A cyclosporin a solid self-emulsifying pharmaceutical composition and a preparation method thereof

Abstract

The application discloses a cyclosporine A solid self-emulsifying drug composition and a preparation method thereof, and the drug composition comprises, in percentage by mass, 60-75% of a carrier material, 16.7-33.3% of a compatibility adjusting material, and the rest of cyclosporine A, and the sum of the mass percentages of all raw materials is 100%. The application is innovative in process from two aspects of selection of a compatibility adjusting material and a drug loading mode by developing a brand-new pre-molecularization-co-extrusion mode, and a ternary amorphous solid dispersion of cyclosporine A with strong compatibility and high stability is designed. The application fully utilizes the dissolution advantage of the amorphous solid dispersion technology, promotes the release of cyclosporine A, and simultaneously, due to the formation of a self-assembled sub-micro emulsion system, the intestinal permeability of cyclosporine A is significantly improved, and therefore, the application is a new preparation strategy which is safe and effective, green and environmentally-friendly, and has application prospects.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical formulation technology, specifically relating to a solid self-emulsifying pharmaceutical composition of cyclosporine A based on amorphous solid dispersion technology and its preparation method. Background Technology

[0002] Self-emulsifying Drug Delivery Systems (SEDDS) are isotropic mixtures composed of an oil phase, surfactants, and co-surfactants. Upon contact with an aqueous medium, they form nanoscale oil-in-water (O / W) emulsion colloidal particles. Due to their simple production and good performance, they are one of the preferred lipid nanocarriers in the pharmaceutical industry. Furthermore, SEDDS offer advantages in protecting drug metabolism (by loading drugs into droplets within the emulsion to prevent hydrolysis by the acidic or enzymatic environment of the gastrointestinal tract) and improving the permeability of the intestinal mucus layer to overcome absorption barriers, making them highly advantageous for the oral delivery of large molecule drugs such as peptides and proteins. While the use of surfactants can effectively solubilize poorly soluble drugs and improve their bioavailability, they often pose potential toxic side effects. In addition, most marketed products based on SEDDS systems are liquid, facing challenges such as limited dosage form selection and drug precipitation. Current research typically employs solidification techniques, such as solid carrier adsorption technology, freeze-drying technology, spray drying technology, and hot-melt extrusion technology, to improve the physicochemical stability of liquid SEDDS systems and reduce production costs.

[0003] Hot-melting extrusion (HME) technology is a novel formulation technology that has been vigorously developed in Europe, America, Japan, and other countries in recent years. Its principle is based on adding two or more materials into a progressively temperature-controlled barrel. A screw element is installed inside the barrel, and the screw element sequentially performs different unit operations from the feeding point to the die head. The materials move forward under the propulsion of the screw, melting or softening in certain sections. The melt is uniformly mixed under the action of shearing and mixing elements, and finally extruded from the die head orifice at a certain pressure, speed, and shape. The basic idea of ​​HME technology is to continuously set up multiple unit operations within an axial space, allowing the particle size of the multi-component materials to continuously decrease as they pass through this space, while simultaneously undergoing symmetrical exchange and penetration of spatial positions, ultimately achieving molecular-level mixing. This process includes both blending modification and extrusion molding. HME technology is a continuous, solvent-free formulation process that is easy to scale up. It can be used to produce various drug dosage forms and is one of the most commonly used industrial production technologies for preparing amorphous solid dispersions (ASDs). It is an important technology for improving the solubility and bioavailability of poorly soluble drugs. It has advantages such as masking drug odor, low cost, significant cost-effectiveness, high consistency and reproducibility, and can also be used to prepare sustained-release formulations.

[0004] Cyclosporin A (CsA) is an immunosuppressant developed by Novartis Pharmaceuticals. It belongs to the cadherin inhibitor class and works by suppressing the activity of the immune system, thereby inhibiting abnormal immune responses. It is used to treat rejection in organ and bone marrow transplant patients and can also be used to treat autoimmune diseases. Because cyclosporin A has good inhibitory effects on both cellular and humoral immunity, it can act on both simultaneously. However, CsA has low oral bioavailability (10%-60%) and high in vivo variability, attributed to its high molecular weight, high lipophilicity, low intestinal permeability, and first-pass metabolism in the liver and gastrointestinal mucosa. Furthermore, it has significant hepatotoxicity, nephrotoxicity, hypertension, and gastrointestinal side effects, thus affecting its further application and development. In commercially available cyclosporin A soft capsules developed and manufactured by Novartis... Cyclosporin A was pre-formulated as a microemulsion pre-concentrate, which forms a homogeneous microemulsion upon contact with gastrointestinal fluids to improve absorption. However, Soft capsules contain surfactants RH can cause allergic reactions and nephrotoxicity, and the co-surfactant ethanol can significantly increase the risk of drug degradation. Furthermore, due to poor permeability and low bioavailability, the daily oral dosage is as high as 15-27 tablets. (25mg) soft capsules can easily cause serious side effects of CsA, such as nephrotoxicity, hepatotoxicity, and neurotoxicity. Among these, the safety issues arising from the use of large amounts of surfactants and the stability risks of drug degradation and / or drug precipitation are common challenges faced by SEDDS formulations.

[0005] Therefore, developing a new formulation strategy that is biosafe, stable, and can effectively improve the oral bioavailability of CsA is of great significance, and can also provide a new approach for the development of oral formulations of other peptide and protein drugs. Summary of the Invention

[0006] One objective of this invention is to provide a solid self-emulsifying pharmaceutical composition of cyclosporine A, comprising, by weight percentage: 60-75% carrier material, 16.7-33.3% compatibility modifier (oil phase), and the remainder being cyclosporine A, wherein the sum of the weight percentages of all raw materials is 100%.

[0007] The carrier material is selected from polyvinylpyrrolidone (such as K12, K17, K25, K30, K60, K90, etc.) and copolyvinylpyrrolidone (such as... HPMC SR, etc.), polyvinyl alcohol (such as 388, 488, 1788, 2488, etc.), Utectic resins (such as EPO, LPO, etc.) and cellulose derivatives (such as methylcellulose MC, hydroxypropylcellulose HPC, hydroxypropyl methylcellulose HPMC, hydroxypropyl methylcellulose acetate succinate HPMC, hydroxypropyl methylcellulose acetate phthalate HPMCP, etc.);

[0008] The compatibility modifiers are small molecule oils (such as trans-anestinene, eugenol, cinnamaldehyde, thymol, vanillin, sennaol, castor oil, hydrogenated castor oil, tea oil, etc.) and medium / long chain oils (such as soybean oil, hydrogenated soybean oil, corn oil, olive oil, coconut oil, fish oil, sesame oil, rapeseed oil, sunflower oil, etc.).

[0009] Furthermore, the amount of the carrier material is 75%, the amount of the compatibility regulating material is 16.7%, and the amount of cyclosporine A is 8.3%.

[0010] Further, the carrier material is a cellulose derivative, preferably methylcellulose MC, hydroxypropylcellulose HPC, hydroxypropyl methylcellulose HPMC, hydroxypropyl methylcellulose acetate succinate HPMC, or hydroxypropyl methylcellulose phthalate HPMCP; more preferably HPC L, HPMC E6, or HPMCP HP-55; the oil phase is a small molecule oil, preferably trans-anenethole (TA).

[0011] A second objective of this invention is to provide a method for preparing the above-mentioned cyclosporine A solid self-emulsifying pharmaceutical composition, comprising the following steps:

[0012] Step 1: Dissolve cyclosporine A in a compatibility modifier.

[0013] Step 2: Mix the mixture from Step 1 with the carrier material;

[0014] Step 3: The mixture from Step 2 is fed into a twin-screw hot melt extruder and extruded into a linear material. After grinding, the solid self-emulsifying drug composition is obtained.

[0015] Further, in step 1, cyclosporine A is mixed with a compatibility modifier and heated to 60-95°C to dissolve cyclosporine A in the compatibility modifier. In one embodiment of the present invention, 80°C is preferred.

[0016] Furthermore, in step 3, the screw speed of the twin-screw hot melt extruder is 10-60 rpm, and the heating temperature is 80-200℃. In one embodiment of the present invention, 20 rpm is preferred.

[0017] First, this invention utilizes hot melt extrusion (HME) technology to prepare a ternary CsA amorphous solid dispersion (ASD). Currently, there are no reports on the preparation of CsA ASD using HME technology; therefore, the significant solubility advantage of ASD suggests a broad market prospect for CsA ASD. CsA exhibits extremely poor compatibility with various polymers suitable for HME development. Only PVP has been reported as a carrier polymer, but it faces the risk of hygroscopic transformation and crystallization. Therefore, it is difficult to develop CsA ASD through binary co-extrusion of CsA and polymers, requiring pre-molecularization treatment with special ligands and processes before extrusion. Furthermore, very few reports indicate that ASD can increase intestinal permeability by generating a liquid-liquid phase separation (LLPS) drug-enriched phase; therefore, preparing a single ASD makes it difficult to increase the in vivo permeability of CsA. This invention improves the compatibility between the drug and polymer by adding trans-anisene (TA), and achieves the construction of a CsA solid-state self-emulsifying drug delivery system based on ASD technology via HME process using a pre-molecularized co-extrusion method. This invention innovates the process by developing a novel pre-molecularized-co-extrusion mode, focusing on both the selection of compatibility-modifying substances and the drug loading mode, resulting in a CsA ternary ASD with strong compatibility and high stability.

[0018] Secondly, this invention combines hot melt extrusion technology with self-microemulsification technology. CsA ASD is prepared using HME technology. During dissolution, ASD undergoes self-microemulsification, forming submicroemulsions with particle sizes between 600-1500 nm, thereby increasing drug supersaturation, prolonging supersaturation time, and significantly enhancing drug permeability. This invention's self-microemulsification technology based on ASD formulations effectively avoids the extensive use of surfactants, and the one-step preparation method solves the complexity of traditional liquid self-emulsification and solidification processes, achieving for the first time the combined application of ASD formulations and self-microemulsification systems.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) By developing a novel pre-molecularization-co-extrusion mode, we have innovated the process from two aspects: the selection of compatibility-modifying substances and the drug loading mode. We have successfully prepared CsA ternary ASD with strong compatibility and high stability.

[0021] (2) Compared with physical mixtures of CsA and various polymers, the novel CsA ternary ASD spontaneously forms a submicroemulsion system during the dissolution process, which has significant advantages in supersaturation and permeability. Attached Figure Description

[0022] Figure 1 Powder X-ray diffraction patterns of CsA ASD prepared in Examples 1-4.

[0023] Figure 2 The results show the content detection of CsA and TA in the CsA ASD prepared in Examples 1-4.

[0024] Figure 3 Liquid forms of CsA, TA, cellulose derivatives (MC, HPC, HPMC, HPMCP) and CsA ASD prepared in Examples 1-4 1 H nuclear magnetic resonance spectrum.

[0025] Figure 4 The dissolution comparison diagram of CsA ASD prepared in Examples 1-4 and physical mixture (PM) prepared in the control example in phosphate buffer medium at pH 6.8 (leaking conditions).

[0026] Figure 5 Dissolution comparison diagram of CsA ASD prepared in Examples 1-4 and physical mixture (PM) prepared in the control example in PBS 6.8 medium (supersaturation conditions).

[0027] Figure 6 Comparison of polarized light microscopy images of the dissolution products of CsAASD prepared in Examples 1-4 in PBS 6.8 medium (under supersaturation conditions).

[0028] Figure 7 A comparison of the permeability coefficients of CsA ASD prepared in Examples 1-4 and PM corresponding to Example 5 in the duodenum, jejunum, and ileum (PBS 6.8 medium, supersaturated conditions).

[0029] Figure 8 This is a schematic diagram showing the particle size comparison of the CsA ASD dissolved in PBS 6.8 medium at different times (under supersaturation conditions) for Examples 1-4.

[0030] Figure 9 A transmission electron microscope (TEM) schematic diagram of the dissolution products of CsA ASD prepared in Examples 1-4 in PBS 6.8 medium (under supersaturation conditions).

[0031] Figure 10 A schematic diagram of small-angle X-ray scattering (under supersaturation conditions) of the CsA-TA-HPMCP ASD dissolved in PBS 6.8 medium for Example 4.

[0032] Figure 11 The time-enthalpy curves and their fitting curves of the isothermal titration calorimetric method of TA with cellulose derivatives (MC, HPC, HPMC, HPMCCP) in PBS 6.8 medium are shown in the figure (under supersaturation conditions).

[0033] Figure 12 This is a schematic diagram of the time-interfacial tension curves of TA and cellulose derivatives (MC, HPC, HPMC, HPMCP) in PBS 6.8 medium (under supersaturation conditions).

[0034] Figure 13 The images show microscopic images of H&E staining of duodenal, jejunal, and ileal tissues prepared in Examples 1-4 4 hours after administration of CsA ASD (magnified 50x, 100x, and 200x). Detailed Implementation

[0035] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0037] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0038] In the following embodiments, the percentage is a mass percentage.

[0039] Example 1

[0040] Step S100: CsA active pharmaceutical ingredient (8.3%) crystal powder and TA (16.7%) liquid are mixed and heated together in an 80°C water bath to fully dissolve the active pharmaceutical ingredient and obtain a liquid mixture of the two.

[0041] Step S200: The above liquid mixture is thoroughly mixed with MC (75%) powder to obtain a homogeneous mixture of the three.

[0042] Step S300: The above uniform mixture is uniformly fed into a twin-screw hot melt extruder. The screw speed is set to 20 rpm, and the heating temperature gradient is set to 100℃, 140℃, and 180℃. A linear material is formed at the die orifice. After cooling, it becomes solid, is ground and sieved for later use, and CsA-TA-MC ASD is obtained.

[0043] Example 2

[0044] Step S100: CsA active pharmaceutical ingredient (8.3%) crystal powder and TA (16.7%) liquid are mixed and heated together in an 80°C water bath to completely dissolve the active pharmaceutical ingredient and obtain a liquid mixture of the two.

[0045] Step S200: The above liquid mixture is thoroughly mixed with HPC L (75%) powder to obtain a homogeneous mixture of the three.

[0046] Step S300: The above uniform mixture is uniformly fed into a twin-screw hot melt extruder. The screw speed is set to 20 rpm, and the heating temperature gradient is set to 100℃, 140℃, and 180℃. A linear material is formed at the die orifice. After cooling, it becomes solid, is ground and sieved for later use, and CsA-TA-HPC ASD is obtained.

[0047] Example 3

[0048] Step S100: CsA active pharmaceutical ingredient (8.3%) crystal powder and TA (16.7%) liquid are mixed and heated together in an 80°C water bath to completely dissolve the active pharmaceutical ingredient and obtain a liquid mixture of the two.

[0049] Step S200: The above liquid mixture is thoroughly mixed with HPMC E6 (75%) powder to obtain a homogeneous mixture of the three.

[0050] Step S300: The above uniform mixture is uniformly fed into a twin-screw hot melt extruder. The screw speed is set to 20 rpm, and the heating temperature gradient is set to 100℃, 140℃, and 180℃. A linear material is formed at the die orifice. After cooling, it becomes solid, is ground and sieved for later use, and CsA-TA-HPMC ASD is obtained.

[0051] Example 4

[0052] Step S100: CsA active pharmaceutical ingredient (8.3%) crystal powder and TA (16.7%) liquid are mixed and heated together in an 80°C water bath to completely dissolve the active pharmaceutical ingredient and obtain a liquid mixture of the two.

[0053] Step S200: The above liquid mixture is thoroughly mixed with HPMCP HP-55 (75%) powder to obtain a homogeneous mixture of the three.

[0054] Step S300: The above-mentioned uniform mixture is uniformly fed into a twin-screw hot melt extruder. The screw speed is set to 20 rpm, and the heating temperature gradient is set to 100℃, 140℃, and 180℃. A linear material is formed at the die orifice. After cooling, it becomes solid, is ground and sieved for later use, and CsA-TA-HPMCP ASD is obtained.

[0055] Comparison Example

[0056] The physical mixture (PM) of CsA active pharmaceutical ingredient (8.3%) crystal powder, TA (16.7%) liquid and MC (75%) powder was directly and thoroughly mixed for analysis.

[0057] The physical mixture (PM) of CsA active pharmaceutical ingredient (8.3%) crystal powder, TA (16.7%) liquid and HPC L (75%) powder was directly and thoroughly mixed for analysis.

[0058] The physical mixture (PM) of CsA active pharmaceutical ingredient (8.3%) crystalline powder, TA (16.7%) liquid, and HPMC E6 (75%) powder was directly and thoroughly mixed for analysis.

[0059] The physical mixture (PM) of CsA active pharmaceutical ingredient (8.3%) crystal powder, TA (16.7%) liquid, and HPMCCP HP-55 (75%) powder was directly and thoroughly mixed for analysis.

[0060] Experimental Example 1

[0061] Solid characterization of CsA ASD prepared by HME technique

[0062] The extruded powders prepared according to steps 1-4 will be subjected to solid-state characterization studies, including PXRD, high-performance liquid chromatography (HPLC) content determination, and Ls. 1 H NMR was used to investigate the solid-state properties of ASD.

[0063] PXRD parameters: Bruker D8 Advance X-ray diffractometer was used. Tube current (100mA); voltage (40kV); scan speed: 4° / min; step size: 0.02°; scan range: 5°-30°.

[0064] HPLC parameters: A Shimadzu LC-2010a HT reversed-phase high-performance liquid chromatograph was used. CsA chromatographic conditions—Mobile phase: 70% Phase A (acetonitrile:methanol 550:50) + 30% Phase B (0.125% phosphate-pure water). Column: Inertsil ODS-SP reversed-phase column (150×4.6mm, 5μm); Column temperature: 60℃; Flow rate: 1.0mL / min; Wavelength: 210nm. TA chromatographic conditions—Mobile phase: 70% Phase A (acetonitrile) + 30% Phase B (pure water-0.05% triethylamine-0.05% acetic acid). Column: Inertsil ODS-SP reversed-phase column (150×4.6mm, 5μm); Column temperature: 30℃; Flow rate: 1.2mL / min; Wavelength: 230nm. Preparation of standard solutions: CsA 40 μg / mL and TA 40 μg / mL (both solvents are methanol); Preparation of test solutions: Weigh an appropriate amount of the above ASD solution (solvent is methanol).

[0065] Ls 1 H NMR parameters: A Bruker VANCE III-HD 600MHz nuclear magnetic resonance spectrometer was used. The X-ray channel and magnetic field were set to 500MHz at 297K. 1 H) and 14.09T. Obtained at a scan width of 36.2kHz. 1 ¹H NMR spectra. Spectral data were analyzed using MestReNova software (version 14.2.0). Sample preparation method: (1) Weigh 20.0 mg of CsA, dissolve it in 0.6 mL of (CD₃)₂S=O at room temperature, and seal it in an NMR tube; (2) Seal 20.0 mg of TA in a quartz capillary tube (Φ=1 mm, (2) 0.6 mL of (CD3)2S=O was sealed in an NMR tube; (3) 10.0 mg of TA was dissolved in 20.0 mg of TA in an 80 °C water bath to prepare a CsA-TA solution, and the prepared solution sample was sealed in a quartz capillary tube (Φ=1.0 mm, (3) Weigh 20.0 mg of MC, HPC, HPMC and HPMC, dissolve them in 0.6 mL of (CD3)2S=O at room temperature, and then seal them in an NMR tube; (4) Weigh 20.0 mg of CsA-TA-MC ASD, CsA-TA-HPC ASD, CsA-TA-HPMC ASD and CsA-TA-HPMCP ASD, dissolve them in 0.6 mL of (CD3)2S=O at room temperature, and then seal them in an NMR tube.

[0066] like Figure 1 As shown, the extrudate powders prepared in Examples 1-4 all lacked any characteristic peaks of drug crystals, indicating that the extrudates existed in an amorphous form within the polymer matrix, proving that they were amorphous solid dispersions.

[0067] like Figure 2 As shown, the contents of CsA and TA in the ASDs prepared in Examples 1-4 are as follows: the samples include CsA-TA-MCASD, CsA-TA-HPC ASD, CsA-TA-HPMC ASD and CsA-TA-HPMCP ASD. The contents of CsA and TA in all samples are approximately consistent with the theoretically calculated contents, which proves that they have not undergone chemical degradation and volatilization and have good physical and chemical stability.

[0068] like Figure 3 As shown, (a) Ls of CsA, TA, CsA-TA, HPMCP, CsA-TA-HPMCP ASD 1 H NMR spectra [(CD3)S=O, 500MHz], (b) CsA, TA, CsA-TA, HPMC, CsA-TA-HPMC ASD Ls 1 H NMR spectra [(CD3)S=O, 500MHz], (c)CsA, TA, CsA-TA, HPC, CsA-TA-HPC ASD Ls 1 H NMR spectra [(CD3)S=O, 500MHz]; (d) Ls of CsA, TA, CsA-TA, MC, CsA-TA-MC ASD 1 H NMR spectrum [(CD3)S=O, 500MHz]. Comparison Figure 4The characteristic chemical shifts of CsA and TA, and the van der Waals interactions between CsA and TA, as well as HPMCP, HPMC, HPC, and MC, are the reasons for the efficient preparation of ASD; and the order of the number of interacting sites in the system is: CsA-TA-MC ASD <CsA-TA-HPC ASD<CsA-TA-HPMC ASD<CsA-TA-HPMCP ASD。

[0069] Experimental Example 2

[0070] Dissolution profile determination under in vitro leak conditions

[0071] Dissolution curves were determined using appropriate amounts of ASD samples from Examples 1-4 and PM prepared from the control examples. Following the second method (paddle method) of the Dissolution and Release Determination Method in the 2020 edition of the Chinese Pharmacopoeia, a Tianjin Tianfa RC-806 dissolution apparatus was used with 900 mL PBS 6.8 as the dissolution medium at a rotation speed of 100 rpm. 0.15 g of ASD and 0.125 g of PM were added to the medium. 5 mL samples were taken at 5, 10, 15, 20, 30, 45, 60, 90, and 120 min, with the same volume of dissolution medium added simultaneously. After filtering through a 0.45 μm microporous membrane, the cumulative dissolution rate was determined and calculated using the HPLC method described above.

[0072] Table 1. Cumulative dissolution of CsAASD under PBS 6.8 conditions at 37°C (n=3, leaky conditions)

[0073]

[0074] Table 2. Cumulative dissolution of PM under PBS 6.8 conditions at 37℃ (n=3, leaky conditions)

[0075]

[0076] As shown in Table 1-2 and Figure 4 As shown, the self-made CsAASD was rapidly released in PBS 6.8 medium and had a significant dissolution advantage compared to PM; meanwhile, HPMCP and HPMC ASD were completely released in the medium, while HPC and MC only achieved 80% drug release; the order of advantages in in vitro drug release rate and dissolution endpoint was: CsA-TA-MC ASD. <CsA-TA-HPC ASD<CsA-TA-HPMC ASD<CsA-TA-HPMCP ASD。

[0077] Experimental Example 3

[0078] Determination of dissolution profile under in vitro supersaturation conditions

[0079] Dissolution curves were determined using excess samples from Examples 1-4 and PM prepared from control examples. Guided by the Dissolution and Release Determination Method in General Chapter 0931 of the Chinese Pharmacopoeia (2020 Edition), a Tianjin Tianfa RC-806 dissolution apparatus was used. The dissolution medium was 100 mL PBS 6.8, and the dissolution speed was 100 rpm. 1.5 g ASD and 1.25 g PM were added to the medium. Samples were taken at 0.167 h, 0.25 h, 0.33 h, 0.5 h, 0.75 h, 1 h, 1.5 h, 2 h, 3 h, 4 h, 6 h, 8 h, and 12 h, with the same volume of dissolution medium added simultaneously. After filtration through a 0.45 μm microporous membrane, the drug dissolution amount was determined and calculated according to the above HPLC method.

[0080] Table 3. Dissolution rate of CsAASD at 37℃ and PBS 6.8 (n=3, supersaturated conditions)

[0081]

[0082] Table 4. Dissolution rate of PM at 37℃ and PBS 6.8 (n=3, supersaturated conditions)

[0083]

[0084] As shown in Table 3-4 and Figure 5 As shown, the self-made CsAASD significantly improved the supersaturation of the drug and maintained a longer supersaturation time in PBS 6.8, exhibiting a much larger improvement in supersaturation compared to PM. Further comparison... Figure 6 (a) is a polarizing microscope (PLM) image of the emulsion collected after 1.5 h of dissolution of CsA-TA-HPMCP; (b) is a PLM image of the emulsion collected after 1.5 h of dissolution of CsA-TA-HPMC ASD; (c) is a PLM image of the emulsion collected after 1.5 h of dissolution of CsA-TA-HPC ASD; and (d) is a PLM image of the emulsion collected after 1.5 h of dissolution of CsA-TA-MC ASD. The results show that the significance of birefringence is ranked as CsA-TA-MC ASD > CsA-TA-HPC ASD > CsA-TA-HPMC ASD > CsA-TA-HPMCP ASD, indicating that CsA-TA-HPMCP ASD has the strongest supersaturation capability and advantage.

[0085] Test Example 4

[0086] In vitro permeability measurement

[0087] Excess samples from Examples 1-4 and PM prepared from control examples were used for in vitro permeation determination. The small intestine (duodenum, jejunum, and ileum) of male SD rats (approximately 250g) was used as a model. The everted intestinal sac method was employed to increase microvilli and mucus layers, ensuring full expansion and contact with the drug for facilitating drug throughput measurement. Using a blunt-tipped syringe, the intestinal segment model was rinsed three times with PBS 6.8, and then gently inverted on a 3mm diameter glass rod. The everted intestinal sac was divided into individual sacs approximately 5.0cm long and filled with approximately 1 mL of HBSS buffer. Following the Dissolution and Release Determination Method II (Paddle Method) of the 2020 edition of the Chinese Pharmacopoeia, a Tianda Tianfa RC-806 dissolution apparatus was used with 100mL PBS 6.8 as the dissolution medium at 100rpm. Each intestinal sac, approximately 1.50g of ASD, and the corresponding 1.25g of PM were placed in separate dissolution apparatus cups. Approximately 6 hours later, the intestinal sacs were removed, rinsed four times with saline solution, and wiped to remove excess water from the surface. The intestinal sacs were cut open to obtain the intestinal contents, and each sac was weighed before and after collecting the intestinal contents to calculate the volume of fluid within it. Simultaneously, the width and length of the intestinal segment were measured to calculate the permeability area of ​​each sac. 100 μL of intestinal contents were vortexed with 200 μL of acetonitrile for 2 min, followed by centrifugation at 12000 rpm for 10 min to obtain the supernatant. The CsA concentration was determined using the above-described HPLC method, and the permeability coefficient was calculated.

[0088] Table 5. In vitro permeability coefficients of CsAASD in different intestinal segments under PBS 6.8 conditions at 37℃.

[0089]

[0090] Table 6. In vitro permeability coefficients of CsAPM in different intestinal segments under PBS 6.8 conditions at 37℃.

[0091]

[0092] The results are shown in Tables 5-6 and Figure 7As shown, where (a) shows the permeability coefficients of CsA-TA-MC ASD, CsA-TA-HPC ASD, CsA-TA-HPMC ASD, and CsA-TA-HPMCP ASD in the duodenum, jejunum, and ileum; (b) shows the permeability coefficients of CsA-TA-MCPM, CsA-TA-HPC PM, CsA-TA-HPMC PM, and CsA-TA-HPMCP PM in the duodenum, jejunum, and ileum. The CsA ASD prepared with various polymers can all increase the drug permeability coefficient in PBS 6.8, showing a significant permeability advantage compared to PM. The advantage ranking is: CsA-TA-MC ASD < CsA-TA-HPC ASD < CsA-TA-HPMC ASD < CsA-TA-HPMCP ASD. Further comparing the ability of different intestinal segments to improve permeability, it is found that the permeability of ASD is the strongest in the duodenum, and the permeability ability ranking is: ileum < jejunum < duodenum.

[0093] Test Example 5

[0094] Particle Size Test of Dissolution Products

[0095] The dissolution products of the samples in Examples 1-4 were subjected to dynamic light scattering particle size testing. Four ASD powders were separately added to PBS 6.8 medium (equivalent to a concentration of 1.125 mg / mL of cellulose derivative in the medium) to prepare the dissolution products. The samples were stirred at a constant speed of 100 rpm in a 37 °C water bath. At 1 h, 1.5 h, 2 h, 3 h, 4 h, 6 h, 8 h, and 12 h, 5 mL of the product was taken with a 0.45 μm filter membrane for detection, and 5 mL of fresh dissolution medium was supplemented. The obtained samples were tested on a Brookhaven NanoBrook Omni particle size analyzer. Each sample was measured 3 times, with each measurement lasting 30 s, and the average of the three measurements was taken as the sample data. The particle size measurement was carried out in a quartz cell at 25 °C with a scattering angle of 90°.

[0096] Table 7 Particle Size Changes of CsA ASD during Dissolution at 37 °C in PBS 6.8

[0097]

[0098] The results are shown in Table 7 and Figure 8As shown, the dissolution products of CsA-TA-MC ASD were too large to be measured in terms of particle size; the particle size of CsA-TA-HPC ASD was between 1560-3960 nm, and significant aggregation occurred during dissolution, indicating that it could not form stable submicroemulsions. In contrast, due to the gradual aggregation of TA, the droplet sizes of CsA-TA-HPMC / HPMCP ASD increased to approximately 1553 nm and approximately 942 nm, respectively, at 1 h. Further, due to the release of CsA molecules from TA, the droplet sizes decreased to approximately 894 nm and approximately 672 nm, respectively, at 4 h, reaching a plateau. Meanwhile, the droplet size in the CsA-TA-HPMCP ASD sample was significantly smaller than that in the CsA-TA-HPMC ASD sample (approximately 600-700 nm). The particle size ranking of the emulsion droplets was CsA-TA-MC ASD>CsA-TA-HPC ASD>CsA-TA-HPMC ASD>CsA-TA-HPMCP ASD, which is opposite to the trend of the apparent permeability coefficient. This shows that the smaller the droplet size, the longer the stabilization time, and the more it can enhance the permeability of the drug in the intestine.

[0099] Experimental Example 6

[0100] ASD dissolution products were analyzed by transmission electron microscopy.

[0101] Samples from Examples 2-4 were subjected to transmission electron microscopy (TEM) analysis after dissolution. The structural characteristics of the ASD dissolution products were evaluated using a Hitachi HT770 TEM with an accelerating voltage of 120 kV. An appropriate amount of ASD was added to 100 mL of PBS 6.8 medium (polymer concentration: 1.125 mg / mL) and stirred at 100 rpm at 37 °C. Approximately 10 μL of sample (1 h, 1.5 h, and 4 h) was added dropwise to a carbon-coated copper mesh and incubated for 10 min. Excess sample was removed from the copper mesh, which was then stained twice with 2% phosphotungstic acid solution. The sample was then dried under infrared light (approximately 1 min) before imaging. The grain size of the obtained images was calculated using ImageJ 2.

[0102] Table 8. Diameter of each submicroemulsion mediated by CsA-TA-HPMC / HPMCP at different time points in the TEM field.

[0103]

[0104] The results are shown in Table 8 and Figure 9As shown, (ab) are TEM images (magnifications of 2.5K and 16K) of emulsion droplets formed by CsA-TA-HPMC ASD after incubation in PBS 6.8 medium for 1 h; (cd) are TEM images (magnifications of 2.5K and 16K) of emulsion droplets formed by CsA-TA-HPMC ASD after incubation in PBS 6.8 medium for 4 h; (ef) are TEM images (magnifications of 2.5K and 16K) of emulsion droplets formed by CsA-TA-HPMCP ASD after incubation in PBS 6.8 medium for 1.5 h; (gh) are TEM images (magnifications of 2.5K and 14K) of emulsion droplets formed by CsA-TA-HPMCP ASD after incubation in PBS 6.8 medium for 4 h; and (ij) are TEM images (magnifications of CsA-TA-HPMC ASD and CsA-TA-HPMCP ASD in PBS 6.8 medium for 1 h. TEM images (2.5K magnification) of emulsion droplets formed after incubation in PBS 6.8 medium for 1 h and 1.5 h; (kl) is a TEM image (2.5K magnification) of emulsion droplets formed after incubation of CsA-TA-HPC ASD in PBS 6.8 medium for 0.5 h and 4 h; (mn) is the droplet size distribution and average diameter of emulsion droplets counted using TEM images of CsA-TA-HPMC ASD (1 and 4 h, 2.5K magnification); (op) is the droplet size distribution and average diameter of emulsion droplets counted using TEM images of CsA-TA-HPMCP ASD (1.5 and 4 h, 2.5K magnification). As shown in Table 8, the width and height of the submicroemulsions are approximately the same, indicating that the three-dimensional structure of the particles is approximately spherical. Based on the particle size distribution and average particle size obtained from Gaussian fitting, ASD showed good particle size distribution (CsA-TA-HPMC ASD: 1h: 1402.5±342.7nm, 4h: 786.3±135.9nm; CsA-TA-HPMCP ASD: 1.5h: 987.7±270.9nm, 4h: 620.5±160.2nm), which was basically consistent with the DLS test results. The emulsion droplets in the CsA-TA-HPC ASD sample showed significant aggregation, exhibiting irregularly shaped aggregates. Further magnification revealed that the center of the spheres was a spherical oil droplet, and the translucent periphery of the spheres consisted of amphiphilic polymers (HPMC or HPMCP) attached to the oil-water interface as surfactants. The spherical oil droplets of the CsA-TA-HPMC / HPMCP ASD sample collected at 4 h were smaller than those of the CsA-TA-HPMC ASD sample collected at 1 h and the CsA-TA-HPMCP ASD sample collected at 1.5 h, which may be due to the release of CsA molecules dissolved in TA.Meanwhile, it can be seen that the particle size of the CsA-TA-HPMCP ASD-mediated submicroemulsion is smaller than that of the CsA-TA-HPMC ASD, and the polymer adsorption layer at the oil-water interface is thicker and denser, indicating that the submicroemulsion has stronger stability.

[0105] Experimental Example 7

[0106] Small-angle X-ray scattering test during ASD dissolution

[0107] A suitable amount of the sample from Example 4 was subjected to small-angle X-ray scattering (SAXS) testing of the dissolution products. The structure of the submicroemulsion formed during the dissolution of CsA-TA-HPMCP ASD was determined using a Xeuss SAXS / WAXS system. Under supersaturated dissolution conditions (the same as the in vitro supersaturated dissolution curve determination test section), approximately 1 mL of CsA-TA-HPMCP ASD sample was taken for SAXS testing at 4 h. Approximately 100 μL of ASD sample was added to a glass plate fixed with polynicotinamide tape and then placed on a support in the room. The obtained SAXS data were analyzed using SasView software (version 4.1.2), and the results are as follows. Figure 10 As shown in the figure. The results show that the scattering curve clearly displays two peaks with symmetrical peak shapes, indicating the existence of different electron density regions, namely, the interface between the oil phase and the amphiphilic polymeric phase, and the interface between the amphiphilic polymeric phase and the solvent phase. Based on the q values ​​corresponding to the scattering peaks, the long diameter (D1 = 571 nm) and short diameter (D2 = 330 nm) were calculated, which are basically consistent with the DLS results (672 nm) and TEM results (620 nm), confirming the formation of submicroemulsion droplets with a "core-shell" structure.

[0108] Experimental Example 8

[0109] Isothermal titration calorimetry between TA and cellulose derivative solutions

[0110] Isothermal titration calorimetry was performed on appropriate amounts of TA, MC, HPC, HPMC, and HPMCP in PBS 6.8 solutions to analyze the driving forces in the self-assembly process of submicroemulsions. The energy of the interaction between the oil phase and the polymer was measured using a MicroCal PEAQ-ITC isothermal titration calorimeter from Malvern. TA was diluted to 1.0% (w / w) with PBS 6.8 medium, and MC, HPC, HPMC, or HPMCP were diluted to 1.125% (w / w), respectively. The calorimeter cell (approximately 220 μL) was filled with the polymer solution (1.125%, w / w), and the calorimeter needle (approximately 40 μL) was filled with the TA suspension (1.0%, w / w). The system was equilibrated at 37 °C. Titration was performed by adding an initial 0.4 μL of injection solution and 19 consecutive 2.0 μL injection solutions, while the mixture was continuously stirred at 750 rpm. Each injection lasted 4 seconds, with a 180-second interval between consecutive injections to obtain the dilution heat. Corrected data was obtained by subtracting the dilution heat data from the raw data.

[0111] Table 9. Specific thermodynamic parameters of each submicron emulsion assembly process fitted by the ITC method.

[0112]

[0113] The results are shown in Table 9 and... Figure 11 As shown, (a) isotherms of the titration of MC, HPC, HPMC, or HPMC / CP PBS 6.8 (1.125%, w / w) solutions with TA suspension (1.0%, w / w) and (be) combined isotherms of the titration of MC, HPC, HPMC, or HPMC / CP PBS 6.8 (1.125%, w / w) solutions with TA suspension (1.0%, w / w). ITC accurately reflects the thermodynamic process of a reaction between two substances by measuring the heat released or absorbed by intermolecular forces. It can be used to determine the interaction between the oil phase and amphiphilic polymers and directly quantify the thermodynamic parameters of the interaction, including the dissociation equilibrium constant (Kc). D The molar binding enthalpy (ΔH) and molar binding entropy (ΔS) were also considered. As shown in Table 9, the titration ΔG values ​​for all polymer solutions were less than 0, indicating that the reaction between the amphiphilic polymer and the oil phase proceeds spontaneously, and the intermolecular forces are the driving force for the self-assembly of the amphiphilic polymer at the oil phase interface. Furthermore, the dissociation constants between different components were ordered as follows: CsA-TA-MC ASD(K D =159×10 -6 ±52.3×10 -6 M)>CsA-TA-HPC ASD(K D =73.6×10 -6 ±30.0×10 -6M)>CsA-TA-HPMC ASD(K D =34.5×10 -6 ±8.84×10 -6 M)>CsA-TA-HPMCP ASD(K D =3.98×10 -6 ±2.70×10 -6 M). K D The inter-component stress strength is a key evaluation indicator; the stronger the interaction and affinity between components, the smaller the dissociation equilibrium constant of the system. In summary, the differences in interactions between different polymers are the main reason for the differences in droplet size and stability. Due to the strong intermolecular forces between HPMCP and TA, HPMCP, as a surfactant, rapidly adsorbs at the oil-water interface, preventing further aggregation of TA and thus forming a stable submicroemulsion system.

[0114] Experimental Example 9

[0115] Interfacial tension test between TA and cellulose derivative solution

[0116] Take an appropriate amount of TA and MC, HPC, HPMC, or HPMC / CP in PBS 6.8 solution (1.125%, w / w), and perform interfacial tension testing using a DataPhysical DCAT 21 automated tensiometer via the Wilhelmy plate method. The contact angle was set to 0°; the motor speed to 0.5 mm / s; and the surface detection threshold to 1 mg. Measurements were stopped when the standard deviation of the last 100 measurements was less than 0.03 mN / m. Approximately 35 mL of TA was added to the sample cell (Φ = 7.0 cm). Buoyancy data for a Wilhelmy plate (side length: 9.95 × 0.20 mm, immersion depth: 12.00 mm) was obtained in a solution with h = 3.5 cm. TA was then removed, and equal volumes of PBS 6.8 solution, MC, HPC, HPMC, or HPMC / PBS 6.8 solution were added to the sample cell, respectively. TA was then slowly added dropwise into the sample cell. When the TA level exceeded the upper edge of the Wilhelmy plate, interfacial tension curves were plotted between TA and the PBS 6.8 solution, MC, HPC, HPMC, or HPMC / PBS 6.8 solution, respectively, based on measurements. The results are shown below. Figure 12As shown, the IFT (Interfacial Tensor Strength) of emulsion droplets in aqueous media is as follows: CsA-TA-MC ASD (IFT = 6.27 mN / m) > CsA-TA-HPCASD (6.24 mN / m) > CsA-TA-HPMC ASD (6.03 mN / m) > CsA-TA-HPMCP ASD (4.79 mN / m). Generally, when one liquid comes into contact with another immiscible liquid, the smaller the interfacial tension at the interface between the two phases, the more stable the droplet distribution in the aqueous phase. In summary, different polymer types affect the structure and stability of submicroemulsions formed by CsAASD, due to the differences in intermolecular forces and interfacial tensions between TA and the polymer in the CsA ASD system.

[0117] Experimental Example 10

[0118] In vivo safety assessment

[0119] Appropriate amounts of samples from Examples 1-4 were taken for in vivo safety assessment. Male SD rats (250±20g) were randomly divided into 4 groups (n=3), and each group was administered CsAASD (CsA dose equivalent to 90mg / kg) by gavage. Each of the four groups of rats was given a single gavage of approximately 3mL of physiological saline suspension containing CsA-TA-MC ASD, CsA-TA-HPC ASD, CsA-TA-HPMC ASD, or CsA-TA-HPMCP ASD. After 6 hours of digestive tract metabolism, the duodenum, jejunum, and ileum were dissected and intestinal rolls were prepared, placed in embedding cassettes, and fixed with 4% paraformaldehyde solution at room temperature for 24 hours. The fixed samples were embedded in paraffin, cut into 5μm thick sections, and stained with hematoxylin and eosin (H&E). Finally, the tissue and cell morphology of the duodenum, jejunum, and ileum were observed under a Nikon Eclipse Ci-POL microscope. The results are as follows: Figure 13 As shown, the cell nuclei are stained purple, and the cytoplasm is stained pink. The duodenal, jejunal, and ileal tissues consist of four layers: the mucosa, submucosa, muscularis propria, and adventitia. Intestinal villi are visible on the mucosal surface, and small intestinal glands are visible in the lamina propria. The epithelial structure remains normal, and no significant atrophy of the glands is observed. There is no obvious degeneration or necrosis of cells, and no significant shortening of villi. It is evident that the submicroemulsions mediated by different ASDs have no significant irritant effect on intestinal tissue, demonstrating that the components within the ASDs have good biocompatibility.

Claims

1. A solid self-emulsifying pharmaceutical composition of cyclosporine A, characterized in that, The carrier material is 60-75% by mass, the compatibility adjusting material is 16.7-33.3% by mass, and the rest is cyclosporine A, and the sum of the mass percentages of all raw materials is 100%. The carrier material is a cellulose derivative, and the cellulose derivative is methyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, or hydroxypropyl methyl cellulate phthalate. The compatibility adjusting material is a small molecule oil, and the small molecule oil is trans-anethole. The preparation method of the cyclosporine A solid self-emulsifying drug composition is: first, dissolve cyclosporine A in the compatibility adjusting material, then mix with the carrier material, and then put the mixture into a double screw hot melt extruder to extrude into a linear material, and then grind to obtain the solid self-emulsifying drug composition.

2. The cyclosporine A solid self-emulsifying pharmaceutical composition according to claim 1, characterized in that, The amount of the carrier material is 75%, the amount of the compatibility adjusting material is 16.7%, and the amount of cyclosporine A is 8.3%.

3. A process for the preparation of a cyclosporin A solid self-emulsifying pharmaceutical composition according to claim 1 or 2, characterized in that, The method comprises the following steps: Step 1: dissolve cyclosporine A in the compatibility adjusting material; Step 2: mix the mixture of step 1 with the carrier material; Step 3: put the mixture of step 2 into a double screw hot melt extruder to extrude into a linear material, and then grind to obtain the solid self-emulsifying drug composition.

4. The production method according to claim 3, characterized by, In Step 1, specifically, cyclosporin A is mixed with a compatibility modifying material and heated to 60-95 o C, to dissolve the cyclosporin A in the compatibility modifying material.

5. The production method according to claim 3, characterized by, In Step 3, the screw rotation speed of the twin-screw hot melt extruder is 10-60 rpm, and the heating temperature is 80-200 o C.

6. A pharmaceutical composition, characterized by, The cyclosporine A solid self-emulsifying drug composition of claim 1 or 2 is used as an effective active ingredient.

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