.88-11.11 mg/cm 3 after freeze-drying.
  • the drug loading of the corresponding freeze-dried powder cake is 6.66, 7.77, 8.33, 8.88, 9.44, 11.11, 13.88 , 16.66, 22.22, 27.77 mg/cm 3 .
  • the content of the compound of formula I in the solution is 4.55-145.45 mg/cm 3 in the drug solution after lyophilization;
  • the content of the compound of formula I in the solution is 8-50 mg/ml in the drug solution, and its powder cake drug loading after freeze-drying is 7.27-45.45 mg/cm 3 ;
  • the content of the compound of formula I in the solution is 7.27-22.73 mg/cm 3 after freeze-drying of the medicine liquid whose content is 8-25 mg/ml;
  • the content of the compound of formula I in the solution is 7.27-13.64 mg/cm 3 after freeze-drying of the medicine liquid whose content is 8-15 mg/ml;
  • the drug solution containing the compound of formula I in the solution is 8-10 mg/ml, and the drug loading amount of the powder cake after freeze-drying is 7.27-9.09 mg/cm 3 .
  • the drug loading of the corresponding freeze-dried powder cake is 5.45, 6.36, 6.82, 7.27, 7.73, 9.09, 11.36 , 13.64, 18.18, 22.73 mg/cm 3 .

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Abstract

本发明提供冻干制剂溶液及冻干制剂、方法和用途。

Description

冻干制剂溶液及冻干制剂、方法和用途 技术领域
本发明涉及TH-302的冻干制剂开发，属于医药制剂技术领域。
背景技术
TH-302为2-硝基咪唑类的前药，是由Threshold制药公司研究人员于2006年(WO2007002931A2，Phosphoramidate alkylator prodrugs)设计合成的具有高细胞毒性的选择性低氧激活型DNA烷化剂。可在肿瘤低氧区或遇酸活化而转化为具烷化剂活性的二溴异磷酰胺氮芥，但在常氧或正常pH条件下几乎无活性。
Threshold制药公司研究人员于2007年(WO2008083101A1，Phosphoramidate alkyla tor prodrugs for the treatment of cancer)设计开发了初步的冻干制剂配方和注射液配方，并在I期临床试验中给药：
将TH-302(100mg)和蔗糖(1g)的溶液(20mL)加入冻干小瓶中并冻干以产生TH-302的冻干单位剂型，载药量不到5mg/cm 3。为了人类施用的目的，将单位剂型溶解在5％葡萄糖注射液中，并向患者施用适量的该溶液；
将TH-302溶解在无水乙醇中以产生5％TH-302的药学上可接受的液体制剂。如本文所用，TH-302的5％溶液在100mL的溶剂(例如，乙醇)中含有5g TH-302；
后续人类患者TH-302的I期临床试验给药方案使用冻干制剂,在100mL玻璃小瓶中制备注射用TH-302冻干制剂，载药量为100mg/100ml，在2-8℃的受控条件下储存，使用时注射到冻干制剂瓶中250mL的5％葡萄糖注射液，并通过输注泵在30分钟内静脉滴注。
Threshold制药公司研究人员于2009年(WO2010048330A1，Treatment of cancer using hypoxia activated prodrugs)设计开发了注射液配方：
液体制剂，含有50mg/ml至约300mg/ml的TH-302，非离子表面活性剂(如吐温80)，载体乙醇，还可以含有二甲基乙酰胺。
临床试验从2007年开始，一直持续到现在，涵盖了多种实体瘤和血液癌，并且用药方案也是多样性的：不同剂量的TH-302单用，或与其他癌症治疗药物联用。目前在美国登记有27项TH-302作为治疗药物来治疗各种癌症、肿瘤的临床试验(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)这些临床试验显示TH-302是一个广谱的候选抗癌药物。
经过上述的I/II期临床试验，Threshold公司发现TH-302的治疗各种适应症有效剂量为(WO2012135757A2，Methods for treating cancer)：
120mg/m 2至460mg/m 2的日剂量来静脉注射给药；
480mg/m 2至大约670mg/m 2或者例如575mg/m 2的周剂量来静脉注射给药。
根据上述有效剂量可知，对于一个普通的人而言(身高175cm，体重75kg)其对应的等效体表面积BSA(m 2)＝([身高(cm)×体重(kg)]/3600) 1/2＝1.90，则对应的剂量为228-1273mg！此种情况下，如果使用载药量不到5mg/cm 3的冻干制剂(将含有TH-302 100mg和蔗糖1g的水溶液20mL加入50ml冻干小瓶中冻干以产生TH-302的冻干单位剂型，载药量不到5mg/cm 3)则需要至少3瓶，如果是较大的剂量则需要13瓶，这种情况不方便临床使用，同时患者用药成本过高。
为此在后续大部分的II/III期临床中，Threshold公司使用的是浓缩注射液(WO2015013448A1，Treatment of pancreatic cancer with a combination of a hypoxia-acti vated prodrug and a taxane)：
用于临床试验的TH-302(用于施用溶液的浓缩物)是TH-302的无菌液体制剂。用70％无水乙醇、25％二甲基乙酰胺和5％聚山梨酯80配制TH-302。它由发起人提供，在具有橡胶塞和flip-off封口的10mL玻璃小瓶中。TH-302药物产品是澄清的、无色至淡黄色的溶液，基本上不含可见颗粒。对于标称总量为650mg的TH-302，每个单次使用的小瓶含有标称填充体积为6.5mL的TH-302药物产品(相当于100mg/mL)，并且被清楚地贴上标签，其公开了批号、施用途径、所需的储存条件、发起人的名称和适用的规定所要求的适宜的预警标记。在施用前需要按照药房手册进行稀释。
在施用前用可商购获得的5％葡萄糖水溶液稀释至总体积为500mL(对于≥1000mg的总剂量为1000mL)施用，以获得所需的终浓度。用不含邻苯二甲酸二(2-乙基己基)酯(无DEHP)的5％葡萄糖水溶液制备每剂TH-302，并使用不含DEHP的静脉输液施用装置静脉滴注。
显然在临床中使用载药量为100mg/ml的浓缩注射液比使用载药量不到5mg/cm 3的冻干制剂方便：前者10ml规格小瓶就可以满足大部分患者的用药要求，而后者要满足大部分患者的用药要求需要常用的100ml规格冻干制剂小瓶13瓶！
然而，Threshold公司发现上述载药量为100mg/ml的浓缩注射液(用70％无水乙醇、25％二甲基乙酰胺和5％聚山梨酯80配制浓缩的TH-302注射液)含有大量的二甲基乙酰胺，这些增加药物溶解度、提高注射液稳定性的辅料在输注到人体后容易引发过敏(WO2015013448A1，Treatment of pancreatic cancer with a combination of a hypoxia-activated prodrug and a taxane)：
已经观察了TH-302的施用反应(主要由二甲基乙酰胺引起)。这些反应的特征在于唇肿胀和荨麻疹，其对类固醇和抗组胺药治疗有响应。推荐施用前在止吐方案中包括类固醇例如地塞米松(或等效物)。超敏反应的症状和迹象包括发热、肌痛、头痛、疹、瘙痒、荨麻疹、血管性水肿、胸部不适(chest discomfort)、呼吸困难、咳嗽、发绀和低血压。如果反应的性质和严重性需要终止治疗，则应当确定反应可能是或可能不是免疫球蛋白E介导的过程。如果存在提示过敏症或过敏样反应的症状例如上气道堵塞或低血压，则研究人员应当酌情考虑使用抗组胺药(例如苯海拉明25-50mg口服、肌内或缓慢i.v.，或等效物)和低剂量类固醇(例如氢化可的松，100mg i.v.，或等效物)治疗。如果事件清楚地是过敏症，则应当考虑肾上腺素(1/1000，皮下给予0.3-0.5mL，或等效物)以及标准治疗方法。在支气管痉挛的情况下，应当考虑吸入β-激动剂。还可以使用抗组胺药和低剂量的类固醇治疗特异质反应，视其严重性而定。应当按照类似方式评价和治疗对施用TH-302的反应。对于对TH-302的所有反应，研究人员应当咨询医疗监护者以确定用于未来治疗的适宜的行动过程。
高浓度的浓缩TH-302注射剂解决了冻干制剂载药量过低的问题，但由于使用上述可能引发不良反应的辅料，会在临床试验中出现相关的不良反应，增加患者的用药风险；
现有开发的冻干制剂使用水和蔗糖溶液溶解TH-302进行冻干，不含有其他辅料，但由于冻干前溶液的药物含量太低，不能得到符合临床用药和后续商业化生产、销售使用的高载药量冻干制剂。
上述现实矛盾的解决需要相关技术人员开发高载药量的TH-302冻干制剂及用于冻干制剂的溶液。
发明内容
本申请的发明人经过多次实验和不断优化，提出了新的高药物浓度的用于生产TH-302等类似药物冻干制剂的溶液处方及相关冻干制剂、方法。
为了便于理解本发明的实质，以下简述发明人的探索过程。
为了筛选溶剂组合、辅料组合来配制高浓度的
Figure PCTCN2022115176-appb-000001
(即TH-302)溶液，发明人首先想到的是对Threshold公司开发的的载药量为100mg/ml的浓缩注射液：用70％无水乙醇、25％二甲基乙酰胺和5％聚山梨酯80配制浓缩的TH-302注射液进行改进：
直接使用无水乙醇和水作为溶剂，并添加蔗糖，结果要么无法冻干，只能得到粘稠的油状物；要么溶液的药物含量提高有限，不满足要求，为此放弃了单纯的乙醇+水方案；
直接使用无水乙醇和水作为溶剂，并添加蔗糖或其他赋形剂，并调节加入增溶剂(PEG、Tween、Span等)的种类和数量，结果发现少量添加提高溶解度但很有限，不能满足高载药量要求；
考虑到pH可能影响溶解度，对TH-302在各种单一常规缓冲溶液中溶解度也进行了研究，发现溶液pH值对TH-302溶解度的影响很小，调节pH值并不能提高水溶液的溶解度，不同pH水溶液、不同添加剂水溶液的TH-302溶解度数据如下表1。
表1：不同pH水溶液、不同添加剂水溶液的TH-302溶解度数据
Figure PCTCN2022115176-appb-000002
注释：1％乙醇水溶液、20％乙醇水溶液、1％N,N-二甲基乙酰胺水溶液、1％聚乙二醇水溶液、1％吐温80水溶液均为体积比。
多番实验尝试证实使用Threshold的单一乙醇、不同pH水溶液、乙醇+水作为溶剂无法开发得到满足要求的高溶解度溶液用来冻干制备高载药量TH-302冻干制剂。
发明人经过多次尝试发现TH-302在单一的水中溶解度较小，在单一的叔丁醇中溶解度也较小，却意外发现TH-302在两者的混合溶剂中溶解度却极大的提升，并且溶解度与两者的混合比有极大的关系，不同叔丁醇+水、叔丁醇+乙醇混合溶剂对TH-302的溶解度数据见下表2。
表2：不同叔丁醇+水、叔丁醇+乙醇混合溶剂对TH-302的溶解度数据
混合溶剂 溶解度(mg/ml)
叔丁醇 8.98
95％叔丁醇水溶液 32.33
90％叔丁醇水溶液 76.41
80％叔丁醇水溶液 88.54
40％叔丁醇水溶液 >40
20％叔丁醇水溶液 10
95％叔丁醇/5％乙醇 9.98
90％叔丁醇/10％乙醇 10.72
注释：表2中的混合溶剂的比例为体积比，TH-302为商业购买。
叔丁醇为无色结晶，易过冷，在少量水存在时则为液体。有类似樟脑的气味，有吸湿性。中文名称2-甲基-2-丙醇、叔丁醇、三甲基甲醇等。熔点为25.7℃，因此在常温下为无色透明液体或无色结晶。
总体来说，其特点如下：
1、凝固点高。纯的叔丁醇在室温下(25℃)就有可能出现结晶，与水混合后也可以在零下几度冻结，在现有的冻干机中都可以完全冻结。
2、叔丁醇的蒸汽压较高。蒸汽压高有利于升华，节省冻干时间。
3、叔丁醇与水可以任意比例混合。这一点极为重要，可以增大一些脂溶性药物在水中的溶解度，同时对一些水溶液中不稳定的药物，加入适量的叔丁醇可以抑制药物的分解，增强药物的稳定性。
4、叔丁醇容易冻干，在制剂中残留量低。在冻干过程中，大部分叔丁醇可在一次干燥阶段升华，在制剂中残留量很低。
5、叔丁醇自身在冻结中形成针状结晶，能改变溶质的结晶方式，利于升华。而当少量的叔丁醇加入到水中形成叔丁醇-水共溶剂后，可以改变水的结晶状态，在冻结过程中形成针状结晶，具有大的表面积,同时冰晶升华后留下了管状通道，使水蒸气流动阻力大大减小，升华速率显著提高，因此可用叔丁醇来加快冷冻干燥过程中的传质过程。
综上，可知叔丁醇+水作为溶剂不仅可以配制高浓度的式I的化合物的溶液，而且，叔丁醇本身的性质也适合作为冻干的辅料。
根据表2的叔丁醇-水混合溶剂结果，可推测TH-302在水溶液中溶解度较低；随着叔丁醇水溶液中叔丁醇浓度的增加，原料药溶解度不断增加；推测在70％叔丁醇水溶液(V/V)中溶解度最高，然后随着叔丁醇水溶液中叔丁醇浓度的增加，溶解度降低。
由此发明人提出了本发明的叔丁醇+水作为溶剂并添加适当的赋形剂能得到高浓度的溶液的方案，并基于此处方开发高载药量的TH-302及其类似化合物的冻干制剂。
进一步的进行了不同叔丁醇体积配比溶剂对TH-302的溶解度实验，得到以下的进一步溶解度数据，如下表3所示。
表3：不同叔丁醇质量配比的叔丁醇-水混合溶剂对TH-302的溶解度数据
Figure PCTCN2022115176-appb-000003
注释：表3中的混合溶剂的比例为质量比，TH-302为申请人自行少量合成。
基于以上的初步实验，本发明提供以下含有TH-302类似化合物的高浓度溶液。
溶液，含有下式I的化合物以及水和叔丁醇：
Figure PCTCN2022115176-appb-000004
其中,R各自独立地选自H、-CH 3、-CH 2CH 3，X各自独立地选自Br、Cl、OMs、OTs，
水和叔丁醇作为混合溶剂，
式I的化合物在溶液中的含量大于或等于1mg/ml而小于或等于500mg/ml。
进一步的，还提供用于制备高载药量冻干制剂的溶液，含有下式I的化合物以及水和叔丁醇、赋形剂：
Figure PCTCN2022115176-appb-000005
其中,R各自独立地选自H、-CH 3、-CH 2CH 3，X各自独立地选自Br、Cl、OMs、OTs，
水和叔丁醇作为混合溶剂，
式I的化合物在溶液中的含量大于或等于5mg/ml而小于或等于500mg/ml，
优选的，式I化合物在溶液中的含量大于或等于5mg/ml而小于或等于160mg/ml，
优选的，式I化合物在溶液中的含量大于或等于8mg/ml而小于或等于50mg/ml，
优选的，式I化合物在溶液中的含量大于或等于8mg/ml而小于或等于25mg/ml；
更优选的，式I化合物在溶液中的含量大于或等于8mg/ml而小于或等于15mg/ml，
进一步优选的，式I化合物在溶液中的含量大于或等于8mg/ml而小于或等于10mg/ml。
由于水与叔丁醇可以任意比混溶，为了提高TH-302等类似化合物的溶解度，选定叔丁醇相对于所述溶液体积百分比为1％-99％，优选为5％-95％，更优选为30％-60％；
或
所述溶液中叔丁醇含量为7.85-777.15mg/ml，选为39.25-745.75mg/ml，更优选为235.5-471mg/ml。
这里叔丁醇相对于所述溶液体积百分比为1％-99％与所述溶液中叔丁醇含量为7.85-777.15mg/ml是对应关系，叔丁醇相对于所述溶液体积百分比为1％对应溶液中叔丁醇含量为7.85mg/ml，换算系数即为叔丁醇的密度，这里申请人使用的叔丁醇的密度为0.785g/ml，实际上不同厂家产品在不同温度下的密度都是不同的，一般在0.775-0.786g/ml之间。
本发明用于制备高载药量冻干制剂的溶液中含有至少一种赋形剂。
药学上可接受的赋形剂是指可有助于原料药在制剂中的稳定性的添加剂或载体，在冻干制剂的制备过程中，向用于冻干的溶液中添加赋形剂或冻干保护剂是必须的。
有些药物溶液能成功地进行冷冻真空干燥，有些则会在冻干后迅速塌陷或熔融成油状物。为了使某些药物溶液能成功地进行冻干，得到稳定的冻干制剂，需要加入一些不与药物反应的辅料，这些辅料本身不会在冻干程序的升华阶段被升华，而是能直接被冻干成为骨架，起到赋予形态的作用，而药物则可以直接被吸附或被填充在骨架的空隙中；或者改善冻干产品的溶解性和稳定性，或使冻干产品有美观的形状等，需要在液体制剂中加入一些附加物质，它们统称为冻干保护剂，有时也叫填充物、填充剂、赋形剂、缓冲物、基础物、骨架等。一般而言，冻干保护剂对药物溶液必须是化学惰性的。
按照冻干保护剂的化学成份可以分为下列几类：
复合物：脱脂乳、明胶、蛋白质及水解物、多肽、酵母、肉汤、糊精、甲基纤维素、血 清、蛋白胨等。
盐类：硫代硫酸钠、乳酸钙、谷氨酸钠、氯化钠、氯化钾、硫酸钠、醋酸氨、氯化铵等。
糖类：蔗糖、乳糖、麦芽糖、葡萄糖、棉子糖、果糖、己糖等。
醇类：山梨醇、乙醇、甘油、甘露醇、肌醇、木糖醇等。
酸类：柠檬酸、磷酸、酒石酸、氨基酸、乙二氨四乙酸(EDTA)等。
碱类：氢氧化钠、碳酸氢钠等。
聚合物：葡聚醣、聚乙二醇、聚山梨醇酯、PVP、泊洛沙姆等。
其他：维生素C、维生素E、维生素K、硫脲等。
上述聚合物的聚合度(分子量)是广泛的。以聚山梨醇酯和聚乙二醇为例。
聚山梨醇酯的平均分子量可以在约500g/mol至约1900g/mol，优选在约800g/mol至约1600g/mol，更优选在约1000g/mol至约1400g/mol的范围内。聚山梨醇酯的非限定性例子包括：聚山梨醇酯-20、聚山梨醇酯-21、聚山梨醇酯-40、聚山梨醇酯-60、聚山梨醇酯-61、聚山梨醇酯-65、聚山梨醇酯-81、聚山梨醇酯-85和聚山梨醇酯-120。优选的聚山梨醇酯包括聚山梨醇酯-20、聚山梨醇酯-80和它们的混合物。
聚乙二醇(PEG)的平均分子量在约200g/mol至约600g/mol，优选在约200g/mol至约500g/mol，更优选在约200g/mol至约400g/mol的范围内。PEG的非限定性例子包括：PEG200、PEG300、PEG400、PEG540和PEG600。
泊洛沙姆，其通式为HO(C 2H 4O) a(C 3H 6O) b(C 2H 4O) cH，其中a和c为2-130，b为15-67。含聚氧乙烯为81.8±1.9％，为聚氧乙烯聚氧丙烯醚嵌段共聚物，其具有不同的牌号:泊洛沙姆182、泊洛沙姆184、泊洛沙姆188、泊洛沙姆407,其对应不同分子量的聚合物,如泊洛沙姆188其分子量为7680-9510。
冻干保护剂的作用较多，具体总结如下：
细菌、病毒等微生物需要在特定的培养介质中生长繁殖，但是这些培养介质与微生物往往难以分离，它们能成功地冻干在这些培养介质之中。例如肉汤、脱脂乳和蛋白质等。
有些冻干制剂浓度很低，干物质含量极少，冻干时已经干燥的部份会被升华气流带走。为了提高浓度，增加干物质的含量，使冻干后的产品能形成较理想的团块，因此需要加入填充物质，使固体物质的浓度在一定范围之间。这些填充物或赋形剂是蔗糖、乳糖、肌醇、脱脂乳、水解蛋白、葡聚糖、山梨醇、聚乙烯吡咯烷酮(PVP)等。
有些生物活性物质特别脆弱，在冷冻和干燥时由于物理或化学原因会受到危害，因此需要加入一些保护剂，以减少在冷冻和干燥中的损害。例如二甲亚砜、甘油、右旋糖苷、糖类和PVP等。
加入某些物质可以提高产品的崩解温度，使产品容易冻干。它们是甘露醇、甘氨酸、右旋糖苷、木糖醇和PVP等。
为了改变冻干制剂的酸碱度，从而提高共晶点，以利于冻干。它们是碳酸氢钠、氢氧化钠等。
为了改进产品贮存的稳定性，提高贮存温度，延长贮存时间。它们是一些抗氧化剂，例如维生素C、维生素E、氨基酸、硫代硫酸钠、硫脲、卵磷脂和水解蛋白等。
加入某些物质可以消除自由基团，增加冻干产品的稳定性。它们是氨基酸、维生素K、维生素C、硫脲、亚硫酸化合物、天门冬氨酸钠等。
可以发现，同样的化合物可以作为冻干保护剂(赋形剂)所起的作用是多样的。
根据本发明的药物的性质，冻干保护剂(赋形剂)选自糖类、多元醇类、聚乙烯吡咯烷酮类、蛋白类、泊洛沙姆或其组合。
糖类选自蔗糖、葡聚糖、环糊精、麦芽糊精、海藻糖、乳糖、麦芽糖、葡萄糖；
多元醇类选自甘油、山梨醇、甘露醇、肌醇、乙二醇、聚乙二醇(PEG)、聚山梨醇酯、 侧金盏花醇；
蛋白类选自白蛋白，优选为牛血清白蛋白、人白蛋白；
泊洛沙姆选自泊洛沙姆182、泊洛沙姆184、泊洛沙姆188、泊洛沙姆407。
优选地，所述赋形剂选自PVP K12、蔗糖、甘露醇、白蛋白或其组合。
一般而言，同类的赋形剂可以组合，相互不反应且不与药物反应的赋形剂可以组合，组合应符合药物配伍要求。
特别地，所述赋形剂选自蔗糖、甘露醇，且蔗糖、甘露醇在所述溶液中的含量为20-300mg/ml，优选为40-100mg/ml，更优选为60-80mg/ml，进一步优选为60-70mg/ml。
蔗糖或甘露醇一般使用一种，特殊情况下可以混合使用。本申请优选的方案是只使用甘露醇或只使用蔗糖。
同理，本发明用于制备高载药量冻干制剂的溶液中的TH-302和赋形剂的质量比为1：(0.5-20)，优选为1:(1-15)，更优选为1:(2-12.5)，进一步优选为1:(5-10)。
TH-302等类似化合物和赋形剂的质量比即冻干用溶液的载药比。根据上述的赋形剂(冻干保护剂、填充剂、骨架)的冻干状态可知，冻干后的冻干体以赋形剂作为骨架，而药物会吸附或负载在骨架上，因此冻干前药物溶液的载药比也就是TH-302和赋形剂的质量比是一个重要的指标。
合适的载药比意味着冻干溶液在冻干后，药物和骨架均匀吸附，药物会较好的分布在骨架的空隙和孔道的表面，如此，在后续的复溶(5％葡萄糖注射液、生理盐水等)过程中，冻干制剂能良好、快速溶解供注射施用。
药学上可接受的缓冲剂是指允许将溶液pH值维持在几乎恒定水平的弱酸或弱碱，用来增强原料药在溶液中的稳定性。
本发明用于制备高载药量冻干制剂的溶液中还可以含有至少一种缓冲剂，所述缓冲剂选自柠檬酸盐缓冲剂、硼酸盐缓冲液、乳酸锂、乳酸钠、乳酸钾、乳酸钙、磷酸锂、磷酸钠、磷酸钾、磷酸钙、马来酸锂、马来酸钠、马来酸钾、马来酸钙、酒石酸锂、酒石酸钠、酒石酸钾、酒石酸钙、琥珀酸锂、琥珀酸钠、琥珀酸钾、琥珀酸钙、乙酸锂、乙酸钠、乙酸钾、乙酸钙或它们的混合物。
优选地，在用于制备高载药量冻干制剂的溶液中使用的缓冲剂是至少一种柠檬酸盐缓冲剂。合适的柠檬酸盐缓冲剂的非限定性例子包括：柠檬酸锂一水合物、柠檬酸钠一水合物、柠檬酸钾一水合物、柠檬酸钙一水合物、柠檬酸锂二水合物、柠檬酸钠二水合物、柠檬酸钾二水合物、柠檬酸钙二水合物、柠檬酸锂三水合物、柠檬酸钠三水合物、柠檬酸钾三水合物、柠檬酸钙三水合物、柠檬酸锂四水合物、柠檬酸钠四水合物、柠檬酸钾四水合物、柠檬酸钙四水合物、柠檬酸锂五水合物、柠檬酸钠五水合物、柠檬酸钾五水合物、柠檬酸钙五水合物、柠檬酸锂六水合物、柠檬酸钠六水合物、柠檬酸钾六水合物、柠檬酸钙六水合物、柠檬酸锂七水合物、柠檬酸钠七水合物、柠檬酸钾七水合物、柠檬酸钙七水合物。
本发明在用于制备高载药量冻干制剂的溶液中还可以含有至少一种pH调节剂。
本发明的pH调节剂是指将根据酸或碱改变的pH进行适当调节所用的缓冲物质或缓冲液，所述pH调节剂包括但不限于盐酸、氢氧化钠、三乙醇胺、磷酸、磷酸氢二钠、磷酸氢二钾、磷酸二氢钠、磷酸二氢钾或磷酸、柠檬酸、乳酸、酒石酸、琥珀酸、富马酸、苹果酸、碳酸氢钠、碳酸钠或它们的混合物。pH调节剂的添加含量使得溶液pH值为4-9，优选为6-8。
由于TH-302及其类似物通常偏酸性:化合物制备工艺中包含酸性环境，同时存在微量的水解，因此制备或购买的原料药会呈现酸性。为此，pH调节剂优选自氢氧化钠、三乙醇胺、碳酸氢钠、碳酸钠等碱或具有碱性的盐。
当然，也有可能因为其他原因导致TH-302及其类似物的pH偏碱性，这时就需要添加 酸或具有酸性的盐(如硫酸铵、氯化铵等)来进行调节。
式I的化合物选自
Figure PCTCN2022115176-appb-000006
(TH-302或I-1)、
Figure PCTCN2022115176-appb-000007
Figure PCTCN2022115176-appb-000008
更优选为TH-302。
式I的化合物的具体合成方法以及相应的波谱数据公开在WO2007002931(对应中国公开文本CN101501054A)中，在此全文引入。
式I的化合物以及这三个具体化合物的相关理化性质、生物活性参见Threshold公司申请的专利(比如WO2016011195A2、WO2004087075A1、WO2007002931A1、WO2008151253A2、WO2009018163A1、WO2009033165A2、WO2010048330A2、WO2012142520A1、WO2008083101A2、WO2020007106A1、WO2020118251A1、WO2014169035A1、WO2013116385A1、WO2019173799A2、WO2016081547A1、WO2014062856A1、WO2015069489A1、WO2012006032A2、WO2018026606A2、WO2010048330A2、WO2015171647A1、WO2013096687A1、WO2013126539A2、WO2013096684A2、WO2012009288A2、WO2012145684A2、WO2016014390A2、WO2019055786A2、WO2012135757A2、WO2015013448A2、WO2016011328A2、WO2013177633A2、WO2016011195A2、WO2015051921A2)，在此本发明将上述相关申请文本信息全部引入。
这三个优选的化合物，理化性质和TH-302相同或类似。
本发明提供用于制备高载药量冻干制剂的溶液，含有
下式I-1化合物以及水、叔丁醇和蔗糖；
或
下式I-1化合物以及水、叔丁醇和甘露醇；
Figure PCTCN2022115176-appb-000009
水和叔丁醇作为混合溶剂，
叔丁醇相对于所述溶液体积含量为30％、40％、60％或所述溶液中叔丁醇对应含量为235.5、314、471mg/ml，
式I-1的化合物在溶液中的含量为6、7、7.5、8、8.5、10、12.5、15、20、25mg/m l，
蔗糖或甘露醇作为赋形剂，在溶液中的含量为40、50、60、70、75、80、90、100mg/ml。
本发明提供用于制备高载药量冻干制剂的溶液，含有
下式化合物I-1以及水、叔丁醇和蔗糖、pH调节剂；
或
下式化合物I-1以及水、叔丁醇和甘露醇、pH调节剂；
Figure PCTCN2022115176-appb-000010
水和叔丁醇作为混合溶剂，
叔丁醇相对于所述溶液体积含量为为30％、40％、60％或所述溶液中叔丁醇含量为235.5、314、471mg/ml，
式I-1的化合物在溶液中的含量为6、7、7.5、8、8.5、10、12.5、15、20、25mg/ml，
蔗糖或甘露醇作为赋形剂，在溶液中的含量为40、50、60、70、75、80、90、100mg/ml，
所述pH调节剂的含量使得溶液的pH值为4-9，优选为6-8，
本发明提供用于制备高载药量冻干制剂的溶液，由
下式I-1化合物以及水、叔丁醇和蔗糖、碳酸氢钠组成；
或
下式I-1化合物以及水、叔丁醇和甘露醇、碳酸氢钠组成；
Figure PCTCN2022115176-appb-000011
水和叔丁醇作为混合溶剂，
叔丁醇相对于所述溶液体积含量为30％、40％、60％或所述溶液中叔丁醇含量为235.5、314、471mg/ml，
式I-1的化合物在溶液中的含量为6、7、7.5、8、8.5、10、12.5、15、20、25mg/ml，
蔗糖或甘露醇作为赋形剂，在溶液中的含量为40、50、60、70、75、80、90、100mg/ml，
碳酸氢钠的含量使得pH值为4-9，优选为6-8。
上述用于制备高载药量冻干制剂的溶液，其中，
所述溶液中化合物与蔗糖或甘露醇的质量比为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。
当然，也可以根据上述说明，结合具体的式I的化合物的性质，添加缓冲液、pH调节剂或其他冻干制剂的辅料。
显然，如果在制备溶液过程中因为容器、管道、工具的接触或污染而检测出别的物质(环境物质)，不在此限。同样的，叔丁醇和蔗糖/甘露醇、式I的化合物的原料必不可少会掺杂有杂质或其他环境中带有的物质(环境物质)，这些杂质或环境物质，同样也不在此限。
所有的因为容器、管道、工具的接触或污染而检测出别的物质(环境物质)、叔丁醇和蔗糖/甘露醇、式I的化合物的原料必不可少会掺杂有杂质或其他环境中带有的物质(环境物质)的含量范围应在法定限度内或符合相应的产品(医药级、医用级或相当质量标准)质量标准。
显然，如果在制备溶液过程中因为容器、管道、工具的接触或污染而检测出别的物质(环境物质)，不在此限。同样的，叔丁醇和蔗糖/甘露醇、碳酸氢钠、式I的化合物的原料必不可少会掺杂有杂质或其他环境中带有的物质(环境物质)，这些杂质或环境物质，同样 也不在此限。
所有的因为容器、管道、工具的接触或污染而检测出别的物质(环境物质)、叔丁醇和蔗糖/甘露醇、碳酸氢钠、式I的化合物的原料必不可少会掺杂有杂质或其他环境中带有的物质(环境物质)的含量范围应在法定限度内或符合相应的产品(医药级、医用级或相当质量标准)质量标准。
因此，上述“由xxx组成”是指配制时刻意、人为的添加的物质(这些物质是必定含有且能被分析检测仪器检出)，除这些物质之外不再含有其他刻意、人为的添加的物质，但依然会不可避免的存在微量的杂质、环境物质等。
以上内容对制备高载药量冻干制剂的溶液的组成(处方)做了说明，以下简要说明其用途。
上述提供的溶液只是作为制备高载药量冻干制剂的中间半成品，不能作为临床制剂的处方使用。一般而言，是在生产现场现配现用，即使用配液容器配制后直接灌装到冻干瓶中，然后批量送去冻干生产设备进行冻干。
因此，制备高载药量冻干制剂的溶液应保证在配制完成后到冻干设备冻干的生产和等待过程中是稳定的，即在室温条件下应至少具有8小时稳定性，更优的，应具有24小时甚至72小时、120小时的稳定性。
这是因为溶液在配制完成后需要经过过滤和灌装才能进入冻干机进行冻干。过滤和灌装一般在8-12小时内完成且一般是在室温下进行操作。后续的冻干虽然是低温，但大型冻干机或冻干系统在将大量灌装在西林瓶的药液降温到设定的低温(-20到-55℃)需要20-60小时，因此冻干制剂的药液应在一定时间内、室温调节下具有稳定性。经过实验，已验证本发明提供的TH-302冻干制剂的药液具有合适的稳定性。
制备高载药量冻干制剂的溶液的用途，其作为冻干制剂溶液，通过冻干过程来制备冻干制剂。
基于上述的制备高载药量冻干制剂的溶液，本发明可以制备高载药量的冻干制剂。
冻干制剂，使用上述的制备高载药量冻干制剂的溶液作为冻干制剂溶液通过冻干过程制备得到。
冻干制剂，含有下式I的化合物以及赋形剂、残留的溶剂成分：
Figure PCTCN2022115176-appb-000012
其中,R各自独立地选自H、-CH 3、-CH 2CH 3，X各自独立地选自Br、Cl、OMs、OTs，
冻干制剂中式I化合物的载药量大于5mg/cm 3而小于或等于555.55mg/cm 3,或
冻干制剂中式I化合物的载药量大于或等于4.55mg/cm 3而小于500mg/cm 3，或
冻干制剂中式I化合物的质量百分比为大于或等于4.39％而小于66.66％，
所述残留的溶剂成分为水和叔丁醇。
冻干制剂，含有下式I的化合物以及赋形剂、残留的溶剂成分、pH调节剂：
Figure PCTCN2022115176-appb-000013
其中,R各自独立地选自H、-CH 3、-CH 2CH 3，X各自独立地选自Br、Cl、OMs、OTs，
冻干制剂中式I化合物的载药量大于5mg/cm 3而小于或等于555.55mg/cm 3,或
冻干制剂中式I化合物的载药量大于或等于4.55mg/cm 3而小于500mg/cm 3，或
冻干制剂中式I化合物的质量百分比为大于或等于4.39％而小于66.66％，
所述残留的溶剂成分为水和叔丁醇。
冻干制剂，含有下式I的化合物以及赋形剂：
Figure PCTCN2022115176-appb-000014
其中,R各自独立地选自H、-CH 3、-CH 2CH 3，X各自独立地选自Br、Cl、OMs、OTs，
冻干制剂中式I化合物的载药量大于5mg/cm 3而小于或等于555.55mg/cm 3,或
冻干制剂中式I化合物的载药量大于或等于4.55mg/cm 3而小于500mg/cm 3，或
冻干制剂中式I化合物的质量百分比为大于或等于4.39％而小于66.66％。
这里冻干制剂中式I化合物的载药量大于5mg/cm 3而小于或等于555.55mg/cm 3、冻干制剂中式I化合物的载药量大于或等于4.55mg/cm 3而小于500mg/cm 3是两种不同的情形。
申请人经过多次实验发现，在50ml西林瓶且最大灌装量为25ml的情况下，对于使用不同的冻干工艺，将前述高载药量冻干制剂溶液进行冻干得到冻干制剂，冻干前溶液的体积和冻干后制剂固体的外表体积存在不超过10％的变化：有可能因冻干后粉饼膨胀而体积变大10％，也有可能因出现粉饼塌陷而体积变小10％。以冻干前高载药量冻干制剂溶液中式I的化合物在溶液中的含量大于或等于5mg/ml而小于或等于500mg/ml为基准，以最大变化10％进行的计算，即膨胀变大使得体积为原体积的1.1倍、塌陷变小使得体积为原先的90％。以冻干后粉饼塌陷体积变小为原先的90％为例进行计算得到此时粉饼的最大载药量为5/0.9＝5.55至500/0.9＝555.55，由于工艺不同有可能出现体积不变化的情况，此时载药量范围为5至500，两个范围取并集，得到5至555.55，即冻干制剂中式I化合物的载药量大于5mg/cm 3而小于或等于555.55mg/cm 3。
同样的道理，当冻干粉饼发生膨胀使得体积变大的情况，则冻干制剂中式I化合物的载药量大于或等于4.55mg/cm 3而小于500mg/cm 3。
本发明提供的冻干制剂载药量是指单位体积所含有的式I的化合物的原料药的量，此处的体积是指单位包装套件内药物的外表总体积，囊括了药物内部空隙。例如，100ml容积的冻干瓶盛装有20ml的冻干溶液，20ml的冻干溶液中含有500mg药物。冻干前制剂溶液经过冻干过程后，20ml的溶液被冻成疏松多孔的冻干制剂，通过测量冻干瓶的内空间底面积为s，冻干制剂在冻干瓶中的高度为h，则这瓶单位包装的冻干制剂的外表观总体积为sh，其载有500mg的原料药，则其载药量为(500/sh)mg/cm 3，一般这个sh的数值会在20cm 3左右，可能冻干后体积膨胀而大于20cm 3，可能冻干后塌陷而体积小于20cm 3。
在测量某个单位包装的冻干制剂的载药量时，先测量计算上述的外表观总体积sh，然后直接将单位包装的冻干制剂全部溶解并检测其含有的药物的质量M，则本发明的冻干制剂的(单位体积)载药量＝M/sh。
这里冻干制剂中式I化合物的质量百分比为大于或等于4.39％而小于66.66％，其换算可以根据冻干后制剂中的API含量、赋形剂以及残留的叔丁醇和水的含量计算得到。
冻干制剂中式I化合物的质量含量是指药物在冻干制剂总质量中的百分比。可以通过如下操作测量计算：
在测量某个单位包装的冻干制剂的药物质量含量时，先称量单位包装的冻干制剂的质量M1，然后直接将单位包装的冻干制剂全部溶解并检测其含有的药物的质量M，溶解后清 洗、烘干并称量空瓶和包装盖的总质量为M2，则冻干制剂的总质量为M1-M2，则本发明的冻干制剂式I化合物的质量含量＝M/(M1-M2)。
上述冻干制剂中，式I的化合物选自
Figure PCTCN2022115176-appb-000015
优选自TH-302。
所述赋形剂为糖类、多元醇类、聚乙烯吡咯烷酮类、蛋白类、泊洛沙姆或其组合，
糖类选自蔗糖、葡聚糖、环糊精、麦芽糊精、海藻糖、乳糖、麦芽糖、葡萄糖，
多元醇类选自甘油、山梨醇、甘露醇、肌醇、乙二醇、聚乙二醇、聚山梨醇酯、侧金盏花醇，
蛋白类选自白蛋白，优选为牛血清白蛋白、人白蛋白，
泊洛沙姆选自泊洛沙姆182、泊洛沙姆184、泊洛沙姆188、泊洛沙姆407。
溶液在冻干后，作为溶剂的水、叔丁醇会升华，因此冻干制剂剩下的就是药物和赋形剂。
显然，冻干的升华过程不可能完全将水、叔丁醇冻干去除，因此水、叔丁醇的残留是不可避免的，实际上水、叔丁醇的残留量多少是一个重要的冻干制剂质量指标：残留越低，冻干制剂质量越好，稳定性也越强，患者在使用后，不良反应也会更少。减少残留量，可以通过调整冻干工艺来解决，但终究不能完全避免残留。
但通过延长冻干时间、提升干燥温度，在不计较成本且满足产品质量要求的情况下，技术上可以将水、叔丁醇的含量降低到检测限以下，此种情况即可以认为冻干制剂中几乎不含有水和叔丁醇。
考虑到生产成本和储存稳定性，水、叔丁醇的残留量有一个合适的值：残留水含量为质量百分比小于或等于6％，优选为小于或等于2％，更优选为小于或等于1％，进一步优选为小于或等于0.5％，
残留叔丁醇含量为质量百分比小于或等于1.75％，优选为小于或等于1％，更优选为小于或等于0.5％。
当然，也可以根据上述说明，结合具体的式I化合物的性质，在溶液中添加缓冲液、pH调节剂或其他冻干制剂的辅料，这样，冻干后的冻干制剂也会相应检测出含有缓冲液、pH调节剂或其他冻干制剂的辅料。
显然，如果在制备溶液过程中因为容器、管道、工具的接触或污染而检测出别的不能升华除去的物质(环境物质)，不在此限。同样的，叔丁醇和蔗糖、式I的化合物的原料必不可少会掺杂有杂质或其他环境中带有的不能升华除去物质(环境物质)，这些杂质或环境物质，同样也不在此限。
所有的因为容器、管道、工具的接触或污染而检测出别的不能升华除去物质(环境物质)、叔丁醇和蔗糖、式I的化合物的原料必不可少会掺杂有杂质或其他环境中带有的不能升华除去物质(环境物质)的含量范围应在法定限度内或符合相应的产品(医药级、医用级或相当质量标准)质量标准。
含有下式I的化合物以及赋形剂指除不可避免的、残留的水和叔丁醇外，冻干制剂能检测出含有式I的化合物、赋形剂以及上述残留的环境物质，除此之外，还可以含有其他辅料。
化合物I与所述赋形剂的质量比为1:(0.5-20)，优选为1:(1-15)，更优选为1:(2-12.5)，进一步优选为1:(5-10)。
冻干制剂中式I化合物的载药量大于或等于5.55mg/cm 3而小于或等于177.77mg/cm 3，优选为大于或等于8.88mg/cm 3而小于或等于55.55mg/cm 3，更优选为大于或等于8.88mg/cm 3而小于或等于27.77mg/cm 3，进一步优选为大于或等于8.88mg/cm 3而小于或等于16.66mg/cm 3，再进一步优选为大于或等于8.88mg/cm 3而小于或等于11.11mg/cm 3；
或者
冻干制剂中式I化合物的载药量大于或等于4.55mg/cm 3而小于或等于145.45mg/cm 3，优选为大于或等于7.27mg/cm 3而小于或等于45.45mg/cm 3，更优选为大于或等于7.27mg/cm 3而小于或等于22.73mg/cm 3，进一步优选为大于或等于7.27mg/cm 3而小于或等于13.64mg/cm 3，再进一步优选为大于或等于7.27mg/cm 3而小于或等于9.09mg/cm 3。
这里的两种情形，前者是冻干后体积塌陷变小的情形，后者是体积膨胀变大的情形。
冻干制剂，实质由下式I-1化合物以及赋形剂、残留水和残留叔丁醇组成：
Figure PCTCN2022115176-appb-000016
冻干制剂中化合物的载药量为6.66、7.77、8.33、8.88、9.44、11.11、13.88、16.66、22.22、27.77mg/cm 3，或者冻干制剂中化合物I-1的载药量为5.45、6.36、6.82、7.27、7.73、9.09、11.36、13.64、18.18、22.73mg/cm 3；
赋形剂为蔗糖或甘露醇；
化合物与所述赋形剂的质量比为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；
残留水含量为质量百分比小于或等于6％，优选为小于或等于2％，更优选为小于或等于1％，进一步优选为小于或等于0.5％；
残留叔丁醇含量为质量百分比小于或等于1.75％，优选为小于或等于1％，更优选为小于或等于0.5％。
冻干制剂，实质由下式I-1化合物以及赋形剂、残留水和残留叔丁醇、pH调节剂组成：
Figure PCTCN2022115176-appb-000017
冻干制剂中化合物的载药量为6.66、7.77、8.33、8.88、9.44、11.11、13.88、16.66、22.22、27.77mg/cm 3，或者冻干制剂中化合物I-1的载药量为5.45、6.36、6.82、7.27、7.73、9.09、11.36、13.64、18.18、22.73mg/cm 3；
赋形剂为蔗糖或甘露醇；
化合物与所述赋形剂的质量比为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；
残留水含量为质量百分比小于或等于6％，优选为小于或等于2％，更优选为小于或等于1％，进一步优选为小于或等于0.5％；
残留叔丁醇含量为质量百分比小于或等于1.75％，优选为小于或等于1％，更优选为小于或等于0.5％；
pH调节剂为碳酸氢钠，含量为0.01-0.10mg/cm 3。
冻干制剂，实质由下式化合物以及赋形剂组成：
Figure PCTCN2022115176-appb-000018
冻干制剂中化合物I-1的载药量为6.66、7.77、8.33、8.88、9.44、11.11、13.88、16.66、22.22、27.77mg/cm 3，或者冻干制剂中化合物I-1的载药量为5.45、6.36、6.82、7.27、7.73、9.09、11.36、13.64、18.18、22.73mg/cm 3；
赋形剂为蔗糖或甘露醇；
化合物I-1与所述赋形剂的质量比为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。
显然，如果在制备溶液过程中因为容器、管道、工具的接触或污染而检测出别的不能升华除去的物质(环境物质)，不在此限。同样的，叔丁醇和蔗糖、式I的化合物的原料必不可少会掺杂有杂质或其他环境中带有的不能升华除去物质(环境物质)，这些杂质或环境物质，同样也不在此限。
所有的因为容器、管道、工具的接触或污染而检测出别的不能升华除去物质(环境物质)、叔丁醇和蔗糖、式I的化合物的原料必不可少会掺杂有杂质或其他环境中带有的不能升华除去物质(环境物质)的含量范围应在法定限度内或符合相应的产品(医药级、医用级或相当质量标准)质量标准。
含有下式I的化合物以及赋形剂指除不可避免的、残留的水和叔丁醇外，冻干制剂能检测出含有I的化合物、赋形剂以及上述残留的环境物质，除此之外，还可以含有其他辅料。
实质由式I的化合物以及赋形剂组成指除不可避免的、残留的水和叔丁醇外，冻干制剂只能检测出含有I的化合物、赋形剂以及上述残留的环境物质，除此之外，不含有其他物质。
本发明提供一种冻干制剂，其为使用上述一系列用于制备高载药量冻干制剂的溶液通过冻干过程制备得到。
药物制剂和其冷冻干燥粉剂可以贮存在药学领域中常用的容器内，其可以包括：塑料容器或玻璃容器，例如标准的USPI型硼硅酸盐玻璃容器。例如，所用的容器可以是注射器或小瓶。
根据背景技术中的式I的化合物的动物模型实验、临床试验剂量可知，不同的物种(人和其他动物)、不同的适应症、不同的患者可能每次给药的量不同，从最小的几毫克到最大的上万毫克，最好是一个单位剂量包装或多个单位剂量包装的组合能满足某次剂量给药，因此，结合上述情况，对应不同物种、不同适应症我们给出了推荐的冻干制剂的制剂单位包装的规格。
本发明还提供盛装有上述冻干制剂的制剂单位包装：
所述冻干制剂被盛装在1000ml容积的密闭容器中，冻干制剂的体积为密闭容器容积的1/5到2/3，优选为1/3到1/2，含有式I的化合物的含量为4000-16500mg；或
所述冻干制剂被盛装在500ml容积的密闭容器中，冻干制剂的体积为密闭容器容积的1/5到2/3，优选为1/3到1/2，含有式I的化合物的含量为2000-8000mg；或
所述冻干制剂被盛装在250ml容积的密闭容器中，冻干制剂的体积为密闭容器容积的1/5到2/3，优选为1/3到1/2，含有式I的化合物的含量为1000-4000mg；或
所述冻干制剂被盛装在100ml容积的密闭容器中，冻干制剂的体积为密闭容器容积的1/5到2/3，优选为1/3到1/2，含有式I的化合物的含量为400-2000mg；或
所述冻干制剂被盛装在50ml容积的密闭容器中，冻干制剂的体积为密闭容器容积的1/5到2/3，优选为1/3到1/2，含有式I的化合物的含量为50-800mg；或
所述冻干制剂被盛装在30ml容积的密闭容器中，冻干制剂的体积为密闭容器容积的1/5到2/3，优选为1/3到1/2，含有式I的化合物的含量为150-600mg；或
所述冻干制剂被盛装在25ml容积的密闭容器中，冻干制剂的体积为密闭容器容积的1/5到2/3，优选为1/3到1/2，含有式I的化合物的含量为125-500mg；或
所述冻干制剂被盛装在20ml容积的密闭容器中，冻干制剂的体积为密闭容器容积的1/5到2/3，优选为1/3到1/2，含有式I的化合物的含量为100-400mg；或
所述冻干制剂被盛装在18ml容积的密闭容器中，冻干制剂的体积为密闭容器容积的1/5到2/3，优选为1/3到1/2，含有式I的化合物的含量为90-360mg；或
所述冻干制剂被盛装在15ml容积的密闭容器中，冻干制剂的体积为密闭容器容积的1/5到2/3，优选为1/3到1/2，含有式I的化合物的含量为75-300mg；或
所述冻干制剂被盛装在10ml容积的密闭容器中，冻干制剂的体积为密闭容器容积的1/5到2/3，优选为1/3到1/2，含有式I的化合物的含量为50-200mg；或
所述冻干制剂被盛装在8ml容积的密闭容器中，冻干制剂的体积为密闭容器容积的1/5到2/3，优选为1/3到1/2，含有式I的化合物的含量为40-160mg；或
所述冻干制剂被盛装在7ml容积的密闭容器中，冻干制剂的体积为密闭容器容积的1/5到2/3，优选为1/3到1/2，含有式I的化合物的含量为35-140mg；或
所述冻干制剂被盛装在5ml容积的密闭容器中，冻干制剂的体积为密闭容器容积的1/5到2/3，优选为1/3到1/2，含有式I的化合物的含量为25-100mg；或
所述冻干制剂被盛装在3ml容积的密闭容器中，冻干制剂的体积为密闭容器容积的1/5到2/3，优选为1/3到1/2，含有式I的化合物的含量为15-60mg；或
所述冻干制剂被盛装在2ml容积的密闭容器中，冻干制剂的体积为密闭容器容积的1/5到2/3，优选为1/3到1/2，含有式I的化合物的含量为10-40mg；或
所述冻干制剂被盛装在1.5ml容积的密闭容器中，冻干制剂的体积为密闭容器容积的1/5到2/3，优选为1/3到1/2，含有式I的化合物的含量为7.5-30mg。
这些小剂量适用于人类中的未成年人或相当体型的动物，比如猪、大鼠、狗等体型、体重小型的动物，适应症不限。
本发明提供的冻干制剂推荐选择静脉输液给药，因此需要将药液复溶，复溶一般选用生理盐水(0.9％)或葡萄糖注射液(5％)，为此提供一种含有TH-302的静脉注射用注射液其溶剂为水，溶质包括TH-302原料药、调节等渗试剂、甘露醇或蔗糖、叔丁醇以及碳酸氢钠，所述调节等渗试剂选自葡萄糖、氯化钠。
本发明还提供一种制备高载药量冻干制剂的溶液的配制方法，包括以下操作：
操作一：称取处方量的TH-302的原料药，加入叔丁醇水溶液中，搅拌至澄清得第一溶液；
操作二：将处方量甘露醇或蔗糖溶解于适量水中，搅拌至澄清后得第二溶液；
操作三：混合第一溶液和第二溶液并最终加入剩下的处方量叔丁醇后加入适量的水定容至预定体积，加入处方量的碳酸氢钠，搅拌混合均匀，
其中，
所述高载药量冻干制剂的溶液中TH-302原料药的含量大于5mg/ml而小于或等于500mg/ml，
叔丁醇相对于所述高载药量冻干制剂的溶液体积百分比为1％-99％，或所述溶液中叔丁醇含量为7.85-777.15mg/ml，
蔗糖或甘露醇在所述高载药量冻干制剂的溶液中的含量为20-300mg/ml，
所述高载药量冻干制剂的溶液中TH-302与蔗糖或甘露醇的质量比为1:(0.5-20)，
所述叔丁醇水溶液中叔丁醇体积比为30-90％，
碳酸氢钠在所述高载药量冻干制剂的溶液中的含量为0.01-0.10mg/ml。
本发明在此提供冻干制剂的制备流程。
高载药量冻干制剂的制备方法，包括以下操作：
预冻，将上述药液置于冻干系统中进行预冻，预冻过程包括0℃保温一段时间和-20到-55℃保温一段时间；
一次干燥，预冻完成后升温至-10到10℃并保温一段时间，并维持真空进行干燥；
二次干燥，一次干燥完成后升温至20到40℃并保温一段时间，并维持真空进行干燥。
由以上的说明可知，冻干制剂是直接将溶液灌装到冻干瓶(西林瓶)中，通过冻干设备直接冻干得到，因此不存在分装环节，即单位包装就是对应的冻干瓶包装，包装的规格与冻干瓶的规格紧密相关。
特别说明，本申请中出现的数字均具有±10％的误差，即在数字的-10％与+10％均应视为在本申请记载的数字范围内，即如本申请中出现“含量大于5mg/ml而小于或等于500mg/ml”这等字样，则实际如测试得到含量大于4.5mg/ml而小于或等于550mg/ml也应被理所当然的确定为与上述范围是等同的。
附图说明
图1为不同质量比的叔丁醇水溶液对TH-302的溶解度曲线；
图2为11种赋形剂制备的冻干制剂在5天内的稳定性曲线，以最右边的圆圈进行区分，自上而下依次为PEG2000、P188、SBECD、甘露醇、DSPE-MPEG2000、果糖、海藻糖、PVPK12、蔗糖、麦芽糖、乳糖；
图3为蔗糖、甘露醇作为赋形剂制备的冻干制剂在10天内的稳定性曲线，图中上部的4条曲线为甘露醇处方的曲线，下部的4条曲线为蔗糖处方的曲线；
图4为100mg/ml蔗糖、80mg/ml甘露醇作为赋形剂制备的冻干样品在高温40℃和室温25℃的稳定性曲线，横坐标为天数，纵坐标为HPLC的百分比纯度；
图5为药液样品置于2-℃环境下底部结晶照片，标签已经打码覆盖；
图6为4批冻干制剂的样品照片，自左到右依次为01、02、03、04批次，标签已经打码覆盖；
图7为4批冻干制剂样品的40ml的5％葡萄糖溶液复溶实验中加入溶液与静置后的对比照片，左边图片为加入溶液后的照片，右边为加入溶液静置后照片，每张照片中自左到右依次为01、02、03、04批次，标签已经打码覆盖；
图8为4批冻干制剂样品的50ml的5％葡萄糖溶液复溶实验中加入溶液与静置后的对比照片，左边图片为加入溶液后的照片，右边为加入溶液静置后照片，每张照片中自左到右依次为01、02、03、04批次，标签已经打码覆盖；
图9为7批冻干制剂的样品照片，自左到右依次为01至07批次，标签已经打码覆盖；
图10为实验冻干机的搁板上西林瓶放置示意图；
图11为冻干制剂样品照片；
图12为冻干制剂中出现“绕颈”样品照片，标签已经打码覆盖。
具体实施方式
以下参照具体的实施例来说明本发明。本领域技术人员能够理解，这些实施例仅用于说明本发明，其不以任何方式限制本发明的范围。
“患者”及“个体”可互换使用，是指需要癌症治疗的哺乳动物。通常，患者是人类。通常，患者是诊断患有癌症的人类。在某些实施例中，“患者”或“个体”可指用于筛选、表征及评估药物及疗法的非人类哺乳动物，例如非人类灵长类动物、狗、猫、兔、猪、小鼠或大鼠。
“前药”是指投与或施用之后经新陈代谢或以其他方式转化为关于至少一种性质的生物学活性或活性更高的化合物(或药物)的化合物。相对于药物，前药以使其相对于药物活性较低或无活性的方式化学修饰，但化学修饰使得在前药投与之后通过代谢或其他生物过程产生相应药物。前药可相对于活性药物具有改变的代谢稳定性或输送特征、较少副作用或较低毒性或经改良的风味。前药可使用除相应药物以外的反应物来合成。
“治疗”或“治疗患者”是指向患者投与、使用或施用本发明相关的治疗有效量的药物。
向患者“投与”或“施用”“使用”药物是指直接投与或施用(其可由医学专业人士向患者投与或施用或者可自投与或施用)及/或间接投与或施用，其可是开处药物的行为。举例而言，指示患者自投与或施用药物及/或将药物的处方提供给患者的医师是向患者投与或施用药物。
药物的“治疗有效量”是指当向患有癌症的患者投与或施用、使用时，将具有预期的治疗效应(例如患者中一或多种癌症的临床表现的缓和、改善、缓解或消除)的药物的量。治疗效应不必通过投与或施用一个剂量而出现，且可仅在投与或施用一系列剂量后出现。因此，治疗有效量可以一或多次来投与或施用。
病况或患者的“治疗”是指采取步骤以获得有益或期望结果(包括临床结果)。出于本发明的目的，有益或期望临床结果包括(但不限于)一或多种癌症症状的缓和或改善；疾病程度的减弱；疾病进展的延迟或减缓；疾病状态的改善、缓解或稳定；或其他有益结果。在一些情形下，癌症的治疗可使得部分反应或稳定疾病。
“肿瘤细胞”是指任何适当物种(例如，哺乳动物，例如鼠类、犬、猫、马或人类)的肿瘤细胞。
以上对本发明具体实施方式的描述并不限制本发明，本领域技术人员可以根据本发明作出各种改变或变形，只要不脱离本发明的精神，均应属于本发明所附权利要求的范围。
通用信息说明
以下各个实验中如未特别说明，其检测或检验的方法、器具等信息如下：
水分含量使用卡尔费休KF法测定，使用仪器为Mettler V10S型卡尔费休水分仪。
残留溶剂叔丁醇含量使用气相色谱法GC测定，使用仪器为Agilent 8860型气相色谱仪，配置7696A型自动顶空进样器、FID检测器，色谱柱为以DB-624填料的毛细管柱。；GC测试参数为载气为N 2；进样口温度为150℃；检测器温度200℃；分流比20:1；升温程序为起始温度60℃，保持5min，再以30℃/min的速率升温至240℃，60℃后运行5min；顶空平衡85℃20min；
TH-302含量和浓度使用高效液相色谱法HPLC测定，使用仪器为Thermo Vanquish高效液相色谱法，色谱柱：YMC pack AQ C18 4.6mm×250mm,5μm，检测方法参见Threshold公司申请的专利WO2008083101A1中的HPLC方法(具体为Example 2.An Ethanol Formulation of TH302)。
如未特殊说明，表格中出现的“/”均表示未检测。
1、TH-302溶解度实验
溶解度采用饱和溶液法实验测定，即将固体TH-302直接投入到溶剂中，直至出现不溶物或浑浊，澄清一段时间后，直接过滤，取澄清的滤液直接检测TH-302的浓度，所得的浓度即为该溶剂体系的TH-302溶解度，具体溶解度数据见表1和表2、表3。
TH-302的浓度(mg/ml)测量方法参见Threshold公司申请的专利WO2008083101A1中的HPLC方法(具体为Example 2.An Ethanol Formulation of TH302)，定量使用外标法。
如浓度过高，对应的澄清溶液应先进行稀释，稀释到HPLC能准确定量检测的程度。
将表3的数据中的叔丁醇质量配比作为X轴，叔丁醇-水混合溶剂对TH-302的溶解度作为Y轴，得到图1的关系曲线：原料药在水溶液中溶解度较低；随着叔丁醇水溶液中叔丁醇浓度的增加，原料药溶解度不断增加；原料药在70％叔丁醇水溶液(w/w)中溶解度最高，然后随着叔丁醇水溶液中叔丁醇浓度的增加，溶解度降低。
2、冻干制剂中赋形剂(冻干保护剂)种类筛选实验
为了探究常用的糖类、多元醇类、非离子聚合物表面活性剂等作为TH-302药物的冻干赋形剂的可行性，分别选择以下赋形剂进行冻干实验并考察稳定性。
糖类选用：蔗糖、乳糖、麦芽糖、果糖、海藻糖
多元醇选用：甘露醇、丙三醇、山梨醇
非离子聚合物表面活性剂选用：PVPK12(分子量为5500的聚乙烯基吡咯烷酮)、PEG2000(聚乙二醇2000)、P188(泊洛沙姆Poloxamer为聚氧乙烯聚氧丙烯醚嵌段共聚物，牌号为188)；
其他类：SBECD(磺丁基醚-β-环糊精)、DSPE-MPEG2000(培化磷脂酰乙醇胺)
按照下列表格4配制冻干用药液。
表4：不同冻干赋形剂配制的冻干药液配制方案
Figure PCTCN2022115176-appb-000019
API：TH-302，TBA：叔丁醇，water：水。
※PEG2000和P188保护剂实验组使用的溶剂为40％TBA/water。
按照上述处方称量TH-302原料药和冻干保护剂，加入处方量的叔丁醇溶液，混合均匀至完全溶解，然后分装(使用5ml西林瓶，每瓶灌装1ml药液，每个处方灌装8瓶)，并在小型快速冻干机上进行快速冷冻干燥:分装制剂在-80℃超低温冰箱中预冻约2.5h，后置于冻干机中冻干约10-90h(-30℃，绝对气压为0.1mbar)，得到冻干制剂。
实验结果
冻干粉状态：除水作为溶剂外，其余溶剂组均成功得到白色冻干粉饼。但丙三醇组在冻干机升温至0℃时成油状物。
复溶状态：除山梨醇组外，其余各组均复溶成功(部分经震摇后溶解)。
将剩下的蔗糖、甘露醇、乳糖、麦芽糖、果糖、PVPK12、海藻糖、DSPE-MPEG2000、SBECD、 PEG2000、P188处方组进行高温加速稳定性实验。
冻干制剂在取出冻干机后于0℃取出1瓶检测原料药的HPLC纯度以及水分、残留溶剂含量，然后置于40℃恒温箱中，分别在第3天、第5天检测HPLC纯度，结果如下表5所示。
表5：不同赋形剂的冻干制剂的高温40℃加速稳定性实验数据。
Figure PCTCN2022115176-appb-000020
ND：未检出
将表5的数据以天数作为X轴，以HPLC纯度作为Y轴，得到图2的曲线。
实验结论
一般而言，注射剂的冻干保护剂选择要兼顾稳定性好，常规使用，对药效、DMPK及毒理不会产生影响。分析图2的曲线，PEG2000非常规使用，P188/SBECD/DSPE-MPEG2000可能对药效、DMPK以及毒理产生影响，总体来讲甘露醇比较理想，蔗糖也具有开发潜力(优化用量和冻干条件有可能提高稳定性以及降低叔丁醇残留)。
为了进一步评估甘露醇和蔗糖的稳定性，对甘露醇和蔗糖样品在放置到第10天后进行HPLC纯度检测，并将结果制作为曲线，如图3所示。
分析图3可知，在表4的初步处方条件下甘露醇处方稳定性优于蔗糖处方。
3、考察不同冻干保护剂(100mg/ml蔗糖、80mg/ml甘露醇)对冻干粉稳定性以及复溶溶液稳定性实验
根据经验以及同类冻干制剂药物，初步确定使用100mg/ml蔗糖、80mg/ml甘露醇作为赋形剂，考察制备的TH-302冻干制剂的稳定性及复溶后溶液的稳定性。
3.1冻干制剂室温和高温加速储存稳定性实验
按照下表6配制冻干药液。配制各40瓶，1ml/瓶，每个处方共配制40ml药液。
表6：100mg/ml蔗糖、80mg/ml甘露醇作为赋形剂配制的冻干药液配制方案
Figure PCTCN2022115176-appb-000021
Figure PCTCN2022115176-appb-000022
配液过程：
1.先将处方量冻干保护剂溶解于处方量水中，完全溶解。
2.将处方量叔丁醇加入冻干保护剂水溶液中，混合均匀。再加入处方量API，搅拌使其完全溶解。
3.蔗糖处方的药液测pH为4.24，使用NaHCO 3注射液10μL，调pH至6.43。再加入10μL，调pH至7.13。
甘露醇处方的药液测pH为4.62，使用NaHCO 3注射液12μL，调pH至7.31。
采用PVDF过滤，分装1ml/瓶。
使用前述的“快速冷冻干燥”类似的操作，并调节相关参数，得到冻干制剂。
冻干结束后，真空压塞，轧盖。得到冻干制剂。检测冻干制剂残留溶剂和水分。
将冻干制剂样品置于25℃、40℃恒温箱中，分别在相应的天数检测HPLC纯度，结果如下表7所示。
表7：100mg/ml蔗糖、80mg/ml甘露醇作为赋形剂制备的冻干样品在高温40℃和室温25℃稳定性实验数据
Figure PCTCN2022115176-appb-000023
NA：未检测
将表7中的HPLC纯度作为Y坐标，时间作为X坐标制作为100mg/ml蔗糖、80mg/ml甘露醇的稳定性曲线，如图4所示。
表7以及图4分析对比可知，使用甘露醇作为赋形剂的药液制备的冻干制剂样品在25℃、40℃的稳定性均优于蔗糖处方。
3.2冻干制剂复溶配伍稳定性实验
取冻干粉一支用D5W(5％质量百分比葡萄糖水溶液)溶解成每1mL含API约5mg的溶液，室温下放置，分别在0、6、24h测定其溶液颜色及澄清度，结果均为无色透明澄清溶液。
分别在0、6、24h用30％乙腈水溶液稀释成每1mL含API约1mg的溶液并检测HPLC纯度，结果如下表8所示。
表8：100mg/ml蔗糖、80mg/ml甘露醇作为赋形剂制备的冻干样品在室温25℃复溶后稳定性实验数据
Figure PCTCN2022115176-appb-000024
Figure PCTCN2022115176-appb-000025
分别取冻干粉一支用D5W溶解成每1mL含API约5mg的溶液，测定其pH值，并测定其渗透压，结果如下表9所示，测定时一并测定5％葡萄糖注射液的pH值和渗透压。
表9：100mg/ml蔗糖、80mg/ml甘露醇作为赋形剂制备的冻干样品在室温25℃使用D5W复溶后pH值和渗透压数据
处方 100mg/ml蔗糖 80mg/ml甘露醇 5％葡萄糖
pH值 5.14 5.05 5.47
渗透压 344mOsmol/kg 374mOsmol/kg 259mOsmol/kg
通过对比发现，冻干制剂复溶配伍稳定性方面，100mg/ml蔗糖与80mg/ml甘露醇处方两者差别不大，均满足要求。
综合冻干制剂室温和高温加速储存稳定性实验和冻干制剂复溶配伍稳定性实验，相对而言，现有快速冻干工艺条件下，使用甘露醇作为赋形剂的药液制备的冻干制剂样品在25℃、40℃的稳定性均优于蔗糖处方，而5％葡萄糖注射液复溶配伍稳定性差别不大。
4、考察药液中间体pH、API浓度对样品复溶影响实验
按照下表10配制冻干药液。
表10：考察API浓度、pH对样品复溶的影响的冻干药液配制方案
Figure PCTCN2022115176-appb-000026
叔丁醇密度ρ＝0.785g/ml，40％体积比对应200ml药液则叔丁醇为80ml，对应的叔丁醇质量为62.80g。
配液过程：
1.在搅拌条件下，先将处方量冻干保护剂溶解于约处方量80％的水中，完全溶解；
2.将处方量API加入叔丁醇和剩余的水中，搅拌溶解；
3.将上述第1、2步的溶液混合均匀；
4.过滤，01、02批不调pH，03、04批调pH至7.0；
5.灌装：01批灌装14mL；02批灌装10.5mL；03批灌装14mL；04批灌装10.5mL。灌装西林瓶为50ml规格。
01批、02批、03批、04批每批1瓶放于医用冷藏箱(2～8℃)，观察结晶现象：发现均有不同程度的结晶，具体如下图5所示。取01批结晶上清液及其复溶液(5％葡萄糖复溶)，检测含量，结果如下表11。
表11：01中间药液2-8℃放置后复溶液、放置后上清液含量及HPLC纯度
Figure PCTCN2022115176-appb-000027
上述分装制剂在真空冷冻干燥机中进行冷冻干燥。冻干条件使用多次优化后的条件，如下表12。冻干结束后，真空压塞，轧盖。得到冻干制剂，如图6所示。
表12：多次优化后的冻干条件
Figure PCTCN2022115176-appb-000028
将上述冻干制剂使用40ml和50ml 5％葡萄糖复溶，结果如下表13。
表13：40ml和50ml 5％葡萄糖复溶后目测现象
Figure PCTCN2022115176-appb-000029
40ml 5％葡萄糖复溶后照片如图7所示，其中批次3(15mg/ml的API浓度，调节pH至7.13)瓶壁无粘附物，瓶底有少许不溶颗粒，静置一段时间，溶液澄清，显示过高的API浓度可能不利于后续冻干制剂复溶。
50ml 5％葡萄糖复溶后照片如图8所示，其中批次3(15mg/ml的API浓度，调节pH至7.03)瓶壁无粘附物，瓶底无不溶颗粒，溶液澄清，显示在增大复溶溶液量的情况下将改善冻干制剂的复溶情况。
实验结论
通过对比实验结果发现，虽然叔丁醇-水混合溶剂体系对API(TH-302)的溶解度可以高达160mg/ml以上，但由于：
其一，冻干过程中的降温过程太高的浓度将导致药液会结晶分层，影响冻干过程；
其二，过高的药物浓度将影响复溶，导致复溶失败。
因此API浓度有一个合适的范围，经过多次试验，发现5-160mg/ml是比较合适的，或者进一步使用8-50mg/ml得到的药液稳定且后续冻干制剂质量较优。
5、考察API浓度、冻干保护剂浓度、叔丁醇浓度对冻干粉残留溶剂的影响实验
考察API浓度、冻干保护剂浓度、叔丁醇浓度对冻干粉残留溶剂的影响，并制备空白冻干粉作为对照。
按照下表14配制冻干药液。每个批次各10瓶，1ml/瓶，共10ml
表14：考察API浓度、冻干保护剂浓度、叔丁醇浓度对冻干粉残留溶剂的影响的冻干药液配制方案
Figure PCTCN2022115176-appb-000030
配液过程：
1.先将处方量冻干保护剂溶解于处方量水中，完全溶解。
2.处方01、02、03、04批将处方量叔丁醇加入冻干保护剂水溶液中，混合均匀。再加入处方量API，搅拌使其完全溶解。
处方05批不加叔丁醇，加入处方量API，搅拌使其完全溶解。
处方06、07批将处方量叔丁醇加入冻干保护剂水溶液中，混合均匀。不加API，作为空白对照。
3.此次实验不调节pH，分装1ml/瓶。
上述分装制剂在-70℃冰箱预冻3h，后置于冻干机中干燥62h。冻干结束后，压塞，轧盖。得到冻干制剂，如图9所示。
将制剂注入D5W进行复溶，01～07批均容易复溶，溶液澄清。
将冻干制剂样品置于40℃恒温箱中，分别在相应的天数检测HPLC纯度，结果如下表15所示。
表15：5批冻干样品在高温40℃时的HPLC纯度数据(％)
批号 0天 第3天 第5天 第10天
01 99.07％ 94.94％ 92.84％ 85.68％
02 98.54％ 98.34％ 98.40％ 98.19％
03 99.25％ 94.95％ 93.02％ 85.83％
04 99.17％ 98.25％ 97.96％ 97.76％
05 98.76％ 92.63％ 90.50％ 83.75％
同时对0天样品测叔丁醇(2瓶)和水分(1瓶)含量，结果如下表16、17所示。
表16：7批冻干样品在0天时测定的残留叔丁醇含量数据
Figure PCTCN2022115176-appb-000031
备注：05中未加入TBA。01中、01下表示同批次药液在冻干机的不同板材(中层和下层)冻干后的冻干制剂样品。
表17：7批冻干样品在0天时测定的残留水含量数据
Figure PCTCN2022115176-appb-000032
实验结论
在小冻干机制备的样品中，每瓶样品中TBA残留不均匀，蔗糖处方中TBA残留比甘露醇处方中TBA残留多。
在40℃条件下，蔗糖处方中有关物质增长较大，甘露醇处方中有关物质增长较小。
API浓度与冻干后制剂中的残留溶剂含量的关系不大，冻干保护剂浓度与冻干后制剂中的残留溶剂含量的关系也不大。
API浓度、冻干保护剂浓度、叔丁醇浓度均影响冻干后制剂中的残留溶剂含量：相较而言，甘露醇作为冻干赋形剂(冻干保护剂)所得的冻干制剂中溶剂残留较低，且在高温加速稳定性实验中，甘露醇处方的稳定性优于蔗糖处方。
6、考察不同API浓度和甘露醇浓度，不同比例叔丁醇，灌装不同体积对样品复溶的影响实验
6.1考察处方(40％叔丁醇+60mg/ml甘露醇+10mg/ml API)不同pH、不同灌装量对样品 复溶的影响
按照表18配制药液600ml。
表18：考察处方(40％叔丁醇+60mg/ml甘露醇+10mg/ml API)不同pH、不同灌装量对样品复溶的影响冻干药液配制方案
Figure PCTCN2022115176-appb-000033
叔丁醇密度ρ＝0.785g/ml，则240ml对应188.4g。
配液过程：
1.在搅拌将处方量叔丁醇加入冻干保护剂水溶液中，混合均匀；再加入处方量API，搅拌溶解；
2.除菌过滤杯过滤。01批：300ml，不调pH；02批pH 4.58，调pH至 7.07；同时分别取6ml溶液置于冷藏冰箱(2～8℃)，观察结晶现象；
3.灌装：
200mg/瓶：01批灌装 21mL，得 6瓶；02批灌装 21mL，得6瓶；
150mg/瓶：01批灌装 16mL，得 6瓶；02批灌装 16mL，得6瓶；
100mg/瓶：01批灌装 10.5mL，得 6瓶；02批灌装 10.5mL，得 6瓶。
6.2考察处方(30％叔丁醇+70mg/ml甘露醇+12.5mg/ml API)不同pH、不同灌装量对样品复溶的影响
按照表19配制药液600ml。
表19：考察处方(30％叔丁醇+70mg/ml甘露醇+12.5mg/ml API)不同pH、不同灌装量对样品复溶的影响冻干药液配制方案
Figure PCTCN2022115176-appb-000034
叔丁醇密度ρ＝0.785g/ml，则180ml对应的质量为141.3g。
配液过程：
1.在搅拌速度500rpm条件下，先将处方量冻干保护剂溶解于约处方量的水中，完全溶解；
2.将处方量叔丁醇加入冻干保护剂水溶液中，混合均匀；再加入处方量API，搅拌溶解；
3.除菌过滤杯过滤。03批：300ml，不调pH；04批pH 4.61，调pH至 7.01；同时分别取6ml溶液置于冷藏冰箱(2～8℃)，观察结晶现象；
4.灌装：
200mg/瓶：03批灌装 17mL，得 8瓶；04批灌装 17mL，得 8瓶；
150mg/瓶：03批灌装 13mL，得 7瓶；04批灌装 13mL，得 7瓶；
100mg/瓶：03批灌装 8.5mL，得 8瓶；04批灌装 8.5mL，得 8瓶；
6.3预冻、冻干
上述分装制剂在真空冷冻干燥机中进行冷冻干燥。使用表12的参数进行冻干，冻干结束后，真空压塞，轧盖。得到冻干制剂。
6.4实验结果
(1)药液中间体结晶现象
01批、02批溶液取6ml置于冷藏冰箱(2～8℃)一段时间(2h)后澄清，未见结晶；
03批、04批溶液取6ml置于冷藏冰箱(2～8℃)一段时间(2h)后有结晶现象。
(2)样品复溶
冻干制剂均为白色块状固体。
样品复溶所使用的溶剂及复溶现象如下表20。
表20：不同API浓度和甘露醇浓度，不同比例叔丁醇，灌装不同体积对样品复溶的影响实验结果
Figure PCTCN2022115176-appb-000035
常压表示将盛装冻干制剂的西林瓶打开铝盖和胶塞与外界相通后注入纯化水复溶；未标注常压表示直接通过注射器将纯化水注入盛装冻干制剂的西林瓶中(此时西林瓶内气压小于外界大气压)进行复溶。
将04(8.5ml、13ml、17ml)样品放40℃条件下10天取样，复溶，结果如下表21。
表21：不同灌装体积的04批次样品在40℃条件下存放10天后的复溶结果
Figure PCTCN2022115176-appb-000036
实验小结：04(8.5ml、13ml、17ml)样品放40℃条件下10天未影响样品复溶，其复溶现象与0天样品复溶现象相差不大。
实验结论：
总体而言，对于容积固定的容器而言，在一定范围内，冻干药液灌装体积较小所得到的冻干制剂其复溶会较容易；
对于药液是否调节pH值，相对而言，调节pH值至7左右所得到的冻干制剂其复溶情况较好。
通过具体对比可以发现，较低的API浓度、较低的甘露醇含量所得的冻干制剂更容易复溶，这提示简单的提高API浓度和甘露醇用量来提高冻干制剂的载药量将导致复溶困难，必须选择合适的API浓度、甘露醇含量来得到较高载药量且复溶容易的冻干药液。
7、考察pH对样品稳定性的影响实验
考察pH对样品稳定性的影响
按照下表22配制药液。共配制6500ml，每瓶灌装25ml，共灌装260瓶。
表22：考察pH对样品稳定性的影响实验药液配制方案
Figure PCTCN2022115176-appb-000037
叔丁醇密度ρ＝0.785g/ml，则1950ml的叔丁醇质量为1530.75g。API纯度为99.26％。
冻干制剂制备过程。
1、配液
①70％叔丁醇水溶液配制：称取叔丁醇 350.81g，水： 150.42g，即得。
②预溶：称取处方量API，在搅拌条件下，将API加入70％叔丁醇水溶液中，搅拌溶解，记为A溶液。
③在搅拌条件下，先将处方量甘露醇溶解于剩余水的70％的水中，搅拌至澄清，记为B溶液。
④混合A、B溶液，搅拌均匀。再用剩余水、剩余叔丁醇荡洗后加入混合液；药液中间体pH为 5.63。
⑤将药液平均分为3份，
药液-01：不调pH；室温下放置24h测pH 4.77。
药液-02：加入碳酸氢钠 200μl，测pH 6.60(pH在6.0～6.5)。室温下放置24h测pH 5.20。
药液-03：加入碳酸氢钠 1000μl，测pH 7.20(pH在7.0)。室温下放置24h测pH 6.90分别取0h药液中间体、室温下放置24h药液，检测含量及有关物质。
2.过滤
采用250ml除菌过滤杯过滤。
3.灌装
药液-01批灌装 24.5g，得 78瓶，即为01批次。
药液-02批灌装 24.5g，得 78瓶，即为02批次。
药液-03批灌装 24.5g，得 78瓶，即为03批次。
4.半加塞
灌装后半加塞，放入冻干机进行冷冻干燥。
5.预冻、冻干
上述分装制剂在真空冷冻干燥机中进行冷冻干燥，该型号冷冻干燥机的西林瓶排列顺序如图10所示。使用表12的冻干参数进行冻干。冻干结束后，真空压塞，轧盖。得到冻干制剂。出料前搁板温度：5.0℃，高真空下手动压塞。
实验结果
药液检测结果如表23所示。
表23：不同批次的药液在调节pH后存放不同时间的pH值数据
Figure PCTCN2022115176-appb-000038
冻干制剂样品状态：白色块状固体，具体样品如图11所示，冻干机中层中后箱样品瓶颈有粉末环绕如图12所示，可能是由于灌装时操作不当引起。
(3)样品复溶检测
分别抽取不不同批次、不同位置的冻干制剂进行复溶，其结果如下表24所示。
表24：不同pH药液冻干后不同批次中位于搁板不同位置的冻干制剂的复溶检测数据
Figure PCTCN2022115176-appb-000039
Figure PCTCN2022115176-appb-000040
注：02批(空白粉)复溶后溶液和水一样澄清；01批、02批、03批复溶后溶液比水稍浑浊。
为了进一步考察与常用的静脉输液溶液-生理盐水的复溶效果：在纯化水复溶到约5mg/ml的API浓度后，再次使用生理盐水稀释到0.5mg/ml(吸取5ml加入50ml氯化钠注射液，0.5mg/ml)：均得到澄清的溶液，然后检测pH值和渗透压数据，结果如下表25所示。
表25：不同pH药液冻干后不同批次中位于搁板不同位置的冻干制剂的复溶检测数据
Figure PCTCN2022115176-appb-000041
在不同时间段取样检测生理盐水复溶后的溶液中API的HPLC纯度，结果如下表26。
表26：不同浓度、不同时间段取样检测生理盐水复溶后的溶液中API的HPLC纯度数据
Figure PCTCN2022115176-appb-000042
(4)样品溶剂残留和稳定性检测
将冻干制剂样品置于40℃、25℃以及2-8℃恒温箱中，分别在相应的时间检测HPLC纯度，结果如下表27所示。
表27：冻干制剂样品置于40℃、25℃以及2-8℃恒温箱中，分别在相应的时间检测HPLC纯度数据
Figure PCTCN2022115176-appb-000043
Figure PCTCN2022115176-appb-000044
残留溶剂检测。
分别对01批、02批、03批0天样品测叔丁醇(2瓶)和水分(2瓶)含量，注意均取冻干机搁板中间位置样品，结果如下表28所示。
表28：3批不同pH药液制备的冻干制剂的溶剂残留含量
批号 水含量质量百分比 叔丁醇含量质量百分比
01 5.19％ 0.75％
02 5.10％ 0.70％
03 5.03％ 0.80％
8、不同叔丁醇浓度、不同赋形剂(蔗糖、甘露醇)、不同赋形剂用量、不同药物含量、不同pH值的溶液在冻干后制备实施例
由于冻干制剂的质量指标包括复溶、稳定性、残留溶剂等三个主要考察指标，而经过上述实验可以发现影响的因素包括溶剂(叔丁醇-水混合溶剂中叔丁醇体积比)、API浓度、赋形剂种类(甘露醇、蔗糖)及用量、药液pH值等，因此进行了多次多因素的实验，结果如下表28。
表28：不同叔丁醇体积比、API浓度、赋形剂种类及用量、药液pH值的药液的冻干制剂实施例数据
Figure PCTCN2022115176-appb-000045
Figure PCTCN2022115176-appb-000046
Figure PCTCN2022115176-appb-000047
备注：
TBA为叔丁醇。
Mannitol为甘露醇。
Sucrose为蔗糖。
API为TH-302原料药。
PBS为磷酸盐缓冲生理盐水，其pH值稳定在7.4左右。
处方中30％TBA/70Mannitol/10API-pH7.0表示30％体积比的叔丁醇，70mg/ml的甘露醇，10mg/ml的API，并最终加入NaHCO 3调节pH为7.0，配制方式通以上实施例；40％TBA/60Mannitol/10API表示40％体积比的叔丁醇，60mg/ml的甘露醇，10mg/ml的API，不进行pH调节(弱酸性)，其余以此类推。
除装量为1ml的批次使用5ml容积的西林瓶外，其余均使用50ml容积的西林瓶。
实验结论
1、将叔丁醇-水混合溶剂体系对TH-302的溶解度数据并结合上述实验和上表28中的85组探索实验结果可推测或验证，化合物在溶液中的含量在5-500mg/ml均是可以稳定存在的，且实验证明大于8mg/ml而小于或等于200mg/ml都是可以进行冻干的，但可能需要调整冻干参数，如进行-80℃的预冻，冻干时更低的绝对气压，更长时间的干燥且干燥温度更低等。优选的，式I化合物在溶液中的含量大于或等于8mg/ml而小于或等于25mg/ml时，冻干制剂稳定性更好，复溶更容易。式I化合物在溶液中的含量大于或等于8mg/ml而小于或等于15mg/ml，这样的含量范围将意味着冻干条件相对温和，冻干周期更短，式I化合物在溶液中的含量大于或等于8mg/ml而小于或等于10mg/ml将进一步意味着冻干制剂在商业上合适的冻干周期和冻干低温条件具有商业大批次生产价值：不合适的API含量将导致商业大批量生产上使用的大型冻干机由于温度不均匀导致的碎瓶、喷粉发生从而产率低于90％的商业生产接受程度。
2、将叔丁醇-水混合溶剂体系对TH-302的溶解度数据并结合上述实验和上表28中的85组探索实验结果可推测或验证，叔丁醇相对于所述溶液体积百分比为1％-99％或所述溶液中叔丁醇含量为7.85-777.15mg/ml(在叔丁醇密度为0.785g/ml条件下)，所制备的TH-302溶液是稳定且澄清的，且实验证明体积比为5％-95％或者溶液中叔丁醇含量为39.25-745.75mg/ml(在叔丁醇密度为0.785g/ml条件下)都是可以进行冻干的，但可能需要调整冻干参数，如进行-80℃的预冻，冻干时更低的绝对气压，更长时间的干燥且干燥温度更低等。优选的，叔丁醇体积比为30％-60％或所述溶液中叔丁醇含量为235.5-471mg/ml(在叔丁醇密度为0.785g/ml条件下)，冻干制剂稳定性更好，复溶更容易。
实际上，叔丁醇-水混合溶剂体系中，过高的叔丁醇含量将影响到后续甘露醇/蔗糖的溶解，而甘露醇/蔗糖做完赋形剂，其用量将直接印象后续冻干制剂的质量：过低的甘露醇/蔗糖将导致药液冻干后无法均匀负载在赋形剂骨架上，甚至导致冻干过程的干燥阶段出现冻干粉饼塌陷或冻干失败，因此混合溶剂中叔丁醇的含量不能为了增加TH-302的溶解性而一味提高。
3、根据叔丁醇-水混合溶剂体系对TH-302的溶解度数据并结合上述实验和上表28中的85组探索实验结果可知，赋形剂使用甘露醇、PEG2000、P188、SBECD、DSPE-MPEG2000可、蔗糖或者其同类物质均是可以得到冻干制剂的，但经过比较考虑到除冻干制剂本身的原因，如是否常规使用且容易商业购买，是否对药效、DMPK以及毒理产生影响等，综合考虑使用蔗糖、甘露醇比较合适，其制备的冻干制剂具有合适的溶液稳定性(室温下至少24小时)、冻干制剂稳定性、复溶性且容易获得符合注射制剂辅料要求的产品。
4、实际上，正如第2条所述，作为赋形剂的甘露醇/蔗糖，其含量也不是越多约好：较多的用量虽然能作为冻干骨架负载更多的药物，且利于冻干生产并提高冻干制剂的稳定 性，但却受到叔丁醇-水混合溶剂对其溶解度的影响，过高的甘露醇/蔗糖用量有可能使得混合溶剂无法完全溶解或溶液不稳定而在冻干的预冻阶段或降温阶段出现析出，因此冻干药液中作为赋形剂的甘露醇/蔗糖的用量也有一个合适的范围而满足以上要求。
蔗糖、甘露醇在所述溶液中的含量为20-300mg/ml，如此的范围能满足以上要求：药液稳定，且不易在冻干的预冻阶段或降温阶段出现析出。进一步的优选为40-100mg/ml，可以得到复溶溶液且冻干制剂稳定性好的冻干制剂。更优选为60-70mg/ml，这样的含量范围将意味着冻干条件相对温和，冻干周期符合商业生产实际(不大于10天即240小时一个生产周期)。
5、如上文第2条所述，过低的甘露醇/蔗糖将导致药液冻干后无法均匀负载在赋形剂骨架上，甚至导致冻干过程的干燥阶段出现冻干粉饼塌陷或冻干失败；而过高的甘露醇/蔗糖含量虽然能得到较好外观和稳定性的冻干制剂，但意味着单位包装单位的冻干制剂中国药物TH-302的含量太低，不符合商业销售和使用要求，因此合适的甘露醇/蔗糖与药物的比例是必要的。溶液中式I的化合物与所述赋形剂的质量比为1：(0.5-20)，如此的比例即能满足上述第1-4条的各种其他成分要求，而且能使得制备的冻干制剂具有较好外观和稳定性；优选为1:(2-12.5)能得到更好的复溶容易的冻干制剂，更优选为1:(5-10)所制备的冻干制剂将具有优异的性质：冻干制剂稳定性好，复溶容易，冻干条件温和，冻干周期短。
6、实验现象显示冻干药液的pH值将直接影响药液的稳定性和冻干制剂的复溶难易程度和冻干制剂的稳定性，实验证明药液如不添加pH调节剂(碱或碱性盐)，药液是偏酸性的，而酸性的药液稳定性较差，冻干制剂复溶不好，且冻干制剂的稳定性差；而在添加pH调节剂(碱或碱性盐)后，这些情况明显改善。
7、值得指出的是，药液在西林瓶的灌装体积也通过各种因素影响最终的冻干粉饼的外观(喷粉或粘附在西林瓶的瓶颈处)和复溶，因此西林瓶中灌装的药液的体积与西林瓶的容积的比例必须合适，以上实验数据并结合生产实际，冻干药液的灌装体积为密闭容器容积的1/3到1/2，对应的冻干制剂的体积为密闭容器容积的1/3到1/2比较合适。
9、药物在D5W溶液中的饱和溶解度(强力振摇条件)
TH-302样品约60mg，精密称定，置透明西林瓶中，加入5％葡萄糖注射液(D5W)6ml，强力振荡2分钟，作为溶解度贮备溶液。观察发现溶解度贮备溶液有不溶物，将溶解度贮备溶液过滤后呈澄清，将过滤后的滤液室温放置24小时，滤液仍然澄清，取样并经适当稀释后经HPLC检测测得该溶液含量为7.25mg/ml，此值即为药物在D5W溶液中的饱和溶解度。
由此可知含有TH-302的静脉注射用注射液其浓度应在0-7.25mg/ml内，实际上考虑到等渗以及静脉注射液中含有的葡萄糖的渗透压，静脉滴注时的输液配方还需要进一步研究确定。
10、药液在冻干前后盛装在50ml西林瓶中高度测量统计结果(计算载药量和药物质量百分比)
使用实验7中的药液进行灌装：25ml药液灌装到50ml西林瓶中，并测量灌装药液后的液位高度，平均为26.5mm。在冻干后分别测量冻干机不同搁板位置上的西林瓶中的冻干粉饼的高度，并据此大概计算体积相对于灌装前25ml的差别。
在进行的多批次实验中发现冻干前后体积有的批次增大，有的批次减小，还有的批次几乎不变，经过在冻干前后标记液位并进行体积变化的统计，发现增大或减小体积的最大幅度为10％，即体积变化在±10％以内。
在冻干前后冻干粉饼体积会减少10％的情况下：
则当药液的API含量为含量大于或等于5mg/ml而小于或等于500mg/ml时，其冻干后的 冻干粉饼载药量为5/0.9至500/0.9即为5.55-555.55mg/cm 3，同样的计算方法：
式I化合物在溶液中的含量在5-160mg/ml的药液其在冻干后的粉饼载药量为5.55-177.77mg/cm 3；
式I化合物在溶液中的含量在8-50mg/ml的药液其在冻干后的粉饼载药量为8.88-55.55mg/cm 3；
式I化合物在溶液中的含量在8-25mg/ml的药液其在冻干后的粉饼载药量为8.88-27.77mg/cm 3；
式I化合物在溶液中的含量在8-15mg/ml的药液其在冻干后的粉饼载药量为8.88-16.66mg/cm 3；
式I化合物在溶液中的含量在8-10mg/ml的药液其在冻干后的粉饼载药量为8.88-11.11mg/cm 3。
如药液的含量为6、7、7.5、8、8.5、10、12.5、15、20、25mg/ml则对应冻干后粉饼载药量为6.66、7.77、8.33、8.88、9.44、11.11、13.88、16.66、22.22、27.77mg/cm 3。
在冻干前后冻干粉饼体积会增大10％的情况下：
则当药液的API含量为含量大于或等于5mg/ml而小于或等于500mg/ml时，其冻干后的冻干粉饼载药量为5/1.1至500/1.1即为4.55-454.55mg/cm 3，同样的计算方法：
式I化合物在溶液中的含量在5-160mg/ml的药液其在冻干后的粉饼载药量为4.55-145.45mg/cm 3；
式I化合物在溶液中的含量在8-50mg/ml的药液其在冻干后的粉饼载药量为7.27-45.45mg/cm 3；
式I化合物在溶液中的含量在8-25mg/ml的药液其在冻干后的粉饼载药量为7.27-22.73mg/cm 3；
式I化合物在溶液中的含量在8-15mg/ml的药液其在冻干后的粉饼载药量为7.27-13.64mg/cm 3；
式I化合物在溶液中的含量在8-10mg/ml的药液其在冻干后的粉饼载药量为7.27-9.09mg/cm 3。
如药液的含量为6、7、7.5、8、8.5、10、12.5、15、20、25mg/ml则对应冻干后粉饼载药量为5.45、6.36、6.82、7.27、7.73、9.09、11.36、13.64、18.18、22.73mg/cm 3。
由于冻干后的药液中含有API药物、赋形剂以及残留的叔丁醇溶剂和水，而申请人制备的冻干制剂冻干制剂的API药物、赋形剂的质量比为1：(0.5-20)，而最大可容许的叔丁醇含量为质量比1.75％，水含量为质量比6％，则冻干制剂中API药物的质量百分比下限为(1/20+1)*(1-6％-1.75％)＝4.76％*92.25％＝4.39％，而上限为(1/0.5+1)*(1-0％-0％)＝66.66％，即冻干制剂中API药物大于或等于4.39％而小于66.66％。

Claims (28)

  1. 溶液，含有下式I的化合物以及水和叔丁醇：
    Figure PCTCN2022115176-appb-100001
    其中,R各自独立地选自H、-CH 3、-CH 2CH 3，X各自独立地选自Br、Cl、OMs、OTs，
    水和叔丁醇作为混合溶剂，
    式I的化合物在溶液中的含量大于或等于1mg/ml而小于或等于500mg/ml。
  2. 用于制备高载药量冻干制剂的溶液，含有下式I的化合物以及水和叔丁醇、赋形剂：
    Figure PCTCN2022115176-appb-100002
    其中,R各自独立地选自H、-CH 3、-CH 2CH 3，X各自独立地选自Br、Cl、OMs、OTs，
    水和叔丁醇作为混合溶剂，
    式I的化合物在溶液中的含量大于或等于5mg/ml而小于或等于500mg/ml。
  3. 根据权利要求1或2所述的溶液，其中，式I的化合物选自
    Figure PCTCN2022115176-appb-100003
  4. 根据权利要求1或2所述的溶液，其中，式I的化合物在溶液中的含量大于或等于5mg/ml而小于或等于160mg/ml，
    优选的，式I化合物在溶液中的含量大于或等于8mg/ml而小于或等于50mg/ml，
    优选的，式I化合物在溶液中的含量大于或等于8mg/ml而小于或等于25mg/ml；
    更优选的，式I化合物在溶液中的含量大于或等于8mg/ml而小于或等于15mg/ml，
    进一步优选的，式I化合物在溶液中的含量大于或等于8mg/ml而小于或等于10mg/ml。
  5. 根据权利要求1或2所述的溶液，其中，
    叔丁醇相对于所述溶液体积百分比为1％-99％，优选为5％-95％，更优选为30％-60％；
    或
    所述溶液中叔丁醇含量为7.85-777.15mg/ml，优选为39.25-745.75mg/ml，更优选为235.5-471mg/ml。
  6. 根据权利要求2所述的溶液，其中，所述赋形剂选自糖类、多元醇类、聚乙烯吡咯烷酮类、蛋白类、泊洛沙姆或其组合，
    糖类选自蔗糖、葡聚糖、环糊精、麦芽糊精、海藻糖、乳糖、麦芽糖、葡萄糖；
    多元醇类选自甘油、山梨醇、甘露醇、肌醇、乙二醇、聚乙二醇、聚山梨醇酯、侧金盏花醇；
    蛋白类选自白蛋白，优选为牛血清白蛋白、人白蛋白；
    泊洛沙姆选自泊洛沙姆182、泊洛沙姆184、泊洛沙姆188、泊洛沙姆407。
  7. 根据权利要求2所述的溶液，其中，所述赋形剂选自蔗糖、甘露醇，
    蔗糖、甘露醇在所述溶液中的含量为20-300mg/ml，优选为40-100mg/ml，更优选为60-80mg/ml，进一步优选为60-70mg/ml。
  8. 根据权利要求2所述的溶液，其中，溶液中式I的化合物与所述赋形剂的质量比为1：(0.5-20)，优选为1:(1-15)，更优选为1:(2-12.5)，进一步优选为1:(5-10)。
  9. 根据权利要求1或2所述的溶液，其中，还含有pH调节剂，pH调节剂选自盐酸、氢氧化钠、三乙醇胺、柠檬酸、磷酸、乳酸、酒石酸、琥珀酸、碳酸氢钠、碳酸钠或它们的混合物。
  10. 用于制备高载药量冻干制剂的溶液，含有
    下式I-1的化合物以及水、叔丁醇和蔗糖，
    或
    下式I-1的化合物以及水、叔丁醇和甘露醇，
    Figure PCTCN2022115176-appb-100004
    其中，
    水和叔丁醇作为混合溶剂，
    叔丁醇相对于所述溶液体积含量为30％、40％、60％或所述溶液中叔丁醇含量为235.5、314、471mg/ml，
    式I-1的化合物在溶液中的含量为6、7、7.5、8、8.5、10、12.5、15、20、25mg/ml。
    蔗糖或甘露醇作为赋形剂，在溶液中的含量为40、50、60、70、75、80、90、100mg/ml。
  11. 用于制备高载药量冻干制剂的溶液，含有
    下式I-1化合物以及水、叔丁醇和蔗糖、pH调节剂，
    或
    下式I-1化合物以及水、叔丁醇和甘露醇、pH调节剂，
    Figure PCTCN2022115176-appb-100005
    其中，
    水和叔丁醇作为混合溶剂，
    叔丁醇相对于所述溶液体积含量为30％、40％、60％或所述溶液中叔丁醇含量为235.5、3 14、471mg/ml，
    式I-1的化合物在溶液中的含量为6、7、7.5、8、8.5、10、12.5、15、20、25mg/ml，
    蔗糖或甘露醇作为赋形剂，在溶液中的含量为40、50、60、70、75、80、90、100mg/ml，
    所述pH调节剂的含量使得溶液的pH值为4-9，优选为6-8。
  12. 用于制备高载药量冻干制剂的溶液，由
    下式I-1的化合物以及水、叔丁醇和蔗糖、碳酸氢钠组成；
    或
    下式I-1的化合物以及水、叔丁醇和甘露醇、碳酸氢钠组成；
    Figure PCTCN2022115176-appb-100006
    水和叔丁醇作为混合溶剂，
    叔丁醇相对于所述溶液体积含量为30％、40％、60％或所述溶液中叔丁醇含量为235.5、314、471mg/ml，
    式I-1的化合物在溶液中的含量为6、7、7.5、8、8.5、10、12.5、15、20、25mg/ml，
    蔗糖或甘露醇作为赋形剂，在溶液中的含量为40、50、60、70、75、80、90、100mg/ml，
    所述碳酸氢钠的含量使得溶液的pH值为4-9，优选为6-8。
  13. 根据权利要求10或11或12所述的溶液，其中，
    所述溶液中化合物I-1与蔗糖或甘露醇的质量比为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。
  14. 冻干制剂，含有下式I的化合物以及赋形剂、残留的溶剂成分：
    Figure PCTCN2022115176-appb-100007
    其中,R各自独立地选自H、-CH 3、-CH 2CH 3，X各自独立地选自Br、Cl、OMs、OTs，
    冻干制剂中式I化合物的载药量大于5mg/cm 3而小于或等于555.55mg/cm 3,或
    冻干制剂中式I化合物的载药量大于或等于4.55mg/cm 3而小于500mg/cm 3，或
    冻干制剂中式I化合物的质量百分比大于或等于4.39％而小于66.66％，
    所述残留的溶剂成分为水和叔丁醇。
  15. 冻干制剂，含有下式I的化合物以及赋形剂、残留的溶剂成分、pH调节剂：
    Figure PCTCN2022115176-appb-100008
    其中,R各自独立地选自H、-CH 3、-CH 2CH 3，X各自独立地选自Br、Cl、OMs、OTs，
    冻干制剂中式I化合物的载药量大于5mg/cm 3而小于或等于555.55mg/cm 3,或
    冻干制剂中式I化合物的载药量大于或等于4.55mg/cm 3而小于500mg/cm 3，或
    冻干制剂中式I化合物的质量百分比为大于或等于4.39％而小于66.66％，
    所述残留的溶剂成分为水和叔丁醇。
  16. 冻干制剂，含有下式I的化合物以及赋形剂：
    Figure PCTCN2022115176-appb-100009
    其中,R各自独立地选自H、-CH 3、-CH 2CH 3，X各自独立地选自Br、Cl、OMs、OTs，
    冻干制剂中式I化合物的载药量大于5mg/cm 3而小于或等于555.55mg/cm 3,或
    冻干制剂中式I化合物的载药量大于或等于4.55mg/cm 3而小于500mg/cm 3，或
    冻干制剂中式I化合物的质量百分比为大于或等于4.39％而小于66.66％。
  17. 权利要求14-16中任意一项所述的冻干制剂，其中，式I的化合物选自
    Figure PCTCN2022115176-appb-100010
  18. 根据权利要求14-16中任意一项所述的冻干制剂，其中，所述赋形剂为糖类、多元醇类、聚乙烯吡咯烷酮类、蛋白类、泊洛沙姆或其组合，
    糖类选自蔗糖、葡聚糖、环糊精、麦芽糊精、海藻糖、乳糖、麦芽糖、葡萄糖，
    多元醇类选自甘油、山梨醇、甘露醇、肌醇、乙二醇、聚乙二醇、聚山梨醇酯、侧金盏花醇，
    蛋白类选自白蛋白，优选为牛血清白蛋白、人白蛋白，
    泊洛沙姆选自泊洛沙姆182、泊洛沙姆184、泊洛沙姆188、泊洛沙姆407。
  19. 根据权利要求14-18中任意一项所述的冻干制剂，其中，
    残留水含量为质量百分比小于或等于6％，优选为小于或等于2％，更优选为小于或等于1％，进一步优选为小于或等于0.5％，
    残留叔丁醇含量为质量百分比小于或等于1.75％，优选为小于或等于1％，更优选为小于或等于0.5％。
  20. 根据权利要求14-18中任意一项所述的冻干制剂，其中，
    化合物I与所述赋形剂的质量比为1:(0.5-20)，优选为1:(1-15)，更优选为1:(2-12.5)，进一步优选为1:(5-10)。
  21. 根据权利要求14-18中任意一项所述的冻干制剂，其中，
    冻干制剂中式I化合物的载药量大于或等于5.55mg/cm 3而小于或等于177.77mg/cm 3，优选为大于或等于8.88mg/cm 3而小于或等于55.55mg/cm 3，更优选为大于或等于8.88mg/cm 3而小于或等于27.77mg/cm 3，进一步优选为大于或等于8.88mg/cm 3而小于或等于16.66mg/cm 3，再进一步优选为大于或等于8.88mg/cm 3而小于或等于11.11mg/cm 3；
    或者
    冻干制剂中式I化合物的载药量大于或等于4.55mg/cm 3而小于或等于145.45mg/cm 3，优选为大于或等于7.27mg/cm 3而小于或等于45.45mg/cm 3，更优选为大于或等于7.27mg/cm 3而小于或等于22.73mg/cm 3，进一步优选为大于或等于7.27mg/cm 3而小于或等于13.64mg/cm 3，再进一步优选为大于或等于7.27mg/cm 3而小于或等于9.09mg/cm 3。
  22. 冻干制剂，实质由以下化合物I-1以及赋形剂、残留水和残留叔丁醇组成：
    Figure PCTCN2022115176-appb-100011
    冻干制剂中化合物I-1的载药量为6.66、7.77、8.33、8.88、9.44、11.11、13.88、16.66、22.22、27.77mg/cm 3，或者冻干制剂中化合物I-1的载药量为5.45、6.36、6.82、7.27、7.73、9.09、11.36、13.64、18.18、22.73mg/cm 3；
    赋形剂为蔗糖或甘露醇；
    化合物I-1与所述赋形剂的质量比为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；
    残留水含量为质量百分比小于或等于6％，优选为小于或等于2％，更优选为小于或等于1％，进一步优选为小于或等于0.5％；
    残留叔丁醇含量为质量百分比小于或等于1.75％，优选为小于或等于1％，更优选为小于或等于0.5％。
  23. 冻干制剂，实质由下式化合物以及赋形剂、残留水和残留叔丁醇、pH调节剂组成：
    Figure PCTCN2022115176-appb-100012
    冻干制剂中化合物I-1的载药量为6.66、7.77、8.33、8.88、9.44、11.11、13.88、16.66、22.22、27.77mg/cm 3，或者冻干制剂中化合物I-1的载药量为5.45、6.36、6.82、7.27、7.73、9.09、11.36、13.64、18.18、22.73mg/cm 3；
    赋形剂为蔗糖或甘露醇；
    化合物I-1与所述赋形剂的质量比为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；
    残留水含量为质量百分比小于或等于6％，优选为小于或等于2％，更优选为小于或等于1％，进一步优选为小于或等于0.5％；
    残留叔丁醇含量为质量百分比小于或等于1.75％，优选为小于或等于1％，更优选为小于或等于0.5％；
    pH调节剂为碳酸氢钠，含量为0.01-0.10mg/cm 3。
  24. 冻干制剂，实质由下式化合物以及赋形剂组成：
    Figure PCTCN2022115176-appb-100013
    冻干制剂中化合物I-1的载药量为6.66、7.77、8.33、8.88、9.44、11.11、13.88、16.66、22.22、27.77mg/cm 3，或者冻干制剂中化合物I-1的载药量为5.45、6.36、6.82、7.27、7.73、9.09、11.36、13.64、18.18、22.73mg/cm 3；
    赋形剂为蔗糖或甘露醇；
    化合物I-1与所述赋形剂的质量比为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。
  25. 盛装有权利要求14-24中任意一项所述的冻干制剂的制剂单位包装，具有以下特征:
    所述冻干制剂被盛装在1000ml容积的密闭容器中，冻干制剂的体积为密闭容器容积的1/5到2/3，优选为1/3到1/2，含有式I的化合物的含量为4000-16500mg；或
    所述冻干制剂被盛装在500ml容积的密闭容器中，冻干制剂的体积为密闭容器容积的1/5到2/3，优选为1/3到1/2，含有式I的化合物的含量为2000-8000mg；或
    所述冻干制剂被盛装在250ml容积的密闭容器中，冻干制剂的体积为密闭容器容积的1/5到2/3，优选为1/3到1/2，含有式I的化合物的含量为1000-4000mg；或
    所述冻干制剂被盛装在100ml容积的密闭容器中，冻干制剂的体积为密闭容器容积的1/5到2/3，优选为1/3到1/2，含有式I的化合物的含量为400-2000mg；或
    所述冻干制剂被盛装在50ml容积的密闭容器中，冻干制剂的体积为密闭容器容积的1/5到2/3，优选为1/3到1/2，含有式I的化合物的含量为50-800mg；或
    所述冻干制剂被盛装在30ml容积的密闭容器中，冻干制剂的体积为密闭容器容积的1/5到2/3，优选为1/3到1/2，含有式I的化合物的含量为150-600mg；或
    所述冻干制剂被盛装在25ml容积的密闭容器中，冻干制剂的体积为密闭容器容积的1/5到2/3，优选为1/3到1/2，含有式I的化合物的含量为125-500mg；或
    所述冻干制剂被盛装在20ml容积的密闭容器中，冻干制剂的体积为密闭容器容积的1/5到2/3，优选为1/3到1/2，含有式I的化合物的含量为100-400mg；或
    所述冻干制剂被盛装在18ml容积的密闭容器中，冻干制剂的体积为密闭容器容积的1/5到2/3，优选为1/3到1/2，含有式I的化合物的含量为90-360mg；或
    所述冻干制剂被盛装在15ml容积的密闭容器中，冻干制剂的体积为密闭容器容积的1/5到2/3，优选为1/3到1/2，含有式I的化合物的含量为75-300mg；或
    所述冻干制剂被盛装在10ml容积的密闭容器中，冻干制剂的体积为密闭容器容积的1/5到2/3，优选为1/3到1/2，含有式I的化合物的含量为50-200mg；或
    所述冻干制剂被盛装在8ml容积的密闭容器中，冻干制剂的体积为密闭容器容积的1/ 5到2/3，优选为1/3到1/2，含有式I的化合物的含量为40-160mg；或
    所述冻干制剂被盛装在7ml容积的密闭容器中，冻干制剂的体积为密闭容器容积的1/5到2/3，优选为1/3到1/2，含有式I的化合物的含量为35-140mg；或
    所述冻干制剂被盛装在5ml容积的密闭容器中，冻干制剂的体积为密闭容器容积的1/5到2/3，优选为1/3到1/2，含有式I的化合物的含量为25-100mg；或
    所述冻干制剂被盛装在3ml容积的密闭容器中，冻干制剂的体积为密闭容器容积的1/5到2/3，优选为1/3到1/2，含有式I的化合物的含量为15-60mg；或
    所述冻干制剂被盛装在2ml容积的密闭容器中，冻干制剂的体积为密闭容器容积的1/5到2/3，优选为1/3到1/2，含有式I的化合物的含量为10-40mg；或
    所述冻干制剂被盛装在1.5ml容积的密闭容器中，冻干制剂的体积为密闭容器容积的1/5到2/3，优选为1/3到1/2，含有式I的化合物的含量为7.5-30mg。
  26. 一种含有TH-302的静脉注射用注射液，其溶剂为水，溶质包括TH-302原料药、调节等渗试剂、甘露醇或蔗糖、叔丁醇以及碳酸氢钠，所述调节等渗试剂选自葡萄糖、氯化钠。
  27. 制备高载药量冻干制剂的溶液的配制方法，包括以下操作：
    操作一：称取处方量的TH-302的原料药，加入叔丁醇水溶液中，搅拌至澄清得第一溶液；
    操作二：将处方量甘露醇或蔗糖溶解于适量水中，搅拌至澄清后得第二溶液；
    操作三：混合第一溶液和第二溶液并最终加入剩下的处方量叔丁醇后加入适量的水定容至预定体积，加入处方量的碳酸氢钠，搅拌混合均匀，
    其中，
    所述高载药量冻干制剂的溶液中TH-302原料药的含量大于5mg/ml而小于或等于500mg/ml，
    叔丁醇相对于所述高载药量冻干制剂的溶液体积百分比为1％-99％，或所述溶液中叔丁醇含量为7.85-777.15mg/ml，
    蔗糖或甘露醇在所述高载药量冻干制剂的溶液中的含量为20-300mg/ml，
    所述高载药量冻干制剂的溶液中TH-302与蔗糖或甘露醇的质量比为1:(0.5-20)，
    所述叔丁醇水溶液中叔丁醇体积比为30-90％，
    碳酸氢钠在所述高载药量冻干制剂的溶液中的含量为0.01-0.10mg/ml。
  28. 高载药量冻干制剂的制备方法，包括以下操作：
    预冻，将权利要求1-13中任意一项所述的的适量溶液置于冻干系统中进行预冻，预冻过程包括0℃保温一段时间和-20到-55℃保温一段时间；
    一次干燥，预冻完成后升温至-10到10℃并保温一段时间，并维持真空进行干燥；
    二次干燥，一次干燥完成后升温至20到40℃并保温一段时间，并维持真空进行干燥。
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