CN119679969A - A teriparatide oral preparation and its preparation method and application - Google Patents
A teriparatide oral preparation and its preparation method and application Download PDFInfo
- Publication number
- CN119679969A CN119679969A CN202510151399.1A CN202510151399A CN119679969A CN 119679969 A CN119679969 A CN 119679969A CN 202510151399 A CN202510151399 A CN 202510151399A CN 119679969 A CN119679969 A CN 119679969A
- Authority
- CN
- China
- Prior art keywords
- teriparatide
- exosomes
- succinylated
- epsilon
- polylysine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention belongs to the field of biological medicine, and in particular relates to a teriparatide oral preparation, a preparation method and application thereof. Specifically, the invention provides an oral preparation of teriparatide, which comprises teriparatide encapsulated by exosomes, wherein succinylated epsilon-polylysine is grafted on the surface of the exosomes. The present invention uses exosomes with surface grafted with succinylated epsilon-polylysine to encapsulate and orally deliver teriparatide, which is found to achieve release of teriparatide in the intestinal tract and also to promote intestinal wall permeability of teriparatide, thereby significantly improving the bioavailability of oral teriparatide.
Description
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to a teriparatide oral preparation, a preparation method and application thereof.
Background
Osteoporosis is a systemic skeletal disease caused by reduced bone mass and deterioration of bone tissue microstructure, and in severe cases, results in osteoporotic fractures. Teriparatide has great potential in treating osteoporosis by stimulating osteoblasts to promote bone formation. However, teriparatide is a polypeptide drug, has poor stability in vivo, is very easily decomposed or modified by enzymes in the digestive tract when orally administered, and has low bioavailability due to difficulty in passing through the intestinal wall, thus limiting the wider clinical application. The currently marketed forms of teriparatide are injection solutions and sterile powder for injection, which are required to be injected daily and have low patient compliance.
Therefore, there is a need to develop an oral formulation that can increase the stability of teriparatide, increase the permeability of the intestinal tract, promote the oral absorption of teriparatide, and increase the bioavailability of teriparatide.
Disclosure of Invention
In order to solve the above-mentioned problems, in a first aspect, the present invention provides an oral preparation of teriparatide comprising an exosome-encapsulated teriparatide, wherein the exosome surface is grafted with succinylated epsilon-polylysine.
In a second aspect, the present invention provides a method of preparing an oral formulation of teriparatide as described herein comprising the steps of:
(1) Preparing exosomes;
(2) Encapsulating teriparatide into the exosomes prepared in step (1) by electroporation;
(3) Preparing succinylated epsilon-polylysine, stirring and mixing the succinylated epsilon-polylysine with the exosomes obtained in the step (2) in a buffer solution, centrifuging to remove supernatant, and re-suspending the obtained precipitate in normal saline to obtain the teriparatide oral preparation.
Further, the exosomes are exosomes derived from human adipose mesenchymal stem cells.
Further, in step (2), the mass ratio of teriparatide to exosome is 1-6:1.
Further, in the step (2), the condition of electroporation is that electroporation is performed 10 to 30 times at 0.5 to 1kV using a pulse of 0.3 to 0.4 s.
Further, in the step (3), the succinylated epsilon-polylysine is prepared by reacting epsilon-polylysine with succinic anhydride in a solvent in the presence of a base, purifying the reaction solution by dialysis, and freeze-drying to obtain succinylated epsilon-polylysine.
Further, the base is 4-dimethylaminopyridine, the solvent is DMSO, and the reaction is carried out at 50-80 ℃ for 15-30 hours with stirring.
Further, in step (3), the buffer is an MES buffer.
Further, step (3) comprises mixing succinylated chitosan with 1-ethyl- (3-dimethylaminopropyl) carbodiimide solution and/or N-hydroxysuccinimide in buffer before mixing with exosomes, and then adding exosomes obtained in step (2).
Further, the mass ratio of succinylated epsilon-polylysine to exosomes is 500-3000:1.
Advantageous effects of the invention
The present invention uses exosomes with surface grafted with succinylated epsilon-polylysine to encapsulate and orally deliver teriparatide, which is found to achieve release of teriparatide in the intestinal tract and also to promote intestinal wall permeability of teriparatide, thereby significantly improving the bioavailability of oral teriparatide.
Drawings
FIG. 1 shows Transmission Electron Microscopy (TEM) images of the individual exosomes prepared in example 1 and succinylated epsilon-polylysine coated exosomes.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Human adipose mesenchymal stem cells hADSC were purchased from Shanghai ze Biotechnology Co., ltd;
Teriparatide was purchased from south Beijing source peptide biotechnology limited;
example 1:
An oral formulation of teriparatide, comprising an exosome-encapsulated teriparatide, wherein the exosome surface is grafted with polylysine.
An oral preparation of teriparatide, prepared as follows:
(1) Preparation of exosomes:
Human adipose-derived mesenchymal stem cells hADSC were cultured in DMEM/F12 complete medium (DMEM/F12: FBS=9:1), and when the cell density was about 70%, the original medium was discarded, washed 3 times with PBS buffer, and further cultured with serum-free DMEM/F12 medium for 48 hours. The cell supernatant was collected and stored in a-80 ℃ refrigerator for subsequent exosome extraction.
Extracting exosomes: the cell supernatant collected above was subjected to extraction of exosomes by ultracentrifugation. The specific procedure was centrifugation at 300 Xg for 10 minutes to remove cell debris and the supernatant was taken. The supernatant was removed by centrifugation at 2000 Xg for 10 minutes. Centrifugation was carried out at 10000 Xg for 30 minutes, and the supernatant was removed by using a 0.22 μm filter. After centrifugation at 120000 Xg for 70 min, the supernatant was removed and the remaining pellet PBS was resuspended and then centrifuged again at 120000 Xg for 70 min, the pellet PBS was collected and resuspended and stored at-80 ℃. All the above steps were centrifuged at 4 ℃.
(2) Exosome encapsulation teriparatide:
mu.L of exosomes (concentration 2. Mu.g/. Mu.l) was added to 390. Mu.L of ice-cold electroporation buffer (21% Opti-MEM TM minus serum medium, 1.15mM potassium phosphate pH 7.2, 25mM potassium chloride), teriparatide powder was added to give a concentration of teriparatide of 1mg/mL, and the exosomes were electroporated 20 times in a 4mM cuvette by an electroporation device using a 0.35s pulse at 0.7 kV. Finally, the mixture was incubated at 37 ℃ for 30 minutes to restore the intact membrane structure. To remove free teriparatide, the supernatant was collected for subsequent drug loading calculations after ultracentrifugation at 120000Xg for 70 min at 4 ℃, and the exosome pellet was resuspended in PBS and stored for later use.
(3) Preparation of succinylated epsilon-polylysine:
0.500g of epsilon-polylysine, 0.803g of succinic anhydride and 0.990g of 4-Dimethylaminopyridine (DMAP) were reacted in 10mL of DMSO at 60℃and 500rpm for 19 hours. The reaction solution was purified in HCl solution (pH 3) using a 3,500Da molecular weight cut-off Snakeikin TM dialysis system. The dialysate was analyzed by ultraviolet spectrophotometry, and when absorbance was not detected, the purification process was considered to be completed. The obtained dialysis solution was freeze-dried for 48 hours to succinylate epsilon-polylysine.
(4) Preparation of succinylated epsilon-polylysine coated exosomes:
40mg of succinylated epsilon-polylysine was dissolved in 4mL of 0.1M MES buffer (pH 6) and stirred at 500 rpm. EDC (4.8 mg) and NHS (3.6 mg) were added and the solution stirred for 1 hour. Dispersing the exosomes obtained in the step (2) in 2mL MES buffer solution to obtain exosome solution with the concentration of 10 mug/mL, then adding the exosomes solution into a reaction mixture, stirring the solution at the speed of 200rpm overnight, centrifuging the solution at 120000g for 70min, removing supernatant, obtaining precipitate, namely succinylated epsilon-polylysine coated exosomes, and re-suspending the precipitate in PBS to obtain the teriparatide oral preparation.
The complete exosomes prepared in step (1) and the succinylated epsilon-polylysine coated exosomes obtained in step (4) were observed by Transmission Electron Microscopy (TEM), and the results are shown in fig. 1A and B, respectively, it can be seen that succinylated epsilon-polylysine coated exosomes retained the vesicle structure of exosomes, indicating that the loading of teriparatide and the simultaneous coating of succinylated epsilon-polylysine did not cause disruption of exosome membrane integrity.
Example 2
The same as in example 1, except that in step (4), the mass ratio of succinylated epsilon-polylysine to exosomes was 1000:1, namely:
(1) - (3) is the same as in example 1.
(4) 40Mg of succinylated epsilon-polylysine was dissolved in 4mL of 0.1M MES buffer (pH 6) and stirred at 500 rpm. EDC (4.8 mg) and NHS (3.6 mg) were added and the solution stirred for 1 hour. Dispersing the exosomes obtained in the step (2) in a buffer solution of 2mLMES to obtain an exosome solution with the concentration of 20 mug/ml, then adding the exosomes solution into a reaction mixture, stirring the solution at the speed of 200rpm overnight, centrifuging the solution of 120000g for 70min, removing the supernatant, obtaining a precipitate, namely succinylated epsilon-polylysine coated exosomes, and re-suspending the precipitate in PBS to obtain the teriparatide oral preparation.
Comparative example 1
The procedure is as in example 1, except that the exosomes are not coated with succinylated epsilon-polylysine, i.e.:
(1) - (2) the same as in example 1.
(3) - (4) Omitted.
The exosomes obtained in the step (2) and resuspended in PBS are teriparatide oral preparations of the comparative example.
Comparative example 2
The same as in comparative example 1, except that bovine milk exosomes were used instead of exosomes derived from mesenchymal stem cells.
(1) Preparation of exosomes bovine milk exosomes were purchased from the cosmosorotics, cat No.UR53202。
(2) As in comparative example 1.
Comparative example 3
The same as in example 1, except that succinylated epsilon-polylysine was replaced with succinylated chitosan, i.e.:
(1) - (2) the same as in example 1.
(3) Preparation of succinylated chitosan:
Preparing a lactic acid solution with the concentration of 5%, adding 0.5g of chitosan into the lactic acid solution, stirring until the chitosan is fully dissolved, adding 160mL of methanol, continuously stirring for 30min, adding 750mg of succinic anhydride, continuously stirring for 24h, adjusting the pH of the solution to 7.0 by using 10M NaOH, filtering by using filter paper, dissolving the collected precipitate in 100mL of deionized water, stirring and dissolving for three days by using a dialysis bag (molecular cutoff of 3500 MW), and freeze-drying to obtain succinylated chitosan.
(4) As in example 1.
Comparative example 4
The same as in example 1, except that in step (4), the mass ratio of succinylated epsilon-polylysine to exosomes was 400:1.
(1) - (3) Is the same as in example 1.
(4) 40Mg of succinylated epsilon-polylysine was dissolved in 4mL of 0.1M MES buffer (pH 6) and stirred at 500 rpm. EDC (4.8 mg) and NHS (3.6 mg) were added and the solution stirred for 1 hour. Dispersing the exosomes obtained in the step (2) in a buffer solution of 2mLMES to obtain an exosome solution with the concentration of 50 mug/ml, then adding the exosomes solution into a reaction mixture, stirring the solution at the speed of 200rpm overnight, centrifuging the solution of 120000g for 70min, removing the supernatant, obtaining a precipitate, namely succinylated epsilon-polylysine coated exosomes, and re-suspending the precipitate in PBS to obtain the teriparatide oral preparation.
Experimental example 1 gastric juice stability study of teriparatide oral preparation
The teriparatide oral preparations prepared in examples 1-2 and comparative examples 1-4 were placed in simulated gastric fluid (ph=1.2), respectively, and after water bath at 37 ℃ for 2 hours, the exosomes were collected by centrifugation, and after washing 3 times, the structural integrity of the exosome membrane was observed to determine whether the exosomes still had the original biological function. The results statistics are shown in Table 1.
TABLE 1
The results of the observations showed that the succinylated epsilon-polylysine coated exosomes prepared in examples 1 and 2 were structurally intact and exhibited good stability when exposed to simulated gastric fluid at 37 ℃ for two hours, whereas the exosomes not coated with succinylated epsilon-polylysine of comparative example 1 were structurally disrupted and no longer had the biological function of exosomes, the exosomes of comparative example 2 were also structurally intact, consistent with the literature report that bovine milk exosomes had excellent gastric fluid stability, the majority of exosomes membrane structures of comparative example 3 were disrupted by gastric fluid, indicating that succinylated chitosan coated exosomes could not increase their stability in gastric fluid, and the minority of exosomes membrane structures of comparative example 4 were disrupted by gastric fluid, indicating that the protective effect of succinylated epsilon-polylysine on exosomes was insufficient to maintain their stability in gastric fluid when the quality of succinylated epsilon-polylysine was low compared to exosomes.
Experimental example 2 bioavailability study of teriparatide oral formulations
Experiments were performed using rhesus monkeys, and diet was prohibited for 12 hours prior to dosing, keeping awake. 2mg of teriparatide or 5mg of the teriparatide oral preparation prepared in examples 1-2 or comparative examples 1-4 are administered orally by intragastric administration, and 15ml of water is taken after administration.
Blood samples were collected at 0, 0.25h, 0.5h, 0.75h, 1h, 1.5h, 2h, 2.5h, 3h, 4h, 5h, 6h after administration. Plasma teriparatide concentrations were determined by chemiluminescent immunoassay with a minimum detectable dose of 5pg/mL or less. The results are shown in Table 2:
TABLE 2
The results show that the active ingredient teriparatide reaches the maximum blood concentration at 1.5 h. Examples 1 and 2 both achieve higher blood levels, indicating that succinylated epsilon-polylysine coated exosomes of the present invention can not only assist in the release of teriparatide in the intestinal environment, but also assist in the penetration of teriparatide through the intestinal wall. In contrast, teriparatide alone and teriparatide encapsulated by exosomes without succinylated epsilon-polylysine on the surface are largely destroyed by the gastrointestinal tract and have very low bioavailability. Bovine milk exosomes, although achieving enteric solubility, are also poorly bioavailable due to the difficulty of the teriparatide itself penetrating the intestinal wall, which also suggests that exosomes themselves have little effect on the penetrability of teriparatide to the intestinal wall.
It should be noted that while the present invention has been illustrated in the drawings and described in connection with the preferred embodiments thereof, it is to be understood that the invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but are to be construed as providing a full breadth of the disclosure. The above technical features are further combined with each other to form various embodiments which are not listed above and are all considered as the scope of the present invention described in the specification, further, the improvement or transformation can be carried out by the person skilled in the art according to the above description, and all the improvements and transformation shall fall within the protection scope of the appended claims.
Claims (10)
1. An oral formulation of teriparatide, comprising an exosome-encapsulated teriparatide, wherein the exosome surface is grafted with succinylated epsilon-polylysine.
2. A process for the preparation of an oral preparation of teriparatide according to claim 1, comprising the steps of:
(1) Preparing exosomes;
(2) Encapsulating teriparatide into the exosomes prepared in step (1) by electroporation;
(3) Preparing succinylated epsilon-polylysine, stirring and mixing the succinylated epsilon-polylysine with the exosomes obtained in the step (2) in a buffer solution, centrifuging to remove supernatant, and re-suspending the obtained precipitate in normal saline to obtain the teriparatide oral preparation.
3. The method of claim 2, wherein the exosomes are human adipose mesenchymal stem cell-derived exosomes.
4. The method according to claim 2, wherein in the step (2), the mass ratio of teriparatide to exosome is 1-6:1.
5. The method according to claim 2, wherein in the step (2), the condition of electroporation is that electroporation is performed 10 to 30 times at 0.5 to 1kV using a pulse of 0.3 to 0.4 s.
6. The process according to claim 2, wherein in the step (3), the succinylated epsilon-polylysine is produced by reacting epsilon-polylysine with succinic anhydride in the presence of a base in a solvent, purifying the reaction solution by dialysis, and freeze-drying the reaction solution.
7. The process according to claim 6, wherein the base is 4-dimethylaminopyridine, the solvent is DMSO, and the reaction is carried out at 50 to 80℃with stirring for 15 to 30 hours.
8. The method of claim 2, wherein in step (3), the buffer is MES buffer.
9. The method according to claim 2, wherein step (3) comprises mixing succinylated chitosan with 1-ethyl- (3-dimethylaminopropyl) carbodiimide solution and/or N-hydroxysuccinimide in buffer before mixing with the exosome, and then adding the exosome obtained in step (2).
10. The method according to claim 2, wherein the mass ratio of succinylated epsilon-polylysine to exosomes is 500-3000:1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202510151399.1A CN119679969A (en) | 2025-02-11 | 2025-02-11 | A teriparatide oral preparation and its preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202510151399.1A CN119679969A (en) | 2025-02-11 | 2025-02-11 | A teriparatide oral preparation and its preparation method and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN119679969A true CN119679969A (en) | 2025-03-25 |
Family
ID=95041002
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202510151399.1A Pending CN119679969A (en) | 2025-02-11 | 2025-02-11 | A teriparatide oral preparation and its preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN119679969A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102470181A (en) * | 2009-07-20 | 2012-05-23 | 默克专利有限公司 | Polylysine conjugates and uses thereof |
| CN113388122A (en) * | 2021-06-29 | 2021-09-14 | 上海艾棵颂生物科技有限公司 | Electropositive surface exosome and preparation method and application thereof |
| CN118403186A (en) * | 2024-04-26 | 2024-07-30 | 华中科技大学 | A composite preparation of functionalized magnetic nanoparticles and exosomes, and its preparation method and application |
| CN118662477A (en) * | 2024-05-27 | 2024-09-20 | 重庆医科大学 | Excritic-encapsulated teriparatide lipid nanoparticle and preparation method thereof |
-
2025
- 2025-02-11 CN CN202510151399.1A patent/CN119679969A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102470181A (en) * | 2009-07-20 | 2012-05-23 | 默克专利有限公司 | Polylysine conjugates and uses thereof |
| CN113388122A (en) * | 2021-06-29 | 2021-09-14 | 上海艾棵颂生物科技有限公司 | Electropositive surface exosome and preparation method and application thereof |
| CN118403186A (en) * | 2024-04-26 | 2024-07-30 | 华中科技大学 | A composite preparation of functionalized magnetic nanoparticles and exosomes, and its preparation method and application |
| CN118662477A (en) * | 2024-05-27 | 2024-09-20 | 重庆医科大学 | Excritic-encapsulated teriparatide lipid nanoparticle and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| YUAN LIU等: "Cell-Specific Impacts of Surface Coating Composition on Extracellular Vesicle Secretion", ACS APPLIED MATERIALS & INTERFACES, vol. 23, no. 16, 28 May 2024 (2024-05-28) * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111569082A (en) | An oral delivery system for protein-encapsulated polypeptide drug exosomes | |
| CN111450258B (en) | Oral administration system for promoting protein drug to permeate across mucus and preparation method thereof | |
| CN101716161A (en) | New thymosin chitosan microsphere type oral medicinal preparation and preparation method thereof | |
| CN107638388B (en) | A kind of asiatic acid chitosan deoxycholic acid graft micelle and preparation method | |
| CN119679969A (en) | A teriparatide oral preparation and its preparation method and application | |
| CN108851084B (en) | Colon positioning micelle loaded with quercetin and preparation method thereof | |
| CN111529695A (en) | Cyclodextrin soluble ACE2 and preparation method and application thereof | |
| CN113797177B (en) | Quercetin oral sustained-release preparation modified by ionic emulsifier chitosan nanoparticles and preparation method thereof | |
| KR101319642B1 (en) | PH sensitive nano complex for drug delivery and preparation method thereof | |
| CN119925694A (en) | A COFs-based nano-implant material for biotin-avidin driven exosome controlled release and a preparation method thereof | |
| CN109662956B (en) | Application of oleanolic acid grafted chitosan drug-loaded nanoparticles | |
| CN103315977B (en) | Folic acid-modified quercetin lipid nanocapsule preparation and preparation method thereof | |
| US11096893B2 (en) | Glucose sensitive compositions for drug delivery | |
| CN103784400B (en) | A kind of pegylated phospholipids contain the oral micellar preparation of insulin | |
| CN109568601A (en) | A kind of protein and peptide drugs dual-microsphere and preparation method thereof and insulin dual-microsphere | |
| CN110251687B (en) | Charge reversal oral chitosan nano-drug preparation and preparation method thereof | |
| CN114010801B (en) | L-ascorbyl palmitate modified small molecule peptide liposome and preparation and application thereof | |
| CN120241655B (en) | Stem cell membrane modified liposome nano drug-loaded particle and preparation method thereof | |
| CN118987236B (en) | Carrier system for achieving targeted drug delivery based on glutathione response to micro-environment active oxygen, and preparation method and application thereof | |
| CN119488503B (en) | Nanometer exosome capsule and application thereof | |
| CN107823185B (en) | An oral drug delivery system using composite nanomaterials as carrier | |
| CN114702608B (en) | An esterase-responsive polymer and its application | |
| CN114668772B (en) | Composition, preparation method and application of bionic nanosystem loaded with amphotericin B | |
| CN120227322A (en) | A nasal administration preparation containing extracellular vesicles and acellular matrix hydrogel and a preparation method thereof | |
| CN114177158A (en) | Nanoparticles for promoting BCS III drug oral absorption and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |