Dexamethasone palmitate nanodisk as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and relates to a dexamethasone palmitate nanodisk as well as a preparation method and application thereof. The dexamethasone palmitate nanodisk disclosed by the invention is prepared from the following components: dexamethasone palmitate, phospholipid and membrane scaffold protein. According to the dexamethasone palmitate eye drops, the dexamethasone palmitate nanodisk still has good stability under the condition that no preservative is added, the residence time of the medicine on the ocular surface of a mouse is remarkably prolonged, and the bioavailability of the eye drops is improved. Results of an ovalbumin (OVA) induced allergic conjunctivitis mouse model show that the dexamethasone palmitate nanodisk can obviously relieve allergic symptoms and shows good safety and biocompatibility.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and relates to a dexamethasone palmitate nanodisc, a preparation method thereof, and an application for preventing or treating allergic diseases. Background Art

[0002] Type I hypersensitivity reaction is a common allergic reaction, and the main diseases involved include allergic conjunctivitis, allergic rhinitis, allergic asthma, atopic dermatitis, etc. Type I hypersensitivity reaction is mainly mediated by IgE. In the early reaction, Th2 cytokines such as IL-4, IL-5, IL-13, etc. are secreted, and IgE antibodies are produced, activating mast cells and causing their degranulation, releasing a series of inflammatory mediators such as histamine, resulting in phenomena such as itchy and swollen eyes. In the late reaction, eosinophils are recruited and activated. For such diseases, the current treatment drugs mainly include corticosteroids, mast cell stabilizers, and antihistamines. Among them, dexamethasone palmitate, as a corticosteroid, can effectively relieve inflammation due to its strong anti-inflammatory effect, such as rheumatoid arthritis, etc., but it is worrying due to its possible toxic and side effects. Therefore, it is necessary to develop appropriate corticosteroid preparations and drug delivery systems to improve the current problems.

[0003] Nanodiscs are small-sized disc-shaped nanocarriers, about 10 nm in size, and have been successfully applied to the fields of treating atherosclerosis, anti-tumor, etc. in recent years. The shape and size of nanoparticles have a significant impact on their in vivo behavior. Some studies have shown that nanodiscs have a strong edge-attaching ability when transported in blood vessels due to their special disc-shaped structure, and tend to roll from the conventional circulation to the blood vessel wall, while spherical nanoparticles tend to follow the streamline. And nanodiscs have a large surface area and a large aspect ratio. Compared with spherical nanoparticles, nanodiscs increase the particle surface area in contact with endothelial cells, allowing more targeting ligand interactions to enhance the binding strength and enhancing the adhesion to the target. In terms of anti-tumor, nanodiscs can effectively penetrate into the tumor microenvironment and effectively accumulate due to their ultra-small size. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a dexamethasone palmitate nanodisc and a preparation method thereof, so as to enhance the residence time of dexamethasone in the ocular mucosal tissue, reduce the administration frequency, and reduce the drug toxicity and side effects. Due to the strong interaction between phospholipids and membrane scaffold protein (peptide 22A), the dexamethasone palmitate nanodisc has good stability. This study found that the nanodisc has a long residence time in the mucosal system. Compared with the commercially available dexamethasone sodium phosphate eye drops, the nanodisc reduces the administration dose of dexamethasone and shows good safety. The results of the study on the mouse model of OVA-induced allergic conjunctivitis show that the dexamethasone palmitate nanodisc can significantly relieve allergic symptoms, thus providing an effective solution for the nanodisc in the field of preventing or treating related allergic diseases.

[0005] The present invention achieves the above object through the following technical solutions:

[0006] The present invention provides a dexamethasone palmitate nanodisc, which is composed of phospholipids, membrane scaffold protein and dexamethasone palmitate; the mass ratio of phospholipids, membrane scaffold protein and dexamethasone palmitate is 50:25:1 - 0.1.

[0007] In the above technical solution, further, the mass ratio of phospholipids, membrane scaffold protein and dexamethasone palmitate is 50:25:1, and the content of dexamethasone palmitate in the nanodisc is 80 μg / mL.

[0008] In the above technical solution, further, the phospholipid material is selected from one or more of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLOPC), egg yolk phosphatidylcholine (EPC), 1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC), 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC), 1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (SMPC) or 1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine (SPPC).

[0009] Furthermore, the phospholipid material is preferably one or a combination of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC).

[0010] Further, DSPE-functionalized phospholipids including but not limited to: dilauroyl phosphatidylethanolamine, dilauroyl phosphatidylethanolamine-polyethylene glycol, dimyristoyl phosphatidylethanolamine, methoxypolyethylene glycol, etc. can be added to the phospholipid material of the nanodisc.

[0011] In the above technical solution, further, the membrane scaffold protein material is selected from one or a mixture of two or more of apoA-I, apoE, apoA-IV, apoA-V, and apolipophorin III; preferably apoA-I.

[0012] Further, the peptide segment of apoA-I is selected from the 22A peptide; the amino acid sequence of the 22A peptide is PVLDLFRELLNELLEALKQKLK.

[0013] In the above technical solution, further, the aforementioned dexamethasone palmitate nanodisc is prepared by the thin film dispersion method, including the following steps:

[0014] (1) Phospholipid and dexamethasone palmitate are separately dissolved in an organic solvent and then mixed, and the organic solvent is removed by rotary evaporation at 37 °C to form a phospholipid film;

[0015] (2) Phosphate buffer solution is added to the phospholipid film obtained in step (1) for hydration, and after sufficient hydration, it is sonicated;

[0016] (3) After the membrane scaffold protein is dissolved in water, it is mixed with the solution sonicated in step (2), and the heating and cooling cycles are repeated to form nanodiscs.

[0017] In the above technical solution, further, the organic solvent in step (1) is selected from at least one or a mixture of two or more of chloroform, dichloromethane, ethyl acetate, and acetone.

[0018] In the above technical solution, further, the rotary evaporation time in step (1) is 15 - 45 minutes.

[0019] In the above technical solution, further, the hydration time in step (2) is 15 - 45 minutes; the hydration temperature is preferably 50 - 60 °C.

[0020] Further, the sonication in step (2) is probe sonication, and the probe sonication power is preferably 90 - 95%.

[0021] In the above technical solution, further, the particle size range of the nanodiscs is 8 - 20 nm.

[0022] The present invention also provides the use of the dexamethasone palmitate nanodiscs in the preparation of a medicament for preventing or treating type I hypersensitivity diseases.

[0023] In the above technical solution, further, the type I hypersensitivity diseases include allergic conjunctivitis, allergic rhinitis, allergic asthma or atopic dermatitis; the administration site of the medicament is the mucosal site of the nasal cavity, oral cavity, gastrointestinal tract or rectum.

[0024] The present invention has the following beneficial effects:

[0025] The present invention provides a dexamethasone palmitate nanodisc. Among them, dexamethasone palmitate is the main drug, and the nanodisc is used as a drug carrier. According to the large specific surface area and aspect ratio of the nanodisc, the drug can better contact with the mucosa, prolong the residence time of the drug on the ocular surface, and promote the absorption of the drug in the eye. Compared with the problem that ordinary eye drops are easily diluted and cleared by tears and have a short residence time in the eye, the dexamethasone palmitate nanodisc improves the bioavailability of the drug. At the same time, compared with the commercially available preparations, the present invention greatly reduces the administration dose of corticosteroids, improves the safety of the product, and effectively reduces the side effects caused by corticosteroids. Through the pharmacodynamic experiment, it was found that the nanodiscs loaded with dexamethasone palmitate significantly inhibited IgE in the body of mice with allergic conjunctivitis, reduced the Th2-type cytokines in the body, and the degranulation rate of mast cells and the number of eosinophils decreased. After ova eye drop stimulation, the scratching phenomenon was not serious, effectively alleviating the ocular surface inflammation and discomfort of the mice. Description of the Drawings

[0026] Figure 1 It is the particle size, appearance and electron microscopy results of the dexamethasone palmitate nanodiscs prepared in Example 1 of the present invention; A. Particle size distribution diagram; B. Appearance of the nanodiscs; C. Electron microscopy result diagram.

[0027] Figure 2 It is the physical stability diagram of the dexamethasone palmitate nanodiscs prepared in Example 1 of the present invention within 6 months.

[0028] Figure 3 It is the fluorescence retention situation of the dexamethasone palmitate nanodiscs and nanoparticles prepared in Example 1 of the present invention in the eyes of BALB / C mice.

[0029] Figure 4 It is the in vitro permeation situation of the dexamethasone palmitate nanodiscs and nanoparticles prepared in Example 1 of the present invention in a horizontal diffusion cell.

[0030] Figure 5 IgE levels in the body after treatment of dexamethasone palmitate nanodiscs and nanoparticles eye drops prepared in Example 1 of the present invention in mice with allergic conjunctivitis.

[0031] Figure 6 Th2 cytokine levels in the body after treatment of dexamethasone palmitate nanodiscs and nanoparticles eye drops prepared in Example 1 of the present invention in mice with allergic conjunctivitis; A shows the IL-4 concentration in the spleens of mice in each group, B shows the IL-5 concentration in the spleens of mice in each group, and C shows the IL-13 concentration in the spleens of mice in each group.

[0032] Figure 7 The number of eosinophils in the eyelids after treatment of dexamethasone palmitate nanodiscs and nanoparticles eye drops prepared in Example 1 of the present invention in mice with allergic conjunctivitis.

[0033] Figure 8 The degranulation rate of mast cells in the eyelids after treatment of dexamethasone palmitate nanodiscs and nanoparticles eye drops prepared in Example 1 of the present invention in mice with allergic conjunctivitis. Detailed implementation manners

[0034] The present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited in any way.

[0035] Example 1: Preparation of dexamethasone palmitate nanodiscs

[0036] Weigh 1 mg of dexamethasone palmitate and dissolve it in 1 mL of chloroform, and take 40% of the chloroform solution; weigh 0.02 g of DMPC and dissolve it in 2 mL of chloroform, and mix the two; evaporate the organic solvent at 37 °C for 30 minutes, add 4 mL of PBS solution to it, hydrate it at 55 °C for 30 minutes, use a probe sonicator to sonicate for 10 minutes, and aspirate 0.8 mL; then weigh 2 mg of 22A peptide, dissolve it in 0.2 mL of distilled water, and aspirate 0.2 mL; finally, mix DMPC and 22A peptide, and heat and cool repeatedly at 20 °C to 50 °C for 3 times, 3 minutes each time, to obtain dexamethasone palmitate nanodiscs. Measure the particle size of the nanodiscs with a Malvern particle size analyzer, and the results are as Figure 1 shown.

[0037] The results show that dexamethasone palmitate nanodiscs with a particle size between 10 - 12 nm were successfully prepared. Its appearance is clear and transparent, and the structure can be clearly observed under the electron microscope.

[0038] Example 2: Investigation of the physical stability of dexamethasone palmitate nanodiscs at 4 °C

[0039] The dexamethasone palmitate nanodiscs prepared according to Example 1 were stored in a 4°C refrigerator, and the particle size was measured once every 1 month. The results are as Figure 2 shown.

[0040] The results showed that the dexamethasone palmitate nanodiscs had good physical stability at 4°C within 6 months.

[0041] Example 3: Ocular retention experiment of dexamethasone palmitate nanodiscs

[0042] After anesthetizing the mice, the same dose but different types of fluorescent eye drops were dropped on the mouse eye surface, including free DSPE-PEG 2000 -FITC eye drops, three different types of dexamethasone palmitate nanoparticles labeled with FITC (DSPE-PEG 2000 , CLS-PEG 2000 -OH, CLS-PEG 2000 -COOH) and the eye drops of the dexamethasone palmitate nanodiscs prepared in Example 1. The free DSPE-PEG2000-FITC eye drops were prepared by dissolving an appropriate amount of DSPE-PEG2000-FITC in pure water. The three different types of dexamethasone palmitate nanoparticles labeled with FITC (DSPE-PEG2000, CLS-PEG2000-OH, CLS-PEG2000-COOH) were prepared by the nanoprecipitation method. The specific preparation process was to dissolve dexamethasone palmitate and DSPE-PEG2000-FITC in methanol, slowly add it to pure water and accelerate stirring, and remove the organic solvent by rotary evaporation. The eye images were taken every 5 minutes using an IVIS imaging system, and the retention of the drug on the mouse eye surface was judged by the fluorescence intensity on the mouse eye surface at different times. The results are as Figure 3 shown.

[0043] The results showed that the dexamethasone palmitate nanodisc eye drops had the longest retention time on the mouse eye surface, which could reach 60 - 65 minutes.

[0044] Example 4: In vitro diffusion test of dexamethasone palmitate nanodiscs

[0045] The Valia-Chien horizontal diffusion cell was used to investigate the permeation of dexamethasone palmitate nanodiscs and dexamethasone palmitate nanoparticles prepared in Example 1. The dexamethasone palmitate nanoparticles were prepared by the nanoprecipitation method. The specific preparation process was to dissolve dexamethasone palmitate and CLS-PEG2000-OH in methanol, slowly add it to pure water and accelerate stirring, and remove the organic solvent by rotary evaporation. The Track-Etch membrane was used to simulate the mouse cornea. The diffusion cell was preheated and maintained at 37 °C; 4 mL of the sample, which was dexamethasone palmitate nanodiscs and dexamethasone palmitate nanoparticles, was added to the donor cell. At the same time, 4 mL of PBS was added to the receiver cell. At 5, 10, 15, 30, 45, and 60 minutes, 200 μL of the sample was collected from the receiver cell and an equal volume of PBS solution was added. The drug concentration in the sample was determined by a high-performance liquid chromatography analyzer. Using the formula to calculate the cumulative permeation amount (Q) at each time point. Q represents the cumulative permeation amount per unit area (μg·cm -2 -2), Cn is the drug concentration (μg / mL) at the nth sampling point. V represents the volume of the receiver cell (4 mL). A is the effective contact area (0.95 cm 2 2).

[0046] The results showed that the in vitro permeation of dexamethasone palmitate nanodiscs was better than that of dexamethasone palmitate nanoparticles.

[0047] Example 5: Establishment of a mouse model of allergic conjunctivitis and the administration protocol of dexamethasone palmitate nanodiscs

[0048] On days 0, 7, and 14, mice were sensitized by intraperitoneal injection of 100 μL of ovalbumin (OVA) (1 mg / mL) and 1 mg of aluminum hydroxide gel. From day 15 to day 18, 10 μL of OVA eye drops (50 μg / μL) were administered three times a day to induce the establishment of an experimental murine allergic conjunctivitis model (EAC), and blank mice were not sensitized. To evaluate the efficacy of different treatment groups, the mice were divided into the following groups: Model group, Blank group, dexamethasone palmitate nanodisc group (DXP-sHDL) prepared in Example 1, dexamethasone palmitate nanoparticles group (DXP-NPs), blank nanodisc group (sHDL), low-dose dexamethasone sodium phosphate group (DSP-L), and high-dose dexamethasone sodium phosphate group (DSP-H). Dexamethasone palmitate nanoparticles (CLS-PEG2000-OH) were prepared by the nanoprecipitation method. The specific preparation process was to dissolve dexamethasone palmitate and CLS-PEG2000-OH in methanol, slowly add it to pure water and accelerate stirring, and remove the organic solvent by rotary evaporation. The preparation method of blank nanodiscs was similar to that of Example 1, except that dexamethasone palmitate was not added at the beginning of the preparation. Dexamethasone sodium phosphate was purchased from Zhengzhou Zhuofeng Pharmaceutical Co., Ltd., and its concentration was 250 μg / mL. The concentration of dexamethasone palmitate nanoparticles was 80 μg / mL. The dose of dexamethasone in the DXP-NPs group, DSP-L group, and DXP-sHDL group was kept consistent, and the administration volume of the DSP-H group and DXP-sHDL group was the same. Specifically, the DSP-L group was administered 3 μL, and the DSP-H group and DXP-sHDL group were both administered 10 μL. Drug administration was performed 1 hour before OVA stimulation, and the blank group received normal saline instead. The mice were euthanized 24 hours after the last OVA stimulation for further experiments.

[0049] Example 6: IgE levels in mice with allergic conjunctivitis after drug administration

[0050] ELISA was used to quantify the level of anti-OVA antibody. A NEST 96-well plate was coated with 100 μL of OVA (10 μg / mL) and incubated overnight at 4 °C. Then it was washed with PBS-T buffer (PBS containing 0.05% Tween-20), and blocked with 5% skim milk dissolved in PBS-T buffer at ambient temperature for 1 hour. After removing the blocking solution, the plate was washed 5 more times with PBS-T buffer. Mouse serum diluted in PBS-T buffer containing 1% BSA was added to the 96-well plate (50 μL / well) and incubated at 37 °C for 1 hour. After incubation, the serum was discarded and the plate was washed 5 times with PBS-T buffer.

[0051] The IgE antibody was detected using HRP-labeled goat anti-mouse IgE. After incubation for 1 hour at ambient temperature in the dark, the chromogenic solution was added. Then 50 μL of 0.2 M H 2 SO 4 was added to terminate the reaction, and the OD value was measured at 450 nm and corrected at 570 nm to avoid interference. The results are as Figure 5 shown. The DXP-sHDL group had a significant inhibitory effect on the production of IgE in mice with allergic conjunctivitis.

[0052] Example 7: The situation of Th2 cytokines in mice with allergic conjunctivitis after administration

[0053] The spleens of the mice were collected, and cells were obtained from the spleens. The cells were seeded onto 24-well plates (5×10 6 cells / well), and OVA was added to a final concentration of 1 mg / mL. After incubation at 37 °C for 96 hours, the supernatant was collected by centrifugation. The concentrations of IL-4, IL-5, and IL-13 were detected using an ELISA kit. The results are as Figure 6 shown. The DXP-sHDL group had a significant inhibitory effect on the production of IL-4, IL-5, and IL-13 in mice with allergic conjunctivitis.

[0054] Example 8: The situation of eosinophils in the eyelids of mice with allergic conjunctivitis after administration

[0055] Histological studies were performed after the last OVA stimulation. The eyelids were removed from each group of mice. Hematoxylin-eosin staining was performed to observe the situation of eosinophils. The results are as Figure 7 shown. The DXP-sHDL group had a significant inhibitory effect on the production of eosinophils in the eyelids of mice with allergic conjunctivitis.

[0056] Example 9: The situation of mast cells in the eyelids of mice with allergic conjunctivitis after administration

[0057] Histological studies were performed after the last OVA stimulation. The eyelids were removed from each group of mice. Toluidine blue staining was used to observe the morphology and degranulation of conjunctival mast cells. The results are as Figure 8 shown. The DXP-sHDL group significantly reduced the degranulation rate of mast cells in the eyelids of mice with allergic conjunctivitis.

Claims

1. A dexamethasone palmitate nanodisc, characterized in that: The nano disk is composed of phospholipids, membrane scaffold protein and dexamethasone palmitate; the mass ratio of the phospholipids, membrane scaffold protein and dexamethasone palmitate is 50:25:1-0.

1.

2. The nanodisk according to claim 1, characterized in that The phospholipid material is selected from 1,2-dioleoyl-sn-glycerol-3-phosphocholine, 1,2-dilauroyl-sn-glycerol-3-phosphocholine, 1,2-dimyristoyl-sn-glycerol-3-phosphocholine, 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine, 1,2-didecanoyl-sn-glycerol-3-phosphocholine, 1,2-didecanoyl-sn-glycerol-3-phosphocholine, 1,2-dicerucyl-sn-glycerol-3-phosphocholine, 1,2-dilinoleoyl-sn-glycerol-3-phosphocholine, egg yolk phosphatidylcholine, 1-myristoyl- One or more of 2-stearoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine, 1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine or 1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine; preferably one or more combinations of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC).

3. The nanodisk according to claim 1, characterized in that The membrane scaffold protein material is selected from one or a mixture of two or more of apoA-Ⅰ, apoE, apoA-IV, apoA-V, and apolipophorinⅢ; preferably apoA-Ⅰ.

4. The nanodisk according to claim 3, characterized in that The peptide segment of apoA-Ⅰ is selected from 22A peptide; the amino acid sequence of 22A peptide is PVLDLFRELLNELLEALKQKLK.

5. The nanodisk according to claim 1, characterized in that The thin film dispersion method is used for preparation, comprising the following steps: (1) Phospholipids and dexamethasone palmitate are dissolved in organic solvents respectively and then mixed, and the organic solvent is removed by rotary evaporation at 37°C to form a phospholipid membrane; (2) adding phosphate buffer to the phospholipid membrane obtained in step (1) for hydration, and sonicating after sufficient hydration; (3) After the membrane scaffold protein is dissolved in water, it is mixed with the solution after ultrasonication in step (2), and the heating and cooling cycles are repeated to form nanodisks.

6. The nanodisk according to claim 5, characterized in that: The organic solvent in step (1) is selected from at least one of chloroform, dichloromethane, ethyl acetate and acetone, or a mixture of two or more thereof.

7. The nanodisk according to claim 5, characterized in that: The hydration time in step (2) is 15-45 minutes; the hydration temperature is 50-60° C.; The ultrasound in step (2) is probe ultrasound, and the power of the probe ultrasound is 90-95%.

8. The dexamethasone palmitate nanodisc according to any one of claims 1 to 7, characterized in that The particle size of the nano disk is in the range of 8-20 nm.

9. Use of the dexamethasone palmitate nanodisc according to any one of claims 1 to 7 in the preparation of a drug for preventing or treating type I hypersensitivity diseases.

10. The use according to claim 9, characterized in that: The type I hypersensitivity diseases include allergic conjunctivitis, allergic rhinitis, allergic asthma or atopic dermatitis; the drug administration site is the mucous membrane of the nasal cavity, oral cavity, gastrointestinal tract or rectum.

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