Tteriparatide oral preparation as well as preparation method and application thereof

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a teriparatide oral preparation as well as a preparation method and application thereof. Specifically, the invention provides an oral preparation of teriparatide, the oral preparation comprises teriparatide encapsulated by exosome, and the surface of the exosome is grafted with succinylated epsilon-polylysine. According to the present invention, the teriparatide is encapsulated and orally delivered by using the exosome with the surface grafted with the succinylated epsilon-polylysine, such that the release of the teriparatide in the intestinal tract can be achieved, and the intestinal wall permeability of the teriparatide can be promoted so as to significantly improve the bioavailability of the oral teriparatide.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a teriparatide oral preparation and a preparation method and application thereof. Background Art

[0002] Osteoporosis is a systemic bone disease caused by decreased bone mass and deterioration of bone tissue microstructure, which can lead to osteoporotic fractures in severe cases. Teriparatide promotes bone formation by stimulating osteoblasts and shows great potential in the treatment of osteoporosis. However, teriparatide is a polypeptide drug with poor stability in the body. It is very easy to be decomposed or modified by enzymes in the digestive tract when administered orally, and it has difficulty passing through the intestinal wall, resulting in low bioavailability, which limits its wider clinical application. Currently, the dosage forms of teriparatide that have been marketed are injection solutions and sterile powders for injection, which need to be injected every day and have low patient compliance.

[0003] Therefore, it is necessary to develop an oral preparation that can increase the stability of teriparatide, improve the permeability of the intestine, promote the oral absorption of teriparatide, and improve the bioavailability of teriparatide. Summary of the invention

[0004] In order to solve the above problems, in a first aspect, the present invention provides an oral preparation of teriparatide, which comprises exosome-encapsulated teriparatide, wherein succinylated ε-polylysine is grafted on the surface of the exosomes.

[0005] In a second aspect, the present invention provides a method for preparing the teriparatide oral formulation as described herein, comprising the following steps:

[0006] (1) Preparation of exosomes;

[0007] (2) encapsulating teriparatide into the exosomes prepared in step (1) by electroporation;

[0008] (3) preparing succinylated ε-polylysine, stirring and mixing it with the exosomes obtained in step (2) in a buffer solution, removing the supernatant by centrifugation, and resuspending the obtained precipitate in physiological saline to obtain the teriparatide oral preparation.

[0009] Furthermore, the exosomes are exosomes derived from human adipose-derived mesenchymal stem cells.

[0010] Furthermore, in step (2), the mass ratio of teriparatide to exosomes is 1-6:1.

[0011] Furthermore, in step (2), the electroporation conditions are 10-30 times of electroporation at 0.5-1 kV using 0.3-0.4 s pulses.

[0012] Furthermore, in step (3), the succinylated ε-polylysine is prepared as follows: ε-polylysine and succinic anhydride are reacted in a solvent in the presence of a base, the reaction solution is purified by dialysis, and succinylated ε-polylysine is obtained after freeze-drying.

[0013] Furthermore, the base is 4-dimethylaminopyridine, the solvent is DMSO, and the reaction is carried out at 50-80° C. with stirring for 15-30 hours.

[0014] Furthermore, in step (3), the buffer is MES buffer.

[0015] Furthermore, step (3) comprises mixing succinylated chitosan with 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution and / or N-hydroxysuccinimide in a buffer solution before mixing with the exosomes, and then adding the exosomes obtained in step (2).

[0016] Furthermore, the mass ratio of succinylated ε-polylysine to exosomes is 500-3000:1.

[0017] Advantageous Effects of the Invention

[0018] The present invention uses exosomes grafted with succinylated ε-polylysine on the surface to encapsulate and orally deliver teriparatide, and it is found that the exosomes can achieve the release of teriparatide in the intestine and can also promote the intestinal wall permeability of teriparatide, thereby significantly improving the bioavailability of oral teriparatide. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Transmission electron microscopy (TEM) images of individual exosomes and succinylated ε-polylysine-coated exosomes prepared in Example 1 are shown. DETAILED DESCRIPTION

[0020] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0021] Human adipose-derived mesenchymal stem cells (hADSCs) were purchased from Shanghai Zeye Biotechnology Co., Ltd.

[0022] Teriparatide was purchased from Nanjing Yuanpeptide Biotechnology Co., Ltd.;

[0023] Embodiment 1:

[0024] A teriparatide oral preparation, characterized in that the oral preparation comprises exosome-encapsulated teriparatide, wherein polylysine is grafted on the surface of the exosomes.

[0025] A teriparatide oral preparation is prepared as follows:

[0026] (1) Preparation of exosomes:

[0027] Collection of exosomes: Human adipose mesenchymal stem cells hADSC were cultured in DMEM / F12 complete medium (DMEM / F12:FBS=9:1). When the cell density reached about 70%, the original medium was discarded, washed three times with PBS buffer, and cultured for 48 hours with serum-free DMEM / F12 medium. The cell supernatant was collected and stored in a -80°C refrigerator for subsequent exosome extraction.

[0028] Extraction of exosomes: Use ultracentrifugation to extract exosomes from the cell supernatant collected above. The specific steps are: centrifuge at 300×g for 10 minutes to remove cell debris and take the supernatant. Centrifuge at 2000×g for 10 minutes to remove macromolecules and take the supernatant. Centrifuge at 10000×g for 30 minutes, take the supernatant, and use a 0.22μm filter to remove large particles. Centrifuge at 120000×g for 70 minutes, remove the supernatant, resuspend the remaining precipitate in PBS, and centrifuge again at 120000×g for 70 minutes. Collect the precipitate and resuspend it in PBS and store it at -80°C. All the above steps are centrifuged at 4°C.

[0029] (2) Exosome encapsulation of teriparatide:

[0030] Add 10 μL of exosomes (at a concentration of 2 μg / μL) to 390 μL of ice-cold electroporation buffer (21% Opti-MEM TM Reduced serum medium, 1.15mM potassium phosphate pH 7.2, 25mM potassium chloride), teriparatide powder was added to make the concentration of teriparatide 1mg / mL, and teriparatide was loaded into exosomes by electroporation device in a 4mm cuvette, using 0.35s pulse at 0.7kV for 20 times. Finally, the mixture was incubated at 37°C for 30 minutes to restore the complete membrane structure. To remove free teriparatide, ultracentrifugation was performed at 4°C and 120000×g for 70 minutes. The supernatant was collected for subsequent drug loading rate calculation, and the exosome precipitate was resuspended in PBS and stored for later use.

[0031] (3) Preparation of succinylated ε-polylysine:

[0032] 0.500 g of ε-polylysine, 0.803 g of succinic anhydride and 0.990 g of 4-dimethylaminopyridine (DMAP) were reacted in 10 mL of DMSO at 60°C and 500 rpm for 19 hours. Snakeskin with a molecular weight cutoff of 3,500 Da was used. TMThe dialysis system purified the reaction solution in HCl solution (pH 3). The dialysate was analyzed by UV spectrophotometry, and the purification process was considered complete when no absorbance was detected. The obtained dialysate was freeze-dried for 48 hours to succinylate ε-polylysine.

[0033] (4) Preparation of succinylated ε-polylysine-coated exosomes:

[0034] 40 mg of succinylated ε-polylysine was dissolved in 4 mL of 0.1 M MES buffer (pH 6) and stirred at 500 rpm. EDC (4.8 mg) and NHS (3.6 mg) were added and the solution was stirred for 1 hour. The exosomes obtained in (2) were dispersed in 2 mL of MES buffer to obtain an exosome solution with a concentration of 10 μg / ml, which was then added to the reaction mixture and stirred at 200 rpm overnight, centrifuged at 120000 g for 70 min, and the supernatant was removed. The precipitate obtained was the succinylated ε-polylysine-coated exosomes, which were resuspended in PBS to obtain the teriparatide oral preparation.

[0035] The intact exosomes prepared in step (1) and the succinylated ε-polylysine-coated exosomes obtained in step (4) were observed by transmission electron microscopy (TEM), and the results were as follows: Figure 1 As shown in A and B, it can be seen that the exosomes coated with succinylated ε-polylysine retain the vesicular structure of the exosomes, indicating that the loading of teriparatide and the simultaneous coating with succinylated ε-polylysine did not cause the destruction of the exosome membrane integrity.

[0036] Example 2

[0037] The same as Example 1, except that in step (4), the mass ratio of succinylated ε-polylysine to exosomes is 1000:1, that is:

[0038] (1)-(3) are the same as in Example 1.

[0039] (4) 40 mg of succinylated ε-polylysine was dissolved in 4 mL of 0.1 M MES buffer (pH 6) and stirred at 500 rpm. EDC (4.8 mg) and NHS (3.6 mg) were added and the solution was stirred for 1 hour. The exosomes obtained in (2) were dispersed in 2 mL of MES buffer to obtain an exosome solution with a concentration of 20 μg / ml, which was then added to the reaction mixture and stirred at 200 rpm overnight. The mixture was centrifuged at 120,000 g for 70 min, and the supernatant was removed. The precipitate obtained was the succinylated ε-polylysine-coated exosomes, which were resuspended in PBS to obtain the teriparatide oral preparation.

[0040] Comparative Example 1

[0041] The same as Example 1, except that succinylated ε-polylysine is not used to coat the exosomes, that is:

[0042] (1)-(2) Same as Example 1.

[0043] (3)-(4) are omitted.

[0044] The exosomes resuspended in PBS obtained in step (2) are the teriparatide oral preparation of this comparative example.

[0045] Comparative Example 2

[0046] The same as comparative example 1, except that bovine milk exosomes were used instead of exosomes derived from mesenchymal stem cells.

[0047] (1) Preparation of exosomes: Bovine milk exosomes were purchased from Yumeibo Biotechnology, catalog number UR53202.

[0048] (2) Same as Comparative Example 1.

[0049] Comparative Example 3

[0050] Same as Example 1, except that succinylated ε-polylysine is replaced by succinylated chitosan, namely:

[0051] (1)-(2) Same as Example 1.

[0052] (3) Preparation of succinylated chitosan:

[0053] Prepare a 5% lactic acid solution, add 0.5g chitosan to the lactic acid solution, stir until fully dissolved, add 160mL methanol, continue stirring for 30min, add 750mg succinic anhydride and continue stirring for 24h, adjust the solution pH to 7.0 with 10M NaOH, filter with filter paper, dissolve the collected precipitate in 100mL deionized water, stir and dissolve overnight, dialyze with a dialysis bag (molecular cutoff of 3500MW) for three days, and then freeze-dry to obtain succinylated chitosan.

[0054] (4) Same as Example 1.

[0055] Comparative Example 4

[0056] The same as Example 1, except that in step (4), the mass ratio of succinylated ε-polylysine to exosomes was 400:1.

[0057] (1)-(3) are the same as in Example 1.

[0058] (4) 40 mg of succinylated ε-polylysine was dissolved in 4 mL of 0.1 M MES buffer (pH 6) and stirred at 500 rpm. EDC (4.8 mg) and NHS (3.6 mg) were added and the solution was stirred for 1 hour. The exosomes obtained in (2) were dispersed in 2 mL of MES buffer to obtain an exosome solution with a concentration of 50 μg / ml, which was then added to the reaction mixture and stirred at 200 rpm overnight. The mixture was centrifuged at 120,000 g for 70 min, and the supernatant was removed. The precipitate obtained was the succinylated ε-polylysine-coated exosomes, which were resuspended in PBS to obtain the teriparatide oral preparation.

[0059] Experimental Example 1: Study on the gastric fluid stability of teriparatide oral formulation

[0060] The teriparatide oral preparations prepared in Examples 1-2 and Comparative Examples 1-4 were placed in simulated gastric fluid (pH = 1.2) and bathed at 37°C for 2 hours; after 2 hours, the exosomes were collected by centrifugation, washed 3 times, and the integrity of the exosome membrane structure was observed to determine whether the exosomes still had the original biological function. The statistical results are shown in Table 1.

[0061] Table 1

[0062]

[0063] The observation results show that after being exposed to simulated gastric juice at 37°C for two hours, the succinylated ε-polylysine-coated exosomes prepared in Examples 1 and 2 have intact structures and show good stability; while the exosomes of Comparative Example 1 that are not coated with succinylated ε-polylysine have destroyed exosome membrane structures and no longer have the biological functions of exosomes; the exosome structure of Comparative Example 2 also remains intact, which is consistent with literature reports that bovine milk exosomes have excellent gastric juice stability; most of the exosome membrane structures of Comparative Example 3 are destroyed by gastric juice, indicating that succinylated chitosan-coated exosomes cannot increase their stability in gastric juice; a small part of the exosome membrane structure of Comparative Example 4 is destroyed by gastric juice, which indicates that when the mass ratio of succinylated ε-polylysine to exosomes is low, the protective effect of succinylated ε-polylysine on exosomes is not sufficient to maintain their stability in gastric juice.

[0064] Experimental Example 2: Bioavailability Study of Oral Teriparatide Formulation

[0065] Rhesus monkeys were used for the experiment, and they were forbidden to eat or drink 12 hours before administration and kept awake. 2 mg of teriparatide or 5 mg of the teriparatide oral preparation prepared in Example 1-2 or Comparative Example 1-4 was administered orally by gavage, and 15 ml of water was drunk after administration.

[0066] Blood samples were collected at 0, 0.25h, 0.5h, 0.75h, 1h, 1.5h, 2h, 2.5h, 3h, 4h, 5h, and 6h after administration. Plasma teriparatide concentration was determined by chemiluminescent immunoassay, and the minimum detectable dose was less than or equal to 5pg / mL. The results are shown in Table 2:

[0067] Table 2

[0068]

[0069]

[0070] The results showed that the active ingredient teriparatide reached the maximum blood concentration in 1.5h. Both Examples 1 and 2 achieved high blood concentrations, indicating that the succinylated ε-polylysine-coated exosomes of the present invention can not only help teriparatide to be released in the intestinal environment, but also help teriparatide to penetrate the intestinal wall. In contrast, most of the teriparatide alone and the teriparatide encapsulated by exosomes without succinylated ε-polylysine on the surface are destroyed by the gastrointestinal tract, and the bioavailability is extremely low. Although bovine milk exosomes can achieve enteric solubility, teriparatide itself is difficult to penetrate the intestinal wall, resulting in low bioavailability, which also shows that the exosomes themselves have little effect on the penetration of teriparatide through the intestinal wall.

[0071] It should be noted that the preferred embodiments of the present invention are given in the specification and drawings of the present invention. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. These embodiments are not used as additional limitations on the content of the present invention. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. In addition, the above-mentioned technical features continue to be combined with each other to form various embodiments not listed above, which are all regarded as the scope of the present invention; further, for ordinary technicians in this field, they can be improved or transformed according to the above description, and all these improvements and transformations should belong to the protection scope of the claims attached to the present invention.

Claims

1. A teriparatide oral preparation, characterized in that: The oral formulation comprises exosome-encapsulated teriparatide, wherein the surface of the exosome is grafted with succinylated ε-polylysine.

2. A method for preparing the teriparatide oral preparation according to claim 1, characterized in that: The following steps are involved: (1) Preparation of exosomes; (2) encapsulating teriparatide into the exosomes prepared in step (1) by electroporation; (3) preparing succinylated ε-polylysine, stirring and mixing it with the exosomes obtained in step (2) in a buffer solution, removing the supernatant by centrifugation, and resuspending the obtained precipitate in physiological saline to obtain the teriparatide oral preparation.

3. The preparation method according to claim 2, characterized in that: The exosomes are exosomes derived from human adipose-derived mesenchymal stem cells.

4. The preparation method according to claim 2, characterized in that: In step (2), the mass ratio of teriparatide to exosomes is 1-6:

1.

5. The preparation method according to claim 2, characterized in that: In step (2), the electroporation conditions are 10-30 times of electroporation at 0.5-1 kV using a 0.3-0.4 s pulse.

6. The preparation method according to claim 2, characterized in that: In step (3), the succinylated ε-polylysine is prepared as follows: ε-polylysine and succinic anhydride are reacted in a solvent in the presence of a base, the reaction solution is purified by dialysis, and the succinylated ε-polylysine is obtained after freeze-drying.

7. The preparation method according to claim 6, characterized in that: The base is 4-dimethylaminopyridine, the solvent is DMSO, and the reaction is carried out at 50-80° C. with stirring for 15-30 hours.

8. The preparation method according to claim 2, characterized in that: In step (3), the buffer is MES buffer.

9. The preparation method according to claim 2, characterized in that: Step (3) comprises mixing succinylated chitosan with 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution and / or N-hydroxysuccinimide in a buffer solution before mixing with the exosomes, and then adding the exosomes obtained in step (2).

10. The preparation method according to claim 2, characterized in that: The mass ratio of succinylated ε-polylysine to exosomes was 500-3000:1.

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