A progesterone lipid liquid crystal, its preparation method and application

Abstract

This invention relates to the field of progesterone preparation technology, specifically to a progesterone lipid liquid crystal suitable for injection, its preparation method, and the pharmaceutical uses of the product. The progesterone lipid liquid crystal injection provided by this invention is a lipid liquid crystal system formed by mixing an oil phase and an aqueous phase. The oil phase includes progesterone, benzyl benzoate, phospholipids, vitamin E, and oleic acid, while the aqueous phase includes water for injection. This invention optimizes the mass ratio of phospholipids to oleic acid in the oil phase to 10:1, and the mass ratio of progesterone, benzyl benzoate, and vitamin E to 2:6:(5-3), resulting in a stable lipid liquid crystal system that improves injection compliance, reduces fat deposition, and achieves sustained drug efficacy for more than 12 hours.

Description

Technical Field

[0001] This invention belongs to the field of progesterone preparation technology, specifically relating to a progesterone lipid liquid crystal suitable for injection, its preparation method, and the pharmaceutical uses of the product. Background Technology

[0002] Progesterone, also known as luteal hormone, has the chemical name gestrin-4-ene-3,20-dione, and its structural formula is shown below. Progesterone is a natural progestin secreted by the corpus luteum of the ovary. Clinically, it is widely used to treat menstrual disorders, dysfunctional uterine bleeding, premenstrual syndrome, endometriosis, threatened miscarriage, and other conditions caused by insufficient luteal function.

[0003]

[0004] Currently available progesterone preparations include injections, capsules, soft capsules, vaginal suppositories, and vaginal sustained-release gels; the routes of administration include intramuscular injection, oral administration, and vaginal mucosal administration. Oral administration of progesterone has a severe first-pass effect in the liver and low bioavailability (<10%); vaginal administration has the same efficacy as intramuscular injection, but the former has a higher incidence of vaginal bleeding during the luteal phase; therefore, intramuscular injection remains the more ideal route of administration for progesterone.

[0005] However, existing progesterone intramuscular injection solutions also have some drawbacks. For example, Mochida Pharmaceuticals' progesterone injection solution, Progeholmon, is for muscle injection. ® Progesterone injections are oil solutions using injectable vegetable oil as a solvent (referred to as "oil injections"). They provide continuous 24-hour effects, but their high viscosity can lead to difficulties in injection, pain and irritation at the injection site, and the formation of local induration. IBSA has developed Progedex, a progesterone injection solution. ® Lubion ® Or Prolutex ® Both are aqueous solutions of progesterone encapsulated by hydroxypropyl-β-cyclodextrin (referred to as "water injections"). They can reduce problems such as difficulty in injection and local irritation. However, their blood drug concentration reaches the Cmax value 0.5-1h after administration, and the drug is completely released within 12h, indicating that the absorption, distribution and metabolism in the body are relatively fast, making it difficult to achieve a sustained effect.

[0006] Therefore, there is a need to further improve progesterone intramuscular injection solutions to improve injection compliance (including difficulty in injection, pain and stimulation at the injection site), reduce fat deposition (i.e., swelling of nodules at the injection site), and achieve a sustained therapeutic effect for more than 12 hours or even longer.

[0007] Lipid liquid crystalline (LLC) systems are systems with unique geometric structures formed by a certain concentration of amphiphilic lipid molecules in polar solvents. Their internal structure includes hydrophilic regions, hydrophobic regions, and lipid layers, exhibiting excellent drug loading capacity. Based on their structure, lipid liquid crystals can be classified into lamellar liquid crystals, cubic liquid crystals, and hexagonal liquid crystals. Drug release rates differ in different liquid crystal structures, enabling sustained-release and controlled-release effects. Therefore, lipid liquid crystals have attracted widespread attention due to their unique structural characteristics and drug loading properties.

[0008] Commonly used amphiphilic lipids are mainly glyceryl monooleate and phytanetriol. Reference 1 (Elgindy NA, Mehanna MM, Mohyeldin SM. Self-assembled nano-architecture liquid crystalline particles as a promising carrier for progesterone transdermal delivery [J]. International Journal of Pharmaceutics, 2016, 501(1-2): 167-179.) discloses a progesterone lipid liquid crystal nanoparticle for transdermal drug delivery. It uses 4.5% w / w glyceryl monooleate as the amphiphilic lipid, 0.5% w / w poloxamer 407 and polyvinyl alcohol as stabilizers, and encapsulates 2% w / w progesterone, forming a cubic phase liquid crystal through emulsification. In in vitro dissolution tests determined by dialysis, the 24-hour dissolution rate was 25–56% w / v; in in vitro permeability tests, the 24-hour drug permeation rate was 28–65% w / v.

[0009] Reference 1 has applied lipid liquid crystals to progesterone drug formulations. However, two issues still need attention: ① Due to their membrane-damping effects, amphiphilic lipids such as monooleate glycerol and phytanetriol are prone to causing adverse immune responses and tissue toxicity. Therefore, the safety risks of applying them via intramuscular injection are higher than those via transdermal administration. ② Transdermal administration requires consideration of transdermal absorption rate. Its release rate and release amount are usually higher than those of intramuscular injection. Therefore, liquid crystal structures suitable for transdermal administration are not suitable for intramuscular injection.

[0010] In summary, selecting a suitable progesterone lipid liquid crystal formulation and its preparation process for injection that can improve injection compliance, reduce fat deposition, and maintain drug efficacy for more than 12 hours or even longer (such as 24 hours or 36 hours) remains a technical challenge that has not yet been solved by those skilled in the art. Summary of the Invention

[0011] The purpose of this invention is to provide a progesterone lipid liquid crystal suitable for injection. By optimizing the types and amounts of amphiphilic lipids, phase modifiers, and other components in the oil phase, a stable lipid liquid crystal system is obtained, which improves injection compliance, reduces fat deposition, and achieves sustained drug efficacy for more than 12 hours.

[0012] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0013] This invention provides a progesterone lipid liquid crystal injection solution, which is formed by mixing an oil phase and an aqueous phase to form a lipid liquid crystal system. The oil phase components include progesterone, benzyl benzoate, phospholipids, vitamin E, and oleic acid; the aqueous phase components include water for injection.

[0014] Preferably, the mass ratio of phospholipid to oleic acid is (10~20):1.

[0015] Preferably, the mass ratio of phospholipid to oleic acid is 10:1.

[0016] Preferably, the mass ratio of progesterone, benzyl benzoate, and vitamin E is 2:(4-7):(3-5).

[0017] Preferably, the mass ratio of progesterone, benzyl benzoate, and vitamin E is 2:6:(3-5).

[0018] More preferably, the mass ratio of benzyl benzoate to vitamin E is 2:1 or 6:5, that is, the mass ratio of progesterone, benzyl benzoate, and vitamin E is 2:6:3 or 2:6:5.

[0019] Preferably, the phospholipid is selected from one or more of phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, and phosphatidylethanolamine.

[0020] More preferably, the phospholipid is selected from soybean phosphatidylcholine or egg yolk phosphatidylcholine.

[0021] Preferably, in the lipid liquid crystal system provided by the present invention, the proportions (w / v) of progesterone, benzyl benzoate, phospholipids, vitamin E and oleic acid in the lipid liquid crystal system are as follows: progesterone 1~5%, benzyl benzoate 3~15%, phospholipids 1.2~10%, vitamin E 1.5~12.5%, and oleic acid 0.12~1%.

[0022] More preferably, the lipid liquid crystal system provided by the present invention has the following weight-volume percentages (w / v): progesterone 2%, benzyl benzoate 6%, phospholipid 2.4%, vitamin E 3-5%, and oleic acid 0.24%.

[0023] The inventors considered the following factors when selecting the key components in this invention:

[0024] Progesterone is used as the active ingredient (main drug), and the dosage is selected from 1 to 5%. This is usually considered to cover some commonly used specifications, such as 1%, 2%, 2.5%, 5%, etc., but it is not limited to these.

[0025] Phospholipids, as amphiphilic lipids, are used to form lipid liquid crystals. If the amount of phospholipids in the lipid liquid crystal system is too low, it will be difficult for micelles to assemble into lipid liquid crystal structures. If the amount is too high, the lipid liquid crystal structure will be easily altered, thus affecting the release effect.

[0026] Benzyl benzoate is used as a solvent to dissolve progesterone. The amount of benzyl benzoate used is related to the amount of progesterone and vitamin E. It affects the high-temperature sterilization tolerance of the lipid liquid crystal system and regulates the particle size change of the lipid liquid crystal system before and after sterilization. This was an unexpected discovery by the inventors.

[0027] Vitamin E and oleic acid are used as phase modifiers to adjust the interface curvature and change the crystal structure to obtain a stable target lipid liquid crystal structure, which can effectively control the sustained release behavior of drugs. The choice of their dosage is related to benzyl benzoate and phospholipids, which will affect the high temperature sterilization resistance of the lipid liquid crystal system and regulate the particle size change of the lipid liquid crystal system before and after sterilization. This was also an unexpected discovery by the inventors.

[0028] In addition, the progesterone lipid liquid crystal injection solution provided by the present invention may, if necessary, have the addition of appropriate amounts of pH adjusters, osmotic pressure adjusters, stabilizers, etc.

[0029] pH adjusters are used to regulate the pH value of lipid liquid crystal systems. There are no special restrictions on their types. They can be selected from one or more of hydrochloric acid, sulfuric acid, acetic acid, acetate, phosphoric acid, phosphate, citric acid, citrate, carbonate, sodium hydroxide, ammonia, ethylenediamine, ethanolamine, tromethamine, disodium hydrogen phosphate or their hydrates, but are not limited to these.

[0030] Osmotic pressure regulators are used to regulate lipid liquid crystal systems to an isotonic state. There are no special restrictions on their types. They can be selected from one or more of glycerol, sucrose, glucose, potassium chloride, and sodium chloride, but are not limited to these.

[0031] Stabilizers are used to improve the stability of lipid liquid crystal dispersions. There are no special restrictions on their types. They can be selected from one or more of polyvinylpyrrolidone, ethylenediaminetetraacetic acid, and disodium ethylenediaminetetraacetate, but are not limited to these.

[0032] The progesterone lipid liquid crystal injection solution provided by the present invention has an average particle size of 200~600nm.

[0033] Preferably, the average particle size of the lipid liquid crystal system is 270~550 nm.

[0034] More preferably, the average particle size of the lipid liquid crystal system is 270~360 nm or 450~550 nm.

[0035] This invention also provides a method for preparing progesterone lipid liquid crystal injection, comprising the following steps: taking the prescribed amounts of benzyl benzoate, vitamin E, oleic acid, phospholipids, and progesterone, heating and stirring to dissolve them, obtaining an oil phase; taking the prescribed amount of water, adding appropriate amounts of stabilizer and osmotic pressure regulator if necessary, heating to dissolve, obtaining an aqueous phase; mixing the oil phase and aqueous phase to form a lipid liquid crystal phase, continuing high-pressure homogenization, obtaining a lipid liquid crystal system; adjusting the pH to a suitable level, filling with nitrogen, and sterilizing by moist heat to obtain the progesterone lipid liquid crystal injection.

[0036] Preferably, the preparation method of the above-mentioned progesterone lipid liquid crystal injection solution includes the following steps:

[0037] 1) Weigh out the prescribed amounts of benzyl benzoate, vitamin E, oleic acid, and phospholipids, heat them in a water bath to dissolve them, and after the phospholipids have dissolved, add the prescribed amount of progesterone, stir to dissolve, and obtain the oil phase;

[0038] 2) Weigh the prescribed amount of water, add appropriate amounts of stabilizer and osmotic pressure regulator if necessary, dissolve in water bath to obtain the aqueous phase;

[0039] 3) The oil phase and the aqueous phase mix under shear conditions to form a lipid liquid crystal phase;

[0040] 4) The sheared lipid liquid crystal phase is subjected to high-pressure homogenization and repeated multiple times to obtain a lipid liquid crystal system with the target particle size.

[0041] 5) Adjust the pH to a suitable level, fill with nitrogen, and sterilize with moist heat to obtain the product.

[0042] Preferably, the water bath temperature in steps 1) and 2) is 55~75℃, and more preferably 60~70℃.

[0043] Preferably, the temperature in steps 3) and 4) is 55~75℃, and more preferably 60~70℃.

[0044] Preferably, in step 5), the pH is adjusted to 5.0~8.0, and more preferably 5.5~7.5.

[0045] Preferably, the conditions for moist heat sterilization in step 5) are: 121℃, sterilization for 8~12min.

[0046] This invention also provides a pharmaceutical use for the progesterone lipid liquid crystal injection as described above. The progesterone drug prepared can be used to treat threatened abortion, prevent premature birth, in vitro fertilization, premenstrual syndrome, dysfunctional uterine bleeding and amenorrhea, and can also be used for luteal support in assisted reproductive technology.

[0047] It should be noted that existing technologies (such as CN118526457A) prepare low-viscosity liquid crystal precursors by combining liquid crystal matrix and liquid crystal modifier. After being administered into the body via subcutaneous or intramuscular injection, the liquid crystal precursors gradually absorb water from body fluids or surrounding tissues to form layered, cubic, or hexagonal liquid crystal phases, thereby slowly releasing the drug at the injection site. However, there may be burst releases, or changes in release behavior due to differences in liquid crystal lattice caused by individual differences.

[0048] The formulation design concept of this invention is to provide a lipid liquid crystal nanopolydisperse, which forms a liquid crystal phase during the preparation process. The appropriate lipid liquid crystal particle size is controlled by high pressure homogenization. After being injected into the body, it is released slowly and steadily, avoiding burst release and achieving sustained drug efficacy for more than 12 hours.

[0049] Therefore, the advantages of the progesterone lipid liquid crystal and its preparation method provided by the present invention compared with the prior art are as follows:

[0050] 1. The progesterone lipid liquid crystal provided by the present invention has a viscosity far lower than that of commercially available progesterone oil injections, which can significantly improve injection compliance during clinical use and solve the shortcomings of oil injections such as difficulty in injection and pain stimulation at the injection site; in addition, the lipid liquid crystal is easy to diffuse, and the fat deposition at the injection site is significantly less than that of oil injections, effectively preventing hardening at the injection site.

[0051] 2. In addition, the present invention obtains a stable lipid liquid crystal system by adjusting the amount of phospholipid, the mass ratio of phospholipid to oleic acid, and the mass ratio of progesterone, benzyl benzoate, and vitamin E. The system can still maintain good stability after high-temperature sterilization and accelerated testing.

[0052] 3. Furthermore, by selecting a lipid liquid crystal system with a particle size range of 270~550 nm, the present invention slows down its metabolism in animals, achieving sustained drug efficacy for more than 12 hours (e.g., 24 hours, 36 hours); it can also achieve pharmacokinetic differences between the uterus and other tissues, thereby reducing adverse effects on other tissues. Attached Figure Description

[0053] Figure 1 The results of the fat deposition test of progesterone lipid liquid crystal injection and progesterone injection (oil injection) are presented.

[0054] Figure 2A comparison of the in vivo pharmacokinetic curves of progesterone lipid liquid crystal injection, progesterone injection (oil injection), and progesterone injection (water injection).

[0055] Figure 2 In the study, Group A was progesterone injection (oil injection), Group B was progesterone injection (water injection), Group C was progesterone lipid liquid crystal injection (Example 1a), Group D was progesterone lipid liquid crystal injection (Example 1b), Group E was progesterone lipid liquid crystal injection (Example 1c), and Group F was progesterone lipid liquid crystal injection (Example 1d).

[0056] Figure 3 A comparison of drug retention rates in the uterus between progesterone lipid liquid crystal injection (Example 1a, Example 1d) and progesterone injection (oil injection). Detailed Implementation

[0057] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments. However, the scope of protection of the present invention includes, but is not limited to, these embodiments. Any changes or equivalent substitutions that do not depart from the concept of the present invention are included within the scope of protection of the present invention.

[0058] The instruments and reagents used in the embodiments of this invention are all commercially available products. As an example, the model, specifications, and manufacturer information of some reagents are as follows:

[0059] Reagents:

[0060] Progesterone injection (oil injection) (specification: 20mg / ml, manufacturer: Zhejiang Xianju Pharmaceutical);

[0061] Progesterone injection (water injection) (specification: 20mg / ml, manufacturer: IBSA);

[0062] Progesterone (Specification: Injectable grade, Manufacturer: Hubei Gedian Renfu Pharmaceutical Co., Ltd.);

[0063] Vitamin E (Specifications: Raw material, Manufacturer: BASF);

[0064] Oleic acid (manufacturer: Nanjing Weier Pharmaceutical Co., Ltd.);

[0065] equipment:

[0066] Agitator (Model: RW20, Manufacturer: IKA, GER);

[0067] High performance liquid chromatograph (model: U3000, manufacturer: Thermo Fisher Scientific, USA).

[0068] Example 1: Formulation and manufacturing process of progesterone lipid liquid crystal injection solution

[0069] The progesterone lipid liquid crystal injection provided by this invention is a lipid liquid crystal system formed by mixing an oil phase and an aqueous phase. Taking the 20 mg / ml specification as an example, the components and their amounts in each 1000 mL are as follows:

[0070]

[0071] Note: The percentages mentioned above refer to the mass-volume percentage of the component in the entire lipid liquid crystal system, that is, the mass-volume percentage of the total formulation.

[0072] Furthermore, based on the above-mentioned prescription, the progesterone lipid liquid crystal injection solution provided by the present invention may, if necessary, add appropriate amounts of pH adjusters, osmotic pressure adjusters, stabilizers, etc.

[0073] pH adjuster, used to adjust pH value, may be selected from one or more of disodium hydrogen phosphate or its hydrate, citric acid in this embodiment, but is not limited to these. It may be one or more of hydrochloric acid, sulfuric acid, acetic acid, acetate, phosphoric acid, disodium hydrogen phosphate or its hydrate, citric acid, citrate, carbonate, sodium hydroxide, ammonia, ethylenediamine, ethanolamine, tromethamine, disodium hydrogen phosphate, as long as it can adjust acidity or alkalinity.

[0074] Osmotic pressure regulators are used to adjust osmotic pressure. In this embodiment, the type of regulator can be selected from glycerol, but is not limited to this. It can be one or more of glycerol, sucrose, glucose, potassium chloride, and sodium chloride, as long as it can achieve the effect of isotonicity.

[0075] Stabilizers are used to improve the dispersion stability of lipid liquid crystals. In this embodiment, the type of stabilizer can be selected from polyvinylpyrrolidone, but is not limited to this. It can be one or more of polyvinylpyrrolidone, ethylenediaminetetraacetic acid, and disodium ethylenediaminetetraacetate, as long as it can achieve the dispersion stability of lipid liquid crystals.

[0076] Furthermore, the progesterone lipid liquid crystal injection solution provided by the present invention can be prepared by referring to the following process:

[0077] 1. Weigh the prescribed amounts of phospholipids, vitamin E, benzyl benzoate, and oleic acid. Heat in a water bath at 60-70°C and stir until completely dissolved (clear and transparent). Then add the prescribed amount of progesterone and stir at high speed for 5-15 minutes until the progesterone is completely dissolved to obtain the oil phase.

[0078] 2. Weigh the prescribed amount of water for injection. If necessary, an appropriate amount of stabilizer and / or osmotic pressure regulator may also be added. Heat in a water bath at 60-70 ℃ and stir until completely dissolved to obtain the aqueous phase.

[0079] 3. The oil phase and the aqueous phase are mixed under shear conditions and sheared at a certain temperature (e.g., 60~70 ℃) for 10~20 min to form a lipid liquid crystal phase; the shearing speed can be adjusted according to the specific parameters of the equipment (depending on the emulsification state).

[0080] 4. Transfer the sheared lipid liquid crystal phase to a high-pressure homogenizer, set the homogenization pressure to 350~450 bar, and cycle the homogenizer multiple times at a certain temperature (e.g., 60~70 ℃) until the lipid liquid crystal system with the target particle size (particle size distribution < 0.2) is obtained.

[0081] 5. Adjust the pH to 5.5~7.5, fill with nitrogen, each bottle contains 1 ml, and sterilize at 121 ℃ for 8~12 min to obtain progesterone lipid liquid crystal injection solution.

[0082] Example 2: Study on viscosity properties

[0083] The viscosity of the progesterone lipid liquid crystal injection obtained in Example 1 and the commercially available progesterone injection (oil injection) was measured using a viscometer. The results are shown in Table 1.

[0084] Table 1. Viscosity test results

[0085]

[0086] As shown in Table 1, compared with commercially available progesterone injections (oil injections), the progesterone lipid liquid crystal injection provided by this invention has a significantly lower viscosity, indicating less injection resistance and less pain at the injection site. Therefore, it can significantly improve injection compliance during clinical use and solve the shortcomings of commercially available "oil injections," such as difficulty in injection and pain at the injection site.

[0087] Example 3: Study on Local Retention

[0088] Currently available progesterone injections (oil injections) often result in hardening at the injection site after administration, which is difficult to resolve and causes significant local retention. However, the progesterone lipid liquid crystal injection provided by this invention can effectively avoid this side effect.

[0089] Fat deposition experiment: Commercially available progesterone injection (oil injection) and the progesterone lipid liquid crystal injection provided by this invention (Example 1) were injected intramuscularly into the lateral thigh of rats, respectively. Six rats were in each group, and each rat received 0.3 mL at each injection site. The administration frequency was once daily for four consecutive days at the same injection site, during which time the rats were allowed normal food and water intake. After the administration period, the rats were sacrificed, and the injection sites were dissected to observe the fat deposition status. Results are shown below. Figure 1 .

[0090] from Figure 1As can be seen from the present invention, the progesterone lipid liquid crystal injection solution is easy to diffuse, and only a small amount of fat is deposited at the injection site, which is significantly less than the fat deposition at the injection site of progesterone injection solution (oil injection), which can effectively prevent hardening at the injection site and significantly reduce adverse reactions at the injection site.

[0091] Example 4: Effect of phospholipid dosage on the stability of lipid liquid crystal system

[0092] Based on preliminary exploratory research, the inventors considered factors such as the physicochemical properties of the active ingredients, the safety and accessibility of excipients for clinical use, and lipid liquid crystal nanopolydispersities. They initially established phospholipids as amphiphilic lipids for forming lipid liquid crystal structures, excluding the use of other commonly used amphiphilic lipids (such as glyceryl monooleate, phytanetriol, etc.).

[0093] Referring to Example 1, assuming that the total amount of the progesterone lipid liquid crystal injection formulation is a constant and the types of each component in the formulation remain unchanged, the effect of phospholipid dosage on the stability of the lipid liquid crystal system is investigated by examining the changes in the amount of phospholipid in the formulation.

[0094] In this embodiment, the proportion (w / v) of phospholipids in the lipid liquid crystal system was screened and set as 1.2%, 2.0%, and 2.4%. The preparation process of the progesterone lipid liquid crystal injection was referenced in Example 1.

[0095] As mentioned above, lipid liquid crystals prepared using different proportions of phospholipids were observed after being placed at 40°C for one week. The results are shown in Table 2.

[0096] Table 2. Stability of the lipid liquid crystal system after one week at 40℃

[0097]

[0098] Furthermore, the stability of the sterilized lipid liquid crystals containing different amounts of phospholipids was studied at 4℃ and 25℃, and the particle size of the lipid liquid crystals on day 0 and day 60 was measured using a nanoparticle size analyzer. The results are shown in Table 3.

[0099] Table 3. Particle size changes of the lipid liquid crystal system after 60 days of storage.

[0100]

[0101] As shown in Tables 2 and 3, when the proportion of phospholipid in the lipid liquid crystal system is 2.4% (i.e., the mass-volume percentage of the total formulation, w / v), the constructed lipid liquid crystal system has better stability and the particle size change fluctuation is the smallest after 2 months of storage, which meets the requirements. However, when the proportion of phospholipid in the lipid liquid crystal system is less than 2.4% (such as 2.0% or 1.2%), the particle size change varies greatly with the storage time, which will affect the stability and does not meet the requirements.

[0102] Therefore, the dosage of phospholipids was determined to be 2.4% (w / v) of the total prescription.

[0103] Example 5: Effect of the mass ratio of phospholipids to oleic acid on the heat resistance of lipid liquid crystal systems to sterilization.

[0104] In this embodiment, it is assumed that the types of each component in the progesterone lipid liquid crystal injection formulation remain unchanged, the mass ratio of progesterone, benzyl benzoate and vitamin E is a certain value (such as 2:5:4 or 2:4:5), and the amount of phospholipid is a certain value. By examining the difference in the mass ratio of phospholipid and oleic acid, the influence of the amount of oleic acid added on the high-temperature sterilization resistance of the constructed lipid liquid crystal system is investigated.

[0105] In this embodiment, the mass ratio of phospholipids to oleic acid was screened and determined to be 10:1 and 20:1. The preparation process of the progesterone lipid liquid crystal injection solution is as described in Example 1.

[0106] As mentioned above, lipid liquid crystal systems prepared by using different mass ratios of phospholipids and oleic acid were investigated to examine the particle size changes before and after sterilization (sterilization conditions: 121℃, 12min). The results are shown in Table 4.

[0107] Table 4. Particle size changes of lipid liquid crystal systems with different mass ratios before and after sterilization

[0108]

[0109] As can be seen from Table 4:

[0110] 1. Comparative analysis of prescriptions 4 and 5 shows that when the mass ratio of progesterone, benzyl benzoate, and vitamin E is 2:5:4, if the amount of phospholipid remains unchanged, as the amount of oleic acid decreases, the fluctuation of particle size before and after sterilization becomes larger and larger, and the particle size stability becomes worse.

[0111] 2. Comparative analysis of prescriptions 6 and 7 shows that when the mass ratio of progesterone, benzyl benzoate, and vitamin E is 2:4:5, if the amount of phospholipid remains unchanged, the particle size stability of the lipid liquid crystal becomes worse as the amount of oleic acid decreases, and even demulsification occurs, making it impossible to form a lipid liquid crystal structure (as in prescription 7).

[0112] It is evident that the ability of the prepared lipid liquid crystal system to withstand high-temperature sterilization changes due to different mass ratios of phospholipids and oleic acid, thus affecting the stability of the lipid liquid crystal system; this was an unexpected discovery made by the inventors through exploratory experimental research.

[0113] Therefore, the mass ratio of phospholipids to oleic acid was determined to be 10:1, and the mass ratio of progesterone, benzyl benzoate, and vitamin E was tentatively set at 2:5:4 or 2:4:5.

[0114] Example 6: Effect of the mass ratio of benzyl benzoate to vitamin E on the heat resistance of the lipid liquid crystal system to sterilization.

[0115] Based on Example 5, assuming that the types of components in the progesterone lipid liquid crystal injection formulation remain unchanged and the mass ratio of phospholipid to oleic acid is 10:1, the mass ratio difference of progesterone, benzyl benzoate and vitamin E is further investigated to explore the effect of the mass ratio of the three on the high-temperature sterilization resistance of the constructed lipid liquid crystal system.

[0116] In this embodiment, the mass ratio of progesterone, benzyl benzoate and vitamin E was screened and investigated, and set as 2:4:5, 2:6:5 and 2:6:3. The preparation process of progesterone lipid liquid crystal injection is as described in Example 1.

[0117] As mentioned above, lipid liquid crystals prepared by using different mass ratios of progesterone, benzyl benzoate and vitamin E were investigated for particle size changes before and after sterilization (sterilization conditions: 121℃, 12min). The results are shown in Table 5.

[0118] Table 5. Particle size changes of lipid liquid crystal systems with different mass ratios before and after sterilization

[0119]

[0120] As can be seen from Table 5,

[0121] 1. When the mass ratio of phospholipid to oleic acid is 10:1, as the amount of benzyl benzoate decreases and the amount of vitamin E increases, the particle size after sterilization increases, but not as significantly as the particle size variation fluctuations in the formulations in Table 4 of Example 3; among them, the particle size variation fluctuation of formulation 6 is the largest, while that of formulations 8 and 9 is relatively smaller, indicating that the lipid liquid crystal systems of formulations 8 and 9 are more stable.

[0122] 2. Taking into account the need to minimize the fluctuation of particle size before and after sterilization, and considering the internal control quality standard that the particle size distribution PDI should be controlled to <0.2, the mass ratio of progesterone, benzyl benzoate and vitamin E is determined to be 2:6:(5-3); further, the preferred mass ratio is 2:6:5 (i.e., prescription 8) or 2:6:3 (i.e., prescription 9).

[0123] In summary, it can be seen that the mass ratio of phospholipids to oleic acid and the mass ratio of progesterone, benzyl benzoate, and vitamin E (especially the mass ratio of benzyl benzoate to vitamin E) interact and both affect the ability of the lipid liquid crystal system to withstand high-temperature sterilization. Furthermore, the amount of phospholipid used also affects the physical stability of the lipid liquid crystal system; these are all unexpected discoveries made by the inventors through exploratory experimental research.

[0124] Therefore, the following was finally determined: the proportion of phospholipids in the lipid liquid crystal system was 2.4% (w / v), the mass ratio of phospholipids to oleic acid was 10:1, and the mass ratio of progesterone, benzyl benzoate, and vitamin E was 2:6:(5-3).

[0125] Specifically, the progesterone lipid liquid crystal injection solution provided by this invention is a lipid liquid crystal system formed by mixing an oil phase and an aqueous phase. The system includes progesterone, benzyl benzoate, phospholipids, vitamin E, oleic acid, and water. If necessary, pH adjusters (such as disodium hydrogen phosphate or its hydrate, citric acid), osmotic pressure adjusters (such as glycerol), and stabilizers (such as polyvinylpyrrolidone) can also be added. The proportions of each component in the oil phase in the system are determined as follows: progesterone 2%, benzyl benzoate 6%, phospholipids 2.4%, vitamin E 3-5%, and oleic acid 0.24% (equivalent to Example 1). If pH adjusters, osmotic pressure adjusters, and stabilizers are added, appropriate amounts are sufficient.

[0126] Example 7 Effect of Homogenization Pressure on Lipid Liquid Crystal Particle Size

[0127] Based on Example 6, assuming that the types and amounts of each component in the progesterone lipid liquid crystal injection formulation remain unchanged, only the homogenization pressure in the preparation process is changed to explore the particle size difference of lipid liquid crystals prepared under different homogenization pressures.

[0128] In this embodiment, the homogenization pressure was screened and tested, and set as follows: 700~800 bar, 500~600 bar, 350~450 bar, 180~250 bar, and 100~150 bar. Other preparation process steps of progesterone lipid liquid crystal injection are as described in Example 1.

[0129] As mentioned above, taking the final formulation determined in Example 6 (equivalent to Example 1) as an example, the lipid liquid crystals obtained by homogenizing using the same homogenizing equipment and controlling different homogenizing pressures were measured with a nanoparticle size analyzer, and the results are shown in Table 6.

[0130] Table 6. Particle size variation of lipid liquid crystal systems under different homogenization pressures (n=3, mean±SD)

[0131]

[0132] Note: When the homogenization pressure is 100~150 bar, the resulting lipid liquid crystal particles are too large, leading to oil-water separation after sterilization and preventing the formation of a stable lipid liquid crystal system.

[0133] Furthermore, the pharmacokinetic characteristics of progesterone lipid liquid crystals with different particle sizes can be analyzed through in vivo pharmacokinetic studies to identify a more suitable particle size range.

[0134] Example 8: Effects of differences in pharmacokinetic properties of progesterone lipid liquid crystals with different particle sizes on the pharmacokinetics of animals.

[0135] As mentioned above, this embodiment aims to investigate the pharmacokinetic differences of progesterone lipid liquid crystal injection with different particle sizes, commercially available progesterone injection (oil injection), and commercially available progesterone injection (water injection) in rats, in order to select a progesterone lipid liquid crystal injection with slow drug metabolism and sustained effectiveness for more than 12 hours.

[0136] The experimental procedure for pharmacokinetic studies is as follows:

[0137] Experimental animals: 36 male SD rats were randomly divided into 6 groups (denoted as A, B, C, D, E, and F), with 6 rats in each group, weighing 180-220 g. During the experiment, they were housed in a standard environment with normal water and food supply.

[0138] Administration method and dosage: SD rats were administered the drug via intramuscular injection on the lateral side of the hind limb at a dose of 10 mg / kg. The drug administration groups were set up as follows:

[0139] Group A was given commercially available progesterone injection (oil injection).

[0140] Group B: Administer commercially available progesterone injection (water injection).

[0141] Group C: The progesterone lipid liquid crystal injection solution provided by the present invention was administered (Example 1a).

[0142] Group D: The progesterone lipid liquid crystal injection solution provided by the present invention was administered (Example 1b).

[0143] Group E: The progesterone lipid liquid crystal injection solution provided by the present invention was administered (Example 1c).

[0144] Group F: The progesterone lipid liquid crystal injection solution provided by the present invention was administered (Example 1d).

[0145] Sampling and testing: Blood samples were collected before administration as blank samples. At 0.5, 1, 2, 4, 8, 12, 24 and 36 h after administration, approximately 0.2 mL of blood was collected and placed in heparinized EP tubes. After centrifugation at 4000 rpm for 10 min, the supernatant was collected and stored at -80 ℃ for testing.

[0146] Detection method: The plasma drug concentration in rats at various time points was detected by HPLC-MS / MS, and drug-time curves were plotted to observe differences in drug metabolism levels. Results are shown below. Figure 2 .

[0147] from Figure 2 From this, we can know that:

[0148] 1. Compared with oil injection and the lipid liquid crystal drug delivery provided by this invention, water injection is metabolized the fastest in animals. The progesterone drug is basically metabolized within 8 hours, and it is impossible to achieve continuous drug release for 12 hours or even longer.

[0149] 2. Lipid liquid crystals with excessively small particle size (such as in Examples 1a and 1b) have higher initial blood drug concentrations and faster metabolism compared to lipid liquid crystals with large particle size, and the drug is basically released within 12 hours.

[0150] 3. Large-particle-size lipid liquid crystals (such as in Example 1c and Example 1d) are metabolized more slowly in animals, and their drug-time curves are closer to those of oil injections, which can achieve sustained drug efficacy for more than 12 hours (such as 24 hours and 36 hours).

[0151] 4. Compared to oil injection, Example 1d showed greater plasma exposure and higher relative bioavailability.

[0152] Example 9: Effect of differences in tissue distribution of progesterone lipid liquid crystals of different particle sizes on animals.

[0153] Female KM mice, approximately 6 weeks old, were used in groups of 18, each weighing 25 g. During the experiment, they were housed in a standard environment with normal access to water and food. Each group of mice was administered progesterone injection (oil injection), progesterone injection (water injection), or progesterone lipid liquid crystal injection (Examples 1a and 1d) at a dose of 80 mg / kg via intramuscular injection on the lateral side of the hind limb.

[0154] At 1, 12, and 48 hours after drug administration, the animals were euthanized, dissected, and major organs such as the heart, spleen, kidney, and uterus were removed. Residual blood and body fluids were absorbed with physiological saline, dried with absorbent paper, weighed, and then ground with grinding beads and homogenizing agent (physiological saline: methanol = 1:1). The mixture was homogenized, the grinding beads were removed, and the mixture was stored at -80℃ for later testing.

[0155] Drug concentrations in various tissues were measured, and drug concentrations (ng / mL) and drug retention rates (%) at 12 hours (h) and 48 hours (h) were calculated to analyze the tissue distribution characteristics and pharmacokinetic features of the formulation. Results are shown in Table 7 and... Figure 3 .

[0156] Table 7. Comparison of drug concentration and retention rate in different tissues

[0157]

[0158] From Table 7 and Figure 3 Analysis of the differences in progesterone drug activity in the uterus (target organ) and other tissues reveals the following:

[0159] 1. Progesterone injections are cleared very quickly in various tissues, almost completely cleared within 12 hours, making it difficult for the drug to maintain its effect.

[0160] 2. The lipid liquid crystal system provided by the present invention (especially large particle size, such as in Example 1d) exhibits the characteristics of slow clearance rate in the uterus and fast clearance rate in other tissues. This can achieve a sustained drug effect and reduce adverse effects on other tissues. This unique differentiated pharmacokinetic characteristic is completely different from commercially available progesterone water injections and progesterone oil injections.

[0161] 3. There are significant differences in drug clearance rates in the uterus among lipid liquid crystal systems with different particle sizes: A comparison of drug retention rates at 12 h and 48 h shows that small-particle-size lipid liquid crystal systems (such as in Example 1a) clear drugs faster than oil injections, while large-particle-size lipid liquid crystal systems (such as in Example 1d) clear drugs slower than oil injections, resulting in longer drug retention times and better long-acting release.

[0162] Based on Examples 7-9, the average particle size of the lipid liquid crystal system was determined to be 270-550 nm, preferably 270-360 nm or 460-550 nm.

Claims

1. A progesterone lipid liquid crystal injection solution, which is formed by mixing an oil phase and an aqueous phase to form a lipid liquid crystal system, characterized in that, The oil phase comprises progesterone, benzyl benzoate, phospholipid, vitamin E and oleic acid; the mass ratio of progesterone, benzyl benzoate and vitamin E is 2:6:(3-5); the mass-volume percentage of phospholipid in the lipid liquid crystal system is 2.4%; the mass ratio of phospholipid to oleic acid is 10:1; The water phase comprises water for injection; the average particle size of the lipid liquid crystal system is 270-550 nm.

2. The progesterone lipid liquid crystal injection according to claim 1, characterized in that, The phospholipid is selected from one or more of phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine and phosphatidylethanolamine.

3. The progesterone lipid liquid crystal injection according to claim 1, characterized in that, The mass ratio of benzyl benzoate to vitamin E is 2:1 or 6:

5.

4. The progesterone lipid liquid crystal injection according to claim 1, characterized in that, The average particle size of the lipid liquid crystal system is 270-360 nm or 450-550 nm.

5. The progesterone lipid liquid crystal injection according to claim 1, characterized in that, The amount of progesterone, benzyl benzoate, phospholipid, vitamin E and oleic acid in the lipid liquid crystal system is 2%, 6%, 2.4%, 3-5% and 0.24%, respectively, in terms of mass-volume percentage.

6. The progesterone lipid liquid crystal injection according to any one of claims 1-5, characterized in that, The lipid liquid crystal system further comprises one or more of a stabilizer, a pH regulator and an osmotic pressure regulator.

7. The progesterone lipid liquid crystal injection according to claim 6, characterized in that, The stabilizer is selected from one or more of polyvinylpyrrolidone, ethylenediaminetetraacetic acid and disodium ethylenediaminetetraacetate; The pH regulator is selected from one or more of hydrochloric acid, sulfuric acid, acetic acid, acetate, phosphoric acid, phosphate, citric acid, citrate, carbonate, sodium hydroxide, ammonia, ethylenediamine, ethanolamine, tromethamine, disodium hydrogen phosphate or a hydrate thereof; The osmotic pressure regulator is selected from one or more of glycerol, sucrose, glucose, potassium chloride and sodium chloride.

8. The method for preparing the progesterone lipid liquid crystal injection according to any one of claims 1-7, characterized in that, The method comprises the following steps: taking a prescribed amount of benzyl benzoate, vitamin E, oleic acid, phospholipid and progesterone, heating and stirring to dissolve them to obtain an oil phase; taking a prescribed amount of water, adding a proper amount of a stabilizer and an osmotic pressure regulator, heating and dissolving to obtain an aqueous phase; mixing the oil phase and the aqueous phase to form a lipid liquid crystal phase, and continuing high-pressure homogenization to obtain a lipid liquid crystal system; adjusting the pH to an appropriate value, filling with nitrogen, filling and filling, and wet heat sterilization to obtain a progesterone lipid liquid crystal injection.

9. The method of preparing the progesterone lipid liquid crystal injection according to any one of claims 1 to 7, wherein The method comprises the following steps: 1) taking a prescribed amount of benzyl benzoate, vitamin E, oleic acid and phospholipid, heating and dissolving in a water bath, adding a prescribed amount of progesterone after the phospholipid is dissolved, stirring and dissolving to obtain an oil phase; 2) taking a prescribed amount of water, adding a proper amount of a stabilizer and an osmotic pressure regulator if necessary, heating and dissolving in a water bath to obtain an aqueous phase; 3) mixing the oil phase and the aqueous phase under shearing to form a lipid liquid crystal phase; 4) performing high-pressure homogenization on the sheared lipid liquid crystal phase for multiple cycles to obtain a lipid liquid crystal system with a target particle size; 5) adjusting the pH to an appropriate value, filling with nitrogen, filling and filling, and wet heat sterilization to obtain the progesterone lipid liquid crystal injection.

10. The production method according to claim 9, wherein The water bath temperature in steps 1) and 2) is 55-75°C.

11. The production method according to claim 10, wherein The water bath temperature in steps 1) and 2) is 60-70°C.

12. The production method according to claim 9, wherein The temperature in steps 3) and 4) is 55-75°C.

13. The production method according to claim 12, wherein The temperature in steps 3) and 4) is 60-70°C.

14. The production method according to claim 9, wherein The pH in step 5) is adjusted to 5.0-8.

0.

15. The production method according to claim 14, wherein The pH in step 5) is adjusted to 5.5-7.

5.

16. The production method according to claim 9, wherein The conditions of the moist heat sterilization in step 5) are 121 ℃ for 8-12 min.

17. Use of the progesterone lipid liquid crystal injection according to any one of claims 1-7 or obtained by the preparation method according to any one of claims 8-16 in the manufacture of a medicament for the treatment of threatened abortion, prevention of premature birth, premenstrual tension syndrome, dysfunctional uterine bleeding and amenorrhea diseases, or for luteal support in assisted reproductive technology.

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