Triprostinil microcrystal / temperature-sensitive gel system as well as preparation method and application of triprostinil microcrystal / temperature-sensitive gel system

Abstract

The invention discloses a treprostinil microcrystal / temperature-sensitive gel system, which is prepared by the following steps: preparing treprostinil and a stabilizer into treprostinil microcrystals, and combining the treprostinil microcrystals with temperature-sensitive gel to obtain the treprostinil microcrystal / temperature-sensitive gel system, the treprostinil microcrystal is prepared from treprostinil and a stabilizer through a film hydration-ultrasonic method. The temperature-sensitive gel is a blank gel solution prepared from methylcellulose, sodium chloride and an additive; the additive is selected from one of polyethylene glycol 4000, polyvinylpyrrolidone K30, hyaluronic acid and bovine serum albumin. The treprostinil microcrystal / temperature-sensitive gel system can slow down burst release of drug microcrystals, prolong drug action time and improve patient compliance and safety. The treprostinil microcrystal-temperature-sensitive gel system can maintain the effective treatment concentration for 3 days after being injected once, and the treatment effect is achieved by reducing the average pulmonary arterial pressure and improving pulmonary vascular remodeling and right heart remodeling.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmaceutical preparations, and particularly relates to a treprostinil microcrystal / warm-sensitive gel system and a preparation method and application thereof. BACKGROUND

[0002] Pulmonary arterial hypertension is a progressive vascular disease, and its clinical manifestations include abnormal increase of pulmonary vascular resistance and pulmonary vascular remodeling, and in severe cases, it can lead to right heart failure and even death. Pulmonary vascular remodeling is an important disease characteristic of pulmonary arterial hypertension. With the deepening of the disease, pulmonary vascular remodeling is the main cause of the increase of pulmonary vascular resistance and pulmonary arterial pressure. At present, the clinical treatment drugs for pulmonary arterial hypertension include prostacyclin analogues, endothelin receptor antagonists, phosphodiesterase-5 inhibitors, and soluble guanylate cyclase agonists, which play a role by dilating pulmonary vessels. However, these drugs can only delay the progression of the disease and are difficult to achieve a cure. In addition, as a chronic disease, pulmonary arterial hypertension requires long-term drug administration, and the existing long-acting drug administration methods are subcutaneous or intravenous infusion, which have the problems of low safety, poor patient compliance, and risk of infection.

[0003] TRE is a prostacyclin analogue, which has the advantages of long half-life and stable chemical structure as a first-line treatment drug. TRE has high affinity for prostacyclin receptors and can increase intracellular cyclic adenosine monophosphate levels, thereby playing a role in dilating blood vessels and inhibiting pulmonary arterial smooth muscle cell proliferation, thereby increasing cardiac output and stroke volume. At present, TRE has four drug administration routes in clinical practice, including intravenous infusion, subcutaneous infusion, inhalation administration, and oral administration. Intravenous infusion requires administration through a permanent central venous catheter, which has the risk of serious catheter-related complications such as sepsis, thromboembolism, or syncope. Subcutaneous infusion can cause pain at the injection site. In addition, intravenous or subcutaneous infusion of TRE needs to be performed in a professional medical institution, and the drug solution needs to be replaced every 3 days, which has poor patient compliance. With inhalation administration, the frequency of TRE administration is high, and it is often accompanied by adverse reactions such as cough, headache, and throat irritation. Oral administration of TRE often leads to premature drug discontinuation due to adverse reactions. Therefore, in order to improve patient medication compliance and quality of life, the development of a long-acting TRE preparation is a potential application direction for the treatment of pulmonary arterial hypertension.

[0004] Drug crystals are nanometer or micrometer particles assembled from stabilizers and pharmaceutically active ingredients, which are suitable for long-acting delivery of poorly soluble drugs. Drug crystals have the advantages of high drug loading, simple prescription composition and easy industrial production, and have unique advantages in drug delivery and clinical transformation. The long-acting drug crystal development strategy is mainly based on the size effect of crystal particles. The particle size of microcrystals is large, the specific surface area is small, and the dissolution rate is relatively low. At the same time, the volume of the surrounding tissue fluid in the subcutaneous and muscle parts is limited, and the microcrystals injected subcutaneously or intramuscularly can form a drug depot at the injection site, slowly release the drug and prolong the drug action time. At present, more than 20 kinds of microcrystal or nanocrystal products have been marketed. Among them, Invega and are used for the treatment of adult schizophrenia, and the action time is up to 3 months and 2 months, respectively. However, the drug microcrystals are released too fast after injection, which easily leads to frequent drug administration, side effects, large loss of drugs and heavier burden on patients.

[0005] Hydrogel is a kind of polymer material with three-dimensional network structure, which has good biocompatibility and biodegradability. According to the different principles of gel formation, it can be divided into temperature-responsive, pH-responsive, enzyme-responsive and light-responsive gels. However, when delivering drugs, hydrogels have problems such as burst release of drugs, limited ability to encapsulate hydrophobic drugs and proteins, and inability to accurately control drug release. SUMMARY

[0006] The purpose of the present application is to develop a long-acting and safe long-acting preparation of treprostinil for the clinical application of treprostinil. After intramuscular injection, the drug microcrystal / thermosensitive gel system can effectively slow down the burst release of drug microcrystals and prolong the release of drugs, thereby reducing systemic side effects and safely and long-acting delivering treprostinil, improving patient compliance and safety. The drug microcrystal / thermosensitive gel system of the present application can provide high drug loading and controlled release of hydrophobic drugs, and is widely applicable to hydrophobic drugs, providing a solution for long-acting treatment of other chronic diseases. In addition, the preparation process of the microcrystal / thermosensitive gel system is simple and safe, minimally invasive, has high drug loading and is easy to scale up production, and has good application prospect.

[0007] The purpose of the present application is achieved by the following technical solutions:

[0008] A treprostinil microcrystal / thermosensitive gel system (TRE-MCs-Gel) is prepared by first preparing treprostinil microcrystals (TRE-MCs) from treprostinil and stabilizers, and then further combining the treprostinil microcrystals with a thermosensitive gel to obtain the treprostinil microcrystal / thermosensitive gel system.

[0009] The ratio of the use amount of the latanoprost microcrystal (calculated by latanoprost) to the temperature-sensitive gel is 4:1-4:4 mg / mL, preferably 4:2 mg / mL.

[0010] The latanoprost microcrystal is prepared by a film hydration-ultrasonic method.

[0011] The stabilizer is selected from one of poloxamer 188, poloxamer 407, Tween 80 and polyvinylpyrrolidone K30.

[0012] Preferably, the stabilizer is poloxamer 188 or polyvinylpyrrolidone K30.

[0013] The latanoprost microcrystal is prepared by a film hydration-ultrasonic method, comprising the following steps:

[0014] Step (1), completely dissolving latanoprost and a stabilizer in anhydrous ethanol; removing the organic solvent by evaporation under reduced pressure, and then drying to remove trace amounts of solvent;

[0015] Step (2), adding ultrapure water to the sample obtained in step (1) to perform hydration treatment to obtain a microcrystal suspension; Step (3), dispersing the microcrystal suspension by ultrasonic crushing treatment, and then freeze-drying to obtain latanoprost microcrystals.

[0016] In step (1), the mass ratio of latanoprost to the stabilizer is 1:2-1:4, preferably 1:4.

[0017] There is no special requirement for the amount of anhydrous ethanol, as long as it can ensure that latanoprost and the stabilizer are completely dissolved. Generally, the ratio of the amount of latanoprost to anhydrous ethanol is 1:0.5-1:1 mg / mL.

[0018] Specifically, latanoprost and the stabilizer are placed in a dry reactor, and anhydrous ethanol is added to completely dissolve latanoprost and the stabilizer.

[0019] The vacuum pump and the rotary evaporator are used for evaporation under reduced pressure. The temperature for evaporation under reduced pressure is 35-50°C, preferably 40°C. The time for evaporation under reduced pressure is 0.5-1 hour, preferably 0.5 hour. The rotation speed for evaporation under reduced pressure is 100 rpm.

[0020] The temperature for drying is 35-40°C, preferably 37°C. The time for drying is 4-8 hours, preferably 6 hours.

[0021] In step (2), the temperature for hydration treatment is 35-40°C, preferably 37°C. The time for hydration treatment is 0.5-2 hours, preferably 1 hour.

[0022] The ratio of the amount of the treprostinil to the amount of the ultrapure water is 1:1-1:2 mg / mL.

[0023] In step (3), the ultrasonic crushing treatment is performed under the following conditions: ultrasonic power 80-100 W, ultrasonic time 5-10 minutes, and ultrasonic mode setting open 2 seconds and off 3 seconds.

[0024] Preferably, the ultrasonic crushing treatment is performed under the following conditions: ultrasonic power 80 W, ultrasonic time 10 minutes, and ultrasonic mode setting open 2 seconds and off 3 seconds.

[0025] The freeze-drying is performed under the following conditions: pre-freezing at -80°C for 12 hours and freeze-drying at -45°C for 48 hours.

[0026] The treprostinil microcrystal / thermosensitive gel system is obtained by physically mixing the treprostinil microcrystal and the thermosensitive gel.

[0027] The thermosensitive gel is a blank gel solution prepared from methyl cellulose, sodium chloride and an additive.

[0028] The additive is selected from one of polyethylene glycol 4000, polyvinylpyrrolidone K30, hyaluronic acid and bovine serum albumin.

[0029] Preferably, the additive is selected from one of polyethylene glycol 4000 and polyvinylpyrrolidone K30.

[0030] More preferably, the additive is polyethylene glycol 4000.

[0031] The thermosensitive gel is prepared by the following method, which comprises the following steps:

[0032] Step (1): preparing a methyl cellulose solution using deionized water;

[0033] Step (2): completely dissolving sodium chloride and an additive in deionized water to prepare a stock solution;

[0034] Step (3): slowly adding the stock solution to the methyl cellulose solution under stirring to obtain a blank gel solution.

[0035] In step (1), the concentration of the methyl cellulose is 15-45 mg / mL, preferably 15 mg / mL.

[0036] Specifically, the methyl cellulose solution is prepared by uniformly dispersing methyl cellulose into 70°C deionized water, adding 25°C deionized water to dilute and stirring to obtain a methyl cellulose solution.

[0037] In step (2), the concentration of sodium chloride in the stock solution is 150-210 mg / mL; when the additive is polyethylene glycol 4000, the concentration of polyethylene glycol 4000 is 240-360 mg / mL; when the additive is polyvinylpyrrolidone K30, the concentration of polyvinylpyrrolidone K30 is 180-240 mg / mL; when the additive is hyaluronic acid, the concentration of hyaluronic acid is 9-21 mg / mL; when the additive is bovine serum albumin, the concentration of bovine serum albumin is 3-30 mg / mL.

[0038] Preferably, in the stock solution, the concentration of sodium chloride is 180 mg / mL; when the additive is polyethylene glycol 4000, the concentration of polyethylene glycol 4000 is 360 mg / mL; when the additive is polyvinylpyrrolidone K30, the concentration of polyvinylpyrrolidone K30 is 240 mg / mL; when the additive is hyaluronic acid, the concentration of hyaluronic acid is 21 mg / mL; when the additive is bovine serum albumin, the concentration of bovine serum albumin is 30 mg / mL.

[0039] In step (3), the volume ratio of the stock solution to the methyl cellulose solution is 1:2.

[0040] Another object of the present application is to provide a preparation method of the said treprostinil microcrystal / thermosensitive gel system, comprising the following steps:

[0041] Step (1), preparing treprostinil microcrystals by thin film hydration-ultrasonic method: completely dissolving treprostinil and stabilizers in anhydrous ethanol; removing the organic solvent by reduced pressure evaporation, and placing the sample treated by reduced pressure evaporation in a drying box to remove trace amounts of solvent; adding ultrapure water to perform hydration treatment to obtain a microcrystal suspension; dispersing the microcrystal suspension by ultrasonic crushing treatment, and then freeze-drying to obtain treprostinil microcrystals;

[0042] Step (2), preparing a thermosensitive gel: preparing a methyl cellulose solution in deionized water; completely dissolving sodium chloride and an additive in deionized water to prepare a stock solution; slowly adding the stock solution to the methyl cellulose solution under stirring to obtain a blank gel solution.

[0043] Step (3), mixing the blank gel solution and the treprostinil microcrystals, and stirring until uniformly dispersed to obtain a treprostinil microcrystal / thermosensitive gel system.

[0044] Another object of the present application is to provide the use of the said treprostinil microcrystal / thermosensitive gel system in the preparation of a long-acting injection for treating pulmonary arterial hypertension.

[0045] Preferably, the long-acting injection is a muscle injection.

[0046] Advantages of the present application:

[0047] The treprostinil microcrystal / warm sensitive gel system significantly slows down the burst release of the drug microcrystal after muscle injection, prolongs the in-vivo action time, reduces the dosage and injection frequency, and improves the effectiveness and safety.

[0048] The treprostinil microcrystal / warm sensitive gel system of the present application can maintain an effective therapeutic concentration for 3 days after a single injection, and plays a therapeutic role by reducing the mean pulmonary arterial pressure and improving pulmonary vascular remodeling and right heart remodeling, and can be used as a substitute for clinical intravenous and subcutaneous infusion osmotic pumps to achieve long-term treatment of pulmonary arterial hypertension.

[0049] The treprostinil microcrystal / warm sensitive gel system of the present application has good biocompatibility. BRIEF DESCRIPTION OF DRAWINGS

[0050] Figure 1 The figure is a particle size and distribution diagram of TRE-MCs.

[0051] Figure 2 The figure is a particle size and distribution diagram of TRE-MCs prepared at different mass ratios.

[0052] Figure 3 The figure is a particle size and distribution diagram of TRE-MCs prepared at different mass ratios at 4℃.

[0053] Figure 4 The figure is a particle size and distribution diagram of TRE-MCs prepared at different mass ratios after freeze-drying.

[0054] Figure 5 The figure is a scanning electron microscope diagram of TRE-MCs.

[0055] Figure 6 The figure is a differential scanning calorimetry analysis spectrum of TRE-MCs.

[0056] Figure 7 The figure is an X-ray diffraction spectrum of TRE-MCs.

[0057] Figure 8 The figure is an infrared spectrum of TRE-MCs.

[0058] Figure 9 The figure is a gelation time result diagram of methylcellulose hydrogel.

[0059] Figure 10 The figure is a scanning electron microscope result diagram of blank hydrogel.

[0060] Figure 11Figure for rheological results of blank hydrogel; wherein, Figure A is linear viscoelastic region; Figure B is gelation temperature.

[0061] Figure 12 Figure for in vitro release behavior of TRE-MCs-Gel.

[0062] Figure 13 Figure for blood concentration-time curve of TRE-MCs-Gel.

[0063] Figure 14 Figure for long-acting antihypertensive effect of TRE-MCs-Gel.

[0064] Figure 15 Figure for pulmonary membrane thickness of TRE-MCs-Gel; wherein, compared with MCT, *** P<0.001, **** P<0.001; compared with TRE-MCs-Gel, #### P<0.0001.

[0065] Figure 16 Figure for right heart hypertrophy index of TRE-MCs-Gel; wherein, compared with MCT, * P<0.05, ** P<0.01, n.s. represents no significant difference.

[0066] Figure 17 Figure for cell biocompatibility of blank hydrogel; wherein, Figure A is cytotoxicity of blank hydrogel extract on normal pulmonary artery smooth muscle cells; Figure B is cytotoxicity of blank hydrogel extract on pathological pulmonary artery smooth muscle cells; wherein, compared with Control, * P<0.05, compared with Control, ## P<0.01, ### P<0.001.

[0067] Figure 18 Figure for injection site safety of TRE-MCs-Gel. DETAILED DESCRIPTION

[0068] The technical solutions of the present application are further described below in combination with specific examples, but the following examples do not limit the protection scope of the claims of the present application.

[0069] The raw materials or reagents used in the present application are commercially available.

[0070] The treprostinil microcrystals are characterized by using a nanoparticle size analyzer, a scanning electron microscope, differential scanning calorimetry, X-ray powder diffraction and Fourier infrared spectroscopy.

[0071] The stability of the treprostinil microcrystals was investigated by high performance liquid chromatography.

[0072] The blank hydrogel solution was characterized by the test tube inversion method, scanning electron microscopy, and a rheometer.

[0073] The in vitro release behavior of the treprostinil microcrystal / / temperature-sensitive gel system was investigated by dynamic membrane dialysis.

[0074] The in vivo pharmacokinetic behavior of the treprostinil microcrystal / / temperature-sensitive gel system was investigated by high performance liquid chromatography-mass spectrometry tandem technology and protein precipitation.

[0075] The long-term antihypertensive effect of the treprostinil microcrystal / / temperature-sensitive gel system was investigated by direct pressure measurement.

[0076] The effects of the treprostinil microcrystal / / temperature-sensitive gel system on improving pulmonary vascular remodeling and right ventricular remodeling were investigated in pulmonary hypertension model rats.

[0077] The safety of the treprostinil microcrystal / / temperature-sensitive gel system was evaluated by cytotoxicity and H&E staining.

[0078] Example 1

[0079] Preparation of treprostinil microcrystals

[0080] According to a mass ratio of 1:3 (w / w), 4 mg of treprostinil (TRE) was placed in a dry flask with 12 mg of poloxamer 188 (P188), poloxamer 407, Tween 80, or polyvinylpyrrolidone K30 (PVP K30), respectively, and 4 mL of anhydrous ethanol was added to completely dissolve the sample. A vacuum pump and a rotary evaporator were used to evaporate the sample at 40°C and 100 rpm for 0.5 hours under reduced pressure. Subsequently, the flask containing the sample was placed in a 37°C drying oven and left to stand for 6 hours to remove trace amounts of solvent. 4 mL of ultrapure water was added to the dried sample, which was hydrated at 37°C for 1 hour to obtain a suspension. 2 mL of the obtained suspension was transferred to a vial, and ultrasonic crushing was performed at an ultrasonic power of 80 W for 10 minutes with an ultrasonic mode setting of 2 seconds on and 3 seconds off to obtain a treprostinil microcrystal (TRE-MCs) suspension.

[0081] The results are shown in Table 1. Figure 1 When P188 or polyvinylpyrrolidone K30 was used as a stabilizer, the particle size of the treprostinil microcrystals was about 1.2 μm and 0.8 μm, respectively, and the particle size distribution was relatively uniform.

[0082] Example 2

[0083] Formulation optimization of treprostinil microcrystals

[0084] Take 4 mg of treprostinil, refer to Example 1 "Preparation of treprostinil microcrystals", only change the amount of P188 according to the mass ratio of TRE to P188 (1:2, 1:3, 1:4), the rest of the amount and conditions remain unchanged, prepare different treprostinil microcrystals, and measure the particle size and its distribution of treprostinil microcrystals (Table 1, Figure 2 ). The freshly prepared treprostinil microcrystal suspension was placed at 4℃ for 24 hours, or the sample was pre-frozen at -80℃ for 12 hours and freeze-dried at -45℃ for 48 hours. The particle size and its distribution of treprostinil microcrystals were measured to screen the optimal mass ratio of TRE to P188.

[0085] The results are shown in Table 1 and Figure 2 The particle size of TRE-MCs prepared according to the mass ratio of TRE to P188 of 1:2, 1:3 and 1:4 was about 1.2 μm, and the particle size distribution was relatively uniform. The particle size of TRE-MCs increased rapidly after being placed at 4℃ (Table 2 and Figure 3 ). After freeze-drying, the particle size and polydispersity index of TRE-MCs prepared according to the mass ratio of TRE to P188 of 1:4 were more close to the freshly prepared sample (Table 3 and Figure 4 ). Therefore, the mass ratio of TRE to P188 of 1:4 is taken as the best prescription for preparing treprostinil microcrystals, and the treprostinil microcrystals are stored after freeze-drying.

[0086] Table 1. Particle size of TRE-MCs prepared at different mass ratios

[0087] TRE:P188 (w / w) Particle size (pm) PDI 1:2 1.36±0.16 0.35±0.05 1:3 1.22±0.15 0.15±0.11 1:4 1.29±0.08 0.25±0.06

[0088] Table 2. Particle size of TRE-MCs prepared at different mass ratios at 4℃

[0089] TRE:P188 (w / w) Particle size (pm) PDI 1:2 4.86±1.55 0.28±0.16 1:3 7.26±1.38 0.83±0.63 1:4 4.64±1.08 0.15±0.14

[0090] Table 3. Particle size of TRE-MCs prepared at different mass ratios after freeze-drying

[0091] TRE:P188 (w / w) Particle size (pm) PDI 1:2 3.65±0.36 0.89±0.25 1:3 7.55±0.31 1.88±1.48 1:4 2.95±0.17 0.41±0.36

[0092] Example 3

[0093] 4 mg of treprostol (TRE) and 16 mg of poloxamer 188 (P188) were placed in a dry flask at a mass ratio of 1:4 (w / w), and 4 mL of anhydrous ethanol was added to completely dissolve them. The mixture was evaporated under reduced pressure at 40 °C and 100 rpm for 0.5 hours using a vacuum pump and rotary evaporator. The flask containing the sample was then placed in a 37 °C drying oven and allowed to stand for 6 hours to remove trace solvents. 4 mL of ultrapure water was added to the dried sample, and the mixture was hydrated at 37 °C for 1 hour to obtain a suspension. 2 mL of the hydrated suspension was transferred to a vial and ultrasonically broken up using an ultrasonic power of 80 W for 10 min, with the ultrasonic mode set to 2 seconds on and 3 seconds off, to obtain a treprostol microcrystal (TRE-MCs) suspension. The treprostol microcrystal suspension was pre-frozen at -80 °C for 12 hours and then freeze-dried at -45 °C for 48 hours to obtain treprostol microcrystals.

[0094] Characterization of TRE-MCs

[0095] 1. The microstructure of TRE-MCs was examined by scanning electron microscopy. The freeze-dried TRE-MCs sample was ultrasonically dispersed in distilled water for 5 minutes, dropped onto a copper sheet with double-sided carbon tape, dried, fixed with conductive adhesive, sputtered with gold under nitrogen atmosphere, and then photographed.

[0096] Scanning electron microscopy results as follows Figure 5 As shown, TRE-MCs have a rod-like structure and a particle size of approximately 2 μm.

[0097] 2. The crystal phase composition of TRE-MCs was investigated by differential scanning calorimetry (DSC) and X-ray diffraction (XRD). For DSC, the freeze-dried TRE-MCs samples were spread evenly in a crucible for testing, with a temperature range of 25-300℃ and a heating rate of 10℃ / min. For XRD, the test conditions for the freeze-dried TRE-MCs samples were a Cu target, a scanning rate of 0.02° / step, a scanning range of 3-40°, and a scanning speed of 10° / min.

[0098] Differential scanning calorimetry analysis results are as follows Figure 6 As shown, TRE-MCs exhibit only one endothermic peak (P188) around 53 °C, while no TRE endothermic peak is observed. It is speculated that TRE may exist in an amorphous state. X-ray diffraction analysis results are as follows... Figure 7 As shown, TRE in TRE-MCs retains some crystallinity, but its diffraction intensity is much lower than that of the TRE active pharmaceutical ingredient. Therefore, TRE in TRE-MCs exists partly in a crystalline form and partly in an amorphous form.

[0099] 3. The intermolecular interactions in TRE-MCs were investigated by Fourier transform infrared spectroscopy. The freeze-dried samples of TRE-MCs were mixed with potassium bromide and compressed into a disc. The detection parameters were as follows: 20 scans, 4000-400 cm -1 , and 1.0 cm -1 .

[0100] The results of Fourier transform infrared spectroscopy are shown in Figure 8 . TRE has characteristic peaks at 3508.45 cm -1 (ν O-H ), 2925.96 cm -1 (ν -COOH ), 1712.75 cm -1 (asymmetric ν C＝O ), 1587.38 cm -1 (symmetric ν C＝O ), and 1265.28 cm -1 (ν C-O-C ). P188 has characteristic peaks at 2889.6 cm -1 (ν C-H ), 1467.80 cm -1 (β O-H ), and 1110.9 cm -1 (ν C-O ). In TRE-MCs, the characteristic peaks of TRE at 3508.45 cm -1 and 2518.98 cm -1 disappeared, indicating that there is a hydrogen bond interaction between TRE and P188.

[0101] Example 4

[0102] The stability of TRE-MCs was investigated by taking different batches of TRE-MCs prepared in Example 3.

[0103] 1. Storage stability test. The freeze-dried samples of TRE-MCs were placed in a sealed container and stored at 25°C and 4°C. Samples were taken on the 0th, 5th, and 10th days, respectively. The particle size and PDI were measured by dynamic light scattering (DLS) instrument, and the content was measured by HPLC.

[0104] 2. Influencing factor test. The particle size and PDI were determined by DLS, and the drug content was determined by HPLC to investigate the influence of high temperature, high humidity and light on the stability of TRE-MCs. The specific test conditions are shown below. High temperature test: the freeze-dried sample of TRE-MCs was placed in a sealed container at 60°C, and samples were taken at 0, 5, 10 days. High humidity test: the freeze-dried sample of TRE-MCs was placed in a sealed container at 25°C, relative humidity 90±5%, and samples were taken at 0, 5, 10 days. Light test: the freeze-dried sample of TRE-MCs was placed in a sealed container under light intensity of 4500±500Lx, and samples were taken at 0, 5, 10 days.

[0105] The particle size change of TRE-MCs is shown in Table 4. In the storage stability test, the particle size of TRE-MCs did not change significantly after 10 days. Similarly, in the high humidity test and the light test, the particle size of TRE-MCs did not change significantly after 10 days. The content change of TRE-MCs is shown in Table 5. In the storage stability test and the high humidity test, the content of TRE did not change significantly. Therefore, the freeze-dried sample of TRE-MCs should be stored at room temperature or 4°C.

[0106] Table 4. Particle size change of TRE-MCs (n=3)

[0107]

[0108] Table 5. Content change of TRE-MCs (n=3)

[0109]

[0110] Example 5

[0111] Preparation of blank hydrogel and prescription screening

[0112] Methyl cellulose was uniformly dispersed in deionized water at 70°C, and then 25°C deionized water was added to dilute and stir uniformly to obtain methyl cellulose solutions with concentrations of 10 mg / mL (i.e. mass fraction of 1%), 20 mg / mL (i.e. 12%), and 30 mg / mL (i.e. 3%), respectively. The 2% and 3% methyl cellulose solutions have high viscosity, which will make it difficult to inject the treprostinil microcrystal / warm-sensitive gel system. Therefore, 1% was selected as the concentration of methyl cellulose in the blank hydrogel solution.

[0113] Methylcellulose was uniformly dispersed into deionized water at 70 °C, then diluted with deionized water at 25 °C and stirred uniformly to obtain a methylcellulose solution. A stock solution consisting of sodium chloride (NaCl) and one of the following additives: polyethylene glycol 4000 (PEG 4000), polyvinylpyrrolidone K30 (PVP K30), hyaluronic acid (HA), and bovine serum albumin (BSA) was prepared using deionized water. In the stock solution, the concentration of NaCl was 150, 180, or 210 mg / mL, the concentration of PEG 4000 was 240, 300, or 360 mg / mL, the concentration of PVP K30 was 180, 210, or 240 mg / mL, and the concentration of HA was 9, 15, or 21 mg / mL. Under stirring, 1 mL of the stock solution was slowly added to 2 mL of the methylcellulose solution (15 mg / mL) and mixed uniformly to obtain a blank hydrogel solution.

[0114] The gelation temperature was determined by the test tube inversion method. 1 mL of the blank hydrogel solution was placed in a 5 mL EP tube and put in a constant temperature water bath, with the temperature ranging from 25 to 60 °C and the heating rate being 0.5 °C / min. The solution was equilibrated at each temperature for 5 min. The tube was inverted for 30 s, and if the content did not flow, the temperature was determined as the gelation temperature.

[0115] The gelation time was determined by the test tube inversion method. 1 mL of the blank hydrogel solution was placed in a 5 mL EP tube and put in a constant temperature water bath at 25 °C. After 10 min, the tube was quickly transferred to a constant temperature water bath at 37 °C and the timing was started. The solution was inverted every 30 s to check the flowability, and the time required for the solution to stop flowing was recorded as the gelation time.

[0116] The results, as shown in Table 6, indicated that the gelation temperature of the blank hydrogel solution decreased with increasing concentrations of methylcellulose and various additives. Considering the flowability of the hydrogel solution, the sol-gel transition at body temperature, and the safety of the injection site, 6% NaCl in 1% methylcellulose was finally selected as the subject for further study.

[0117] The results, as shown in Table 7, indicated that the gelation time of the blank hydrogel solution decreased with increasing concentrations of various additives. Rapid gelation of the gel solution can reduce the diffusion of free drugs, so 1% BSA, 12% PEG 4000, 8% PVP K30, and 0.7% HA were selected as the subjects for further study. Figure 9 Table 6. Gelation temperature of methylcellulose hydrogels containing different additives (n = 3)

[0118]

[0119]

[0120] ​Example 6

[0121] Preparation of Blank Gel (BSA), Blank Gel (PEG 4000), Blank Gel (PVP K30) and Blank Gel (HA).

[0122] Preparation of Blank Gel (BSA): Methyl cellulose was uniformly dispersed into deionized water at 70 °C, then deionized water at 25 °C was added to dilute and stirred uniformly to obtain a methyl cellulose solution with a concentration of 15 mg / mL. A stock solution with a NaCl concentration of 180 mg / mL and a BSA concentration of 30 mg / mL was prepared using deionized water. Under stirring conditions, 1 mL of the stock solution was slowly added to 2 mL of the methyl cellulose solution (concentration of 15 mg / mL), and a Blank Gel (BSA) solution was obtained after uniform mixing.

[0123] Preparation of Blank Gel (PEG 4000): Methyl cellulose was uniformly dispersed into deionized water at 70 °C, then deionized water at 25 °C was added to dilute and stirred uniformly to obtain a methyl cellulose solution with a concentration of 15 mg / mL. A stock solution with a NaCl concentration of 180 mg / mL and a PEG 4000 concentration of 360 mg / mL was prepared using deionized water. Under stirring conditions, 1 mL of the stock solution was slowly added to 2 mL of the methyl cellulose solution (concentration of 15 mg / mL), and a Blank Gel (PEG 4000) solution was obtained after uniform mixing.

[0124] Preparation of Blank Gel (PVP K30): Methyl cellulose was uniformly dispersed into deionized water at 70 °C, then deionized water at 25 °C was added to dilute and stirred uniformly to obtain a methyl cellulose solution with a concentration of 15 mg / mL. A stock solution with a NaCl concentration of 180 mg / mL and a PVP K30 concentration of 240 mg / mL was prepared using deionized water. Under stirring conditions, 1 mL of the stock solution was slowly added to 2 mL of the methyl cellulose solution (concentration of 15 mg / mL), and a Blank Gel (PVP K30) solution was obtained after uniform mixing.

[0125] Preparation of Blank Gel (HA): Methyl cellulose was uniformly dispersed into deionized water at 70 °C, then deionized water at 25 °C was added to dilute and stirred uniformly to obtain a methyl cellulose solution with a concentration of 15 mg / mL. A stock solution with a NaCl concentration of 180 mg / mL and a HA concentration of 21 mg / mL was prepared using deionized water. Under stirring conditions, 1 mL of the stock solution was slowly added to 2 mL of the methyl cellulose solution (concentration of 15 mg / mL), and a Blank Gel (HA) solution was obtained after uniform mixing.

[0126] Characterization of blank hydrogel

[0127] 1. The microstructure of the hydrogel was observed by scanning electron microscope. 1 mL blank hydrogel solution was added into 5 mL EP tube, which was placed in 37℃ water bath until the gel was completely solidified. The freeze-drying was performed after the sample was frozen at -80℃ for 24 hours. The freeze-dried sample of the hydrogel was fixed with conductive glue, and then was taken pictures after gold spraying treatment in nitrogen environment.

[0128] The results, as shown in Figure 10 , Blank Gel(PEG 4000) and Blank Gel(PVP K30) both had three-dimensional porous network structure, and the structures of the two were obviously different. Blank Gel(PEG 4000) presented larger and irregular pores, and the network structure was looser with a wider pore size distribution range. Blank Gel(PVP K30) presented smaller, more uniform and denser pores, and the network structure was more fine and uniform.

[0129] 2. Rheological study. The rheological properties of blank hydrogel solution were determined using MCR102e Anton Paar oscillatory rheometer. The linear viscoelastic region was determined first. The gelation temperature was determined in oscillation mode, the sample was added dropwise on the sample stage, the instrument parameters were set, and the curves of storage modulus and loss modulus of the sample with temperature were detected. Parameter settings: angular frequency: 1 rad / s; strain: 3.2%; temperature range: 10-40℃; heating rate: 2℃ / min; plate spacing: 0.104 mm.

[0130] The results, as shown in Figure 11 , the limit value of the linear viscoelastic region of Blank Gel(PEG 4000) was 3.54%. The higher the limit value, the more conducive to the stability of the structure of the hydrogel at the injection site. The gelation temperature of Blank Gel(PEG 4000) was 25℃, and the gel was formed when the environmental temperature was higher than 25℃. It showed that the blank hydrogel could form a gel at body temperature.

[0131] Example 7

[0132] In vitro release behavior of TRE-MCs-Gel

[0133] Preparation of treprostinil microcrystals / warm hydrogel system TRE-MCs-Gel(BSA), TRE-MCs-Gel(PEG 4000), TRE-MCs-Gel(PVP K30) and TRE-MCs-Gel(HA).

[0134] Preparation of TRE-MCs-Gel (BSA): Blank Gel (BSA) was prepared according to Example 6, 2 mL Blank Gel (BSA) was added to the lyophilized sample of 4 mL drug microcrystal suspension of treprostinil (Example 3, with 4 mg treprostinil as raw material), and stirred to uniform dispersion to obtain the TRE-MCs-Gel (BSA) system.

[0135] Preparation of TRE-MCs-Gel (PEG 4000): Blank Gel (PEG 4000) was prepared according to Example 6, 2 mL Blank Gel (PEG 4000) was added to the lyophilized sample of 4 mL drug microcrystal suspension of treprostinil (Example 3), and stirred to uniform dispersion to obtain the TRE-MCs-Gel (PEG 4000) system.

[0136] Preparation of TRE-MCs-Gel (PVP K30): Blank Gel (PVP K30) was prepared according to Example 6, 2 mL Blank Gel (PVP K30) was added to the lyophilized sample of 4 mL drug microcrystal suspension of treprostinil (Example 3), and stirred to uniform dispersion to obtain the TRE-MCs-Gel (PVP K30) system.

[0137] Preparation of TRE-MCs-Gel (HA): Blank Gel (HA) was prepared according to Example 6, 2 mL Blank Gel (HA) was added to the lyophilized sample of 4 mL drug microcrystal suspension of treprostinil (Example 3), and stirred to uniform dispersion to obtain the TRE-MCs-Gel (HA) system.

[0138] Preparation of free TRE: 2.11 mg of TRE sodium salt was accurately weighed and dissolved in 1 mL of ultrapure water, and completely dissolved by ultrasonic dissolution in water bath to obtain free TRE.

[0139] The in vitro release behavior of free TRE, TRE-MCs (2 mL of ultrapure water was added to 4 mL of lyophilized treprostinil microcrystal suspension (Example 3), and the mixture was ultrasonically dispersed in a water bath for 5 minutes) and TRE-MCs-Gel was investigated using dynamic membrane dialysis. To simulate the physiological environment of muscle tissue, a PBS buffer solution (containing 0.5% SDS) at pH 7.2 was prepared as the release medium. 0.8 mL of free TRE, TRE-MCs, and four types of TRE-MCs-Gel (TRE content 1.6 mg) were placed in a dialysis bag (MWCO 3500 Da) and then in 30 mL of release medium. The mixture was kept at 37°C and shaken at 100 rpm. 1 mL of release medium was removed at 0, 0.25, 0.5, 1, 2, 4, 6, 9, 12, 24, and 48 hours, and an equal amount of fresh release medium was added promptly. The extracted solution was filtered through a 0.22 μm filter membrane, and the TRE content was determined by high-performance liquid chromatography (HPLC). The cumulative release rate of TRE was calculated, and a release curve was plotted. HPLC conditions: chromatographic column: A 5 μm C18 column (250 mm × 4.6 mm) was used, with a detection wavelength of 216 nm, a column temperature of 35 °C, a flow rate of 1 mL / min, an injection volume of 10 μL, and a mobile phase of methanol: 0.1% trifluoroacetic acid aqueous solution (78:22, v / v).

[0140] The results are as follows Figure 12 As shown, free TRE, TRE-MCs, TRE-MCs-Gel (PEG 4000), TRE-MCs-Gel (HA), TRE-MCs-Gel (PVP K30), and TRE-MCs-Gel (BSA) reached release equilibrium at 6, 24, 24, 48, 48, and 24 hours, respectively. Compared with free TRE and TRE-MCs, the cumulative release rate of the four TRE-MCs-Gels was lower at 0.5 hours, indicating that the hydrogels can reduce the burst release of microcrystals. Among them, TRE-MCs-Gel (PEG 4000) and TRE-MCs-Gel (PVP K30) showed more significant effects in reducing the burst release of microcrystals.

[0141] Example 8

[0142] Pharmacokinetic behavior of TRE-MCs-Gel

[0143] The formulations of TRE-MCs group, TRE-MCs-Gel (PEG 4000) group and TRE-MCs-Gel (PVPK30) group were prepared according to Example 7.

[0144] Preparation of the formulation of the free TRE group: The formulation of the free TRE group was prepared using deionized water to prepare the free TRE at a concentration of 1.6 mg / mL, NaCl at a concentration of 5.3 mg / mL, and sodium citrate at a concentration of 6.3 mg / mL.

[0145] Twelve healthy SD rats were randomly divided into four groups: the free TRE group, the TRE-MCs group, the TRE-MCs-Gel (PEG 4000) group (Example 7), and the TRE-MCs-Gel (PVP K30) group (Example 7), and the dose of each group was 0.8 mg / kg of TRE. Each group was injected with the corresponding formulation into the muscle of the right hind limb of the rat, and the orbital blood of the rat was taken at 5 minutes, 15 minutes, 30 minutes, 1 hour, 3 hours, 5 hours, 7 hours, 9 hours, 1 day, 2 days, and 3 days after injection. 3.3% sodium citrate solution (sodium citrate: blood volume ratio of 1:9) was added to the blood sample as an anticoagulant. The blood sample was centrifuged at 3500 rpm for 10 minutes, and the supernatant was obtained to obtain the plasma sample. The TRE plasma sample was treated by protein precipitation method. 100 μL of plasma was taken, 10 μL of internal standard solution (1-naphthoxyacetic acid, 500 ng / mL) was added, vortex mixed, 20 μL of isopropyl alcohol was added, and vortex mixed again. 300 μL of acetonitrile was added to the plasma sample, vortexed for 5 minutes, and centrifuged at 4°C (12000 x g, 6 minutes). The supernatant was measured by triple quadrupole liquid chromatography-mass spectrometry to obtain the plasma concentration-time curve, and the pharmacokinetic parameters were calculated by non-compartmental non-oral model in PKSolver software.

[0146] The AB QTRAP4500 triple quadrupole liquid chromatography-mass spectrometer equipped with an AB SCIEX liquid chromatography system was used to detect the content of TRE. The liquid chromatography system conditions of TRE: the chromatographic column was ACE Excel 3 μm C18 column (50 x 2.1 mm, SHISEIDO), column temperature was 35°C, flow rate was 0.5 mL / min, sample plate temperature was 4°C, sample volume was 5 μL, and mobile phase conditions are shown in Table 7. The mass spectrometry conditions of TRE: using electrospray ionization technology, using negative ion detection method, the parent ion of TRE was 389.2, the daughter ion was 331.1, the declustering voltage was -122.19 V, and the collision energy was 35.08 eV; the parent ion of the internal standard was 200.9, the daughter ion was 143.1, the declustering voltage was -73.87 V, and the collision energy was 30.48 eV.

[0147] Table 7. Mobile phase gradient conditions

[0148]

[0149]

[0150] TRE-MCs-Gel blood concentration-time curves and pharmacokinetic parameters were as shown in Table 8. The effective blood concentrations of free TRE, TRE-MCs, TRE-MCs-Gel (PVP K30) and TRE-MCs-Gel (PEG 4000) were maintained for 7 hours, 24 hours, 48 hours and 72 hours, respectively. Compared with free TRE and TRE-MCs, TRE-MCs-Gel increased the drug half-life and area under the curve, and reduced the maximum plasma concentration and clearance, indicating that the gel can reduce the burst release of drug crystals and further prolong the in vivo release of the drug. Among them, TRE-MCs-Gel (PEG 4000) has the lowest maximum plasma concentration and the longest drug release time, which is conducive to avoiding adverse reactions caused by high drug concentration and maintaining a relatively stable blood drug concentration. Therefore, TRE-MCs-Gel (PEG 4000) was selected for subsequent in vivo pharmacodynamic evaluation. Figure 13 and Table 8. The effective blood concentrations of free TRE, TRE-MCs, TRE-MCs-Gel (PVP K30) and TRE-MCs-Gel (PEG 4000) were maintained for 7 hours, 24 hours, 48 hours and 72 hours, respectively. Compared with free TRE and TRE-MCs, TRE-MCs-Gel increased the drug half-life and area under the curve, and reduced the maximum plasma concentration and clearance, indicating that the gel can reduce the burst release of drug crystals and further prolong the in vivo release of the drug. Among them, TRE-MCs-Gel (PEG 4000) has the lowest maximum plasma concentration and the longest drug release time, which is conducive to avoiding adverse reactions caused by high drug concentration and maintaining a relatively stable blood drug concentration. Therefore, TRE-MCs-Gel (PEG 4000) was selected for subsequent in vivo pharmacodynamic evaluation.

[0151] Table 8. Pharmacokinetic parameters of each preparation in rats (mean ± SD, n = 3)

[0152]

[0153] Note: compared with TRE-MCs-Gel (PEG 4000), * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns represents no significant difference.

[0154] Example 9

[0155] Long-acting antihypertensive effect of TRE-MCs-Gel

[0156] Male SD rats were subcutaneously injected with a solution of monocrotaline (dissolved in dimethyl sulfoxide and stored in the dark, 60 mg / kg) to establish a pulmonary arterial hypertension model. After three weeks of modeling, the treatment experiment was performed. Twenty-four pulmonary arterial hypertension model rats were randomly divided into groups, with 3 rats in each group. The pulmonary arterial hypertension rats were treated with a single intramuscular injection of TRE-MCs-Gel (PEG 4000) (Example 7, TRE dose of 0.8 mg / kg). The mean pulmonary arterial pressure (mPAP) of the rats was measured at 1 hour, 3 hours, 5 hours, 1 day, 2 days, 3 days, 4 days and 5 days after drug treatment by direct pressure measurement to investigate the long-term antihypertensive effect of the preparation.

[0157] The results are as shown in Table 9. Figure 14As shown, 1 hour after administration of TRE-MCs-Gel, the mean pulmonary arterial pressure of the rats rapidly decreased to the normal range, and the antihypertensive effect can be maintained for 3-4 days, with good long-acting antihypertensive ability.

[0158] Example 10

[0159] In vivo pharmacodynamics of TRE-MCs-Gel

[0160] Male SD rats were subcutaneously injected with a solution of monocrotaline to establish a pulmonary arterial hypertension model. After three weeks of modeling, the rats were randomly divided into groups. The pulmonary arterial hypertension model rats were randomly divided into 5 groups: MCT group, free TRE group, TRE-MCs group, low-dose TRE-MCs-Gel group (TRE-MCs-Gel (Low) group), and high-dose TRE-MCs-Gel group (TRE-MCs-Gel (High) group), with 5 rats in each group. The rats in the MCT group were intramuscularly injected with normal saline, the rats in the free TRE group were intramuscularly injected with free TRE (free TRE was dissolved in normal saline), the rats in the TRE-MCs group were intramuscularly injected with TRE-MCs (the freeze-dried sample of the treprostinil drug microcrystal suspension of Example 3 was dispersed by water bath ultrasonic), and the rats in the low-dose TRE-MCs-Gel group and the high-dose TRE-MCs-Gel group were intramuscularly injected with the TRE-MCs-Gel (PEG 4000) of Example 7. The TRE dose in the high-dose TRE-MCs-Gel group was 1.2 mg / kg, the TRE dose in the free TRE group, the TRE-MCs group, and the low-dose TRE-MCs-Gel group was 0.8 mg / kg, and the administration was once every 3 days, with a treatment cycle of 2 weeks. A control group (WT group) was also set up, in which normal rats were intramuscularly injected with the same volume of normal saline each time. After the treatment, the pulmonary membrane thickness and right ventricular hypertrophy index of the rats in each group were investigated.

[0161] The formula for calculating the pulmonary arterial media thickness is: pulmonary arterial media thickness (%) = external wall thickness of blood vessel / external diameter of blood vessel x 100%

[0162] The formula for calculating the right ventricular hypertrophy index (RVHI) is: right ventricular hypertrophy index (%) = right ventricular weight / (left ventricular + interventricular septum weight) x 100%

[0163] The results are shown in Figure 15 and Figure 16 As shown, the pulmonary media thickness and right ventricular hypertrophy index of each treatment group were reduced to varying degrees. The pulmonary media thickness and right ventricular hypertrophy index of the TRE-MCs-Gel group were closest to the level of normal rats, indicating that TRE-MCs-Gel can play a role in resisting pulmonary arterial hypertension by inhibiting pulmonary vascular remodeling and right ventricular remodeling.

[0164] Example 11

[0165] Safety evaluation of TRE-MCs-Gel

[0166] Blank Gel (PEG 4000), Blank Gel (PVP K30) were prepared according to Example 6, and sterilized by UV irradiation for 30 min. Then, 200 μL of the blank gel solution was added to a 24-well plate, and transferred to a cell incubator at 37°C to form a gel. Subsequently, 1.8 mL of RPMI 1640 complete medium was added, and the cells were incubated for 24, 48 and 72 hours. The supernatant was then removed to obtain the gel extract. Pulmonary arterial hypertension-pulmonary arterial smooth muscle cells (PAH-PASMCs) and normal-pulmonary arterial smooth muscle cells (N-PASMCs) in the logarithmic growth phase were seeded into a 96-well plate at a cell density of 5000 cells per well, and incubated in a cell incubator for 24 hours. The old medium was removed, and 200 μL of the gel extract was added to each well of the test group, which was then incubated for 24, 48 and 72 hours, respectively. Meanwhile, the cells were treated with complete medium as a negative control group (Control group), and PBS as a zero hole, and incubated in a cell incubator for 24, 48 and 72 hours, respectively. Subsequently, 20 μL of thiazolyl blue solution (5 mg / mL) was added to each well, and incubated in a cell incubator for 4 hours in the dark. The old medium was removed, and 200 μL of dimethyl sulfoxide was added to each well, and shaken at a low speed for 10 minutes to fully dissolve the formazan crystals. The absorbance (Abs) at 570 nm was detected using a microplate reader, and the cell survival rate was calculated.

[0167]

[0168] wherein Abs (treated), Abs (control) and Abs (blank) represent the absorbance values of the test hole, the negative control hole and the PBS zero hole, respectively.

[0169] The results are shown in Table 1. Figure 17 The survival rates of both types of cells after 24 hours of incubation in the extract were greater than 70%, indicating that the blank gel had good cell compatibility.

[0170] The tissue safety of TRE-MCs-Gel was investigated by H&E staining. 9 SD rats were randomly divided into 3 groups, namely: TRE-MCs-Gel (PEG 4000) group, TRE-MCs-Gel (PVP K30) group, and control group, 3 rats in each group. The TRE-MCs-Gel (PEG 4000) group and the TRE-MCs-Gel (PVP K30) group were single-muscle injected with TRE-MCs-Gel (PEG 4000) and TRE-MCs-Gel (PVP K30) prepared in Example 7 (the dose of TRE was 0.8 mg / kg), and the control group was muscle injected with the same amount of normal saline. The rats were euthanized on the 3rd, 7th, and 14th day after administration. The muscle tissue at the injection site was taken out, fixed, paraffin-embedded, sectioned, deparaffinated, hydrated, and H&E stained, and then observed and photographed using the bright field of an upright fluorescence microscope.

[0171] The results are shown in Figure 18 After administration of TRE-MCs-Gel, neutrophils and inflammatory cells were first observed to infiltrate, and then to subside over time, indicating that TRE-MCs-Gel has good biological safety.

Claims

1. A treprostinil microcrystal / thermosensitive gel system, characterized in that: It is a system in which treprostane is first made into treprostane microcrystals by combining treprostane and stabilizer, and then combined with thermosensitive gel to obtain treprostane microcrystal / thermosensitive gel system.

2. The treprostene microcrystal / thermosensitive gel system according to claim 1, characterized in that: The treprostone microcrystals are prepared by thin-film hydration-ultrasound method using treprostone and a stabilizer; the stabilizer is selected from one of poloxamer 188, poloxamer 407, Tween 80 and polyvinylpyrrolidone K30, preferably poloxamer 188 or polyvinylpyrrolidone K30; the mass ratio of treprostone to stabilizer is 1:2 to 1:4, preferably 1:

4.

3. The treprostene microcrystal / thermosensitive gel system according to claim 1, characterized in that: The aforementioned treprostene microcrystals were prepared by a thin-film hydration-ultrasonic method, comprising the following steps: Step (1): Dissolve treprostone and stabilizer completely in anhydrous ethanol; evaporate under reduced pressure to remove organic solvent, and then dry to remove trace amounts of solvent; Step (2): Add ultrapure water to the sample obtained in step (1) and perform hydration treatment to obtain a microcrystalline suspension; the hydration treatment temperature is 35-40℃ and the hydration treatment time is 0.5-2 hours. Step (3): The microcrystalline suspension is dispersed by ultrasonic disruption and then freeze-dried to obtain treprostone microcrystals; the conditions for ultrasonic disruption are: ultrasonic power 80-100W, ultrasonic time 5-10 minutes, and ultrasonic mode set to 2 seconds on and 3 seconds off.

4. The treprostene microcrystal / thermosensitive gel system according to claim 1, characterized in that: The thermosensitive gel is a blank gel solution made of methylcellulose, sodium chloride and additives; the additives are selected from polyethylene glycol 4000, polyvinylpyrrolidone K30, hyaluronic acid and bovine serum albumin.

5. The treprostene microcrystal / thermosensitive gel system according to claim 4, characterized in that: Preferably, the additive is selected from polyethylene glycol 4000 and polyvinylpyrrolidone K30, with polyethylene glycol 4000 being the most preferred.

6. The treprostene microcrystal / thermosensitive gel system according to claim 4, characterized in that: The temperature-sensitive gel is prepared by the following method, including the following steps: Step (1): Prepare a methylcellulose solution using deionized water, wherein the concentration of the methylcellulose is 15-45 mg / mL; Step (2): Dissolve sodium chloride and additives completely in deionized water to prepare a stock solution; the concentration of sodium chloride in the stock solution is 150-210 mg / mL; when the additive is polyethylene glycol 4000, the concentration of polyethylene glycol 4000 is 240-360 mg / mL; when the additive is polyvinylpyrrolidone K30, the concentration of polyvinylpyrrolidone K30 is 180-240 mg / mL; when the additive is hyaluronic acid, the concentration of hyaluronic acid is 9-21 mg / mL; when the additive is bovine serum albumin, the concentration of bovine serum albumin is 3-30 mg / mL. Step (3): Under stirring conditions, add the stock solution to the methylcellulose solution and mix evenly to obtain a blank gel solution.

7. The treprostnier microcrystal / thermosensitive gel system according to claim 6, characterized in that: In step (1), the concentration of methylcellulose is 15 mg / mL; in step (2), the concentration of sodium chloride in the stock solution is 180 mg / mL; when the additive is polyethylene glycol 4000, the concentration of polyethylene glycol 4000 is 360 mg / mL; when the additive is polyvinylpyrrolidone K30, the concentration of polyvinylpyrrolidone K30 is 240 mg / mL; when the additive is hyaluronic acid, the concentration of hyaluronic acid is 21 mg / mL; when the additive is bovine serum albumin, the concentration of bovine serum albumin is 30 mg / mL; in step (3), the volume ratio of the stock solution to the methylcellulose solution is 1:

2.

8. The treprostnier microcrystal / thermosensitive gel system according to claim 1, characterized in that: The ratio of treprostone microcrystals (calculated as treprostone) to thermosensitive gel is 4:1 to 4:4 mg / mL, preferably 4:2 mg / mL.

9. A method for preparing the treprostinil microcrystalline / thermosensitive gel system according to claim 1, characterized in that: Includes the following steps: Step (1): Preparation of treprostone microcrystals by thin-film hydration-ultrasound method: treprostone and stabilizer are completely dissolved in anhydrous ethanol; organic solvent is removed by vacuum evaporation, and the sample after vacuum evaporation is placed in a drying oven and allowed to stand to remove trace amounts of solvent; ultrapure water is added for hydration treatment to obtain a microcrystal suspension; the microcrystal suspension is dispersed by ultrasonic crushing treatment and then freeze-dried to obtain treprostone microcrystals; Step (2) Preparation of temperature-sensitive gel: Prepare methylcellulose solution in deionized water; completely dissolve sodium chloride and additives in deionized water to prepare a stock solution; under stirring conditions, slowly add the stock solution to the methylcellulose solution and mix evenly to obtain a blank gel solution; Step (3): Mix the blank gel solution and treprostene microcrystals and stir until uniformly dispersed to obtain the treprostene microcrystal / thermosensitive gel system.

10. The application of the treprostinil microcrystal / thermosensitive gel system according to claim 1 in the preparation of a long-acting injectable for the treatment of pulmonary hypertension.

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