Method for industrially preparing vitamin K1 micelle injection preparation

Abstract

The invention discloses a method for industrially preparing fat-soluble vitamin / cholate / phospholipid micelles, which comprises the following steps: sequentially dissolving phospholipid, cholic acid and vitamin in ethanol, removing most of ethanol from a solution system through reduced pressure evaporation, then adding an alkaline solution to adjust the pH value, removing ethanol and most of water through reduced pressure evaporation, and drying to obtain the fat-soluble vitamin / cholate / phospholipid micelles. The polar structure of the amphiphilic surfactant consisting of cholic acid and phospholipid is arranged outside, and the vitamin K1 is encapsulated inside the non-polar structure. The method provided by the invention is suitable for lab-scale tests in laboratories and industrial large-scale production, and can be used for preparing the vitamin K1 solution with consistent indexes and stable quality. The encapsulation efficiency of the obtained vitamin K1 micelle solution is 95% or above, and 6-month accelerated stability results show that the properties of the solution, such as the particle size, the Zeta potential and the content of related substances, meet the medicinal requirements.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to a method for the industrial preparation of fat-soluble vitamin micelles, and more particularly to a method for the industrial preparation of vitamin K1 micelle injection formulations. Background Technology

[0002] Vitamin K1 is essential for the liver to synthesize prothrombin, and a deficiency can cause coagulation disorders. When the blood lacks prothrombin, blood clotting is slowed down. Supplementing with an appropriate amount of vitamin K1 can promote the liver to synthesize prothrombin, thus achieving the effect of blood clotting and hemostasis.

[0003] Vitamin K1 is mainly used clinically for vitamin K1 deficiency, hypothrombin syndrome, prevention and treatment of neonatal spontaneous hemorrhage, and bleeding symptoms caused by bile leakage, obstructive jaundice, and chronic diarrhea. In addition, vitamin K1 also has analgesic and bronchospasm-relieving effects, and has a significant therapeutic effect on colic caused by visceral smooth muscle colic, bile duct spasm, and intestinal spasm.

[0004] Vitamin K1 is a fat-soluble vitamin, appearing as a yellow to orange-yellow transparent viscous liquid. It is easily decomposed by light, readily soluble in chloroform, ether, or vegetable oil, slightly soluble in ethanol, and insoluble in water. Therefore, surfactants and organic solvents, such as Tween-80, propylene glycol, and ethanol, are added to vitamin K1 injection solutions to aid dissolution. However, these solubilizers can easily cause vascular irritation and hemolysis after entering the bloodstream, resulting in a high rate of adverse reactions. For example, Ma Liyuan et al. pointed out in their literature (Analysis of the Correlation between Adverse Reactions / Events of Vitamin K1 Injection and its Processing Solubilizer Tween. Pharmaceutical and Clinical Research. 2014, pp. 187-190.) that adverse reactions to vitamin K1 injection are related to organic solubilizers such as Tween. Song Haibo et al. from the Center for Drug Evaluation of the State Food and Drug Administration, in their paper "Safety Analysis of Tween 80 and its Application in Injection. Proceedings of the Second Academic Conference (2017) of the Professional Committee of Toxicology of Traditional Chinese Medicine and Natural Products of the Chinese Society of Toxicology," proposed that attention should be paid to the safety research and rational use of Tween-containing injections. The original product, Konakion MM (CHEPLAPHARM Arzneimittel GmbH / Roche Pharma AG), a vitamin K1 injection, is prepared using a mixed micelle process. It uses a combination of bile salts and phosphatidylcholine to form micelles as drug carriers, encapsulating vitamin K1 within them to form a clear solution, greatly improving clinical safety. Therefore, the preparation of vitamin K1 micelles has become a research and development hotspot for many domestic pharmaceutical companies.

[0005] For example, patent document CN105997869A discloses a vitamin K1 micelle injection and its preparation method. The method uses an organic solvent (2-methyl-2-propanol, 2-methyl-2-butanol) freeze-drying process, employing a mixed micelle composed of phospholipids and bile salts as a carrier to encapsulate vitamin K1 within the hydrophobic core of the mixed micelles. However, this method uses freeze-drying to remove the organic solvent, making industrial scale-up difficult. The prepared product cannot be used directly and requires reconstitution with a suitable solvent. Patent document CN110876719A discloses a vitamin K1 injection and its preparation method. This method involves mixing and dissolving vitamin K1, sodium glycocholate, phospholipids, and ethanol, purging with nitrogen to remove the ethanol, mixing the resulting semi-solid mixture with water, adjusting the pH, and then dissolving. However, during the implementation of this patent, as ethanol evaporates, the viscosity of the ethanol solution gradually increases. Repeating the experiment reveals that when the ethanol concentration drops to 5% (V / V), the viscosity of the ethanol solution increases significantly, limiting the solution's fluidity. When the ethanol concentration drops to 3% (V / V), the solution has virtually no fluidity, and ethanol purging can no longer uniformly affect the solution. In other words, the ethanol removal method described in this patent results in a solution with a high ethanol concentration, failing to meet the regulatory requirement of below 5000 ppm. Patent document CN112870160A reports a novel fat-soluble vitamin mixed micelle injection and its preparation method. In preparing the vitamin K1 micelle injection, no organic solvent is used for dissolution; instead, a microfluidic homogenizer is used to obtain the micelle solution through a mechanical method involving high-pressure shearing. Reportedly, this process can only form emulsions and cannot prepare mixed micelles. Summary of the Invention

[0006] The existing technologies for preparing vitamin K1 / cholesterol / phospholipid micelles all share a common characteristic: they are only feasible and reproducible on a small-scale laboratory basis. However, when the scale is industrialized, the repeatability of the results decreases significantly, the quality of the micelle products fluctuates greatly, the process is difficult to control stably, and the quality requirements of micelle injection formulations cannot be met. It is difficult to produce commercial products with the same quality as those produced on a small-scale laboratory basis, and therefore, they are not feasible for industrial application.

[0007] To explore a truly industrial-scale production process for vitamin K1 / cholesterol / phospholipid micelle injections, achieving stable pilot-scale and industrial-scale production of tens of thousands or even hundreds of thousands of vials of vitamin K1 injections, we innovated existing process technologies and preparation methods. During the micelle injection production process, we strictly controlled residual oxygen and dissolved oxygen to ensure stable production of products with consistent indicators as the original drug at industrial scale. Furthermore, to reduce production costs, improve economic efficiency, and achieve commercial success, the new process utilizes commonly used chemical equipment and apparatus, avoiding the use of relatively expensive specialized equipment such as microfluidic homogenizers / devices / machines, thus saving on hardware investment. Specifically, this invention provides the following technical solution.

[0008] An industrial method for preparing fat-soluble vitamin / cholesterol salts (or phospholipid micelles) includes the following steps:

[0009] (1) Add phospholipids, cholic acid or their salts and more than one kilogram of fat-soluble vitamins to a mixing tank containing ethanol in proportion, and stir at room temperature in the dark to form a clear ethanol solution A;

[0010] (2) Vacuum the mixing tank in step (1), heat it up, and concentrate the ethanol solution A by vacuum distillation below 50°C until 90%-97% of the ethanol is removed, to obtain concentrated ethanol solution B.

[0011] (3) Add the alkaline solution to the ethanol solution B obtained in step (2) and stir until the system is clear to obtain an ethanol aqueous solution C. The alkaline solution is selected from the aqueous solutions of sodium hydroxide, potassium hydroxide, sodium carbonate and sodium bicarbonate. The pH value of the ethanol aqueous solution C is 5.0-6.5, preferably 5.5-6.2. The alkaline solution is preferably a sodium hydroxide solution.

[0012] (4) Vacuum the mixing tank in step (3), heat it up, and concentrate the ethanol aqueous solution C by vacuum distillation at 20℃-70℃ until the residual ethanol content is not higher than 5000ppm. Add water to make the vitamin concentration 5-20mg / ml, preferably 8-15mg / ml, more preferably 10mg / ml, to obtain a fat-soluble vitamin / cholesterol / phospholipid micelle solution.

[0013] In one embodiment, the fat-soluble vitamin is selected from vitamin A, vitamin D, vitamin E, vitamin K1, or a mixture of two or more thereof, preferably vitamin K1;

[0014] The cholic acid is selected from glycocholic acid, taurine cholic acid, glycodeoxycholic acid, taurine deoxycholic acid, glycochenodeoxycholic acid, taurine chenodeoxycholic acid, porphyric acid, porphyric deoxycholic acid, or a mixture of two or more thereof, preferably glycocholic acid; and / or

[0015] The phospholipid is selected from soybean phospholipid (soybean phosphatidylcholine), egg yolk lecithin, hydrogenated soybean phospholipid, hydrogenated egg yolk lecithin, or a mixture of two or more thereof, preferably soybean phospholipid or egg yolk lecithin, and more preferably soybean phospholipid (soybean phosphatidylcholine).

[0016] Preferably, the fat-soluble vitamin / cholesterol / phospholipid micelles meet the requirements of ICH Q3C; the ethanol concentration is not higher than 5000 ppm, preferably not higher than 2000 ppm, not higher than 1000 ppm, preferably not higher than 500 ppm, preferably not higher than 200 ppm, and more preferably not higher than 100 ppm.

[0017] In one embodiment, the water mentioned in step (4) above is water for injection.

[0018] Preferably, the weight percentage of vitamin K1 in step (1) is about 0.5-5 wt%, preferably 0.6-4 wt%, preferably 0.7-3 wt%, preferably 0.8-2 wt%, and more preferably about 1.0 wt%, the weight percentage of sodium cholate is about 3-7 wt%, preferably 3.5-6.5 wt%, preferably 4.0-6.0 wt%, preferably 4.5-5.5 wt%, and more preferably about 5.5 wt%, and the weight percentage of phospholipid is about 4.0-10.0 wt%, preferably 5.0-9.0 wt%, preferably 5.5-8.5 wt%, preferably 6.0-8.0 wt%, and more preferably about 7.5 wt%.

[0019] It should be understood that in this article, when describing numerical characteristics, the terms "approximately" or "around" mean that the expressed number may have an error range or fluctuation range of ±10%, ±9%, ±8%, ±7%, ±6%, or ±5%.

[0020] Optionally, step (2) above may involve evacuating the mixing tank / mixing vessel / reactor in step (1) to a vacuum level of -0.95 bar or higher, heating the ethanol solution A to boiling point, at which point the solution temperature is not greater than 50°C, and rapidly evaporating the ethanol until 90%-97% of the ethanol is removed, resulting in a concentrated viscous ethanol solution B.

[0021] Further, step (4) above involves, for example, evacuating the mixing tank / mixing vessel / reactor in step (3) to a vacuum degree above -0.95 bar, heating it to boiling point of the ethanol aqueous solution C, at which point the solution temperature is not greater than 70°C, evaporating the ethanol and water until the ethanol content is below 5000 ppm, adding water to make up the volume, and obtaining a fat-soluble vitamin / cholesterol / phospholipid micelle solution with a fat-soluble vitamin concentration of 5-20 mg / ml.

[0022] In a preferred embodiment, in steps (2) to (4) above, the dissolved oxygen concentration of the liquid materials and solutions, such as ethanol solution, sodium hydroxide solution and water for injection, is controlled to be less than 0.1 ppm, preferably less than 0.05 ppm, more preferably less than 0.05 ppm, and more preferably less than 0.01 ppm, thereby avoiding the damage of oxygen to the micelle components and micelle quality, and ensuring the physicochemical properties of the micelle product, such as thermal stability.

[0023] Furthermore, in steps (2) to (4) above, the headspace oxygen is controlled to be less than 1%, preferably less than 0.5%, preferably less than 0.1%, and more preferably less than 0.05%, in order to control the residual oxygen and dissolved oxygen in the micelle product.

[0024] In one embodiment, when the fat-soluble vitamin is vitamin K1, the cholic acid is glycocholic acid, and the phospholipid is soybean phospholipid, the pH of the ethanol aqueous solution C in step (3) is pH 5.0-6.5.

[0025] Preferably, when the fat-soluble vitamin is vitamin K1, the cholic acid is glycocholic acid, and the phospholipid is soybean phospholipid, the average particle size of more than 80%, preferably more than 85%, more preferably more than 90% of the fat-soluble vitamin / cholate / phospholipid micelles is 1-20 nm, preferably 8-15 nm.

[0026] The micelle solution encapsulation efficiency is 92% or higher, preferably 95% or higher, more preferably 96% or higher, and even more preferably 98% or higher;

[0027] The zeta potential is -30mV to -60mV, preferably -40mV to -50mV; and / or

[0028] The micelle solution is clear and transparent in appearance, with a light transmittance of not less than 90%, preferably not less than 92%, more preferably not less than 92%, and remains clear and transparent after being sterilized at 121°C for 15 minutes, and preferably meets the requirements of ICH Q3C.

[0029] Furthermore, the above method preferably includes the following steps:

[0030] (5) The fat-soluble vitamin / cholesterol / phospholipid micelle solution obtained in step (4) is filtered through a microporous membrane with a diameter of 0.45 μm or less, preferably 0.40 μm or less, and more preferably 0.35 μm or less. If necessary, it is stored and quality inspected, dispensed, and sterilized by autoclaving. If necessary, it is sterilized and then quality inspected to obtain an injection.

[0031] In one embodiment, the above-mentioned dispensing refers to filling the filtrate into ampoules and sealing them.

[0032] The heat sterilization mentioned above can refer to sterilization at around 121°C for 12-20 minutes, for example, 15 minutes.

[0033] Preferably, the above-mentioned ampoules are treated with the following steps before use: cleaned with sterile water such as double-distilled water, dried and / or sterilized.

[0034] Optionally, the ampoule is, for example, a borosilicate glass ampoule.

[0035] In one embodiment, preferably, the filtrate stored in step (5) above is filtered by a secondary microporous membrane with a diameter of less than 0.22 μm, preferably less than 0.20 μm, before being dispensed.

[0036] For factory production purposes, in the production of fat-soluble vitamins / cholates / phospholipid micelles, the production workshop area is arranged according to the following air supply level / cleanliness level to achieve GMP management that ensures product quality and controls industrial costs:

[0037] Step (5) of the filtrate filling and sealing process, namely the filtrate filling into ampoules and the sealing process, is a Class A air supply area, namely a Class A operation area;

[0038] The blank ampoule processing step (5), namely the ampoule cleaning and drying / sterilization process, is a Class D air supply zone, i.e., a Class D operating zone; and / or

[0039] The processes in steps (1) to (4) and the filtration and storage processes in step (5) are Class C air supply zones, i.e. Class C operating zones.

[0040] The method for producing fat-soluble vitamin / cholate / phospholipid micelle solutions developed in this invention overcomes the shortcomings of laboratory-scale technology, such as difficulty in scaling up and high economic costs. A single batch of vitamin K1 is fed in quantities of kilograms or more, resulting in vitamin K1 micelle solutions with an encapsulation rate exceeding 95%, a narrow particle size distribution, and liquid transparency / transmittance, thermal stability, zeta potential, and related substance content all meet the best laboratory data reported in existing technical literature. This method meets the pharmaceutical requirements for micelle injections and promotes the industrial-scale production and application of vitamin K1 micelle formulations. Attached Figure Description

[0041] Figure 1 A process flow diagram for preparing vitamin K1 micelle injection formulations for factory production workshops.

[0042] Figure 2 To display AFM (Atomic Force Microscope) images of the vitamin K1 / sodium glycocholate / soybean lecithin micelle product prepared in Example 1.

[0043] Figure 3AFM image of the original product Konakion MM (CHEPLAPHARM Arzneimittel GmbH / Roche Pharma AG).

[0044] Figure 4 In vitro release curve of vitamin K1 injection (original formulation / self-made formulation) Detailed Implementation

[0045] In the chemical, bioengineering, and pharmaceutical fields, it is well known that scaling up chemical reactions from laboratory pilot-scale trials to factory pilot-scale trials often suffers from a "scale-up effect"—a significant deterioration in laboratory results and a substantial reduction in reproducibility. This "scale-up effect" hinders the practical application of laboratory research findings in factories, leading to insufficient industrial production feasibility for many laboratory research results. Given that existing technologies, such as those disclosed in CN105997869A, CN110876719A, and CN112870160A, demonstrate strong scale-up effects in their laboratory production of vitamin K1 / cholate / phospholipid micelle injections, and considering the high market demand for vitamin K1 micelle injection formulations, we have innovated a micelle injection preparation process after exploring laboratory pilot-scale and factory pilot-scale trials, taking into account economic costs. This innovative process effectively overcomes the bottleneck problem. Furthermore, the innovative production process easily complies with GMP management requirements for pharmaceutical manufacturing companies, enabling the smooth commercial production of vitamin K1 micelle products.

[0046] In improving the production process of vitamin Kl / cholate / phospholipid micelle injections, this invention primarily references the laboratory techniques disclosed in documents CN110876719A and CN112870160A. Compared to CN110876719A, the advantages of this invention's method include at least the following: significantly improved production efficiency, enabling large-scale production; elimination of the need for a proprietary microfluidic homogenizer, significantly reducing production costs, and ensuring stable finished product quality.

[0047] The principle of this industrial preparation of vitamin K1 micelles is as follows: the excipients phospholipids and cholic acid / cholate salts in the formulation, as well as the raw material vitamin K1, are sequentially dissolved in the organic solvent ethanol. Most of the organic solvent ethanol is gradually removed from the solution system by vacuum evaporation. Then, sodium hydroxide solution is added to adjust the pH, and ethanol and most of the water are removed by vacuum evaporation. At this point, the amphiphilic surfactant composed of cholic acid and phospholipids can form a micelle structure with a polar structure on the outside in the water, and encapsulate the fat-soluble vitamin K1 inside the non-polar structure, thereby forming a target micelle solution with vitamin K1 as the effective drug component.

[0048] In the actual production process of micelles, the automatic control system composed of bubble sensors can realize dynamic automatic adjustment of vacuum degree, effectively solving the problem of a large number of bubbles being generated and overflowing in the solution, avoiding the loss of active ingredients, and ensuring the smooth progress of the process.

[0049] As is well known to those skilled in the art, the term "micelle" refers to an amphiphilic substance in a solvent (e.g., water) whose concentration exceeds a certain critical value, forming a solid core-shell structure due to the mutual attraction between its solvophytic (e.g., hydrophobic) or solvophilic (e.g., hydrophilic) portions. The core is composed of the solvophytic (e.g., hydrophobic) portion of the molecule, and the shell is composed of the solvophilic (e.g., hydrophilic) portion of the molecule. Micellar systems can be used to encapsulate / load drugs, achieving drug solubilization.

[0050] When the process of this invention is used on a pilot-scale basis with a vitamin K1 feed amount of over kilograms, the ethanol concentration of the finished vitamin K1 / cholate / phospholipid micelle injection formulation remains below 5000 ppm, meeting the requirements of regulation (ICH Q3C). The micelle particle size distribution is narrow, maintaining an average particle size of 1-20 nm, and the appearance is clear and transparent. Testing showed that the encapsulation efficiency of the obtained vitamin K1 micelle solution was over 95%, and accelerated stability testing after 6 months showed that the physical properties of the solution (particle size, zeta potential, and related substances, etc.) were comparable to the formulations reported in CN110876719A and CN112870160A.

[0051] In this article, the term "related substances" refers to impurities introduced into a drug during the manufacturing process, such as starting materials, intermediates, polymers, by-reaction products, and degradation products during storage. Because the chemical structures of these impurities are generally similar to or related to the active ingredient, they are commonly referred to as related substances. The term "related substances" can also refer to the content of the aforementioned impurities in the drug.

[0052] In this document, the term "injectable preparation" or "injection" refers to a sterile preparation made from an active pharmaceutical ingredient (API), such as vitamin K1, and suitable excipients, intended for injection into the body. This includes sterile solutions, suspensions, and emulsions, as well as sterile powders or concentrated solutions intended for reconstitution into solutions, suspensions, or emulsions immediately before use. In this document, the term "injection solution" refers to a liquid injection.

[0053] The industrial-scale preparation process of vitamin K1 micelles of this invention requires no new specialized equipment, and the resulting solution is homogeneous and stable. By controlling the vacuum level inside the tank, the entire preparation process can be carried out at room temperature, avoiding the influence of high temperatures on the solution. Furthermore, this process is highly compatible with production batches, allowing for adjustments to production volume as needed. In addition, the finished product production time is significantly shorter than processes such as freeze drying, resulting in higher efficiency and lower costs.

[0054] It is anticipated that the micelle production process of the present invention is not only suitable for preparing vitamin Kl / cholate / phospholipid micelle injection formulations, but should also be suitable for preparing other fat-soluble vitamin / cholate / phospholipid micelle injection solutions (injectable formulations).

[0055] In this article, for the sake of convenience, the term "vitamin K1 / cholate / phospholipid micelles (solution / injection)" can be abbreviated as "vitamin K1 micelles"; similarly, the term "fat-soluble vitamin / cholate / phospholipid micelles" can be abbreviated as "fat-soluble vitamin micelles" or "vitamin micelles".

[0056] Since the fat-soluble vitamin micelles of the present invention contain multiple components, they belong to the category of mixed micelles. It should be understood that the term "mixed micelles" refers to a micelle system formed by the compounding of surface-active substances, which can enhance the solubilizing properties of a single surface-active substance.

[0057] The vitamin K1 micelle preparation process developed by the inventors can already be put into actual production in the factory workshop. According to the GMP specifications of the pharmaceutical industry, corresponding cleanroom layouts are implemented in different sections to achieve optimized quality control and thereby maximize economic benefits. For example, the filtrate filling and sealing process in step (5), i.e., the filtrate filling into ampoules and the sealing process, is a Class A air supply area, i.e., a Class A operating area; the blank ampoule processing process, i.e., the ampoule cleaning and drying / sterilization process, is a Class D air supply area, i.e., a Class D operating area; and / or the processes in steps (1) to (4) and the filtration and storage process in step (5) are Class C air supply areas, i.e., Class C operating areas. See also Figure 1 .

[0058] As is known to those skilled in the art, GMP workshop cleanliness levels are typically divided into four grades: A, B, C, and D, with each grade corresponding to different operating areas and production requirements.

[0059] Class A: High-risk operation areas, such as filling areas. A unidirectional flow control panel (hood) should be used to maintain the environmental conditions in this area, with an air velocity of 0.36-0.54 m / s (guideline value). The airborne particle level for Class A clean areas is ISO 4.8.

[0060] Grade B: Refers to the background area of ​​a Grade A clean area where high-risk operations such as aseptic preparation and filling are performed. The airborne particle level of a Grade B clean area (static) is ISO 5.

[0061] Grade C and Grade D: These refer to clean areas for less critical operational steps in the production of sterile pharmaceuticals. Grade C clean areas (static and dynamic) have airborne particle levels of ISO 7 and ISO 8, respectively; Grade D clean areas (static) have an airborne particle level of ISO 8.

[0062] In the description of the technical solutions of this invention, when referring to the performance indicators and / or preparation process characteristics of vitamin K1 micelles, the term "and / or" used in phrases such as "A and / or B" or "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to cover each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); C (alone).

[0063] In this invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the laboratory procedures described herein are all standard procedures widely used in the relevant fields.

[0064] Example

[0065] The present invention is further illustrated below by way of examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the invention. Various changes or modifications made by those skilled in the art based on the concept of the present invention should fall within the protection scope of the present invention.

[0066] This article involves the addition amount, content and concentration of various substances. Unless otherwise specified, the percentage content mentioned refers to the mass percentage.

[0067] In the embodiments described herein, unless otherwise specified, the operating temperature generally refers to room temperature (10-30°C).

[0068] The micelle detection method in the embodiments, referring to CN110876719A and CN112870160A, includes, for example:

[0069] Appearance: Visually, qualified micelles should be a pale yellow, clear liquid, without stratification, flocculation, or sedimentation.

[0070] Transmittance: Measured at 600 nm using a Shimadzu UV2600 UV-Vis spectrophotometer, in accordance with General Chapter 0401 of the 2020 edition of the Chinese Pharmacopoeia.

[0071] Morphological examination: The diluted mixed micelle solution was dropped onto a copper grid, counterstained with 2% sodium phosphotungstenate solution, and observed and photographed under a transmission electron microscope.

[0072] Particle size and zeta potential determination: The micelle solution was diluted to 0.5 mg / mL according to General Chapter 0982 of the 2020 edition of the Chinese Pharmacopoeia and determined using a ZETASIZER LAB Malvern nanoparticle size and zeta potential analyzer.

[0073] pH value: determined using a sevenExcellence S470-k / uMix Mettler pH meter according to General Chapter 0631 of the 2020 edition of the Chinese Pharmacopoeia.

[0074] Content and related substances determination: The content was determined using an Agilent 1260 liquid chromatograph according to General Chapters 3100 and 2400 of the 2020 edition of the Chinese Pharmacopoeia. Chromatographic conditions: C18 column (150*4.6mm, S-5nm, 12nm), mobile phase 90% ethanol, detection wavelength 254nm.

[0075] Thermal stability test: Compare the micelles placed at 60℃ for 30 days, under accelerated conditions of 40℃±2℃ / RH75% for 6 months, with those placed on day 0.

[0076] Raw materials and reagents: All raw materials and reagents used in the embodiments of this invention are pharmaceutical grade and were purchased from Aladdin Shanghai Co., Ltd.

[0077] Example 1: Preparation of Vitamin K1 / Sodium Glycinecholate / Soybean Phosphatidylcholine Micelles

[0078] In a 500-liter (500L) reactor, 50L of anhydrous ethanol was added, followed by 15kg of soybean lecithin, 10.5kg of glycocholic acid and 2kg of vitamin K1. The mixture was stirred for 15 minutes at room temperature in the dark to obtain a clear yellow ethanol solution A.

[0079] Meanwhile, prepare 150L of 0.56% wt sodium hydroxide aqueous solution for later use.

[0080] The reactor was evacuated to -0.95 bar and heating was started. The ethanol solution inside the reactor boiled, and the solution temperature was 20°C. The ethanol evaporated rapidly, and the solution volume was reduced to about 30 L, yielding a viscous yellow ethanol solution B.

[0081] Add 150L of the prepared sodium hydroxide solution to the reaction vessel and react with stirring for 30 minutes until the reaction solution system is completely clear, to obtain an ethanol aqueous solution C with a pH of 6.0.

[0082] The reaction vessel was evacuated to -0.95 bar and heating was started. The ethanol-water solution C inside the vessel boiled, and the temperature of the solution inside the vessel was 20°C. After the ethanol-water solution evaporated to about 80 L, water for injection was added, and the volume was adjusted to 200 L to obtain the target vitamin K1 / sodium glycocholate / soybean phosphatidylcholine micelle solution, which was a pale yellow and clear solution.

[0083] AFM images of the obtained vitamin K1 micelle products are as follows: Figure 2 As shown. Figure 3An AFM image of the original product, Konakion MM, is shown. Based on the AFM image comparison, the product prepared by the process of this invention is at least visually similar to the original product, Konakion MM.

[0084] The obtained micelle solution was tested and found to contain 800 ppm ethanol and approximately 1 wt% vitamin K1. The solution was clear and transparent with 95% transmittance. The pH was 6.0. The encapsulation efficiency was 95%. Over 90% of the micelles had an average particle size of 1-10 nm. The zeta potential was -45 mV. The residual ethanol concentration was less than 5000 ppm, and the solution remained clear and transparent after sterilization at 121°C for 15 minutes. Accelerated stability tests at 30 days and 6 months showed that the physicochemical properties of the micelle solution were largely equivalent to the laboratory products disclosed in literature CN110876719A and CN112870160A. Furthermore, the correlation curves of the in vitro release test (IVRT), used to characterize drug release properties and simulate in vivo release behavior, were also obtained. Figure 4 The results showed that the obtained micelle solution exhibited similar release behavior to the original reagent.

[0085] Example 2: Preparation of Vitamin K1 / Sodium Glycinecholate / Soybean Phosphatidylcholine Micelles

[0086] Vitamin K1 / sodium glycocholate / soybean phosphatidylcholine micelles were prepared using essentially the same method as in Example 1, except that the amounts of the three raw materials were 2 kg of vitamin K1, 9 kg of glycocholic acid and 12 kg of soybean phosphatidylcholine, and the concentration of the 150 L sodium hydroxide solution was 0.45% wt.

[0087] Testing revealed that the obtained micelle solution contained 1000 ppm ethanol and approximately 1 wt% vitamin K1; the solution was clear and transparent with a transmittance of 93%; the pH was 6.1; the encapsulation efficiency was 94%; over 90% of the micelles had an average particle size of 9-15 nm; the zeta potential was -48 mV; and it remained clear and transparent after sterilization at 121°C for 15 minutes. Accelerated stability tests at high temperatures for 30 days and 6 months showed that the properties of the micelle solution were substantially equivalent to the micelle product of Example 1.

[0088] Example 3: Preparation of Vitamin K1 / Sodium Glycinecholate / Soybean Phosphatidylcholine Micelles

[0089] Vitamin K1 / sodium glycocholate / soybean phosphatidylcholine micelles were prepared using essentially the same method as in Example 1, except that: the pH of the ethanol aqueous solution C was adjusted to 6.5, glycocholic acid was glycodeoxycholic acid (10.5 kg), egg yolk lecithin (15 kg) was selected as the phospholipid, and the concentration of 150 L sodium hydroxide solution was 0.62% wt.

[0090] Testing revealed that the obtained micelle solution contained 700 ppm ethanol and approximately 1 wt% vitamin K1; the solution was clear and transparent with 90% light transmittance; the pH was 6.5; the encapsulation efficiency was 93%; over 90% of the micelles had an average particle size of 5-15 nm; the zeta potential was -50 mV; and it remained clear and transparent after sterilization at 121°C for 15 minutes. Accelerated stability tests at high temperatures for 30 days and 6 months showed that the properties of the micelle solution were substantially equivalent to the micelle product of Example 1.

[0091] Example 4: Preparation of Vitamin K1 / Sodium Glycinecholate / Soybean Phosphatidylcholine Micelles

[0092] Vitamin K1 / sodium glycocholate / soybean phosphatidylcholine micelles were prepared using essentially the same method as in Example 1, except that: the distillation temperature of the ethanol aqueous solution C was set to 70°C, glycocholic acid was glycodeoxycholic acid (10.5 kg), hydrogenated soybean phospholipids (15 kg) were selected, and the concentration of 150 L sodium hydroxide solution was 0.62% wt.

[0093] Testing revealed that the obtained micelle solution contained 550 ppm ethanol and approximately 1 wt% vitamin K1; the solution was clear and transparent with 90% light transmittance; the pH was 6.0; the encapsulation efficiency was 93%; over 90% of the micelles had an average particle size of 5-10 nm; the zeta potential was -50 mV; and it remained clear and transparent after sterilization at 121°C for 15 minutes. Accelerated stability tests at high temperatures for 30 days and 6 months showed that the properties of the micelle solution were substantially equivalent to the micelle product of Example 1.

[0094] Example 5: Preparation of Vitamin K1 / Sodium Glycinecholate / Soybean Phosphatidylcholine Micelles

[0095] Vitamin K1 / sodium glycocholate / soybean phosphatidylcholine micelles were prepared using essentially the same method as in Example 1, except that the distillation temperature of the ethanol aqueous solution C was set to 70°C, the pH of the ethanol aqueous solution C was adjusted to 6.5, and hydrogenated egg yolk lecithin (15 kg) was used as the phospholipid.

[0096] Testing revealed that the obtained micelle solution contained 500 ppm ethanol and approximately 1 wt% vitamin K1; the solution was clear and transparent with a transmittance of 94%; the pH was 6.5; the encapsulation efficiency was 91%; over 90% of the micelles had an average particle size of 5-20 nm; the zeta potential was -35 mV; and it remained clear and transparent after sterilization at 121°C for 15 minutes. Accelerated stability tests at high temperatures for 30 days and 6 months showed that the properties of the micelle solution were substantially equivalent to the micelle product of Example 1.

[0097] Example 6: Preparation of Vitamin K1 / Sodium Glycinecholate / Soybean Phosphatidylcholine Micelles

[0098] Vitamin K1 / sodium glycocholate / soybean phosphatidylcholine micelles were prepared using essentially the same method as in Example 1, except that: the distillation temperature of ethanol solution B was set to 50°C, glycocholic acid was glycodeoxycholic acid (10.5 kg), hydrogenated egg yolk lecithin (15 kg) was selected as the phospholipid, and the concentration of 150 L sodium hydroxide solution was 0.62% wt.

[0099] Testing revealed that the obtained micelle solution contained 300 ppm ethanol and approximately 1 wt% vitamin K1; the solution was clear and transparent with a transmittance of 94%; the pH was 5.8; the encapsulation efficiency was 91%; over 90% of the micelles had an average particle size of 5-20 nm; the zeta potential was -35 mV; and it remained clear and transparent after sterilization at 121°C for 15 minutes. Accelerated stability tests at high temperatures for 30 days and 6 months showed that the properties of the micelle solution were substantially equivalent to the micelle product of Example 1.

[0100] Example 7: Preparation of Vitamin K1 / Sodium Glycinecholate / Soybean Phosphatidylcholine Micelles

[0101] Vitamin K1 / sodium glycocholate / soybean phosphatidylcholine micelles were prepared using essentially the same method as in Example 1, except that: the distillation temperature of ethanol solution B was set to 50°C, the pH of ethanol aqueous solution C was adjusted to 6.5, and hydrogenated soybean phosphatidylcholine (15 kg) was used as the phosphatidylcholine.

[0102] The obtained micelle solution was tested and found to contain 350 ppm ethanol and approximately 1 wt% vitamin K1. The solution was clear and transparent with a transmittance of 91%. The pH was 6.5, the encapsulation efficiency was 92%, and over 90% of the micelles had an average particle size of 1-15 nm. The zeta potential was -42 mV. The solution remained clear and transparent after sterilization at 121°C for 15 minutes. Accelerated stability tests at high temperatures for 30 days and 6 months showed that the properties of the micelle solution were substantially equivalent to those of the micelle product in Example 1.

[0103] Example 8: Preparation of Vitamin K1 / Sodium Glycinecholate / Soybean Phosphatidylcholine Micelles

[0104] Vitamin K1 / sodium glycocholate / soybean phosphatidylcholine micelles were prepared using essentially the same method as in Example 1, except that the distillation temperature of ethanol solution B was set to 50°C, the distillation temperature of ethanol aqueous solution C was set to 70°C, and egg yolk lecithin (15 kg) was used as the phospholipid.

[0105] The obtained micelle solution was tested and found to contain 270 ppm ethanol and approximately 1 wt% vitamin K1. The solution was clear and transparent with a transmittance of 93%. The pH was 5.5, the encapsulation efficiency was 95%, and over 90% of the micelles had an average particle size of 1-10 nm. The zeta potential was -32 mV. The solution remained clear and transparent after sterilization at 121°C for 15 minutes. Accelerated stability tests at high temperatures for 30 days and 6 months showed that the properties of the micelle solution were substantially equivalent to those of the micelle product in Example 1.

[0106] Example 9: Preparation of Vitamin K1 / Sodium Glycinecholate / Soybean Phosphatidylcholine Micelles (Part 9)

[0107] Vitamin K1 / sodium glycocholate / soybean phosphatidylcholine micelles were prepared using essentially the same method as in Example 1, except that: the distillation temperature of ethanol solution B was set to 50°C, the distillation temperature of ethanol aqueous solution C was set to 70°C, the pH of ethanol aqueous solution C was adjusted to 6.5, glycocholic acid was used as glycodeoxycholic acid (10.5 kg), and the concentration of 150 L sodium hydroxide solution was 0.62% wt.

[0108] The obtained micelle solution was tested and found to contain 230 ppm ethanol and approximately 1 wt% vitamin K1. The solution was clear and transparent with a transmittance of 91%. The pH was 6.6, the encapsulation efficiency was 94%, and over 90% of the micelles had an average particle size of 1-10 nm. The zeta potential was -30 mV. The solution remained clear and transparent after sterilization at 121°C for 15 minutes. Accelerated stability tests at high temperatures for 30 days and 6 months showed that the properties of the micelle solution were substantially equivalent to those of the micelle product in Example 1.

[0109] Comparative Example

[0110] In a 500-liter (500L) reactor, 50L of anhydrous ethanol was added, followed by 10.5kg of glycocholic acid, 15kg of soybean lecithin, and 2kg of vitamin K1. The mixture was stirred for 15 minutes at room temperature in the dark to obtain ethanol solution A, which was not clear.

[0111] Meanwhile, prepare 150L of a 0.25wt% sodium hydroxide aqueous solution for later use.

[0112] The reactor was evacuated to -0.55 bar and heating was started. The ethanol solution inside the reactor boiled, and the solution temperature was 80°C. The ethanol evaporated rapidly, and the solution volume was reduced to about 50 L, yielding a viscous yellow ethanol solution B.

[0113] Add 150L of the prepared sodium hydroxide solution to the reaction vessel and react with stirring for 30 minutes to obtain an ethanol aqueous solution C.

[0114] The reaction vessel was evacuated to -0.55 bar and heating was started. The ethanol-water solution C inside the vessel boiled, and the temperature of the solution inside the vessel was 90℃. After the ethanol-water solution evaporated to about 80L, water for injection was added, and the volume was adjusted to 200L to obtain the target vitamin K1 / sodium glycocholate / soybean phosphatidylcholine micelle solution, which was a pale yellow and slightly turbid solution.

[0115] Testing revealed that the obtained micelle solution contained 800 ppm ethanol and approximately 1 wt% vitamin K1; the solution was turbid with a transmittance of 25%; the pH was 6.0; the encapsulation efficiency was 20%; over 90% of the micelles had an average particle size of 100-500 nm; the zeta potential was -18 mV; and the solution remained turbid even after sterilization at 121°C for 15 minutes. This does not meet the quality standards and is inconsistent with the original formulation. The main reasons for the sample's failure to meet the requirements in this case are: 1. An incorrect order of adding glycocholic acid and soybean lecithin resulted in incomplete dissolution of glycocholic acid, affecting the morphology of the drug-loaded micelles; 2. Insufficient sodium hydroxide solution concentration led to an excessively low pH, affecting the dissolution of excipients; 3. An excessively high distillation temperature affected the zeta potential.

[0116] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for the industrial-scale preparation of fat-soluble vitamin / cholate / phospholipid micelles, characterized in that, Includes the following steps: (1) Add phospholipids, cholic acid or their salts and more than one kilogram of fat-soluble vitamins to a mixing tank containing ethanol in proportion, and stir at room temperature in the dark to form a clear ethanol solution A; (2) Vacuum the mixing tank in step (1), heat it up, and concentrate the ethanol solution A by vacuum distillation below 50°C to remove 90%-97% of the ethanol and obtain concentrated ethanol solution B. (3) Add the alkaline solution to the ethanol solution B obtained in step (2) and stir until the system is clear to obtain an ethanol aqueous solution C, wherein the alkaline solution is selected from the aqueous solutions of sodium hydroxide, potassium hydroxide, sodium carbonate and sodium bicarbonate, and the pH value of the obtained ethanol aqueous solution C is 5.0-6.

5. (4) Vacuum the mixing tank in step (3), heat it up, and concentrate the ethanol aqueous solution C under reduced pressure at 20℃-70℃ until the residual ethanol content is not higher than 5000ppm. Add water to make the vitamin concentration 5-20mg / ml to obtain a fat-soluble vitamin / cholesterol / phospholipid micelle solution.

2. The method according to claim 1, characterized in that, The fat-soluble vitamin is selected from vitamin A, vitamin D, vitamin E, vitamin K1, or a mixture of two or more of them, preferably vitamin K1; The cholic acid is selected from glycocholic acid, taurine cholic acid, glycodeoxycholic acid, taurine deoxycholic acid, glycochenodeoxycholic acid, taurine chenodeoxycholic acid, porphyric acid, porphyric deoxycholic acid, or a mixture of two or more thereof, preferably glycocholic acid; and / or The phospholipid is selected from soybean phospholipid, egg yolk lecithin, hydrogenated soybean phospholipid, hydrogenated egg yolk lecithin, or a mixture of two or more thereof, preferably soybean phospholipid or egg yolk lecithin, and more preferably soybean phospholipid (soybean phosphatidylcholine).

3. The method according to claim 1, characterized in that, In step (2), ethanol solution A is concentrated by vacuum distillation at 20℃-50℃; and / or In step (3), the pH value of the ethanol aqueous solution C is 5.5-6.2; and / or In step (4), water is added until the vitamin concentration is 8-15 mg / ml, preferably 10 mg / ml.

4. The method according to claim 1, characterized in that, The water mentioned in step (4) is water for injection.

5. The method according to claim 1, characterized in that, The weight percentage of vitamin K1 mentioned in step (1) is 0.5-5 wt%, the weight percentage of sodium cholate is 3-7 wt%, and the weight percentage of phospholipids is 4.0-10.0 wt%.

6. The method according to claim 1, characterized in that, Step (2) involves evacuating the mixing tank from step (1) to a vacuum level of -0.95 bar or higher.

7. The method according to claim 1, characterized in that, Step (4) involves evacuating the mixing tank from step (3) to a vacuum level of -0.95 bar or higher, heating it to boiling point of the ethanol-water solution C, evaporating the ethanol and water, reducing the ethanol content to below 5000 ppm, adding water to make up the volume, and obtaining a fat-soluble vitamin / cholesterol / phospholipid micelle solution with a fat-soluble vitamin concentration of 5-20 mg / ml.

8. The method according to claim 1, characterized in that, In steps (2) to (4), the dissolved oxygen concentration of the liquid material and solution is controlled to be below 0.1 ppm and the headspace oxygen to be below 1%.

9. The method according to claim 1, characterized in that, When the fat-soluble vitamin is vitamin K1, the cholic acid is glycocholic acid, and the phospholipid is soybean phospholipid, the pH of the ethanol aqueous solution C in step (3) is pH 5.0-6.

5.

10. The method according to claim 1, characterized in that, It also includes the following steps: (5) The fat-soluble vitamin / cholesterol / phospholipid micelle solution obtained in step (4) is filtered through a microporous membrane with a diameter of less than 0.45 μm, dispensed, and sterilized by autoclaving to obtain an injectable formulation.

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