Pharmaceutical composition containing progesterone and application

Abstract

The invention discloses a pharmaceutical composition containing progesterone and application, and belongs to the technical field of pharmaceutical preparations. The pharmaceutical composition comprises progesterone serving as an active ingredient, a sustained-release carrier, a pharmaceutically acceptable solvent and an optional fat-soluble nonionic surfactant, the pharmaceutical composition has a long-acting slow-release effect, can play a more excellent drug effect, remarkably reduces the administration frequency and injection irritation, greatly improves the compliance of a patient in a clinical use process in the future, and effectively reduces the treatment operation.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and more specifically, relates to a pharmaceutical composition containing progesterone and its applications. Background Art

[0002] Progesterone is a natural progestogen secreted by the corpus luteum of the ovary and the placenta. It is a white or almost white crystalline powder, odorless, very soluble in chloroform, soluble in ethanol, ether or vegetable oil, and insoluble in water. Progesterone is widely used in all stages of pregnancy, including luteal support in assisted reproductive technology, threatened abortion, recurrent abortion, and prevention of premature birth, etc. As the gestational age gradually increases, the demand for assisted reproduction is increasing day by day. As one of the main progestogen drugs in the process of assisted reproduction, the clinical demand for progesterone is also increasing day by day.

[0003] Progesterone is a BCS Class IV drug (low solubility, low permeability). Currently, commercially available progesterone preparations mainly include oral capsules, oil-based injections, and vaginal gels, etc. Oral progesterone is restricted by low solubility and the first-pass effect of the liver, and its bioavailability is only about 5% (Stanczyk F Z, Hapgood J P, Winer S, et al. Progestogens used in postmenopausal hormone therapy: differences in their pharmacological properties, intracellular actions, and clinical effects [J]. Endocr Rev, 2013, 34(2): 171-208). After oral administration, the plasma concentration of progesterone is significantly lower than that of intramuscular injection and is very unstable. The blood drug concentration reaches its peak 1-3 hours after oral administration and then gradually decreases. The blood drug concentration is unstable and completely disappears in about 72 hours. Due to its low bioavailability, a relatively large dose often needs to be used, which leads to serious side effects, including adverse reactions such as dizziness, drowsiness, and liver damage. Current research shows that oral progesterone capsules cannot fully support endometrial development, and their effectiveness in luteal support during assisted reproduction is lower than that of intramuscular injection and vaginal administration of progesterone, while the side effects are more than those of intramuscular injection and vaginal administration. Therefore, oral progesterone capsules are not recommended as a routine luteal support drug in embryo transfer (IVF). Progesterone injection uses oil as a solvent. After intramuscular injection, the blood drug concentration increases significantly, reaches its peak at 6-8 hours, then gradually decreases, can last for 24 hours, and disappears in 48 hours. The irritation at the injection site is relatively strong, prone to form local induration, and occasionally local abscesses and sciatic nerve injuries occur. The absorption and recovery of local induration and aseptic abscesses take a long time and are difficult to eliminate. At the same time, due to the solvent diffusion effect, the drug is directly released, which not only often causes local pain and irritation at the injection site, but also causes adverse reactions such as headache and abdominal pain due to too high peak drug concentration (C max ). When progesterone is used in assisted reproduction, according to the guidelines, 40-60 mg needs to be injected daily for more than 8 consecutive weeks. The long-term high-frequency injection causes great inconvenience to the patient's life. Progesterone vaginal gel provides an additional option for patients. The content of progesterone in it is 8%. After administration through the vaginal route, the vaginal epithelial cells quickly absorb and spread to the cervix and uterine body, and complete the diffusion from the endometrium to the myometrium. Although it improves the therapeutic effect to a certain extent, it is prone to cause adverse reactions such as vaginal bleeding and abdominal pain. The randomized controlled study led by the Third Hospital of Peking University showed that the use of The incidence of luteal phase bleeding is about 12.14%, which is much higher than that of the progesterone injection group (about 3%). When bleeding occurs during the implementation of assisted reproduction, clinically, vaginal administration can only be stopped and replaced by injection. This situation not only poses corresponding safety risks to patients, but also brings great psychological pressure to patients and medical staff. Although it greatly improves the convenience of drug administration, its bioavailability is only about 5%, so in order to ensure clinical effect, it needs to be taken twice a day. The main ingredients in the formula that play a sustained-release role are polycarbophil and carbomer, which will gradually melt and be discharged from the body in the vaginal environment, causing inconvenience to patients. Considering the above physiological structure and long-term medication, although The compliance problem (irritation and daily uninterrupted injection) of frequent injection of progesterone injection has been solved, but it still brings great inconvenience to work and life, and does not solve the problem of reducing the frequency of administration, so it cannot be used as an ideal choice for long-term medication. Therefore, reducing the frequency of administration while reducing injection irritation, thereby improving patient compliance is a key problem that needs to be solved clinically.

[0004] In order to reduce the irritation of drug administration, a progesterone ethosome and its preparation method are disclosed in the patent document of Chinese patent application publication number CN102397255A, wherein progesterone, lipid material, cholesterol and short-chain alcohol are mixed to obtain an alcohol phase, a nonionic surfactant (Tween-60, Tween-80, benzyl-35, sodium cholate, etc.) and water are mixed to obtain an aqueous phase, the alcohol phase is added to the aqueous phase to obtain colostrum, and then high-pressure emulsification, secondary emulsification, cooling and solidification are performed to obtain progesterone ethosome, and the proportion of water in the system is about 50%. The patent shows that the prepared progesterone ethosome has a high encapsulation rate, a small particle size, fast transdermal absorption, and a large permeation rate, which is beneficial to improve the bioavailability and therapeutic index of the drug, and can avoid the use of organic solvents harmful to the human body, and has low irritation. However, the ethosome has the following shortcomings: (1) The progesterone content is only 0.05-0.5%, and the drug loading is low, which means that the administration volume is relatively large, which is not convenient for clinical use. (2) In the in vitro cumulative release experiment of the ethosome, there was an obvious burst release phenomenon at 1 hour, and the cumulative drug release rate reached 90% at only 12 hours. Therefore, the existing technology is difficult to solve the problem of reducing the frequency of progesterone administration.

[0005] To improve patient compliance, a preparation method of progesterone nanocrystal injection is disclosed in the patent document with the Chinese patent application publication number CN109223722A. The progesterone raw material is emulsified to obtain an oil-in-water emulsion, and then the oil-in-water emulsion is directly freeze-dried and redissolved to form nanocrystals. Nanocrystal injections often have physical stability problems, including sedimentation, aggregation, crystal growth, and crystal transformation, which may lead to changes in drug release behavior. Although nanocrystals are one of the optional technologies for long-acting preparations, they may reduce the problems related to the dosing frequency of progesterone. At the same time, nanocrystals require aseptic production, with expensive production equipment and facilities, complex processes, and relatively small batch sizes, making it difficult to meet the current market demand.

[0006] To achieve the slow release of progesterone, a progesterone thermosensitive gel injection and its preparation method are disclosed in the patent document with the Chinese patent application publication number CN113520990A. Progesterone is included in the cavity of cyclodextrin to form a complex and dissolved in water, and then the complex is mixed with a thermosensitive gel (i.e., poloxamer or a chitosan / carboxymethyl chitosan / sodium glycerophosphate system hydrogel) to embed the complex into the thermosensitive gel. However, the injection obtained by this method only achieves slow release within 12 hours. In addition, the cyclodextrin inclusion complex injection of progesterone Although it has been marketed abroad, it is rapidly absorbed after intramuscular and subcutaneous injections, with large fluctuations in blood drug concentration and prone to adverse reactions (Cometti B. Pharmaceutical and clinical development of a novel progesterone formulation[J]. Acta Obstet Gynecol Scand, 2015, 94 Suppl 161: 28-37.).

[0007] In addition, a long-acting reversible thermosensitive gel preparation of locally injected progesterone is disclosed in the patent document with the Chinese patent application publication number CN117064845A. This patent uses poloxamer as the gel matrix, adds polysorbate 80 as a solubilizer, polyethylene glycol 400 as a suspending agent, benzyl alcohol as an analgesic and preservative, and water as a dispersion solvent. In this patent, micronized progesterone is directly added to the gel matrix and then ground continuously. Progesterone is suspended in the system in a crystal state, which is a non-true solution preparation similar to the nanocrystal technology and is consistent with the technical route adopted in the patent with the Chinese patent application publication number CN109223722A.

[0008] In order to achieve the slow release of progesterone, the patent document of Chinese patent application publication number CN108635330A discloses a long-acting sustained-release progesterone gel composition, which is to add water-insoluble phospholipids, cholesterol, surfactants and progesterone into water, disperse into a uniform suspension through microjet, form a progesterone delivery liquid, and then disperse the delivery liquid in the gel matrix of poloxamer 407. Since progesterone is poorly soluble in water, with a solubility of about 8 μg / mL in water, it is difficult to reach the clinical administration concentration under the solubilization of commonly used surfactants. The invention is essentially a composite technology of liposomes and nanocrystals, using phospholipids to embed a part of progesterone, and then using surfactant to solubilize a part of progesterone, and another part of progesterone is transformed into tiny crystals under high pressure by microjet, and then the mixture is dispersed in the gel matrix. Therefore, the progesterone in the preparation exists in both molecular state and crystalline state, which causes the drug release of administration to be uncontrollable, the molecular state is released first, and the crystal particle size also affects the release rate. Therefore, the present invention is similar to nanocrystals in that the progesterone is micronized by physical means and then suspended in a gel matrix, which is basically consistent with the technical route adopted in the patent document CN109223722A. However, the progesterone in the technical route adopted in the technical solution of the present invention exists in a molecular state and appears to be a clear liquid, which is essentially different from the technical route in the patent document CN108635330A.

[0009] In order to reduce the frequency of administration, a polyethylene glycol (PEG) modified progesterone nanoparticle for injection is disclosed in the patent document of Chinese patent application publication number CN107441061A. Progesterone and polymer are dissolved in an organic solvent, and then the final emulsion is obtained by shear emulsification with an aqueous solution containing an emulsifier. After low temperature solidification, washing, concentration, and finally mixing with a protective agent and freeze drying, progesterone sustained-release nanoparticles are obtained. The progesterone sustained-release injection containing the above-mentioned progesterone sustained-release nanoparticles has a higher bioavailability than commercially available preparations and traditional progesterone nanoparticles, and achieves a sustained-release effect of more than one week. This prior art increases the compliance of patients with medication to a certain extent, but since PEGylated drugs easily stimulate the body to produce anti-PEG antibodies after entering the body, thereby targetedly removing substances containing PEG structures, as the use time increases, the drug efficacy will gradually decrease, which has been confirmed in doxorubicin long-circulating liposomes, and the degradation mechanism in the body is still unclear, and this scheme still has potential renal toxicity in clinical application.

[0010] Based on the above, the development of a progesterone dosage form with good safety and long-acting sustained-release effect is a technical problem that needs to be solved urgently in this field. Summary of the invention

[0011] 1. Problem to be solved

[0012] The first object of the present invention is to provide a progesterone pharmaceutical composition with a long-acting and sustained-release effect and low irritation, which can greatly reduce the administration frequency of progesterone preparations and improve the compliance of patients.

[0013] Based on the above object, the second object of the present invention is to further provide a progesterone composition with a reduced administration volume, which can maintain a single-dose administration at a lower volume to further improve the compliance of patients.

[0014] The third object of the present invention is to provide a progesterone injection with a long-acting and sustained-release effect and low irritation.

[0015] The fourth object of the present invention is to provide a subcutaneous progesterone injection with a small single-dose administration volume, a long-acting and sustained-release effect, and low irritation.

[0016] 2. Technical solution

[0017] To solve the problem of high administration frequency, a drug delivery system with a sustained-release effect can be used as a carrier to deliver progesterone. In-situ gel is a very promising drug delivery system, which can undergo a phase change at the injection site and form a local drug delivery depot due to changes in the external environment (such as light, temperature, pH value, and solvent exchange, etc.), and has good tissue compatibility and the ability to release drugs for a long time at the administration site. The gel matrix of in-situ gel generally uses phospholipids, chitosan, poloxamer, and polyethylene glycol block copolymers, etc. Among them, phospholipids, as one of the components of cell membranes, are easy to fuse with cell membranes to deliver drugs into cells to play a role, and have the best biocompatibility and safety. At the same time, the self-assembly characteristics of phospholipids also determine the property that phospholipids self-assemble into vesicles with a bilayer structure when encountering water. Vesicles, as an excellent drug depot, can store both lipophilic drugs and water-soluble drugs. Therefore, lipid in-situ gel prepared with phospholipids as the main excipient can interact with phospholipids using the water in tissue fluid after injection to form a multi-layer vesicle structure, thereby forming a drug depot and achieving a sustained-release effect.

[0018] However, it was found during the exploration of the present invention that when using only phospholipids as the sustained-release carrier of progesterone, due to its generally low phase transition temperature, the bilayer of the formed vesicles is easy to flow and not dense, and the drug release rate is relatively fast, and the sustained-release effect is not ideal. For example, in some embodiments, when the ratio of progesterone to phospholipids is 1:4, the release rate at 48 h is about 72.3%. Based on this calculation, it should be completely released at 72 - 96 h. Although it has a certain sustained-release effect, due to the low strength of the bilayer structure formed by phospholipids, the sustained-release effect on progesterone is difficult to achieve a long-term release of more than 144 h.

[0019] In order to further improve the sustained-release duration of progesterone by phospholipids, cholesterol was considered to be added to the sustained-release system to enhance the strength of the phospholipid bilayer structure. Cholesterol and phospholipids are the basic substances that make up cell membranes and usually jointly form cell membranes. Cholesterol can regulate the fluidity and permeability of the phospholipid bilayer, so it can effectively change the drug release rate and avoid the burst release effect caused by the rapid release of drugs.

[0020] Adding cholesterol to the sustained-release system achieved the purpose of extending the sustained-release time. However, the unexpected discovery is that The addition of cholesterol unexpectedly increased the drug-loading concentration of progesterone.

[0021]

Drug Composition

[0022] On this basis, the first aspect of the present invention provides a drug composition, comprising the following components:

[0023] a. Active ingredient;

[0024] b. Sustained-release carrier;

[0025] c. Lipid-soluble non-ionic surfactant;

[0026] d. Pharmaceutically acceptable solvent;

[0027] Among them, the active ingredient includes progesterone;

[0028] The sustained-release carrier includes phospholipids and cholesterol;

[0029] The pharmaceutically acceptable solvent includes a dispersion solvent;

[0030] The dispersion solvent includes NMP and short-chain alcohols;

[0031] The active ingredient accounts for 5-13% (w / w) of the total weight of the drug composition;

[0032] The ratio of the phospholipids to the active ingredient is (3.5-10):1 (w / w)

[0033] The ratio of the cholesterol to the active ingredient is (0.5-3.5):1 (w / w);

[0034] The ratio of the dispersion solvent to the active ingredient is (2-8):1 (w / w);

[0035] The ratio of the lipid-soluble non-ionic surfactant to the active ingredient is (0-1.2):1 (w / w);

[0036] The pharmaceutically acceptable solvent optionally includes a phase transition regulator, and the weight proportion of the phase transition regulator in the composition is 0-4%.

[0037] In the present invention, "optionally" means that this component may or may not be included. When the weight percentage is 0, it means that this component is not included. For example, in the above-mentioned composition, when the weight percentages of the fat-soluble nonionic surfactant and the phase transition regulator are 0, it means that these components are not included.

[0038] In the composition of the present invention, the sustained-release carrier refers to a carrier of the active ingredient, which is a preparation that enables the active ingredient to be slowly released in a specified release medium as required after administration. Compared with the corresponding ordinary preparation without a sustained-release carrier, the dosing frequency is reduced, and the compliance of patients can be significantly increased. In some cases, the side effects of the drug can be reduced. The sustained-release carrier includes phospholipids and cholesterol. Phospholipids are amphiphilic and can spontaneously self-assemble into lyotropic gels in the presence of water, thereby exerting a sustained-release effect.

[0039] As a preference for any technical solution of the first aspect of the present invention, the ratio of the phospholipid to the active ingredient is (4-8):1 (w / w).

[0040] As a preference for any technical solution of the first aspect of the present invention, in the composition of the present invention, the phospholipid is a natural phospholipid, including soybean phospholipid, lecithin or a combination thereof. Preferably, the content of phosphatidylcholine in the natural phospholipid is not less than 80%. The drug loading concentration in the liquid composition is affected by multiple factors. For example, the solubility of the active ingredient in the dispersion solvent, the volume of the dispersion solvent, etc. In the liquid composition, in order to obtain a uniform and stable system, the solubility of other non-liquid components such as the sustained-release carrier in the dispersion solvent should also be considered simultaneously. In the system of the first aspect of the present invention, the dispersion solvent is used to dissolve the active ingredient and the sustained-release carrier. Under the combined influence of NMP and short-chain alcohol as the dispersion solvent and cholesterol, on the one hand, the proportion of the dispersion solvent is reduced, and on the other hand, the solubility of progesterone is increased. Finally, the drug loading concentration of progesterone can be increased to a maximum of 130 mg / mL. When the drug loading concentration is increased, in some application scenarios, such as subcutaneous injection, the reduction of the single injection dose can be achieved, and the compliance of patients in drug use can be improved from multiple aspects such as convenience and irritation.

[0041] While increasing the drug loading concentration, the addition of cholesterol effectively enhances the sustained-release effect of progesterone compared with the phospholipid system. The in vitro release results show that within a certain range, as the amount of cholesterol used increases, the release of progesterone in the early stage slows down, reducing the burst release. At the same time, surprisingly, the addition of cholesterol also unexpectedly increases the drug loading concentration of progesterone in the system, which can further reduce the dosing volume and improve the compliance in clinical use.

[0042] Based on the technical solution of the first aspect of the present invention, the applicant further surprisingly found that the promoting effect of cholesterol on the solubility of progesterone does not have a proportional relationship as expected. When the ratio of cholesterol to progesterone in the system exceeds a certain range, the drug-loading concentration of progesterone in the system decreases.

[0043] Therefore, as a preference for any technical solution of the first aspect of the present invention, the ratio of cholesterol to the active ingredient is (1 - 2.5):1 (w / w), preferably the ratio of cholesterol to the active ingredient is (1.5 - 2.5):1 (w / w). When the ratio of cholesterol to the active ingredient is less than 0.5:1, the promoting effect of cholesterol on the dissolution of progesterone in the system is not ideal. When it is between 1:1 and 2.5:1, the promoting effect of cholesterol on the dissolution of progesterone in the system is very significant, and the solution remains a light yellow clear liquid after standing for 60 days; when the ratio of cholesterol to the active ingredient is greater than or equal to 4:1, the promoting effect of cholesterol on the dissolution of progesterone in the system disappears. Although a clear solution can be obtained, a small amount of fine crystals precipitate after the solution stands for 24 hours; a large amount of fine crystals precipitate after the solution stands for 60 days.

[0044] The selection of the dispersion medium is crucial for injectables. The dispersion medium can play the role of dissolving the raw and auxiliary materials and solve the problem of drug administration convenience. However, at the same time, the dispersion medium will also cause corresponding irritation. Therefore, the selection of the dispersion medium needs to consider both solubility and irritation to ensure the safety and effectiveness of the drug. In long-acting injectables, co-solvents are often used in in-situ forming dosage forms. Co-solvents can inhibit gel formation, reduce viscosity, and ensure the injectability of the drug. Once the drug reaches the injection site, the co-solvent combines with the surrounding tissue fluid to form an in-situ gel. The in-situ gel based on phospholipids undergoes a phase change after interacting with the tissue fluid to form a drug reservoir with bilayer characteristics. The speed of this phase change depends on the miscibility of the dispersion medium with water. The higher the miscibility, the faster the phase change speed, and the faster the drug reservoir is formed, which can effectively reduce the burst release of the drug. Commonly used co-solvents include NMP, ethanol, 1,2-propanediol, glycerol, etc. NMP is a water-soluble organic solvent with strong biocompatibility. Due to its water solubility, it can quickly disperse with the tissue fluid in the tissue at the injection site after injection, enabling the preparation to form an in-situ gel. NMP not only has good water solubility but also has good solubility for progesterone. Therefore, the present invention selects it as the first choice for the dispersion solvent. However, the solubility of other excipients such as cholesterol and phospholipids in NMP is limited. Therefore, one or more other co-solvents are needed to solve the dissolution problem of other excipients.

[0045] In the composition of the present invention, the short-chain alcohol in the dispersion solvent (dispersion medium) refers to a fatty alcohol with no more than 3 carbon atoms, including straight-chain or branched-chain saturated or unsaturated fatty alcohols, and the fatty alcohol can be a monohydric alcohol or a polyhydric alcohol. For example, ethanol, propanol, propylene glycol, glycerol, etc.

[0046] Preferably, as any one of the technical solutions of the first aspect of the present invention, the short-chain alcohol includes one or more of ethanol, propylene glycol, or glycerol.

[0047] Preferably, as any one of the technical solutions of the first aspect of the present invention, the short-chain alcohol contains at least ethanol. Ethanol is a commonly used solvent in the preparation, which has good biological safety and good diffusibility, and can accelerate the rate of gel formation when the preparation meets the aqueous phase. The solubility of phospholipids in the present invention in ethanol is greater than its solubility in NMP. Therefore, the present invention selects NMP and ethanol as co-solvents to solve the dissolution problem of progesterone and excipients (phospholipids, cholesterol, surfactants).

[0048] The toxicity of the solvent determines its maximum dosage in the preparation. In order to further improve the safety of the preparation and reduce the impact of the solvent on the injection site after injection, the present invention screens the solvent dosage based on considering the safety of the composition and the drug loading concentration to obtain the solvent dosage suitable for the composition of the present invention.

[0049] Preferably, as any one of the technical solutions of the first aspect of the present invention, the dispersion solvent contains NMP and ethanol, and the ratio of NMP to ethanol is (2-4):1 (w / w).

[0050] Preferably, as any one of the technical solutions of the first aspect of the present invention, the ratio of the dispersion solvent to the active ingredient is (2-8):1 (w / w), preferably (3-6):1 (w / w).

[0051] In order to further increase the drug loading concentration and reduce the irritation caused by the dispersion solvent, based on any one of the technical solutions of the first aspect of the present invention, another object of the present invention is to further reduce the proportion of the dispersion solvent in the composition.

[0052] As a preference for any technical solution of the first aspect of the present invention, a surfactant is added to the pharmaceutical composition to reduce the amount of the dispersion solvent while ensuring the drug loading concentration. Substantially, the addition of the surfactant reduces the amount of the dispersion solvent. On the one hand, while ensuring the drug loading amount, the overall volume of the composition is reduced, achieving a substantial increase in the mass-volume concentration of the drug loading; during the research process of the present invention, with the addition of the surfactant, the progesterone drug loading amount in the lipid gel constructed by phospholipids and cholesterol is effectively increased. On the other hand, the reduction in the amount of the dispersion solvent can simultaneously reduce the irritation during administration. In some application scenarios, such as subcutaneous injection or intramuscular injection, it can reduce symptoms such as local redness, pain, and induration at the injection site.

[0053] As a preference for any technical solution of the first aspect of the present invention, the lipophilic nonionic surfactant is GMS.

[0054] Since GMS itself is a solid and needs to be dissolved in the dispersion solvent first before enhancing the dissolution of progesterone, its addition amount is also limited by the amount of the dispersion solvent. When the addition ratio of GMS is too high, although it can reduce the amount of the dispersion solvent more, the stability of the composition becomes poor, and crystals are likely to precipitate in the system after dissolution and standing for a period of time. Therefore, as a preference for any technical solution of the first aspect of the present invention, the ratio of GMS to the active ingredient is (0 - 1.2):1 (w / w), more preferably (0 - 1):1 (w / w).

[0055] Another unexpected discovery is that, compared with the composition without GMS added, on the premise of meeting the stability requirements of the composition, the addition of GMS as a surfactant further extends the sustained-release time of the active ingredient.

[0056] As a preference for any technical solution of the first aspect of the present invention, the pharmaceutical composition comprises the following components:

[0057] a. An active ingredient;

[0058] b. A sustained-release carrier;

[0059] c. A lipophilic nonionic surfactant;

[0060] d. A pharmaceutically acceptable solvent;

[0061] Wherein, the active ingredient comprises progesterone;

[0062] The sustained-release carrier comprises phospholipids and cholesterol;

[0063] The pharmaceutically acceptable solvent comprises a dispersion solvent;

[0064] The dispersion solvent comprises an organic solvent NMP and a short-chain alcohol;

[0065] The active ingredient accounts for 5-13% (w / w) of the total weight of the pharmaceutical composition;

[0066] The ratio of the phospholipid to the active ingredient is (4-8):1 (w / w);

[0067] The ratio of the cholesterol to the active ingredient is (1-2.5):1 (w / w);

[0068] The ratio of the dispersion solvent to the active ingredient is (3-6):1 (w / w);

[0069] The ratio of the lipophilic non-ionic surfactant to the active ingredient is (0-1):1 (w / w).

[0070] As a preference of any technical solution of the first aspect of the present invention, the phase transition regulator includes water. Preferably, the water is water for injection.

[0071] Water for injection can adjust the phase transition time of the in-situ gel preparation formed by the pharmaceutical composition. In order to maximize the phase transition speed after in-situ gel injection, a certain proportion of water for injection can be added in the present invention.

[0072] As a preference of any technical solution of the first aspect of the present invention, the percentage of the water in the total weight of the composition is 0-4%. Within this range, the pharmaceutical composition does not undergo phase transition during storage and is suitable as an injection.

[0073] As a preference of any technical solution of the first aspect of the present invention, the percentage of the water in the total weight of the composition is 4%-7%, excluding 4%. Within this percentage range, the pharmaceutical composition undergoes phase transition and is suitable as a gel.

[0074] As a preference of any technical solution of the first aspect of the present invention, the pharmaceutical composition is a drug administered by injection, or a drug administered by subcutaneous implantation, or a drug administered by topical and vaginal administration.

[0075] According to the preference of any technical solution of the first aspect of the present invention, as the form (dosage form) of the pharmaceutical composition with preventive effect and / or therapeutic effect, it is mainly in liquid form and can be directly formulated or prepared into a form that can be used as raw materials for drugs, quasi-drugs, etc. Preferably, the dosage form of the pharmaceutical composition includes sustained-release agents, controlled-release agents, pulsatile release agents, injection solutions. The formulation and preparation method of the pharmaceutical composition of the present invention in these pharmaceutical preparation forms can be achieved by methods and experiences well-known to those skilled in the art.

[0076] As a preference of any technical solution of the first aspect of the present invention, the dosage form of the pharmaceutical composition is an injection solution.

[0077] Preferably, for any technical solution of the first aspect of the present invention, the injection solution includes a subcutaneous injection solution or an intramuscular injection solution.

[0078] Preferably, for any technical solution of the first aspect of the present invention, the mass percentage content of progesterone in the pharmaceutical composition is any value selected from any of the following numerical ranges:

[0079] 5.0 - 12.0 wt%;

[0080] 5.0 - 11.0 wt%;

[0081] 5.0 - 10.0 wt%, 5.5 - 10.0 wt%, 6.0 - 10.0 wt%, 6.5 - 10.0 wt%, 7.0 - 10.0 wt%, 7.5 - 10.0 wt%, 8.0 - 10.0 wt%, 8.5 - 10.0 wt%, 9.0 - 10.0 wt%, 9.5 - 10.0 wt%;

[0082] 5.0 - 9.0 wt%, 5.5 - 9.0 wt%, 6.0 - 9.0 wt%, 6.5 - 9.0 wt%, 7.0 - 9.0 wt%, 7.5 - 9.0 wt%, 8.0 - 9.0 wt%, 8.5 - 9.0 wt%;

[0083] 5.0 - 8.0 wt%, 5.5 - 8.0 wt%, 6.0 - 8.0 wt%, 6.5 - 8.0 wt%, 7.0 - 8.0 wt%, 7.5 - 8.0 wt%.

[0084] Preferably 5.5 - 10.0 wt%; More preferably 6.0 - 9.0 wt%; Most preferably 7.5 - 8.5 wt%.

[0085] Preferably, for any technical solution of the first aspect of the present invention, the concentration of progesterone (drug - loading concentration) in the pharmaceutical composition is 55 - 130 mg / mL.

[0086] Preferably 55 - 100 mg / mL; Preferably 60 - 100 mg / mL; More preferably 60 - 80 mg / mL.

[0087]

Injection Solution

[0088] The second aspect of the present invention provides an injection solution comprising the pharmaceutical composition described in any technical solution of the first aspect of the present invention.

[0089] For the injection solution according to any technical solution of the second aspect of the present invention, the injection solution is a subcutaneous injection solution or an intramuscular injection solution.

[0090]

In - situ Gel

[0091] The third aspect of the present invention provides an in-situ gel, which comprises the pharmaceutical composition described in any one of the technical solutions of the first aspect of the present invention.

[0092]

Sustained-release depot drug

[0093] The fourth aspect of the present invention provides a sustained-release depot drug, which comprises the pharmaceutical composition described in any one of the technical solutions of the first aspect of the present invention. It should be noted here that the in-situ gel can be regarded as a specific dosage form in the sustained-release depot drug.

[0094]

Preparation method of pharmaceutical composition

[0095] The fifth aspect of the present invention provides a preparation method of the pharmaceutical composition described in any one of the technical solutions of the first aspect of the present invention, comprising the following steps:

[0096] Prepare a sustained-release carrier solution: dissolve the sustained-release carrier in a pharmaceutically acceptable solvent;

[0097] Dissolve the active ingredient: dissolve the active ingredient in the sustained-release carrier solution to obtain a pharmaceutical composition.

[0098] As a preference of any one of the technical solutions of the fifth aspect of the present invention, before dissolving the active ingredient in the sustained-release carrier solution, dissolve a surfactant in the sustained-release carrier solution.

[0099] As a preference of any one of the technical solutions of the fifth aspect of the present invention, filter the solution obtained after dissolving the active ingredient with a filter membrane; the filter membrane is preferably a filter membrane with a pore size not greater than 0.22 μm.

[0100] As a preference of any one of the technical solutions of the fifth aspect of the present invention, add water for injection to the solution obtained after dissolving the active ingredient. Preferably, the water for injection is filtered with a filter membrane; the filter membrane is preferably a filter membrane with a pore size not greater than 0.22 μm.

[0101] As a preference of any one of the technical solutions of the fifth aspect of the present invention, the pharmaceutical composition is stored in a sterile vial. Preferably, the pharmaceutical composition is stored in a light-proof environment at a storage temperature not higher than 30°C.

[0102] As a preference of any one of the technical solutions of the fifth aspect of the present invention, the pharmaceutical composition can be sterilized by a moist heat sterilization process.

[0103] Specifically, the preparation method of the pharmaceutical composition includes:

[0104] (1) Weigh the prescribed amount of phospholipid, add the prescribed amount of absolute ethanol, stir or dissolve by ultrasound, and after dissolution, add NMP and mix evenly;

[0105] (2) Add the prescribed amount of cholesterol. If GMS is included in the formulation, then add the prescribed amount of GMS and stir or dissolve by ultrasonic wave.

[0106] (3) Add the prescribed amount of active ingredient and dissolve.

[0107] (4) Filter with a 0.22 μm filter membrane. If water is included in the formulation, then add the prescribed amount of injection water filtered through 0.22 μm to the filtered sample and stir evenly.

[0108] (5) Subpackage into sterile vials, fill with nitrogen, plug, crimp the cap, and store at room temperature in the dark.

[0109] (6) Alternatively, sterilize the above sample after crimping the cap by using the moist heat sterilization process at 121 °C for 12 min, and store the sterilized finished product in the dark.

[0110]

Pharmaceutical applications of pharmaceutical composition, injection, in-situ gel, and sustained-release depot drug

[0111] The sixth aspect of the present invention provides the pharmaceutical composition of the first aspect of the present invention, or the injection of the second aspect of the present invention, or the in-situ gel of the third aspect of the present invention, or the sustained-release depot drug of the fourth aspect of the present invention, and the application of any one of their technical solutions in the preparation of a drug for preventing or treating menstrual disorders, such as amenorrhea, functional uterine bleeding, luteal insufficiency, threatened abortion, and habitual abortion.

[0112] As a preference of any one of the technical solutions of the sixth aspect of the present invention, the concentration of progesterone in the pharmaceutical composition is 55 - 130 mg / mL.

[0113] Preferably 55 - 100 mg / mL; preferably 60 - 100 mg / mL; more preferably 60 - 80 mg / mL.

[0114] When administered at the above drug-loading concentration, satisfactory sustained-release and other effects can be obtained.

[0115] Any implementation scheme (technical solution) of any aspect of the present invention can be combined with other implementation schemes without contradiction. In addition, in any implementation scheme of any aspect of the present invention, any technical feature can be applied to the corresponding technical feature in other implementation schemes without contradiction.

[0116] Without contradiction, any technical feature possessed by any aspect of the present invention or any implementation scheme of that aspect is equally applicable to any other implementation scheme or any implementation scheme of any other aspect. Of course, when applicable to each other, the corresponding features can be appropriately modified if necessary. The following further describes each aspect and feature of the present invention.

[0117] In addition, various terms and phrases used in the present invention have general meanings well-known to those skilled in the art. Even so, the present invention still hopes to provide more detailed explanations and interpretations of these terms and phrases herein. In case of any inconsistency between the mentioned terms and phrases and their well-known meanings, the meanings expressed in the present invention shall prevail.

[0118] In the present invention, the term "comprising" or "containing" means that various components can be applied together to the compositions of the present invention.

[0119] The compositions of the present invention also include isomers, solvates of the specific compounds in the above compositions, or pharmaceutically acceptable salts thereof, provided that they also have the same or substantially the same functions as the specific compounds in the compositions.

[0120] In the present invention, the term "in-situ gel" refers to a preparation in which progesterone undergoes a phase transition at the site of administration after being administered in a solution state and solidifies from a liquid to form a semi-solid gel.

[0121] In the present invention, the term "sustained-release depot drug" means that an injection solution composed of phospholipids, cholesterol, GMS, NMP, and ethanol with progesterone as the active ingredient forms a drug depot at the injection site and slowly releases to produce a drug effect.

[0122] In the present invention, the term "phase transition regulator" refers to water, which is a substance that can accelerate the speed of the composition changing from a clear liquid to a light yellow or white semi-solid gel state after being added.

[0123] The actual dosage level and administration method of the active ingredient in the pharmaceutical compositions of the present invention can be changed, for example, by increasing the drug loading concentration, or by increasing the administration volume, or by increasing the administration frequency, so that the amount of the active substance obtained can effectively achieve the desired therapeutic response for a specific patient. The dosage level must be selected according to the activity of the specific active substance, the administration route, the severity of the condition to be treated, and the condition and medical history of the patient to be treated.

[0124] Therefore, compared with the prior art, the present invention constructs a progesterone pharmaceutical composition using phospholipids and cholesterol as the main excipients, effectively increasing the drug loading concentration of progesterone and enabling the slow release of progesterone. In-vivo experiments show that the sustained-release duration of the pharmaceutical composition 3 in the examples reaches more than 168 hours. Especially when the pharmaceutical composition is administered as a subcutaneous injection of progesterone injection solution, due to the high drug loading concentration, the injection solution can achieve the dual purposes of forming a drug depot for long-term release and reducing irritation with a minimum injection volume, and is an ideal long-acting preparation with a high drug loading capacity, a long release time, low injection irritation, and good patient compliance.

[0125] 3. Beneficial effects

[0126] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0127] (1) The design process of the present invention always focuses on the dissolution and release of progesterone. Through a large number of experimental studies, a progesterone pharmaceutical composition has been invented. This pharmaceutical composition can be used as a lipid in-situ gel, with a long-acting and sustained-release effect, significantly reducing the frequency of drug administration and injection irritation. Further, in Example 14, through pharmacokinetic testing, the duration of the pharmaceutical composition 3 in rabbits can reach more than 168 hours. In the endometrial conversion pharmacodynamic experiment of rabbits, the progesterone pharmaceutical composition in the experimental group was administered only once a week, and repeated administration was carried out 3 times, while the control group of progesterone oil injection was continuously administered for 21 days. Surprisingly, the experimental results found that the progesterone pharmaceutical composition in the experimental group effectively increased the concentration of progesterone in the uterus. The concentration of progesterone in the experimental group was about 15 times that of the control group, and the uterine coefficient was nearly 2 times that of the control group. The results of uterine staining sections showed that the number of glands in the progesterone pharmaceutical composition of the experimental group was also significantly more than that of the control group. At the same time, in Example 11, compared with the progesterone oil injection, no irritating reaction occurred at the injection site of the progesterone pharmaceutical composition, while erythema occurred 7 days after administration at the injection point in the progesterone oil injection group, showing mild irritation. Therefore, compared with the prior art, the pharmaceutical composition prepared by the present invention has a long-acting and sustained-release effect, exerts better pharmacodynamic effects, and at the same time significantly reduces the frequency of drug administration and injection irritation, which will greatly improve the compliance of patients during clinical use in the future and effectively reduce treatment operations.

[0128] (2) For the purpose of long-acting and sustained release, cholesterol is added to the composition. Cholesterol can change the fluidity of the membrane formed after the phase transition of the phospholipid reservoir, making the phospholipid membrane after phase transition more stable, effectively controlling the burst release of the drug, and playing a long-acting role together with phospholipids. However, the present invention surprisingly finds that the addition of cholesterol in the composition effectively increases the concentration of the drug. For example, on the basis of a formulation without cholesterol, a certain amount of cholesterol is added so that the weight ratio of cholesterol to progesterone is 1:1, and the drug loading concentration of the pharmaceutical composition is increased from about 68 mg / ml to about 116 mg / ml. At the same time, the increase in the drug loading concentration promoted by cholesterol can only be achieved at a specific ratio, and the increase in the drug loading concentration significantly reduces the administration volume or frequency of administration of the pharmaceutical composition preparation of the present invention, improving the clinical convenience of the preparation.

[0129] (3) The present invention further discovers that the fat-soluble nonionic surfactant GMS unexpectedly exhibits the effect of delaying drug release in the composition. As shown by the data in Example 8, the cumulative release rate of the composition without GMS at 72 h is approximately 58.3%, while the cumulative release rate of the composition with GMS at 72 h is approximately 39.8%, indicating that it further significantly reduces the drug release rate of the phospholipid reservoir, enables the composition to have a longer release time, and effectively achieves the invention purpose of long-acting and sustained release. At the same time, the present invention uses GMS as the fat-soluble nonionic surfactant, effectively increases the drug concentration in the composition, thereby further reducing the administration volume or frequency of the preparation, and is more conducive to improving the clinical use convenience of the preparation.

[0130] (4) In a further composition scheme of the present invention, a small amount of water can be contained. As a phase transition regulator, water can effectively regulate the formation of the reservoir. In the in vitro experiment of Example 12, compared with the composition without water, when a total amount of about 7% of water is added to the system, the composition changes from a clear liquid to a milky white semi-solid. Therefore, adding water to the system can accelerate the speed of forming an in-situ gel at the injection site and reduce the burst release to a certain extent. Description of the Drawings

[0131] Figure 1 They are the appearance of the drug composition 3 in Example 12 and the appearance after phase change.

[0132] Figure 2 They are the safety and in vivo forming diagrams of the solvent group and composition 1 in Example 13 after injection for 24 h and 168 h respectively (the right figure is a partial enlarged view of the left figure).

[0133] Figure 3 They are the irritation and in vivo forming diagrams at the injection site in the rabbit endometrial transformation experiment of Example 14.

[0134] Figure 4 They are the uterine weight (A) and progesterone concentration (B) in the uterine tissue in the rabbit endometrial transformation experiment of Example 14.

[0135] Figure 5 They are the pathological section diagrams of the uterus in the rabbit endometrial transformation experiment of Example 14. Detailed Embodiments

[0136] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this invention belongs; the term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0137] For those not specified in the examples, they are carried out under conventional conditions or conditions recommended by the manufacturer. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0138] As used herein, the term "about" is used to provide flexibility and imprecision associated with a given term, measurement, or value. A person skilled in the art can easily determine the degree of flexibility of a specific variable.

[0139] Concentrations, amounts, and other numerical data may be presented herein in a range format. It should be understood that such a range format is used only for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range but also all individual numerical values or sub-ranges subsumed within the stated range as if each numerical value and sub-range were explicitly recited. For example, a numerical range of about 1 to about 4.5 should be interpreted to include not only the explicitly recited limits of 1 to about 4.5 but also the individual numbers (such as 2, 3, 4) and sub-ranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges that recite only one numerical value, such as "less than about 4.5", which should be interpreted to include all of the above values and ranges. In addition, this interpretation should apply regardless of the breadth of the range or feature being described.

[0140] The present invention will be further described below in conjunction with specific embodiments.

[0141] Example 1

[0142] Study on the solubility of solvents for active ingredients and phospholipids

[0143] Combined with the solubility of the drug in the solvent and the solubilization effect of surfactants, combined means are used to increase the solubility of the target drug, and a solvent or solvent composition is used to screen the optimal system for dissolving progesterone. The results are shown in Table 1.

[0144] Table 1 Solubility of progesterone in different solvents or solvent compositions and rat toxicity data of the solvents

[0145] Solvent or composition Solubility (mg / mL) <![CDATA[Toxicity data of the dispersion medium (LD 50 , subcutaneous injection)]]> Water ≈0.008 / NMP ≈154.20 2.00 g / kg Ethanol ≈31.75 8.29 g / kg 1,2 - Propylene glycol ≈25.10 22.5 g / kg Ethanol - phospholipid (1:1) ≈125.50 /

[0146] As can be seen from the above table, among the single solvents, the solubility of progesterone in NMP is the largest, followed by ethanol. In addition, the animal toxicity data show that each dispersion medium has good biosafety when used for subcutaneous injection. According to the dosing calculation of the present invention, the dosage of each dispersion solvent during injection is lower than LD 50 , so using NMP and ethanol as dispersion solvents has good safety.

[0147] Phospholipids have the property of self-assembly. They can spontaneously form vesicle structures in water and effectively encapsulate active ingredients. As can be seen from the above table, the addition of phospholipids can increase the solubility of active ingredients in ethanol, and the type and dosage of phospholipids directly affect their solubility in solvents. To further screen for suitable phospholipids, solubility experiments were carried out on phospholipids with different types and different phosphatidylcholine (PC) contents. Take 0.5 g of phospholipids and 1 g of solvent (ethanol or NMP) and place them in a container. Seal it and place it in a water bath at 50 °C. Observe the dissolution time and state of different phospholipids. The results are shown in Table 2.

[0148] Table 2 Solubility Experiment of Phospholipids

[0149] Phospholipid type Solvent Time min (50 °C) Appearance 50% Soybean phospholipid Ethanol 5.18 Deep yellow clear liquid 70% Soybean phospholipid Ethanol 4.25 Yellow clear liquid 95% Soybean phospholipid Ethanol 1.33 Light yellow clear liquid 80% Egg yolk lecithin Ethanol 1.51 Light yellow clear liquid DPPC (>95%) Ethanol / Not completely dissolved HSPC (>95%) Ethanol / Not completely dissolved 95% Soybean phospholipid NMP 13.01 Light yellow clear liquid 80% Egg yolk lecithin NMP 28.12 Dark brown clear liquid

[0150] According to the above results, compared with NMP, ethanol is more suitable as a good solvent for phospholipids. Natural-source soybean phospholipids and egg yolk lecithin have good solubility in ethanol, while synthetic dipalmitoyl phosphatidylcholine (DPPC) and hydrogenated soybean phospholipid (HSPC) cannot be completely dissolved in ethanol. As the PC content in soybean phospholipids increases, its dissolution rate in ethanol becomes faster. Based on this, the present invention preferably selects soybean phospholipids with a purity of 95% or egg yolk lecithin with a content of 80% as the sustained-release carrier.

[0151] In summary, through solubility studies, it can be known that the target active ingredient progesterone has the largest solubility in NMP, followed by ethanol. Phospholipids, as the sustained-release carrier, have the largest solubility in ethanol. Therefore, NMP and ethanol are preferably used as co-solvents to dissolve the target active ingredient and phospholipids.

[0152] Example 2

[0153] Screening of Phospholipid Ratio

[0154] Prepare samples containing progesterone according to the prescription in Table 3 and observe the dissolution of progesterone at room temperature.

[0155] Table 3 Prescription Composition and Solution Properties

[0156]

[0157] The experimental results show that due to the amphiphilic nature of phospholipids, they have a certain solubilizing effect on the lipophilic drug progesterone. Therefore, the present invention preferably selects a ratio of progesterone:phospholipid of 1:(3.5 - 8), and further preferably 1:(4 - 8).

[0158] Example 3

[0159] Screening of the Ratio of Dispersing Solvents NMP and Ethanol

[0160] Prepare samples according to the prescription in Table 4 and observe the dissolution of progesterone at room temperature.

[0161] Table 4 Prescription composition and dissolution

[0162]

[0163] The experimental phenomenon shows that when NMP: absolute ethanol is (2 - 4):1, the sample is a yellow clear liquid. Therefore, this ratio range is preferably selected for the prescription of the present invention.

[0164] Example 4

[0165] Screening of cholesterol ratio

[0166] In order to explore the effect of cholesterol on the dissolution of progesterone, samples containing progesterone were prepared according to the prescription in Table 5, placed at room temperature for 24 h and 60 d, and the properties were observed.

[0167] Table 5 Prescription composition and properties

[0168]

[0169] In the above ratio prescriptions, crystals precipitated in the group of progesterone: cholesterol = 1:4. No crystals precipitated in the other groups after 60 days. Therefore, the preferred ratio of progesterone to cholesterol in the present invention is 1:(1 - 2.5).

[0170] Example 5

[0171] Screening of surfactant types and optimization of content

[0172] The addition of surfactants is beneficial to improving the solubility of substances in solvents and is a commonly used means for solubilization in pharmaceutical preparations. In order to further improve the solubility of progesterone by adding surfactants, samples were prepared as shown in Table 6, and the change in the dissolution of progesterone was investigated.

[0173] Table 6 Prescription composition and dissolution

[0174]

[0175]

[0176] The experimental results show that as a fat-soluble nonionic surfactant, the addition of GMS can effectively dissolve progesterone under reduced solvent conditions.

[0177] The change in the dissolution of progesterone in the system was investigated after adding different ratios of GMS. Samples containing progesterone were prepared according to Table 7, placed at room temperature for 24 h, 60 d, and at a constant temperature of 25 °C for 60 d, and the dissolution of progesterone was investigated.

[0178] Table 7 Prescription composition and dissolution

[0179]

[0180] The above samples were placed in a sample retention room at room temperature and 25°C for 60 days and then observed. The results showed that no crystals were precipitated when the progesterone:GMS=1:0 and 1:1, proving that the system was clear, stable and uniform within this range. Therefore, the preferred weight ratio of progesterone to GMS in the present invention is 1:(0-1.2), and more preferably 1:(0-1).

[0181] Example 6

[0182] Effect of phospholipid ratio on sustained release effect

[0183] Samples containing different proportions of phospholipids were prepared according to the formulation in Table 8, and the in vitro release was investigated.

[0184] In vitro release experiment: about 1.0 g of the composition sample was taken and placed in a dialysis bag (3000 Da), the dialysis bag was placed in 250 mL of phosphate buffer (pH 7.4), kept at 37°C, stirred at 100 rpm, and samples were taken at different time points for measurement.

[0185] Table 8 Prescription composition and cumulative release data

[0186]

[0187]

[0188] The experimental results show that the more phospholipids there are in the prescription, the slower the release of progesterone in the sample, proving that the increase in phospholipid content can effectively delay the release of progesterone. However, as the proportion of phospholipids in the sample increases, the viscosity of the solution increases, which may also cause progesterone to be wrapped too tightly and release too slowly, causing the release concentration in the body to be lower than the effective concentration.

[0189] Example 7

[0190] Effect of cholesterol ratio on sustained release effect

[0191] The samples were prepared according to the formulation in Table 9, and the cumulative release data were determined according to the method in Example 6.

[0192] Table 9 Formulation composition and cumulative release data

[0193]

[0194] The results showed that, surprisingly, the release rate of the composition slowed down after adding cholesterol compared with the cholesterol-free formulation. At the same time, as the cholesterol content increased, the burst release of the active ingredient could be effectively reduced. Cholesterol within this range can change the fluidity and integrity of the phospholipid molecular layer, improve stability, and avoid the rapid release of the active ingredient in the first 4 hours.

[0195] Example 8

[0196] Effect of GMS Ratio on Sustained Release Effect

[0197] Surprisingly, compared with the composition without GMS, on the premise of meeting the stability requirements of the composition, the addition of GMS as a surfactant further prolongs the sustained release time of the active ingredient.

[0198] Samples were prepared according to the prescription in Table 10, and the cumulative release data were measured according to the method in Example 6.

[0199] Prescription Composition and Cumulative Release Data of Table 10

[0200]

[0201] The results showed that with the addition of GMS, the release rate of progesterone could be slowed down. In summary, adding GMS when constructing the lipid in-situ gel can not only increase the solubility of progesterone while reducing the solvent dosage, but also delay the release rate of progesterone.

[0202] Example 9

[0203] To further verify the proportion of water in the total weight of the composition, samples were prepared according to the prescription in Table 11 and placed at room temperature for 24 h, and the properties were observed.

[0204] Prescription Composition and Properties of Table 11

[0205]

[0206] The results showed that the addition of water could change the state of the system. When the addition amount of water was too high, the system would no longer be stable, manifested as the solution changing from clear to turbid. This experiment simulated the phenomenon of in-situ gel injection into the body in vitro, manifested as its utilization of the water in the tissue fluid to interact with phospholipids, thereby forming an in-situ gel-type sustained release drug depot at the injection site.

[0207] Example 10

[0208] To further verify the effect of the composition of the present invention, a pharmacokinetic study on rats was carried out.

[0209] Compositions 1 and 2 were prepared according to the prescription in Table 12. Among them, the progesterone concentration of Composition 1 was 66 mg / mL, and the mass percentage was 7.04%. The progesterone concentration of Composition 2 was 66 mg / mL, and the mass percentage was 6.67%. The preparation method of Composition 1 or Composition 2 was:

[0210] (1) Weigh the prescription amount of phospholipids, add the prescription amount of absolute ethanol, stir to dissolve, and add NMP after dissolution;

[0211] (2) Add the prescribed amount of cholesterol and active ingredient and dissolve them.

[0212] (3) Filter using a 0.22 μm filter membrane.

[0213] (4) Dispense into sterile vials, stopper, and place in a light-protected environment.

[0214] The storage temperature is below 30°C.

[0215] Table 12 Prescription composition of Composition 1 and Composition 2

[0216] Prescription composition Composition 1 Composition 2 Progesterone concentration (mg / mL) 66 66 Progesterone: phospholipid (w / w) 1:6 1:7.5 Progesterone: cholesterol (w / w) 1:1.2 1:1.5 Progesterone: dispersion solvent (w / w) 1:6 1:5 NMP: absolute ethanol (w / w) 2:1 2:1

[0217] The experimental animals were male SD rats. The rats were randomly divided into 3 groups and received the following dosing regimens by subcutaneous administration. Production License Number: SCXK(Zhe)2019 - 0001; Certificate Number: 20230106Aazz0619000180.

[0218] Table 13 Dosing regimens

[0219] Drug Progesterone injection Composition 1 Composition 2 Progesterone concentration (mg / mL) 20 66 66 Administration volume (mL / animal) 0.33 0.1 0.1 Drug dose (mg / animal) 6.6 6.6 6.6

[0220] * 20 mg / mL, Zhejiang Xianju Pharmaceutical Co., Ltd.

[0221] Using commercially available progesterone injection (20 mg / mL, also known as progesterone oil injection) as a control, the experiment was conducted. The dosing regimen is shown in Table 14. Blood samples were collected from the fundus venous plexus at 30 min, 1 h, 2 h, 4 h, 8 h, 24 h, 48 h, 72 h, 96 h, and 120 h after dosing for content detection. The results are shown in Tables 14 - 16:

[0222] Table 14 Plasma progesterone concentration - time data (ng / mL) after subcutaneous injection of progesterone oil injection

[0223]

[0224] Note: BLQ: Below the limit of detection.

[0225] Table 15 Plasma progesterone concentration - time data (ng / mL) after subcutaneous injection of Composition 1

[0226]

[0227] Table 16 Plasma progesterone concentration - time data (ng / mL) after subcutaneous injection of Composition 2

[0228]

[0229] The processed data is shown in Table 14 - 16:

[0230] Table 17 Pharmacokinetic parameters of progesterone in plasma after subcutaneous administration of progesterone injection

[0231]

[0232]

[0233] Table 18 Pharmacokinetic parameters of progesterone in plasma after subcutaneous administration of Composition 1

[0234]

[0235] Table 19 Pharmacokinetic parameters of progesterone in plasma after subcutaneous administration of Composition 2

[0236]

[0237] After subcutaneous injection of progesterone oil injection, progesterone in the plasma of rats was basically completely eliminated about 72 hours after administration; while after subcutaneous injection of Composition 1 and Composition 2, the concentration of progesterone in the plasma of rats could still be detected until 120 hours after administration, and maintained at a relatively stable level from 24 hours to 96 hours after administration. The half-life was significantly improved compared with progesterone oil injection, indicating a better sustained-release effect.

[0238] Composition 1 and Composition 2 relative to the control group of progesterone oil injection t 1 / 2 Increased by 7.75 times and 3.22 times respectively, showing an obvious sustained-release effect. C max Were only 29.4% and 34.8% of the control group respectively, showing a mild release compared with the control group and no burst release behavior occurred. The AUC were 58.4% and 63.3% of the control group respectively, probably because the detection time was stopped at 120h and not fully determined.

[0239] Example 11

[0240] The experimental animals were male beagle dogs (source: Animal Center of Southeast University School of Medicine). Each beagle dog was subcutaneously injected with Composition 3 in Table 20. The progesterone concentration of Composition 3 was 66 mg / mL, the mass percentage was 6.06%, and the administration dose was 2 mg / kg, with only one injection.

[0241] The preparation method of Composition 3 is as follows:

[0242] (1) Weigh the prescription amount of phospholipids, add the prescription amount of absolute ethanol, stir to dissolve, and add NMP after dissolution;

[0243] (2) Add the prescribed amount of cholesterol and GMS, and stir or dissolve by ultrasonic wave;

[0244] (3) Add the prescribed amount of active ingredient and dissolve;

[0245] (4) Filter through a 0.22 μm filter membrane;

[0246] (5) Subpackage into sterile vials, stopper, and place in a light-proof environment.

[0247] The storage temperature is below 30°C.

[0248] Table 20 Prescription composition of composition 3

[0249] Prescription composition Composition 3 Progesterone concentration (mg / mL) 66 Progesterone: phospholipid (w / w) 1:8 Progesterone: cholesterol (w / w) 1:1.5 Progesterone: GMS (w / w) 1:1 Progesterone: dispersion solvent (w / w) 1:5 NMP: absolute ethanol (w / w) 4:1

[0250] At 30 min, 1 h, 2 h, 4 h, 8 h, 24 h, 32 h, 48 h, 72 h, 96 h, 120 h, and 144 h after administration, blood samples were collected from the cephalic vein of the forelimb for content detection. The data are shown in Table 21:

[0251] Table 21 Concentration-time data of progesterone in plasma after subcutaneous injection of composition 3 (ng / mL)

[0252]

[0253] The processed data are shown in Tables 22 - 23.

[0254] Table 22 Pharmacokinetic parameters of progesterone in Beagle dog plasma

[0255]

[0256] As can be seen from Table 22, compared with the reference progesterone oil injection (intramuscular injection), the average MRT of progesterone composition 3 is 41.55 h, while the average MRT of the progesterone oil injection recorded in the literature is 14.69 h (Rui Li, Chen Ying, Chen Xun, Zhang Ziqiang. Pharmacokinetics study of progesterone preparations in Beagle dog [J]. Journal of Pharmaceutical Research, 2022, 41: 709 - 712.); the elimination half-life of composition 3 is 64.34 h, while the elimination half-life of the progesterone oil injection is only 12.11 h, indicating that composition 3 has a longer retention time in the body and a longer pharmacological effect. Compared with the progesterone oil injection, C of composition 3 max decreases, T maxThe extension further proves that the composition of the present invention has a sustained-release effect.

[0257] Table 23 Drug concentration at different time points as a percentage of C max Ratio

[0258]

[0259] The blood drug concentrations at different collection points in Table 23 and the ratio of C max also further prove the sustained-release effect of the composition. The drug concentration can still be monitored at 144 h, and its intensity is about 8.76% of C max , further indicating that its sustained-release effect can reach more than 144 h.

[0260] Example 12

[0261] Application of the pharmaceutical composition as an in-situ gel or a sustained-release depot drug

[0262] Figure 1 shows the changes in the appearance properties of the samples before and after adding a certain amount of water (the water accounts for about 7% of the total weight of the composition) to the pharmaceutical composition 3 in Example 11.

[0263] The above experiment simulates the change in the properties of the pharmaceutical composition after phase transition when the tissue fluid in the body exchanges with the pharmaceutical composition during subcutaneous injection. The above results can further show that the pharmaceutical composition of the present invention can form a gel after interacting with the tissue fluid at the injection site and become a sustained-release depot drug, slowly releasing the active ingredient.

[0264] Example 13

[0265] To further prove the safety of the pharmaceutical composition, 4 rats, 2 males and 2 females, were taken and divided into two groups. One group was subcutaneously injected with 1 mL of the solvent (NMP: absolute ethanol = 2:1), and the other group was subcutaneously injected with 1 mL of the composition 1 in Example 10. The animals were dissected at 24 h and 168 h after injection respectively to observe whether there was bleeding under the skin and whether the tissue color changed, and at the same time, the changes in the administration group were observed.

[0266] The results show that there were no red or swollen phenomena in the subcutaneous tissues of the rats after 24 h and 168 h of administration of the solvent and the composition, indicating that both the composition and the solvent were non-irritating at this dose. The results are shown in Figure 2 .

[0267] Example 14

[0268] Pharmacodynamic data

[0269] In this example, the so-called "progesterone pharmaceutical composition group", "pharmaceutical composition 3" or "composition 3" all refer to the composition formula of the pharmaceutical composition 3 in Example 11.

[0270] Through the rabbit endometrial transformation experiment, the biological activity of the progesterone pharmaceutical composition was further verified. The experimental animals were 15 immature New Zealand female rabbits, 6 weeks old, with a body weight of 0.5 - 0.8 kg. Original source: Hubei Yizhicheng Biotechnology Co., Ltd., Production License Number: SCXK(E)2021 - 0020, Certificate Number: 42817300002822.

[0271] In the progesterone oil injection group, it was injected intramuscularly into the hind legs at 5 mg / kg, with alternating administration of both legs, once a day for a total of 21 times. The progesterone pharmaceutical composition group was administered subcutaneously into the neck at 35 mg / kg with Pharmaceutical Composition 3, and the injection frequency was once a week for a total of 3 times. The vehicle group was Pharmaceutical Composition 3 without active ingredients, with the same injection site, dose, and frequency as the Pharmaceutical Composition 3 group. At 1, 3, 7, 14, and 21 days after administration, the vehicle group, Pharmaceutical Composition 3 group, and progesterone oil injection group were observed for irritation at the injection site, and the injection site was photographed and scored. After 21 days, the rabbits were euthanized and the uterus was taken. The wet weights of the 2 uteri of each female rabbit were weighed. One was stored at 4°C for the detection of drug tissue distribution; for the other, the middle section of the uterus was fixed with Bouin's solution and stained with H&E for observation and photography.

[0272] Since the progesterone oil injection was administered daily, the last two detection time points were set 1 h before administration on the sixth and seventh days. The results in Tables 24 - 25 showed that although it was injected daily, the blood drug concentration was only 1.922 ± 0.642 ng / mL and 2.920 ± 1.026 ng / mL after less than 24 h. While Pharmaceutical Composition 3 was only injected once, the blood drug concentrations at 144 h and 168 h were 2.688 ± 1.291 ng / mL and 1.640 ± 0.411 ng / mL, indicating that it had a certain sustained-release effect.

[0273] Table 24 Plasma concentration-time data of progesterone after subcutaneous injection of Pharmaceutical Composition 3 (ng / mL) (once every 7 days)

[0274]

[0275] Table 25 Plasma concentration-time data of progesterone after intramuscular injection of progesterone oil injection (ng / mL) (once a day)

[0276]

[0277] *Data at 143 h and 167 h were the data before the next injection, and the data at other times were the data after administration.

[0278] Figure 3The injection site of the progesterone oil injection group showed erythema 7 days after administration, indicating mild irritation. However, neither the progesterone pharmaceutical composition group nor the solvent group showed irritation, suggesting that it greatly reduced the irritation at the injection site.

[0279] The results of the uterine weight gain experiment are as Figure 4 (A) and Table 26 show that the uterine weight of the solvent group was 0.228 ± 0.066 g, and the uterine coefficient was 0.017 ± 0.004. The uterine weight of the progesterone pharmaceutical composition group was 1.550 ± 0.513 g, and the uterine coefficient was 0.116 ± 0.039. The uterine weight of the progesterone oil injection group was 0.792 ± 0.506 g, and the uterine coefficient was 0.059 ± 0.039. Figure 4 (B) shows that the progesterone concentration in the rabbit uterus in the solvent group was 1.780 ± 1.933 ng / g, and the progesterone concentration in the rabbit uterus in the progesterone pharmaceutical composition group was 107.54 ± 60.922 ng / g, which was much higher than the progesterone concentration of 7.140 ± 4.294 ng / g in the progesterone oil injection group. This strongly proves that the progesterone pharmaceutical composition group can maintain a relatively high concentration of progesterone in the uterus while greatly reducing the dosing frequency, thereby effectively exerting the role of progesterone in promoting endometrial development.

[0280] Table 26 Rabbit uterine weight and uterine coefficient

[0281]

[0282] As Figure 5 shown, the red circled part indicates the uterine glands. In the solvent group, the number of glands was small, and only scattered glands under the endometrium were visible, and there was no obvious uterine gland structure in the deeper stroma. In the pharmaceutical composition 3 group, severe glandular hyperplasia was visible, with an increased number, crowded arrangement, and the proliferated glands extended to near the muscular layer, showing co-walls and back-to-back phenomena. In the progesterone oil injection group, moderate glandular hyperplasia was observed, with a relatively large number, and the uterine glands extended to the deeper stroma, showing co-walls and back-to-back phenomena.

[0283] In addition, as shown in Table 27, the number of rabbit uterine glands in the solvent group was approximately 1.333 ± 1.269, the number of uterine glands in the pharmaceutical composition 3 group was approximately 30.933 ± 8.719, and the number of glands in the progesterone oil injection group was approximately 22.200 ± 13.146. The number of glands in the pharmaceutical composition 3 was higher than that in the progesterone oil injection group, indicating that the progesterone pharmaceutical composition of the present invention can more effectively exert the role of progesterone and promote the increase of uterine glands.

[0284] Table 27 Rabbit uterine gland number

[0285]

[0286]

[0287] The above results further verify that the preparation has achieved the invention object of reducing the injection frequency, reducing the injection irritation and exerting excellent pharmacological effects proposed by the present invention.

[0288] Example 15

[0289] Moist heat sterilization

[0290] The sample of Composition 3 in Example 11 (the prescription composition and preparation process are shown in detail in Example 11) was subjected to moist heat sterilization using a sterilization process of 121 °C / 12 min. The comparison of the properties and contents of the samples before and after sterilization is shown in the following table.

[0291] Table 28 Comparison results of the properties and contents of Composition 3 before and after sterilization

[0292] Item Sample before sterilization Sample after sterilization Appearance Yellow clear liquid Yellow clear liquid Content (%) 100.9 101.3

[0293] The inspection results show that there are no obvious changes in the properties and contents of the composition before and after sterilization, indicating that the pharmaceutical composition in the present invention can be subjected to moist heat sterilization treatment at 121 °C / 12 min. Compared with aseptic treatment, moist heat sterilization has the advantages of reliable sterilization, high efficiency, convenience and easy control, and can minimize the risk of contamination to the greatest extent.

[0294] The above content is a schematic description of the present invention and its implementation manners. This description is not restrictive. What is shown in the examples is only one of the implementation manners of the present invention, and the actual implementation manners are not limited thereto. Therefore, if those of ordinary skill in the art are inspired by it and design similar implementation manners and examples without creative work without departing from the gist of the present invention, they shall fall within the protection scope of the present invention.

Claims

1. A pharmaceutical composition comprising the following components: a.Active ingredients; b. sustained release carrier; c. Fat-soluble nonionic surfactant; d. a pharmaceutically acceptable solvent; in, The active ingredients include progesterone; The sustained-release carrier includes phospholipids and cholesterol; The pharmaceutically acceptable solvent includes a dispersing solvent; The dispersing solvent includes an organic solvent NMP and a short-chain alcohol; The active ingredient accounts for 5-13% (w / w) of the total weight of the pharmaceutical composition; The ratio of the phospholipid to the active ingredient is (3.5-10):1 (w / w); The ratio of cholesterol to active ingredient is (0.5-3.5):1 (w / w); The ratio of the dispersing solvent to the active ingredient is (2-8):1 (w / w); The ratio of the fat-soluble nonionic surfactant to the active ingredient is (0-1.2):1 (w / w); The pharmaceutically acceptable solvent optionally includes a phase transition regulator, and the weight proportion of the phase transition regulator in the composition is 0-4%.

2. The pharmaceutical composition according to claim 1, wherein the ratio of cholesterol to the active ingredient is (1-2.5):1 (w / w), more preferably the ratio of cholesterol to the active ingredient is (1.5-2.5):1 (w / w).

3. The pharmaceutical composition according to any one of claims 1 or 2, wherein the ratio of the organic dispersion solvent to the active ingredient is (3-6):1 (w / w).

4. The pharmaceutical composition according to claim 1 or 2, wherein the short-chain alcohol comprises one or more of ethanol, propylene glycol or glycerol. The pharmaceutical composition according to claim 3 , wherein the short-chain alcohol comprises at least ethanol.

6. The pharmaceutical composition according to claim 5, wherein the ratio of NMP to ethanol in the organic dispersion solvent is (2-4):1 (w / w).

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the fat-soluble nonionic surfactant is selected from glyceryl monostearate (GMS).

8. The pharmaceutical composition according to claim 7, wherein the ratio of GMS to active ingredient is (0-1):

1.

9. The pharmaceutical composition according to claim 1, comprising the following components: a.Active ingredients; b. sustained release carrier; c. Fat-soluble nonionic surfactant; d. a pharmaceutically acceptable solvent; in, The active ingredients include progesterone; The sustained-release carrier includes phospholipids and cholesterol; The pharmaceutically acceptable solvent includes a dispersing solvent; The dispersing solvent includes an organic solvent NMP and a short-chain alcohol; The active ingredient accounts for 5-13% (w / w) of the total weight of the pharmaceutical composition; The ratio of the phospholipid to the active ingredient is (4-8):1 (w / w); The ratio of cholesterol to active ingredient is (1-2.5):1 (w / w); The ratio of the dispersing solvent to the active ingredient is (3-6):1 (w / w); The ratio of the fat-soluble nonionic surfactant to the active ingredient is (0-1):1 (w / w).

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the phase transition regulator comprises water.

11. The pharmaceutical composition according to claim 10, wherein the water accounts for 0-4% of the total weight of the composition.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the phospholipid is a natural phospholipid, including soybean lecithin, egg phospholipid or a combination thereof.

13. The pharmaceutical composition according to claim 12, wherein the content of phosphatidylcholine in the natural phospholipid is not less than 80%.

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein the dosage form of the pharmaceutical composition is selected from injection, preferably, the injection comprises subcutaneous injection or intramuscular injection.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the concentration of progesterone in the pharmaceutical composition is 55 to 130 mg / mL; preferably 60 to 100 mg / mL; more preferably 60 to 80 mg / mL.

16. An injection comprising the pharmaceutical composition according to any one of claims 1 to 15, preferably, the injection is a subcutaneous injection or an intramuscular injection.

17. An in situ gel comprising the pharmaceutical composition according to any one of claims 1 to 15.

18. A sustained-release depot drug comprising the pharmaceutical composition according to any one of claims 1 to 15.

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