Stable ast-3424 injection preparation and preparation method

Abstract

A stable AST-3424 injection preparation, being a solution containing 0.1-200 mg / ml or 1-200 mg / ml of AST-3424 active ingredients. A solvent in the solution contains a C2-C8 monohydric alcohol. The preparation method comprises the following steps: performing first dissolution preparation on the AST-3424 active ingredients and a partial prescription amount of ethanol; adding a prescription amount of propylene glycol for the second-time dissolution preparation; and then, adding the remaining prescription amount of ethanol for mixing and dissolving to obtain a solution containing 1-200 mg / ml of AST-3424 active ingredients. The solvent of the AST-3424 injection is water, or a glucose solution, ethanol, and a propylene glycol solute consist of the AST-3424 active ingredients, an isotonic regulator, and a pH regulator. The concentration of the AST-3424 active ingredients of the injection is 0.001-1.000 mg / ml, the pH of the injection is 6.8-10.5, and the solution is an isotonic solution.

Description

Stable AST-3424 injection formulation and preparation method Technical Field

[0001] This invention relates to the development of an injection solution of the compound disclosed in patent application PCT / US2016 / 021581, publication number WO2016145092A1, corresponding to Chinese application number 2016800150788, publication number CN107530556A, belonging to the field of formulation development of cancer treatment compounds. Background Technology

[0002] Our company has developed AST-3424, a DNA alkylation cancer therapeutic drug targeting the overexpression of aldehyde-ketone reductase 1C3 (AKR1C3). (See patent application: DNA alkylating agent, corresponding PCT application number PCT / US2016 / 021581, publication number WO2016 / 145092, corresponding Chinese application number 2016800150788, publication number CN107530556A, which discloses compound TH2870; (R)- and (S)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxy-4-nitrophenyl)-1-ethyl-N,N'-bis(ethyl)aminophosphate). The composition, its use, and preparation method, corresponding to PCT application number PCT / US2016 / 062114, publication number WO2017087428A1, and Chinese application number 2016800446081, publication number CN108290911A (S-configuration compound), with the Chinese name (S)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxy-4-nitrophenyl)-1-ethyl-N,N'-bis(ethylene)aminophosphate, also known as the S-configuration compound of OBI-3424 and TH-2870, CAS number 2097713-69-2, has the following structure:

[0003]

[0004] Chemical structural formula of AST-3424

[0005] There are already authoritative industry literature (Kathryn Evans, Jian Xin Duan, Tara Pritchard, et al. OBI-3424, a novel AKR1C3-activated prodrug, exhibits potent efficacy against preclinical models of T-ALL[J], Clinical Cancer Research, 2019, DOI:10.1158 / 1078-0432.CCR-19-0551; Richard B. Lock, Kathryn Evans, Raymond Yung, Tara Pritchard, Beverly A. Teicher, Jian Xin Duan, Yuelong Guo, Stephen W. Erickson and Malcolm A. Smith, Abstract LB -B16: The AKR1C3-Activated Prodrug OBI-3424 Exerts Profound In Vivo Efficacy Against Preclinical Models of T-Cell Acute Lymphoblastic Leukemia (T-ALL); a Pediatric Preclinical Testing Consortium Study [C], AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; October 26-30, 2017; Philadelphia, PA, DOI: 10.1158 / 1535-7163.) confirmed that this compound is a broad-spectrum small molecule anticancer prodrug with efficacy against various solid tumors and hematological malignancies.

[0006] In order to conduct subsequent clinical trials, it is necessary to prepare a suitable dosage form for human administration: usually oral or injectable administration.

[0007] During the synthesis process, the substance was found to be a pale yellow liquid, which presents several challenges in storage, transportation, and formulation. Due to its amide and phosphate ester structure, it is difficult to develop oral dosage forms such as tablets and oral liquids. However, preliminary experiments by the research team revealed that conventional water-based injection solutions are not stable enough to meet the requirements of subsequent multi-center, multi-sample long-term clinical trials and commercial production and sales.

[0008] Summary of the Invention

[0009] This invention provides a stable AST-3424 injection formulation, its preparation method, and related technologies. The injection formulation provided by this technology meets the requirements for long-term clinical trials and commercial production and sales. Specifically, this application discloses the following technical solutions.

[0010] The present invention discloses a storage-stable concentrated form of AST-3424 injection, which is a concentrated injection of a pharmaceutical composition. This composition needs to be diluted before administration, and medical personnel, pharmacists, pharmacies, or factories need to dilute or prepare it according to requirements before use.

[0011] The concentrated injection solution of the present invention can be diluted with any medically acceptable isotonic adjusting reagent (as solute) and water for injection (as solvent).

[0012] Suitable isotonic adjusting agents include, but are not limited to, anhydrous or aqueous forms of sodium chloride, glucose, sucrose, fructose, xylitol, glycerol, sorbitol, mannitol, potassium chloride, mannose, calcium chloride, magnesium chloride, and other inorganic salts. Preferably, the osmotic agent is glucose or sodium chloride.

[0013] A stable AST-3424 injection formulation, which is a solution containing 0.1-200 mg / ml of AST-3424 active pharmaceutical ingredient, wherein the solvent of the solution contains a C2-C8 monohydric alcohol.

[0014] Furthermore, the solvent of the solution is a liquid solvent composed of a C2-C8 monohydric alcohol and a C2-C8 dihydric alcohol or trihydric alcohol.

[0015] Obviously, to further enhance stability to time and heat, other harmless substances that do not react with the AST-3424 active pharmaceutical ingredient can be added. Generally, these substances include:

[0016] Protective gas is used to greatly reduce the content of certain active gases such as O2 and CO2 in the injection solution by evacuating the injection solution. Then, an inert gas that does not react with the raw drug, monohydric alcohol, dihydric alcohol or trihydric alcohol, such as N2 or other inert gases, is introduced. These protective gases will dissolve in the injection solution.

[0017] Antioxidants, such as vitamin E, vitamin C or glutathione, are added to the injection solution to further improve stability.

[0018] To improve patient compliance, anesthetic or analgesic drugs in doses permitted by the pharmacopoeia or formulary can be added.

[0019] The injection solution disclosed in this invention may also contain another therapeutic agent to form a compound preparation, thereby enhancing the therapeutic effect through synergy. In particular, these drugs are recommended to regulate the expression level of the AKR1C3 enzyme or its corresponding gene.

[0020] In some cases, in order to cope with special environments, the injection solution disclosed in this invention may also contain antibacterial agents and antifungal agents.

[0021] In addition, electrolytes such as NaCl and KCl, which are commonly used in injection solutions, as well as substances such as glucose and amino acids that regulate water-salt, electrolyte, acid-base balance, and osmotic pressure, can also be added as needed.

[0022] Substances that adjust pH include alkali metal and alkaline earth metal salts of weak acids such as carbonic acid, phosphoric acid, citric acid, and acetic acid, as well as inorganic bases such as hydroxides of alkali metals and alkaline earth metals such as Na and K: sodium citrate, potassium 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. Some embodiments disclosed in this invention clarify that certain organic amine-based organic bases can cause instability in injection solutions. Although the mechanism of instability is not yet clear, these experimental results guide or inspire against adding organic amine-based organic bases, such as triethylamine and triethanolamine, without rigorous stability testing.

[0023] Obviously, the amounts of the protective gases, antioxidants, anesthetic or analgesic drugs, antibacterial agents, and antifungal agents added are very small, and they do not affect the solubility or other properties of the AST-3424 raw material or can enhance its stability. However, the amounts of other therapeutic agents, substances that regulate water, salt, electrolyte and acid-base balance, osmotic pressure, and pH need to be added according to the actual situation such as purpose, prescription dosage, pharmacopoeia, and formulary.

[0024] C2-C8 monohydric alcohols, including straight-chain, branched-chain, and cyclic fatty alcohols and aromatic alcohols (excluding phenols and highly reactive benzyl alcohols).

[0025] C2-C8 diols or triols, including straight-chain, branched-chain, and cyclic fatty alcohols and aromatic alcohols (excluding phenols and highly reactive benzyl alcohols).

[0026] Obviously, the mixed solvent of C2-C8 monohydric alcohols and C2-C8 dihydric or trihydric alcohols as defined in this invention means that the aforementioned monohydric alcohol and dihydric or trihydric alcohol should each be liquid at room temperature and differential pressure, or the mixed solvent should be liquid. The polyhydric alcohols are preferably ethylene glycol, propylene glycol, glycerol, mannitol, sorbitol, or mixtures thereof.

[0027] Furthermore, the solvent of the solution is a liquid or semi-liquid mixture of a monohydric alcohol containing C2-C8 and a suitable water-soluble polymer for pharmaceutical use.

[0028] In pharmaceutical preparations, liquid or semi-liquid mixtures of suitable water-soluble polymers include many types: polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, poloxamer, polysorbate, dextran, etc.

[0029] The monohydric alcohol (preferably ethanol) contained in the injectable formulation provided by the present invention mainly serves to dissolve and dilute the viscous, oily AST-3424 active pharmaceutical ingredient (during synthesis, it was verified that monohydric alcohols such as ethanol have good solubility for the active pharmaceutical ingredient, but experiments have shown that the stability of the ethanol solution is relatively poor).

[0030] Since monohydric alcohols such as ethanol have poor solution stability, it is speculated that this is due to their volatility, which causes changes in concentration in the solution; or because the solvent ethanol molecules are too reactive, the movement of the active pharmaceutical ingredient molecules is unimpeded, resulting in rapid changes and thus poor stability.

[0031] When diols or triols, water-soluble polymers, etc., are added to monohydric alcohols, the solution becomes viscous, thus thickening the solution. At the same time, these added substances also hinder the movement of the active pharmaceutical ingredient molecules, thereby enhancing stability.

[0032] Furthermore, the solvent of this stable AST-3424 injection is a mixture of C2-C4 monohydric alcohols and C2-C3 dihydric alcohols, more preferably a mixture of ethanol and propylene glycol.

[0033] Furthermore, the volume ratio of monohydric alcohol in the mixed solvent of this stable AST-3424 injection solution is not less than 50%.

[0034] As a preferred embodiment, the mixed solvent of this stable AST-3424 injection solution consists of 75% ethanol and 25% propylene glycol by volume.

[0035] Considering factors such as stability and ease of use, the stable AST-3424 injection disclosed in this invention is a solution containing 10 mg / ml of AST-3424 active pharmaceutical ingredient. This concentration is the recommended value of the research and development team, and it has shown good properties in comparative experiments.

[0036] Furthermore, the stable AST-3424 injection disclosed in this document does not contain added water, and the water content is controlled within 0.5% by mass.

[0037] Obviously, "no water added" means that no additional water is added during the preparation process, and other reagents and solvents used are anhydrous reagents, such as anhydrous ethanol and anhydrous propylene glycol. In other words, the water content needs to be controlled during the preparation of the injection solution of this invention. Theoretically, the lower the water content of the injection solution, the better the stability. However, considering the technical difficulty and the realization of large-scale industrial production, the research team believes that a water content (determined by Karl Fischer method) within 0.5% by mass is better.

[0038] The stable AST-3424 injection consists of 0.75 ml of anhydrous ethanol, 0.25 ml of anhydrous propylene glycol, and 10 mg of AST-3424 active pharmaceutical ingredient.

[0039] The above-mentioned 1.0 ml injection containing 10 mg AST-3424 raw material is a specific embodiment. It is filled into a 2 ml (or 5 ml) volume borosilicate glass tube (brown) injection vial, then filled with inert protective gas, capped with a rubber stopper and sealed with an aluminum-plastic combination cap for antibiotic vials.

[0040] Obviously, the volume of the 0.75 ml anhydrous ethanol and 0.25 ml anhydrous propylene glycol will change after mixing (it has been shown that it will decrease), so reasonable volume changes are also known to those skilled in the art (researchers or medical staff in the fields of pharmaceutical research and development, organic chemical synthesis, formulation development, etc.).

[0041] In particular, the concentration of AST-3424 active pharmaceutical ingredient in the injection solution of this embodiment will be specified as 10 mg / ml. Similarly, the actual tested content may vary in pharmaceutical standards. The concentration is considered acceptable within the corresponding range of the pharmacopoeia, formulary, or drug standard. In other words, the concentration within the corresponding range mentioned above is technically equivalent to the specified 10 mg / ml.

[0042] A stable AST-3424 injection formulation product includes a packaging container and an AST-3424 injection formulation contained in the container, wherein the injection formulation is the aforementioned injection formulation, wherein the AST-3424 active pharmaceutical ingredient content in the product is 1-200 mg, preferably 10 mg or 20 mg, and is filled in a light-proof glass bottle made of borosilicate glass filled with a protective gas.

[0043] The stable AST-3424 injection formulation consists of 0.75 ml of anhydrous ethanol, 0.25 ml of anhydrous propylene glycol, and 10 mg of AST-3424 active pharmaceutical ingredient, and is packaged in light-proof glass vials of 2, 5, and 10 ml volume made of borosilicate glass filled with protective gas.

[0044] A method for preparing a stable AST-3424 injection solution, comprising the following steps:

[0045] The AST-3424 active pharmaceutical ingredient was first dissolved and prepared with a portion of the prescribed amount of ethanol.

[0046] Add the prescribed amount of propylene glycol for a second dissolution and preparation;

[0047] Add the remaining amount of ethanol to the mixture and dissolve to obtain a solution containing 1-200 mg / ml or 1-200 mg / ml of AST-3424 active pharmaceutical ingredient.

[0048] In the above preparation method, the AST-3424 raw material must first be dissolved with a portion of the prescribed amount of ethanol to prepare a relatively concentrated solution, and then propylene glycol and the remaining prescribed amount of ethanol are added for dilution.

[0049] This procedure is based on the viscous, oily properties of the AST-3424 active pharmaceutical ingredient. Preferably, the amount of ethanol used for the first dissolution is 50% of the prescribed volume.

[0050] Alternatively, the AST-3424 active pharmaceutical ingredient can be first dissolved and prepared with ethanol; then, the prescribed amount of propylene glycol can be added for a second dissolution and preparation to obtain a solution containing 0.1-200 mg / ml or 1-200 mg / ml of AST-3424 active pharmaceutical ingredient.

[0051] Furthermore, the present invention also discloses an AST-3424 injection solution, which is a ready-to-use pharmaceutical composition injection. The composition does not need to be diluted before administration and is ready for administration during production, so medical personnel do not need to dilute or prepare it before use.

[0052] Obviously, AST-3424 injection solution can be a ready-to-use injection solution in various ways, such as intradermal, subcutaneous, intramuscular, and intravenous injection.

[0053] This invention discloses an AST-3424 injection solution, the solvent of which is water, and the solute is composed of AST-3424 active pharmaceutical ingredient, isotonicity adjusting reagent, ethanol, propylene glycol and pH adjuster. The concentration of AST-3424 active pharmaceutical ingredient in the injection solution is 0.001-1.000 mg / ml, the pH of the injection solution is 6.8-10.5, and it is an isotonic solution.

[0054] The isotonic adjusting agent, relative to the injectable solution of the present invention, can be any medically acceptable osmotic agent. Suitable osmotic agents include, but are not limited to, anhydrous or aqueous sodium chloride, glucose, sucrose, fructose, xylitol, glycerol, sorbitol, mannitol, potassium chloride, mannose, calcium chloride, magnesium chloride, and other inorganic salts or mixtures thereof.

[0055] Isotonic solutions, generally defined for humans as having a concentration of 308 mmol / L, include 0.9% saline and 5% glucose solutions. However, concentrations between 280-320 mmol / L can also be considered isotonic.

[0056] Of course, if the target of treatment is other animals, such as other primates, the concentration of the isotonic solution should be adjusted accordingly.

[0057] Furthermore, there is an AST-3424 injection solution, wherein the concentration of the AST-3424 active pharmaceutical ingredient in the injection solution is 0.004-0.94 mg / ml.

[0058] Preferably, AST-3424 intravenous injection solution has a pH of 7.4-10.5. Intravenous injection solutions at this pH value have good stability and can ensure storage for a certain period of time (e.g., when patients cannot take the medication on time after it has been prepared on-site).

[0059] The AST-3424 injection solution provided by this invention contains a pH adjuster that is 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.

[0060] An AST-3424 intravenous injection solution is provided, wherein the solvent is water, and the solute is composed of AST-3424 active pharmaceutical ingredient, glucose, ethanol, propylene glycol, and sodium bicarbonate as a pH adjuster. The concentration of AST-3424 active pharmaceutical ingredient in the injection solution is 0.004-0.94 mg / ml, the pH of the injection solution is 7.4, the glucose content in the injection solution is 4.5-5.0% by mass, and it is an isotonic solution.

[0061] The method for preparing a stable AST-3424 injection solution for intravenous injection is as follows: an appropriate amount of sodium bicarbonate solution as a pH adjuster is added to 5% glucose injection solution to make the pH of the mixed solution 7.4; the above-mentioned stable AST-3424 injection solution is then added to the above-mentioned mixed solution and mixed to dissolve.

[0062] The amounts and concentrations of sodium bicarbonate solution, 5% glucose injection, and AST-3424 injection used in the above dilution preparation process were calculated. Attached Figure Description

[0063] Figure 1 is a flowchart of the manufacturing process of AST-3424 pharmaceutical formulation. Detailed Implementation

[0064] One embodiment of the present invention is a non-ready-to-use concentrated injection solution.

[0065] The present invention discloses a storage-stable concentrated form of AST-3424 injection, which is a concentrated injectable pharmaceutical composition. This composition needs to be diluted before administration, and medical personnel, pharmacists, pharmacies, or factories need to dilute or prepare it according to requirements before use.

[0066] The concentrated injection solution of the present invention can be diluted with any medically acceptable isotonic adjusting reagent (as solute) and water for injection (as solvent).

[0067] Suitable isotonic adjusting agents include, but are not limited to, anhydrous or aqueous forms of sodium chloride, glucose, sucrose, fructose, xylitol, glycerol, sorbitol, mannitol, potassium chloride, mannose, calcium chloride, magnesium chloride, and other inorganic salts. Preferably, the osmotic agent is glucose or sodium chloride.

[0068] A stable AST-3424 injection is a solution containing 1-200 mg / ml of AST-3424 active pharmaceutical ingredient, in which the solvent is a mixture of C2-C8 monohydric alcohols and C2-C8 dihydric or trihydric alcohols.

[0069] Obviously, to further enhance stability to time and heat, other harmless substances that do not react with the AST-3424 active pharmaceutical ingredient can be added. Generally, these substances include:

[0070] Protective gas is used to greatly reduce the content of certain active gases such as O2 and CO2 in the injection solution by evacuating the injection solution. Then, an inert gas that does not react with the raw drug, monohydric alcohol, dihydric alcohol or trihydric alcohol, such as N2 or other inert gases, is introduced. These protective gases will dissolve in the injection solution.

[0071] Antioxidants, such as vitamin E, vitamin C or glutathione, are added to the injection solution to further improve stability.

[0072] To improve patient compliance, anesthetic or analgesic drugs in doses permitted by the pharmacopoeia or formulary can be added.

[0073] The injection solution disclosed in this invention may also contain another therapeutic agent to form a compound preparation, thereby enhancing the therapeutic effect through synergy. In particular, these drugs are recommended to regulate the expression level of the AKR1C3 enzyme or its corresponding gene.

[0074] In some cases, in order to cope with special environments, the injection solution disclosed in this invention may also contain antibacterial agents and antifungal agents.

[0075] In addition, electrolytes such as NaCl and KCl, which are commonly used in injection solutions, as well as substances such as glucose and amino acids that regulate water-salt, electrolyte, acid-base balance, and osmotic pressure, can also be added as needed.

[0076] Substances that adjust pH include alkali metal and alkaline earth metal salts of weak acids such as carbonic acid, phosphoric acid, citric acid, and acetic acid, as well as inorganic bases such as hydroxides of alkali metals and alkaline earth metals such as Na and K. Some embodiments disclosed in this invention clarify that certain organic amine bases can cause instability in injection solutions. Although the mechanism of instability is not yet clear, these experimental results guide or inspire against adding organic amine bases, such as triethylamine and triethanolamine, without rigorous stability testing.

[0077] Obviously, the amounts of the protective gases, antioxidants, anesthetic or analgesic drugs, antibacterial agents, and antifungal agents added are very small, and they do not affect the solubility or other properties of the AST-3424 raw material or can enhance its stability. However, the amounts of other therapeutic agents, substances that regulate water, salt, electrolyte and acid-base balance, osmotic pressure, and pH-regulating substances need to be added according to the actual situation such as purpose, prescription dosage, pharmacopoeia, and formulary.

[0078] C2-C8 monohydric alcohols, including straight-chain, branched-chain, and cyclic fatty alcohols and aromatic alcohols (excluding phenols and highly reactive benzyl alcohols).

[0079] C2-C8 diols or triols, including straight-chain, branched-chain, and cyclic fatty alcohols and aromatic alcohols (excluding phenols and highly reactive benzyl alcohols).

[0080] Obviously, the mixed solvent of C2-C8 monohydric alcohol and C2-C8 dihydric alcohol or trihydric alcohol as defined in this invention means that the monohydric alcohol and dihydric alcohol or trihydric alcohol should each be liquid at room temperature and differential pressure, or the mixed solvent should be liquid.

[0081] Obviously, the solvent for this solution can also be a liquid or semi-liquid mixture of a monohydric alcohol containing C2-C8 molecules and a suitable water-soluble polymer for pharmaceutical use. The following explanation uses anhydrous ethanol and anhydrous propylene glycol as examples.

[0082] Considering factors such as stability and ease of use, the stable AST-3424 injection disclosed in this invention is a solution containing 10 mg / ml of AST-3424 active pharmaceutical ingredient. This concentration is the recommended value of the research and development team, and it has shown good properties in comparative experiments.

[0083] Furthermore, the stable AST-3424 injection disclosed in this document does not contain added water, and the water content is controlled within 0.5% by mass.

[0084] Obviously, "no water added" means that no additional water is added during the preparation process, and other reagents and solvents used are anhydrous reagents, such as anhydrous ethanol and anhydrous propylene glycol. In other words, the water content needs to be controlled during the preparation of the injection solution of this invention. Theoretically, the lower the water content of the injection solution, the better the stability. However, considering the technical difficulty and the realization of large-scale industrial production, the research team believes that a water content (determined by Karl Fischer method) within 0.5% by mass is better.

[0085] The stable AST-3424 injection consists of 0.75 ml of anhydrous ethanol, 0.25 ml of anhydrous propylene glycol, and 10 mg of AST-3424 active pharmaceutical ingredient.

[0086] The above-mentioned 1.0ml injection containing 10mg AST-3424 raw material is a specific embodiment. It is filled into a 2ml (or 5ml) volume borosilicate glass vial (brown, light-proof), then filled with inert protective gas, capped with a rubber stopper, and sealed with an aluminum-plastic combination cap for antibiotic vials.

[0087] Obviously, the volume of the 0.75 ml anhydrous ethanol and 0.25 ml anhydrous propylene glycol will change after mixing (it has been shown that it will decrease), so reasonable volume changes are also known to those skilled in the art (researchers or medical staff in the fields of pharmaceutical research and development, organic chemical synthesis, formulation development, etc.).

[0088] In particular, the concentration of AST-3424 active pharmaceutical ingredient in the injection solution of this embodiment will be specified as 10 mg / ml. Similarly, the actual tested content may vary in pharmaceutical standards. The concentration is considered acceptable within the corresponding range of the pharmacopoeia, formulary, or drug standard. In other words, the concentration within the corresponding range mentioned above is technically equivalent to the specified 10 mg / ml.

[0089] The excipients used in this invention include solubilizers, antioxidants (such as vitamin E, vitamin C or glutathione), buffers, salts, glucose, stabilizers, and electrolytes.

[0090] The excipients used in this invention include other therapeutic agents, alkalizing agents, antibacterial agents, antifungal agents, and combinations thereof.

[0091] This invention addresses the need for a stable mixed AST-3424 injection solution, which is a commercially available concentrated product.

[0092] In some embodiments, the concentrated injection solution provided by the present invention still retains more than 90% of its original content after being stored in an accelerated test at room temperature (25±2℃, protected from light, in a brown vial, with a humidity of 60±5%RH) for 6 months.

[0093] In some embodiments, the concentrated injection solution provided by the present invention still retains a content of over 95% after being stored in an accelerated test at room temperature (5±2℃, protected from light, in a brown vial, with a humidity of 60±5%RH) for 6 months.

[0094] In some embodiments, the concentrated injection solution provided by the present invention still retains a content of over 98% after being stored in an accelerated test at room temperature (-20±2℃, protected from light, in a brown vial, with a humidity of 60±5%RH) for 6 months.

[0095] Furthermore, since the AST-3424 active pharmaceutical ingredient is of the S configuration, stability is not only about the stability of the compound itself, but also requires examination of whether chiral inversion occurs during storage (examining the enantiomeric excess (EE) value). Among the relatively stable concentrated injection solutions obtained during this examination:

[0096] In some embodiments, the concentrated injection solution provided by the present invention still has an EE value of over 97% after being stored in an accelerated test at room temperature (25±2℃, protected from light, in a brown vial, with a humidity of 60±5%RH) for 6 months.

[0097] In some embodiments, the concentrated injection solution provided by the present invention still has an EE value of over 97% after being stored in an accelerated test at room temperature (5±2℃, protected from light, in a brown vial, at a humidity of 60±5%RH) for 6 months.

[0098] In some embodiments, the concentrated injection solution provided by the present invention still has an EE value of over 97% after being stored in an accelerated test at room temperature (-20±2℃, protected from light, in a brown vial, with a humidity of 60±5%RH) for 6 months.

[0099] In particular, in some embodiments, the addition of an organic amine (triethanolamine) to the concentrated injection resulted in a less stable injection compared to the injection without the addition of an organic amine (triethanolamine): lower content, more impurities, and the appearance of new impurities.

[0100] The pH value of the compositions of the present invention can be adjusted using a suitable pH adjuster containing acid or base groups. A suitable pH adjuster typically comprises at least one acid or a salt thereof, or at least one base or a salt thereof. An acid or base is added to adjust the pH value to the desired value. For example, if the pH value is lower than the desired pH value, a base (or some salt) is added to raise the pH value to the desired pH value.

[0101] Amino acids that can be used in this invention include, for example, arginine, glycine, methionine, or lysine. In some embodiments, the amino acid has at least one basic group with a pKa value greater than 5, 6, 7, 8, or 8.5, or a mixture thereof, or a salt thereof, or a mixture of the amino acid and its salt. The amount of amino acid used in this invention is 0.1–100 mg / mL, 1–50 mg / mL, or 5–25 mg / mL.

[0102] In some embodiments, the concentrated injection solution of the present invention is sterile, such as by terminal sterilization.

[0103] The injectable composition of the present invention is packaged in a medically acceptable packaging container, which may be an intravenous infusion bag or bottle. The infusion bag and bottle may be made of glass, suitable plastic, or polymeric material. The entirety or most of the packaging container may comprise materials such as polyvinyl chloride, polyolefin, polyester, polypropylene, or combinations thereof. In other embodiments, only the surface material in contact with the injectable drug contains these materials. The present invention is preferably a vial.

[0104] The provided injection solution is surrounded by a protective sheath (such as foil or paper) to prevent the active material from being exposed to light. In other embodiments, nitrogen gas is filled between the injection solution packaging bag and the protective sheath to prevent oxidation of the composition. In other embodiments, the packaging container (such as glass or plastic) may be light-resistant.

[0105] The concentrated injection solution of the present invention may also contain a certain amount of opioid analgesics.

[0106] Opioid analgesics that can be selected include: alfentanil, allprodrug, afarotin, antralidine, apomorphine, apocodeine, benzylmorphine, benzoylmide, buffentanil, buprenorphine, butorphanol, carfentanil, malamidophos, codeine, cyclopolychlorinated biphenyls, cipronorphine, dihydrodeoxymorphine, dezocine, dienprofen, dihydrocodeine, demetadine, demetonol, dimethylthiamethoxam, dichlorophenylbutyrate, dipiperazine, ethazocine, ethylthiamethoxam, ethylmorphine, etonicillin. Fentanyl, heroin, hydrocodone, hydroxymethylmorphine, hydromorphone hydroxy, ketomidone, isomethadone, levorpheneline, levorpheneline, rofentanyl, pethidine, mepital, metoprolol, metazoxine, methadone, methylmorphine, metoprolol, mifentanyl, morphine, morphine-6-glucuronic acid, merophenone, nalbuphine, papaverine, nicotinic acid, norlevorpheneline, normethadone, allylmorphine, orphanone / orphanone FQ (N / OFQ), normorphine, nopiperidone Oxymethylfentanyl, opium, oxycodone, hydroxydihydromorphone, total opioids, tebuconazole, benzoxone, fenofosine, phenazocine, phenazocine, phenvalerate, hydroxydihydromorphone, teratin, nitroxymethylene, propoxur, dimethomorphine, profloxacin, propofol, propipiridine, propoxyphene, remifentanil, sufentanil, tapentathol, tramadol, trafentanil, nalbuphine; any opioid with agonistic activity at opioid receptors belonging to the phenanthrene, morphine, phenylmorphine, and methadone classes. Opioids of ketones, phenylpiperidines, propionamide-4-phenylaminopiperidines, 4-arylpiperidines, and 4-isoarylpiperidines; any pentacyclic nuclear opioid having agonistic activity on opioid receptors, having the same properties as nalmefene, naltrexone, buprenorphine, levonorphine, mepitafen, pentazocine, and dezocine; any fentanyl analogues, precursors, derivatives, or medically acceptable salts thereof, and mixtures thereof in racemic or enantiomeric forms, that are active on opioid receptors.

[0107] The following examples are provided to aid in understanding the present invention, but should not be construed as limiting the invention. For those skilled in the art, based on the ideas presented in this invention, there will be changes in specific implementation methods and application scope. The content of this specification should not be construed as limiting the invention, and any changes made in accordance with the design concept of this invention are within the protection scope of this invention.

[0108] The following are the specific experiments and embodiments of the present invention.

[0109] The following experiments will reveal some physicochemical properties of the AST-3424 developed by the applicant that are related to the stability of the concentrated injection and ready-to-use injection of the present invention. The applicant hereby declares that the rights to the following experimental data belong to the applicant.

[0110] I. Study on the solubility and solution stability of AST-3424

[0111] 1.1 Buffer / Solution Preparation

[0112] As long as the target concentration remains unchanged, different volumes of stock solutions and buffer solutions than specified can be used.

[0113] Sodium hydroxide solution, 0.2 mol / L: Take 8.00 g of sodium hydroxide [NaOH], dissolve it in water, and dilute it to 1000 ml.

[0114] Potassium dihydrogen phosphate, 0.2 mol / L: Dissolve 27.22 g of potassium dihydrogen phosphate [KH2PO4] in water and dilute to 1000 ml.

[0115] Acetic acid, 2 mol / L: Measure 114.4 ml of acetic acid, dilute with water to 1000 ml, and mix well.

[0116] Boric acid and potassium chloride, 0.2 mol / L: Take 12.37 g of boric acid [H3BO3] and 14.91 g of potassium chloride (KCl), dissolve them in water, and dilute with water to 1000 ml.

[0117] pH 4.5 acetate buffer. Take 99g of sodium acetate (NaC2H3O2·3H2O2) and place it in a 1000ml volumetric flask. Add 14.0ml of acetic acid solution, then add water to the mark and mix.

[0118] pH 6.8 phosphate buffer. Place 50 ml of potassium dihydrogen phosphate solution in a 200 ml volumetric flask, add 22.4 ml of sodium hydroxide solution, and then add water to the mark.

[0119] pH 7.4 phosphate buffer. Take 50 ml of potassium dihydrogen phosphate solution and place it in a 200 ml volumetric flask. Add 39.1 ml of sodium hydroxide solution, and then add water to the mark.

[0120] pH 10.0 alkaline borate buffer. Take 50 ml of boric acid and potassium chloride solution and place it in a 200 ml volumetric flask. Add 43.7 ml of sodium hydroxide solution, and then add water to the mark.

[0121] 1.2 Solubility Test

[0122] Take an appropriate amount of AST-3424 (hereinafter referred to as the active pharmaceutical ingredient or API) and place it in a suitable container containing 40 ml of the above solution (20 ml for organic solvents) until there are excess AST-3424 oil droplets in the solution.

[0123] Place the sample in a constant temperature shaking incubator, maintain the temperature at 25°C, and shake at a suitable speed (100 rpm). Take samples at each predetermined time point according to the specifications in Table 1, check the pH, and then centrifuge (10000 rpm, 10 minutes). Dilute with the appropriate solution or organic solvent (to dissolve the API for solubility studies) to the appropriate concentration for HPLC analysis (Note: accurately record the dilution ratio after the experiment) to obtain solubility data.

[0124] If a significant decrease in solubility is observed, there is no need to test the solubility at 48h and 72h.

[0125] Table 1: Sampling Time and Solubility Test Items

[0126] Solution / Buffer 1h 4h 8h 24h 48h 72h Ethanol AAAAAA Propylene Glycol AAAAAA pH 4.5 Sodium Acetate Buffer A, PA, PA, PA, PA, PA, P pH 6.8 Phosphate Buffer A, PA, PA, PA, PA, PA, P pH 7.4 Phosphate Buffer A, PA, PA, PA, PA, P pH 10.0 Alkaline Borate Buffer A, PA, PA, PA, PA, PA, P Purified Water A, PA, PA, PA, PA, PA, P

[0127] Note: A = content determination, measuring the content of AST-3424 in the solution; P = pH

[0128] 1.3 Solution Stability Study

[0129] Take approximately 107.32 mg of AST-3424 (50% v / v, ethanol) and place it in a 50 mL volumetric flask. Add solvent (organic solvent, buffer solution, or purified water) to the mark. At each predetermined time point, take 1 mL of sample for HPLC analysis. If the API is stable in solutions of different pH values, extend the sampling time, for example, to 5 days or longer. See Table 2 below for specific sampling times and detection items.

[0130] Table 2: Sample analysis time for solution stability studies

[0131] Solution / Buffer 0h 1h 4h 8h 24h 48h 72h Ethanol AAAAAAA Propylene Glycol AAAAAAA pH 4.5 Sodium Acetate Buffer A, PAAAAAA, P pH 6.8 Phosphate Buffer A, PAAAAAA, P pH 7.4 Phosphate Buffer A, PAAAAAA, P pH 10.0 Alkaline Borate Buffer A, PAAAAAA, P Purified Water A, PAAAAAA, P

[0132] Note: A = content determination, determining the content of AST-3424 in the solution, as well as the HPLC peak purity and total impurities; P = pH

[0133] 1.4 Test Methods

[0134] For solubility studies involving content determination, measure approximately 1 ml of culture medium, centrifuge at 10,000 rpm for 10 minutes, and collect the clear lower layer for HPLC analysis. For solution stability studies, the sample can be directly injected into the HPLC system for analysis.

[0135] The content was determined by HPLC: AST-3424 was used as an external standard for quantification.

[0136] The UVDAD detector has a wavelength of 230 nm, a C18 column, and a column temperature of 25 °C.

[0137] Mobile phase:

[0138] A: Ammonium acetate is dissolved in a 10 mmol / L ammonium acetate solution in a mixed solvent of 95% water and 5% acetonitrile (volume ratio).

[0139] B: An 8 mmol / L ammonium acetate solution in a mixed solvent of 95% acetonitrile and 5% water (by volume).

[0140] Gradient elution is performed.

[0141] 1.5 Test Results

[0142] Table 3 summarizes the solution stability data for AST-3424. The solution stability results for AST-3424 indicate that, at room temperature, the API is stable for at least 72 hours in ethanol, ethanol / propylene glycol (1 / 1), pH 7.4, and pH 10.0 buffer solutions, and stable for at least 24 hours in pH 6.8 solution. The API is unstable in pH 4.5 solution and water, particularly in pH 4.5 acetate buffer.

[0143] Table 3: Results of solution stability studies of AST-3424 in different solutions

[0144]

[0145] Based on solution stability results, AST-3424 is highly unstable in pH 4.5 acetate buffer. Therefore, solubility tests were performed using pH 6.8 phosphate buffer, pH 7.4 phosphate buffer, pH 10.0 alkaline borate buffer, and purified water. Weigh an appropriate amount of AST-3424 into 40 mL of the medium (8 mL of ethanol / propylene glycol, 50:50 V / V) until excess flocculent material is present in the solution. If the API solubility in aqueous solution is >2% (20 mg / mL), no further API addition is necessary.

[0146] Table 4: Results of Solubility Study of AST-3424

[0147]

[0148] AST-3424 has a solubility greater than 270 mg / mL in an ethanol / propylene glycol (50:50, v / v) solution. API has a solubility of approximately 23 mg / mL at pH 6.8, pH 7.4, and pH 10.0, and approximately 20 mg / mL in water. Because API is unstable at pH 6.8 and in water, its solubility gradually decreases over time. The pH of API solutions in media at pH 6.8, pH 7.4, and pH 10.0 remains constant over different time periods. In aqueous solutions containing API, the pH value gradually increases from pH 5.004 to pH 6.512 within 48 hours.

[0149] 1.6 Summary of Physicochemical Properties Related to Injectable Solutions

[0150] Solubility. Table 5 summarizes the solubility of AST-3424 in different solvents at 25°C. AST-3424 is readily soluble in alcohol solvents such as ethanol / propylene glycol. Researchers have also preliminarily investigated other monohydric alcohols such as methanol, propanol, and butanol, as well as ethylene glycol, propylene glycol, glycerol, 1,3-butanediol, and 1,2-butanediol. These solvents all exhibit good solubility for the active pharmaceutical ingredient.

[0151] AST-3424 is slightly soluble in water, pH 6.8 phosphate buffer, pH 7.4 phosphate buffer, and pH 10.0 alkaline borate buffer.

[0152] In addition, AST-3424 is stable in pH 7.4 phosphate buffer, but its stability is poor in water and pH 6.8 phosphate buffer. Therefore, the water content should be minimized during the production and storage of the injection (the inventors speculate that the N-containing three-membered ring structure in AST-3424 is prone to ring-opening hydrolysis and deterioration in the presence of water).

[0153] Table 5: Solubility of AST-3424

[0154]

[0155] Optical rotation

[0156] Optical rotation of AST-3424[a] 24 D =-30.04° (c=0.006, EtOH).

[0157] II. Formulation design, preparation and stability study of AST-3424 injection

[0158] Based on the solubility and solution stability studies revealed above, the stability of AST-3424 under alkaline conditions was investigated, and formulation, preparation, and stability studies were conducted.

[0159] 2.1 Prescription Design and Preparation

[0160] The formulation study of AST-3424 pharmaceutical preparations included selecting various solvents and preparing different formulations.

[0161] Considering the toxicity of the substances and the safety and availability of the solvents for the injections, the inventors' team selected formulation solvents composed of ethanol, propylene glycol, and triethanolamine (an organic amine used to make the pH alkaline) to prepare different formulations.

[0162] Then, based on solubility, dosages of 10 and 200 mg / mL were selected to prepare different formulations for research.

[0163] Stability studies of these different formulations were then conducted at -20°C, 2-8°C, and 25°C.

[0164] HPLC was used to monitor the chemical properties of the product, including content, related substances, and ee value (enantiomer excess), to determine the optimal formulation of AST-3424.

[0165] Table 6 below lists the different formulations of 10 mg / mL AST-3424 injection.

[0166] Table 6: Different formulations of AST-3424 injection (10 mg / mL)

[0167]

[0168] Table 7 below lists the different formulations of 200 mg / mL AST-3424 injection.

[0169] Table 7: Different formulations of AST-3424 injection (200 mg / mL)

[0170]

[0171] Accurately weigh AST-3424 (dissolved in ethanol) and place it in a suitable volumetric flask. Then, add different solvents to prepare a final API concentration of 10 mg / mL or 200 mg / mL. Different formulation compositions are listed in Tables 3 and 4. Measure one mL of the bulk solution and fill it into a 6 mL amber vial. After sealing with a rubber stopper and aluminum cap, store the drug preparation at -20±2℃, 5±2℃, or 25±2℃ in a light-protected amber vial at 60±5% RH for different durations. At predetermined times, remove the sample and test it. The test method is the same as that for solubility and solution stability described in Section 1 above.

[0172] 2.2 Stability Test Results

[0173] At each predetermined time point, samples were taken out, and the content of the active pharmaceutical ingredient and related substances (i.e., impurities) in the injection solution were analyzed by HPLC. Table 8-10 shows the sampling schedule analysis data and the stability test data of related substances.

[0174] Table 8: Summary of stability data for different formulations at 25℃ (25±2℃)

[0175]

[0176] Table 9: Summary of stability data for different formulations at 5℃ (2-8℃)

[0177]

[0178] Table 10: Summary of stability data for different formulations at -20℃ (-22 to -18℃)

[0179]

[0180] The EE values ​​of seven relatively stable prescriptions were measured and recorded in Table 11 below:

[0181] Table 11: EE% values ​​for relatively stable prescriptions

[0182]

[0183] Notes: / indicates no test; TBD indicates not detected; * indicates the data is bad; ND indicates below the instrument's detection limit and not detected; N / A indicates not containing

[0184] 2.3 Results and Discussion

[0185] The stability results of different formulations of AST-3424 injection are shown in Table 8-11. For the stability results of different formulations stored at 25°C, it was found that the stability of the formulation increased with the increasing proportion of ethanol in the formulation. For formulations containing triethanolamine, the stability was lower than that of formulations without triethanolamine.

[0186] At a dosage strength of 10 mg / ml, F9 is the most stable formulation among the other formulations.

[0187] For formulations stored at 2-8℃ and -20℃, the stability of the samples was significantly increased. Samples stored at -20℃ were more stable than those stored at 2-8℃. Enantiomer excess (EE) was tested for stable formulations (i.e., F1 (10 mg / ml), F7 (10 mg / ml), F9 (10 mg / ml), F1-1 (10 mg / ml), F1 (200 mg / ml), F3 (200 mg / ml), and F2-1 (200 mg / ml)). The EE values ​​for the relatively stable formulations are shown in Table 11. The EE values ​​for all formulations remained unchanged for 6 months under different storage conditions, indicating that the active ingredients in these formulations did not undergo isomerization.

[0188] By comparing the stability test results of different formulations, it can be concluded that:

[0189] (1) According to the results of the prescription screening study in the table above, the stability of the drug preparation increases with the increase of the proportion of ethanol in the prescription. On the other hand, if triethanolamine is added to the preparation, the drug preparation becomes unstable.

[0190] (2) Stability studies showed that the pharmaceutical preparations were more stable at a storage temperature of -20°C than at 2-8°C or 25°C. Storage temperature has a significant impact on the stability of pharmaceutical preparations.

[0191] (3) Through AST-3424 drug formulation concentration screening, candidate formulation with code F9 was identified as the most stable among the candidate formulations. Six-month stability results showed no significant changes in related substances and ee values.

[0192] (4) Based on the results of the prescription review, prescription F9 (75.0% ethanol and 25.0% propylene glycol) was selected as the final candidate prescription for the AST-3424 drug formulation.

[0193] III. Photostability Study of AST-3424 Injection Solution

[0194] 3.1 Sample preparation and test results

[0195] After preparing the AST-3424 injection solution (1 mL: 10 mg, Formula F9), it was placed in 1.1 mL clear vials and brown vials respectively, and sealed. Before starting the photostability study, the light intensity of the stability test chamber was measured to ensure that the total illuminance of the test samples was greater than 1.2 × 10⁻⁶. 6 Lux·hrs was used to calculate the end time of the photostability experiment (the light intensity in the photostability chamber where the sample was located was 5200 Lux, and the experiment period was no less than 10 days). All samples were placed in the photostability chamber and samples were taken at 5, 10 and 20 days to check the properties, content and related substances of each sample.

[0196] The transparent vials are pharmaceutical grade vials made of borosilicate glass, and the brown vials are also pharmaceutical grade vials made of borosilicate glass. During testing, considering the influence of acidity and alkalinity, the composition and pH of the borosilicate glass tubing were chosen to suit the stability of the AST-3424 injection solution of this invention under slightly alkaline conditions.

[0197] The initially prepared AST-3424 injection solution was placed in a brown vial as a test sample control and stored at -20°C. It was analyzed together with the sample stored in the photostable chamber at each sampling time point. A transparent vial wrapped in aluminum foil was used as a control group for photostable studies, and samples were taken and analyzed according to the same sampling schedule (HPLC method in Section 1.4). The results are shown in Table 12 below.

[0198] Table 12: Results of photostable study of AST-3424 injection solution

[0199]

[0200] Note: Content refers to the relative percentage content of the corresponding substance as determined by HPLC; related substances refer to impurities in the active pharmaceutical ingredient (API).

[0201] 3.2 Conclusion

[0202] Photostability results of AST-3424 injection stored in transparent vials showed that after 5 days of light exposure, the appearance of the AST-3424 injection changed from pale yellow to brown. The content of the active ingredient decreased at all sampling points, and by the end of 20 days of light exposure, the content of the active ingredient had decreased from 99.9% to 56.6%.

[0203] AST-3424 injection samples stored in transparent vials wrapped in aluminum foil remained essentially unchanged in terms of active ingredient content and impurities after 20 days of light exposure.

[0204] The content and related substances of AST-3424 injection stored in brown vials remained unchanged after 10 days of light exposure, but the content of active ingredient decreased from 99.9% to 97.3% after 20 days of light exposure.

[0205] The results of photostability studies indicate that AST-3424 injection is unstable under light conditions and should be stored in amber (brown) vials to avoid direct exposure to light sources.

[0206] IV. Temperature Cyclic Stability Study of AST-3424 Injection Solution

[0207] To assess the stability of the active ingredient AST-3424 during temperature cycling, the temperature cycling stability of AST-3424 injection will be studied to provide guidance on the storage methods for this product during transportation, storage, and use.

[0208] Since the second part above has clearly stated that the optimal storage temperature for AST-3424 injection is -20℃, when using it, the injection needs to be taken out of the low-temperature environment and placed in a normal temperature environment of 25℃, or the injection needs to be placed back from the normal temperature environment of 25℃ to the low-temperature environment of the optimal storage temperature of -20℃. Such temperature cycling shock may affect the stability of the injection, so it is necessary to conduct a study on the temperature cycling stability of the injection.

[0209] 4.1 Test Operation

[0210] The prepared AST-3424 injection solution (1 ml: 10 mg) was first stored at -20°C for 2 days, then the sample was transferred to an environment of 2–8°C and stored for 2 days, which constituted one temperature cycle. Three temperature cycles were performed, and the sample was analyzed after each temperature cycle. The initially prepared AST-2424 injection solution was stored at -20°C as a control. The assay items included appearance, content, and related substances. The above process was repeated to perform temperature cycles from -20°C to 25°C.

[0211] 4.2 Test Results

[0212] Two samples (4 vials) were prepared for each temperature cycle. One sample (2 vials) was tested, and the other sample (2 vials) was kept as a backup. Table 13 lists the cycle times in the original test records, and Table 14 summarizes the results of temperature cycling stability.

[0213] Table 13: Sampling Schedule for Temperature Cycling Studies

[0214]

[0215] Vials: Indicates the number of vials containing the medication; Pull: Indicates the number of samples taken.

[0216] Table 14: Temperature Cyclic Stability Results of AST-3424 Injection

[0217]

[0218] Contraol: Control group; first cycle, second cycle, third cycle: First, second, and third cycles.

[0219] 4.3 Test Results

[0220] Three temperature cycles of AST-3424 injection from -20℃ to 2-8℃ showed that the content and impurities in the sample remained unchanged during this period. Temperature cycling tests of AST-3424 injection from -20℃ to 25℃ also showed no change in the content of impurities in the sample, with only a slight increase in related substances (from 1.3% to 1.4%). These results indicate that AST-3424 injection is stable in the three temperature cycles from -20℃ to 2-8℃ and from -20℃ to 25℃.

[0221] V. Preparation process and specific examples of concentrated injection solutions

[0222] The production process flow chart of AST-3424 drug formulation is shown in Figure 1.

[0223] Step 1: Dissolving and Mixing

[0224] Step 2-1: Add ethanol solution

[0225] Weigh the prescribed amount of AST-3424 raw material (pyrogen-free) into a beaker and put it into the mixing tank. Add 50% of the prescribed amount of pharmaceutical-grade anhydrous ethanol (pyrogen-free) and stir until dissolved (dissolution time 15 min, stirring speed 50 Hz, i.e., 50 revolutions per minute).

[0226] Step 2-2: Add propylene glycol

[0227] Add the prescribed amount of propylene glycol (already depyrogenated) and stir until dissolved (dissolution time 15 min, stirring speed 50 Hz, i.e., 50 revolutions per minute).

[0228] Steps 2-3: Mixing

[0229] Add 50% of the prescribed amount of anhydrous ethanol (after pyrogen removal) and stir until dissolved (dissolution time 15 min, stirring speed 50 Hz, i.e., 50 revolutions per minute).

[0230] Step 3: Sterilization

[0231] Perform sterilization on the solution obtained in step 2.

[0232] Step 4: Aseptic filling

[0233] Perform aseptic filling, with a filling volume of 1.0-1.2 ml (0.860-1.032 g).

[0234] Step 5: Crimping and Visual Inspection

[0235] The filled medicine bottles are conveyed to the capping room via a conveyor belt for capping. A visual inspection is then performed.

[0236] Step 6: Release Inspection

[0237] Samples of AST-3424 injection solution were taken for QC testing. After QA release, the solution was stored at -20°C for clinical use.

[0238] The above studies investigated the formulation design, stability, and other properties of the concentrated injection solution, as well as specific preparation examples. The following will reveal the preparation process of the ready-to-use injection solution and conduct a stability study.

[0239] VI. Combination and Stability Study of Ready-to-Use Intravenous Injection Solutions

[0240] To provide information on the compatibility of AST-3424 injection with its reconstituted solutions, the short-term stability of the injection in the commonly used intravenous diluent, 5% glucose injection, needs to be investigated. Considering the instability of APIs in acidic environments, the pH of the 5% glucose injection was first adjusted to 7.4 using sodium bicarbonate solution.

[0241] 6.1 Experimental methods and procedures

[0242] Preparation of 5% glucose injection

[0243] Add 0.22 mL of sodium bicarbonate solution to 5% glucose injection (250 mL: 12.5 g) using a 1 mL syringe, mix well, and the pH of the mixed solution will be approximately 7.4.

[0244] Preparation of AST-3424 compound solution

[0245] Compound solutions with concentrations of 0.004 mg / mL, 0.071 mg / mL, and 0.94 mg / mL were prepared using AST-3424 injection (1 mL: 10 mg, clinical batch number: 20170701) and 5% glucose injection.

[0246] Preparation of 0.004 mg / mL compound solution

[0247] Take one vial of AST-3424 injection (1 mL: 10 mg), and use a 1 mL syringe to draw 0.1 mL of the solution into a mixing bag containing 250 mL of 5% glucose injection (adjusted to pH 7.4 with a pH adjuster). Before storage and sampling, invert the mixing bag several times to ensure the mixture is thoroughly mixed.

[0248] Preparation of 0.071 mg / mL compound solution

[0249] Using a 2.5 mL syringe, draw 1.8 mL of sample from each of the two vials of AST-3424 injection and add it to a mixing bag containing 250 mL of 5% glucose injection (pre-adjusted to pH 7.4 with a pH adjuster). Ensure the mixture is thoroughly mixed by repeatedly inverting the mixing bag before storage and sampling.

[0250] Preparation of 0.94 mg / mL compound solution

[0251] Using a 30mL syringe, draw 26mL of sample from each of the 26 vials of AST-3424 injection and add it to a mixing bag containing 250mL of 5% glucose injection (pre-adjusted to pH 7.4 with a pH adjuster). Ensure the mixture is thoroughly mixed by repeatedly inverting the mixing bag before storage and sampling.

[0252] Temperature and light conditions

[0253] All compound solution samples were stored at room temperature (25±3℃) under natural light for 24 hours.

[0254] Sampling time point and test items

[0255] Using a 20mL syringe, take two 10mL samples of the compound solution from each bag at 0, 1, 2, 4, 8, and 24 hours. One sample will be used for testing, and the other will serve as a backup. The test items are as follows:

[0256] 1) Visual inspection;

[0257] 2) Osmotic pressure (initial and final sampling time points);

[0258] 3) pH value of the compound solution;

[0259] 4) Content (tested using the test method in Section 1.4);

[0260] 5) Related substances (tested using the test methods in Section 1.4);

[0261] 6)ee

[0262] Table 15: Sampling Time Points and Test Items for AST-3424 Compound Solution

[0263]

[0264] Notes: I = Visual inspection; O = Osmotic pressure; P = pH value; A = Content; R = Related substances; E = ee value.

[0265] 6.2 Experimental Results

[0266] All experimental data will be recorded in Tables 16-20, which summarize the stability data of AST-3424 compound solutions diluted to different concentrations.

[0267] Table 16: Summary of stability data for compound solutions of AST-3424 injection (1 mL: 10 mg) and 5% glucose injection at a concentration of 0.004 mg / mL

[0268] Date: 7-8 March 2018 Temperature: 25.6-26.2℃ Relative Humidity: 26.8-33.8%

[0269]

[0270] Table 17: Summary of stability data for the compound solution of AST-3424 injection (1 mL: 10 mg) and 5% glucose injection at a concentration of 0.071 mg / mL

[0271] Date: 8-9 March 2018 Temperature: 23.9-26.6℃ Relative Humidity: 18.1-28.9%

[0272]

[0273] Table 18: Summary of related substance data for AST-3424 injection (1 mL: 10 mg) and 5% glucose injection compound solution at a concentration of 0.071 mg / mL

[0274]

[0275] Table 19: Summary of stability data for the compound solution of AST-3424 injection (1 mL: 10 mg) and 5% glucose injection at a concentration of 0.94 mg / mL

[0276] Date: 09MAR2018-10MAR2018 Temperature: 22.1-27.2℃ Relative Humidity: 18.1-25.2%

[0277]

[0278] Table 20: Summary of related substance data for AST-3424 injection (1 mL: 10 mg) and 5% glucose injection compound solution at a concentration of 0.94 mg / mL

[0279]

[0280] Remark:

[0281] Regarding the content of the compound solution

[0282] 1) Preparing a 0.004 mg / mL compound solution requires 0.1 mL of AST-3424 injection solution, but 0.1 mL of sample is not easily measured accurately using a 1 mL syringe. 0.0055 mg / mL is the actual measurement result at the initial time point, which is approximately 138% of the theoretical value.

[0283] 2) The actual volume of 5% glucose injection is approximately 268 mL, while the theoretical volume is 250 mL. Therefore, when preparing samples with concentrations of 0.071 g / mL and 0.94 g / mL, the actual results at the initial time points are 0.067 mg / mL and 0.86 mg / mL, respectively.

[0284] 3) PRT, retention time, in minutes, is the retention time of the chromatographic peak of a specific impurity detected in the HPLC method described in Section 1.4.

[0285] 4) Initial, initial value.

[0286] 6.3 Experimental Conclusions

[0287] 1) No significant changes were observed in any of the samples after storage at room temperature (25±3℃) under natural light conditions for 24 hours. The compound solutions with concentrations of 0.004 mg / mL and 0.071 mg / mL were colorless and transparent, while the compound solution with concentration of 0.96 mg / mL was a pale yellow and transparent solution.

[0288] 2) The osmotic pressure of the low-concentration (0.004 mg / mL) compound solution is lower than isotonic, and it is almost isotonic at a concentration of 0.071 mg / mL (284-288 mmol / kg) (0.074 mg / mL is an isotonic solution). The high-concentration (0.94 mg / mL) compound solution is hypertonic (561-571 mmol / kg). Osmotic pressure data from sampling points at 0, 8, and 24 hours show that the osmotic pressure of the compound solution remains constant over 24 hours at all three concentrations.

[0289] 3) During the 24-hour observation period, the pH values ​​of the compound solutions with concentrations of 0.004 mg / mL, 0.071 mg / mL, and 0.94 mg / mL fluctuated very little during storage.

[0290] 4) The content determination results showed that the compound solutions with concentrations of 0.004 mg / mL, 0.071 mg / mL and 0.94 mg / mL were stable during the 24-hour storage period.

[0291] 5) Regarding related substances, the total impurities in the compound solutions with concentrations of 0.071 mg / mL and 0.94 mg / mL did not exceed 2.0% within 24 hours and remained essentially unchanged at room temperature (25±3℃) for 8 hours. However, the total impurities in the compound solution with a concentration of 0.94 mg / mL increased within 24 hours. A new impurity peak with a content of approximately 0.26-0.35% was observed in the compound solutions with concentrations of 0.071 mg / mL and 0.94 mg / mL after 8 hours and 4 hours of storage, respectively. Furthermore, a new impurity peak with a content of approximately 0.10-0.13% was observed in the compound solutions with concentrations of 0.071 mg / mL and 0.94 mg / mL after 24 hours of storage. Related substances were not determined for the low-concentration compound solution (0.004 mg / mL) because the content of AST-3434 was below the quantification limit.

[0292] 6) At concentrations of 0.071 mg / mL and 0.94 mg / mL, the ee value of the compound solution remained unchanged during the 24-hour storage period. The ee value of the low-concentration compound solution (0.004 mg / mL) was not determined because its low content did not meet the method requirements.

[0293] 7) The above results indicate that, under natural light and room temperature (25±3℃), the compound solution of AST-3424 injection (1mL:10mg preparation) and 5% glucose injection (adjusted to pH 7.4) (concentration range: 0.004mg / mL-0.94mg / mL) is best used within 8 hours.

[0294] When preparing the compound, based on the experimental conclusions above regarding the stability of AST-3424 solution under alkaline conditions, it is necessary to first adjust the pH of 5% glucose injection (pharmacopoeia specifies pH 3.2-6.5) or physiological saline (pharmacopoeia specifies pH 4.5-7.0) to 6.8-10.5 using alkaline solutions such as NaHCO3, preferably slightly alkaline, ideally 7.4. Only in this way can the subsequent preparation of concentrated AST-3424 injection solution yield a qualified injection solution for field use.

Claims

1. A stable AST-3424 injection preparation, which is a solution containing 0.1 - 200 mg / ml or 1 - 200 mg / ml of AST-3424 bulk drug substance, and the solvent of the solution contains a C2 - C8 monohydric alcohol.

2. The stable AST-3424 injection preparation according to claim 1, wherein the solvent of the solution is a liquid solvent obtained by mixing a C2 - C8 monohydric alcohol with a C2 - C8 polyhydric alcohol, and these polyhydric alcohols are preferably a liquid solvent obtained by mixing a dihydric alcohol or a trihydric alcohol and a hexahydric alcohol.

3. The stable AST-3424 injection preparation according to claim 1, wherein the solvent of the solution is a liquid or semi-liquid mixture obtained by mixing a C2 - C8 monohydric alcohol with a pharmaceutically suitable water-soluble high molecular polymer.

4. The stable AST-3424 injection preparation according to claim 3, wherein the water-soluble high molecular polymer contained in the solvent of the solution is preferably polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, Poloxamer, Polysorbate, dextran.

5. The stable AST-3424 injection preparation according to claim 2, wherein the polyhydric alcohol contained in the solvent of the solution is preferably ethylene glycol, propylene glycol, glycerol, mannitol, sorbitol.

6. The stable AST-3424 injection preparation according to claim 1, wherein, the solvent of the solution is preferably a mixture of a C2 - C4 monohydric alcohol and a C2, C3 dihydric alcohol.

7. The stable AST-3424 injection preparation according to claims 1 - 6, wherein the monohydric alcohol contained in the solvent of the solution is preferably ethanol.

8. The stable AST-3424 injection preparation according to claim 1, wherein, the solvent of the solution is a mixture of ethanol and propylene glycol.

9. The stable AST-3424 injection preparation according to claim 1, wherein, the volume ratio of the monohydric alcohol in the mixed solvent is not less than 50%.

10. The stable AST-3424 injection preparation according to claim 1, wherein, the mixed solvent is composed of 75% ethanol and 25% propylene glycol by volume.

11. The stable AST-3424 injection preparation according to claim 1, which is a solution containing 10 mg / ml of AST-3424 bulk drug substance.

12. The stable AST-3424 injection preparation according to claim 1, which does not add water, and the water content is controlled within 0.5% by mass.

13. A stable AST-3424 injection preparation, which is substantially composed of 0.75 ml of absolute ethanol, 0.25 ml of absolute propylene glycol, and 10 mg of AST-3424 bulk drug substance.

14. A stable AST-3424 injection preparation product, which includes a packaging container and the AST-3424 injection preparation contained in the container, and the injection preparation is the injection preparation described in any one of the above 1 - 13, wherein, The content of AST-3424 bulk drug in this product is 1-200 mg, preferably 10 mg and 20 mg, and it is filled in a light-resistant glass bottle made of medium borosilicate glass filled with protective gas.

15. A stable AST-3424 injection preparation product, which is essentially composed of 0.75 ml of absolute ethanol, 0.25 ml of anhydrous propylene glycol and 10 mg of AST-3424 bulk drug, and is filled in a light-resistant glass bottle made of medium borosilicate glass with a volume of 2, 5, 10 ml filled with protective gas.

16. A preparation method of a stable AST-3424 injection preparation, which includes the following operations: The AST-3424 bulk drug is first dissolved and prepared with ethanol; the prescribed amount of propylene glycol is added for the second dissolution and preparation to obtain a solution containing 0.1-200 mg / ml or 1-200 mg / ml of AST-3424 bulk drug.

17. A preparation method of a stable AST-3424 injection preparation, which includes the following operations: The AST-3424 bulk drug is first dissolved and prepared with a partial prescribed amount of ethanol; the prescribed amount of propylene glycol is added for the second dissolution and preparation; then the remaining prescribed amount of ethanol is added for mixing and dissolution to obtain a solution containing 1-200 mg / ml or 1-200 mg / ml of AST-3424 bulk drug.

18. An AST-3424 injection solution for injection, the solvent of which is water, and the solute includes AST-3424 bulk drug, an isotonicity adjusting reagent, ethanol, propylene glycol and a pH adjusting agent. The concentration of AST-3424 bulk drug in this injection solution is 0.001-1.000 mg / ml, the pH of this injection solution is 6.8-10.5, and it is an isotonic solution.

19. The AST-3424 injection solution for injection according to claim 18, the concentration of AST-3424 bulk drug in this injection solution is 0.004-0.94 mg / ml.

20. The AST-3424 injection solution for injection according to claim 18, the pH of this injection solution is 7.4-10.

5.

21. The AST-3424 injection solution for injection according to claim 18, wherein the pH adjusting agent is one or a mixture of sodium citrate, potassium 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, potassium carbonate.

22. The AST-3424 injection solution for injection according to claim 18, wherein the isotonicity adjusting reagent is sodium chloride, glucose, sucrose, fructose, xylitol, glycerol, sorbitol, mannitol, potassium chloride, mannose, calcium chloride or magnesium chloride.

23. An AST-3424 injection for intravenous injection, with water as the solvent and the solute composed of AST-3424 raw material drug, glucose, ethanol, propylene glycol, and the pH regulator sodium bicarbonate. The concentration of the AST-3424 raw material drug in this injection is 0.004 - 0.94 mg / ml, the pH of this injection is 7.4, the content of glucose in this injection is 4.5 - 5.0% by mass ratio, and it is an isotonic solution.

24. A method for preparing a stable AST-3424 injection as an injection for intravenous injection. Add an appropriate amount of sodium bicarbonate solution of the pH regulator to 5% glucose injection to make the pH value of the mixed solution 7.4; add the injection preparation in the stable AST-3424 injection preparation described in any one of claims 1 - 12 or the injection preparation product in the stable AST-3424 injection preparation described in claim 13 or 14 to the above-mentioned mixed solution and mix and dissolve to obtain.

Images