Fingolimod hydrochloride sustained-release microspheres and a method for preparing the same

Abstract

The application relates to a fingolimod hydrochloride sustained-release microsphere and a preparation method thereof. The application adopts an emulsification-solvent evaporation process, selects PLGA as the base material of the microsphere, and uses low-molecular-weight PVA as an emulsifier; the fingolimod hydrochloride and the PLGA are dissolved in an organic solvent to form a uniform oil-phase solution, and high and low concentration PVA aqueous solutions are configured as water phases; the oil-phase solution is slowly added into the high-concentration water phase, homogenization emulsification is carried out by using high-speed stirring to form an oil-water emulsion, the oil-water emulsion is transferred into the low-concentration PVA aqueous solution, and the solvent in the emulsion is volatilized by low-speed stirring to evaporate the solvent, so that the fingolimod hydrochloride sustained-release microsphere is formed. The fingolimod hydrochloride sustained-release microsphere has high drug loading and encapsulation rates, can control the release rate of the drug, reduce the peak concentration of the drug in the body, thereby reducing the occurrence of side effects, can realize long-acting release, reduces the drug administration frequency, and improves the drug use compliance of patients.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to pharmaceutical preparations, specifically to fingolimod hydrochloride sustained-release microspheres and their preparation method. Background Technology

[0002] Fingolimod hydrochloride, the first novel immunosuppressant administered orally, is the only oral medication available for children aged 10 and older with relapsing-remitting multiple sclerosis. The marketed product is fingolimod hydrochloride capsules (0.5 mg / capsule), launched by Novartis in the United States in 2010 under the brand name GILENYA, specifically for the treatment of relapsing-remitting multiple sclerosis.

[0003] Fingolimod hydrochloride is a non-selective modulator of sphingosine-1-phosphate receptor 1. This receptor has multiple subtypes widely distributed in various organ cells and has diverse biological effects. Furthermore, it requires high daily oral doses, and high therapeutic doses can result in individual differences in blood drug concentrations of up to 10 times. These factors lead to various toxic side effects associated with the administration of fingolimod hydrochloride oral capsules. Therefore, current fingolimod hydrochloride oral capsules face challenges in long-term treatment of multiple sclerosis, including low patient compliance, significant individual variability, and systemic toxicity. Summary of the Invention

[0004] Based on this, the present invention provides fingolimod hydrochloride sustained-release microspheres, which have high drug loading and encapsulation efficiency; can control the drug release rate, reduce the peak drug concentration in the body, thereby reducing the occurrence of side effects; can achieve long-acting release, reduce the frequency of administration, and improve patient medication compliance.

[0005] The present invention includes the following technical solutions.

[0006] In the first aspect, the present invention provides a sustained-release microsphere of fingolimod hydrochloride, which is prepared by encapsulating fingolimod hydrochloride in polylactic acid-glycolic acid copolymer under the action of polyvinyl alcohol;

[0007] The weight-average molecular weight of the polyvinyl alcohol is less than 65,000.

[0008] The mass ratio of the polylactic acid-glycolic acid copolymer to fingolimod hydrochloride is 8-15:1.

[0009] Secondly, the present invention provides a method for preparing the fingolimod hydrochloride sustained-release microspheres, comprising the following steps:

[0010] (1) Fingolimod hydrochloride and polylactic acid-glycolic acid copolymer were dissolved in an organic solvent to obtain an oil phase;

[0011] (2) Polyvinyl alcohol was dissolved in water to prepare high-concentration aqueous phase and low-concentration aqueous phase respectively;

[0012] (3) The oil phase is added to a high-concentration aqueous phase and homogenized to obtain an emulsion;

[0013] (4) Pour the low-concentration aqueous phase into the emulsion, evaporate the organic solvent under stirring, collect the microspheres and dry them to obtain the fingolimod hydrochloride sustained-release microspheres.

[0014] The present invention has the following beneficial effects:

[0015] This invention selects polylactic acid-glycolic acid copolymer (PLGA) as the substrate of microspheres and low molecular weight polyvinyl alcohol (PVA) as the emulsifier, so that the prepared fingolimod hydrochloride sustained-release microspheres have high drug loading and encapsulation efficiency, and achieve stable and long-term drug release. They can stably deliver fingolimod hydrochloride in vivo for up to 5 weeks, improving in vivo absorption and therapeutic efficacy. These microspheres can precisely control the drug release rate, reducing peak drug concentration in vivo and thus reducing the occurrence of side effects. Furthermore, these microspheres can reduce the frequency of administration, requiring only once-monthly subcutaneous injection for continuous and effective treatment. Compared with daily oral capsules, this effectively improves patient compliance, reduces patients' daily focus on the disease, and improves their quality of life.

[0016] The preparation method of fingolimod hydrochloride sustained-release microspheres of the present invention employs an emulsification-solvent evaporation process. Polylactic acid-glycolic acid copolymer (PLGA) is selected as the substrate of the microspheres, and low molecular weight polyvinyl alcohol (PVA) is used as the emulsifier. Fingolimod hydrochloride and PLGA polymer are dissolved in an organic solvent (preferably dichloromethane containing anhydrous ethanol) to form a homogeneous oil phase solution. High and low concentration PVA aqueous solutions are prepared as the aqueous phase. The oil phase solution is slowly added to the high concentration aqueous phase, and homogenization and emulsification are carried out using high-speed stirring to form an oil-water emulsion. The oil-water emulsion is then transferred to a low concentration PVA aqueous solution, and the solvent is evaporated by low-speed stirring, causing the solvent in the emulsion to evaporate and forming fingolimod hydrochloride sustained-release microspheres. Through this specific preparation process, combined with further condition optimization, the prepared fingolimod hydrochloride sustained-release microspheres can achieve more uniform particle size, higher encapsulation efficiency and drug loading, obtain a more accurate drug release curve, and further improve the in vivo absorption and therapeutic effect of the drug. Attached Figure Description

[0017] Figure 1 The particle size distribution diagram is shown for the fingolimod hydrochloride sustained-release microspheres prepared in Example 1.

[0018] Figure 2 Scanning electron microscope image of the fingolimod hydrochloride sustained-release microspheres prepared in Example 1.

[0019] Figure 3In vitro drug release profiles of fingolimod hydrochloride sustained-release microspheres prepared with emulsifiers of different concentrations.

[0020] Figure 4 The particle size distribution diagram is shown for the fingolimod hydrochloride sustained-release microspheres prepared in Comparative Example 1.

[0021] Figure 5 The image shows a scanning electron microscope (SEM) image of the fingolimod hydrochloride sustained-release microspheres prepared in Comparative Example 1.

[0022] Figure 6 The in vitro cumulative drug release curve of the fingolimod hydrochloride sustained-release microspheres prepared in Example 1.

[0023] Figure 7 This is the LC-MS standard curve for FIN.

[0024] Figure 8 The figures show the plasma concentration-time curves of FIN in SD rats. A represents a single intravenous injection of FIN solution; B represents a single subcutaneous injection of microspheres.

[0025] Figure 9 The cumulative in vivo absorption-time curve of FIN in the fingolimod hydrochloride sustained-release microspheres prepared in Example 1 in SD rats. Detailed Implementation

[0026] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0027] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0028] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0029] Furthermore, as used herein, the term "or" is an inclusive "or" sign and is equivalent to the term "and / or" unless the context clearly specifies otherwise. The term "based on" is not exclusive and allows for basing on other factors not described unless the context clearly specifies otherwise. Additionally, throughout the specification, the meanings of "an," "a," and "the" include plural indicators. The meaning of "in" includes both "in" and "on."

[0030] Some embodiments of the present invention relate to a sustained-release microsphere of fingolimod hydrochloride, which is prepared by encapsulating fingolimod hydrochloride in polylactic acid-glycolic acid copolymer under the action of polyvinyl alcohol;

[0031] The weight-average molecular weight of the polyvinyl alcohol is less than 65,000.

[0032] The mass ratio of the polylactic acid-glycolic acid copolymer to fingolimod hydrochloride is 8-15:1.

[0033] In some embodiments, the weight-average molecular weight of the polyvinyl alcohol is 30,000-60,000, more preferably 40,000-50,000, and even more preferably 45,000-49,000.

[0034] In some embodiments, the mass ratio of the polylactic acid-glycolic acid copolymer to fingolimod hydrochloride is 9-12:1, more preferably 10-11:1.

[0035] In some embodiments, the polylactic acid-glycolic acid copolymer is a carboxyl-terminated polylactic acid-glycolic acid copolymer.

[0036] In some embodiments, the polylactic acid-hydroxyacetic acid copolymer has a weight-average molecular weight of 25k-35k.

[0037] In some embodiments, the polylactic acid-glycolic acid copolymer has a weight-average molecular weight of 28k-32k.

[0038] In some embodiments, the monomer molar ratio of lactic acid to glycolic acid in the polylactic acid-glycolic acid copolymer is 45:55-55:45, preferably 50:50.

[0039] In some embodiments, the fingolimod hydrochloride sustained-release microspheres are prepared by a water / oil monoemulsion solvent evaporation method.

[0040] The inventors of this invention discovered during the preparation of fingolimod hydrochloride sustained-release microspheres that using conventional polyvinyl alcohol 1788 as an emulsifier resulted in very low drug loading and encapsulation efficiency of the microspheres. Further research revealed that replacing conventional polyvinyl alcohol 1788 with a low molecular weight polyvinyl alcohol as the emulsifier could significantly improve the drug loading and encapsulation efficiency of the microspheres. Therefore, this invention selects low molecular weight polyvinyl alcohol as the emulsifier to prepare fingolimod hydrochloride sustained-release microspheres.

[0041] By further optimizing the ratio of polymer to drug, the encapsulation efficiency and drug loading of the drug were further improved.

[0042] Furthermore, it was found that the preparation method and homogenization conditions also have a significant impact on the particle size distribution, morphology, encapsulation efficiency, and drug loading of the obtained microspheres.

[0043] Based on this, some embodiments of the present invention relate to a method for preparing the aforementioned fingolimod hydrochloride sustained-release microspheres, comprising the following steps:

[0044] (1) Fingolimod hydrochloride and polylactic acid-glycolic acid copolymer were dissolved in an organic solvent to obtain an oil phase;

[0045] (2) Polyvinyl alcohol was dissolved in water to prepare high-concentration aqueous phase and low-concentration aqueous phase respectively;

[0046] (3) The oil phase is added to a high-concentration aqueous phase and homogenized to obtain an emulsion;

[0047] (4) Pour the low-concentration aqueous phase into the emulsion, evaporate the organic solvent under stirring, collect the microspheres and dry them to obtain the fingolimod hydrochloride sustained-release microspheres.

[0048] Through this specific preparation process, combined with further condition optimization, the prepared fingolimod hydrochloride sustained-release microspheres can have more uniform particle size, higher encapsulation efficiency and drug loading, and obtain a more accurate drug release curve, thereby further improving the in vivo absorption and therapeutic effect of the drug.

[0049] In some embodiments, the organic solvent in step (1) is a mixture of ethanol and dichloromethane, wherein the mass ratio of ethanol to dichloromethane is preferably 1:8-10, more preferably 1:9.

[0050] In some embodiments, the concentration of fingolimod hydrochloride in the oil phase is 45 mg / mL to 55 mg / mL, more preferably 49 mg / mL to 51 mg / mL.

[0051] In some embodiments, the concentration of polyvinyl alcohol in the high-concentration aqueous phase is 6 mg / mL-60 mg / mL, preferably 10 mg / mL-50 mg / mL, more preferably 15 mg / mL-25 mg / mL, and even more preferably 18 mg / mL-22 mg / mL.

[0052] In some embodiments, the concentration of polyvinyl alcohol in the low-concentration aqueous phase is 2 mg / mL to 5 mg / mL, more preferably 3 mg / mL.

[0053] In some embodiments, the volume ratio of the oil phase to the high-concentration aqueous phase is 1:8-12, more preferably 1:10.

[0054] In some embodiments, the homogenization time is 1 minute to 2 minutes, more preferably 80 seconds to 100 seconds, and even more preferably 85 seconds to 95 seconds.

[0055] In some embodiments, the homogenization speed is 5000rpm-10000rpm, more preferably 7000rpm-8000rpm, and even more preferably 7400rpm-7600rpm.

[0056] In some embodiments, the homogenization temperature is 0°C-8°C.

[0057] In some embodiments, step (3) includes: transferring the oil phase into a syringe and injecting the oil phase dropwise into the high-concentration aqueous phase using an injection pump at a injection rate of 25-35 mm / min.

[0058] In some embodiments, the volume of the low-concentration aqueous phase is 4-6 times that of the high-concentration aqueous phase, more preferably 5 times.

[0059] In some embodiments, the stirring speed in step (4) is 500 rpm to 700 rpm, and the time is 4 hours to 6 hours.

[0060] In some embodiments, step (4) of collecting and drying microspheres includes: centrifuging, washing, sieving, collecting microspheres between 150 and 600 mesh, redispersing them in water, and then freeze-drying them to obtain the fingolimod hydrochloride sustained-release microspheres.

[0061] In some embodiments, the centrifugation conditions in step (4) include: a temperature of 0°C-8°C, a rotation speed of 3000rpm-5000rpm, and a time of 8 minutes-12 minutes.

[0062] In some embodiments, the centrifugation conditions in step (4) include: a temperature of 0°C-5°C, a rotation speed of 3500 rpm-4500 rpm, and a time of 9 minutes-11 minutes.

[0063] The English translation of the main abbreviations used in this invention is shown in the table below.

[0064]

[0065]

[0066] The present invention will be further described in detail below with reference to specific embodiments.

[0067] The instruments used in the following examples are as follows:

[0068] Analytical balance (Mettler-Toledo, Switzerland, model: MS105DU / A)

[0069] Magnetic stirrer (Changzhou Yuexin Instrument Manufacturing Co., Ltd., Model: WS-2)

[0070] High-speed homogenizer (Hangzhou Jingfei Instrument Technology Co., Ltd., Model: FSH-2A)

[0071] Multi-tube vortex mixer (Beijing Yousheng, Model: UMV-2)

[0072] Laboratory water purification system (Zhiang Instruments Shanghai Co., Ltd., Model: Simple-Q5)

[0073] High-speed centrifuge (Shanghai Anting Scientific Instrument Factory, Model: DL-4000B)

[0074] Injection pump (Beijing Zhongyuan Taihe Biotechnology Co., Ltd., Model: QHZS-001A)

[0075] Dual-function air bath constant temperature oscillator (Changzhou Jintan Liangyou Instrument Co., Ltd., Model: ZD-85) and vacuum drying oven (Shanghai Lichen Bangxi Instrument Technology Co., Ltd., Model: DZF)

[0076] Freeze dryer (BUCHI Instruments GmbH, Switzerland, model: Lyovapor™ L-200pro) and high performance liquid chromatograph (Agilent Technologies, USA, model: 1260)

[0077] Laser particle size analyzer (Sympatec GmbH, Germany, model: HELOS & INHALER)

[0078] High-resolution cold field scanning electron microscope (Hitachi, Japan, model: SU1000)

[0079] Ion sputtering instrument (Beijing Zhongke Kemei Technology Co., Ltd., Model: SBC-12)

[0080] Triple Quad LC-MS / MS (AB SCIEX, model: Triple Quad 4500)

[0081] The formulations and materials used in the following examples are as follows:

[0082] PLGA-COOH (50 / 50 molar ratio of lactic acid and glycolic acid monomers, weight-average molecular weight of 30k) (Xi'an Ruixi Biotechnology Co., Ltd., batch number: RA0240328)

[0083] Fingolimod Hydrochloride (Shanghai Aladdin, Batch No.: K2218537)

[0084] Dichloromethane, analytical grade (Guangdong Guanghua, batch number: 20230613)

[0085] Methanol, analytical grade (Anhui Zesheng Technology Co., Ltd., batch number: P6DR8EPD)

[0086] Anhydrous ethanol, analytical grade (Guangdong Guanghua, batch number: 20230701)

[0087] Polyvinyl alcohol type 1788 (Shanghai Jizhi Biochemical Technology Co., Ltd., batch number: P2253389F)

[0088] Polyvinyl alcohol Mw~47000 (Shanghai Maclean, degree of alcoholysis 98.0~99.0% (mol / mol), batch number: C16024912)

[0089] Acetonitrile, analytical grade (Guangdong Guanghua, batch number: 20230307)

[0090] Acetonitrile chromatographic grade (Shanghai Aladdin, batch number: L2406737)

[0091] Methanol chromatographic grade (Shanghai Maclean, batch number: C16249958)

[0092] Anhydrous formic acid, analytical grade (Shanghai Maclean's, batch number: C16229261).

[0093] Twain 80 (Shanghai Maclean's, batch number: C14134830)

[0094] Twain 20 (Shanghai Maclean's, batch number: C16923144)

[0095] PBS (Wuhan Sewell, batch number: GA0075191)

[0096] Phosphoric acid, analytical grade (Shanghai Maclean, batch number: C14217572)

[0097] Potassium dihydrogen phosphate, analytical grade (Shanghai Maclean, batch number: C16463550)

[0098] Mannitol, analytical grade (Shanghai Maclean's, batch number: C5190375)

[0099] Sodium carboxymethyl cellulose, analytical grade (Shanghai Aladdin, batch number: K2119519)

[0100] Isoflurane (Shenzhen Ruiward Life Technology Co., Ltd., batch number: 20240405)

[0101] Chloral hydrate, analytical grade (Shanghai Maclean, batch number: C12685891)

[0102] Animal testing depilatory cream (Reckitt Benckiser Group, Veet France, batch number: C220131)

[0103] Example 1: Preparation of Fingolimod Hydrochloride Sustained-Release Microspheres by Water / Oil (W / O) Monoemulsion Solvent Evaporation Method

[0104] (1) Preparation of the oil phase

[0105] Weigh out fingolimod hydrochloride (50 mg) and carboxyl-terminated polylactic acid-glycolic acid copolymer (PLGA-COOH) (500 mg), and dissolve them in dichloromethane (1 mL) containing 10% w / w anhydrous ethanol to obtain the oil phase.

[0106] (2) Preparation of the aqueous phase

[0107] Weigh out 100 mL of deionized water and 2 g of polyvinyl alcohol (PVAMw ~ 47000). Stir and mix at 70 °C until PVA is completely dissolved. Then cool to room temperature to obtain a 20 mg / mL PVA solution.

[0108] Weigh out 100 mL of deionized water and 0.3 g of polyvinyl alcohol (PVAMw ~ 47000). Stir and mix at 70 °C until PVA is completely dissolved. Then cool to room temperature to obtain a 3 mg / mL PVA solution.

[0109] (3) Emulsification

[0110] Transfer the oil phase to a 1 mL syringe and inject it dropwise into 10 mL of 20 mg / mL PVA solution using a syringe pump at a injection rate of 30 mm / min. Stir the mixture for 1.5 minutes at 7500 rpm using a high-speed homogenizer in an ice bath to form a single emulsion.

[0111] (4) Solvent evaporation

[0112] Pour 50 mL of 3 mg / mL PVA solution into the emulsion to form a microsphere mixture, and transfer it to a magnetic stirrer and stir at 600 rpm for 6 hours at room temperature.

[0113] (5) Collection and drying of microspheres

[0114] The mixture obtained in step (4) was centrifuged at 4°C and 4000 rpm for 10 minutes using a high-speed centrifuge. The separated precipitated microspheres were washed three times with ultrapure water to remove excess PVA and unencapsulated drug. The microspheres were then sieved through 150-mesh and 600-mesh stainless steel meshes. Microspheres between 150-mesh and 600-mesh were collected, redispersed and suspended in water, and lyophilized to obtain fingolimod hydrochloride sustained-release microspheres.

[0115] (6) Microsphere particle size determination

[0116] Particle size distribution was determined using a SYMPATEC laser particle size analyzer. A suitable amount of microspheres was used for measurement in dry testing mode, and results are reported as mean ± SD. Particle size uniformity was quantified by calculating the Span value using Equation 4-1.

[0117]

[0118] The smaller the Span value, the more uniform the size distribution of the microspheres, and the smaller the particle size (D). 10 D 50 and D 90 These correspond to the particle sizes at the 10th, 50th, and 90th percentiles of the particle size distribution, respectively.

[0119] The results are shown in Table 1 and Figure 1 As shown: The sustained-release microspheres of fingolimod hydrochloride prepared in this embodiment have uniform particle size.

[0120] Table 1. Particle size distribution of fingolimod hydrochloride sustained-release microspheres

[0121]

[0122] (7) Morphological characterization of microspheres

[0123] The shape and surface morphology of the microspheres were examined using a scanning electron microscope. Conductive double-sided black carbon tape was attached to the stage, and an appropriate amount of microspheres were coated onto the tape. The microspheres were sputtered with gold at a current of 10 mA for 60 seconds using a gold sputtering instrument. The surface morphology of the nanoparticles was observed using a scanning electron microscope. Subsequently, the coated microspheres were imaged using SEM.

[0124] The results are as follows Figure 2 As shown: The fingolimod hydrochloride sustained-release microspheres prepared in this embodiment have a uniform spherical structure.

[0125] (8) Determination of drug loading and encapsulation efficiency of microspheres

[0126] 5 mg of fingolimod hydrochloride microspheres were dispersed in 5 mL of acetonitrile and vortexed at high speed for 5 minutes until a clear solution was obtained. Then, 5 mL of ultrapure water was added to extract fingolimod hydrochloride, yielding the test sample solution. The solution was filtered, and the concentration of fingolimod hydrochloride was analyzed by high-performance liquid chromatography (HPLC) under the following conditions.

[0127] Chromatographic conditions: A Shim-pack GIST C18 column (250 × 4.6 mm, 5 μm) was used, with the column temperature set at 40 °C. Elution was performed isocratically with 60% acetonitrile and 40% 0.025 mol / L phosphate buffer (pH = 3.0). The flow rate was set to 1 mL / min, and the UV absorbance signal was monitored at 215 nm. The retention time was approximately 4.1 minutes.

[0128] The drug loading (DL%) and encapsulation efficiency (EE%) of the microspheres were calculated using Formulas 1 and 2:

[0129]

[0130] Where a is the actual weight of the drug encapsulated in the microspheres, and b is the weight of the drug added to the microspheres;

[0131]

[0132] Where m is the actual weight of drug encapsulated in the microspheres, and n is the theoretical weight of drug encapsulated in the microspheres.

[0133] Test results: The DL% of the fingolimod hydrochloride sustained-release microspheres prepared in this example is 6.73±0.37, and the EE% is 72.94±0.08.

[0134] Example 2: Preparation of sustained-release fingolimod hydrochloride microspheres with different amounts of emulsifier

[0135] In the emulsification step, the concentrations of PVA solution were 10 mg / mL, 20 mg / mL, and 50 mg / mL, respectively. The amounts of other reagents were the same as in Example 1. Fingolimod hydrochloride sustained-release microspheres were prepared according to the method in Example 1.

[0136] 1. The drug loading (DL%) and encapsulation efficiency (EE%) were tested according to the method in Example 1. The results are shown in Table 2. The concentration of PVA solution within a certain range had no significant effect on the drug loading and encapsulation efficiency of fingolimod hydrochloride sustained-release microspheres.

[0137] Table 2. Drug loading and encapsulation efficiency of fingolimod hydrochloride sustained-release microspheres prepared with different amounts of emulsifier.

[0138]

[0139] 2. Test the in vitro release of microspheres prepared with different concentrations of PVA under real-time conditions simulating the physiological environment in vivo.

[0140] Accurately weigh 5 mg of fingolimod hydrochloride sustained-release microspheres into a 7 mL centrifuge tube, and add 4 mL of release medium (PBS containing 0.02% Tween 80). Place the centrifuge tube containing the microspheres and release medium in a constant-temperature shaker at 37.0 °C and 100 rpm. Samples are taken for analysis at 4 hours and at 1, 2, 4, 7, 14, 21, 28, 35, 48, 56, and 62 days. To obtain samples for analysis, the centrifuge tubes are centrifuged at 4000 rpm for 5 minutes. Then, carefully collect 3 mL of the supernatant as the test sample, and add an equal volume of fresh release medium to maintain the sample volume. Subsequently, the test sample is filtered through a 0.22 μm microporous membrane and analyzed by HPLC to determine the drug concentration in the supernatant.

[0141] The calculation method is as follows: the drug release amount at each test point represents the difference between the total drug content present in the microsphere and the remaining drug content; the cumulative release amount is the drug release amount at each time point divided by the total drug content.

[0142]

[0143] Where Release(t) represents the cumulative release at time point t, C is the concentration of fingolimod in the medium at time point t, V0 is the total volume of the release medium, C1, C2, C3, etc. are the concentrations of fingolimod in the medium at different time points before time point t, V is the volume of samples taken at different time points, M is the total mass of microspheres, and DL is the actual drug loading of fingolimod in the microspheres.

[0144] The results are as follows Figure 3 As shown, the three types of fingolimod hydrochloride sustained-release microspheres prepared in this embodiment all exhibited similar biphasic drug release curves, with release periods exceeding 60 days and no obvious burst release phase observed. The cumulative release amount after 7 days was less than 25%. Among them, the microspheres prepared with 10 mg / mL PVA showed a significant burst release at 28 days and entered a plateau phase at 35 days. The microspheres in the 20 mg / mL and 50 mg / mL PVA groups continued to release until entering a plateau phase of one week at 35 days, after which they continued to release slowly, reaching a cumulative release amount of 45% and 40% respectively at 60 days.

[0145] Example 3: Preparation of fingolimod hydrochloride sustained-release microspheres with different amounts of polymer

[0146] In the preparation steps of the oil phase, the amount of PLGA-COOH used was 150 mg, 250 mg, 500 mg, and 1 g (that is, the mass ratio of PLGA-COOH to fingolimod hydrochloride was 3:1, 5:1, 10:1, and 20:1, respectively). The amount of other reagents used was the same as in Example 1. Fingolimod hydrochloride sustained-release microspheres were prepared according to the method in Example 1.

[0147] The drug loading (DL%) and encapsulation efficiency (EE%) were tested according to the method in Example 1, and the results are shown in Table 3. The mass ratio of PLGA-COOH to fingolimod hydrochloride has a significant impact on the drug loading and encapsulation efficiency of the obtained microspheres. Within a certain range, the lower the drug ratio, the higher the drug loading and encapsulation efficiency of the microspheres. When the mass ratio of PLGA-COOH to fingolimod hydrochloride is 10:1, the drug loading and encapsulation efficiency of the microspheres are the highest. However, if the proportion of PLGA-COOH is too high, the drug loading will be reduced due to the small proportion of drug, and the microsphere surface pores will be too large, resulting in a short sustained-release time. Therefore, the optimal mass ratio of PLGA-COOH to fingolimod hydrochloride is 10:1.

[0148] Table 3. Drug loading and encapsulation efficiency of fingolimod hydrochloride sustained-release microspheres prepared with different amounts of polymer.

[0149]

[0150] Comparative Example 1

[0151] In Example 1, PVAMw~47000 was replaced with an equal amount of polyvinyl alcohol 1788, and other reagents and amounts were the same as in Example 1. Fingolimod hydrochloride sustained-release microspheres were prepared according to the method of Example 1.

[0152] The drug loading (DL%) and encapsulation efficiency (EE%) were tested according to the method in Example 1, and the results are shown in Table 6. The molecular weight of PVA has a significant impact on the drug loading and encapsulation efficiency of fingolimod hydrochloride sustained-release microspheres. When the PVA AMw ~ 47000 of the present invention is replaced with PVA 1788 with a higher molecular weight, the drug loading and encapsulation efficiency of the fingolimod hydrochloride sustained-release microspheres will be significantly reduced.

[0153] Table 6. Drug loading and encapsulation efficiency of fingolimod hydrochloride sustained-release microspheres prepared from PVA with different molecular weights.

[0154]

[0155] Comparative Example 2: Preparation of Fingolimod Hydrochloride Sustained-Release Microspheres by Water / Oil / Water (W / O / W) Dual Emulsion Solvent Evaporation Method

[0156] (1) Preparation of the oil phase

[0157] Take 500 mg of carboxyl-terminated polylactic acid-glycolic acid copolymer (PLGA-COOH) and dissolve it in 0.5 mL of dichloromethane to obtain the oil phase.

[0158] (2) Preparation of the aqueous phase

[0159] Dissolve 50 mg of fingolimod hydrochloride in 0.5 mL of water to obtain a fingolimod hydrochloride solution;

[0160] Weigh out 100 mL of deionized water and 2 g of polyvinyl alcohol (PVAMw ~ 47000). Stir and mix at 70 °C until PVA is completely dissolved. Then cool to room temperature to obtain a 20 mg / mL PVA solution.

[0161] Weigh out 100 mL of deionized water and 0.3 g of polyvinyl alcohol (PVAMw ~ 47000). Stir and mix at 70 °C until PVA is completely dissolved. Then cool to room temperature to obtain a 3 mg / mL PVA solution.

[0162] (3) Emulsification

[0163] Add the fingolimod hydrochloride solution to the oil phase and sonicate in an ultrasonic instrument for 3 minutes to emulsify the two phases and obtain a primary emulsion;

[0164] The primary emulsion was mixed with 10 mL of 20 mg / mL PVA solution and stirred at 7500 rpm for 1.5 minutes in an ice bath using a high-speed homogenizer to form a double emulsion.

[0165] (4) Solvent evaporation

[0166] Pour 50 mL of 3 mg / mL PVA solution into the double emulsion to form a microsphere mixture, and transfer it to a magnetic stirrer and stir at 600 rpm for 6 hours at room temperature.

[0167] (5) Collection and drying of microspheres

[0168] The mixture obtained in step (4) was centrifuged at 4°C and 4000 rpm for 10 minutes using a high-speed centrifuge. The separated precipitated microspheres were washed three times with ultrapure water to remove excess PVA and unencapsulated drug. The microspheres were then sieved through 150-mesh and 600-mesh stainless steel meshes. Microspheres between 150-mesh and 600-mesh were collected, redispersed and suspended in water, and lyophilized to obtain fingolimod hydrochloride sustained-release microspheres.

[0169] The particle size of the fingolimod hydrochloride sustained-release microspheres prepared in this comparative example was determined according to the method of Example 1, and the shape and surface morphology of the microspheres were examined using a scanning electron microscope.

[0170] The results are shown in Table 7. Figure 4 and Figure 5As shown, the microspheres prepared in this comparative example have small particle sizes and large ranges; the microspheres exhibit various morphologies, and some microspheres failed to separate into individual spheres before solidification, resulting in uneven size distribution. Microspheres with smaller particle sizes are prone to burst drug release, and the uneven particle size of the microspheres affects the uniformity of subsequent drug release.

[0171] Table 7. Particle size distribution of fingolimod hydrochloride sustained-release microspheres prepared by the dual emulsion solvent evaporation method.

[0172]

[0173] Example 6: In vitro release of fingolimod hydrochloride sustained-release microspheres

[0174] Given that fingolimod hydrochloride has lower solubility in PBS than in water, this example uses an aqueous solution containing 0.02% Tween 20 as the release medium to investigate the in vitro release behavior of the fingolimod hydrochloride sustained-release microspheres prepared in Example 1. In vitro release experiments were conducted under real-time conditions: 20 mg of precisely weighed microspheres were placed in a 50 mL centrifuge tube, and 20 mL of release medium (an aqueous solution containing 0.02% Tween 20) was added. The centrifuge tube containing the microspheres and release medium was placed in an air bath constant-temperature shaker at 37.0 °C and 100 rpm. Samples were taken for analysis at 4, 8, and 24 hours, and at 2, 4, 7, 10, 14, 17, 21, 28, and 35 days. To obtain samples for analysis, the centrifuge tubes were centrifuged at 4000 rpm for 5 minutes. Then, 10 mL of the supernatant was carefully collected as the test sample, and an equal volume of fresh release medium was added to maintain the sample volume. Subsequently, the test sample was filtered through a 0.22 μm microporous membrane and analyzed by HPLC to determine the drug concentration in the supernatant. Calculations were performed according to the method in Example 2.

[0175] The results are as follows Figure 6 As shown, the cumulative in vitro release of fingolimod hydrochloride microspheres was close to 100% on day 35, indicating that the prepared drug-loaded microspheres have the effect of long-acting sustained-release drug.

[0176] Example 7: Pharmacokinetics of Fingolimod Hydrochloride Extended-Release Microspheres in Rats

[0177] 1. Laboratory animals

[0178] Male Sprague-Dawley (SD) rats (160-180g) were provided by the Guangdong Provincial Center for Laboratory Animal Science. The rats were housed at room temperature with free access to food and water for 7 days to acclimatize. The animal research protocol was approved by the Animal Ethics Review Committee of Jinan University, protocol code 20200314-07, on March 13, 2020.

[0179] 2. Establishment of methods for determining in vivo content

[0180] 10.0 mg of fingolimod hydrochloride was weighed and 2.0 mL of methanol was added to prepare a 5.0 mg / mL solution, thereby preparing the calibration standard. A calibration curve was created by adding a known amount of the calibration standard to blank rat plasma and performing LC-MS analysis using the plasma processing method described in section 2.1.

[0181] 2.1 Plasma Treatment

[0182] Methods: 50 μL of methanol and 50 μL of acetonitrile were added to 100 μL of plasma sample, vortexed for 10 min, and then centrifuged at 14000 g for 5 min. The supernatant was transferred to a polypropylene centrifuge tube and vacuum dried overnight at room temperature. The dried residue was reconstituted in 100 μL of mobile phase (0.2% formic acid aqueous solution / 0.2% acetonitrile: 95 / 5, v / v), vortexed for 10 mL, and centrifuged. The supernatant was transferred to a liquid chromatography bottle for LC-MS analysis (Table 8).

[0183] Table 8. LC-MS gradient elution method for FIN in rat plasma.

[0184]

[0185] Analytical methods: LC-MS / MS was performed on an AB Sciex QTRAP 4500 triple quadrupole LC-MS system using a Waters ACQUITYUPLC HSS T3 column (100 × 2.1 mm, 1.8 μm). Gradient elution was performed at a column temperature of 40°C. The injection volume was 8 μL. Mobile phase A was an aqueous solution containing 0.2% formic acid. Mobile phase B was an acetonitrile solution containing 0.2% formic acid. The mobile phase solution was pumped in at a flow rate of 0.3 mL / min, with the concentration of B at 5%, increasing from 5% to 65% over 0.11 to 4 minutes, and then from 65% to 95% over 4 to 6 minutes. FIN eluted at approximately 5.1 minutes.

[0186] The mass spectrometer operated in positive ion mode with a collision energy of 19.6 V, an electrospray voltage of 5500 V, and a temperature of 550 °C. Single reaction monitoring (SRM) mode was switched to quantitative detection of the m / z 308.3→255.2 transition and qualitative detection of the m / z 308.8→105.10 transition. Peak areas were measured using the same Analyst software as the control mass spectrometer. The FIN peak area was plotted against known drug concentrations, and linear regression was performed.

[0187] The standard calibration curve constructed in the concentration range of 0.1–10 μg / mL is shown below. Figure 7 As shown, the equation for the linear fit is y = 1123.5x + 70900 (R²). 2=0.9993), indicating good linearity, which suggests that the established LC-MS analytical method can be used for subsequent determination of fingolimod hydrochloride concentration in blood samples.

[0188] 3. Pharmacokinetics: Dosing, Sampling, and Sample Processing

[0189] FIN and FRET MS@FIN (fingolimod hydrochloride sustained-release microspheres prepared in Example 1) were accurately weighed and added to physiological saline and physiological saline containing 0.29 wt% mannitol and 0.06 wt% sodium carboxymethyl cellulose, respectively. The mixture was vortexed to prepare a 0.5 mg / mL FIN solution and a 2 mg / mL FRET MS@FIN microsphere suspension.

[0190] 3.1 Administration

[0191] To investigate the sustained-release behavior of microspheres in vivo, 11 male Sprague-Dawley (SD) rats that had been fasted for 12 hours were randomly divided into two groups: an intravenous injection group (n=3) and a subcutaneous injection group (n=8). In the subcutaneous injection group, the fur on the back of the rats was removed and the skin on their backs was disinfected with 75% ethanol for later use.

[0192] Intravenous injection group: FIN solution was injected directly into the tail vein in a single dose of 0.5 mg / kg. Blood samples were collected from the retroorbital venous plexus of rats at regular intervals of 0.5, 1, 2, 4, 8, 12, 24, 48, and 72 hours after injection.

[0193] Subcutaneous injection group: The prepared microsphere suspension was subcutaneously injected into the dorsal skin of rats anesthetized with isoflurane gas using a 25G needle at a dose of 5 mg / kg. Blood samples were collected from the retroorbital venous plexus of the rats at regular intervals, and small animal in vivo imaging was performed at the injection site on days 1, 2, 4, 7, 10, 14, 19, 25, 30, and 35 post-injection.

[0194] 3.2 Sampling

[0195] Methods: Blood samples were collected from the retroorbital venous plexus of rats using blood collection tubes, placed in heparinized centrifuge tubes, and temporarily stored in an ice box. The blood samples were centrifuged at 4000 rpm for 10 minutes, and the supernatant plasma was separated and transferred to centrifuge tubes, stored at -80°C for further determination of FIN concentration in plasma using LC-MS. Data were processed and pharmacokinetic parameters analyzed using Origin and Phoenix software.

[0196] 3.3 Results

[0197] The pharmacokinetic parameters for different routes of administration are shown in Table 9. These data indicate that fingolimod hydrochloride sustained-release microspheres can significantly prolong the half-life of FIN in SD rats, and FIN in the microsphere group is significantly enriched in tissues and organs.

[0198] Table 9. Pharmacokinetic parameters of fingolimod hydrochloride in rats

[0199]

[0200] Blood drug concentration curves for different routes of administration are as follows: Figure 8 As shown, the microspheres exhibited the ability to deliver FIN continuously for 5 weeks, and their plasma concentration curves showed a biphasic curve.

[0201] The PK parameters were obtained by fitting the blood drug concentration of the intravenously injected rats using the Phoenix PK model, as shown in Table 10: the fast phase distribution rate constant α was higher than the slow phase elimination rate constant β. The rapid distribution time of FIN in SD rats was 0.08 h in the early stage, meaning that after rapid distribution within 5 minutes, the terminal elimination half-life required 4.5 hours for metabolism or excretion.

[0202] Table 10 Parameters of the FIN two-compartment model in rats of the intravenous injection group

[0203]

[0204] Based on the two-compartment model parameters of the intravenous injection group, the pharmacokinetic curves were deconvolved using the Loo-Riegelman deconvolution method in the Phoenix pharmacokinetic software. The cumulative absorption curve of FIN in the microspheres in SD rats was calculated, yielding the in vivo absorption-time curve. The formula for calculating the in vivo absorption fraction is as follows:

[0205] Loo-Riegelman method:

[0206] Wherein, Cp, Ct, K10 and AUC are the central compartment plasma concentration, apparent tissue compartment concentration, elimination rate constant and area under the plasma-time curve, respectively.

[0207] See results Figure 9 The FIN in the microspheres entered the absorption plateau phase in SD rats on day 30, with an in vivo absorption rate of 93.27%.

[0208] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A sustained-release microsphere of fingolimod hydrochloride, characterized in that, It was prepared by loading fingolimod hydrochloride onto a polylactic acid-glycolic acid copolymer in the presence of polyvinyl alcohol; The weight-average molecular weight of the polyvinyl alcohol is 40,000-50,000; The mass ratio of the polylactic acid-glycolic acid copolymer to fingolimod hydrochloride is 8-15:1; The preparation method of the fingolimod hydrochloride sustained-release microspheres includes the following steps: (1) Fingolimod hydrochloride and polylactic acid-glycolic acid copolymer were dissolved in an organic solvent to obtain an oil phase; (2) Polyvinyl alcohol was dissolved in water to prepare high-concentration aqueous phase and low-concentration aqueous phase respectively; (3) The oil phase is added to a high-concentration aqueous phase and homogenized to obtain an emulsion; (4) Pour the low-concentration aqueous phase into the emulsion, evaporate the organic solvent under stirring, collect the microspheres and dry them to obtain the fingolimod hydrochloride sustained-release microspheres; The organic solvent in step (1) is a mixture of ethanol and dichloromethane in a mass ratio of 1:8-10.

2. The fingolimod hydrochloride sustained-release microspheres according to claim 1, characterized in that, The weight-average molecular weight of the polyvinyl alcohol is 45,000-49,000.

3. The fingolimod hydrochloride sustained-release microspheres according to claim 1, characterized in that, The mass ratio of the polylactic acid-glycolic acid copolymer to fingolimod hydrochloride is 9-12:1; And / or, the polylactic acid-glycolic acid copolymer is a carboxyl-terminated polylactic acid-glycolic acid copolymer; And / or, the polylactic acid-glycolic acid copolymer has a weight-average molecular weight of 25k-35k; And / or, the molar ratio of lactic acid to glycolic acid monomers in the polylactic acid-glycolic acid copolymer is 45:55-55:

45.

4. The fingolimod hydrochloride sustained-release microspheres according to claim 3, characterized in that, The mass ratio of the polylactic acid-glycolic acid copolymer to fingolimod hydrochloride is 10-11:1; And / or, the weight-average molecular weight of the polylactic acid-glycolic acid copolymer is 28k-32k; And / or, the molar ratio of lactic acid to glycolic acid monomers in the polylactic acid-glycolic acid copolymer is 50:

50.

5. The fingolimod hydrochloride sustained-release microspheres according to claim 4, characterized in that, The polylactic acid-hydroxyacetic acid copolymer has a weight-average molecular weight of 30k.

6. A method for preparing fingolimod hydrochloride sustained-release microspheres according to any one of claims 1-5, characterized in that, Includes the following steps: (1) Fingolimod hydrochloride and polylactic acid-glycolic acid copolymer were dissolved in an organic solvent to obtain an oil phase; (2) Polyvinyl alcohol was dissolved in water to prepare high-concentration aqueous phase and low-concentration aqueous phase respectively; (3) The oil phase is added to a high-concentration aqueous phase and homogenized to obtain an emulsion; (4) Pour the low-concentration aqueous phase into the emulsion, evaporate the organic solvent under stirring, collect the microspheres and dry them to obtain the fingolimod hydrochloride sustained-release microspheres; The organic solvent in step (1) is a mixture of ethanol and dichloromethane in a mass ratio of 1:8-10.

7. In the method for preparing fingolimod hydrochloride sustained-release microspheres according to claim 6, the concentration of fingolimod hydrochloride in the oil phase is 45 mg / mL-55 mg / mL.

8. In the method for preparing fingolimod hydrochloride sustained-release microspheres according to claim 7, the concentration of fingolimod hydrochloride in the oil phase is 49 mg / mL-51 mg / mL.

9. The method for preparing fingolimod hydrochloride sustained-release microspheres according to claim 6, characterized in that, The concentration of polyvinyl alcohol in the high-concentration aqueous phase is 6 mg / mL-60 mg / mL; And / or, the concentration of polyvinyl alcohol in the low-concentration aqueous phase is 2 mg / mL-5 mg / mL.

10. The method for preparing fingolimod hydrochloride sustained-release microspheres according to claim 9, characterized in that, The concentration of polyvinyl alcohol in the high-concentration aqueous phase is 10 mg / mL-50 mg / mL; And / or, the concentration of polyvinyl alcohol in the low-concentration aqueous phase is 3 mg / mL.

11. The method for preparing fingolimod hydrochloride sustained-release microspheres according to claim 10, characterized in that, The concentration of polyvinyl alcohol in the high-concentration aqueous phase is 15 mg / mL to 25 mg / mL.

12. The method for preparing fingolimod hydrochloride sustained-release microspheres according to claim 11, characterized in that, The concentration of polyvinyl alcohol in the high-concentration aqueous phase is 18 mg / mL-22 mg / mL.

13. The method for preparing fingolimod hydrochloride sustained-release microspheres according to claim 6, characterized in that, The volume ratio of the oil phase to the high-concentration aqueous phase is 1:8-12; And / or, the homogenization time is 1 minute to 2 minutes; And / or, the rotational speed of the homogeneous fluid is 5000 rpm to 10000 rpm; And / or, the temperature of the homogenization is 0°C-8°C; And / or, step (3) includes: transferring the oil phase into a syringe and injecting the oil phase dropwise into the high-concentration aqueous phase using a syringe pump at a injection rate of 25-35 mm / min.

14. The method for preparing fingolimod hydrochloride sustained-release microspheres according to claim 13, characterized in that, The volume ratio of the oil phase to the high-concentration aqueous phase is 1:10; And / or, the homogenization time is 80-100 seconds; And / or, the rotational speed of the homogeneous fluid is 7000rpm-8000rpm.

15. The method for preparing fingolimod hydrochloride sustained-release microspheres according to claim 14, characterized in that, The homogenization time is 85-95 seconds; And / or, the rotational speed of the homogeneous fluid is 7400 rpm to 7600 rpm.

16. The method for preparing fingolimod hydrochloride sustained-release microspheres according to any one of claims 6-15, characterized in that, The volume of the low-concentration aqueous phase is 4-6 times that of the high-concentration aqueous phase; And / or, the stirring speed in step (4) is 500 rpm-700 rpm, and the time is 4 hours-6 hours; And / or, step (4) of collecting and drying microspheres includes: centrifuging, washing, sieving, collecting microspheres between 150 and 600 mesh, redispersing them in water, and then freeze-drying them to obtain the fingolimod hydrochloride sustained-release microspheres.

17. The method for preparing fingolimod hydrochloride sustained-release microspheres according to claim 16, characterized in that, The centrifugation conditions include: a temperature of 0℃-8℃, a rotation speed of 3000 rpm-5000 rpm, and a time of 8 minutes-12 minutes.

18. The method for preparing fingolimod hydrochloride sustained-release microspheres according to claim 17, characterized in that, The centrifugation conditions include: a temperature of 0℃-5℃, a rotation speed of 3500 rpm-4500 rpm, and a time of 9 minutes-11 minutes.

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