WO2024179467A1 - 溶液、冻干制剂、冻干制剂单位包装、注射液及注射液配制方法 - Google Patents

溶液、冻干制剂、冻干制剂单位包装、注射液及注射液配制方法 Download PDF

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WO2024179467A1
WO2024179467A1 PCT/CN2024/078807 CN2024078807W WO2024179467A1 WO 2024179467 A1 WO2024179467 A1 WO 2024179467A1 CN 2024078807 W CN2024078807 W CN 2024078807W WO 2024179467 A1 WO2024179467 A1 WO 2024179467A1
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volume
lyophilized preparation
freeze
content
solution
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English (en)
French (fr)
Inventor
于继兵
段建新
蔡晓宏
房启龙
申广彬
阮美珍
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Ascentawits Pharmaceuticals Ltd
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Ascentawits Pharmaceuticals Ltd
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Priority to KR1020257031999A priority Critical patent/KR20250160163A/ko
Priority to EP24763148.4A priority patent/EP4674418A1/en
Priority to CN202480015234.5A priority patent/CN120835787A/zh
Publication of WO2024179467A1 publication Critical patent/WO2024179467A1/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
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • 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

Definitions

  • the invention relates to the development of a freeze-dried preparation of TH-302, and belongs to the technical field of pharmaceutical preparations.
  • TH-302 is a 2-nitroimidazole prodrug, designed and synthesized by researchers at Threshold Pharmaceuticals in 2006 (WO2007002931A2, Phosphoramidate alkylator prodrugs) as a selective hypoxia-activated DNA alkylating agent with high cytotoxicity. It can be converted into dibromoisophosphoramide nitrogen mustard with alkylating agent activity in tumor hypoxic areas or when activated by acid, but it is almost inactive under normoxic or normal pH conditions.
  • a solution (20 mL) of TH-302 (100 mg) and sucrose (1 g) was added to a lyophilization vial and lyophilized to produce a lyophilized unit dosage form of TH-302 with a drug load of less than 5 mg/cm 3 .
  • the unit dosage form was dissolved in 5% glucose injection and an appropriate amount of the solution was administered to the patient;
  • TH-302 is dissolved in anhydrous 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 a solvent (e.g., ethanol);
  • the dosing regimen for the subsequent Phase I clinical trial of TH-302 in human patients used a lyophilized preparation.
  • the lyophilized preparation of TH-302 for injection was prepared in a 100-mL glass vial with a drug loading of 100 mg/100 mL. It was stored under controlled conditions at 2-8°C and injected into 250 mL of 5% glucose injection in the lyophilized preparation bottle during use and intravenously dripped within 30 minutes via an infusion pump.
  • the liquid preparation contains 50 mg/mL to about 300 mg/mL of TH-302, a nonionic surfactant (such as Tween 80), a carrier ethanol, and may also contain dimethylacetamide.
  • TH-302 as a therapeutic drug to treat various cancers and tumors (NCT02402062, NCT02020226, NCT02076230, NCT01381822, NCT02093962, NCT01440088, NCT02255110, NCT02342379, NCT01864538, NCT01149915, NCT02433639, NCT00743379, NCT01485042, NCT01721941, NCT02047500, NCT00742963, NCT01497444, NCT00495144, NCT01746979, NCT01144455, NCT01403610, NCT01522872, NCT01833546, NCT02598687, NCT03098160, NCT024968
  • Threshold found that the effective dose of TH-302 for treating various indications is (WO2012135757A2, Methods for treating cancer):
  • the drug may be administered intravenously in a weekly dose of about 480 mg/ m2 to about 670 mg/ m2 or, for example, 575 mg/ m2 .
  • a lyophilized preparation with a drug loading of less than 5 mg/cm 3 is used (adding 20 mL of an aqueous solution containing 100 mg of TH-302 and 1 g of sucrose to a 50 mL lyophilized vial for lyophilization to produce a lyophilized unit dosage form of TH-302, with a drug loading of less than 5 mg/cm 3 ), at least 3 bottles are required, and if a larger dose is used, 13 bottles are required, which is not convenient for clinical use, and the cost of medication for patients is too high.
  • Threshold used a concentrated injection (WO2015013448A1, Treatment of pancreatic cancer with a combination of a hypoxia-activated prodrug and a taxane):
  • TH-302 (concentrate for administration solution) used in clinical trials is a sterile liquid formulation of TH-302.
  • TH-302 is formulated with 70% anhydrous ethanol, 25% dimethylacetamide and 5% polysorbate 80. It is provided by the sponsor in a 10 mL glass vial with a rubber stopper and flip-off seal.
  • the TH-302 drug product is a clear, colorless to pale yellow solution that is essentially free of visible particles.
  • each single-use vial contains a nominal fill volume of 6.5 mL of TH-302 drug product (equivalent to 100 mg/mL) and is clearly labeled, which discloses the batch number, route of administration, required storage conditions, name of the sponsor and appropriate warning labels required by applicable regulations. Dilution is required according to the pharmacy manual before administration.
  • TH-302 Before administration, dilute to a total volume of 500 mL (1000 mL for a total dose of ⁇ 1000 mg) with a commercially available 5% glucose aqueous solution to obtain the desired final concentration.
  • Each dose of TH-302 was prepared with a 5% glucose aqueous solution without di(2-ethylhexyl) phthalate (DEHP-free) and administered intravenously using a DEHP-free intravenous infusion administration device.
  • DEHP-free di(2-ethylhexyl) phthalate
  • TH-302 Reactions to administration of TH-302 (primarily caused by dimethylacetamide) have been observed. These reactions are characterized by lip swelling and urticaria, which respond to treatment with steroids and antihistamines. It is recommended to include a steroid such as dexamethasone (or equivalent) in the antiemetic regimen prior to administration. 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 require discontinuation of treatment, it should be determined that the reaction may or may not be an immunoglobulin E-mediated process.
  • an antihistamine e.g., diphenhydramine 25-50 mg orally, intramuscularly, or slowly i.v., or equivalent
  • a low-dose steroid e.g., hydrocortisone, 100 mg i.v., or equivalent
  • 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, depending on their severity. Reactions to the administration of TH-302 should be evaluated and treated in a similar manner. For all reactions to TH-302, the investigator should consult with the medical monitor to determine the appropriate course of action for future treatment.
  • the high-concentration concentrated TH-302 injection solves the problem of low drug loading in freeze-dried preparations.
  • excipients that may cause adverse reactions
  • related adverse reactions may occur in clinical trials, increasing the risk of medication for patients.
  • the currently developed lyophilized preparation uses water and sucrose solution to dissolve TH-302 for lyophilization and does not contain other excipients.
  • the drug content of the solution before lyophilization is too low, it is impossible to obtain a high-drug-load lyophilized preparation that is suitable for clinical use and subsequent commercial production and sales.
  • TH-302 i.e. TH-302
  • Threshold Company with a drug loading of 100 mg/mL: using 70% anhydrous ethanol, 25% dimethylacetamide and 5% polysorbate 80 to prepare a concentrated injection solution of TH-302:
  • Table 1 TH-302 solubility data in aqueous solutions with different pH values and different additives
  • 1% ethanol aqueous solution, 20% ethanol aqueous solution, 1% N,N-dimethylacetamide aqueous solution, 1% polyethylene glycol aqueous solution, and 1% Tween 80 aqueous solution are all in volume ratio.
  • the ratio of the mixed solvents in Table 2 is by volume, and TH-302 was purchased commercially.
  • Tert-butyl alcohol is a colorless crystal that is easily supercooled and becomes a liquid in the presence of a small amount of water. It has a camphor-like odor and is hygroscopic. Its Chinese name is 2-methyl-2-propanol, tert-butyl alcohol, trimethylmethanol, etc. Its melting point is 25.7°C, so it is a colorless transparent liquid or colorless crystal at room temperature.
  • tert-butyl alcohol may crystallize at room temperature (25°C), and can also freeze at several degrees below zero after mixing with water. It can be completely frozen in existing freeze dryers.
  • Tert-butyl alcohol has a high vapor pressure. High vapor pressure is conducive to sublimation and saves freeze-drying time.
  • Tert-butyl alcohol and water can be mixed in any proportion. This is extremely important. It can increase the solubility of some fat-soluble drugs in water. At the same time, for some unstable drugs in aqueous solution, adding an appropriate amount of tert-butyl alcohol can inhibit the decomposition of the drug and enhance the stability of the drug.
  • Tert-butyl alcohol is easy to freeze-dry, and the residual amount in the preparation is low. During the freeze-drying process, most of the tert-butyl alcohol can be sublimated in the primary drying stage, and the residual amount in the preparation is very low.
  • Tert-butyl alcohol itself forms needle-shaped crystals during freezing, which can change the crystallization mode of the solute and facilitate sublimation.
  • a small amount of tert-butyl alcohol is added to water to form a tert-butyl alcohol-water co-solvent, the crystal state of water can be changed, and needle-shaped crystals with a large surface area are formed during the freezing process.
  • tubular channels are left after the ice crystals sublimate, which greatly reduces the flow resistance of water vapor and significantly increases the sublimation rate. Therefore, tert-butyl alcohol can be used to accelerate the mass transfer process during freeze-drying.
  • tert-butanol + water as a solvent can not only prepare a high concentration solution of the compound of formula I, but also the properties of tert-butanol itself are also suitable as a lyophilization auxiliary material.
  • TH-302 has a low solubility in aqueous solution; as the concentration of tert-butanol in the tert-butanol aqueous solution increases, the solubility of the raw material increases continuously; it is inferred that the solubility is highest in 70% tert-butanol aqueous solution (V/V), and then as the concentration of tert-butanol in the tert-butanol aqueous solution increases, the solubility decreases.
  • the inventors proposed the scheme of the present invention that tert-butyl alcohol + water is used as a solvent and appropriate excipients are added to obtain a high-concentration solution, and based on this prescription, a lyophilized preparation of TH-302 and its similar compounds with a high drug loading was developed.
  • the present invention provides the following high-concentration solution containing TH-302-like compounds.
  • R is independently selected from H, -CH 3 , -CH 2 CH 3 , and 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-drug-load lyophilized preparation comprising a compound of the following formula I, water, tert-butyl alcohol, and an excipient:
  • R is independently selected from H, -CH 3 , -CH 2 CH 3 , and 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 volume percentage of tert-butyl alcohol relative to the solution is selected to be 1%-99%, preferably 5%-95%, and more preferably 30%-60%;
  • the content of tert-butyl alcohol in the solution is 7.85-777.15 mg/mL, preferably 39.25-745.75 mg/mL, and more preferably 235.5-471 mg/mL.
  • the volume percentage of tert-butanol relative to the solution is 1%-99% and the tert-butanol content in the solution is 7.85-777.15 mg/mL, which is a corresponding relationship.
  • the volume percentage of tert-butanol relative to the solution is 1%, and the corresponding tert-butanol content in the solution is 7.85 mg/mL.
  • the conversion coefficient is the density of tert-butanol.
  • the density of tert-butanol used by the applicant here is 0.785 g/mL. In fact, the density of products from different manufacturers at different temperatures is different, generally between 0.775-0.786 g/mL.
  • the solution for preparing the high-drug-loaded lyophilized preparation of the present invention contains at least one excipient.
  • compositions refer to additives or carriers that can contribute to the stability of the API in the preparation. In the preparation of lyophilized preparations, it is necessary to add excipients or lyoprotectants to the solution used for lyophilization.
  • Some drug solutions can be successfully freeze-dried, while others will quickly collapse or melt into oily substances after freeze-drying. In order to successfully freeze-dry certain drug solutions and obtain stable freeze-dried preparations, it is necessary to add some excipients that do not react with the drug.
  • freeze-dried protectants sometimes also called fillers, fillers, excipients, buffers, bases, skeletons, etc.
  • freeze-dried protectants must be chemically inert to drug solutions.
  • freeze-drying protective agents According to the chemical composition of freeze-drying protective agents, they can be divided into the following categories:
  • Salts sodium thiosulfate, calcium lactate, sodium glutamate, sodium chloride, potassium chloride, sodium sulfate, ammonium acetate, ammonium chloride, etc.
  • Carbohydrates sucrose, lactose, maltose, glucose, raffinose, fructose, hexose, etc.
  • Alcohols sorbitol, ethanol, glycerol, mannitol, inositol, xylitol, etc.
  • Acids citric acid, phosphoric acid, tartaric acid, amino acids, ethylenediaminotetraacetic acid (EDTA), etc.
  • Alkali sodium hydroxide, sodium bicarbonate, etc.
  • Polymers dextran, polyethylene glycol, polysorbate, PVP, poloxamer, etc.
  • Vitamin C Vitamin C
  • Vitamin E Vitamin E
  • Vitamin K Vitamin K
  • thiourea etc.
  • the degree of polymerization (molecular weight) of the above polymers is wide, taking polysorbate and polyethylene glycol as examples.
  • the molecular weight of polysorbate can be 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.
  • the non-limiting example of polysorbate includes: 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.
  • PEG polyethylene glycol
  • Non-limiting examples of PEG include: PEG200, PEG300, PEG400, PEG540 and PEG600.
  • Poloxamer has the general formula of HO(C 2 H 4 O) a (C 3 H 6 O) b (C 2 H 4 O) c H, wherein a and c are 2-130, and b is 15-67. It contains 81.8 ⁇ 1.9% polyoxyethylene and is a polyoxyethylene polyoxypropylene ether block copolymer. It has different grades: poloxamer 182, poloxamer 184, poloxamer 188, and poloxamer 407, which correspond to polymers of different molecular weights, such as poloxamer 188, whose molecular weight is 7680-9510.
  • freeze-drying protective agents There are many functions of freeze-drying protective agents, which are summarized as follows:
  • Microorganisms such as bacteria and viruses need to grow and reproduce in specific culture media, but these culture media are often difficult to separate from the microorganisms. They can be successfully freeze-dried in these culture media, such as broth, skim milk and protein.
  • freeze-dried preparations have very low concentrations and very little dry matter content.
  • the dried part during freeze-drying will be carried away by the sublimation airflow.
  • fillers In order to increase the concentration and the content of dry matter so that the freeze-dried product can form a more ideal mass, it is necessary to add fillers to keep the concentration of solid matter within a certain range.
  • These fillers or excipients are sucrose, lactose, inositol, skim milk, hydrolyzed protein, dextran, sorbitol, polyvinyl pyrrolidone (PVP), etc.
  • bioactive 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 the damage during freezing and drying, such as dimethyl sulfoxide, glycerol, dextran, sugars and PVP.
  • Adding certain substances can increase the disintegration temperature of the product and make it easier to freeze-dry the product. 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, sulfite compounds, sodium aspartate, etc.
  • the lyoprotectant is selected from sugars, polyols, polyvinyl pyrrolidones, proteins, poloxamers or a combination thereof.
  • the carbohydrate is selected from sucrose, dextran, cyclodextrin, maltodextrin, trehalose, lactose, maltose, and glucose;
  • the polyols are selected from glycerol, sorbitol, mannitol, inositol, ethylene glycol, polyethylene glycol (PEG), polysorbate, and adonitol;
  • the protein is selected from albumin, preferably bovine serum albumin and human albumin;
  • the poloxamer is selected from poloxamer 182, poloxamer 184, poloxamer 188, and 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 with the drug can be combined, and the combination should meet the requirements for drug compatibility.
  • the excipient is selected from sucrose and mannitol, and the content of sucrose and mannitol in the solution is 20-300 mg/mL, preferably 40-100 mg/mL, more preferably 60-80 mg/mL, and further preferably 60-70 mg/mL.
  • Sucrose or mannitol is generally used alone, and can be used in combination under special circumstances.
  • the preferred solution of the present application is to use only mannitol or only sucrose.
  • the mass ratio of TH-302 to excipient in the solution of the present invention for preparing a high drug loading lyophilized preparation is 1(0.5-20), preferably 1:(1-15), more preferably 1:(2-12.5), and further preferably 1:(5-10).
  • the mass ratio of TH-302 and similar compounds to excipients is the drug loading ratio of the lyophilized solution.
  • the lyophilized body after lyophilization uses the excipients as the skeleton, and the drug will be adsorbed or loaded on the skeleton. Therefore, the drug loading ratio of the drug solution before lyophilization, that is, the mass ratio of TH-302 and excipients, is an important indicator.
  • the appropriate drug loading ratio means that after freeze-drying, the drug and the skeleton are evenly adsorbed, and the drug will be better distributed on the surface of the voids and pores of the skeleton. In this way, in the subsequent reconstitution process (5% glucose injection, normal saline, etc.), the freeze-dried preparation can be well and quickly dissolved for injection.
  • a pharmaceutically acceptable buffer is a weak acid or base that allows the pH of a solution to be maintained at a nearly constant level and is used to enhance the stability of the drug substance in solution.
  • the solution used for preparing the high drug load lyophilized preparation of the present invention may also contain at least one buffer, and the buffer is selected from citrate buffer, borate buffer, lithium lactate, sodium lactate, potassium lactate, calcium lactate, lithium phosphate, sodium phosphate, potassium phosphate, calcium phosphate, lithium maleate, sodium maleate, potassium maleate, calcium maleate, lithium tartrate, sodium tartrate, potassium tartrate, calcium tartrate, lithium succinate, sodium succinate, potassium succinate, calcium succinate, lithium acetate, sodium acetate, potassium acetate, calcium acetate or a mixture thereof.
  • the buffer is 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,
  • the buffer used in the solution for preparing the high drug load 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, sodium citrate dihydrate, potassium citrate dihydrate, calcium citrate dihydrate, lithium citrate trihydrate, sodium citrate trihydrate, potassium citrate trihydrate, calcium citrate trihydrate, lithium citrate tetrahydrate, sodium citrate tetrahydrate, potassium citrate tetrahydrate, calcium citrate tetrahydrate, lithium citrate pentahydrate, sodium citrate pentahydrate, potassium citrate pentahydrate, calcium citrate pentahydrate, lithium citrate hexahydrate, sodium citrate hexahydrate, potassium citrate hexahydrate, calcium citrate hexahydrate, lithium citrate heptahydrate, sodium citrate heptahydrate, potassium citrate heptahydrate, calcium citrate heptahydrate.
  • the solution for preparing the high drug loading lyophilized preparation of the present invention may further contain at least one pH regulator.
  • the pH regulator of the present invention refers to a buffer substance or buffer used to appropriately adjust the pH changed by acid or alkali
  • the pH regulator includes but is not limited to hydrochloric acid, sodium hydroxide, triethanolamine, phosphoric acid, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate or phosphoric acid, citric acid, lactic acid, tartaric acid, succinic acid, fumaric acid, malic acid, sodium bicarbonate, sodium carbonate or a mixture thereof.
  • the added content of the pH regulator makes the solution pH value 4-9, preferably 6-8.
  • the compound preparation process includes an acidic environment and there is a trace of hydrolysis, so the prepared or purchased API will be acidic.
  • the pH regulator is preferably selected from alkalis such as sodium hydroxide, triethanolamine, sodium bicarbonate, sodium carbonate, or alkaline salts.
  • the compound of formula I is selected from
  • TH-302 is more preferred.
  • Threshold such as WO2016011195A2, WO2004087075A1, WO2007002931A1, WO2008151253A2, WO2009018163A1, WO2009033165A2, WO2010 048330A2, WO2012142520A1, WO2008083101A2, WO2020007106A1, WO2020118251A1, WO2014169035A1, WO2013116385A1, WO2019173799A2, WO2016081547A1, WO2014062856A1, W O2015069489A1, WO2012006032A2, WO2018026606A2, WO2010048330A2, WO2015171647A1, WO2013096687A1, WO2013126539A2, WO2013096684A2, WO201200928 8A2 ⁇ WO201214568 4A2, WO2016014390A2,
  • the three preferred compounds have the same or similar physical and chemical properties as TH-302.
  • the present invention provides a solution for preparing a high-drug-load lyophilized preparation, comprising
  • the volume content of tert-butanol relative to the solution is 30%, 40%, 60% or the corresponding content of tert-butanol in the solution is 235.5, 314, 471 mg/mL,
  • the content of the compound of formula I-1 in the solution is 6, 7, 7.5, 8, 8.5, 10, 12.5, 15, 20, 25 mg/mL,
  • 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 present invention provides a solution for preparing a high-drug-load lyophilized preparation, comprising
  • the compound of the following formula I-1 water, tert-butyl alcohol, sucrose, and a pH regulator;
  • the volume content of tert-butanol relative to the solution is 30%, 40%, 60% or the content of tert-butanol in the solution is 235.5, 314, 471 mg/mL,
  • the content of the compound of formula I-1 in the solution is 6, 7, 7.5, 8, 8.5, 10, 12.5, 15, 20, 25 mg/mL,
  • 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 content of the pH adjuster is such that the pH value of the solution is 4-9, preferably 6-8.
  • the present invention provides a solution for preparing a high-drug-load 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, 471 mg/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, 25 mg/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 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, and 1:9.375.
  • a buffer, a pH adjuster or other auxiliary materials of the lyophilized preparation can be added.
  • the above-mentioned “consisting of xxx” refers to substances that are intentionally and artificially added during preparation (these substances must be contained and can be detected by analytical testing instruments). Apart from these substances, it no longer contains other intentionally and artificially added substances, but there will still inevitably be trace amounts of impurities, environmental substances, etc.
  • composition (prescription) of the solution for preparing the high drug loading lyophilized preparation, and the following briefly describes its use.
  • the solution provided above is only used as an intermediate semi-finished product for preparing high-drug-loaded lyophilized preparations and cannot be used as a prescription for clinical preparations. Generally speaking, it is prepared and used immediately at the production site, that is, it is directly filled into lyophilized bottles after being prepared in a liquid preparation container, and then sent in batches to the lyophilization production equipment for lyophilization.
  • the solution for preparing high-drug-load lyophilized preparations should be stable during the production and waiting process from preparation to lyophilization in the lyophilization equipment, that is, it should have stability for at least 8 hours at room temperature, and preferably, it should have stability for 24 hours, or even 72 hours or 120 hours.
  • the solution needs to be filtered and filled after preparation before it can enter the freeze dryer for freeze drying.
  • Filtration and filling are generally completed within 8-12 hours and are generally performed at room temperature.
  • the subsequent freeze drying is low temperature, it takes 20-60 hours for a large freeze dryer or freeze drying system to cool a large amount of liquid medicine filled in a vial to a set low temperature (-20 to -55°C). Therefore, the liquid medicine of the freeze-dried preparation should be stable within a certain period of time and under room temperature regulation. After experiments, it has been verified that the liquid medicine of the TH-302 freeze-dried preparation provided by the present invention has suitable stability.
  • the invention discloses a method for preparing a solution for preparing a lyophilized preparation with a high drug loading, wherein the solution is used as a lyophilized preparation solution and is used to prepare a lyophilized preparation through a lyophilization process.
  • the present invention can prepare a freeze-dried preparation with high drug loading.
  • the lyophilized preparation is prepared by a lyophilization process using the above-mentioned solution for preparing the lyophilized preparation with high drug loading as the lyophilized preparation solution.
  • a lyophilized preparation comprising a compound of the following formula I, excipients, and residual solvent components:
  • R is independently selected from H, -CH 3 , -CH 2 CH 3 , and X is independently selected from Br, Cl, OMs, OTs,
  • 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 , or
  • 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 , 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 residual solvent components are water and tert-butyl alcohol.
  • a lyophilized preparation comprising a compound of the following formula I, an excipient, a residual solvent component, and a pH adjuster:
  • R is independently selected from H, -CH 3 , -CH 2 CH 3 , and X is independently selected from Br, Cl, OMs, OTs,
  • 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 , or
  • 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 , 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 residual solvent components are water and tert-butyl alcohol.
  • a lyophilized preparation comprising a compound of the following formula I and an excipient:
  • R is independently selected from H, -CH 3 , -CH 2 CH 3 , and X is independently selected from Br, Cl, OMs, OTs,
  • 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 , or
  • 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 , 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 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 are two different situations.
  • the volume of the solution before freeze-drying and the surface volume of the preparation solid after freeze-drying vary by no more than 10%: the volume may increase by 10% due to the expansion of the powder cake after freeze-drying, or it 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 increases the volume to 1.1 times the original volume, and the collapse decreases the volume to 90% of the original volume.
  • the drug loading range is 5 to 500.
  • the union of the two ranges is 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 freeze-dried preparation is greater than or equal to 4.55 mg/cm 3 and less than 500 mg/cm 3 .
  • the drug loading of the lyophilized preparation refers to the amount of the raw material drug of the compound of formula I contained in a unit volume.
  • the volume here refers to the total volume of the drug in the unit packaging kit, including the internal space of the drug.
  • a lyophilized bottle with a volume of 100 mL contains 20 mL of lyophilized solution, and 20 mL of lyophilized solution contains 500 mg of drug. After the lyophilization process, the 20 mL of solution is frozen into a loose and porous lyophilized preparation.
  • the total volume of the lyophilized preparation in the unit packaging is sh, which contains 500 mg of raw material drug, and its drug loading is (500/sh) mg/cm 3.
  • sh contains 500 mg of raw material drug, and its drug loading is (500/sh) mg/cm 3.
  • the value of sh will be around 20 cm 3.
  • the volume may expand after lyophilization and be greater than 20 cm 3 , and the volume may collapse after lyophilization and be less than 20 cm 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%, which can be calculated based on the content of API, excipients, and residual tert-butyl alcohol and water in the lyophilized preparation.
  • the mass content of the compound of formula I in the lyophilized preparation refers to the percentage of the drug in the total mass of the lyophilized preparation. It can be measured and calculated by the following operation:
  • the compound of formula I is selected from
  • the excipient is a sugar, a polyol, a polyvinyl pyrrolidone, a protein, a poloxamer or a combination thereof.
  • the sugar is selected from sucrose, dextran, cyclodextrin, maltodextrin, trehalose, lactose, maltose, glucose,
  • the polyols are selected from glycerol, sorbitol, mannitol, inositol, ethylene glycol, polyethylene glycol, polysorbate, and adonitol.
  • the protein is selected from albumin, preferably bovine serum albumin, human albumin,
  • the poloxamer is selected from poloxamer 182, poloxamer 184, poloxamer 188, and poloxamer 407.
  • the sublimation process of freeze-drying cannot completely remove water and tert-butyl alcohol, so the residue of water and tert-butyl alcohol is inevitable.
  • the amount of residual water and tert-butyl alcohol is an important quality indicator of freeze-dried preparations: the lower the residue, the better the quality of the freeze-dried preparation, the stronger the stability, and the fewer adverse reactions patients will have after use. Reducing the residue can be solved by adjusting the freeze-drying process, but it cannot be completely avoided after all.
  • the freeze-dried preparation contains almost no water and tert-butanol.
  • the residual amount of water and tert-butyl alcohol has a suitable value: the residual water content is less than or equal to 6% by mass, preferably less than or equal to 2%, more preferably less than or equal to 1%, and further preferably less than or equal to 0.5%.
  • the residual tert-butyl alcohol content is less than or equal to 1.75% by mass, preferably less than or equal to 1%, and more preferably less than or equal to 0.5% by mass.
  • a buffer, a pH adjuster or other auxiliary materials of the lyophilized preparation can be added to the solution.
  • the lyophilized preparation after lyophilization can also be detected to contain a buffer, a pH adjuster or other auxiliary materials of the lyophilized preparation.
  • the compound of the following formula I and the excipients means that, except for the inevitable, residual water and tert-butyl alcohol, the lyophilized preparation can be detected to contain the compound of formula I, the excipients and the above-mentioned residual environmental substances, and in addition, it may also contain other excipients.
  • the mass ratio of compound I to the excipient is 1:(0.5-20), preferably 1:(1-15), more preferably 1:(2-12.5), and further preferably 1:(5-10).
  • 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 greater than or equal to 8.88 mg/cm 3 and less than or equal to 27.77 mg/cm 3 , further preferably greater than or equal to 8.88 mg/cm 3 and less than or equal to 16.66 mg/cm 3 , and further preferably greater than or equal to 8.88 mg/cm 3 and less than or equal to 11.11 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 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 greater than or equal to 7.27 mg/cm 3 and less than or equal to 22.73 mg/cm 3 , further preferably greater than or equal to 7.27 mg/cm 3 and less than or equal to 13.64 mg/cm 3 , and further preferably greater than or equal to 7.27 mg/cm 3 and less than or equal to 9.09 mg/cm 3 .
  • the former is the situation where the volume collapses and becomes smaller after freeze-drying, and the latter is the situation where the volume expands and becomes larger.
  • a lyophilized preparation which is essentially composed of the compound of the following formula I-1, excipients, residual water and residual tert-butyl alcohol:
  • the drug loading of the compound in the lyophilized formulation 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 of compound I-1 in the lyophilized formulation is 5.45, 6.36, 6.82, 7.27, 7.73, 9.09, 11.36, 13.64, 18.18, 22.73 mg/cm 3 ;
  • excipients are sucrose or mannitol
  • the mass ratio of the compound to the excipient is 1:2, 1:3, 1:3.5, 1:4, 1:5, 1:5.333, 1:5.6, 1:6, 1:7, 1:8, 1:8.235, 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%, and further preferably less than or equal to 0.5% by mass;
  • the residual tert-butyl alcohol content is less than or equal to 1.75% by mass, preferably less than or equal to 1%, and more preferably less than or equal to 0.5% by mass.
  • the lyophilized preparation is essentially composed of the compound of the following formula I-1, excipients, residual water, residual tert-butyl alcohol, and a pH adjuster:
  • the drug loading of the compound in the lyophilized formulation 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 of compound I-1 in the lyophilized formulation is 5.45, 6.36, 6.82, 7.27, 7.73, 9.09, 11.36, 13.64, 18.18, 22.73 mg/cm 3 ;
  • excipients are sucrose or mannitol
  • the mass ratio of the compound to the excipient is 1:2, 1:3, 1:3.5, 1:4, 1:5, 1:5.333, 1:5.6, 1:6, 1:1.67, 1:7, 1:8, 1:8.235, 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%, and further preferably less than or equal to 0.5% by mass;
  • the residual tert-butyl alcohol content is less than or equal to 1.75% by mass, preferably less than or equal to 1%, and more preferably less than or equal to 0.5% by mass;
  • the pH adjuster is sodium bicarbonate, and the content is 0.01-0.10 mg/cm 3 .
  • the lyophilized preparation is essentially composed of the following compound and excipients:
  • the drug loading of compound I-1 in the lyophilized formulation 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 of compound I-1 in the lyophilized formulation is 5.45, 6.36, 6.82, 7.27, 7.73, 9.09, 11.36, 13.64, 18.18, 22.73 mg/cm 3 ;
  • excipients are sucrose or mannitol
  • the mass ratio of compound I-1 to the excipient is 1:2, 1:3, 1:3.5, 1:4, 1:5, 1:5.333, 1:5.6, 1:6, 1:1.67, 1:7, 1:8, 1:8.235, 1:8.75, and 1:9.375.
  • the compound and excipients containing the following formula I refer to that, except for the inevitable, residual water and tert-butyl alcohol, the lyophilized preparation can detect the compound containing I, the excipients and the above-mentioned residual environmental substances, and in addition, it can also contain other excipients.
  • lyophilized preparation can only be detected to contain the compound of formula I, excipients and the above-mentioned residual environmental substances, and does not contain other substances.
  • the present invention provides a lyophilized preparation, which is prepared by a lyophilization process using the above series of solutions for preparing lyophilized preparations with high drug loading.
  • the pharmaceutical preparation and its freeze-dried powder can be stored in a container commonly used in the pharmaceutical field, which can include: a plastic container or a glass container, such as a standard USPI type borosilicate glass container.
  • a container commonly used in the pharmaceutical field can include: a plastic container or a glass container, such as a standard USPI type borosilicate glass container.
  • the container used can be a syringe or a vial.
  • the present invention also provides a preparation unit package containing the above-mentioned freeze-dried preparation:
  • the lyophilized preparation is contained in a sealed container with a volume of 1000 mL, the volume of the lyophilized preparation is 1/5 to 2/3 of the volume of the sealed container, preferably 1/3 to 1/2, and the content of the compound of formula I is 4000-16500 mg; or
  • the lyophilized preparation is contained in a sealed container with a volume of 500 mL, the volume of the lyophilized preparation is 1/5 to 2/3 of the volume of the sealed container, preferably 1/3 to 1/2, and the content of the compound of formula I is 2000-8000 mg; or
  • the lyophilized preparation is contained in a sealed container with a volume of 250 mL, the volume of the lyophilized preparation is 1/5 to 2/3 of the volume of the sealed container, preferably 1/3 to 1/2, and the content of the compound of formula I is 1000-4000 mg; or
  • the lyophilized preparation is contained in a sealed container with a volume of 100 mL, the volume of the lyophilized preparation is 1/5 to 2/3 of the volume of the sealed container, preferably 1/3 to 1/2, and the content of the compound of formula I is 400-2000 mg; or
  • the lyophilized preparation is contained in a sealed container with a volume of 50 mL, the volume of the lyophilized preparation is 1/5 to 2/3 of the volume of the sealed container, preferably 1/3 to 1/2, and the content of the compound of formula I is 50-800 mg; or
  • the lyophilized preparation is contained in a sealed container with a volume of 30 mL, the volume of the lyophilized preparation is 1/5 to 2/3 of the volume of the sealed container, preferably 1/3 to 1/2, and the content of the compound of formula I is 150-600 mg; or
  • the lyophilized preparation is contained in a sealed container with a volume of 25 mL, the volume of the lyophilized preparation is 1/5 to 2/3 of the volume of the sealed container, preferably 1/3 to 1/2, and the content of the compound of formula I is 125-500 mg; or
  • the lyophilized preparation is contained in a sealed container with a volume of 20 mL, the volume of the lyophilized preparation is 1/5 to 2/3 of the volume of the sealed container, preferably 1/3 to 1/2, and the content of the compound of formula I is 100-400 mg; or
  • the lyophilized preparation is contained in a sealed container with a volume of 18 mL, the volume of the lyophilized preparation is 1/5 to 2/3 of the volume of the sealed container, preferably 1/3 to 1/2, and the content of the compound of formula I is 90-360 mg; or
  • the lyophilized preparation is contained in a sealed container with a volume of 15 mL, the volume of the lyophilized preparation is 1/5 to 2/3 of the volume of the sealed container, preferably 1/3 to 1/2, and the content of the compound of formula I is 75-300 mg; or
  • the lyophilized preparation is contained in a sealed container with a volume of 10 mL, the volume of the lyophilized preparation is 1/5 to 2/3 of the volume of the sealed container, preferably 1/3 to 1/2, and the content of the compound of formula I is 50-200 mg; or
  • the lyophilized preparation is contained in a sealed container with a volume of 8 mL, the volume of the lyophilized preparation is 1/5 to 2/3 of the volume of the sealed container, preferably 1/3 to 1/2, and the content of the compound of formula I is 40-160 mg; or
  • the lyophilized preparation is contained in a sealed container with a volume of 7 mL, the volume of the lyophilized preparation is 1/5 to 2/3 of the volume of the sealed container, preferably 1/3 to 1/2, and the content of the compound of formula I is 35-140 mg; or
  • the lyophilized preparation is contained in a sealed container with a volume of 5 mL, the volume of the lyophilized preparation is 1/5 to 2/3 of the volume of the sealed container, preferably 1/3 to 1/2, and the content of the compound of formula I is 25-100 mg; or
  • the lyophilized preparation is contained in a sealed container with a volume of 3 mL, the volume of the lyophilized preparation is 1/5 to 2/3 of the volume of the sealed container, preferably 1/3 to 1/2, and the content of the compound of formula I is 15-60 mg; or
  • the lyophilized preparation is contained in a sealed container with a volume of 2 mL, the volume of the lyophilized preparation is 1/5 to 2/3 of the volume of the sealed container, preferably 1/3 to 1/2, and the content of the compound of formula I is 10-40 mg; or
  • the lyophilized preparation is contained in a sealed container with a volume of 1.5 mL.
  • the volume of the lyophilized preparation is 1/5 to 2/3 of the volume of the sealed container, preferably 1/3 to 1/2, and the content of the compound of formula I is 7.5-30 mg.
  • small doses are suitable for minors among humans or animals of comparable size, such as pigs, rats, dogs and other small animals in size and weight, and the indications are not limited.
  • the lyophilized preparation provided by the present invention is recommended to be administered by intravenous infusion, so the drug solution needs to be reconstituted, and physiological saline (0.9%) or glucose injection (5%) is generally used for reconstitution.
  • physiological saline 0.8% or glucose injection (5%) is generally used for reconstitution.
  • an intravenous injection solution containing TH-302 is provided, wherein the solvent is water, and the solutes include TH-302 raw material, an isotonicity adjusting agent, mannitol or sucrose, tert-butanol and sodium bicarbonate, and the isotonicity adjusting agent is selected from glucose and sodium chloride.
  • the present invention also provides a method for preparing a solution for preparing a high drug loading lyophilized preparation, comprising the following operations:
  • Operation 1 Weigh the prescribed amount of TH-302 API, add it to the tert-butyl alcohol aqueous solution, and stir until it is 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 amount of tert-butyl alcohol, then add an appropriate amount of water to the predetermined volume, add the prescribed amount of sodium bicarbonate, stir and mix evenly,
  • the content of TH-302 API in the solution of the high drug loading lyophilized preparation is greater than 5 mg/mL and less than or equal to 500 mg/mL,
  • the volume percentage of tert-butanol relative to the high drug loading lyophilized preparation is 1%-99%, or the content of tert-butanol in the solution is 7.85-777.15 mg/mL,
  • the content of sucrose or mannitol in the solution of the high drug loading lyophilized preparation is 20-300 mg/mL
  • the mass ratio of TH-302 to sucrose or mannitol in the solution of the high drug loading lyophilized preparation is 1:(0.5-20),
  • the volume ratio of tert-butanol in the tert-butanol aqueous solution is 30-90%
  • the content of sodium bicarbonate in the solution of the high drug loading lyophilized preparation is 0.01-0.10 mg/mL.
  • the present invention provides a preparation process of a lyophilized preparation.
  • the method for preparing a high drug loading lyophilized preparation comprises the following operations:
  • Pre-freezing placing the above-mentioned drug solution in a freeze-drying system for pre-freezing, the pre-freezing process includes keeping at 0°C for a period of time and keeping at -20 to -55°C for a period of time;
  • the temperature is raised to -10 to 10°C after pre-freezing and kept warm for a period of time, and vacuum drying is maintained;
  • freeze-dried preparation is obtained by directly filling the solution into the freeze-dried bottle (vial) and directly freeze-drying it through the freeze-drying equipment. Therefore, there is no subpackaging link, that is, the unit packaging is the corresponding freeze-dried bottle packaging, and the specifications of the packaging are closely related to the specifications of the freeze-dried bottle.
  • the numbers appearing in this application have an error of ⁇ 10%, that is, numbers within -10% and +10% should be considered to be within the range of numbers recorded in this application. That is, if the words "content is greater than 5 mg/mL and less than or equal to 500 mg/mL" appear in this application, then if the actual test results in a content greater than 4.5 mg/mL and less than or equal to 550 mg/mL, it should naturally be determined to be equivalent to the above range.
  • the corresponding range is 7.20-8.80. If the actual test result shows that the content is greater than or equal to 6.48 and less than or equal to 9.68, it should be naturally determined to be equivalent to the above range.
  • the applicant proposed the following recommended scheme: Based on certain considerations, the applicant has used the following recommended scheme to carry out relevant batch production of freeze-dried preparations.
  • the finished freeze-dried preparations produced meet the requirements of subsequent clinical trials and are expected to become candidate drugs for subsequent large-scale clinical trials and even for commercial production in the future.
  • a solution for preparing a high drug loading lyophilized preparation comprising a compound of the following formula I-1, water, tert-butyl alcohol and mannitol:
  • tert-butanol Water and tert-butanol are used as a mixed solvent, the volume percentage of tert-butanol relative to the solution is (30 ⁇ 3)% or the mass percentage is (24 ⁇ 2.4)%, or the content of tert-butanol in the solution is (235.5 ⁇ 23.55) mg/mL;
  • the content of the compound of formula I-1 in the solution is (8.16 ⁇ 0.82) mg/g or (8.00 ⁇ 0.80) mg/mL;
  • Mannitol is an excipient, and its mass percentage in the solution is (7.14 ⁇ 0.71) % or the mannitol content in the solution is (70 ⁇ 7) mg/mL,
  • the solution has a pH of 4-9.
  • TH-302 (Compound I-1) itself and the relationship between production batches, it is possible that the pH of the solution containing Compound I-1, water, tert-butyl alcohol and mannitol is not 4-9 under a specific temperature and environment. Therefore, it is necessary to add a pH additive for adjustment.
  • the recommended pH adjusters are Na 2 CO 3 , NaHCO 3 , K 2 CO 3 , KHCO 3 and the like commonly used in medicine, preferably NaHCO 3 . Therefore, the above solution also contains a pH adjuster, sodium bicarbonate.
  • the pH value of the solution is 6-8.
  • a lyophilized preparation comprising a compound of the following formula I-1 and mannitol:
  • the drug loading of the compound of formula I-1 in the lyophilized preparation is (8.00 ⁇ 0.80) mg/cm 3 or the mass percentage of the compound of formula I-1 in the lyophilized preparation is (10.23 ⁇ 1.02)%,
  • the content of mannitol in the lyophilized preparation is (70 ⁇ 7) mg/cm 3 , or the content of mannitol in the lyophilized preparation is the remainder of (10.23 ⁇ 1.02)% of the mass percentage of the compound of formula I-1.
  • the volume of the freeze-dried solid obtained after freeze-drying basically did not change: no detailed collapse (resulting in a smaller volume) and spray bottle (resulting in an larger volume) were found in the freeze-dried preparation. Therefore, as the water and tert-butyl alcohol used as solvents were evaporated and removed during the freeze-drying process, only TH-302 and mannitol were left in the freeze-dried preparation.
  • the drug loading of the compound of formula I-1 in the freeze-dried preparation was 8.00 mg/cm 3 , and its corresponding mass percentage was 10.23%; the content of mannitol in the freeze-dried preparation was 70 mg/cm 3 , and considering the 10% difference in the filling amount during the filling process, the above values all had a variation range of ⁇ 10%.
  • a lyophilized preparation comprising a compound of the following formula I-1, mannitol, and a pH adjuster:
  • the drug loading of the compound of formula I-1 in the lyophilized preparation is (8.00 ⁇ 0.80) mg/cm 3 or the mass percentage of the compound of formula I-1 in the lyophilized preparation is (10.23 ⁇ 1.02)%,
  • the content of mannitol in the lyophilized preparation is (70 ⁇ 7) mg/cm 3 , or the content of mannitol in the lyophilized preparation is the remainder of (10.23 ⁇ 1.02)% of the mass percentage of the compound of formula I-1.
  • a lyophilized preparation comprising a compound of the following formula I-1, mannitol, residual solvent components, and a pH adjuster:
  • the drug loading of the compound of formula I-1 in the lyophilized preparation is (8.00 ⁇ 0.80) mg/cm 3 or the mass percentage of the compound of formula I-1 in the lyophilized preparation is (10.23 ⁇ 1.02)%,
  • the content of mannitol in the freeze-dried preparation is (70 ⁇ 7) mg/cm 3 ,
  • the residual solvent components are water and tert-butyl alcohol.
  • the lyophilized preparation is dissolved in water for injection into a 5.0 mg/mL aqueous solution with a pH value of 4.0-7.0.
  • the recommended testing methods are as follows:
  • the pH regulator is sodium bicarbonate, and its content is 0.01-0.10 mg/cm 3 .
  • the residual water content is less than or equal to 6% by mass, preferably less than or equal to 2%, more preferably less than or equal to 1%, and further preferably less than or equal to 0.5%.
  • the residual tert-butyl alcohol content is less than or equal to 1795 ppm by mass, preferably 1000 ppm, and more preferably less than or equal to 500 ppm.
  • Tert-butyl alcohol is used as a pharmaceutical excipient in the production of TH-302 for injection. There may be a small amount of residue after freeze-drying. Tert-butyl alcohol has low toxicity to the human body. In order to ensure the safety and quality of drugs, a limit control should be established to control the residual amount of tert-butyl alcohol in TH-302 for injection.
  • the PDE (permissible daily exposure) of tert-butyl alcohol is 35 mg/day.
  • the maximum daily dose of TH-302 is estimated to be 1.5 g. According to 2 g, the corresponding maximum daily dose of injection is 2 g.
  • the content of TH-302 is 0.2 g/bottle (50 mL vial), and 10 bottles are required.
  • Each bottle of freeze-dried powder is 1.95 g (1.75 g mannitol and 0.20 g raw material TH-302).
  • the limit of tert-butanol can also be calculated according to the labeled amount of TH-302.
  • the residual tert-butanol content is less than or equal to 1795 ppm by mass, preferably 1000 ppm, and more preferably less than or equal to 500 ppm, which is completely equivalent to: the residual tert-butanol content is less than or equal to 1.75% by mass of the labeled amount, preferably less than or equal to 1%, and more preferably less than or equal to 0.5%.
  • the mass per unit volume of the lyophilized preparation was (79.2 ⁇ 7.9) mg/cm 3 .
  • the unit volume mass is similar to density.
  • the freeze-dried preparation is a porous cake in which the TH-302 API is fully fused with mannitol and sodium bicarbonate (residual solvent water, tert-butanol) to form a honeycomb structure, forming a cavity inside the honeycomb.
  • the size of the unit volume mass is a comprehensive indicator, which is related to the drug loading, mannitol content, residual solvent water, and tert-butanol of the freeze-dried preparation. Studies have shown that uniform and high-quality freeze-dried preparations have good rapid dissolution characteristics, which is convenient for subsequent compatibility.
  • freeze-dried preparation is loose and porous and the degree of looseness and porosity can be qualitatively studied at the microscopic level through direct observation or microscopic observation, and at the macroscopic level, it can be characterized by the unit volume mass of the above-mentioned freeze-dried preparations.
  • the above-mentioned lyophilized preparation can be prepared by freeze-drying the above-mentioned solution for preparing the lyophilized preparation with high drug loading.
  • the lyophilized preparation is contained in a sealed container with a volume of 1000 mL, the volume of the lyophilized preparation is 1/5 to 2/3 of the volume of the sealed container, preferably 1/3 to 1/2, and the content of the compound of formula I-1 is 1600-5333 mg; or
  • the lyophilized preparation is contained in a sealed container with a volume of 500 mL, the volume of the lyophilized preparation is 1/5 to 2/3 of the volume of the sealed container, preferably 1/3 to 1/2, and the content of the compound of formula I-1 is 800-2666 mg; or
  • the lyophilized preparation is contained in a sealed container with a volume of 250 mL, the volume of the lyophilized preparation is 1/5 to 2/3 of the volume of the sealed container, preferably 1/3 to 1/2, and the content of the compound of formula I-1 is 400-1333 mg; or
  • the lyophilized preparation is contained in a sealed container with a volume of 100 mL, the volume of the lyophilized preparation is 1/5 to 2/3 of the volume of the sealed container, preferably 1/3 to 1/2, and the content of the compound of formula I-1 is 160-533 mg; or
  • the lyophilized preparation is contained in a sealed container with a volume of 50 mL, the volume of the lyophilized preparation is 1/5 to 2/3 of the volume of the sealed container, preferably 1/3 to 1/2, and the content of the compound of formula I-1 is 80-266 mg; or
  • the lyophilized preparation is contained in a sealed container with a volume of 30 mL, the volume of the lyophilized preparation is 1/5 to 2/3 of the volume of the sealed container, preferably 1/3 to 1/2, and the content of the compound of formula I-1 is 48-160 mg; or
  • the lyophilized preparation is contained in a sealed container with a volume of 25 mL, the volume of the lyophilized preparation is 1/5 to 2/3 of the volume of the sealed container, preferably 1/3 to 1/2, and the content of the compound of formula I-1 is 40-133 mg; or
  • the lyophilized preparation is contained in a sealed container with a volume of 20 mL, the volume of the lyophilized preparation is 1/5 to 2/3 of the volume of the sealed container, preferably 1/3 to 1/2, and the content of the compound of formula I-1 is 32-107 mg; or
  • the lyophilized preparation is contained in a sealed container with a volume of 18 mL, the volume of the lyophilized preparation is 1/5 to 2/3 of the volume of the sealed container, preferably 1/3 to 1/2, and the content of the compound of formula I-1 is 29-96 mg; or
  • the lyophilized preparation is contained in a sealed container with a volume of 15 mL, the volume of the lyophilized preparation is 1/5 to 2/3 of the volume of the sealed container, preferably 1/3 to 1/2, and the content of the compound of formula I-1 is 24-79 mg; or
  • the lyophilized preparation is contained in a sealed container with a volume of 10 mL, the volume of the lyophilized preparation is 1/5 to 2/3 of the volume of the sealed container, preferably 1/3 to 1/2, and the content of the compound of formula I-1 is 16-53 mg; or
  • the lyophilized preparation is contained in a sealed container with a volume of 8 mL, the volume of the lyophilized preparation is 1/5 to 2/3 of the volume of the sealed container, preferably 1/3 to 1/2, and the content of the compound of formula I-1 is 13-42 mg; or
  • the lyophilized preparation is contained in a sealed container with a volume of 7 mL, the volume of the lyophilized preparation is 1/5 to 2/3 of the volume of the sealed container, preferably 1/3 to 1/2, and the content of the compound of formula I-1 is 11-40 mg; or
  • the lyophilized preparation is contained in a sealed container with a volume of 5 mL, the volume of the lyophilized preparation is 1/5 to 2/3 of the volume of the sealed container, preferably 1/3 to 1/2, and the content of the compound of formula I-1 is 8-27 mg; or
  • the lyophilized preparation is contained in a sealed container with a volume of 3 mL, the volume of the lyophilized preparation is 1/5 to 2/3 of the volume of the sealed container, preferably 1/3 to 1/2, and the content of the compound of formula I-1 is 5-16 mg; or
  • the lyophilized preparation is contained in a sealed container with a volume of 2 mL, the volume of the lyophilized preparation is 1/5 to 2/3 of the volume of the sealed container, preferably 1/3 to 1/2, and the content of the compound of formula I-1 is 3-11 mg; or
  • the lyophilized preparation is contained in a sealed container with a volume of 1.5 mL.
  • the volume of the lyophilized preparation is 1/5 to 2/3 of the volume of the sealed container, preferably 1/3 to 1/2, and the content of the compound of formula I-1 is 2-8 mg.
  • the above compound content is calculated based on 8.0 mg/ cm3 .
  • the value is the labeled amount, and the actual content is within the range of ⁇ 10% of the labeled amount.
  • the invention discloses an injection for intravenous injection, wherein the solvent is water, and the solutes include TH-302 raw material medicine, mannitol, tert-butyl alcohol, sodium bicarbonate and an isotonicity adjusting agent, wherein the content of TH-302 raw material medicine is 0.1-4.0 mg/mL, the osmotic pressure of the injection is 260-320 mOsmol/kg, and the pH value is 4.0-9.0.
  • the solute may also include an isotonicity adjusting agent selected from glucose and sodium chloride.
  • the above-mentioned lyophilized preparation is placed at room temperature until it returns to room temperature;
  • the above-mentioned lyophilized preparation restored to room temperature is prepared into an aqueous solution for injection with a TH-302 raw material drug content of (5 ⁇ 0.5) mg/mL using water for injection;
  • aqueous solution for injection is injected into 0.9% saline injection or 5% glucose injection to dilute the solution to a content of TH-302 raw material drug of 0.1-4.0 mg/mL.
  • the preparation method of the above injection is printed in the instructions attached to the unit package of the freeze-dried preparation, which is used to guide the preparation and use of the preparation after clinical preparation.
  • the prepared injection should be injected within 8 hours, including the following operations:
  • the lyophilized preparation according to any one of claims 4 to 12 is placed at room temperature for 30 to 120 minutes until it returns to room temperature;
  • FIG1 is a solubility curve of TH-302 in aqueous tert-butyl alcohol solutions of different mass ratios
  • Figure 2 shows the stability curves of lyophilized preparations prepared with 11 excipients within 5 days, which are distinguished by the rightmost circle, from top to bottom: PEG2000, P188, SBECD, mannitol, DSPE-MPEG2000, fructose, trehalose, PVPK12, sucrose, maltose, and lactose;
  • FIG3 is a stability curve of a lyophilized preparation prepared with sucrose and mannitol as excipients within 10 days, wherein the 4 curves in the upper part of the figure are curves of the mannitol prescription, and the 4 curves in the lower part are curves of the sucrose prescription;
  • Figure 4 is a stability curve of a freeze-dried sample prepared with 100 mg/mL sucrose and 80 mg/mL mannitol as excipients at a high temperature of 40°C and a room temperature of 25°C, with the horizontal axis representing days and the vertical axis representing the percentage purity of HPLC;
  • Figure 5 is a photo of the bottom crystallization of the liquid medicine sample placed in an environment of 2-8°C, and the label has been coded and covered;
  • Figure 6 shows sample photos of four batches of freeze-dried preparations, from left to right they are batches 01, 02, 03, and 04, and the labels have been coded;
  • FIG7 is a comparison photo of 4 batches of freeze-dried preparation samples after adding solution and standing in a 40 mL 5% glucose solution reconstitution experiment.
  • the left picture is a photo after adding solution
  • the right picture is a photo after adding solution and standing. From left to right in each photo are batches 01, 02, 03, and 04, and the labels have been coded and covered;
  • FIG8 is a comparison photo of 4 batches of freeze-dried preparation samples after adding solution and standing in a 50 mL 5% glucose solution reconstitution experiment.
  • the left picture is a photo after adding solution
  • the right picture is a photo after adding solution and standing. From left to right in each photo are batches 01, 02, 03, and 04, and the labels have been coded and covered;
  • FIG9 is a sample photo of 7 batches of freeze-dried preparations, from left to right, batches 01 to 07, and the labels have been coded;
  • FIG10 is a schematic diagram of the placement of vials on the shelf of the experimental freeze dryer
  • FIG11 is a photo of a freeze-dried preparation sample
  • Figure 12 is a photo of a sample with "neck wrapping" in a freeze-dried preparation, and the label has been coded and covered;
  • FIG13 is a schematic diagram showing the angle at which the syringe needle penetrates the vial stopper when the lyophilized preparation is reconstituted
  • FIG14 is a schematic diagram showing that when a lyophilized preparation is reconstituted, the syringe needle pierces the stopper of a vial and then injects the injection solution along the inner wall of the vial.
  • a patient and “subject” are used interchangeably and refer to a mammal in need of cancer treatment.
  • the patient is a human.
  • the patient is a human diagnosed with cancer.
  • a "patient” or “subject” may refer to a non-human mammal, such as a non-human primate, dog, cat, rabbit, pig, mouse, or rat, used for screening, characterization, and evaluation of drugs and therapies.
  • Prodrug refers to a compound that, after administration or application, is metabolized or otherwise converted to a biologically active or more active compound (or drug) with respect to at least one property.
  • a prodrug is chemically modified relative to a drug in a manner that renders it less active or inactive relative to the drug, but the chemical modification allows the production of the corresponding drug by metabolism or other biological processes following administration of the prodrug.
  • a prodrug may have altered metabolic stability or delivery characteristics, fewer side effects or lower toxicity, or improved flavor relative to an active drug.
  • Prodrugs may be synthesized using reactants other than the corresponding drug.
  • Treatment or “treating a patient” refers to administering, using or applying to a patient a therapeutically effective amount of a drug related to the present invention.
  • administering or “applying” a drug to a patient refers to direct administration or administration (which may be administered or administered to a patient by a medical professional or may be self-administered or administered) and/or indirect administration or administration, which may be the act of prescribing a drug.
  • direct administration or administration which may be administered or administered to a patient by a medical professional or may be self-administered or administered
  • indirect administration or administration which may be the act of prescribing a drug.
  • a physician who instructs a patient to self-administer or administer a drug and/or provides a prescription for a drug to a patient is administering or administering a drug to a patient.
  • a “therapeutically effective amount” of a drug refers to an amount of the drug that, when administered or used to a patient suffering from cancer, will have the desired therapeutic effect (e.g., alleviation, improvement, remission, or elimination of clinical manifestations of one or more cancers in the patient).
  • the therapeutic effect does not necessarily occur by administering or applying one dose, and may only occur after administering or applying a series of doses. Therefore, a therapeutically effective amount can be administered or applied one or more times.
  • Treatment of a condition or patient refers to taking steps to obtain beneficial or desired results (including clinical results).
  • beneficial or desired clinical results 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; improvement, remission, or stabilization of the disease state; or other beneficial results.
  • treatment of cancer may result in a partial response or stabilization of the disease.
  • Tumor cell refers to a tumor cell of any appropriate species (eg, mammalian, such as murine, canine, feline, equine, or human).
  • mammalian such as murine, canine, feline, equine, or human.
  • the moisture content was determined using the Karl Fischer (KF) method using a Mettler V10S Karl Fischer titrator.
  • the residual solvent tert-butyl alcohol content was determined by gas chromatography GC, using an Agilent 8860 gas chromatograph equipped with a 7696A automatic headspace injector and an FID detector, and a capillary column with DB-624 filler.
  • GC test parameters were as follows: carrier gas was N 2 ; injection port temperature was 150°C; detector temperature was 200°C; split ratio was 20:1; the heating program was a starting temperature of 60°C, maintained for 5 minutes, then heated to 240°C at a rate of 30°C/min, and then operated at 60°C for 5 minutes; headspace equilibrium was 85°C for 20 minutes;
  • the content and concentration of TH-302 were determined by high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • the instrument used was Thermo Vanquish high performance liquid chromatography.
  • the chromatographic column was YMC pack AQ C18 4.6mm ⁇ 250mm, 5 ⁇ m.
  • the detection method refers to the HPLC method in patent WO2008083101A1 applied by Threshold Company (specifically Example 2. An Ethanol Formulation of TH302).
  • the residual solvent (tert-butyl alcohol) index values in the freeze-dried preparation test items all represent the residual tert-butyl alcohol content as a mass percentage relative to the API labeled amount of the freeze-dried preparation.
  • the solubility was determined by the saturated solution method, that is, solid TH-302 was directly put into the solvent until insoluble matter or turbidity appeared. After a period of clarification, it was directly filtered and the concentration of TH-302 was directly tested in the clarified filtrate. The obtained concentration was the solubility of TH-302 in the solvent system.
  • the specific solubility data are shown in Tables 1, 2 and 3.
  • TH-302 concentration mg/mL
  • HPLC method for the measurement method of TH-302 concentration (mg/mL)
  • Threshold Corporation specifically Example 2.
  • An Ethanol Formulation of TH302 and the external standard method is used for quantification.
  • the concentration is too high, the corresponding clear solution should be diluted first to a level that can be accurately quantitatively detected by HPLC.
  • the mass ratio of tert-butanol in the data of Table 3 is used as the X-axis, and the solubility of TH-302 in the tert-butanol-water mixed solvent is used as the Y-axis, and the relationship curve of Figure 1 is obtained: the solubility of the raw material drug in the aqueous solution is low; with the increase of the tert-butanol concentration in the tert-butanol aqueous solution, the solubility of the raw material drug continues to increase; the solubility of the raw material drug is the highest in 70% tert-butanol aqueous solution (w/w), and then with the increase of the tert-butanol concentration in the tert-butanol aqueous solution, the solubility decreases.
  • Non-ionic polymer surfactants include: PVPK12 (polyvinyl pyrrolidone with a molecular weight of 5500), PEG2000 (polyethylene glycol 2000), and P188 (Poloxamer is a polyoxyethylene polyoxypropylene ether block copolymer with a brand number of 188);
  • SBECD sulfobutyl ether- ⁇ -cyclodextrin
  • DSPE-MPEG2000 phosphatidylethanolamine
  • API TH-302
  • TBA tert-butyl alcohol
  • water water.
  • the solvent used in the PEG2000 and P188 protective agent experimental groups is 40% TBA/water.
  • TH-302 API and lyophilization protective agent were weighed, the prescribed amount of tert-butanol solution was added, mixed evenly until completely dissolved, then packaged (using 5 mL vials, each bottle was filled with 1 mL of drug solution, and 8 bottles were filled for each prescription), and rapidly freeze-dried in a small rapid freeze dryer: the packaged preparation was pre-frozen in a -80°C ultra-low temperature refrigerator for about 2.5 h, and then placed in a freeze dryer for freeze drying for about 10-90 h (-30°C, absolute air pressure of 0.1 mbar) to obtain a freeze-dried preparation.
  • Freeze-dried powder state Except for water as solvent, the other solvent groups successfully obtained white freeze-dried powder cakes. However, the glycerol group turned into oily substance when the freeze dryer was heated 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 experiments.
  • Table 5 High temperature 40°C accelerated stability test data of lyophilized preparations with different excipients.
  • lyoprotectants for injections should take into account good stability, routine use, and no effect on efficacy, DMPK, and toxicology. Analyzing the curves in Figure 2, PEG2000 is not routinely used, and P188/SBECD/DSPE-MPEG2000 may have an effect on efficacy, DMPK, and toxicology. In general, mannitol is ideal, and sucrose also has development potential (optimizing dosage and lyophilization conditions may improve stability and reduce tert-butyl alcohol residues).
  • the mannitol and sucrose samples were subjected to HPLC purity tests after being placed for 10 days, and the results were plotted as a curve, as shown in FIG3 .
  • the pH of the sucrose solution was 4.24. 10 ⁇ L of NaHCO 3 injection was used to adjust the pH to 6.43. Another 10 ⁇ L was added to adjust the pH to 7.13.
  • the pH of the mannitol prescription solution was measured to be 4.62, and 12 ⁇ L of NaHCO 3 injection was used to adjust the pH to 7.31.
  • the freeze-dried preparation was obtained by using a similar operation to the aforementioned "quick freeze drying" and adjusting relevant parameters.
  • freeze drying After freeze drying, vacuum plugging and capping were performed to obtain the freeze-dried preparation. The residual solvent and water content of the freeze-dried preparation were tested.
  • Table 7 Stability test data of freeze-dried samples prepared with 100 mg/mL sucrose and 80 mg/mL mannitol as excipients at high temperature 40°C and room temperature 25°C
  • the stability curve of 100 mg/mL sucrose and 80 mg/mL mannitol was prepared using the HPLC purity in Table 7 as the Y coordinate and the time as the X coordinate, as shown in FIG. 4 .
  • the solution was diluted with 30% acetonitrile aqueous solution to a solution containing about 1 mg of API per 1 mL and the HPLC purity was tested.
  • the results are shown in Table 8 below.
  • Table 8 Stability test data of freeze-dried samples prepared with 100 mg/mL sucrose and 80 mg/mL mannitol as excipients after reconstitution at room temperature 25°C
  • Table 9 pH and osmotic pressure data of freeze-dried samples prepared with 100 mg/mL sucrose and 80 mg/mL mannitol as excipients after reconstitution with D5W at room temperature 25°C
  • a volume ratio of 40% corresponds to 80 mL of tert-butanol for 200 mL of the drug solution, and the corresponding mass of tert-butanol is 62.80 g.
  • Batch 01 is filled with 14mL; Batch 02 is filled with 10.5mL; Batch 03 is filled with 14mL; Batch 04 is filled with 10.5mL.
  • the filled vials are 50mL in size.
  • the above-mentioned subpackaged preparations were freeze-dried in a vacuum freeze dryer.
  • the freeze-drying conditions were optimized multiple times, as shown in Table 12. After freeze-drying, the vacuum plug was pressed and the cap was rolled to obtain a freeze-dried preparation, as shown in Figure 6.
  • the above-mentioned subpackaged preparation was pre-frozen in a -70°C refrigerator for 3 hours, and then placed in a freeze dryer for 62 hours. After freeze drying, the plug was pressed and the cap was rolled to obtain a freeze-dried preparation, as shown in FIG9 .
  • the preparations were reconstituted by injecting into D5W. Batches 01 to 07 were easily reconstituted and the solutions were clear.
  • Table 15 HPLC purity data of 5 batches of freeze-dried samples at high temperature 40°C (%)
  • Table 16 Residual tert-butyl alcohol content data of 7 batches of freeze-dried samples measured at 0 days
  • TBA is not added to 05.
  • 01 in the middle and 01 below represent freeze-dried preparation samples of the same batch of drug solution after freeze-drying on different plates (middle and lower layers) of the freeze dryer.
  • Table 17 Residual water content data of 7 batches of freeze-dried samples measured at 0 days
  • the related substances in the sucrose prescription increased significantly, while the related substances in the mannitol prescription increased slightly.
  • the concentration of API, lyoprotectant, and tert-butanol all affect the residual solvent content in the freeze-dried preparation: in comparison, the residual solvent in the freeze-dried preparation obtained by using mannitol as a freeze-drying excipient (lyoprotectant) is lower, and in the high-temperature accelerated stability experiment, the stability of the mannitol prescription is better than that of the sucrose prescription.
  • Table 18 Effects of different pH and filling volumes on sample reconstitution for the formulation (40% tert-butyl alcohol + 60 mg/mL mannitol + 10 mg/mL API)
  • Batch 01 is filled with 21 mL, and 6 bottles are obtained;
  • Batch 02 is filled with 21 mL, and 6 bottles are obtained;
  • Batch 01 is filled with 10.5 mL, resulting in 6 bottles;
  • Batch 02 is filled with 10.5 mL, resulting in 6 bottles.
  • Table 19 Effects of different pH and filling volumes on sample reconstitution for the formulation (30% tert-butyl alcohol + 70 mg/mL mannitol + 12.5 mg/mL API)
  • Batch 03 is filled with 17 mL, resulting in 8 bottles;
  • Batch 04 is filled with 17 mL, resulting in 8 bottles;
  • Batch 03 is filled with 13 mL, resulting in 7 bottles;
  • Batch 04 is filled with 13 mL, resulting in 7 bottles;
  • Batch 03 is filled with 8.5 mL, resulting in 8 bottles
  • Batch 04 is filled with 8.5 mL, resulting in 8 bottles
  • the above-mentioned subpackaged preparations were freeze-dried in a vacuum freeze dryer.
  • the freeze drying was performed using the parameters in Table 12. After the freeze drying was completed, the vacuum plug was pressed and the cap was rolled to obtain the freeze-dried preparation.
  • the freeze-dried preparations are all white solid blocks.
  • Normal pressure means that the aluminum cap and rubber stopper of the vial containing the lyophilized preparation are opened to the outside world, and then purified water is injected for reconstitution; no normal pressure is marked, which means that purified water is directly injected into the vial containing the lyophilized preparation through a syringe (at this time, the air pressure in the vial is lower than the external atmospheric pressure) for reconstitution.
  • Table 21 Reconstitution results of 04 batches of samples with different filling volumes after storage at 40°C for 10 days
  • the lyophilized preparation obtained by adjusting the pH value to about 7 has a better reconstitution condition.
  • the API purity is 99.26%.
  • Solution 01 No pH adjustment; pH was measured at 4.77 after being placed at room temperature for 24 hours.
  • Solution-02 Add 200 ⁇ l of sodium bicarbonate and measure pH 6.60 ( pH 6.0-6.5). Place at room temperature for 24 hours and measure pH 5.20.
  • Drug solution-03 Add 1000 ⁇ l of sodium bicarbonate and measure pH 7.20 ( pH at 7.0). Place at room temperature for 24 hours and measure pH 6.90. Take the intermediate of the drug solution at 0 hours and the drug solution placed at room temperature for 24 hours and test the content and related substances.
  • Solution-01 batch is filled with 24.5 g, and 78 bottles are obtained, which is batch 01.
  • the drug solution - Batch 02 is filled with 24.5 g, and 78 bottles are obtained, which is Batch 02.
  • the drug solution - Batch 03 is filled with 24.5 g, and 78 bottles are obtained, which is Batch 03.
  • half stopper is added and placed in freeze dryer for freeze drying.
  • the above-mentioned subpackaged preparations were freeze-dried in a vacuum freeze dryer.
  • the arrangement order of the vials of this type of freeze dryer is shown in FIG10. Freeze-drying was performed using the freeze-drying parameters of Table 12. After freeze-drying, vacuum plugging and capping were performed to obtain a freeze-dried preparation. Shelf temperature before discharging: 5.0°C, manual plugging under high vacuum.
  • Table 23 pH data of different batches of drug solution after pH adjustment and storage for different time periods
  • the state of the freeze-dried preparation sample white block solid, the specific sample is shown in Figure 11, and the bottleneck of the sample in the middle and rear box of 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 test data of freeze-dried preparations located at different positions on the shelf in different batches after freeze-drying of different pH solutions
  • Table 25 Reconstitution test data of freeze-dried preparations located at different positions on the shelf in different batches after freeze-drying of different pH solutions
  • Table 26 HPLC purity data of API in solutions reconstituted with saline at different concentrations and time periods
  • the quality indicators of freeze-dried preparations include three main evaluation indicators such as reconstitution, stability, and residual solvents, and the above experiments show that the influencing factors include solvent (volume ratio of tert-butanol in tert-butanol-water mixed solvent), API concentration, excipient type (mannitol, sucrose) and dosage, pH value of the drug solution, etc., multiple multi-factor experiments were conducted, and the results are shown in Table 28.
  • Table 28 Data of freeze-dried preparation examples of drug solutions with different tert-butyl alcohol volume ratios, API concentrations, excipient types and amounts, and drug solution pH values
  • TBA is tert-butyl alcohol.
  • Mannitol is mannitol.
  • sucrose sucrose
  • API is TH-302 raw material drug.
  • PBS is phosphate buffered saline, and its pH value is stable at around 7.4.
  • 30% TBA/70Mannitol/10API-pH7.0 means 30% by volume of tert-butanol, 70 mg/mL of mannitol, and 10 mg/mL of API, and finally NaHCO3 is added to adjust the pH to 7.0, and the preparation method is the same as the above embodiment;
  • 40% TBA/60Mannitol/10API means 40% by volume of tert-butanol, 60 mg/mL of mannitol, and 10 mg/mL of API, and no pH adjustment is performed (weakly acidic), and the rest are similar.
  • the solubility data of TH-302 in the tert-butyl alcohol-water mixed solvent system can be inferred or verified by combining the above experiment and the 85 groups of exploratory experimental results in Table 28 above.
  • the compound can be stably present in the solution at a content of 5-500 mg/mL, and the experiment proves that it can be lyophilized when it is greater than 8 mg/mL and less than or equal to 200 mg/mL, but the lyophilization parameters may need to be adjusted, such as pre-freezing at -80°C, lower absolute air pressure during lyophilization, longer drying time and lower drying temperature, etc.
  • the lyophilized preparation has better stability and is easier to redissolve.
  • the content of the compound of formula I in the solution is greater than or equal to 8 mg/mL and less than or equal to 15 mg/mL. Such a content range will mean that the freeze-drying conditions are relatively mild and the freeze-drying cycle is shorter.
  • the content of the compound of formula I in the solution is greater than or equal to 8 mg/mL and less than or equal to 10 mg/mL, which will further mean that the freeze-dried preparation has commercially suitable freeze-drying cycle and freeze-drying low temperature conditions and has commercial large-scale production value: inappropriate API content will lead to large-scale freeze dryers used in commercial large-scale production. Due to uneven temperature, broken bottles and powder spraying will occur, resulting in a yield lower than 90% of the commercial production acceptance level.
  • the solubility data of TH-302 in the tert-butanol-water mixed solvent system can be inferred or verified by combining the above experiments and the 85 groups of exploratory experimental results in Table 28 above.
  • the volume percentage of tert-butanol relative to the solution is 1%-99% or the content of tert-butanol in the solution is 7.85-777.15 mg/mL (under the condition that the density of tert-butanol is 0.785 g/mL)
  • the prepared TH-302 solution is stable and clear.
  • the volume ratio is 5%-95% or the content of tert-butanol in the solution is 39.25-745.75 mg/mL (under the condition that the density of tert-butanol is 0.785 g/mL) and can be freeze-dried, but the freeze-drying parameters may need to be adjusted, 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 tert-butanol content in the solution is 235.5-471 mg/mL (under the condition that the density of tert-butanol is 0.785 g/mL), the freeze-dried preparation has better stability and is easier to reconstitute.
  • tert-butanol-water mixed solvent system too high tert-butanol content will affect the subsequent dissolution of mannitol/sucrose.
  • mannitol/sucrose When mannitol/sucrose is used as an excipient, its dosage will directly affect the quality of the subsequent freeze-dried preparation: too low mannitol/sucrose will result in the inability to evenly load the drug solution on the excipient skeleton after freeze-drying, and may even cause the freeze-dried powder cake to collapse or freeze-drying failure during the drying stage of the freeze-drying process. Therefore, the tert-butanol content in the mixed solvent cannot be increased blindly in order to increase the solubility of TH-302.
  • the more mannitol/sucrose the better: although a larger amount can serve as a freeze-dried skeleton to load more drugs, and is beneficial to freeze-drying production and improves the stability of freeze-dried preparations, it is affected by its solubility in the tert-butyl alcohol-water mixed solvent. Too high a dosage of mannitol/sucrose may make the mixed solvent unable to completely dissolve or the solution unstable and precipitate in the pre-freezing stage or cooling stage of freeze-drying. Therefore, the amount of mannitol/sucrose as an excipient in the freeze-dried solution also has an appropriate range to meet the above requirements.
  • the content of sucrose and mannitol in the solution is 20-300 mg/mL, and such a range can meet the above requirements: the liquid medicine is stable and is not easy to precipitate in the pre-freezing stage or the cooling stage of freeze-drying. It is further preferred to be 40-100 mg/mL, which can obtain a reconstituted solution and a freeze-dried preparation with good stability of the freeze-dried preparation. More preferably, it is 60-70 mg/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 (no more than 10 days, i.e., a production cycle of 240 hours).
  • too low mannitol/sucrose will result in the inability to evenly load the drug solution on the excipient skeleton after freeze-drying, and even cause freeze-dried powder cake collapse or freeze-drying failure in the drying stage of the freeze-drying process; and too high mannitol/sucrose content can obtain freeze-dried preparations with better appearance and stability, but it means that the content of the freeze-dried preparation of Chinese drug TH-302 per unit packaging unit is too low, which does not meet the requirements of commercial sales and use. Therefore, a suitable ratio of mannitol/sucrose to the drug is necessary.
  • the mass ratio of the compound of formula I to the excipient in the solution is 1: (0.5-20), which can meet the various other component requirements of items 1-4 above, and can make the prepared freeze-dried preparation have a better appearance and stability; preferably 1: (2-12.5) can obtain a better freeze-dried preparation that is easy to reconstitute, and more preferably 1: (5-10)
  • the prepared freeze-dried preparation will have excellent properties: the freeze-dried preparation has good stability, easy reconstitution, mild freeze-drying conditions, and a short freeze-drying cycle.
  • the filling volume of the drug solution in the vial also affects the appearance (powder spraying or adhering to the bottleneck of the vial) and reconstitution of the final lyophilized powder through various factors. Therefore, the ratio of the volume of the drug solution filled in the vial to the volume of the vial must be appropriate. Based on the above experimental data and combined with actual production, the filling volume of the lyophilized drug solution is 1/3 to 1/2 of the volume of the closed container, and the corresponding volume of the lyophilized preparation is 1/3 to 1/2 of the volume of the closed container.
  • TH-302 sample was accurately weighed and placed in a transparent vial. 6 mL of 5% glucose injection (D5W) was added and vigorously shaken for 2 minutes to prepare the solubility stock solution. It was observed that the solubility stock solution contained insoluble matter. The solubility stock solution was filtered and became clear. The filtered filtrate was placed at room temperature for 24 hours and the filtrate was still clear. After sampling and appropriate dilution, the content of the solution was measured by HPLC to be 7.25 mg/mL, which is the saturated solubility of the drug in the D5W solution.
  • D5W 5% glucose injection
  • the concentration of the intravenous injection solution containing TH-302 should be within the range of 0-7.25 mg/mL.
  • the infusion formula for intravenous drip needs further research and determination.
  • the API content of the drug solution is greater than or equal to 5 mg/mL and less than or equal to 500 mg/mL
  • the drug loading of the freeze-dried powder after freeze-drying is 5/0.9 to 500/0.9, that is, 5.55-555.55 mg/cm 3 .
  • the same calculation method is:
  • the drug content of the compound of formula I in the solution is 5-160 mg/mL, and the drug loading of the powder after freeze-drying is 5.55-177.77 mg/cm 3 ;
  • the drug content of the compound of formula I in the solution is 8-50 mg/mL, and the drug loading of the powder after freeze-drying is 8.88-55.55 mg/cm 3 ;
  • the drug content of the compound of formula I in the solution is 8-25 mg/mL, and the drug loading of the powder after freeze-drying is 8.88-27.77 mg/cm 3 ;
  • the drug content of the compound of formula I in the solution is 8-15 mg/mL, and the drug loading of the powder after freeze-drying is 8.88-16.66 mg/cm 3 ;
  • the drug content of the compound of formula I in the solution is 8-10 mg/mL, and the drug loading of the powder after freeze-drying is 8.88-11.11 mg/cm 3 .
  • the corresponding drug loading of the freeze-dried powder is 6.66, 7.77, 8.33, 8.88, 9.44, 11.11, 13.88, 16.66, 22.22, 27.77 mg/cm 3 .
  • the API content of the drug solution is greater than or equal to 5 mg/mL and less than or equal to 500 mg/mL
  • the drug loading of the freeze-dried powder after freeze-drying is 5/1.1 to 500/1.1, that is, 4.55-454.55 mg/cm 3 .
  • the same calculation method is:
  • the drug content of the compound of formula I in the solution is 5-160 mg/mL, and the drug loading of the powder after freeze-drying is 4.55-145.45 mg/cm 3 ;
  • the drug content of the compound of formula I in the solution is 8-50 mg/mL, and the drug loading of the powder after freeze-drying is 7.27-45.45 mg/cm 3 ;
  • the drug content of the compound of formula I in the solution is 8-25 mg/mL, and the drug loading of the powder after freeze-drying is 7.27-22.73 mg/cm 3 ;
  • the drug content of the compound of formula I in the solution is 8-15 mg/mL, and the drug loading of the powder after freeze-drying is 7.27-13.64 mg/cm 3 ;
  • the drug content of the compound of formula I in the solution is 8-10 mg/mL, and the drug loading of the powder after freeze-drying is 7.27-9.09 mg/cm 3 .
  • the corresponding drug loading of the freeze-dried powder is 5.45, 6.36, 6.82, 7.27, 7.73, 9.09, 11.36, 13.64, 18.18, 22.73 mg/cm 3 .
  • freeze-dried preparations Based on the previous exploratory experiments and taking certain considerations into account, the applicant has used the following recommended scheme to carry out relevant batch production of freeze-dried preparations.
  • the finished freeze-dried preparations produced meet the requirements of subsequent clinical trials and are expected to become candidate drugs for subsequent large-scale clinical trials and even for commercial production in the future.
  • API Predissolution of API: Weigh part of tert-butyl alcohol and water for injection, stir and mix evenly to prepare a 70% (w/w) tert-butyl alcohol solution. Then weigh the prescribed amount of API, add it to the 70% (w/w) tert-butyl alcohol solution and stir until it is visually completely dissolved.
  • half stopper is added and placed in freeze dryer for freeze drying.
  • the above-mentioned subpackaged preparation is freeze-dried in a vacuum freeze dryer, and freeze-dried using the freeze-drying parameters in Table 12 after adjustment. After freeze-drying, vacuum plugging and capping are performed to obtain a freeze-dried preparation. Shelf temperature before discharging: 5.0°C, manual plugging under high vacuum.
  • the drug solution was prepared and freeze-dried to finally obtain 174 bottles of freeze-dried preparations, with a specification of 0.20g/bottle (i.e., the bottle of freeze-dried preparation was labeled to contain 0.20g of TH-302 raw material), and an average filling amount of 1.98g/bottle (i.e., the average powder mass of this batch of freeze-dried preparations was 1.98g, and 25cm3 of freeze-dried powder contained 1.75g mannitol, 0.20g of TH-302 raw material and others).
  • 0.20g/bottle i.e., the bottle of freeze-dried preparation was labeled to contain 0.20g of TH-302 raw material
  • an average filling amount of 1.98g/bottle i.e., the average powder mass of this batch of freeze-dried preparations was 1.98g, and 25cm3 of freeze-dried powder contained 1.75g mannitol, 0.20g of TH-302 raw material and others).
  • the intermediate drug solution also contains mannitol as an excipient. Due to the relatively stable properties of mannitol, mannitol is not tested during quality control.
  • Table 30 Test results of freeze-dried preparations in batch 1202206001
  • pH test method Take 1 bottle of this product, add 38mL of injection water to dissolve, make a solution containing 5.0mg of TH-302 per 1mL, use a pH meter to measure, measure two samples in parallel, and take the average value.
  • the tert-butanol content test is carried out using the gas chromatography method specified in the pharmacopoeia. Take 1 bottle of freeze-dried preparation, fully warm it to room temperature, add about 30mL of water with a disposable syringe while maintaining the vacuum, shake well to dissolve, open the lid (note that the action of opening the stopper should be gentle and slow to avoid solution loss), transfer the solution to a 50mL volumetric flask, wash the test bottle and the inner surface of the stopper with water at least 3 times each, add the washing liquid to the volumetric flask, dilute with water to the scale, and shake well. Prepare 2 copies in parallel, use gas chromatography to detect two samples, and finally take the average value.
  • the tert-butanol project refers to the mass percentage of tert-butanol relative to the labeled amount of 200mg of raw materials calculated according to the labeled amount of TH-302 200mg.
  • the moisture content test is determined by the Karl Fischer method. To ensure the uniformity and representativeness of sampling, the following operations are used for sampling: Take a bottle of freeze-dried preparation, return to room temperature, wipe off the water attached to the surface of the bottle, open the lid, release the pressure in the bottle, open the bottle stopper, stir with a spoon to prevent the sample from adhering to the inner wall and bottom of the bottle, cover the bottle stopper, place the bottle horizontally and rotate it for no less than 5 times, turn the bottle upside down for no less than 5 times, open the bottle stopper, stir the sample with a spoon for no less than 5 times, cover the bottle stopper (seal the bottle mouth with a sealing film if necessary), and set aside.
  • Total impurities refer to the total amount of impurities other than TH-302 in the API except for the auxiliary materials tert-butyl alcohol and mannitol, and are determined using the above-mentioned TH-302 API HPLC purity test method.
  • Table 31 Quality index test data of three batches of freeze-dried preparations at different time periods of long-term and accelerated storage
  • the values of the content test items are: the first value is the percentage of the amount of TH-302 contained in the labeled amount (the amount of TH-302 contained is 0.20g); the second value is the mass percentage of TH-302 contained in the product is 9.8% (based on the average loading amount of 1.98g).
  • the freeze-thaw test was performed by placing the test sample in a dark place (-20°C ⁇ 5°C) for 2 days and (25 ⁇ 5°C) for 2 days as one cycle, and testing the HPLC purity of TH-302 in the freeze-dried sample.
  • Table 32 HPLC purity test data of the raw material TH-302 and two batches of lyophilized preparations prepared after three freeze-thaw cycles (%)
  • the data of the API project indicates the HPLC test purity of the TH-302 raw material itself used to prepare these two batches of lyophilized preparations.
  • the data under 1202206001-0 day represents the purity of the lyophilized formulation of batch 1202206001 when tested on day 0.
  • the 5.0 mg/mL solution diluted with the above-mentioned water for injection was used to prepare 5 mg/mL sterile water for injection solution, 4 mg/mL 5% glucose injection solution, 0.5 mg/mL 5% glucose injection solution, 4 mg/mL 0.9% sodium chloride injection solution, and 0.5 mg/mL 0.9% sodium chloride injection solution, and tested them respectively.
  • the test items included solution properties, osmotic pressure, pH, solution color, content, and impurity HPLC percentage (total impurities). The results are shown in Table 33.
  • the content, related substances, pH, osmotic pressure, solution color, properties and other test items of each compatible solution are in compliance with the regulations, which shows that the above lyophilized preparations can be reconstituted and compatible with the commonly used clinical water for injection, 5% glucose injection, and 0.9% sodium chloride injection to meet the requirements of intravenous injection.
  • Exposure to light for 5 days* indicates that the vial containing the lyophilized preparation is without outer packaging, while exposure to light for 5 days indicates the normal situation, in which the vial is with outer packaging.
  • 3 means the color is lighter than the No. 0.5 standard colorimetric solution; 4 means the color is between the No. 0.5 and No. 1 standard colorimetric solutions.
  • Table 36 Stability test results of reconstituted solution of GMP batch 296220901 lyophilized preparation

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Abstract

提供TH-302的溶液、冻干制剂、冻干制剂单位包装、注射液及注射液配制方法。用于制备高载药量冻干制剂的溶液，含有TH-302以及水和叔丁醇、甘露醇：水和叔丁醇作为混合溶剂，叔丁醇相对于所述溶液体积百分比为(30±3)％或质量百分比为(24±2.4)％，或所述溶液中叔丁醇含量为(235.5±23.55)mg/mL；TH-302在溶液中的含量为(8.16±0.82)mg/g或(8.00±0.80)mg/mL；甘露醇为赋形剂，其在溶液中的质量百分比为(7.14±0.71)％或所述溶液中甘露醇含量为(70±7)mg/mL，所述溶液pH为4-9。冻干制剂，含有TH-302以及甘露醇，冻干制剂中TH-302的载药量为(8.00±0.80)mg/cm3或冻干制剂中TH-302的质量百分比为(10.23±1.02)％，冻干制剂中甘露醇的含量为(70±7)mg/cm3，或冻干制剂中甘露醇的含量为TH-302的质量百分比(10.23±1.02)％的百分比余量。

Description

溶液、冻干制剂、冻干制剂单位包装、注射液及注射液配制方法 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 alkylat or 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/cm3。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 usi ng 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/m2至460mg/m2的日剂量来静脉注射给药；
480mg/m2至大约670mg/m2或者例如575mg/m2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(m2)＝([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/cm3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/cm3)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 pr odrug 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/cm3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-acti vated 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(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
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
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
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：
其中,R各自独立地选自H、-CH3、-CH2CH3，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：
其中,R各自独立地选自H、-CH3、-CH2CH3，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：葡聚糖、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(C2H4O)a(C3H6O)b(C2H4O)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
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；
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/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。
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；
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；
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。
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：
其中,R各自独立地选自H、-CH3、-CH2CH3，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/cm3而小于或等于555.55mg/cm3,或
冻干制剂中式I化合物的载药量大于或等于4.55mg/cm3而小于500mg/cm3，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：
其中,R各自独立地选自H、-CH3、-CH2CH3，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/cm3而小于或等于555.55mg/cm3,或
冻干制剂中式I化合物的载药量大于或等于4.55mg/cm3而小于500mg/cm3，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：
其中,R各自独立地选自H、-CH3、-CH2CH3，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/cm3而小于或等于555.55mg/cm3,或
冻干制剂中式I化合物的载药量大于或等于4.55mg/cm3而小于500mg/cm3，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/cm3而小于或等于555.55mg/cm3、冻干制剂中式I化合物的载药量大于或等于4.55mg/cm3而小于500mg/cm3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/cm3而小于或等于555.55mg/cm3。
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/cm3而小于500mg/cm3。
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/cm3，一般这个sh的数值会在20cm3左右，可能冻干后体积膨胀而大于20cm3，可能冻干后塌陷而体积小于20cm3。
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
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、冻干制剂过程中因为容器、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/cm3而小于或等于177.77mg/cm3，优选为大于或等于8.88mg/cm3而小于或等于55.55mg/cm3，更优选为大于或等于8.88mg/cm3而小于或等于27.77mg/cm3，进一步优选为大于或等于8.88mg/cm3而小于或等于16.66mg/cm3，再进一步优选为大于或等于8.88mg/cm3而小于或等于11.11mg/cm3；
或者
冻干制剂中式I化合物的载药量大于或等于4.55mg/cm3而小于或等于145.45mg/cm3，优选为大于或等于7.27mg/cm3而小于或等于45.45mg/cm3，更优选为大于或等于7.27mg/cm3而小于或等于22.73mg/cm3，进一步优选为大于或等于7.27mg/cm3而小于或等于13.64mg/cm3，再进一步优选为大于或等于7.27mg/cm3而小于或等于9.09mg/cm3。
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：
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/cm3，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/cm3；
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：
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/cm3，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/cm3；
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/cm3。
冻干制剂，实质由下式化合物以及赋形剂组成：
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/cm3，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/cm3；
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、冻干制剂过程中因为容器、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。
特别的，对于(8.00±0.80)这种情况，其范围对应为7.20-8.80，则实际如测试得到含量大于或等于6.48而小于或等于9.68也应被理所当然的确定为与上述范围是等同的。
在前期探索实验基础上，申请人提出以下的推荐方案：基于某些方面的考量，申请人已使用以下的推荐方案进行了冻干制剂的相关批量生产，生产的成品冻干制剂满足了后续临床试验的要求，有望成为后续大规模临床试验的候选药物甚至将来的商业化上市生产。
用于制备高载药量冻干制剂的溶液，含有下式I-1的化合物以及水和叔丁醇、甘露醇：
水和叔丁醇作为混合溶剂，叔丁醇相对于所述溶液体积百分比为(30±3)％或质量百分比为(24±2.4)％，或所述溶液中叔丁醇含量为(235.5±23.55)mg/mL；
式I-1的化合物在溶液中的含量为(8.16±0.82)mg/g或(8.00±0.80)mg/mL；
甘露醇为赋形剂，其在溶液中的质量百分比为(7.14±0.71)％或所述溶液中甘露醇含量为(70±7)mg/mL，
所述溶液pH为4-9。
由于TH-302(化合物I-1)自身的性质以及生产批次的关系，有可能上述含有I-1化合物以及水和叔丁醇、甘露醇的溶液在特定的温度、环境下其pH并不是在4-9，因此需要添加pH添加剂进行调节，推荐的pH调节剂为医药上常用的Na2CO3、NaHCO3、K2CO3、KHCO3等，优选为NaHCO3，因此上述溶液还含有pH调节剂碳酸氢钠。
进一步的，所述溶液的pH值为6-8。
冻干制剂，含有下式I-1的化合物以及甘露醇：
冻干制剂中式I-1化合物的载药量为(8.00±0.80)mg/cm3或冻干制剂中式I-1化合物的质量百分比为(10.23±1.02)％，
冻干制剂中甘露醇的含量为(70±7)mg/cm3，或冻干制剂中甘露醇的含量为式I-1化合物的质量百分比(10.23±1.02)％的百分比余量。
经过实验，使用上述溶液在调整冻干工艺后，冻干后所得的冻干固体其体积基本不会发生变化：冻干制剂没有发现明细的塌陷(导致体积变小)和喷瓶(导致体积变大)，因此随着冻干过程中作为溶剂的水、叔丁醇被蒸发除去，冻干制剂中只有TH-302以及甘露醇，因此冻干制剂中式I-1化合物的载药量为8.00mg/cm3，其对应的质量百分比为10.23％；冻干制剂中甘露醇的含量为70mg/cm3，考虑到灌装过程中的10％的装量差异，上述数值均有±10％的变动范围。
冻干制剂，含有下式I-1的化合物以及甘露醇、pH调节剂：
冻干制剂中式I-1化合物的载药量为(8.00±0.80)mg/cm3或冻干制剂中式I-1化合物的质量百分比为(10.23±1.02)％，
冻干制剂中甘露醇的含量为(70±7)mg/cm3，或冻干制剂中甘露醇的含量为式I-1化合物的质量百分比(10.23±1.02)％的百分比余量。
冻干制剂，含有下式I-1的化合物以及甘露醇、残留的溶剂成分、pH调节剂：
冻干制剂中式I-1化合物的载药量为(8.00±0.80)mg/cm3或冻干制剂中式I-1化合物的质量百分比为(10.23±1.02)％，
冻干制剂中甘露醇的含量为(70±7)mg/cm3，
所述残留的溶剂成分为水和叔丁醇。
进一步的，冻干制剂被注射用水溶解为5.0mg/mL的水溶液其pH值为4.0-7.0。
推荐的测试方法如下：
于室温下精密称量，加入注射用水溶解并定容配制成每1mL含TH-302 5.0mg的溶液，使用pH计进行测定，平行测定至少两个样，取平均值。
所述pH调节剂为碳酸氢钠，其含量为0.01-0.10mg/cm3。
优选的，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1795ppm，优选为1000ppm，更优选为小于或等于500ppm。
叔丁醇作为药用辅料用于注射用TH-302的生产，在冻干后可能会有少量残留。叔丁醇对人体有低毒性，为保证药品安全和质量，应制定限度控制注射用TH-302中叔丁醇的残留量。根据ICHO3C(R8)文件中规定叔丁醇的PDE(每日允许暴露量)为35mg天，根据文献预计TH-302最大日剂量1.5g，按2g从严计算，对应注射最大日剂量2g，以TH-302的含量为0.2g/瓶(50mL西林瓶)需要10瓶，每瓶冻干粉饼为1.95g(1.75g甘露醇以及0.20g原料药TH-302)，则10瓶的冻干粉饼总质量为19.5g，则残留叔丁醇含量为质量百分比小于或等于35mg/19.5g＝0.1795％即为1795ppm。
基于处方筛选过程中处方的一直调整、叔丁醇检测方法的操作性以及计算方便性，以及考虑工艺过程的差异性可能导致每瓶装量的变化，叔丁醇的限度也可以选择按标示的TH-302量进行计算，则残留叔丁醇含量相对于标示的原料药TH-302质量百分比小于或等于35mg/2.0g＝1.75％。
根据换算，如以含TH-302原料药API为200mg规格的冻干制剂为例，上述残留叔丁醇含量为质量百分比小于或等于1795ppm，优选为1000ppm，更优选为小于或等于500ppm将完全等同于：残留叔丁醇含量为标示量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％。
进一步，冻干制剂的单位体积质量为(79.2±7.9)mg/cm3。
单位体积质量类似于密度，冻干制剂为多孔的饼状，其内TH-302原料药与甘露醇、碳酸氢钠(残留的溶剂水、叔丁醇)充分融合而形成蜂窝状结构，在蜂窝内部形成空腔。单位体积质量的大小是一个综合指标，与冻干制剂的载药量、甘露醇含量、残留的溶剂水、叔丁醇均有关系。研究认为，均匀且的冻干制剂具有较好的快速溶解特性，便于后续配伍使用。冻干制剂是否疏松多孔以及疏松多孔的程度微观层面可以通过直接观察或显微观察定性研究，宏观层面则可以通过上述冻干制剂的单位体积质量进行表征。
经实验验证，上述冻干制剂的单位体积质量为(79.2±7.9)mg/cm3是比较合适的：疏松多孔的程度合适，载药量也满足要求，而且冻干工艺在大规模生产中比较容易实现。
显然，上述冻干制剂其可由上述用于制备高载药量冻干制剂的溶液冷冻干燥而制备。
盛装上述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-1的化合物的含量为1600-5333mg；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-1的化合物的含量为800-2666mg；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-1的化合物的含量为400-1333mg；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-1的化合物的含量为160-533mg；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-1的化合物的含量为80-266mg；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-1的化合物的含量为48-160mg；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-1的化合物的含量为40-133mg；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-1的化合物的含量为32-107mg；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-1的化合物的含量为29-96mg；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-1的化合物的含量为24-79mg；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-1的化合物的含量为16-53mg；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-1的化合物的含量为13-42mg；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-1的化合物的含量为11-40mg；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-1的化合物的含量为8-27mg；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-1的化合物的含量为5-16mg；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-1的化合物的含量为3-11mg；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-1的化合物的含量为2-8mg。
上述化合物含量是依据8.0mg/cm3计算而得的，数值为标识的量，其实际的含量在标识量±10％的范围内。
一种静脉注射用注射液，其溶剂为水，溶质包括TH-302原料药、甘露醇、叔丁醇以及碳酸氢钠、调节等渗试剂，其中，TH-302原料药的含量为0.1-4.0mg/mL，注射液的渗透压为260～320mOsmol/kg，pH值为4.0～9.0。
同理，作为静脉注射用溶液，需要调节渗透压，常用的注射液为5％质量百分比的葡萄糖注射液和0.9％质量百分比的氯化钠注射液，因此溶质中可能还包括调节等渗试剂，其选自葡萄糖、氯化钠。
进一步，还提供上述注射液的配制方法，包括以下操作：
将上述的冻干制剂于室温下放置直至恢复室温；
使用注射用水将上述恢复室温的冻干制剂配制为TH-302原料药含量为(5±0.5)mg/mL的注射用水溶液；
将适量的上述注射用水溶液注入到0.9％生理盐水注射液或5％葡萄糖注射液中稀释到TH-302原料药的含量为0.1-4.0mg/mL即得。
上述注射液的配制方法，其被印刷在冻干制剂的制剂单位包装附带的说明书中，用于指导临床配药后现配现用，所配制的注射液应在8小时内注射完毕，包括以下操作：
将权利要求4-12中任意一项所述的冻干制剂于室温下放置30-120分钟直至恢复室温；
使用注射器抽取适量体积的注射用水，针管斜面向上且针头与瓶塞呈约60°进针后沿瓶内壁注入，注入完成后摇动药瓶至少20秒，确保所有冻干块/粉完全溶解且混合均匀，静置消除气泡，得到TH-302原料药含量为(5±0.5)mg/mL的注射用水溶液；
从盛装0.9％生理盐水注射液或5％葡萄糖注射液容器中抽取计算量的溶液，将与抽取量等量的上述注射用水溶液注入到盛装0.9％生理盐水注射液或5％葡萄糖注射液中以稀释到TH-302原料药的含量为0.1-4.0mg/mL即得。
补充的，如静置消除气泡后，溶液中具有混悬物，应在30～35℃下振摇2分钟至澄清。
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2-8℃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；
图13为冻干制剂复溶时注射器针头刺入西林瓶胶塞的角度示意图；
图14为冻干制剂复溶时注射器针头刺入西林瓶胶塞后，针头沿着瓶内壁注入注射液的示意图。
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N2；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。
如未特殊说明，为计算方便，冻干制剂检测项目中的残留溶剂(叔丁醇)的指标数值均表示残留叔丁醇含量为相对于冻干制剂的API标示量的质量百分比。
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

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。
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
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NaHCO3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NaHCO3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
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
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
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
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
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
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
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
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(％)
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

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
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
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pH4.58，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
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pH4.61，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

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
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
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pH4.77。
药液-02：加入碳酸氢钠200μl，测pH6.60(pH在6.0～6.5)。室温下放置24h测pH5.20。
药液-03：加入碳酸氢钠1000μl，测pH7.20(pH在7.0)。室温下放置24h测pH6.90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

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
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
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

(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
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
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


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NaHCO3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/cm3，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/cm3；
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/cm3；
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/cm3；
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/cm3；
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/cm3。
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/cm3。
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/cm3，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/cm3；
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/cm3；
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/cm3；
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/cm3；
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/cm3。
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/cm3。
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％。
在前期探索实验基础上，申请人基于某些方面的考量，已使用以下的推荐方案进行了冻干制剂的相关批量生产，生产的成品冻干制剂满足了后续临床试验的要求，有望成为后续大规模临床试验的候选药物甚至将来的商业化上市生产。
11、小批次冻干制剂制备、稳定性及复溶实验
11.1参照“7、考察pH对样品稳定性的影响实验”的相关制备及表22的处方制定以下的实验室模拟生产及生产线上生产流程制备冻干制剂
1、配液
①原料药预溶：称取部分叔丁醇和注射用水，搅拌混合均匀，制备成70％(w/w)的叔丁醇溶液。再称量处方量的原料药，加入70％(w/w)叔丁醇溶液中搅拌，搅拌至目视完全溶解。
②在配液罐中加入注射用水总量的约70％(w/w)的注射用水，加入处方量甘露醇，搅拌溶解。
③将处方总量中剩余叔丁醇加入配液罐中，开启搅拌，混合均匀。
④将预溶好的原料药加入配液罐，用注射用水加至配制总量。开启搅拌，混合均匀。
⑤加入碳酸氢钠溶液调节pH值至4.0-9.0。碳酸氢钠溶液的质量百分比为5％。
2、过滤，使用除菌过滤器过滤。
3、灌装
使用50mL西林瓶灌装，理论装量为25mL/瓶(密度按0.98g/mL记为24.5g/瓶)，装量控制在±2％以内，则可接受的实际灌装量为(25.0±0.5)mL/瓶，质量控制在(24.5±0.49)g/瓶。
4、半加塞
灌装后半加塞，放入冻干机进行冷冻干燥。
5、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的冻干参数在进行调整后进行冻干。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。
根据以上的流程进行药液配制和冻干最终得到174瓶冻干制剂，规格为0.20g/瓶(即标识该瓶冻干制剂含有TH-302原料药为0.20g)，装量平均为1.98g/瓶(即该批次冻干制剂的粉饼质量平均为1.98g，25cm3的冻干粉饼含1.75g甘露醇、0.20g的TH-302原料药以及其他)。
上述批次的中间药液即调节pH后的药液的取样检测结果如下表29所示。
表29：1202206001批次中间药液检测结果
中间药液除作为溶剂的水、叔丁醇外，还含有赋形剂甘露醇，由于甘露醇性质比较稳定，在质量控制中不进行甘露醇的检测。
上述批次的冻干制剂抽样进行检测的结果如下表30所示。
表30：1202206001批次冻干制剂检测结果
酸碱度测试方法：取本品1瓶，加入38mL注射用水溶解，制成每1mL含TH-302 5.0mg的溶液，使用pH计进行测定，平行测定两个样，取平均值。
叔丁醇含量测试使用药典中规定的气相色谱法进行检测。取冻干制剂1瓶，充分复温至室温，在保持真空度的情况下，用一次性注射器加入水约30mL，摇匀溶解后，启盖(注意开启胶塞的动作要轻柔缓慢，避免溶液损失)，将溶液转移至50mL量瓶中，用水洗涤供试品瓶和胶塞内表面各至少3次，洗液并入量瓶中，再用水稀释至刻度，摇匀。平行制备2份，使用气相色谱法检测两份样品，最后取平均值。叔丁醇项目是指按标示的TH-302量200mg进行计算得出叔丁醇相对于标示量200mg原料药的质量百分比。
水分含量测试使用卡尔费休法测定。为确保取样均一性及代表性，采用以下操作进行取样：取冻干制剂1瓶，恢复至室温，擦干瓶子表面附着水，启盖，释放瓶内压力，打开瓶塞，用勺子搅拌使样品不附着于瓶内壁和瓶底，盖上瓶塞，横置瓶子并转动不少于5圈，上下倒置瓶子不少于5次，打开瓶塞，用勺子搅拌样品不少于5圈，盖上瓶塞(必要时用封口膜密封瓶口)，待用。精密称取约0.20g的冻干制剂样品(实际由于无法完全将瓶内壁的冻干粉末转移到水分仪的滴定池中，实际质量是少于0.20g的，通过质量差法精密称取)并加入到滴定池中，预先搅拌2分钟后开始滴定。或参照USP<921>方法进行测定得出水分质量百分比数值。
总杂即除辅料叔丁醇、甘露醇外原料药中TH-302之外的杂质的总量，使用上述TH-302原料药HPLC纯度测试方法测定。
同样的流程，在冻干盘中制备另一批次的冻干制剂(批号1202206002)共529.54g，并用内层为双层低密度聚乙烯(PE)自封袋，外层为单层300mm无印刷复合膜(符合相关要求)热封口。
同样的流程，制备另一批次的冻干制剂(批号F00106301)共600瓶，装量平均为1.97g/瓶进行稳定性研究。
11.2冻干制剂在拟定的长期贮藏条件下(-20℃±5℃)、加速条件下(25±5℃)贮藏的稳定性研究
考察TH-302冻干制剂在拟定的长期贮藏条件下(-20℃±5℃)、高温加速条件下(25±5℃)相关检测指标随时间变化的规律，为冻干制剂的质量稳定性和存储时间(有效期)提供依据。
将上述两批样品(1202206002和1202206001)置于-20±5℃避光环境中存储进行长期稳定性实验，将F00106301批进行长期稳定性实验和25±5℃避光环境中加速稳定性实验，具体实验结果如下表31。
表31：三批次冻干制剂长期和加速贮藏不同时间的质量指标测试数据
鉴别，HPLC谱图与对照品溶液保留时间是否一致。
含量检测项目的数值，第一个数值为含TH-302的量为标示量(含有TH-302量为0.20g)的百分比数值；第二个为含TH-302的质量百分比为9.8％(以平均装量1.98g计)。
根据以上长期数据可知，在长期贮藏条件下(-20℃±5℃)总杂质含量百分比变化缓慢，其在3个月、6个月的存储后杂质、pH、水分、含量变化均不明显；另根据另一批次的使用较高纯度的TH-302原料药的F00106301批次的数据可知，在加速条件下(25±5℃)存储的情况下，上述指标依然变化不明显，进一步根据加速条件推测这些结果相当于在-20℃±5℃条件下存储18月以上的结果。
11.3冻干制剂冻融循环稳定性研究
冻融试验是将供试品于避光(-20℃±5℃)放置2天、(25±5℃)放置2天作为一次循环，并测试冻干样品中TH-302的HPLC纯度。
表32：原料药TH-302、以及制备的两批次冻干制剂、三次冻融循环后的HPLC纯度测试数据(％)
API项目的数据标示制备这两批冻干制剂的TH-302原料药本身的HPLC测试纯度。
1202206001-0天项下的数据表示1202206001批次冻干制剂在0天检测时的纯度。
根据以上长期数据可知，在长期贮藏条件下(-20℃±5℃)拿出后放置在室温环境(25±5℃)，即使在经历3此操作后，冻干制剂中原料药TH-302的纯度变化极小，证明冻干制剂具有较好的抗温度突变冲击的稳定性，为冻干制剂实际转运、运输过程中较长时间的常温暴露和运输的稳定性提供了实验证据。
11.4冻干制剂配伍(0.9％NaCl、5％D5W)稳定性实验
从-20℃±5℃处取出注射用冻干制剂，充分恢复至室温，使用一次性无菌注射器(50mL)抽取灭菌注射用水38mL，针管斜面向上，针头与瓶塞呈约60°进针(见图1)以避免产生针刺落屑，沿瓶内壁转圈注入(见图2)，注入完成后剧烈摇动药瓶至少20秒，确保所有冻干块/粉完全溶解且混合均匀，得到约40mL溶液，每1mL溶液中约含TH-302 5.0mg。如遇剧烈摇动后20秒冻干块/粉仍未能完全溶解，可在30℃～35℃水浴下，振摇直至完全溶解。
使用上述注射用水稀释的5.0mg/mL浓度的溶液分别配制成5mg/mL灭菌注射用水配伍溶液、4mg/mL 5％葡萄糖注射液配伍溶液、0.5mg/mL 5％葡萄糖注射液配伍溶液、4mg/mL 0.9％氯化钠注射液配伍溶液、0.5mg/mL 0.9％氯化钠注射液配伍溶液，并分别进行测试，测试项目包括溶液性状、渗透压、酸碱度pH、溶液颜色、含量、杂质HPLC百分比(总杂)结果如表33所示。
表33：不同配伍溶液检测结果

根据以上配伍实验结果，各配伍溶液含量、有关物质、pH、渗透压、溶液颜色、性状等检测项目均符合规定，这显示上述冻干制剂与临床常用的注射用水、5％葡萄糖注射液、0.9％氯化钠注射液进行复溶配伍均可以达到静脉注射要求。
12、大批次冻干制剂制备、稳定性及复溶配伍实验
在以上第11部分制备冻干制剂实验的基础上进行更大规模的冻干制剂制备，其批号分别为F00106301、F00115101进行高温加速条件下(25±5℃)的稳定性测试和冻融实验，结果如下表表34。
表34：两批次(F00106301、F00115101)冻干制剂高温加速贮藏和多次冻融试验结果
表34的结果显示放大后制备的冻干制剂的质量几乎没有变化，各项指标均合格。
为此使用暂定质量标准的API原料药在GMP条件下生产了GMP批次，该批次将考虑在后续的临床试验中使用，对该批次296220901进行了高温加速稳定性实验和复配实验，具体实验数据如下表35、表36。
表35：GMP批次296220901冻干制剂高温加速贮藏和多次冻融实验结果

光照5天*表示盛装有冻干制剂的西林瓶不带有外包装，而光照5天表示为正常情形，其带有外包装。
1，表示白色疏松块状物；2，表示淡黄色疏松块状物。
3，表示颜色淡于0.5号标准比色液；4，表示在颜色0.5号和1号标准比色液之间。
不溶性微粒1，表示粒径≥10um的微粒数目；不溶性微粒2表示粒径≥25um的微粒数目。
表36：GMP批次296220901冻干制剂复配溶液稳定性检测结果

以上实验数据证明，上述冻干制剂处方在大规模制备后所得的冻干制剂的稳定性、复溶配伍均是合格的，另外通过加速实验的结果，可以推测冻干制剂在拟定的-20±5℃避光环境可以稳定存储18个月。

Claims (17)

  1. 用于制备高载药量冻干制剂的溶液，含有下式I-1的化合物以及水和叔丁醇、甘露醇：
    水和叔丁醇作为混合溶剂，叔丁醇相对于所述溶液体积百分比为(30±3)％或质量百分比为(24±2.4)％，或所述溶液中叔丁醇含量为(235.5±23.55)mg/mL；
    式I-1的化合物在溶液中的含量为(8.16±0.82)mg/g或(8.00±0.80)mg/mL；
    甘露醇为赋形剂，其在溶液中的质量百分比为(7.14±0.71)％或所述溶液中甘露醇含量为(70±7)mg/mL，
    所述溶液pH为4-9。
  2. 根据权利要求1所述的溶液，其还含有pH调节剂碳酸氢钠。
  3. 根据权利要求1所述的溶液，其中，所述溶液的pH值为6-8。
  4. 冻干制剂，含有下式I-1的化合物以及甘露醇：
    冻干制剂中式I-1化合物的载药量为(8.00±0.80)mg/cm3或冻干制剂中式I-1化合物的质量百分比为(10.23±1.02)％，
    冻干制剂中甘露醇的含量为(70±7)mg/cm3，或冻干制剂中甘露醇的含量为式I-1化合物的质量百分比(10.23±1.02)％的百分比余量。
  5. 冻干制剂，含有下式I-1的化合物以及甘露醇、pH调节剂：
    冻干制剂中式I-1化合物的载药量为(8.00±0.80)mg/cm3或冻干制剂中式I-1化合物的质量百分比为(10.23±1.02)％，
    冻干制剂中甘露醇的含量为(70±7)mg/cm3，或冻干制剂中甘露醇的含量为式I-1化合物的质量百分比(10.23±1.02)％的百分比余量。
  6. 冻干制剂，含有下式I-1的化合物以及甘露醇、残留的溶剂成分、pH调节剂：
    冻干制剂中式I-1化合物的载药量为(8.00±0.80)mg/cm3或冻干制剂中式I-1化合物的质量百分比为(10.23±1.02)％，
    冻干制剂中甘露醇的含量为(70±7)mg/cm3，
    所述残留的溶剂成分为水和叔丁醇。
  7. 根据权利要求4-6中任意一项所述的冻干制剂，
    冻干制剂被注射用水溶解为5.0mg/mL的水溶液，其pH值为4.0-7.0。
  8. 根据权利要求5或6所述的冻干制剂，其中，
    所述pH调节剂为碳酸氢钠，其含量为0.01-0.10mg/cm3。
  9. 根据权利要求6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1795ppm，优选为1000ppm，更优选为小于或等于500ppm。
  10. 根据权利要求4-6中任意一项所述的冻干制剂，其中，
    冻干制剂的单位体积质量为(79.2±7.9)mg/cm3。
  11. 根据权利要求4-6中任意一项所述的冻干制剂，其由权利要求1中的溶液冷冻干燥而制备。
  12. 盛装有权利要求4-11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-1的化合物的含量为1600-5333mg；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-1的化合物的含量为800-2666mg；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-1的化合物的含量为400-1333mg；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-1的化合物的含量为160-533mg；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-1的化合物的含量为80-266mg；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-1的化合物的含量为48-160mg；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-1的化合物的含量为40-133mg；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-1的化合物的含量为32-107mg；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-1的化合物的含量为29-96mg；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-1的化合物的含量为24-79mg；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-1的化合物的含量为16-53mg；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-1的化合物的含量为13-42mg；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-1的化合物的含量为11-40mg；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-1的化合物的含量为8-27mg；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-1的化合物的含量为5-16mg；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-1的化合物的含量为3-11mg；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-1的化合物的含量为2-8mg。
  13. 一种静脉注射用注射液，其溶剂为水，溶质包括TH-302原料药、甘露醇、叔丁醇以及碳酸氢钠、调节等渗试剂，其中，TH-302原料药的含量为0.1-4.0mg/mL，注射液的渗透压为260～320mOsmol/kg，pH值为4.0～9.0。
  14. 根据权利要求13所述的注射液，所述调节等渗试剂选自葡萄糖、氯化钠。
  15. 上述权利要求14的注射液的配制方法，包括以下操作：
    将权利要求4-12中任意一项所述的冻干制剂于室温下放置直至恢复室温；
    使用注射用水将上述恢复室温的冻干制剂配制为TH-302原料药含量为(5±0.5)mg/m L的注射用水溶液；
    将适量的上述注射用水溶液注入到0.9％生理盐水注射液或5％葡萄糖注射液中稀释到TH-302原料药的含量为0.1-4.0mg/mL即得。
  16. 上述权利要求14的注射液的配制方法，其被印刷在冻干制剂的制剂单位包装附带的说明书中，用于指导临床配药后现配现用，所配制的注射液应在8小时内注射完毕，包括以下操作：
    将权利要求4-12中任意一项所述的冻干制剂于室温下放置30-120分钟直至恢复室温；
    使用注射器抽取适量体积的注射用水，针管斜面向上且针头与瓶塞呈约60°进针后沿瓶内壁注入，注入完成后摇动药瓶至少20秒，确保所有冻干块/粉完全溶解且混合均匀，静置消除气泡，得到TH-302原料药含量为(5±0.5)mg/mL的注射用水溶液；
    从盛装0.9％生理盐水注射液或5％葡萄糖注射液容器中抽取计算量的溶液，将与抽取量等量的上述注射用水溶液注入到盛装0.9％生理盐水注射液或5％葡萄糖注射液中以稀释到TH-302原料药的含量为0.1-4.0mg/mL即得。
  17. 根据权利要求16所述的配制方法，如静置消除气泡后，溶液中具有混悬物，应在30～35℃下振摇2分钟至澄清。
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