A method for preparing a liquid formulation of a taxane

Abstract

The application provides a preparation method of a taxane liquid preparation, which can effectively improve the solubility and stability of the drug by forming an inclusion compound of a taxane drug and a beta-cyclodextrin substance, can eliminate the allergic reaction and hypersensitivity reaction in clinical use and improve the drug efficacy, thereby reducing the occurrence of inflammatory reactions of patients, improving the tolerance of patients and reducing the risk of tumor metastasis, and creates conditions for increasing the drug use dosage. Through improvement on the preparation process of the taxane liquid preparation, the application can significantly improve the properties and applicability of the preparation, provides a safer, more effective and more convenient treatment option for clinic, can meet the needs of patients and medical staff, and has very great economic value and social value.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to a method for preparing a taxane liquid formulation, and also to a taxane liquid formulation prepared using the method of this invention. Background Technology

[0002] Taxanes are diterpenoids widely used in chemotherapy. They exert their anticancer activity by inhibiting mitosis through the inhibition of microtubule depolymerization in cells. Among taxanes, the most well-known are paclitaxel and its semi-synthetic analogue, docetaxel. Docetaxel has slightly better water solubility than paclitaxel, but its oral bioavailability is only 8%, so it is often prepared as an injectable formulation.

[0003] The original drug, Taxotere, uses Tween-80 for solubilization and ethanol for further solubilization. The injectable concentrate and solvent are packaged in two separate vials. The injectable concentrate is a Tween-80 solution containing the active pharmaceutical ingredient (40 mg / mL), and the solvent is 13% (w / w) ethanol for injection. Before use, the drug is removed from the refrigerator and allowed to warm to room temperature for 5 minutes. The solvent is then added to the concentrate, and the mixture is manually shaken for at least 15 seconds, followed by 5 minutes to defoam. The pre-use procedure involves numerous steps. Before use, the drug is diluted with physiological saline or 5% glucose injection, resulting in a final concentration of 0.3-0.9 mg / mL. The stability time of the diluted solution is generally less than 4 hours. On the other hand, the use of Tween-80 increases the toxicity of the injectable drug, including hypersensitivity reactions, allergic reactions, and other serious side effects. This limits the duration and completion of taxane treatment, thus limiting its effectiveness.

[0004] Chinese patent CN 111728940A discloses a taxane pharmaceutical composition without the solubilizer Tween 80. However, in actual clinical applications, it still has several shortcomings, specifically as follows: First, regarding the stability of the pharmaceutical composition after dilution, the patent claims a stability of approximately 6 hours after dilution. However, in busy clinical environments, this stability duration is often insufficient to meet practical needs. Second, the storage stability of the pharmaceutical composition is also a problem that urgently needs to be solved. A storage stability of 6 months is relatively short, which to some extent limits the long-term storage and use of the drug. In addition, although the patent mentions that using a β-cyclodextrin derivative to replace Tween 80 can reduce the occurrence of allergic reactions, the patent does not provide specific experimental data to quantify this effect. Moreover, the patent discloses a "taxane complex of a β-cyclodextrin derivative," without further explaining how the two are combined.

[0005] Chinese patent CN 113456586A discloses an injectable docetaxel composition that does not contain polysorbate or ethanol, which can reduce side effects such as allergies, irritation, addiction, and the risk of intoxication. The reconstitution stability time of the composition disclosed in this patent can reach more than 24 hours. However, since it does not use ethanol, a large amount of polyethylene glycol is required as a cosolvent. On the one hand, the reduction of ethanol reduces the solubility of docetaxel, thereby reducing its bioavailability and affecting efficacy. On the other hand, the large amount of polyethylene glycol increases the burden on the kidneys and affects the distribution of the drug in the body. Summary of the Invention

[0006] To address a series of problems encountered by taxane drugs in clinical applications, such as insufficient reconstitution stability, short storage time, and frequent allergic reactions, one objective of this invention is to provide a method for preparing a taxane liquid formulation. By forming a taxane inclusion complex, its solubility and stability can be effectively improved, thereby improving its dilution stability and storage stability. Furthermore, the encapsulation of taxane can reduce allergic reactions, thereby reducing inflammation. For patients undergoing multiple chemotherapy sessions, this can continuously reduce inflammation, lower the likelihood of developing an inflammatory constitution, and thus reduce the risk of tumor metastasis, thereby improving the safety and efficacy of the medication.

[0007] Another object of the present invention is to provide a taxane liquid formulation.

[0008] The first aspect of the present invention provides a method for preparing a taxane liquid formulation, comprising the following steps:

[0009] S1: Dissolve β-cyclodextrin in water containing alcohol to obtain a saturated aqueous solution of β-cyclodextrin, and dissolve taxane in polyethylene glycol to obtain a taxane solution;

[0010] S2: Mix the saturated aqueous solution of the β-cyclodextrin and the taxane solution to obtain a first mixed solution, then add povidone to the mixture and disperse it evenly to obtain a second mixed solution; and

[0011] S3: Remove the solids from the second mixed solution, and add water to the remaining solution until the final volume of the preparation is reached;

[0012] The weight ratio of taxane, β-cyclodextrin, polyethylene glycol, alcohol, and povidone is 1:20-80:10-25:10-35:2-10.

[0013] The preparation method provided by this invention first dissolves β-cyclodextrin in water containing alcohol to obtain a saturated aqueous solution. The addition of alcohol at this stage helps with solubility and enhances inclusion. Simultaneously, taxane is pre-dissolved in polyethylene glycol, which improves the solubility of taxane and increases subsequent inclusion efficiency. The taxane, in the form of a polyethylene glycol solution, contacts the saturated aqueous solution of β-cyclodextrin, thereby allowing the β-cyclodextrin to encapsulate the drug. The resulting inclusion complex has a high inclusion rate. Then, povidone is added and uniformly dispersed, which better utilizes the stabilizing effect of povidone and works synergistically with polyethylene glycol to further improve the stability of the formulation. In the taxane liquid formulation prepared by the method of the present invention, the taxane drug is well encapsulated in β-cyclodextrin-like substances, which can improve the water solubility of the drug and protect the drug from external environmental factors such as light, heat, and oxygen, thereby improving the chemical stability of the drug and preventing its degradation during storage and use. In addition, the formation of inclusion complexes can not only improve the stability of the drug itself, but also reduce the direct contact between the drug and the human immune system, reduce the immune system's recognition and response to the drug, and thus reduce the occurrence of allergic reactions.

[0014] In the preparation method provided by this invention, when taxane is 1 part by weight, the β-cyclodextrin can be 20-80 parts by weight depending on the properties required, for example, about 20 parts by weight, about 30 parts by weight, about 40 parts by weight, about 50 parts by weight, about 60 parts by weight, about 70 parts by weight, about 80 parts by weight, or any weight range. The polyethylene glycol can be 10-25 parts by weight depending on the properties required, for example, about 10 parts by weight, about 12 parts by weight, about 15 parts by weight, about 18 parts by weight, about 20 parts by weight, about 22 parts by weight, about 25 parts by weight, or any weight range. The alcohols can be 10 to 35 parts by weight, depending on their properties, for example, about 10 parts by weight, about 12 parts by weight, about 15 parts by weight, about 18 parts by weight, about 20 parts by weight, about 22 parts by weight, about 25 parts by weight, about 28 parts by weight, about 30 parts by weight, about 32 parts by weight, about 35 parts by weight, or any range of weight. The povidones can be 2 to 10 parts by weight, depending on their properties, for example, about 2 parts by weight, about 3 parts by weight, about 4 parts by weight, about 5 parts by weight, about 6 parts by weight, about 7 parts by weight, about 8 parts by weight, about 9 parts by weight, about 10 parts by weight, or any range of weight. In some preferred embodiments, the weight ratio of taxane, β-cyclodextrin, polyethylene glycol, alcohols, and povidones can be 1:50–80:15–25:20–35:5–10. In some preferred embodiments, the weight ratio of taxane, β-cyclodextrin, polyethylene glycol, alcohol, and povidone can be 1:50-60:15-20:20-25:5-8.

[0015] In the preparation method provided by the present invention, the taxane can be selected from common taxane drugs in the art, such as one or more of paclitaxel, docetaxel, cabazitaxel, oxalitaxel, and docetaxel.

[0016] In the preparation method provided by this invention, the β-cyclodextrin-like substance can be selected from β-cyclodextrins and their derivatives commonly found in the art, such as sulfonyl ether-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, hydroxypropyl-sulfonyl ether-β-cyclodextrin, acetyl-β-cyclodextrin, methyl-β-cyclodextrin, 2,6-dimethyl-β-cyclodextrin, and one or more of β-cyclodextrin. In some preferred embodiments, the β-cyclodextrin-like substance can be selected from sulfonyl ether-β-cyclodextrin or hydroxypropyl-sulfonyl ether-β-cyclodextrin. These β-cyclodextrin derivatives can more effectively encapsulate drugs, thereby providing better isolation.

[0017] In the preparation method provided by the present invention, the polyethylene glycol can be selected from common low molecular weight polyethylene glycols in the art, such as polyethylene glycols with a molecular weight of 200 to 2000, such as one or more of PEG 200, PEG 300, PEG 400, PEG 600 and PEG 800.

[0018] In the preparation method provided by the present invention, the alcohol can be selected from one or more of ethanol, n-propanol, isopropanol, and benzyl alcohol, which are suitable for parenteral administration.

[0019] In the preparation method provided by the present invention, the polyvinyl alcohol can be selected from common types in the art, such as one or more of PVP K12, PVP K15, PVP K17, PVP K25, PVP K30, and PVP K45.

[0020] In the preparation method provided by the present invention, the taxane liquid formulation preferably includes the following components: docetaxel, sulfobutyl ether-β-cyclodextrin, PEG 300, ethanol and PVP K12.

[0021] In the preparation method provided by this invention, the concentration of taxane in the taxane liquid formulation can be 10–100 mg / mL, for example, about 10 mg / mL, about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, or any concentration range. In some preferred embodiments, the concentration of taxane in the taxane liquid formulation can be 30–50 mg / mL.

[0022] In the preparation method provided by the present invention, step S2 may further include the following steps:

[0023] S201: At a temperature of 20–60°C (e.g., approximately 20°C, approximately 30°C, approximately 40°C, approximately 50°C, approximately 60°C, or any temperature range), a saturated aqueous solution of the β-cyclodextrin and a taxane solution are mixed and stirred to obtain the first mixed solution; and

[0024] S202: At 20–60°C (for example, about 20°C, about 30°C, about 40°C, about 50°C, about 60°C, or any temperature range), povidone is added to the first mixed solution and stirred or sonicated to disperse it evenly, to obtain the second mixed solution.

[0025] In the preparation method provided by this invention, the stirring in step S201 enables the β-cyclodextrin and taxane to come into sufficient contact to form a good inclusion complex. The stirring time can be 1–5 h, and the stirring speed can be 100–500 rpm. In some preferred embodiments, the stirring temperature can be 40–60 °C, the stirring time can be 1–3 h, and the stirring speed can be 100–300 rpm.

[0026] In the preparation method provided by this invention, the stirring or sonication in step S202 enables the povidone to be uniformly dispersed in the solution system, thereby exerting its stabilizing effect. The stirring time can be 0.5–5 h, and the stirring speed can be 100–500 rpm. In some preferred embodiments, the stirring temperature can be 40–60 °C, the stirring time can be 0.5–1 h, and the stirring speed can be 100–300 rpm.

[0027] In the preparation method provided by the present invention, in step S202, the ultrasonic time can be 10-60 min, and the ultrasonic power can be 100-500 W. In some preferred embodiments, in step S202, the ultrasonic temperature can be 30-50℃, the ultrasonic time can be 10-30 min, and the ultrasonic power can be 100-300 W.

[0028] In the preparation method provided by the present invention, ultrasonic treatment can make the polyvinylpyrrolidone more uniformly dispersed in the solution system, thus exerting a better stabilizing effect.

[0029] In the preparation method provided by this invention, in step S3, solids (e.g., large particulate impurities) in the second mixed solution can be removed by membrane filtration or centrifugation. In some preferred embodiments, the membrane filtration can use a 3-5 μm filter membrane.

[0030] In the preparation method provided by this invention, step S3 may further include: adding water to the final volume of the preparation, and adjusting its pH value to 4-5. This step is unnecessary when the pH value of the solution system is already within the range of 4-5. In some preferred embodiments, an organic acid can be used to adjust the pH value; the organic acid may be selected from one or more of citric acid, acetic acid, and phosphoric acid.

[0031] The preparation method provided by the present invention may include common process steps in the art after adjusting the pH value, such as sterile filtration, dispensing, nitrogen filling, stoppering, capping, etc.

[0032] A second aspect of the present invention provides a taxane liquid formulation prepared by any one of the above-described technical solutions. In some preferred embodiments, the taxane liquid formulation may be a taxane injection.

[0033] The technical solution provided by this invention has the following advantages:

[0034] (1) The preparation method provided by the present invention enables taxane drugs to form good inclusion complexes with β-cyclodextrins, thereby effectively improving the solubility and stability of the drug. The drug reconstitution stability time can reach 24 hours or even longer, and it is not easy to deteriorate under long-term storage. The impurity limit meets the United States Pharmacopeia (USP) standard within 24 months, which can significantly extend the effective use period of the drug.

[0035] (2) Because the taxane liquid preparation prepared by the present invention has better stability, it can effectively control impurities in the drug, thereby eliminating allergic and hypersensitivity reactions during clinical use. This not only reduces the risk that patients need to receive additional anti-allergy medication during treatment, but also reduces the complexity of treatment and the economic burden on patients. As a result, it can significantly improve the quality of life of patients and bring better efficacy. It not only helps to improve the patient's treatment experience, but also enhances the patient's compliance with the treatment plan, thereby optimizing the overall treatment effect.

[0036] (3) The preparation method of the present invention is simple, mild, easy to operate and control, and low in cost, thus having good industrial applicability.

[0037] In summary, by improving the preparation process of taxane liquid formulations, this invention can significantly improve the properties and applicability of the formulations, providing a safer, more effective, and more convenient treatment option for clinical use, and meeting the needs of patients and medical staff. Therefore, it has significant economic and social value. Attached Figure Description

[0038] Figure 1A graph showing the plasma histamine concentrations in beagle dogs after a single intravenous bolus injection of each formulation in Test Example 6.

[0039] Figure 2 A graph showing the plasma prostaglandin D2 concentration in beagle dogs after a single intravenous bolus injection of each formulation in Test Example 6.

[0040] Figure 3 Image of the tumor in the mouse in test example 7. Detailed Implementation

[0041] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0042] Unless otherwise specified, all raw materials or reagents used in the embodiments and comparative examples of this invention are commercially available products, and all water used is purified water for injection unless otherwise specified.

[0043] Unless otherwise specified, all percentages used in the embodiments and comparative examples of this invention are mass percentages.

[0044] Example 1: Preparation of Docetaxel Injection

[0045] The formulation composition of docetaxel injection is shown in Tables 1 and 2:

[0046] Table 1 Composition of Prescriptions 1 to 6

[0047] Prescription 1 Prescription 2 Prescription 3 Prescription 4 Prescription 5 Prescription 6 API(g) 0.8 0.8 0.8 0.8 0.8 0.8 SBECD(g) 16 40 64 40 40 40 PEG300(g) 12.8 12.8 12.8 8 20 12.8 PVP K12(g) 4.8 4.8 4.8 4.8 4.8 1.6 Ethanol (g) 17.6 17.6 17.6 17.6 17.6 17.6 Citric acid (mg) 4 4 4 4 4 4 WPI Up to 20mL Up to 20mL Up to 20mL Up to 20mL Up to 20mL Up to 20mL

[0048] Table 2 Composition of Prescriptions 7 to 12

[0049] Prescription 7 Prescription 8 Prescription 9 Prescription 10 Prescription 11 Prescription 12 API(g) 0.8 0.8 0.8 0.8 0.8 0.8 SBECD(g) 40 40 40 40 40 64 PEG300(g) 12.8 12.8 12.8 12.8 12.8 20 PVP K12(g) 8 4.8 4.8 4.8 4.8 8 Ethanol (g) 17.6 8 28 17.6 17.6 28 Citric acid (mg) 4 4 4 0 8 8 WPI Up to 20mL Up to 20mL Up to 20mL Up to 20mL Up to 20mL Up to 20mL

[0050] The preparation steps for docetaxel injection are as follows:

[0051] (1) Dissolve sulfobutyl ether-β-cyclodextrin (SBECD) in ethanol-containing pure water for injection to form a saturated solution.

[0052] (2) Dissolve API docetaxel in PEG300 and then slowly add it to the saturated solution of sulfobutyl ether-β-cyclodextrin obtained in step (1). Stirring is carried out during the addition process to ensure that sulfobutyl ether-β-cyclodextrin and API are fully in contact and encapsulated. The stirring speed is controlled at 100-300 rpm, the temperature is maintained at 40-60℃, and the stirring time is 1-3 hours.

[0053] (3) After stirring, add PVP K12 and continue stirring for 30-60 minutes to make PVP K12 evenly dispersed.

[0054] (4) Cool the solution to room temperature or lower, filter or centrifuge through a 3-5 μm filter membrane to remove large particulate impurities, and wash the impurities with a small amount of water or ethanol. Combine the washing liquid with the filtrate or centrifuged supernatant (the total amount of ethanol used should not exceed the prescription amount).

[0055] (5) Add pure water to the filtrate or centrifugation supernatant obtained in step (4) to obtain the required volume of inclusion complex solution. Test the pH value of the solution and add citric acid as needed to adjust the pH value of the solution to between 4 and 5.

[0056] (6) The inclusion complex solution obtained in step (5) was filtered for sterilization through microporous membranes of 0.45 μm and 0.22 μm, respectively.

[0057] (7) Fill the sterilized inclusion complex solution obtained in step (6) into ampoules or vials, fill the vials with nitrogen gas during the filling process, and seal them.

[0058] Example 2

[0059] The formulation of docetaxel injection is the same as in Example 1, and the preparation steps are as follows:

[0060] (1) Dissolve sulfobutyl ether-β-cyclodextrin in an aqueous solution containing ethanol for injection to form a saturated solution.

[0061] (2) Dissolve API docetaxel in PEG300 and then slowly add it to the saturated solution of sulfobutyl ether-β-cyclodextrin obtained in step (1). Stirring is carried out during the addition process to ensure that sulfobutyl ether-β-cyclodextrin and API are fully in contact and encapsulated. The stirring speed is controlled at 100-300 rpm, the temperature is maintained at 20-30℃, and the stirring time is 0.5-1 hour.

[0062] (3) After stirring, add PVP K12 to the mixed solution obtained in step (2), and then place it in an ultrasonic device for ultrasonic treatment. The ultrasonic power is 100-300W, the ultrasonic time is 10-30 minutes, and the temperature is controlled at 30-50℃.

[0063] (4) After ultrasonic treatment, cool the solution to room temperature or lower, filter it through a 3-5 μm filter membrane or centrifuge it to remove large particulate impurities, and wash the impurities with a small amount of water or ethanol. Combine the washing liquid with the filtrate or centrifugation supernatant (the total amount of ethanol used should not exceed the prescription amount).

[0064] (5) Add pure water to the filtrate or centrifugation supernatant obtained in step (4) to obtain the required volume of inclusion complex solution. Test the pH value of the solution and add citric acid as needed to adjust the pH value of the solution to between 4 and 5.

[0065] (6) The inclusion complex solution obtained in step (5) was filtered for sterilization through microporous membranes of 0.45 μm and 0.22 μm, respectively.

[0066] (7) Fill the sterilized inclusion complex solution obtained in step (6) into ampoules or vials, fill the vials with nitrogen gas during the filling process, and seal them.

[0067] Comparative Example 1

[0068] The preparation process differs from that in Example 2. An ethanol-free saturated aqueous solution of SBECD is prepared. Ethanol is added after the API PEG300 solution is mixed with the saturated aqueous solution of SBECD in step (2). The remaining steps are the same as in Example 2.

[0069] Comparative Example 2

[0070] Unlike the preparation process in Example 2, API is not soluble in PEG300. API and PEG300 are added to the saturated solution of sulfobutyl ether-β-cyclodextrin obtained in step (1), and the remaining steps are the same as in Example 2.

[0071] Comparative Example 3

[0072] The injection was prepared according to the preparation method disclosed in Chinese Patent CN 111728940A, paragraphs 0054-0058. The specific steps are as follows:

[0073] (1) Dissolve docetaxel in ethanol to form a docetaxel solution;

[0074] (2) PEG300 was dispersed in water for injection to form a dispersion, and sulfobutyl ether-β-cyclodextrin was dissolved in the dispersion;

[0075] (3) Disperse PVP K12 in the dispersion obtained in step (2);

[0076] (4) Combine the docetaxel solution obtained in step (1) with the dispersion obtained in step (3) to form a homogeneous solution; and

[0077] (5) Adjust the homogenized solution obtained in step (4) to the final volume with pure water for injection, test the pH value, and add an appropriate amount of citric acid as needed to adjust the pH value of the solution to between 4 and 5.

[0078] Comparative Example 4

[0079] The injection was prepared according to the preparation method disclosed in Chinese Patent CN 111728940A, paragraphs 0060-0065, and the specific steps are as follows:

[0080] (1) Combine docetaxel with a portion of ethanol and mix until docetaxel dissolves;

[0081] (2) Dissolve polyethylene glycol in water for injection;

[0082] (3) Add sulfobutyl ether-β-cyclodextrin to the polyethylene glycol / water dispersion obtained in step (2) and mix until sulfobutyl ether-β-cyclodextrin dissolves;

[0083] (4) Disperse PVP K12 in the mixture obtained in step (3);

[0084] (5) Add the remaining ethanol to the mixture obtained in step (4) and mix to form a solution; and

[0085] (6) While mixing, add the docetaxel / alcohol mixture obtained in step (1) to the solution obtained in step (5) to obtain a homogeneous solution. Test the pH value and add an appropriate amount of citric acid as needed to adjust the pH value of the solution to between 4 and 5.

[0086] Test Example 1 Inclusion Rate

[0087] The inclusion rate was tested using high-performance liquid chromatography (HPLC), as detailed below:

[0088] (1) Test Experiment Principle

[0089]

[0090] (2) Sample pretreatment

[0091] Determination of total docetaxel concentration:

[0092] Objective: To determine the total docetaxel content (free + included) in the inclusion complex.

[0093] Method: Take an appropriate amount of inclusion complex solution, add an organic solvent (such as methanol, acetonitrile) or a strong acid (such as 0.1M HCl), sonicate for 10-15 minutes to destroy the SBECD inclusion structure and release all the drug, centrifuge (10,000 rpm, 10 minutes), take the supernatant and filter it through a 0.22 μm filter membrane, and perform HPLC analysis.

[0094] Determination of free docetaxel concentration:

[0095] Objective: To determine the concentration of only the unencapsulated drug.

[0096] Method (avoiding damage to the inclusion complex):

[0097] a. Ultrafiltration:

[0098] Add the inclusion complex solution to an ultrafiltration centrifuge tube (molecular weight cutoff 2.5 kDa), centrifuge (4000 rpm, 30 minutes), collect the filtrate (containing free drug), and directly inject the filtrate into the HPLC system.

[0099] b. Dialysis:

[0100] The inclusion complex solution was placed in a dialysis bag (molecular weight cutoff 1-3 kDa) and dialyzed in pure water for 24 hours (the water was changed every 4 hours). The drug in the liquid outside the dialysis bag was the free fraction, and the concentration of the liquid outside the dialysis bag was detected by HPLC.

[0101] (3) HPLC analysis method

[0102] a. Chromatographic conditions

[0103] Chromatographic column: Reversed-phase C18 column (Agilent ZORBAX SB-C18, 250mm × 4.6mm, 5μm).

[0104] Mobile phase: acetonitrile-water (containing 0.1% phosphoric acid or 0.1% triethylamine), gradient elution (acetonitrile from 40% to 70%, 15 minutes).

[0105] Flow rate: 1.0 mL / min.

[0106] Detection wavelength: 227nm (maximum absorption wavelength of docetaxel).

[0107] Column temperature: 30℃.

[0108] Injection volume: 20 μL.

[0109] b. Preparation of standard curves

[0110] Prepare docetaxel standard solutions (concentration range: 1-50 μg / mL), inject by HPLC, and plot a standard curve with peak area versus concentration (linearity must be satisfied R2>0.999).

[0111] c. Specificity verification

[0112] Blank interference: HPLC chromatograms of excipients such as SBECD, PEG300, and ethanol were tested to confirm that they did not interfere with the docetaxel peak.

[0113] Inclusion complex destruction verification: Compare the HPLC chromatograms of the inclusion complex before and after destruction to confirm that the destruction method releases all the drug.

[0114] d. Calculation of inclusion rate

[0115] The inclusion rate is calculated using the formula described in the experimental principle.

[0116] The results of testing each formulation involved in Example 2, and the preparations obtained according to Formulation 2 using the processes described in Comparative Examples 1-4, are shown in Table 3:

[0117] Table 3. Results of Inclusion Rate Test

[0118] Prescription 1 Prescription 2 Prescription 3 Prescription 4 Prescription 5 Prescription 6 Inclusion rate 87.5％ 92.3％ 96.9％ 91.7％ 95.5％ 92.1％ Prescription 7 Prescription 8 Prescription 9 Prescription 10 Prescription 11 Prescription 12 Inclusion rate 93.3％ 90.5％ 92.8％ 92.6％ 91.6％ 96.1％ Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Inclusion rate 75.5％ 60.2％ 32.5％ 28.8％

[0119] The results in Table 3 show that all the formulations prepared according to the preparation method provided by the present invention achieved good inclusion rates, which were significantly better than those of the comparative examples. This indicates that the preparation method of the present invention can enable docetaxel and β-cyclodextrin to form stable inclusion complexes, thereby improving their properties, such as solubility and stability.

[0120] Test Example 2: Reconstitution Stability

[0121] The reconstitution stability time test method is as follows: The injection prepared in the examples is reconstituted into a solution containing approximately 0.5 mg / mL of API using 5% glucose injection or 0.9% sodium chloride injection. The solution is then visually inspected under a light to check for particulate matter precipitation or obvious opalescence. Observations are generally made every 30 minutes. The time point preceding the last observed drug precipitation time is considered the reconstitution stability time. If visible particulate matter precipitation or opalescence is observed, the product is deemed unqualified; if no visible particulate matter precipitation or opalescence is observed, the product is deemed qualified. The formulations involved in Example 2, as well as the formulations obtained according to Formulation 2 using the processes described in Comparative Examples 1-4, were tested. The results are shown in Table 4.

[0122] Table 4 Results of Reconstitution Stability Time Test

[0123]

[0124] Table 4 shows that all formulations achieved a reconstitution stability time of over 12 hours. When the PVP K12 content in the formulation reached 80 mg / mL, the reconstitution stability time could reach over 24 hours, or even longer, significantly better than the comparative example. This indicates that the present invention, through improvements in the formulation process, can significantly extend the reconstitution stability time. This good reconstitution stability allows the drug to more flexibly meet patients' medication needs in clinical applications, especially in situations where long-term advance preparation of the drug is required, ensuring the safety and efficacy of the drug during use.

[0125] Test Example 3 Long-term stability

[0126] The changes in impurities of the injection of Formulation 2 in Example 2 were tested by HPLC under long-term storage at room temperature.

[0127] The components in the sample were separated using a Waters Symmetry (3.5 μm 4.6 × 150 mm) column and gradient elution. The column temperature was 45 °C, the mobile phase A was water, the mobile phase B was acetonitrile, the flow rate was 1.2 mL / min, the injection volume was 20 μL, the solvent:acetonitrile:water = 1:1, and the detection wavelength was 232 nm.

[0128] A batch of injectable solution was prepared. Five vials were taken at each of the following time points: 0, 2, 5, 8, 12, 16, and 24 months. The state of the injection solution was observed, and the content of various impurities in each vial was measured, and the average value of each impurity was calculated. Simultaneously, three additional vials were taken, and their reconstitution stability time was examined by visual inspection following the method described in Test Example 2. The results are shown in Table 5.

[0129] Table 5. Results of Long-Term Stability Tests

[0130]

[0131] The composition of impurity AE in Table 5 is shown in Table 6:

[0132] Table 6 Comparison of Impurity Components

[0133] serial number name Impurity A 10-Deacetylbassin Impurity B 2-Debenzoyl-2-pentenyldosetaxel Impurity C 10-Oxygenated Docetaxel Impurity D 6-Oxydrated Docetaxel Impurity E 7-Table Dorset impurity F 7-Table-10-Oxygenated Docetaxel

[0134] The results in Table 5 show that the injection prepared by the method of the present invention remained a colorless and clear liquid after 24 months of storage, and the content of single impurities and total impurities both met the USP standard. This indicates that the injection prepared by the present invention has significant advantages in long-term stability.

[0135] Test Example 4 Process Comparison

[0136] Based on Formulation 2 in Example 1, three different formulation processes were used to study the long-term stability and reconstitution stability of the formulations obtained by different processes.

[0137] Process 1: The saturated aqueous solution method described in Example 1 is adopted.

[0138] Process 2: The improved method using saturated aqueous solution described in Example 2 is adopted.

[0139] Process 3: The preparation method described in Comparative Example 1 is adopted.

[0140] Process 4: The preparation method described in Comparative Example 2 is used.

[0141] Process 5: The preparation method described in Comparative Example 3 using patent CN 111728940A is employed.

[0142] The long-term stability and reconstitution stability of the formulations obtained under the five processes were tested according to Test Example 2 and Test Example 3. The results are shown in Tables 7-9:

[0143] Table 7 Results of room temperature stability tests for formulations from processes 1 and 2

[0144]

[0145] Table 8. Results of room temperature stability tests for formulations from processes 3 and 4.

[0146]

[0147]

[0148] Table 9 Results of room temperature stability test of formulation in process 5

[0149]

[0150] The results in Tables 7-9 show that the preparation method of the present invention is significantly superior to the prior art in improving drug stability. In particular, the preparation method described in Example 2 of the present invention has a better effect in improving drug stability. Even after 24 months of storage, the reconstitution stability time did not decrease significantly, and the increase in impurity content was the lowest.

[0151] Test Example 5: Guinea Pig Allergic Reaction

[0152] To verify the effectiveness of the injection prepared by the method of the present invention in avoiding hypersensitivity and allergic reactions, Hengxing Pharmaceutical was commissioned to conduct a systemic active sensitization experiment on guinea pigs.

[0153] Drug grouping:

[0154] G1: The formulation of process 1 described in Test Example 4, to be immediately reconstituted and used;

[0155] G2: Formulation of Process 2 as described in Test Example 4, to be immediately reconstituted and used;

[0156] G3: The formulation of process 3 described in Test Example 4 is to be reconstituted and used immediately;

[0157] G4: Formulation of Process 4 as described in Test Example 4, to be immediately reconstituted and used;

[0158] G5: Formulation 5 as described in Test Example 4, to be immediately reconstituted and used;

[0159] G6: The formulation of process 1 described in test example 4, after being stored at room temperature for 24 months, is used after being reconstituted for 24 hours;

[0160] G7: The formulation of process 2 described in test example 4, after being stored at room temperature for 24 months, is used after being reconstituted for 24 hours.

[0161] G8: The formulation of process 3 described in test example 4, after being stored at room temperature for 24 months, is used after being reconstituted for 24 hours;

[0162] G9: The formulation of process 4 described in test example 4 is used after being stored at room temperature for 24 months and then reconstituted for 24 hours.

[0163] G10: The formulation of process 5 described in Test Example 4, after being stored at room temperature for 24 months, is used after being reconstituted for 24 hours.

[0164] G11: Physiological saline.

[0165] The active sensitization assay in guinea pigs used 132 guinea pigs, divided into 11 groups (12 animals per group, half male and half female). The clinically recommended dose of docetaxel is 75 mg / m². 2 Based on the equivalent dose conversion between animals and humans, the dosage for guinea pigs is approximately 10 mg / kg. Animal group and dosage information are shown in Tables 10 and 11.

[0166] Table 10 Animal Groups and Dosing Information

[0167]

[0168] Table 11 Animal group and drug administration information

[0169]

[0170] Table 12 Evaluation Table of the Occurrence of Whole-Body Active Sensitization Test

[0171]

[0172] Table 13 Results of the guinea pig whole-body active sensitization experiment

[0173]

[0174]

[0175] Table 14 Results of the Active Sensitization Experiment in Guinea Pigs

[0176]

[0177] Without additional anti-allergy treatment, the results after a single intravenous administration of 10 mg / kg showed:

[0178] The negative rate for groups G1, G2, G7, and G11 was 100%, with no allergic reactions.

[0179] A small number of weak positive reactions were observed in groups G3, G4, G5, and G6.

[0180] Groups G8 and G9 had a weak positive rate of 33.33%.

[0181] The negative rate in the G10 group was only 50%, with 41.67% weakly positive and 8.33% positive reactions, and a total of 50% of the animals showed allergic reactions.

[0182] This demonstrates that the preparation method of the present invention is significantly superior to the prior art in reducing drug allergic reactions. In particular, the preparation method described in Example 2 of the present invention has better effects, and no allergic reactions were caused even after 24 months of storage and long-term reconstitution.

[0183] Test Case 6: Allergic Reaction in a Beagle

[0184] Histamine and prostaglandin D2 (PGD2) are common allergy mediators. Reducing the production of allergy mediators can improve the tumor microenvironment (TME), inhibit pro-metastatic signaling pathways, and restore anti-tumor immunity, thereby reducing the persistent inflammatory response in cancer patients and reducing the risk of tumor metastasis.

[0185] To further verify the effectiveness of the injection prepared by the method of the present invention in avoiding hypersensitivity and allergic reactions and reducing inflammation, Hengxing Pharmaceutical was commissioned to conduct an allergy test on beagle dogs to examine the changes of histamine and prostaglandin D2, two allergy markers, in healthy beagle dogs.

[0186] The clinically recommended dose of docetaxel injection is 75 mg / m². 2 Based on the equivalent dose conversion between animals and humans, the beagle dog dose is approximately 3.75 mg / kg. In previous experiments, referring to the publicly available information on docetaxel injection (Reviews for NDA020449, FDA), the docetaxel injection dosage was designed to be 1.5 mg / kg (the highest dosage for canine pharmacokinetic studies). During the administration, beagle dogs showed a strong positive allergic reaction at a dose of 1.5 mg / kg. Therefore, in this experiment, the docetaxel injection dosage was 0.5 mg / kg, which is lower than the clinical dose.

[0187] This experiment used 66 beagle dogs, divided into 11 groups (6 animals per group, half male and half female). The animal groups and drug administration information are shown in Tables 15 and 16.

[0188] Table 15 Animal Groups and Dosing Information

[0189]

[0190]

[0191] Table 16 Animal Groups and Dosing Information

[0192]

[0193] On the day of the experiment, the animals' weight was measured and recorded. The dosage of the drug administered to each animal and the time of plasma sample collection were also recorded. Animals were allowed free access to food and water during the experiment. On the day of the experiment, approximately 0.6 mL of blood was collected from the limb veins at 0.0833h, 0.167h, 0.25h, 0.5h, 1h, 2h, 4h, and 6h after drug administration (based on the start time of the infusion pump). The blood samples were placed in labeled EDTA-2K anticoagulant tubes.

[0194] All whole blood samples were placed in an ice-water bath and centrifuged for 1 hour (centrifugation conditions: 1200g, 10min, 4℃) to separate plasma. After centrifugation, each plasma sample was divided into two portions: 200μL of plasma was quantitatively transferred to test tube 1 (containing 400μL of acetonitrile containing internal standard) (the amount can be transferred proportionally according to the actual plasma volume of the sample), and the remaining plasma was placed in test tube 2. All samples were stored in a refrigerator at a temperature not higher than -60℃.

[0195] Histamine and prostaglandin D2 were extracted from plasma by protein precipitation and quantitatively analyzed using a triple quadrupole tandem mass spectrometer in multiple reaction monitoring mode. The concentrations of histamine and prostaglandin D2 in the biological samples obtained in this experiment were determined using a validated LC-MS / MS method. The detection time points for different analytes are shown in Table 17.

[0196] Table 17 Analytes and Detection Time Points

[0197] Analytes Detection tube Detection time point histamine Detection tube 2 0h, 0.0833h, 0.167h, 0.25h, 0.5h, 1h Prostaglandin D2 Detection tube 1 0h, 0.0833h, 0.167h, 0.25h, 0.5h, 1h, 2h, 4h, 6h

[0198] Histamine: The components in the sample were separated using a Sepax BR-C18 (4.6×50mm, 5μm) column and gradient elution. The mobile phase was 0.1% formic acid-10mM ammonium acetate-water and acetonitrile. The flow rate was 0.5000mL / min, and the quantification range was 0.500-200ng / mL.

[0199] Prostaglandin D2: Utilizing Welch Ultimate The components in the sample were separated using an XB-C18 (2.1×100mm, 5μm) column and gradient elution method. The mobile phase was 2mM ammonium bicarbonate-water and methanol, the flow rate was 0.5000mL / min, and the quantification range was 0.500-200ng / mL.

[0200] Test results are as follows Figure 1 and Figure 2As shown, without additional anti-allergy treatment, histamine levels in all groups of beagle dogs increased slightly 10-15 minutes after intravenous injection. This may be due to a slight change in histamine levels caused by a combined stress response. The trends in histamine concentration showed that groups G10, G9, and G8 had the highest histamine levels, indicating that formulations from processes 3, 4, and 5 with long shelf lives and reconstitution times resulted in more pronounced allergic reactions. In contrast, the histamine concentrations in groups G1-G7 were comparable and closer to the control group G11, demonstrating excellent performance in preventing allergic reactions. The results of prostaglandin D2 detection showed that no prostaglandin D2 was detected in groups G1, G2, G7, and G11. Figure 2 In the study, the curves of groups G1, G2, G7, and G11 overlapped, further verifying the excellent performance of process 2 formulation in avoiding allergic reactions due to its long shelf life and long reconstitution time.

[0201] Test Case 7: Tumor Treatment Efficacy

[0202] The in vivo therapeutic effects of the drugs in groups G1, G2, G3, G5 and G11 described in the above test cases were examined.

[0203] Six-week-old female BALB / c mice (purchased from Hunan Slack Jingda Experimental Animal Co., Ltd.) were purchased and divided into 5 groups of 4 mice each. 4T1 cell suspension (100 μL, 1×10⁻⁶ cells) was added. 6 Four T1 cells (purchased from Wuhan Shangen Biotechnology Co., Ltd., culture medium: 1640 + 10% FBS + 1% P / S) were subcutaneously injected into the back of mice. When the tumor volume in the mice reached 100 mm... 3 At that time, all mice received a single intravenous infusion of the drug at a dose of 10 mg / kg. On days 3, 7, 14, and 21 after treatment, one mouse from each group was sacrificed, and tumor masses were collected, recorded, and photographed. The results are as follows: Figure 3 As shown, groups G1 and G2 showed better tumor control, with tumors completely disappearing, indicating that the inclusion complex helps the drug release and take effect.

[0204] As demonstrated by the test examples above, the liquid formulation preparation method provided by this invention enables excellent encapsulation of taxane drugs, thereby effectively reducing impurity generation, improving drug stability, and achieving a reconstitution stability time of over 24 hours and long-term stability of up to 24 months. Furthermore, the taxane liquid formulation prepared by this invention allows patients to avoid allergic reactions without additional anti-allergy treatment, thus reducing inflammatory responses, improving efficacy to some extent, reducing the risk of tumor metastasis, and creating conditions for increasing drug dosage.

[0205] Unless otherwise specified, the terms used in this invention have the meanings commonly understood by those skilled in the art.

[0206] The embodiments described in this invention are for illustrative purposes only and are not intended to limit the scope of protection of this invention. Those skilled in the art can make various other substitutions, changes and improvements within the scope of this invention. Therefore, this invention is not limited to the above embodiments, but is only defined by the claims.

Claims

1. A method for preparing a taxane liquid formulation, characterized in that, Includes the following steps: S1: Dissolve β-cyclodextrin in water containing alcohol to obtain a saturated aqueous solution of β-cyclodextrin, and dissolve taxane in polyethylene glycol to obtain a taxane solution; S2: Mix the saturated aqueous solution of the β-cyclodextrin and the taxane solution to obtain a first mixed solution, then add povidone to the mixture and disperse it evenly to obtain a second mixed solution; and S3: Remove the solids from the second mixed solution, and add water to the remaining solution until the final volume of the preparation is reached; The weight ratio of taxane, β-cyclodextrin, polyethylene glycol, alcohol, and povidone is 1:20-80:10-25:10-35:2-10.

2. The preparation method according to claim 1, characterized in that, The weight ratio of taxane, β-cyclodextrin, polyethylene glycol, alcohol, and povidone is 1:50-80:15-25:20-35:5-10.

3. The preparation method according to claim 1 or 2, characterized in that, The taxane is selected from one or more of paclitaxel, docetaxel, cabazitaxel, oxalitaxel, and docetaxel; and / or The β-cyclodextrin is selected from one or more of sulfobutyl ether-β-cyclodextrin, hydroxypropyl-β-cyclodextrin, hydroxypropyl-sulfobutyl ether-β-cyclodextrin, acetyl-β-cyclodextrin, methyl-β-cyclodextrin, 2,6-dimethyl-β-cyclodextrin, and β-cyclodextrin; and / or The polyethylene glycol is selected from one or more of PEG 200, PEG 300, PEG 400, PEG 600, and PEG 800; and / or The alcohols are selected from one or more of ethanol, n-propanol, isopropanol, and benzyl alcohol; and / or The polyvinylpyrrolidone is selected from one or more of PVP K12, PVP K15, PVP K17, PVP K25, PVP K30, and PVP K45.

4. The preparation method according to any one of claims 1-3, characterized in that, The concentration of taxane in the taxane liquid formulation is 10–100 mg / mL.

5. The preparation method according to any one of claims 1-4, characterized in that, Step S2 further includes the following steps: S201: At 20–60°C, a saturated aqueous solution of the β-cyclodextrin and a taxane solution are mixed and stirred to obtain the first mixed solution; and S202: At 20-60°C, add povidone to the first mixed solution and stir or sonicate to disperse it evenly to obtain the second mixed solution.

6. The preparation method according to claim 5, characterized in that, In step S201, the stirring time is 1 to 5 hours and the stirring speed is 100 to 500 revolutions per minute.

7. The preparation method according to claim 5, characterized in that, In step S202, the stirring time is 0.5–5 hours, and the stirring speed is 100–500 rpm; and / or The ultrasound duration is 10–60 min, and the ultrasound power is 100–500 W.

8. The preparation method according to any one of claims 1-7, characterized in that, In step S3, solids in the second mixed solution are removed by membrane filtration or centrifugation; preferably, the membrane filtration uses a 3-5 μm filter membrane.

9. The preparation method according to any one of claims 1-8, characterized in that, Step S3 further includes: adding water to the final volume of the preparation and adjusting its pH value to 4-5; preferably, an organic acid is used to adjust the pH value, wherein the organic acid is selected from one or more of citric acid, acetic acid, and phosphoric acid.

10. A taxane liquid formulation, characterized in that, The preparation is made by any one of the preparation methods according to claims 1-9; preferably, the taxane liquid formulation is a taxane injection.

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