Lyophilized preparation solution of AST-3424, lyophilized preparation, method, and use

Abstract

Provided are a lyophilized preparation solution of AST-3424, a lyophilized preparation, a method, and use. The lyophilized preparation solution comprises a compound AST-3424 of the following formula, water, an excipient, and a pH regulator, wherein the water is used as a solvent; the excipient is selected from trehalose, sorbitol, water-soluble modified β-cyclodextrin, and a phospholipid-polyethylene glycol conjugate; the content of AST-3424 in the solution is 1 mg / ml to 20 mg / ml; the pH value of the solution is 7.4 to 10.5. The lyophilized preparation comprises a compound AST-3424 of the following formula, an excipient, and a pH regulator, wherein the excipient is selected from trehalose, sorbitol, water-soluble modified β-cyclodextrin, and a phospholipid-polyethylene glycol conjugate; the drug loading capacity of AST-3424 in the lyophilized preparation is 1 mg / cm3 to 20 mg / cm3; a mass ratio of the excipient to AST-3424 is (10-20):1.

Description

AST-3424 lyophilized formulation solution and lyophilized formulation, methods and uses TECHNICAL FIELD

[0001] The present application relates to the development of AST-3424 lyophilized formulation, belonging to the technical field of pharmaceutical preparations. BACKGROUND

[0002] DNA alkylating agent prodrug AST-3424 (WO2016145092, WO2017087428) targeting overexpression of aldehyde ketone reductase 1C3 (AKR1C3), CAS number 2097713-69-2, structure as follows:

[0003] AST-3424 (also known as OBI-3424, TH-3424) is activated by AKR1C3 enzyme overexpressed in cancer cells to release metabolite AST-2660 (also known as AST-2660) inside cancer cells. AST-3424 itself has little toxicity to cancer cells, and its pharmacological effect in animal models and in vitro pharmacological experiments is related to the expression of AKR1C3 enzyme: prodrug AST-3424 is metabolized to AST-2660 under the action of AKR1C3 enzyme and NADPH, and the expression of the enzyme is positively correlated with the drug efficacy.

[0004] Currently, the drug has entered I / II phase clinical trials in China and the United States respectively (U.S. NCT03592264, indications: liver cancer, pancreatic cancer and other solid tumors, sponsor: Taiwan, China, Haiding Biotechnology Co., Ltd. OBI Pharma Inc (4174), drug named OBI-3424; U.S. NCT04315324, T-ALL / T-LBL (acute T lymphoblastic leukemia / T lymphoblastic lymphoma), drug named OBI-3424; China CTR20191371, indications: various solid tumors, sponsor: Shenzhen Aixin Dawei Pharmaceutical Technology Co., Ltd. Ascentawits Pharmaceuticals, LTD., drug named AST-3424; CTR20201915, indications: acute T lymphoblastic leukemia and acute B lymphoblastic leukemia, sponsor: Shenzhen Aixin Dawei Pharmaceutical Technology Co., Ltd. Ascentawits Pharmaceuticals, LTD., drug named AST-3424).

[0005] In these I / II phase clinical trials in China and the United States, AST-3424 has been preliminarily proven to have therapeutic effects on advanced solid tumors and hematological tumors.

[0006] The drug administered in the above-mentioned clinical trial is a concentrated solution of AST-3424 for injection. According to the patent application PCT / CN2020 / 101870, publication number WO2021008520, and the above-mentioned clinical trial information, it can be known that:

[0007] The concentrated solution of AST-3424 for injection, 1ml specification, contains 10mg AST-3424, is composed of 0.75ml of anhydrous ethanol and 0.25ml of anhydrous propylene glycol and 10mg of AST-3424 raw material, and is filled in a 2ml brown light-proof glass bottle made of borosilicate glass. The storage condition is light-proof, -20℃.

[0008] In the subsequent phase III clinical trial and commercial sales, the above-mentioned concentrated solution of AST-3424 for injection needs to be stored and transported at -20℃ cold chain, and needs to be specially equipped with special low-temperature equipment for storage (general household level refrigerator cannot meet the requirements), and also puts forward higher requirements for cold chain transportation.

[0009] Therefore, it is necessary to develop a new dosage form to meet the easy storage and transportation requirements. SUMMARY

[0010] The inventors of the present application have prepared a lyophilized preparation of AST-3424 through multiple experiments and continuous optimization. The lyophilized preparation has certain stability at high temperature 40℃, and can be stored for a long time under the condition of ordinary cold chain transportation and household level refrigerator refrigeration temperature 2-8℃. That is, the prepared lyophilized preparation of AST-3424 has good stability, and the lyophilized preparation is preliminarily verified to be suitable for subsequent intravenous injection administration, and has the potential to be further developed to replace the above-mentioned concentrated solution of AST-3424 for injection. Therefore, the present application proposes a new solution formula of AST-3424 lyophilized preparation and related lyophilized preparation and preparation method.

[0011] In order to facilitate understanding of the essence of the present application, the exploration process of the inventors is briefly described as follows.

[0012] In the patent application PCT / CN2020 / 101870, publication number WO2021008520, the solubility data of AST-3424 (Table 1 and Table 2) are disclosed,

[0013] Table 1: Solubility of AST-3424

[0014] Table 2: Solubility / stability (room temperature storage) research results of AST-3424 (content unit: mg / ml)

[0015] According to the solubility / stability data of AST-3424 in different solvents or solvent combinations:

[0016] 1. The solubility of AST-3424 in ethanol / propylene glycol (50:50, v / v) solution is greater than 270 mg / mL.

[0017] 2. The solubility of AST-3424 in pH 6.8, pH 7.4 and pH 10.0 is about 23 mg / mL, and the solubility in water is about 20 mg / mL.

[0018] 3. According to the stability, AST-3424 is unstable in pH 6.8 buffer aqueous solution and water, the content of AST-3424 in the solution gradually decreases with time, and the pH value changes obviously; in particular, the pH value of the aqueous solution containing AST-3424 gradually increases from pH 5.004 to pH 6.512 within 48 hours, indicating that AST-3424 aqueous solution at room temperature has undergone chemical changes.

[0019] 4. According to the stability, AST-3424 has good stability in pH 7.4 and pH 10.0 buffer aqueous solution, the content of AST-3424 in the solution does not change significantly with time, and the pH value also does not change much, and the solution pH remains unchanged.

[0020] 5. According to 3 and 4, the stability of AST-3424 aqueous solution is affected by pH: AST-3424 degrades in neutral or acidic pH water environment (significant decrease in content and dramatic change in buffer pH are observed); and AST-3424 is stable in weakly alkaline (pH range of 7.4 to 10.5 according to the above experimental results and the properties of pH buffer, combined with subsequent examples) water environment at room temperature within 48 hours.

[0021] Based on the above preliminary facts and the properties of AST-3424 aqueous solution, it can be determined that it is possible to develop a solution of AST-3424 with appropriate excipients into a lyophilized preparation.

[0022] The inventors' team prepared solutions without adding excipients and with different excipients (mannitol, sucrose, trehalose, sorbitol, lactose, povidone K12, hydroxypropyl β-cyclodextrin, sulfobutyl β-cyclodextrin, and injection-processed phosphatidyl ethanolamine) respectively, and then lyophilized, and investigated the appearance, clarity after dilution of intravenous injection, and high-temperature accelerated stability to finally determine the preliminary qualified lyophilized preparation solution and lyophilized preparation formula.

[0023] Therefore, the present application provides the following technical solutions of AST-3424 lyophilized preparation solution and lyophilized preparation, method and use.

[0024] AST-3424 solution contains the following compound AST-3424, as well as water, excipients, and pH adjuster:

[0025] Water is used as the solvent, and the excipients are selected from trehalose, sorbitol, water-soluble modified β-cyclodextrin, or phospholipid-polyethylene glycol conjugates.

[0026] The concentration of AST-3424 in solution ranges from 1 mg / ml to 20 mg / ml.

[0027] The pH of the solution ranges from 7.4 to 10.5.

[0028] The present invention also provides the use of the above solution as a solution for the lyophilized formulation of AST-3424, which, after lyophilization, becomes the lyophilized formulation:

[0029] AST-3424 lyophilized formulation solution contains the following compound AST-3424, as well as water, excipients, and pH adjuster:

[0030] Water is used as the solvent, and the excipients are selected from trehalose, sorbitol, water-soluble modified β-cyclodextrin, or phospholipid-polyethylene glycol conjugates.

[0031] The concentration of AST-3424 in solution ranges from 1 mg / ml to 20 mg / ml.

[0032] The pH of the solution ranges from 7.4 to 10.5.

[0033] The AST-3424 lyophilized solution is used as a lyophilized formulation solution to prepare lyophilized formulations through a lyophilization process.

[0034] Based on the above-mentioned AST-3424 lyophilized solution, the present invention can prepare AST-3424 lyophilized formulations.

[0035] The lyophilized formulation was prepared by lyophilizing the above-mentioned AST-3424 lyophilization solution.

[0036] The lyophilized formulation contains the following compound AST-3424, as well as excipients and pH adjusters:

[0037] The excipients are selected from trehalose, sorbitol, water-soluble modified β-cyclodextrin, or phospholipid-polyethylene glycol conjugates.

[0038] The drug loading of AST-3424 in the lyophilized formulation was 1 mg / cm³. 3 Up to 20 mg / cm 3 ,

[0039] The mass ratio of the excipient to AST-3424 is (10-20):1.

[0040] The solution used in this invention for preparing the lyophilized formulation of AST-3424 contains at least one excipient.

[0041] Pharmaceutically acceptable excipients are additives or carriers that contribute to the stability of the active pharmaceutical ingredient in the formulation. In the preparation of lyophilized formulations, it is necessary to add excipients or lyophilization protectants to the solution used for lyophilization.

[0042] Some drug solutions can be successfully freeze-dried, while others collapse or melt into an oily substance after lyophilization: experiments have shown that AST-3424 aqueous solution remains oily after lyophilization. To enable successful lyophilization of certain drug solutions and obtain stable lyophilized formulations, excipients that do not react with the drug are added. These excipients themselves do not sublimate during the sublimation stage of the lyophilization process but are directly lyophilized into a framework, acting as a morphological agent, while the drug can be directly adsorbed or filled into the gaps in the framework. Alternatively, to improve the solubility and stability of the lyophilized product, or to give it an aesthetically pleasing shape, additional substances need to be added to the liquid formulation. These are collectively called lyophilization protectants, sometimes also called fillers, excipients, buffers, bases, or frameworks. Generally, lyophilization protectants must be chemically inert to the drug solution.

[0043] Through experimental screening and exploration, it was determined that excipients selected from trehalose, sorbitol, water-soluble modified β-cyclodextrin, or phospholipid-polyethylene glycol conjugates can be freeze-dried to obtain AST-3424 freeze-dried formulation with good appearance, clear reconstituted solution, and good stability.

[0044] Trehalose, sorbitol, water-soluble modified β-cyclodextrin, or phospholipid-polyethylene glycol conjugate may be used individually, and in special cases, they may be used in combination. The preferred embodiment of this application is the use of only one of trehalose, sorbitol, water-soluble modified β-cyclodextrin, or phospholipid-polyethylene glycol conjugate.

[0045] Water-soluble modified β-cyclodextrins are a class of β-cyclodextrin derivatives. These derivatives are prepared by chemically reacting the hydroxyl groups on the outer surface of the β-cyclodextrin molecular cavity with modifying reagents through etherification, esterification, oxidation, and cross-linking. This process introduces new functional groups onto the outer surface of the cyclodextrin molecular cavity, resulting in new β-cyclodextrin derivatives. These new derivatives exhibit higher solubility than natural β-cyclodextrin (1.85 g dissolved in 100 ml of water at a solubility of 25%). Commonly introduced groups include hydrophilic carboxyl, amino, hydroxyl, sulfonyl, sulfonate, and quaternary ammonium groups, resulting in carboxymethyl-β-cyclodextrin, glucosyl-β-cyclodextrin, ethylenediamine-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, sulfonate-β-cyclodextrin, and quaternary ammonium-β-cyclodextrin.

[0046] Phospholipid-polyethylene glycol (PEG) conjugates are a class of PEG-phosphatidylethanolamine (PE) conjugates obtained through the artificial reaction of PEG and phosphatidylethanolamine (PE). Natural phosphatidylethanolamine (PE), CAS: 39382-08-6, Molecular formula: C 41 H 78 NO8P is distearate phosphatidylethanolamine, but synthetically produced versions may contain other acyl groups. To increase its water solubility, groups such as hydroxyl, amino, carboxyl, and amide groups may be introduced.

[0047] Experiments have shown that the stable solubility of AST-3424 in aqueous solution is 20 mg / ml. Therefore, the concentration of the above solution should not exceed 20 mg / ml. Considering the requirements of the lyophilized formulation, the content of AST-3424 in the solution is determined to be 1 mg / ml-20 mg / ml, preferably 10 mg / ml-20 mg / ml.

[0048] The solution of the AST-3424 lyophilized formulation of the present invention may also contain at least one pH adjuster.

[0049] The pH adjuster of this invention refers to a buffer substance or buffer solution used to appropriately adjust the pH according to changes in acidity or alkali. The pH adjuster includes, but is not limited to, hydrochloric acid, sodium hydroxide, triethanolamine, phosphoric acid, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, or phosphoric acid, citric acid, lactic acid, tartaric acid, succinic acid, fumaric acid, malic acid, sodium bicarbonate, sodium carbonate, or mixtures thereof. The amount of pH adjuster added results in a solution pH of 7.4 to 10.5, preferably 7.4.

[0050] According to patent application PCT / CN2022 / 129548, publication number WO2024092614, the AST-3424 compound raw material (API) actually used is a relatively viscous oily substance and is prepared as a 30-40% (w / w) ethanol solution. Furthermore, because the final purification process uses silica gel column chromatography, its pH is slightly acidic (Table 2 shows that the pH of the AST-3424 aqueous solution is less than 7, i.e., acidic). Therefore, to adjust its pH to a stable, slightly alkaline state, the pH adjuster is preferably a base or an alkaline salt such as sodium hydroxide, triethanolamine, sodium bicarbonate, or sodium carbonate, and should not be an acid. For cases where the aqueous solution of the AST-3424 compound raw material itself is weakly acidic, the preferred pH adjuster is selected from one or a mixture of sodium citrate, sodium acetate, potassium acetate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, potassium phosphate, sodium phosphate, sodium hydroxide, potassium hydroxide, sodium bicarbonate, potassium bicarbonate, sodium carbonate, and potassium carbonate.

[0051] If the process of preparing AST-3424 is changed or the column chromatography is adjusted (such as using alumina column chromatography), resulting in the pH of the aqueous solution of the AST-3424 compound raw material being greater than 10.5, then dilute hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, lactic acid, tartaric acid, succinic acid, fumaric acid, malic acid, potassium dihydrogen phosphate, sodium dihydrogen phosphate, or the corresponding acidic salt should be selected.

[0052] In some embodiments, the pH adjuster is selected from one or a mixture of sodium citrate, sodium acetate, potassium acetate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, potassium phosphate, sodium phosphate, sodium hydroxide, potassium hydroxide, sodium bicarbonate, potassium bicarbonate, sodium carbonate, and potassium carbonate.

[0053] Preferably, the water-soluble modified β-cyclodextrin is selected from hydroxyalkyl β-cyclodextrin, sulfoalkyl β-cyclodextrin or their sodium or potassium salts.

[0054] More preferably, the hydroxyl group in the hydroxyl-alkyl β-cyclodextrin is selected from C1-C6 hydroxyl groups, preferably from C2-C5 hydroxyl groups; the sulfonyl group in the sulfonyl β-cyclodextrin is selected from C1-C6 sulfonyl groups, preferably from C2-C5 sulfonyl groups.

[0055] In some embodiments, the water-soluble modified β-cyclodextrin is selected from hydroxypropyl β-cyclodextrin, hydroxyethyl β-cyclodextrin, and sodium sulfonyl β-cyclodextrin. In particular, hydroxypropyl β-cyclodextrin and sodium sulfonyl β-cyclodextrin are excipients approved by drug regulatory authorities in various countries for use in injectable formulations.

[0056] Preferably, the phospholipid-polyethylene glycol conjugate is selected from distearylphosphatidylethanolamine-polyethylene glycol (DSPE-MPEG), distearylphosphatidylethanolamine-polyethylene glycol-amino (DSPE-PEG-NH2), distearylphosphatidylethanolamine-polyethylene glycol-carboxyl (DSPE-PEG-COOH), distearylphosphatidylethanolamine-polyethylene glycol-maleimide (DSPE-PEG-MAL), distearylphosphatidylethanolamine-polyethylene glycol-succinimide ester (DSPE-PEG-NHS), distearylphosphatidylethanolamine-polyethylene glycol-mercapto (DSPE-PEG-SH), or their sodium / potassium salts.

[0057] More preferably, the phospholipid-polyethylene glycol conjugate is selected from distearylphosphatidylethanolamine-polyethylene glycol (DSPE-MPEG) or its sodium / potassium salt.

[0058] Specifically, polyethylene glycol has various degrees of polymerization, such as 2000 and 5000. In some embodiments, the phospholipid-polyethylene glycol conjugate is selected from distearylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-MPEG2000) or its sodium salt. The sodium salt of distearylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-MPEG2000) is registered in China as "Peihua Phosphatidylethanolamine (for injection)," with the chemical name N-(carbonyl-methoxy polyethylene glycol 2000)-1,2-distearyl-SN-glycerol-3-phosphatidylethanolamine, sodium salt, and the English name DSPE-MPEG2000, CAS number 147867-65-0.

[0059] The mass ratio of AST-3424 to the excipient is the drug loading ratio of the lyophilization solution. Based on the lyophilization state of the excipient (also known as a lyophilization protectant, filler, or matrix), the lyophilized body uses the excipient as a matrix, and the drug is adsorbed or loaded onto the matrix. Therefore, the drug loading ratio of the drug solution before lyophilization, i.e., the mass ratio of AST-3424 to the excipient, is an important indicator.

[0060] A suitable drug loading ratio means that after lyophilization, the drug and matrix are uniformly adsorbed, and the drug is well distributed on the surface of the voids and pores of the matrix. In this way, the lyophilized preparation can be well and quickly dissolved for injection in the subsequent reconstitution process (5% glucose injection, physiological saline, etc.).

[0061] As a preferred embodiment, in some embodiments of the present invention, the mass ratio of excipient to AST-3424 is (10-20):1.

[0062] In some embodiments, the AST-3424 lyophilized formulation solution contains the compound AST-3424 of the following formula, as well as water, excipients, and pH adjusters:

[0063] in,

[0064] Water as a solvent

[0065] The excipients are selected from trehalose, sorbitol, distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-MPEG 2000) sodium salt, sodium sulfonyl β-cyclodextrin, and hydroxypropyl β-cyclodextrin.

[0066] The concentration of AST-3424 in solution is 10 mg / ml-20 mg / ml.

[0067] The mass ratio of the excipient to AST-3424 is (10-20):1.

[0068] The pH adjuster is selected from one or a mixture of sodium citrate, sodium acetate, potassium acetate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, potassium phosphate, sodium phosphate, sodium hydroxide, potassium hydroxide, sodium bicarbonate, potassium bicarbonate, sodium carbonate, and potassium carbonate.

[0069] The pH of the solution ranges from 7.4 to 10.5.

[0070] In some embodiments, the AST-3424 lyophilized formulation solution contains the compound AST-3424 of the following formula, as well as water, excipients, and pH adjusters:

[0071] in,

[0072] Water as a solvent

[0073] The excipients are selected from trehalose, sorbitol, distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-MPEG 2000) sodium salt, sodium sulfonyl β-cyclodextrin, and hydroxypropyl β-cyclodextrin.

[0074] The concentration of AST-3424 in solution is 10 mg / ml-20 mg / ml.

[0075] The mass ratio of the excipient to AST-3424 is (10-20):1.

[0076] The pH adjuster is selected from sodium bicarbonate.

[0077] The pH of the solution ranges from 7.4 to 10.5.

[0078] In some embodiments, the AST-3424 lyophilized formulation solution contains the compound AST-3424 of the following formula, as well as water and the excipient trehalose or sorbitol, and the pH adjuster sodium bicarbonate:

[0079] in,

[0080] The concentration of AST-3424 in the solution is 10 mg / ml, and the concentration of the excipients trehalose or sorbitol in the solution is 200 mg / ml.

[0081] The pH of the solution ranges from 7.4 to 10.5.

[0082] In some embodiments, the AST-3424 lyophilized formulation solution contains the compound AST-3424, water, and the excipient distearate-phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-MPEG2000) sodium salt, and the pH adjuster sodium bicarbonate.

[0083] in,

[0084] The concentration of AST-3424 in the solution is 10 mg / ml, and the concentration of the excipient distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-MPEG2000) sodium salt in the solution is 100 mg / ml.

[0085] The pH of the solution ranges from 7.4 to 10.5.

[0086] In some embodiments, the lyophilized formulation solution of AST-3424 contains the compound AST-3424 of the following formula, as well as water and the excipient sodium sulfobutyl β-cyclodextrin and the pH adjuster sodium bicarbonate:

[0087] in,

[0088] The concentration of AST-3424 in the solution is 10 mg / ml, and the concentration of the excipient sodium sulfobutyl β-cyclodextrin in the solution is 200 mg / ml or 100 mg / ml.

[0089] The pH of the solution ranges from 7.4 to 10.5.

[0090] In some embodiments, the AST-3424 lyophilized formulation solution contains the compound AST-3424 of the following formula, as well as water and the excipient hydroxypropyl β-cyclodextrin and the pH adjuster sodium bicarbonate:

[0091] in,

[0092] The concentration of AST-3424 in solution is 10 mg / ml, and the concentration of the excipient hydroxypropyl β-cyclodextrin in solution is 200 mg / ml.

[0093] The pH of the solution ranges from 7.4 to 10.5.

[0094] The phrase "composed of xxx" or "contains" refers to substances that are intentionally or artificially added during the preparation process (these substances are certain to be present and can be detected by analytical instruments). Apart from these substances, no other intentionally or artificially added substances are present, but trace amounts of impurities, environmental substances, etc., will still inevitably exist.

[0095] This limitation does not apply if other substances (environmental substances) are detected during the solution preparation process due to contact or contamination with containers, pipes, or tools. Similarly, this limitation also does not apply to water, pH adjusters, excipients, and AST-3424 active pharmaceutical ingredient, which inevitably contain impurities or other substances present in the environment.

[0096] All substances detected due to contact or contamination with containers, pipes, or tools (environmental substances), water, pH adjusters, excipients, and AST-3424 active pharmaceutical ingredient that are inevitably contaminated with impurities or other substances present in the environment (environmental substances) must be within the legal limits or comply with the corresponding product (pharmaceutical grade, medical grade, or equivalent quality standards) quality standards.

[0097] Since AST-3424 is a viscous oily substance at room temperature, the AST-3424 active pharmaceutical ingredient used in the embodiments of the present invention is an ethanol solution containing 30-40% by mass of AST-3424. For the specific preparation method, please refer to patent application PCT / CN2022 / 129548, publication number WO / 2024 / 092614. Therefore, the AST-3424 lyophilized solution prepared in the embodiments of this application contains ethanol in addition to the water, pH adjuster, excipient, and AST-3424 mentioned above.

[0098] In the embodiments of the present invention, the AST-3424 active pharmaceutical ingredient used is an ethanol solution with a mass percentage of 30.3%. Therefore, it can be deduced that the mass ratio of ethanol to AST-3424 in the above-mentioned AST-3424 lyophilized formulation solution is 69.7:30.3. If the content of AST-3424 in the solution is 10 mg / ml, then the corresponding ethanol content in the solution is calculated to be 23 mg / ml.

[0099] The above content describes the composition (formulation) of the solution for preparing high drug loading lyophilized formulations. The following is a brief description of its uses.

[0100] The solutions provided above are only intermediate semi-finished products for the preparation of AST-3424 lyophilized formulation and cannot be used as prescriptions for clinical formulations. Generally, they are prepared and used on-site, that is, after being prepared using a dispensing container, they are directly filled into lyophilization bottles and then sent in batches to the lyophilization production equipment for lyophilization.

[0101] Therefore, the solution used to prepare the AST-3424 lyophilized formulation should be stable during the freeze-drying process and waiting period after preparation, i.e., it should have stability for at least 8 hours at room temperature, and preferably 24 hours or even 48 hours.

[0102] This is because the solution needs to be filtered and filled before entering the freeze dryer for lyophilization after preparation. Filtration and filling are generally completed within 8-12 hours and are usually carried out at room temperature. Although the subsequent lyophilization is at a low temperature, large freeze dryers or systems require 20-40 hours to cool a large number of vials of the drug solution to the set low temperature (-20 to -55°C) (the temperature of the freeze dryer plates cannot be completely uniform, so the cooling time for a single vial is a wide range). Therefore, the lyophilized formulation should have stability at room temperature for a certain period of time. Through experiments, the data in Table 2 preliminarily confirm that the AST-3424 solution prepared by this invention has stability at room temperature for 48 hours or even longer.

[0103] The mass of AST-3424 per unit volume of lyophilized formulation is the drug loading of the lyophilized formulation. Based on the lyophilization state of the excipients (also known as lyophilization protectants, fillers, or matrix), it is known that the lyophilized body uses the excipients as a matrix, and the drug is adsorbed or loaded onto the matrix. Therefore, the drug loading of the drug solution before lyophilization is an important indicator: a higher drug loading means a higher drug content, resulting in economic advantages in subsequent production costs and storage and transportation.

[0104] Through multiple experiments, the applicant discovered that, with a 2ml vial and a maximum filling volume of 1ml, for different lyophilization solution compositions and processes, the volume of the aforementioned AST-3424 solution before lyophilization and the external volume of the solid formulation after lyophilization exhibit a change of no more than 5%. This change could be due to the expansion of the powder cake after lyophilization (increasing volume by 5%), or the collapse of the powder cake (increasing volume by 5%). Alternatively, optimization of the lyophilization process could result in no change in volume. Therefore, 1ml of the drug solution after lyophilization will change by almost proportionally to 1cm. 3 The lyophilized formulation, namely, a lyophilized solution containing 1 mg / ml to 20 mg / ml of AST-3424 after being lyophilized into a lyophilized formulation, wherein the excipients are selected from trehalose, sorbitol, water-soluble modified β-cyclodextrin, or phospholipid-polyethylene glycol conjugate, and the AST-3424 content is 1 mg / ml. 3 -20mg / cm 3 .

[0105] The drug loading of the lyophilized formulation provided by this invention refers to the amount of ASAT-3424 contained per unit volume. Here, volume refers to the total apparent volume of the drug within a unit package (e.g., a vial), encompassing the internal voids of the drug. For example, a 2ml lyophilized vial contains 1ml of lyophilized solution, and 1ml of lyophilized solution contains 10mg of drug. After the lyophilization process, the 1ml solution is frozen into a loose, porous lyophilized formulation. By measuring the bottom area s of the internal space of the lyophilized vial and the height h of the lyophilized formulation within the vial, the apparent total volume of this unit package of lyophilized formulation is sh, which contains 10mg of active pharmaceutical ingredient. Therefore, its drug loading per unit volume is (10 / sh)mg / cm³. 3 The value of this sh will be at 1cm 3 It is approximately 1 cm in size, and its volume may expand beyond 1 cm after freeze-drying. 3 It may collapse after freeze-drying and its volume may be less than 1 cm³. 3 .

[0106] When measuring the drug loading of a unit package of lyophilized formulation, first measure and calculate the apparent total volume sh, then directly dissolve the entire unit package of lyophilized formulation and detect the mass M of the drug it contains. Then, the drug loading (per unit volume) of the lyophilized formulation of the present invention = M / sh.

[0107] The excipients provided by this invention are selected from trehalose, sorbitol, water-soluble modified β-cyclodextrin or phospholipid-polyethylene glycol conjugate, and the lyophilized solution with AST-3424 content of 1 mg / ml-20 mg / ml has an almost unchanged total volume after being lyophilized into a lyophilized formulation (the drug cake is regular and does not collapse, so the volume of the drug cake in the lyophilized formulation is almost equal to the volume of the solution before lyophilization).

[0108] Preferably, the AST-3424 loading in the lyophilized formulation is 10 mg / cm³. 3 Up to 20 mg / cm 3 .

[0109] In some embodiments, the lyophilized formulation contains the following compound AST-3424, as well as excipients and pH adjusters:

[0110] in,

[0111] The excipients are selected from trehalose, sorbitol, distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-MPEG 2000) sodium salt, and sodium sulfobutyl β-cyclodextrin.

[0112] The drug loading of AST-3424 in the lyophilized formulation was 10 mg / cm³. 3 Up to 20 mg / cm 3 ,

[0113] The mass ratio of the excipient to AST-3424 is (10-20):1.

[0114] pH adjuster is selected from one or a mixture of sodium citrate, sodium acetate, potassium acetate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, potassium phosphate, sodium phosphate, sodium hydroxide, potassium hydroxide, sodium bicarbonate, potassium bicarbonate, sodium carbonate, and potassium carbonate.

[0115] As a preferred lyophilized formulation, it contains the following compound AST-3424, as well as excipients and pH adjusters:

[0116] in,

[0117] The excipients are selected from trehalose, sodium sorbitol distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-MPEG2000), sodium sulfobutyl β-cyclodextrin, and hydroxypropyl β-cyclodextrin.

[0118] The drug loading of AST-3424 in the lyophilized formulation was 10 mg / cm³. 3 Up to 20 mg / cm 3 ,

[0119] The mass ratio of the excipient to AST-3424 is (10-20):1.

[0120] The pH adjuster is sodium bicarbonate.

[0121] As a preferred lyophilized formulation, it contains the following compound AST-3424, as well as the excipient trehalose or sorbitol and the pH adjuster sodium bicarbonate:

[0122] in,

[0123] The drug loading of AST-3424 in the lyophilized formulation was 10 mg / cm³. 3 The content of excipients trehalose or sorbitol in the freeze-dried preparation is 200 mg / cm³. 3 .

[0124] As a preferred lyophilized formulation, it contains the following compound AST-3424, the excipient distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-MPEG2000) sodium salt, and the pH adjuster sodium bicarbonate:

[0125] in,

[0126] The drug loading of AST-3424 in the lyophilized formulation was 10 mg / cm³. 3The content of the excipient distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-MPEG2000) in the lyophilized formulation is 100 mg / cm³. 3 .

[0127] As a preferred lyophilized formulation, it contains the following compound AST-3424, as well as the excipient sodium sulfobutyl β-cyclodextrin and the pH adjuster sodium bicarbonate:

[0128] in,

[0129] The drug loading of AST-3424 in the lyophilized formulation was 10 mg / cm³. 3 The content of sodium sulfobutyl β-cyclodextrin, an excipient in the lyophilized formulation, is 200 mg / cm³. 3 Or 100mg / cm 3 .

[0130] As a preferred lyophilized formulation, it contains the following compound AST-3424, as well as the excipient hydroxypropyl β-cyclodextrin and the pH adjuster sodium bicarbonate:

[0131] in,

[0132] The drug loading of AST-3424 in the lyophilized formulation was 10 mg / cm³. 3 The content of the excipient hydroxypropyl β-cyclodextrin in the lyophilized formulation is 200 mg / cm³. 3 .

[0133] The mass ratio of AST-3424 to excipients is the drug loading ratio of the lyophilized formulation. Based on the lyophilization state of the excipients described above, the lyophilized body uses the excipients as a framework, and the drug is adsorbed or loaded onto the framework. Therefore, the drug loading ratio of the drug solution before lyophilization, i.e., the mass ratio of AST-3424 to excipients, is an important indicator.

[0134] After the solution is lyophilized, the water and a small amount of ethanol (the AST-3424 active pharmaceutical ingredient used in the embodiments of the present invention is an ethanol solution of AST-3424) will sublimate, so what remains of the lyophilized formulation is the drug, excipients, and pH adjuster.

[0135] Obviously, the sublimation process of lyophilization cannot completely remove water and ethanol. Therefore, the residue of water and ethanol is unavoidable. In fact, the amount of residual water and ethanol is an important quality indicator of lyophilized formulations: the lower the residue, the better the quality of the lyophilized formulation, the stronger its stability, and the fewer adverse reactions patients experience after use. Reducing the amount of residue can be achieved by adjusting the lyophilization process, but ultimately, it cannot completely eliminate residue.

[0136] However, by extending the freeze-drying time and increasing the drying temperature, without considering costs and while meeting product quality requirements, it is technically possible to reduce the content of water and ethanol to below the detection limit. In this case, the freeze-dried formulation can be considered to contain almost no water and ethanol.

[0137] Considering production costs and storage stability, there is a suitable value for the residual amounts of water and ethanol. In actual production, as long as this residual value meets the requirements of regulatory agencies / pharmacographies, it is acceptable.

[0138] Of course, based on the above description and the specific properties of AST-3424, other excipients for lyophilized formulations can be added to the solution. In this way, the lyophilized formulation will also be detected to contain the excipients of other lyophilized formulations.

[0139] Pharmaceutical formulations (such as the lyophilized formulations of this invention) can be stored in containers commonly used in the pharmaceutical field, which may include: plastic containers or glass containers, such as standard USPI type borosilicate glass containers. For example, the container used may be a vial.

[0140] Based on the AST-3424 clinical trial dosage in the background technology, it is known that different species (humans and other animals), different indications, and different patients may require different dosages per administration, such as 5 mg or 10 mg. Ideally, a single-dose package or a combination of multiple single-dose packages can meet the requirements of a single dose. Therefore, considering the above, multiple specifications should be designed for different species and indications, such as 2 mg, 5 mg, 10 mg, and 15 mg. As a result, the lyophilized (preparation) solutions filled in the vials are different, and the AST-3424 in a single vial is also different. The final amount of AST-3424 in the lyophilized preparation is also different: containing 2 mg, 5 mg, 10 mg, or 15 mg of AST-3424. In this way, one or more vials can meet the single-dose requirements of a patient, making administration convenient.

[0141] The lyophilized formulation provided by this invention is recommended for intravenous infusion. Therefore, the drug solution needs to be reconstituted. Reconstitution is generally carried out using physiological saline (0.9%) or glucose injection (5%). For this purpose, an intravenous injection solution containing AST-3424 is provided. Its solvent is water, and the solute includes AST-3424, an isotonicity adjusting agent, an excipient, and sodium bicarbonate. The isotonicity adjusting agent is selected from glucose and sodium chloride. The excipient is selected from trehalose, sorbitol, distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-MPEG2000) sodium salt, sodium sulfobutyl β-cyclodextrin, and sodium hydroxypropyl β-cyclodextrin. The pH of this injection solution is 6.8-10.5, and it is an isotonic solution.

[0142] The above-mentioned intravenous injection solution containing AST-3424 is prepared by dissolving the above-mentioned lyophilized AST-3424 formulation:

[0143] Before administering the medication, add 0.1 ml of 5% sodium bicarbonate injection to 100 ml of commercially available sterile 0.9% saline solution for injection (or 5% glucose injection) in an intravenous infusion bag that does not contain DEHP (di(2-ethylhexyl) phthalate) to adjust the pH. At this point, the pH value of the 0.9% saline solution for injection should be between 6.8 and 10.5.

[0144] Using a disposable syringe free of DEHP (di(2-ethylhexyl) phthalate), accurately draw 2 ml of physiological saline from the pH-adjusted saline bag and inject it into a vial containing 10 mg (or other strengths, such as 2 mg, 5 mg, or 15 mg) of the lyophilized formulation for reconstitution. After reconstitution, add the calculated amount (accurate to 0.01 ml) of the AST-3424 lyophilized formulation reconstitution solution to the pH-adjusted saline bag to prepare AST-3424 injection solution for intravenous infusion.

[0145] This invention also provides a method for preparing the above-mentioned AST-3424 lyophilized solution, comprising the following steps:

[0146] Step 1: Prepare an aqueous solution of the pH adjuster;

[0147] Step 2: Dissolve the prescribed amount of excipient and AST-3424 in an appropriate amount of water, add an appropriate amount of water and an aqueous solution of the pH adjuster, make up the volume, and adjust the pH to 7.4 to 10.5.

[0148] Taking NaHCO3 as an example of a pH adjuster, Operation 1 prepares a 5% (by mass) aqueous solution of NaHCO3. If other pH adjusters are used, their concentrations need to be adjusted accordingly: adjusted so that the pH value of the solution is equivalent to that of a 5% (by mass) aqueous solution of NaHCO3.

[0149] Operation 2 involves dissolving the prescribed amount of excipient and AST-3424 in an appropriate amount of water, then adding an appropriate amount of the pH adjuster aqueous solution prepared in Operation 1 and the remaining prescribed amount of water to finally adjust the pH value to 7.4 to 10.5.

[0150] The present invention also provides a method for preparing the above-mentioned AST-3424 lyophilized formulation, comprising the following steps:

[0151] Pre-freezing: Place an appropriate amount of the above-mentioned AST-3424 lyophilized solution into a lyophilization system for pre-freezing. The pre-freezing process includes holding at 0-5℃ for a period of time and holding at -40 to -50℃ for a period of time.

[0152] After the first drying and pre-freezing process, the temperature is raised to -20 to -40°C and kept at that temperature for a period of time, while maintaining a vacuum for drying.

[0153] Secondary drying: After the first drying is completed, the temperature is raised to 20-40℃ and kept at that temperature for a period of time, while maintaining a vacuum for drying.

[0154] Pre-freezing is the process of rapidly freezing a solution into a solid. During pre-freezing, the drug solution needs to be rapidly cooled from room temperature to the pre-freezing temperature to complete the rapid freezing. Therefore, the cooling time from room temperature to the pre-freezing temperature should be as short as possible (e.g., within 10 minutes). To achieve this, the following measures should be taken: i. After the drug solution enters the freeze dryer, the machine should be cooled at full speed; ii. After the drug solution enters the freeze dryer, the plate temperature should be maintained at approximately 0°C (to prevent the drug solution from freezing), keeping the drug solution at a relatively low liquid temperature, and then the machine should be cooled at full speed; iii. The plates should be cooled to -30 to -35°C first, and then the product should be placed in, with the machine cooling at full speed. The specific measures taken to achieve rapid freezing depend on the properties of the excipients and drugs in the drug solution.

[0155] For a single drying process, the heating rate should not be too fast, as excessively rapid heating may cause material spraying during the sublimation process. The specific drying temperature, heating rate, and holding time should be determined based on the chemical solution.

[0156] Secondary drying involves further removing and drying the solvent after the primary vacuum drying has removed most of the solvent. This is done under vacuum and at near-room temperature, further reducing the amount of moisture and organic solvent, thus giving the freeze-dried solid higher stability. The specific drying temperature, heating rate, and holding time are determined based on the chemical solution.

[0157] In some embodiments, trehalose or sorbitol (trehalose is D(+)-trehalose dihydrate, CAS Registry No. 6138-23-4; sorbitol is D-sorbitol, CAS Registry No. 50-70-4) are used as excipients, and the specific freeze-drying conditions are as follows:

[0158] The corresponding pre-freezing process includes holding at 0-5℃ for 60 minutes and holding at -45℃ for 300 minutes. The rapid freezing measure adopted is the second one mentioned above: after the liquid medicine enters the freeze dryer, the plate temperature is maintained at about 0℃ (so that the liquid medicine drops from room temperature to 0℃ within 1 minute), so that the liquid medicine is in a low liquid temperature. Then the machine is cooled at full speed (so that the liquid medicine drops from room temperature to 0℃ to -45℃ within 5 minutes or even 1 minute).

[0159] The corresponding drying process involves pre-freezing, followed by a 30-minute period of heating from -45°C to -32°C while maintaining a vacuum (absolute pressure 0.2 mbar) for drying, with a drying time of 900 minutes.

[0160] The corresponding secondary drying process involves raising the temperature from -32°C to 30°C and maintaining a vacuum (absolute pressure 0.2 mbar) for 120 minutes after the primary drying is completed, with the drying time being 300 minutes.

[0161] In some embodiments, hydroxypropyl beta-cyclodextrin (hydroxypropyl β-cyclodextrin, HP-β-CD, CAS Registry No. 128446-35-5) is used as an excipient, and the specific lyophilization conditions are as follows:

[0162] The corresponding pre-freezing process includes holding at 0-5℃ for 60 minutes and holding at -45℃ for 300 minutes. The rapid freezing measure adopted is the second one mentioned above: after the liquid medicine enters the freeze dryer, the plate temperature is maintained at about 0℃ (so that the liquid medicine drops from room temperature to 0℃ within 1 minute), so that the liquid medicine is in a low liquid temperature. Then the machine is cooled at full speed (so that the liquid medicine drops from room temperature to 0℃ to -45℃ within 5 minutes or even 1 minute).

[0163] The corresponding drying process involves pre-freezing, followed by a 30-minute period of heating from -45°C to -35°C while maintaining a vacuum (absolute pressure 0.2 mbar) for drying, with a drying time of 800 minutes.

[0164] The corresponding secondary drying process involves raising the temperature from -35°C to 30°C and maintaining a vacuum (absolute pressure 0.2 mbar) for 120 minutes after the primary drying is completed, with the drying time being 300 minutes.

[0165] In some embodiments, sodium sulfobutyl β-cyclodextrin (trade name: sulfobutyl beta-cyclodextrin, SBE-β-CD, CAS Registry No. 182410-00-0) is used as an excipient, and the specific lyophilization conditions are as follows:

[0166] The corresponding pre-freezing process includes holding at 0-5℃ for 60 minutes and holding at -45℃ for 300 minutes. The rapid freezing measure adopted is the second one mentioned above: after the liquid medicine enters the freeze dryer, the plate temperature is maintained at about 0℃ (so that the liquid medicine drops from room temperature to 0℃ within 1 minute), so that the liquid medicine is in a low liquid temperature. Then the machine is cooled at full speed (so that the liquid medicine drops from room temperature to 0℃ to -45℃ within 5 minutes or even 1 minute).

[0167] The corresponding drying process involves pre-freezing, followed by a 30-minute period of heating from -45°C to -32°C while maintaining a vacuum (absolute pressure 0.2 mbar) for drying, with a drying time of 700 minutes.

[0168] The corresponding secondary drying process involves raising the temperature from -32°C to 30°C and maintaining a vacuum (absolute pressure 0.2 mbar) for 120 minutes after the primary drying is completed, with a total drying time of 270 minutes.

[0169] In some embodiments, distearylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-MPEG2000) sodium salt (trade name: Peyrene phosphatidylethanolamine (for injection), CAS Registry No. 147867-65-0) is used as an excipient, and the specific lyophilization conditions are as follows:

[0170] The corresponding pre-freezing process includes holding at 0-5℃ for 60 minutes and holding at -45℃ for 300 minutes. The rapid freezing measure adopted is the second one mentioned above: after the liquid medicine enters the freeze dryer, the plate temperature is maintained at about 0℃ (so that the liquid medicine drops from room temperature to 0℃ within 1 minute), so that the liquid medicine is in a low liquid temperature. Then the machine is cooled at full speed (so that the liquid medicine drops from room temperature to 0℃ to -45℃ within 5 minutes or even 1 minute).

[0171] The corresponding drying process involves pre-freezing, followed by a 30-minute period of heating from -45°C to -32°C while maintaining a vacuum (absolute pressure 0.2 mbar) for drying, with a drying time of 700 minutes.

[0172] The corresponding secondary drying process involves raising the temperature from -32°C to 30°C and maintaining a vacuum (absolute pressure 0.2 mbar) for 120 minutes after the primary drying is completed, with a total drying time of 270 minutes.

[0173] As can be seen from the above explanation, lyophilized formulations are obtained by directly filling the solution into lyophilized vials (vials) and directly lyophilizing it using lyophilization equipment. Therefore, there is no repackaging process, meaning that the unit packaging is the corresponding lyophilized vial packaging, and the packaging specifications are closely related to the specifications of the lyophilized vial.

[0174] In some cases, the lyophilized solid can also be used as a cured AST-3424 active pharmaceutical ingredient. Specifically, some embodiments also provide a lyophilized mixture containing the following compound, AST-3424, as well as excipients and pH adjusters:

[0175] The excipients are selected from trehalose, sorbitol, water-soluble modified β-cyclodextrin, or phospholipid-polyethylene glycol conjugates.

[0176] The AST-3424 loading in the lyophilized mixture was 1 mg / cm³. 3 Up to 20 mg / cm 3 ,

[0177] The mass ratio of the excipient to AST-3424 is (10-20):1.

[0178] In other words, when preparing drugs containing AST-3424, the above-mentioned solidified lyophilized mixture is used instead of the oily AST-3424 to facilitate storage, weighing and transfer during the formulation process.

[0179] In addition to the lyophilized mixture described in the above embodiments, the resulting drug may also contain pharmaceutically acceptable excipients or excipients. The drug can be any dosage form for clinical use, such as tablets, suppositories, dispersible tablets, enteric-coated tablets, chewable tablets, orally disintegrating tablets, capsules, sugar-coated tablets, granules, dry powders, oral solutions, small injections, or large-volume infusions. Depending on the specific dosage form and administration method, the pharmaceutically acceptable excipients or excipients in the drug may include one or more of the following: diluents, solubilizers, disintegrants, suspending agents, lubricants, binders, fillers, flavoring agents, sweeteners, antioxidants, surfactants, preservatives, encapsulating agents, and pigments, etc.

[0180] It should be noted that all figures appearing in this application have an error margin of ±10%. That is, both -10% and +10% of the figures should be considered within the range of the figures recorded in this application. For example, if the figure states that "the drug loading of AST-3424 in the lyophilized formulation is 10 mg / cm³", it is not considered part of the numerical range. 3 "Such wording indicates that the actual drug loading, as measured by testing, is (9-11) mg / cm³." 3 It should also be reasonably determined that it is equivalent to the above-mentioned scope. Attached Figure Description

[0181] Figure 1 shows photos of the drug solutions before and after filtration in the first freeze-dried formulation screening experiment with 5 different excipients. The top image is before filtration and the bottom image is after filtration. From left to right, 01-05 in the figure represent F00206501 to F00206505 respectively.

[0182] Figure 2 shows photos of five groups of different excipient solutions after freeze-drying in the first freeze-dried formulation screening experiment. From left to right, they represent F00206501 to F00206505, respectively.

[0183] Figure 3 shows photos of the drug solutions before and after filtration in two groups of different excipients during the second freeze-dried formulation screening experiment. The top image is before filtration, and the bottom image is after filtration. From left to right in the figure, 05402 and 05401 represent F00205402 and F00205401, respectively.

[0184] Figure 4 shows photographs of solutions of different concentrations after reconstitution of the lyophilized formulation with water for injection. In the figure, 05402 and 05401 represent F00205402 and F00205401, respectively.

[0185] Figure 5 shows photographs of different concentrations of solutions after reconstitution of the lyophilized preparation using 5% glucose injection. In the figure, 05402 and 05401 represent F00205402 and F00205401, respectively. The two upper figures do not have NaHCO3 added to adjust the pH value, while the two lower figures have NaHCO3 added to adjust the pH value.

[0186] Figure 6 shows photographs of solutions of different concentrations after reconstitution of the lyophilized formulation with physiological saline. In the figure, 05402 and 05401 represent F00205402 and F00205401, respectively.

[0187] Figure 7 shows photos of three groups of different excipient solutions after freeze-drying in the first freeze-dried formulation screening experiment. From left to right, they represent F00207001 to F00207003, respectively.

[0188] Figure 8 shows photographs of different concentrations of solutions after reconstitution of lyophilized preparations using 5% glucose injection. In the figure, 07001 to 07003 represent F00207001 to F00207003, respectively. Other vials without concentration markings are standard turbidity solutions of different numbers.

[0189] Figure 9 shows photographs of different concentrations of solutions after reconstitution of the lyophilized preparation using 5% glucose injection. In the figure, 07401 and 07402 represent F00207401 and F00207402, respectively. Other vials without concentration markings are standard turbidity solutions of different numbers. Detailed Implementation

[0190] The present invention will be described below with reference to specific embodiments. Those skilled in the art will understand that these embodiments are for illustrative purposes only and do not limit the scope of the invention in any way.

[0191] The terms "patient" and "individual" are used interchangeably and refer to a mammal in need of cancer treatment. Typically, a patient is a human being diagnosed with cancer. In some embodiments, "patient" or "individual" may refer to a non-human mammal used to screen, characterize, and evaluate drugs and therapies, such as a non-human primate, dog, cat, rabbit, pig, mouse, or rat.

[0192] "Treatment" or "treatment of a patient" refers to administering, using, or applying to a patient a therapeutically effective amount of the medicine relating to this invention.

[0193] "Administering" or "using" a drug to a patient refers to the direct administration or application of a drug (which may be done by a medical professional to a patient or by the doctor himself) and / or indirect administration or application, and may include the act of prescribing a drug. For example, instructing a patient to administer or use a drug himself and / or providing a prescription for a drug to a patient constitutes administering or using a drug to a patient.

[0194] The "therapeutic effective dose" of a drug refers to the amount of drug that, when administered or used to a patient with cancer, will have the expected therapeutic effect (e.g., alleviation, improvement, relief, or elimination of clinical manifestations of one or more cancers in the patient). The therapeutic effect does not necessarily occur with a single dose, but may occur only after a series of doses. Therefore, a therapeutic effective dose can be administered or used once or multiple times.

[0195] "Treatment" for a condition or patient refers to steps taken to achieve a beneficial or desired outcome (including clinical outcomes). For the purposes of this invention, beneficial or desired clinical outcomes include (but are not limited to) the alleviation or improvement of one or more cancer symptoms; a reduction in disease severity; a delay or slowing of disease progression; an improvement, relief, or stabilization of the disease state; or other beneficial outcomes. In some cases, cancer treatment may result in a partial response or stabilization of the disease.

[0196] The above description of specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention, and as long as they do not depart from the spirit of the present invention, they should all fall within the scope of the appended claims.

[0197] General Information

[0198] Unless otherwise specified, the methods and equipment used in the following experiments are as follows:

[0199] The content and concentration of AST-3424 were determined by high performance liquid chromatography (HPLC). The instrument used was a Thermo Vanquish HPLC system with a YMC pack AQ C18 column of 4.6 mm × 250 mm and 5 μm. The detection method is described in patent application PCT / CN2020 / 101870, publication number WO2021008520 (specifically section 1.4, Test Methods).

[0200] Unless otherwise specified, any " / " in the table indicates that the data was not detected or added.

[0201] Screening experiment on the types of excipients (lyophilization protectants) in lyophilized formulations

[0202] To explore the feasibility of using commonly used sugars, polyols, and nonionic polymer surfactants as lyophilization excipients for AST-3424, lyophilization experiments were conducted on eight representative excipients, and the appearance of the lyophilized products was examined.

[0203] Sugars to choose from: sucrose, trehalose, lactose

[0204] Polyols selected: mannitol, sorbitol;

[0205] The nonionic polymer surfactant selected is: polyvinylpyrrolidone K12 (PVPK12);

[0206] Other options include: hydroxypropyl β-cyclodextrin, sulfobutyl β-cyclodextrin, and phosphatidylethanolamine.

[0207] The specific reagent details are shown in Table 3 below.

[0208] Table 3: Market Suppliers of Major Reagents Used in the Experiment

[0209] The lyophilized solution is packaged in colorless borosilicate glass vials, each with a volume of 2 ml.

[0210] After freeze-drying, the vials are sealed with chlorinated butyl rubber stoppers coated with polytetrafluoroethylene / ethylene copolymer film and then capped (for antibiotic vials, use aluminum-plastic combination caps).

[0211] The vacuum freeze dryer is a Tofflon LYO-0.5.

[0212] The pH meter is a METTLER TOLEDO FE28.

[0213] Ultrapure water system is Lab water uses its freshly produced ultrapure water.

[0214] I. First screening experiment for lyophilized formulation

[0215] The active pharmaceutical ingredient AST-3424 was prepared by a CDMO in accordance with PCT / CN2022 / 129548, publication number WO2024092614, with batch number 03-03P-01-37-01. It is an ethanol solution with a mass percentage of 30.3%.

[0216] The first formulation screening experiment was conducted using a simple aqueous solution and excipients mannitol, sucrose 1, trehalose, and sorbitol.

[0217] 1.1 Preparation of lyophilized solution

[0218] Based on a 1ml:10mg specification and an excipient ratio of 20:1 (i.e., 200mg), to prepare 20ml of AST-3424 solution from 20 vials of the drug solution, theoretically, 4g of excipient is needed. The mass of 30.3% AST-3424 ethanol solution is 0.66g. Finally, 5% NaHCO3 solution is added to adjust the pH and the volume is brought to 20ml.

[0219] The specific preparation method of the medicinal solution is as follows:

[0220] Operation 1: Prepare a 5% (w / w) NaHCO3 aqueous solution.

[0221] Weigh 0.5g of sodium bicarbonate, add water to 10.0g (sodium bicarbonate weight: 0.501g, weight after volume adjustment: 10.312g), mix well and set aside.

[0222] Step 2: Dissolve the prescribed amount of excipient and AST-3424 in an appropriate amount of water, add an appropriate amount of water and the aqueous solution of the pH adjuster, make up the volume and adjust the pH to 7.4 to 10.5 to obtain the final product.

[0223] Weigh the prescribed amount of excipients and add approximately 19 ml of ultrapure water, stirring until homogeneous. Then add the prescribed amount of the active pharmaceutical ingredient (API) ethanol solution and stir until homogeneous. Adjust the pH to approximately 7.5 using a 5% sodium bicarbonate solution, then add the remaining ultrapure water to a final volume of 20 ml. Record the amount of 5% NaHCO3 solution added during the process, and use a pH meter to measure the pH value before and after adjustment using the added NaHCO3 solution.

[0224] The solution was filtered using a needle filter equipped with a 0.22 μm PVDF membrane to obtain a clear solution. The clarity of the solution before and after filtration was recorded.

[0225] After filtration and clarification, the liquid solution was filled into 20 bottles at a rate of 1 ml per bottle.

[0226] Table 4 below records the actual mass of the AST-3424 ethanol solution, the name and mass of the excipients added, the volume of NaHCO3 solution added, the pH value before and after adjustment with NaHCO3 solution, and the visual clarity of the solution before and after filtration. For the specific condition of the solution before and after filtration (in a broad sense, it may show turbidity due to some insoluble substances), please refer to Figure 1.

[0227] Table 4: Preparation of drug solution for the first lyophilized formulation screening experiment

[0228] 1.2 Freeze-drying

[0229] After being frozen dry in trays and partially plugged, the trays are then freeze-dried according to a predetermined procedure.

[0230] After freeze-drying, the product is fully plugged and capped to obtain the finished product, which is then removed from the freeze dryer and stored in a warehouse.

[0231] The freezing and determination procedures for the above 5 groups of solutions are shown in Table 5 below.

[0232] Table 5: Freeze-drying parameter settings for the first freeze-dried formulation screening experiment

[0233] The set time refers to the time required for the freeze dryer to reach the set temperature.

[0234] The sample was removed from the box, capped, and freeze-dried, as shown in Figure 2.

[0235] Visual inspection revealed five groups of lyophilized products prepared after freeze-drying the drug solutions:

[0236] The formulation without excipient F00206501 was a failure, resulting in a yellow, viscous solid.

[0237] The formulation containing 20 times the weight of the active pharmaceutical ingredient, mannitol F00206502, showed good solid appearance after lyophilization.

[0238] Formula F00206503, which contains 20 times the weight of the raw drug sucrose, shrank severely after freeze-drying.

[0239] The formulation containing trehalose F00206504 at 20 times the weight of the raw material showed moderate shrinkage after freeze-drying.

[0240] The formulation containing 20 times the weight of the raw material sorbitol F00206505 shrank significantly after freeze-drying.

[0241] 1.3 Dilution and compatibility of lyophilized formulations

[0242] The lyophilized formulation prepared in 1.2 was immediately diluted and compatibility studied using 5% glucose injection (pH adjusted to 7.5 with sodium bicarbonate).

[0243] In an intravenous infusion bag that does not contain DEHP (di(2-ethylhexyl) phthalate), add 0.1 ml of 5% sodium bicarbonate injection to 100 ml of commercially available sterile 5% glucose injection (D5W) to complete pH adjustment. At this point, the pH value of D5W is approximately 7.5.

[0244] Using a disposable syringe free of DEHP (di(2-ethylhexyl) phthalate), accurately draw 1 ml of the injection solution from the pH-adjusted intravenous infusion bag and inject it into the 10 mg lyophilized formulation vial for reconstitution. The resulting reconstituted solution has a concentration of approximately 10 mg / ml. Further dilution of this solution yields reconstituted solutions of approximately 5 mg / ml and 1 mg / ml, respectively.

[0245] The degree of turbidity was examined for different formulations and different reconstituted concentrations, and visually compared with turbidity standard solutions ranging from 0.5# to 4#.

[0246] At room temperature, place the test solution and an equal volume of turbidity standard solution separately in colorless borosilicate glass vials (used for lyophilization). Five minutes after the turbidity standard solution is prepared, place them vertically under a 1000 lx illuminance lamp in a dark room and visually observe and compare the clarity of the test solution and the turbidity standard solution from a horizontal perspective. The test solution should be examined immediately after dissolution. The results are shown in Table 6 below.

[0247] Table 6: Turbidity of lyophilized formulations after reconstitution at different concentrations in the first lyophilized formulation screening experiment. = indicates equivalent to...

[0248] Relatively speaking, the formulations containing 20 times the weight of the active pharmaceutical ingredient (API) – mannitol F00206502 and sucrose F00206503 – were better among the five formulations, while trehalose F00206504 and sorbitol F00206504 barely met the requirements. Therefore, it was decided to conduct further research.

[0249] II. Second Freeze-Dried Formulation Screening Experiment

[0250] Considering that the amount of excipients, the manufacturers of raw materials and excipients, and the lyophilization process settings may all affect the appearance of the lyophilized formulation and the clarity after dilution and reconstitution, the second lyophilization experiment used mannitol (manufacturer same as the first) and sucrose 2 (manufacturer different from the first) as excipients for the second formulation screening experiment, and reduced the amount of excipients to 10 times the amount of raw materials.

[0251] The active pharmaceutical ingredient AST-3424 was prepared by a CDMO in accordance with PCT / CN2022 / 129548, publication number WO2024092614, with batch number 03-03P-01-37-01. It is a 100 mg / ml ethanol solution prepared by diluting a 30.3% ethanol solution.

[0252] Based on a 1ml:10mg specification and an excipient ratio of 10:1 (i.e., 100mg), to prepare 20ml of AST-3424 solution from 20 vials of drug solution, theoretically, 2g of excipient and 2ml of 100mg / ml AST-3424 ethanol solution are needed. Finally, 5% (w / w) NaHCO3 solution is added to adjust the pH and the volume is brought to 20ml.

[0253] Similar to operations 1.1, 1.2, and 1.3, the relevant results are listed in Tables 7, 8, and 9 below.

[0254] Table 7 below records the actual volume of AST-3424 ethanol solution added, the name and mass of the excipients added, the volume of NaHCO3 solution added, the pH value before and after adjustment with NaHCO3 solution, and the visual clarity of the solution before and after filtration. For the specific condition of the solution before and after filtration (in a broad sense, it may show turbidity due to some insoluble substances), please refer to Figure 3.

[0255] Table 7: Preparation of drug solutions for the second lyophilized formulation screening experiment

[0256] Table 8: Freeze-drying parameter settings for the second freeze-dried formulation screening experiment

[0257] After the sample is removed from the box and capped, it is visually inspected after freeze-drying.

[0258] The bottom of batch F00205401, which had 10 times the weight of the raw drug sucrose added, shrank slightly.

[0259] The mannitol F00205402 batch, with 10 times the weight of the raw material, has a full appearance.

[0260] To further consider the impact of the type of diluent used and whether NaHCO3 solution was added to adjust the pH value on the clarity of the solution during the dilution and preparation process, the clarity of the two batches was tested after different diluents (ultrapure water, 5% glucose injection D5W, 0.9% physiological saline) and whether the pH was adjusted for reconstitution. The results are shown in Table 9 below.

[0261] Table 9: Turbidity of lyophilized formulations after reconstitution at different concentrations in the second lyophilized formulation screening experiment. = indicates equivalent to...

[0262] For more detailed information on the clarification of the reconstituted solution, please refer to Figures 4, 5, and 6.

[0263] Based on the second experiment and in conjunction with the first experiment, we can conclude that:

[0264] Reducing / increasing the amount of excipients and changing the lyophilization procedure can improve the appearance of lyophilized formulations using sucrose as an excipient, but have little effect on the use of mannitol as an excipient, demonstrating the advantage of mannitol as an excipient for AST-3424 lyophilized formulations that is not affected by the amount of excipients or the lyophilization procedure.

[0265] The solution obtained by directly reconstituted the lyophilized preparation with purified water or by adjusting the pH value with NaHCO3 solution and then reconstituted with 5% glucose injection has good clarity.

[0266] Similarly, adjusting the amounts of excipients trehalose and sorbitol, as well as the freeze-drying process, may improve the appearance of the final freeze-dried product.

[0267] III. Third Freeze-Dried Formulation Screening Experiment

[0268] Based on the initial lyophilized formulation screening experiment, experiments were continued using low-endotoxin lactose, povidone K12, and hydroxypropyl betacyclodextrin.

[0269] Based on a 1ml:10mg specification and an excipient ratio of 20:1 (i.e., 200mg), to prepare 20ml of AST-3424 solution from 20 vials of the drug solution, theoretically, 4g of excipient is needed. The mass of 30.3% AST-3424 ethanol solution is 0.66g. Finally, 5% NaHCO3 solution is added to adjust the pH and the volume is brought to 20ml.

[0270] Similar to operations 1.1, 1.2, and 1.3, the relevant results are listed in Tables 10, 11, and 12 below.

[0271] Table 10 below records the actual volume of AST-3424 ethanol solution added, the name and mass of the excipients added, and the volume of NaHCO3 solution added.

[0272] Table 10: Preparation of drug solutions in the third lyophilized formulation screening experiment

[0273] * The actual amount of lactose weighed was 4.006g, but due to an operational error, twice the amount of ultrapure water (40ml) was added. After the operator discovered the error, they stirred it thoroughly and took out 20ml, then added the prescribed amount of raw material and stirred it thoroughly. Therefore, the amount of lactose added was calculated to be 2.003g, and the final solution had an excipient ratio of 10:1.

[0274] Table 11: Freeze-drying parameter settings for the third freeze-dried formulation screening experiment

[0275] After the sample is removed from the box and capped, it is visually inspected after freeze-drying.

[0276] The sample was removed from the box, capped, and freeze-dried, as shown in Figure 7.

[0277] Visual observation reveals the following freeze-dried products prepared from the three groups of drug solutions:

[0278] Formula F00207001, which contains 10 times the weight of the raw material, suffers severe shrinkage at the bottom after freeze-drying.

[0279] The formulation containing 20 times the weight of the raw material povidone K12 F00207002 showed severe shrinkage at the bottom.

[0280] The lyophilized formulation of F00207003, containing 20 times the weight of the active pharmaceutical ingredient, showed good appearance.

[0281] The lyophilized formulation prepared above was immediately diluted and compatibility studied using 5% glucose injection (pH adjusted to 7.5 with sodium bicarbonate). The results of the clarification of the reconstituted solution are shown in Table 12 below.

[0282] Table 12: Turbidity of lyophilized formulations after reconstitution at different concentrations in the third lyophilized formulation screening experiment

[0283] For a more detailed description of the clarification of the reconstituted solution, please refer to Figure 8.

[0284] Relatively speaking, the formulation with 20 times the mass of the active pharmaceutical ingredient added (F00206503) was better among the three formulations. Further investigation is warranted.

[0285] IV. Fourth Freeze-Dried Formulation Screening Experiment

[0286] Based on the second lyophilized formulation screening experiment, experiments were continued using phosphatidylethanolamine and sulfobutyl betacyclodextrin.

[0287] Based on a 1ml:10mg specification and an excipient ratio of 10:1 (i.e., 100mg), to prepare 20ml of AST-3424 solution from 20 vials of drug solution, theoretically, 2g of excipient is needed, along with 0.66g of 30.3% AST-3424 ethanol solution. Finally, 5% NaHCO3 solution is added to adjust the pH and the volume is brought to 20ml.

[0288] Similar to operations 1.1, 1.2, and 1.3, the relevant results are listed in Tables 13, 14, and 15 below.

[0289] Table 10 below records the actual volume of AST-3424 ethanol solution added, the name and mass of the excipients added, and the volume of NaHCO3 solution added.

[0290] Table 13: Preparation of drug solutions in the third lyophilized formulation screening experiment

[0291] * Phosphatidylethanolamine is difficult to dissolve in water. After prolonged stirring, it produces a large number of bubbles that cannot be eliminated over a long period of time.

[0292] Table 14: Freeze-drying parameter settings for the fourth freeze-dried formulation screening experiment

[0293] After the sample is removed from the box and capped, it is visually inspected after freeze-drying.

[0294] The lyophilized formulations containing 10 times the mass of the active pharmaceutical ingredient (API) of sulfobutyl betacyclodextrin F00207401 and 10 times the mass of the API of phosphatidylethanolamine F00207402 showed good appearance.

[0295] The lyophilized formulation prepared above was immediately diluted and compatibility studied using 5% glucose injection (pH adjusted to 7.5 with sodium bicarbonate). The results of the clarification of the reconstituted solution are shown in Table 15 below.

[0296] Table 15: Turbidity of lyophilized formulations after reconstitution at different concentrations in the fourth lyophilized formulation screening experiment

[0297] For a more detailed description of the clarification of the reconstituted solution, please refer to Figure 9.

[0298] Relatively speaking, the formulations with 10 times the weight of the active pharmaceutical ingredient (API) of sulfobutyl betacyclodextrin (F00207401) and phosphatidylethanolamine (F00207402) are both better and worthy of further exploration.

[0299] V. Accelerated Stability Test of Lyophilized Formulations at High Temperature

[0300] Through the experiments in the above four parts, combined with factors such as the cost and availability of excipients in the production stage and whether they would cause post-injection allergies, it was confirmed that the following formulations have further research value: sucrose F00206503+F00205401, mannitol F00206502+F00205402, trehalose F00206504, sorbitol F00206505, hydroxypropyl β-cyclodextrin F00207003, sulfobutyl β-cyclodextrin F00207401, and phosphatidylethanolamine F00207402.

[0301] 5.1 Visually assess the clarity of the diluted and prepared solutions after high-temperature accelerated processing.

[0302] Therefore, high-temperature accelerated stability tests were conducted on these 7 batches of lyophilized preparations: the corresponding samples were placed in the dark at 40℃±2℃ and 75%±5%RH for 3, 5 and 10 days, and then diluted with 5% glucose injection (pH adjusted to 7.52) to examine the clarity of the diluted solution. For specific procedures, please refer to Section 1.3.

[0303] Table 16: Turbidity of different batches of lyophilized formulations after reconstitution at different concentrations after high-temperature storage for different times. = indicates equivalent to...

[0304] In particular, after high-temperature stability testing, batches F00206503 / 04 / 05 all showed varying degrees of melting.

[0305] 5.2 Investigate the changes in impurity content in the formulation after high-temperature acceleration.

[0306] Take freshly prepared lyophilized formulations and the formulations placed at high temperature in Section 5.1 above (F00206503 for sucrose, F00206502 for mannitol, F00206504 for trehalose, F00206505 for sorbitol, F00207003 for hydroxypropyl β-cyclodextrin, F00207401 for sulfobutyl β-cyclodextrin, and F00207402 for phosphatidylethanolamine) diluted to 10 mg / ml using D5W. Filter the solutions using a 0.2 μm PVDF membrane. Wash the membrane three times with 2 ml of D5W, and then repeat the washing process three times with 2 ml of anhydrous ethanol. Analyze the washed anhydrous ethanol using HPLC. The results are shown in Table 17 below.

[0307] Table 17: HPLC purity test results of different batches of lyophilized formulations after reconstitution following high-temperature storage for different times.

[0308] * indicates that the highest concentration of a single impurity in this batch has the same elution time in the HPLC chromatogram, suggesting that it is the same impurity compound.

[0309] Comparison of impurity changes shows that the trehalose formulation (F00206504), sorbitol formulation (F00206505), hydroxypropyl β-cyclodextrin formulation (F00207003), sulfobutyl β-cyclodextrin formulation (F00207401), and phosphatidylethanolamine formulation (F00207402) exhibit relatively good stability. In particular, the hydroxypropyl β-cyclodextrin formulation (F00207003), sulfobutyl β-cyclodextrin formulation (F00207401), and phosphatidylethanolamine formulation (F00207402) are exceptionally stable, and the solutions exhibit good clarity after reconstitution, making them suitable for optimization and development.

[0310] Considering the changes in impurities, the maximum single impurity species of the phosphatidylethanolamine formulation F00207402 changed after high-temperature storage, while the maximum single impurity of the hydroxypropyl β-cyclodextrin formulation F00207003 and the sulfobutyl β-cyclodextrin formulation F00207401 remained unchanged at 0, 3, and 5 days, still being the same impurity. This means that the stability of the hydroxypropyl β-cyclodextrin and sulfobutyl β-cyclodextrin formulations may be better.

[0311] Based on the experiments in Part II, it is known that adjusting the amount of excipients and modifying the lyophilization process helps improve the clarity and appearance of the lyophilized formulation after reconstitution. Therefore, it is necessary to further optimize the formulations based on trehalose (F00206504), sorbitol (F00206505), hydroxypropyl β-cyclodextrin (F00207003), sulfobutyl β-cyclodextrin (F00207401), and phosphatidylethanolamine (F00207402): change the amount of excipients to prepare new lyophilized solutions and optimize the lyophilization process of the solutions, thereby preparing AST-3424 lyophilized formulations with good appearance, clear reconstitution, and greater stability.

Claims

1. An AST-3424 solution containing the compound AST-3424, water, excipients, and a pH adjuster: in, Water is used as the solvent, and the excipients are selected from trehalose, sorbitol, water-soluble modified β-cyclodextrin, or phospholipid-polyethylene glycol conjugates. The concentration of AST-3424 in solution ranges from 1 mg / ml to 20 mg / ml. The pH of the solution ranges from 7.4 to 10.

5.

2. AST-3424 lyophilized formulation solution, containing the compound AST-3424 of the following formula, as well as water, excipients, and pH adjuster: in, Water is used as the solvent, and the excipients are selected from trehalose, sorbitol, water-soluble modified β-cyclodextrin, or phospholipid-polyethylene glycol conjugates. The concentration of AST-3424 in solution ranges from 1 mg / ml to 20 mg / ml. The pH of the solution ranges from 7.4 to 10.

5.

3. The solution according to claim 1 or 2, wherein, Water-soluble modified β-cyclodextrin is selected from hydroxyalkyl β-cyclodextrin, sulfoalkyl β-cyclodextrin, or their sodium or potassium salts.

4. The solution according to claim 3, wherein, The hydroxyl group in hydroxyalkyl β-cyclodextrin is selected from C1-C6 hydroxyl groups, preferably from C2-C5 hydroxyl groups; the sulfonyl group in sulfonyl β-cyclodextrin is selected from C1-C6 hydroxyl groups, preferably from C2-C5 sulfonyl groups.

5. The solution according to claim 1 or 2, wherein, The water-soluble modified β-cyclodextrin is selected from hydroxypropyl β-cyclodextrin, hydroxyethyl β-cyclodextrin, and sodium sulfobutyl β-cyclodextrin.

6. The solution according to claim 1 or 2, wherein, The phospholipid-polyethylene glycol conjugate is selected from distearylphosphatidylethanolamine-polyethylene glycol (DSPE-MPEG), distearylphosphatidylethanolamine-polyethylene glycol-amino (DSPE-PEG-NH2), distearylphosphatidylethanolamine-polyethylene glycol-carboxyl (DSPE-PEG-COOH), distearylphosphatidylethanolamine-polyethylene glycol-maleimide (DSPE-PEG-MAL), distearylphosphatidylethanolamine-polyethylene glycol-succinimide ester (DSPE-PEG-NHS), distearylphosphatidylethanolamine-polyethylene glycol-mercapto (DSPE-PEG-SH), or their sodium / potassium salts.

7. The solution according to claim 6, wherein, The phospholipid-polyethylene glycol conjugate is selected from distearylphosphatidylethanolamine-polyethylene glycol (DSPE-MPEG) or its sodium / potassium salt.

8. The solution according to claim 6, wherein, The phospholipid-polyethylene glycol conjugate is selected from distearylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-MPEG2000) or its sodium salt.

9. The solution according to claim 1 or 2, wherein, The concentration of AST-3424 in solution is 10 mg / ml-20 mg / ml.

10. The solution according to claim 1 or 2, wherein, The mass ratio of the excipient to AST-3424 is (10-20):

1.

11. The solution according to claim 1 or 2, wherein, The pH adjuster is selected from one or a mixture of sodium citrate, sodium acetate, potassium acetate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, potassium phosphate, sodium phosphate, sodium hydroxide, potassium hydroxide, sodium bicarbonate, potassium bicarbonate, sodium carbonate, and potassium carbonate.

12. AST-3424 lyophilized formulation solution, containing the compound AST-3424 of the following formula, as well as water, excipients, and pH adjuster: in, Water as a solvent The excipients are selected from trehalose, sorbitol, distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-MPEG 2000) sodium salt, sulfonyl β-cyclodextrin sodium salt, and hydroxypropyl β-cyclodextrin. The concentration of AST-3424 in solution is 10 mg / ml-20 mg / ml. The mass ratio of the excipient to AST-3424 is (10-20):

1. The pH adjuster is selected from one or a mixture of sodium citrate, sodium acetate, potassium acetate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, potassium phosphate, sodium phosphate, sodium hydroxide, potassium hydroxide, sodium bicarbonate, potassium bicarbonate, sodium carbonate, and potassium carbonate. The pH of the solution ranges from 7.4 to 10.

5.

13. AST-3424 lyophilized formulation solution, containing the compound AST-3424 of the following formula, as well as water, excipients, and pH adjuster: in, Water as a solvent The excipients are selected from trehalose, sorbitol, distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-MPEG 2000) sodium salt, sodium sulfonyl β-cyclodextrin, and hydroxypropyl β-cyclodextrin. The concentration of AST-3424 in solution is 10 mg / ml-20 mg / ml. The mass ratio of the excipient to AST-3424 is (10-20):

1. The pH adjuster is selected from sodium bicarbonate. The pH of the solution ranges from 7.4 to 10.

5.

14. AST-3424 lyophilized formulation solution, containing the compound AST-3424 of the following formula, as well as water, excipient trehalose or sorbitol, and pH adjuster sodium bicarbonate: in, The concentration of AST-3424 in the solution is 10 mg / ml, and the concentration of the excipients trehalose or sorbitol in the solution is 200 mg / ml. The pH of the solution ranges from 7.4 to 10.

5.

15. AST-3424 lyophilized formulation solution, containing the compound AST-3424, water, and excipient distearate-phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-MPEG2000) sodium salt, and pH adjuster sodium bicarbonate: in, The concentration of AST-3424 in the solution is 10 mg / ml, and the concentration of the excipient distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-MPEG2000) sodium in the solution is 100 mg / ml. The pH of the solution ranges from 7.4 to 10.

5.

16. AST-3424 lyophilized formulation solution, containing the following compound AST-3424, water, and excipients sodium sulfobutyl β-cyclodextrin and pH adjuster sodium bicarbonate: in, The concentration of AST-3424 in the solution is 10 mg / ml, and the concentration of the excipient sodium sulfobutyl β-cyclodextrin in the solution is 200 mg / ml or 100 mg / ml. The pH of the solution ranges from 7.4 to 10.

5.

17. AST-3424 lyophilized formulation solution, containing the compound AST-3424 of the following formula, as well as water, excipient hydroxypropyl β-cyclodextrin, and pH adjuster sodium bicarbonate: in, The concentration of AST-3424 in solution is 10 mg / ml, and the concentration of the excipient hydroxypropyl β-cyclodextrin in solution is 200 mg / ml. The pH of the solution ranges from 7.4 to 10.

5.

18. A lyophilized formulation containing the following compound AST-3424, as well as excipients and pH adjusters: in, Excipients are selected from trehalose, sorbitol, water-soluble modified β-cyclodextrin, or phospholipid-polyethylene glycol conjugates. The drug loading of AST-3424 in the lyophilized formulation was 1 mg / cm³. 3 Up to 20 mg / cm 3 , The mass ratio of the excipient to AST-3424 is (10-20):

1.

19. The lyophilized formulation according to claim 18, wherein, Water-soluble modified β-cyclodextrin is selected from hydroxyalkyl β-cyclodextrin, sulfoalkyl β-cyclodextrin, or their sodium or potassium salts.

20. The lyophilized formulation according to claim 19, wherein, The hydroxyl group in hydroxyalkyl β-cyclodextrin is selected from C1-C6 hydroxyl groups, preferably from C2-C5 hydroxyl groups; the sulfonyl group in sulfonyl β-cyclodextrin is selected from C1-C6 sulfonyl groups, preferably from C2-C5 sulfonyl groups.

21. The lyophilized formulation according to claim 18, wherein, The water-soluble modified β-cyclodextrin is selected from hydroxypropyl β-cyclodextrin, hydroxyethyl β-cyclodextrin, and sodium sulfobutyl β-cyclodextrin.

22. The lyophilized formulation according to claim 18, wherein, The phospholipid-polyethylene glycol conjugate is selected from distearylphosphatidylethanolamine-polyethylene glycol (DSPE-MPEG), distearylphosphatidylethanolamine-polyethylene glycol-amino (DSPE-PEG-NH2), distearylphosphatidylethanolamine-polyethylene glycol-carboxyl (DSPE-PEG-COOH), distearylphosphatidylethanolamine-polyethylene glycol-maleimide (DSPE-PEG-MAL), distearylphosphatidylethanolamine-polyethylene glycol-succinimide ester (DSPE-PEG-NHS), distearylphosphatidylethanolamine-polyethylene glycol-mercapto (DSPE-PEG-SH), or their sodium / potassium salts.

23. The lyophilized formulation according to claim 18, wherein, The phospholipid-polyethylene glycol conjugate is selected from distearylphosphatidylethanolamine-polyethylene glycol (DSPE-MPEG) or its sodium / potassium salt.

24. The lyophilized formulation according to claim 18, wherein, The phospholipid-polyethylene glycol conjugate is selected from distearylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-MPEG2000) or its sodium salt.

25. The lyophilized formulation according to claim 18, wherein, The drug loading of AST-3424 in the lyophilized formulation was 10 mg / cm³. 3 Up to 20 mg / cm 3 .

26. The lyophilized formulation according to claim 18, wherein, The pH adjuster is selected from one or a mixture of sodium citrate, sodium acetate, potassium acetate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, potassium phosphate, sodium phosphate, sodium hydroxide, potassium hydroxide, sodium bicarbonate, potassium bicarbonate, sodium carbonate, and potassium carbonate.

27. A lyophilized formulation containing the following compound AST-3424, as well as excipients and pH adjusters: in, The excipients are selected from trehalose or sorbitol, distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-MPEG 2000) sodium salt, and sodium sulfonyl β-cyclodextrin. The drug loading of AST-3424 in the lyophilized formulation was 10 mg / cm³. 3 Up to 20 mg / cm 3 , The mass ratio of the excipient to AST-3424 is (10-20):

1. pH adjuster is selected from one or a mixture of sodium citrate, sodium acetate, potassium acetate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, potassium phosphate, sodium phosphate, sodium hydroxide, potassium hydroxide, sodium bicarbonate, potassium bicarbonate, sodium carbonate, and potassium carbonate.

28. A lyophilized formulation containing the following compound AST-3424, as well as excipients and pH adjusters: in, The excipients are selected from trehalose, sorbitol, distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-MPEG 2000) sodium salt, sodium sulfonyl β-cyclodextrin, and hydroxypropyl β-cyclodextrin. The drug loading of AST-3424 in the lyophilized formulation was 10 mg / cm³. 3 Up to 20 mg / cm 3 , The mass ratio of the excipient to AST-3424 is (10-20):

1. The pH adjuster is sodium bicarbonate.

29. A lyophilized formulation containing the following compound AST-3424, as well as the excipient trehalose or sorbitol and the pH adjuster sodium bicarbonate: in, The drug loading of AST-3424 in the lyophilized formulation was 10 mg / cm³. 3 The content of excipients trehalose or sorbitol in the freeze-dried preparation is 200 mg / cm³. 3 .

30. A lyophilized formulation containing the following compound AST-3424, the excipient distearate-phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-MPEG2000) sodium salt, and the pH adjuster sodium bicarbonate: in, The drug loading of AST-3424 in the lyophilized formulation was 10 mg / cm³. 3 The content of the excipient distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-MPEG2000) in the lyophilized formulation is 100 mg / cm³. 3 .

31. A lyophilized formulation containing the following compound AST-3424, as well as the excipient sodium sulfobutyl β-cyclodextrin and the pH adjuster sodium bicarbonate: in, The drug loading of AST-3424 in the lyophilized formulation was 10 mg / cm³. 3 The content of sodium sulfobutyl β-cyclodextrin, an excipient in the lyophilized formulation, is 200 mg / cm³. 3 Or 100mg / cm 3 .

32. A lyophilized formulation containing the following compound AST-3424, as well as the excipient hydroxypropyl β-cyclodextrin and the pH adjuster sodium bicarbonate: in, The drug loading of AST-3424 in the lyophilized formulation was 10 mg / cm³. 3 The content of the excipient sodium hydroxypropyl β-cyclodextrin in the lyophilized formulation is 200 mg / cm³. 3 .

33. An intravenous injection solution containing AST-3424, wherein the solvent is water, and the solute includes AST-3424, an isotonicity adjusting agent, an excipient, and sodium bicarbonate, wherein the isotonicity adjusting agent is selected from glucose and sodium chloride, and the excipient is selected from trehalose, sorbitol, distearate phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-MPEG2000) sodium salt, sodium sulfobutyl β-cyclodextrin, and hydroxypropyl β-cyclodextrin, wherein the pH of the injection solution is 6.8-10.5 and is an isotonic solution.

34. The method for preparing the AST-3424 lyophilized formulation solution according to claims 2-17, comprising the following operations: Step 1: Prepare an aqueous solution of the pH adjuster; Step 2: Dissolve the prescribed amount of excipient and AST-3424 in an appropriate amount of water, add an appropriate amount of water and an aqueous solution of the pH adjuster, make up the volume, and adjust the pH to 7.4 to 10.

5.

35. The preparation method of AST-3424 lyophilized formulation includes the following steps: Pre-freezing involves placing an appropriate amount of the solution described in claim 34 into a freeze-drying system for pre-freezing. The pre-freezing process includes holding at 0°C for a period of time and holding at -40 to -50°C for a period of time. After the first drying and pre-freezing process, the temperature is raised to -20 to -40°C and kept at that temperature for a period of time, while maintaining a vacuum for drying. Secondary drying: After the first drying is completed, the temperature is raised to 20-40℃ and kept at that temperature for a period of time, while maintaining a vacuum for drying.

36. A lyophilized mixture containing the following compound AST-3424, as well as excipients and pH adjusters: in, Excipients are selected from trehalose, sorbitol, water-soluble modified β-cyclodextrin, or phospholipid-polyethylene glycol conjugates. The AST-3424 loading in the lyophilized mixture was 1 mg / cm³. 3 Up to 20 mg / cm 3 , The mass ratio of the excipient to AST-3424 is (10-20):1.

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