Teriparatide oral formulation, and preparation method and application thereof

Abstract

The present application belongs to the field of biological medicine, and particularly relates to a teriparatide oral preparation, a preparation method and application thereof. Specifically, the present application provides a teriparatide oral preparation, which comprises exosome-encapsulated teriparatide, wherein the surface of the exosome is grafted with succinylated epsilon-polylysine. The present application uses exosomes grafted with succinylated epsilon-polylysine to encapsulate and orally deliver teriparatide, and it is found that the exosomes can realize the release of teriparatide in the intestinal tract, and can also promote the intestinal wall permeability of teriparatide, thereby significantly improving the bioavailability of the oral teriparatide.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to an oral teriparatide preparation, its preparation method, and its application. Background Technology

[0002] Osteoporosis is a systemic skeletal disease caused by decreased bone mass and deterioration of bone microstructure, which can lead to osteoporotic fractures in severe cases. Teriparatide, by stimulating osteoblasts to promote bone formation, shows great potential in the treatment of osteoporosis. However, teriparatide is a polypeptide drug with poor stability in vivo. When administered orally, it is easily broken down or modified by enzymes in the digestive tract, and its passage through the intestinal wall is difficult, resulting in low bioavailability and limiting its wider clinical application. Currently, teriparatide is available in injectable solutions and sterile powder for injection, requiring daily injections, leading to low patient compliance.

[0003] Therefore, there is a need to develop an oral formulation that can increase the stability of teriparatide, improve intestinal permeability, promote oral absorption of teriparatide, and enhance the bioavailability of teriparatide. Summary of the Invention

[0004] To address the aforementioned problems, in a first aspect, the present invention provides an oral formulation of teriparatide comprising teriparatide encapsulated in exosomes, wherein the surface of the exosomes is grafted with succinylated ε-polylysine.

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

[0006] (1) Preparation of exosomes;

[0007] (2) Teriparatide was encapsulated into the exosomes obtained in step (1) by electroporation;

[0008] (3) Prepare succinylated ε-polylysine, mix it with the exosomes obtained in step (2) in a buffer solution, centrifuge to remove the supernatant, and resuspend the precipitate in physiological saline to obtain the oral teriparatide formulation.

[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 to use a 0.3-0.4s pulse to electroporate 10-30 times at 0.5-1kV.

[0012] Further, 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 by freeze drying.

[0013] Further, 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] Further, in step (3), the buffer solution is an MES buffer solution.

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

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

[0017] Beneficial effects of the invention

[0018] This invention uses exosomes with succinylated ε-polylysine grafted on their surface to encapsulate and orally deliver teriparatide. It was found that this method 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 orally administered teriparatide. Attached Figure Description

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

[0020] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

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

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

[0023] Example 1:

[0024] A teriparatide oral formulation, characterized in that the oral formulation comprises teriparatide encapsulated in exosomes, wherein the surface of the exosomes is grafted with polylysine.

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

[0026] (1) Preparation of exosomes:

[0027] Exosome collection: Human adipose-derived mesenchymal stem cells (hADSCs) were cultured in DMEM / F12 complete medium (DMEM / F12:FBS = 9:1). When the cell density reached approximately 70%, the original medium was discarded, and the cells were washed three times with PBS buffer. The cells were then cultured in serum-free DMEM / F12 medium for another 48 hours. The cell supernatant was collected and stored at -80°C for subsequent exosome extraction.

[0028] Exosome extraction: Exosomes were extracted from the collected cell supernatant using ultracentrifugation. The specific steps were as follows: Centrifuge at 300×g for 10 minutes to remove cell debris and collect the supernatant. Centrifuge at 2000×g for 10 minutes to remove large molecules and collect the supernatant. Centrifuge at 10000×g for 30 minutes, collect the supernatant, and remove large particles using a 0.22μm filter. Centrifuge at 120000×g for 70 minutes, discard the supernatant, resuspend the remaining precipitate in PBS, and centrifuge again at 120000×g for 70 minutes. Collect the precipitate, resuspend in PBS, and store at -80℃. All centrifugation steps were performed at 4℃.

[0029] (2) Exosome encapsulation of teriparatide:

[0030] Add 10 μL of exosomes (concentration 2 μg / μl) to 390 μL of ice-cold electroporation buffer (21% Opti-MEM). TM Teriparatide powder was added to serum-depleted medium (1.15 mM pH 7.2 potassium phosphate, 25 mM potassium chloride) to achieve a teriparatide concentration of 1 mg / mL. Teriparatide was loaded into exosomes by electroporation 20 times at 0.7 kV with 0.35 s pulses in a 4 mm cuvette. The mixture was then incubated at 37 °C for 30 minutes to restore the intact membrane structure. To remove free teriparatide, the mixture was ultracentrifuged at 120,000 × g for 70 minutes at 4 °C. The supernatant was collected for subsequent drug loading calculations. 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 ultraviolet spectrophotometry, and the purification process was considered complete when no absorbance was detected. The obtained dialysate was freeze-dried for 48 hours and ε-polylysine was succinylated.

[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. The solution was then added to the reaction mixture and stirred overnight at 200 rpm. After centrifugation at 120,000 g for 70 min, the supernatant was removed, and the precipitate obtained was the succinylated ε-polylysine-coated exosome. The precipitate was resuspended in PBS to obtain the oral teriparatide formulation.

[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). The results are as follows: Figure 1 As shown in A and B, it can be seen that the succinylated ε-polylysine-coated exosomes retain the vesicle structure of the exosomes, indicating that the loading of teriparatide and the simultaneous succinylated ε-polylysine coating did not cause damage to the exosome membrane integrity.

[0036] Example 2

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

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

[0039] (4) Dissolve 40 mg of succinylated ε-polylysine in 4 mL of 0.1 M MES buffer (pH 6) and stir at 500 rpm. Add EDC (4.8 mg) and NHS (3.6 mg) and stir the solution for 1 hour. Disperse the exosomes obtained in (2) in 2 mL of MES buffer to obtain an exosome solution with a concentration of 20 μg / mL. Then add it to the reaction mixture and stir overnight at 200 rpm. Centrifuge at 120,000 g for 70 min, remove the supernatant, and the precipitate obtained is the succinylated ε-polylysine-coated exosome. Resuspend it in PBS to obtain the oral teriparatide formulation.

[0040] Comparative Example 1

[0041] Same as Example 1, except that succinylated ε-polylysine was not used to coat the exosomes, i.e.:

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

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

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

[0045] Comparative Example 2

[0046] Similar to 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, product 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 with succinylated chitosan, i.e.:

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

[0052] (3) Preparation of succinylated chitosan:

[0053] Prepare a 5% lactic acid solution, add 0.5g of chitosan to the lactic acid solution, stir until fully dissolved, add 160mL of methanol, continue stirring for 30min, add 750mg of succinic anhydride and continue stirring for 24h, adjust the pH of the solution to 7.0 using 10M NaOH, filter using filter paper, dissolve the collected precipitate in 100mL of deionized water, stir to dissolve overnight, dialyze using 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] Same as in Example 1, except that in step (4), the mass ratio of succinylated ε-polylysine to exosomes is 400:1.

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

[0058] (4) Dissolve 40 mg of succinylated ε-polylysine in 4 mL of 0.1 M MES buffer (pH 6) and stir at 500 rpm. Add EDC (4.8 mg) and NHS (3.6 mg) and stir the solution for 1 hour. Disperse the exosomes obtained in (2) in 2 mL of MES buffer to obtain an exosome solution with a concentration of 50 μg / mL. Then add it to the reaction mixture and stir overnight at 200 rpm. Centrifuge at 120,000 g for 70 min, remove the supernatant, and the precipitate obtained is the succinylated ε-polylysine-coated exosome. Resuspend it in PBS to obtain the oral teriparatide formulation.

[0059] Experimental Example 1: Gastric stability study of oral teriparatide formulation

[0060] The oral teriparatide formulations prepared in Examples 1-2 and Comparative Examples 1-4 were placed in simulated gastric fluid (pH=1.2) and incubated at 37°C for 2 hours. After 2 hours, exosomes were collected by centrifugation, washed three times, and the integrity of the exosome membrane structure was observed to determine whether the exosomes still retained their original biological functions. The results are shown in Table 1.

[0061] Table 1

[0062]

[0063] The observation results showed that after two hours of exposure to simulated gastric juice at 37°C, the succinylated ε-polylysine-coated exosomes prepared in Examples 1 and 2 maintained intact structures and exhibited good stability. However, the exosomes in Comparative Example 1, which were not coated with succinylated ε-polylysine, had their membrane structures disrupted and lost their biological functions. The exosomes in Comparative Example 2 also maintained intact structures, consistent with literature reports of excellent gastric juice stability in bovine milk exosomes. In Comparative Example 3, most of the exosome membrane structures were disrupted by gastric juice, indicating that succinylated chitosan-coated exosomes could not increase their stability in gastric juice. In Comparative Example 4, only a small portion of the exosome membrane structures were disrupted by gastric juice, suggesting that when the mass ratio of succinylated ε-polylysine to exosomes is low, the protective effect of succinylated ε-polylysine on exosomes is insufficient to maintain their stability in gastric juice.

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

[0065] The experiment was conducted using rhesus monkeys. The monkeys were kept conscious and fasted for 12 hours prior to administration. 2 mg of teriparatide or 5 mg of the oral teriparatide formulation prepared in Examples 1-2 or Comparative Examples 1-4 was administered orally by gavage, followed by 15 ml of water.

[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, with a minimum detectable dose of ≤5 pg / mL. The results are shown in Table 2.

[0067] Table 2

[0068]

[0069]

[0070] The results showed that the active ingredient teriparatide reached its maximum plasma concentration at 1.5 h. Examples 1 and 2 both achieved high plasma concentrations, indicating that the succinylated ε-polylysine-coated exosomes of the present invention not only facilitate the release of teriparatide in the intestinal environment but also aid in its penetration of the intestinal wall. In contrast, teriparatide alone, as well as teriparatide encapsulated by exosomes without succinylated ε-polylysine coating, was largely destroyed by the gastrointestinal tract, resulting in extremely low bioavailability. Although bovine milk exosomes can achieve enteric coagulation, teriparatide itself has difficulty penetrating the intestinal wall, leading to very low bioavailability. This also demonstrates that exosomes themselves have almost no effect on teriparatide's penetration of the intestinal wall.

[0071] It should be noted that while the preferred embodiments of the present invention are given in the specification and accompanying drawings, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. These embodiments are not intended to impose additional limitations on the content of the present invention; their purpose is to provide a more thorough and comprehensive understanding of the disclosure of the present invention. Furthermore, the above-described technical features can be combined with each other to form various embodiments not listed above, all of which are considered to be within the scope of the present invention specification. Moreover, those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A method for preparing an oral teriparatide formulation, characterized in that, Includes the following steps: (1) Preparation of exosomes; (2) Teriparatide was encapsulated into the exosomes obtained in step (1) by electroporation; (3) Prepare succinylated ε-polylysine, mix it with the exosomes obtained in step (2) in a buffer solution, centrifuge to remove the supernatant, and resuspend the precipitate in physiological saline to obtain the oral teriparatide preparation, wherein the mass ratio of succinylated ε-polylysine to exosomes is 500-3000:

1.

2. The preparation method according to claim 1, characterized in that, The exosomes are derived from human adipose-derived mesenchymal stem cells.

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

1.

4. The preparation method according to claim 1, characterized in that, In step (2), the electroporation is performed using a 0.3-0.4s pulse at 0.5-1kV for 10-30 electroporations.

5. The preparation method according to claim 1, 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 succinylated ε-polylysine is obtained by freeze drying.

6. The preparation method according to claim 5, 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.

7. The preparation method according to claim 1, characterized in that, In step (3), the buffer solution is MES buffer.

8. The preparation method according to claim 1, characterized in that, Step (3) involves mixing succinylated ε-polylysine with a solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in a buffer solution before adding the exosomes obtained in step (2).

9. A teriparatide oral formulation, characterized in that, The oral teriparatide formulation is obtained by the preparation method according to any one of claims 1-8.

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