Busulfan nanomicelles, their lyophilized powder injections, their preparation methods, and applications

Abstract

This invention discloses a busulfan nanomicelle, its lyophilized powder for injection, its preparation method, and its applications. The busulfan nanomicelle comprises busulfan, phospholipids, bile salts, stabilizers, and protectants. The lyophilized powder for injection is prepared by freeze-drying the busulfan nanomicelle prepared according to this invention. This invention aims to reduce the in vivo toxicity of busulfan, reduce the use of organic solvents, and improve the solubility and bioavailability of busulfan by preparing nanomicelles. This invention provides a novel formulation of busulfan, namely, a lyophilized micelle formulation, which improves drug stability, reduces drug toxicity, increases drug solubility, and enhances its in vivo bioavailability.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical formulation technology, and provides busulfan nanomicelles, their lyophilized powder injection, their preparation method and application, especially in the treatment of hematological diseases and / or anti-tumor applications. Background Technology

[0002] Busulfan is a bifunctional alkylating agent in which two unstable methanesulfonate groups are attached to both ends of a four-carbon alkyl chain. In an aqueous medium, busulfan hydrolyzes to release the methanesulfonate groups. This produces reactive carbohydrate ions that can alkylate DNA. Therefore, DNA damage is considered the cause of most of busulfan's cytotoxicity. It was synthesized in 1951; in 1953, scientists Galton et al. used it for chronic myeloid leukemia and demonstrated that busulfan was more effective than radiotherapy for chronic myeloid leukemia; in 1954, GlaxoSmithKline developed Myleran tablets. ® Busulfan (Malleyan) was marketed in the United States; in 1999, the U.S. Food and Drug Administration (FDA) approved Otsuka Pharmaceutical Co., Ltd.'s Busulfan injection. ® The domestically produced Busulfan injection was approved for marketing starting in 2018; its main components are shown in Table 1 below:

[0003]

[0004] Busulfan is primarily used for the treatment of chronic myeloid leukemia in the chronic phase and as a pre-treatment regimen before allogeneic hematopoietic stem cell transplantation (busulfan in combination with cyclophosphamide). It can also be used to treat three main types of myeloproliferative neoplasms (MPNs): polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis (MF). Its pharmacokinetic behavior in vivo is as follows:

[0005] 1. Absorption: Busulfan is easily absorbed through the gastrointestinal tract and has good oral absorption, but some patients have poor bioavailability. The bioavailability of busulfan in adults is approximately 47% to 103%, and in children it is approximately 22% to 120%.

[0006] 2. Distribution: Both oral and intravenous formulations conform to a one-compartment model. Busulfan binds to plasma proteins at a rate of 30%-50% and to erythrocytes at a rate of 47%.

[0007] 3. Metabolism: Busulfan can be hydrolyzed in the body to 4-methanesulfonyloxybutanol, which then undergoes cyclization to form substances such as 4-hydroxyfuran. Its metabolism mainly occurs in the liver, where it forms a conjugate with glutathione under the catalysis of glutathione transferase and is excreted from the body. The polymorphism of the most common active form of glutathione transferase in the human body is an important factor influencing individual differences in the pharmacokinetics of oral busulfan.

[0008] 4. Excretion: 90% of the drug disappears from the blood within 2-3 minutes after intravenous injection. The vast majority of the drug is metabolized into methanesulfonic acid, which is excreted in the urine via the kidneys. However, due to slow excretion, repeated use may lead to gradual accumulation in the body.

[0009] Busulfan is recognized by the World Health Organization as a Group 1 carcinogen, ranking 23rd on the list of Group 1 carcinogens due to its drug side effects. However, it is only likely to be carcinogenic when used in large quantities over a long period of time, and the carcinogenic effect varies from person to person (individual differences).

[0010] Busulfan has serious adverse reactions, mainly including:

[0011] 1. Bone marrow suppression: The most common side effect of busulfan is bone marrow suppression. Excessive dosage or prolonged use may cause long-term bone marrow suppression. During treatment, blood cell counts should be monitored, and the medication should be temporarily discontinued if the blood cell count drops sharply.

[0012] 2. May trigger epilepsy: Because busulfan is a highly lipophilic small molecule, it can be rapidly distributed to various tissues and easily cross the blood-brain barrier, which may cause some patients to experience epilepsy when using busulfan. Precautions are necessary.

[0013] 3. Potential side effects include cataracts, gonadal atrophy, infertility, and hepatic vein obstruction syndrome: Because busulfan is metabolized slowly in the body, excessive accumulation of the drug may lead to all of the above side effects. When using busulfan, attention should be paid to the drug clearance rate, and efforts should be made to accelerate the metabolism of busulfan in the body. Patients can reduce the dosage of busulfan or take other medications to accelerate metabolism; specific assessment by the attending physician is required.

[0014] Nanomicelles utilize micelle solubilization to improve the solubility and oral bioavailability of poorly soluble drugs. Based on the relative molecular mass of the constituent carrier materials, nanomicelles can be classified into low-molecular-weight micelles and polymeric micelles. They not only significantly improve the solubility of poorly soluble drugs but also enhance drug efficacy and stability, making them highly biocompatible drug delivery carriers.

[0015] Currently, among the reported carrier materials for polymers, commonly used ones include ethylene oxide-based polymers such as Pluronic, which are characterized by good biocompatibility and safety. Studies have shown that combining polyvinylpyrrolidone (PVP) with low-molecular-weight binary bile salt / phospholipid polymeric micelles using physical methods to construct PPVP-phospholipid-cholate compositions, which, as carriers for poorly soluble drugs, exhibit good solubilizing effects. Furthermore, due to their small particle size and large molecular weight, polymer micelles are not phagocytosed by the endothelial reticulum system or effectively avoid renal excretion, thus allowing them to remain in the bloodstream for a longer period. Simultaneously, because micelle systems have good tissue permeability, especially in tissues with leaky blood vessels (such as tumors or infarcted areas), micelle drug delivery systems possess natural passive targeting properties.

[0016] In recent years, there has been an increasing number of studies on structural modification or attachment of other targeting groups to polymer micelles to increase drug loading, stability and targeting effect. More and more receptor-mediated and physicochemically targeted micelle drug delivery systems are being used in the development of drugs for the treatment of diseases such as tumors.

[0017] Currently, there is no technology for preparing nanomicelle formulations using busulfan as a raw material, which has significant practical implications for safe clinical applications. Summary of the Invention

[0018] Based on the aforementioned technical objectives, this invention provides busulfan nanomicelles, their lyophilized powder for injection, their preparation method, and applications, aiming to reduce the in vivo toxicity of busulfan, decrease the use of organic solvents, and improve the solubility and bioavailability of busulfan. Existing commercially available busulfan formulations are conventional liquid injections, which use dimethylacetamide and polyethylene glycol 400 as solubilizers and co-solvents, respectively. The raw materials themselves have high toxicity and side effects, and the use of organic solvents such as solubilizers and co-solvents exacerbates the harm to the user's body. This invention provides a new formulation, namely, a micelle lyophilized formulation, which improves drug stability, reduces drug toxicity, increases drug solubility, and enhances its in vivo bioavailability.

[0019] In a first aspect, the present invention provides a busulfan nanomicelle comprising busulfan, phospholipids, bile salts, stabilizers, and protectants.

[0020] In some embodiments, the stabilizer is at least one of polyvinylpyrrolidone (the type of polyvinylpyrrolidone may be at least one of C30, K15, K29 / 32, and K60, preferably K29 / 32), polyvinyl alcohol, polyethylene glycol, diethanolamine, ferric chloride, inositol, sodium gluconate, creatine, glycerol, nicotinamide, sodium saccharin, sodium caprylate, arginine, methionine, and cysteine.

[0021] In some embodiments, the protective agent is at least one selected from mannitol, glucose, sucrose, maltose, galactose, trehalose, and glycine. In some embodiments, the protective agent is preferably mannitol, specifically a freeze-drying grade mannitol with a purity of ≥98% and a particle size range of D. 90 The particle size is preferably between 3 μm and 50 μm, with a purity >99% and a particle size range D. 90 <10 μm.

[0022] Stabilizers and protectants can modify the surface of micelles to increase their stability.

[0023] In some embodiments, the busulfan nanomicelles, based on 1 part by weight of busulfan, contain 1.75 to 3.55 parts by weight of phospholipids, 1.3 to 3.55 parts by weight of bile salts (preferably 1.75 to 3.55), 0.78 to 2.2 parts by weight of stabilizer, and 1.2 to 3.6 parts by weight of protectant. Optimized weight proportions facilitate control of micelle size; bile salts have a solubilizing effect and can also reduce micelle size; phospholipids provide structural rigidity, which is beneficial for encapsulating busulfan.

[0024] In some embodiments, the busulfan nanomicelles contain, based on 1 part by weight of busulfan, 2.17-2.5 parts by weight of phospholipids, 1.91-2.2 parts by weight of bile salts, 0.78-2.2 parts by weight of stabilizer, and 1.2-3.6 parts by weight of protectant.

[0025] In some embodiments, the phospholipid is selected from at least one of soybean phospholipids, lecithin, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA), and sphingomyelin (SM), preferably lecithin. The amount of phospholipid present in the nanomicelles can vary and depends on the nature and type of the drug and the amount of drug to be administered, the nature and type of the phospholipid, and the type of interaction between the drug and the phospholipid molecules in the aqueous phase.

[0026] In some embodiments, the cholate is selected from any or a combination of sodium taurocholate, sodium glycocholate, and sodium deoxycholate, preferably sodium taurocholate.

[0027] In some embodiments, the particle size range D of the sulfanilamide nanomicelles is... 90The preferred nanoparticle size range is 10 nm to 30 nm, with D being the most desirable. 90 The wavelength range is 10 nm to 20 nm.

[0028] Secondly, the present invention provides a method for preparing sulfan nanomicelles, comprising the following steps:

[0029] Step 1: Mix busulfan, phospholipids and solvent to obtain the first mixture;

[0030] Step 2: Mix bile salts, stabilizer, protectant and water for injection to obtain a second mixture;

[0031] Step 3: Add the first mixed solution to the second mixed solution, homogenize the solution, remove the solvent by evaporation, filter, adjust the volume, and fill to obtain sulfan nano micelles or their solvates.

[0032] In some embodiments, the solvent is selected from at least one of ethyl acetate, ethanol, chloroform, methanol, acetone, and dichloromethane.

[0033] In some embodiments, the homogenization includes at least one of ultrasonic homogenization and high-pressure homogenization.

[0034] In some embodiments, the ultrasonic homogenization time is 10-45 min, preferably 10, 15, 20, 25, 30, 35, 40, or 45 min.

[0035] In some embodiments, the frequency of the ultrasonic homogenization is 10W-1000W, preferably 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000W.

[0036] In some embodiments, the high-pressure homogenization is performed using a high-pressure homogenizer, with a homogenization pressure of 500 bar to 1500 bar, preferably 850 bar, and the number of homogenization cycles is 3 to 7, preferably 4. In some embodiments, the feed rate of the solution during homogenization is 50 mL / min to 300 mL / min.

[0037] Homogenization by ultrasound or high pressure can overcome the energy barrier, promote the formation of nanoscale micelles, effectively optimize the particle size distribution, and facilitate the acquisition of uniform nanomicelles.

[0038] In some embodiments, the temperature of the solution in step 3 is controlled at 5 °C to 45 °C, preferably 25 °C to 35 °C.

[0039] In some embodiments, the solvent removal is carried out by vacuum rotary evaporation concentration at a temperature of 40°C to 50°C.

[0040] In some embodiments, the filtration removes insoluble particles and bacteria. In some embodiments, the filtration is selected from at least one of the following methods: 0.45 µm organic membrane pre-filtration, 0.22 µm organic membrane redundant filtration, and 0.22 µm organic membrane sterilization filtration.

[0041] In some embodiments, the medium for volume adjustment is water for injection, and the volume adjustment concentration is 6 mg / mL.

[0042] In some embodiments, the filling container is a borosilicate glass vial for injection (hereinafter referred to as a vial), a chlorinated butyl rubber stopper with a polytetrafluoroethylene / ethylene copolymer film for injection, and an aluminum-plastic composite cap, and the filling volume is 10 mL to 30 mL, preferably 10 mL.

[0043] Thirdly, the present invention provides a lyophilized nanomicelle powder, comprising the lyophilized nanomicelles or their solvates described in the present invention.

[0044] In some embodiments, the lyophilized sodium bicarbonate nanomicelle powder is prepared by freeze-drying the sodium bicarbonate nanomicelles or their solvates described in this invention.

[0045] In some embodiments, the freeze-drying method includes a pre-freezing stage, a sublimation stage, and a desorption stage.

[0046] The pre-freezing stage effectively removes water from busulfan nanomicelles or their solvates, the sublimation stage effectively removes free water, and the desorption stage removes bound water, thus gradually reducing the mechanical damage to the micelle structure caused by ice crystals. The freeze-dried powder prepared by the staged freeze-drying method is conducive to rapid restoration of the original nano-morphology (particle size and PDI remain basically unchanged) after subsequent reconstitution.

[0047] Fourthly, the present invention provides a method for preparing sulfadiazine nanomicelle lyophilized powder, comprising the following steps:

[0048] Step S1: Pre-freezing stage, after holding the sulfanilamide nanomicelles or their solvates at a first predetermined temperature for a first predetermined time, the temperature is controlled to a second predetermined temperature and held for a second predetermined time to obtain a frozen sample;

[0049] Step S2: Sublimation stage, control the plate temperature to not exceed 8°C, control the temperature of the frozen sample to the third predetermined temperature, maintain it for the third predetermined time, and control the first vacuum degree; after reaching the third predetermined time, raise the temperature to the fourth predetermined temperature, maintain it for the fourth predetermined time, and control the second vacuum degree; obtain the sublimated sample;

[0050] Step S3: Desorption stage: After reaching the fourth predetermined time, the temperature of the sublimation sample is raised from the fourth predetermined temperature to the fifth predetermined temperature and maintained for the fifth predetermined time; after reaching the fifth predetermined time, the fifth predetermined temperature is raised to the sixth predetermined temperature, the vacuum degree is maintained at the second vacuum degree, and maintained for the sixth predetermined time, requiring the moisture content to reach less than 1.0%, and the lyophilized nano micelle powder is obtained.

[0051] In some embodiments, the first predetermined temperature is 2°C to 5°C, preferably 3°C.

[0052] In some embodiments, the first predetermined time is 1 h to 3 h, preferably 2 h.

[0053] In some embodiments, the second predetermined temperature is -45 ℃ to -35 ℃, preferably -40 ℃.

[0054] In some embodiments, the second predetermined time is 4 h to 6 h, preferably 4.5 h.

[0055] In some embodiments, the third predetermined temperature is -60 ℃ to -45 ℃, preferably -53 ℃.

[0056] In some embodiments, the third predetermined time is 20 h to 24 h, preferably 22 h.

[0057] In some embodiments, the first vacuum level is 0.1 mbar to 0.2 mbar, preferably 0.18 mbar.

[0058] In some embodiments, the fourth predetermined temperature in step S2 is -45 ℃ to -30 ℃, preferably 36 ℃.

[0059] In some embodiments, the fourth predetermined time in step S2 is 16 h to 20 h, preferably 18.5 h.

[0060] In some embodiments, the second vacuum level in step S2 is 0.3 mbar to 0.6 mbar, preferably 0.5 mbar.

[0061] In some embodiments, the fifth predetermined temperature in step S3 is 18 ℃ ± 2 ℃.

[0062] In some embodiments, the fifth predetermined time in step S3 is 3 h to 5 h, preferably 4 h.

[0063] In some embodiments, the sixth predetermined temperature in step S3 is 30 ℃ ± 4 ℃.

[0064] In some embodiments, the sixth predetermined time in step S3 is 1 h to 2 h, preferably 1 h.

[0065] Fifthly, the present invention provides the application of the busulfan nanomicelles or busulfan nanomicelle lyophilized powder described herein in the preparation of drugs for related diseases.

[0066] The relevant diseases include hematologic disorders and / or neoplastic diseases, selected from at least one of myeloproliferative neoplasms (MPNs): polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis (MF).

[0067] This invention leverages the self-assembly properties of amphiphilic molecules—phospholipids and bile salts—combined with the synergistic effect of the specific stabilizer polyvinylpyrrolidone and the protectant mannitol, to form core-shell structured nanoparticles encapsulating busulfan. Both phospholipids and bile salts possess hydrophobic tails and hydrophilic heads. In water for injection, through hydrophobic interactions and hydrogen bonding between water molecules via the hydrophilic heads, a thermodynamically stable micelle structure spontaneously forms. Busulfan, as a hydrophobic drug, is encapsulated within the hydrophobic region of the micelle, preventing water contact. The hydrophilic heads of the phospholipids and bile salts align outwards, forming a hydration layer that ensures the water solubility and stability of the micelles. The stabilizer adsorbs onto the micelle surface or embeds itself in the phospholipid layer, reducing interfacial tension and improving physical stability. The protectant forms an exfoliated matrix during lyophilization or storage, preventing micelle collapse and protecting busulfan from degradation.

[0068] Beneficial effects

[0069] 1. Existing busulfan formulations are conventional aqueous injections. This new formulation is prepared using micellar lyophilization. The route of administration remains unchanged. The lyophilized powder is reconstituted with sodium chloride injection before clinical administration. This formulation improves the stability of busulfan, reduces its toxicity, decreases the use of organic solvents, increases drug solubility, and enhances its bioavailability. Compared to existing technologies that use organic solvents to prepare aqueous injections, this invention effectively reduces toxicity by avoiding the use of toxic solvents in the micellar lyophilization process.

[0070] 2. In the preparation process of the nanomicelles of this invention, a high-pressure homogenization method is used, along with reasonable formulation steps and formulation, to replace N,N-dimethylacetamide (DMA) and polyethylene glycol 400, which are originally required for highly toxic injections, with low-toxicity or non-toxic excipients such as phospholipids and polyvinylpyrrolidone. In the preparation of the nanomicelle lyophilized powder, this invention reduces the use of organic reagents while adjusting the lyophilization technology to reduce the toxicity of busulfan and lower the incidence of adverse reactions. The nanomicelles and their lyophilized powder system mentioned in this invention improve the bioavailability of busulfan, reduce the use of organic solvents, lower toxicity to humans, and increase bioavailability in vivo. The preparation method is simple, and the micelles are stable, facilitating large-scale industrial production.

[0071] 3. The formula and method used in this invention are simple and feasible, require no special equipment, and are easy to mass-produce industrially. Attached Figure Description

[0072] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0073] Figure 1 This is a real photo of the lyophilized nano micelle powder of bleach.

[0074] Terminology Explanation

[0075] Certain embodiments of the invention will now be described in detail, examples of which are illustrated by the accompanying structural and chemical formulas. The invention is intended to cover all alternatives, modifications, and equivalents, all of which are included within the scope of the invention as defined in the claims. Those skilled in the art will recognize that many similar or equivalent methods and materials can be used to practice the invention. The invention is by no means limited to the methods and materials described herein. In the event that one or more of the incorporated documents, patents, and similar materials differ from or contradict this application (including, but not limited to, defined terminology, application of terminology, described techniques, etc.), this application shall prevail.

[0076] It should be further appreciated that certain features of the invention, for clarity, have been described in multiple independent embodiments, but may also be provided in combination in a single embodiment. Conversely, various features of the invention, for brevity, have been described in a single embodiment, but may also be provided individually or in any suitable sub-combination.

[0077] Unless otherwise stated, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. All patents and publications related to this invention are incorporated herein by reference in their entirety.

[0078] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0079] In the following content, all numbers disclosed herein, whether or not they use words such as "approximately" or "about," are approximate values. The value of each number may vary by 1%, 2%, 5%, 7%, 8%, 10%, 15%, or 20%. Whenever a number with a value of N is disclosed, any numbers with values ​​of N+ / -1%, N+ / -2%, N+ / -3%, N+ / -5%, N+ / -7%, N+ / -8%, N+ / -10%, N+ / -15%, or N+ / -20% will be explicitly disclosed, where "+ / -" indicates addition or subtraction.

[0080] "3M" stands for 3 Months. Detailed Implementation

[0081] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0082] All reagents used in this invention can be purchased commercially or prepared by the methods described in this invention.

[0083] The preparation method of the lyophilized nanomicelle powder of the present invention includes the following steps:

[0084] S1: Dissolve 1 part by weight of sulfadiazine and 1.75 ~ 3.55 parts by weight of phospholipids in an appropriate amount of acetone to obtain the first mixture;

[0085] S2: 0.78 ~ 2.2 parts by weight of polyvinylpyrrolidone, 1.3 ~ 3.2 (preferably 1.75 ~ 3.55) parts by weight of bile salt, and 1.2 ~ 3.6 parts by weight of mannitol are mixed with water for injection to obtain a second mixture; the preferred proportions of the materials in S1 and S2 are as follows: based on 1 part by weight of busulfan, the proportions of phospholipids are 2.17 ~ 2.5, the proportions of bile salt are 1.91 ~ 2.2, the proportions of stabilizer are 0.78 ~ 2.2, and the proportions of protectant are 1.2 ~ 3.6.

[0086] S3: Add the first mixture to the second mixture and sonicate for 10 min to 45 min, preferably 30 min; the sonication frequency is 10W-1000W;

[0087] S4: High-pressure homogenization, with a homogenization pressure of 500 bar ~ 1500 bar, preferably 850 bar, and the number of high-pressure homogenization cycles is 3 ~ 7, preferably 4. The temperature control range of the drug solution is 5 °C ~ 45 °C, preferably 25 °C ~ 35 °C, and the feed rate is 50 mL / min ~ 300 mL / min.

[0088] S5: Remove acetone by rotary evaporation under reduced pressure at a temperature of 40℃ ~ 50℃;

[0089] S6: Filtration, consisting of three filtration stages: 0.45 µm organic membrane pre-filtration, 0.22 µm organic membrane redundant filtration, and 0.22 µm organic membrane sterilization filtration.

[0090] S7: Adjust the volume with water for injection to a concentration of 6 mg / mL;

[0091] S8: Filling, using borosilicate glass vials, chlorinated butyl rubber stoppers coated with polytetrafluoroethylene / ethylene copolymer film for injection, and aluminum-plastic composite caps for combined filling, with a filling volume of 10 mL to 30 mL, preferably 10 mL, to obtain sulfanilamide nanomicelles or their solvates, with a nanoparticle size range D90 of 10 nm to 30 nm, preferably 10 nm to 20 nm;

[0092] S9: Place the sulfanilamide nanomicelles or their solvates from step S8 into a freeze dryer for freeze drying, which consists of three stages: pre-freezing, sublimation, and desorption drying.

[0093] Pre-freezing stage: After the equipment is turned on, the temperature is lowered. Vials containing medicine are placed into the chamber at 2℃ ~ 5℃. After the contents are placed into the chamber, the temperature is maintained for 1h ~ 3h. Then, the temperature is lowered from 2℃ ~ 5℃ to -45℃ ~ -35℃ and maintained for 4h ~ 6h. The pre-freezing stage is then complete.

[0094] Sublimation stage: The plate temperature is controlled at less than 8 ℃, so that the temperature of the pre-frozen vials is maintained at -60℃ ~ -45℃. This condition is maintained for 20 h ~ 24 h, and the vacuum degree is controlled at 0.1 mbar ~ 0.2 mbar. After that, the temperature is raised to -45℃ ~ -30℃, and this condition is maintained for 16 h ~ 20 h, and the vacuum degree is controlled at 0.3 mbar ~ 0.6 mbar.

[0095] Desorption stage: The temperature is raised to 18 ℃ ± 2 ℃ and maintained for 3 h ~ 5 h. Then the temperature is raised to 30 ℃ ± 4 ℃ and the vacuum degree is kept constant. This process is continued for 1 h ~ 2 h, and the moisture content is required to be below 1.0%.

[0096] The resulting product is a lyophilized nanomicelle powder.

[0097] Example 1: White fungicide nanomicelle freeze-dried powder

[0098] Preparation steps of sulfanilamide nanomicelles:

[0099] 1 part by weight of busulfan and 2.17 parts by weight of phospholipids were dissolved in 55 mL of acetone to obtain the first mixture; 1.04 parts by weight of polyvinylpyrrolidone, 1.91 parts by weight of cholate, and 1.74 parts by weight of mannitol were mixed with 50 mL of water for injection to obtain the second mixture; the first mixture was added to the second mixture and sonicated for 30 min at a frequency of 500 W.

[0100] Add to a high-pressure homogenizer (APV Gaulin 1000) for high-pressure homogenization, set to 850 bar, control the temperature of the drug solution within 25 °C, feed rate at 115 mL / min, and cycle 4 times;

[0101] Acetone was removed by rotary evaporation under reduced pressure in a 45℃ water bath; the remaining solution after evaporation was passed sequentially through a 0.45 µm organic filter membrane, a 0.22 µm organic filter membrane, and a 0.22 µm organic filter membrane; the filtrate was added to a 1000 mL sterile volumetric flask and diluted to 1000 mL with water for injection, and the concentration was measured to be 6.19 mg / mL.

[0102] Accurately dispense 10 mL of the solution into a 20 mL borosilicate glass vial for injection, add a chlorobutyl rubber stopper with a polytetrafluoroethylene / ethylene copolymer membrane, and determine the nanoparticle size range D. 90It is 15.7 nm.

[0103] Freeze-dried powder preparation steps:

[0104] The above samples were placed in a freeze dryer at 3°C ​​and pre-frozen for 1.5 h. Then, the temperature was lowered from 3°C to -40°C and maintained for 4.5 h. The plate temperature was controlled at 5°C to maintain the temperature of the pre-frozen samples at -53°C. This condition was maintained for 22 h with the vacuum degree controlled at 0.18 mbar. The temperature was then raised to -36°C and maintained for 18.5 h with the vacuum degree controlled at 0.5 mbar. The temperature was then raised to 18°C ​​and maintained for 4 h. The temperature was then raised to 30°C with the vacuum degree remaining unchanged. This condition was maintained for another h, and the moisture content was measured to be 0.79%.

[0105] The self-made formulation was obtained by following the steps described above using the formula in Table 2.

[0106] Table 2 Prescription information table used in Example 1

[0107]

[0108] Examples 2-9:

[0109] The preparation process was the same as in Example 1. Using 6.9g of busulfan as one unit, the mass fractions of each component in the example are shown in Table 3 below:

[0110] Table 3. Component proportions for each embodiment

[0111]

[0112] Examples 10-11

[0113] The formulations of Examples 10-11 differ from those of Example 1 in that the preparation process and parameters were modified according to the preparation process of Example 1. The parameter changes are shown in Table 4 below.

[0114] Table 4. Table of Changed Preparation Process Parameters

[0115]

[0116] Comparative Examples 1-4

[0117] The preparation processes of Comparative Examples 1-4 were the same as those in Example 1, except that, taking 6.9g of busulfan as one part, the mass fractions of each component in the comparative examples were calculated as shown in Table 5 below:

[0118] Table 5. Distribution ratios of each group in the comparative example.

[0119]

[0120] Comparative Example 5

[0121] The difference from Example 1 is that the sample of sulfanilamide nanomicelles was directly freeze-dried at -53°C for 27 hours with the vacuum level controlled at 0.18 mbar. Then the temperature was raised to 18°C ​​and maintained for 4 hours. The temperature was then raised to 30°C while maintaining the vacuum level, and this process was continued for 1 hour. This yielded lyophilized sulfanilamide nanomicelle powder.

[0122] Example 1

[0123] The busulfan formulations prepared in the examples and comparative examples were subjected to accelerated stability testing for 3 months (3M), as shown in Tables 6 and 7 below:

[0124] Table 6 Accelerated 3M Stability Data Table of Examples

[0125]

[0126] Table 7. Accelerated 3M Stability Data for Comparative Examples

[0127]

[0128] A comparison of Tables 6 and 7 shows that the phospholipid content in Comparative Examples 1 and 2 exceeds the scope of patent protection, resulting in excessive content and total impurity content in the prepared lyophilized powder. In Comparative Examples 3 and 4, sodium cholate exceeds the protection scope, leading to excessive total impurities in the resulting lyophilized powder. Comparative Example 5 did not employ a segmented lyophilization method, resulting in excessive moisture content in the lyophilized powder. After 3 months, the maximum single impurity and total impurities in the related substances exceeded the limits, indicating poor stability. The appearance, pH, particle size, labeled content, and total impurities of the samples from Examples 1-10 did not show significant changes. These results demonstrate that the micelle lyophilized powders of Examples 1-10 of this invention exhibit good stability.

[0129] Example 2: Particle size and zeta potential of the prepared nanomicelles were measured.

[0130] method:

[0131] (1) Particle size determination: The nano micelles prepared in the example of this application were diluted with 0.9% sodium chloride injection and their particle size was determined by laser particle size analyzer (Malvin 3000 laser particle size analyzer).

[0132] (2) ζ potential determination: The potential of the busulfan nanomicelles prepared in the example of this application was measured by ELS method after being appropriately diluted with 0.9% sodium chloride injection. The potential was measured by Malvern zetasizer.

[0133] The results are shown in Table 8 below.

[0134] Table 8. Size and potential of white smoke-containing nanomicelles

[0135]

[0136] In the notes, "mean±SD" means "mean ± SD".

[0137] The zeta potential is negative and has a large absolute value, indicating that the particle surface carries a large amount of negative charge. This usually means that the particles will exhibit strong electrostatic repulsion in the solution, thus helping to maintain the stability of the colloid. As can be seen from the results in Table 8, the micelle lyophilized powders prepared in Examples 1-10 of this invention have better stability compared to the comparative examples.

[0138] Example 3: Relative Bioavailability Experiment

[0139] Experimental reagents: the lyophilized nanomicelle powder prepared in this application and commercially available injection solutions.

[0140] Drug processing: Reconstitute the nano-micelle lyophilized powder with 0.9% sodium chloride injection; commercially available injection solutions can be used directly.

[0141] Animal drug administration and blood sample processing

[0142] Thirty-six healthy SD rats, half male and half female, four weeks old, were randomly divided into six groups of six each. The rats were fasted for 8 hours before administration of the drug, but had free access to water.

[0143] The relevant drug was injected via the tail vein. At 0.16, 0.33, 0.5, 0.67, 0.83, 1, 1.5, 2, 3, 4, 6, 8, 12, and 24 hours post-administration, approximately 0.1 mL of blood was collected from the posterior ocular vein of the rat and placed in heparin anticoagulant tubes. The blood was then incubated at 4000 r·min. -1 After centrifugation for 10 min, separate the plasma and store it frozen at -20°C until analysis. Take 60 μL of plasma and place it in a 5 mL stoppered centrifuge tube, add 150 μL of water for injection, and add 50 μL of internal standard solution (10 μg∙mL⁻¹). -1N-(2,6-difluorobenzoyl)-N'-[3,5-dichloro-4-(3-chloro-5-trifluoromethylpyridin-2-oxy)phenyl]urea (methanol solution), 50 μL of 5% DEDC solution, followed by 120 μL of ethanol, vortexed for 10 min, the supernatant was transferred to a clean centrifuge tube and dried under nitrogen in a 37°C water bath. The residue was dissolved in 100 μL of methanol and vortexed for 1 min, centrifuged at 3000 rpm for 5 min, and the solid-phase extraction column (pre-treated with 1 mL × 3 of methanol and 1 mL × 2 of water) was washed with 1 mL × 2 of 50% methanol, and the sample was eluted with 2.5 μL × 2 of methanol in a test tube. 20 μL of the supernatant was injected into the HPLC for analysis.

[0144] The chromatographic conditions are as follows:

[0145] Chromatographic column: MicroPak-SP-Cx column (15 cm × 4.0 mm, 3.0 μm)

[0146] Mobile phase: Acetonitrile-water-tetrahydrofuran (55:25:20)

[0147] Flow rate: 0.8 mL / min

[0148] Detection: UV, 278 nm.

[0149] Plasma drug-time curve and relative bioavailability

[0150] Pharmacokinetic parameters were presented as mean ± SD, and the results are shown in Table 9.

[0151] Table 9 Pharmacokinetic parameters

[0152]

[0153] As can be seen from the results in Table 9, the bioavailability of Examples 1 and 3 of this application is increased by 2.3 times compared with commercially available injectable solutions.

[0154] The method of this invention has been described through preferred embodiments. Those skilled in the art will readily be able to modify or appropriately alter and combine the methods and applications described herein within the scope, spirit, and context of this invention to implement and apply the technology of this invention. Those skilled in the art can refer to the content herein to appropriately improve process parameters. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of this invention.

Claims

1. A busulfan nanomicelle, characterized in that, including busulfan, phospholipid, cholate, stabilizer and protective agent; In the busulfan nanomicelles, the amount of busulfan is 1 part by weight, and the mass of phospholipid is 1.75-3.55, the mass of cholate is 1.3-3.55, the mass of stabilizer is 0.78-2.2, and the mass of protective agent is 1.2-3.6; The busulfan nanomicelles are in the form of micelle lyophilized preparation; The particle size range D of the busulfan nanomicelles is between 10 nm and 30 nm. 90 10 nm ~ 30 nm, The phospholipid is at least one selected from soybean phospholipid, lecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, and sphingomyelin; The cholate is sodium taurocholate; The stabilizer is polyvinylpyrrolidone, and the type of polyvinylpyrrolidone is at least one selected from C30, K15, K29 / 32, and K60; The protective agent is mannitol; The preparation method of the busulfan nanomicelles comprises the following steps: Step 1: mixing busulfan, phospholipid, and solvent to obtain a first mixed solution; Step 2: mixing cholate, stabilizer, protective agent, and water for injection to obtain a second mixed solution; Step 3: adding the first mixed solution to the second mixed solution, and then performing high-pressure homogenization on the obtained solution, and then removing the solvent by evaporation, filtering, constant volume, and filling to obtain busulfan nanomicelles or solvate thereof; the high-pressure homogenization is performed by a high-pressure homogenizer, the homogenization pressure is 500 bar-1500 bar, the homogenization cycle number is 3-7 times, and the feeding speed of the solution during homogenization is 50 mL / min-300 mL / min; Step 4: placing the busulfan nanomicelles or solvate thereof into a freeze dryer for freeze drying; the freeze drying comprises the following steps: Step S1: pre-freezing stage, keeping the busulfan nanomicelles or solvate thereof at 2 ℃-5 ℃ for 1 h-3 h, controlling the temperature to-45 ℃--35 ℃, and keeping for 4 h-6 h to obtain a frozen sample; Step S2: sublimation stage, controlling the plate layer temperature to be not more than 8 ℃, controlling the temperature of the frozen sample to-60 ℃--45 ℃, keeping for 20 h-24 h, and controlling the vacuum degree to be 0.1 mbar-0.2 mbar; after reaching the predetermined time, the temperature is increased to-45 ℃--30 ℃, keeping for 16 h-20 h, and the vacuum degree is controlled to be 0.3 mbar-0.6 mbar; a sublimated sample is obtained; Step S3: desorption stage: after reaching the predetermined time, the temperature of the sublimated sample is increased to 18 ℃ ± 2 ℃, keeping for 3 h-5 h; after reaching the predetermined time, the temperature is increased to 30 ℃ ± 4 ℃, the vacuum degree is kept unchanged, keeping for 1 h-2 h, and the moisture content is required to be less than 1.0%, to obtain busulfan nanomicelle lyophilized powder.

2. The nanoscale bilosomes according to claim 1, wherein, The phospholipid is lecithin; The cholate is sodium taurocholate; The protective agent is mannitol.

3. The busulfan nanomicelles according to claim 1, wherein The mass of the phospholipid is 2.17-2.5, the mass of the cholic acid salt is 1.91-2.2, the mass of the stabilizer is 0.78-2.2, and the mass of the protective agent is 1.2-3.6, based on 1 part by weight of busulfan.

4. The nanoscale bilosomes according to claim 1, wherein, The particle size range D of the busulfan nanomicelles is between 10 nm and 20 nm. 90 is between 10 nm and 20 nm.

5. A method of preparing the nanosize busulfan micelles of claim 1, comprising: The method comprises the following steps: Step 1: mixing busulfan, phospholipid and solvent to obtain a first mixed solution; Step 2: mixing cholic acid salt, stabilizer, protective agent and water for injection to obtain a second mixed solution; Step 3: adding the first mixed solution into the second mixed solution, and then performing high-pressure homogenization on the obtained solution, and then removing the solvent from the solution by evaporation, and then performing filtration, and then performing constant volume, and then performing filling to obtain busulfan nanomicelles or solvates thereof; the high-pressure homogenization is performed by using a high-pressure homogenizer, the homogenization pressure is 500 bar-1500 bar, the homogenization cycle number is 3-7, and the feeding speed of the solution during homogenization is 50 mL / min-300 mL / min; Step 4: placing the busulfan nanomicelles or solvates thereof into a freeze dryer to perform freeze drying; the freeze drying comprises the following steps: Step S1: pre-freezing stage, keeping the busulfan nanomicelles or solvates thereof at 2 ℃-5 ℃ for 1 h-3 h, controlling the temperature to-45 ℃--35 ℃, and keeping the temperature for 4 h-6 h to obtain a frozen sample; Step S2: sublimation stage, controlling the plate temperature to be not more than 8 ℃, controlling the temperature of the frozen sample to be-60 ℃--45 ℃, keeping the temperature for 20 h-24 h, and controlling the vacuum degree to be 0.1 mbar-0.2 mbar; after a predetermined time is reached, the temperature is increased to-45 ℃--30 ℃, keeping the temperature for 16 h-20 h, and controlling the vacuum degree to be 0.3 mbar-0.6 mbar; a sublimated sample is obtained; Step S3: desorption stage: after a predetermined time is reached, the temperature of the sublimated sample is increased to 18 ℃ ± 2 ℃, keeping the temperature for 3 h-5 h; after a predetermined time is reached, the temperature is increased to 30 ℃ ± 4 ℃, the vacuum degree is kept unchanged, keeping the temperature for 1 h-2 h, and the moisture content is required to be less than 1.0%, to obtain a freeze-dried powder of busulfan nanomicelles.

6. The preparation method according to claim 5, characterized in that, The solvent is at least one selected from ethyl acetate, ethanol, chloroform, methanol, acetone and dichloromethane; The temperature of the solution in step 3 is controlled to be 5 ℃-45 ℃, The solvent is removed by using reduced-pressure rotary evaporation, and the evaporation temperature is 40 ℃-50 ℃; The filtration is at least one selected from 0.45 µm organic filter pre-filtration, 0.22 µm organic filter redundancy filtration and 0.22 µm organic filter sterilization filtration; The constant-volume medium is water for injection, and the constant-volume concentration is 6 mg / mL; The filling container is a borosilicate glass tube injection vial, a polytetrafluoroethylene / ethylene copolymer film chlorobutyl rubber stopper for injection liquid, and an aluminum-plastic combined cover, and the filling volume is 10 mL-30 mL.

7. Use of the nanosize buccolome or the nanosize buccolome prepared by the method of any one of claims 5-6 in the preparation of a medicament for the treatment of a related disease, wherein the related disease comprises at least one of polycythemia vera, essential thrombocythemia, myelofibrosis, and chronic myeloid leukemia chronic phase. ​

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