USE OF MILK EXOSOME IN PREPARATION OF DRUG CARRIER
20250134815 ยท 2025-05-01
Assignee
Inventors
Cpc classification
A61K47/10
HUMAN NECESSITIES
A61K9/148
HUMAN NECESSITIES
A61K9/006
HUMAN NECESSITIES
A61K47/24
HUMAN NECESSITIES
International classification
A61K9/14
HUMAN NECESSITIES
A61K9/127
HUMAN NECESSITIES
A61K9/00
HUMAN NECESSITIES
A61K47/10
HUMAN NECESSITIES
A61K47/26
HUMAN NECESSITIES
Abstract
The present disclosure relates to the field of pharmaceutical preparations, and in particular to use of a milk exosome in the manufacture of a drug carrier. The present disclosure uses milk exosomes as a drug carrier. The exosomes are derived from milk with safety and reliability, and has the functional properties of milk exosomes. Using the exosomes as a drug carrier can achieve oral administration of protein drugs. Using the milk exosomes to load liraglutide can achieve oral administration of liraglutide and treat type 2 diabetes.
Claims
1-10. (canceled)
11. A method for treating a disease, comprising administering to a subject a drug loaded in a milk exosome, wherein the milk exosome is used as a drug carrier.
12. The method according to claim 11, wherein the drug includes a liraglutide, insulin degludec, insulin detemir, semaglutide or tirzepatide.
13. (canceled)
14. The method according to claim 12, wherein a sublingual route is used to administer to a subject.
15. The method according to claim 14, wherein adaptation disease includes type 2 diabetes.
16. The method according to claim 11, wherein ultrasonication, electroporation or co-incubation approach is used for loading the drug into milk exosomes.
17. The method according to claim 16, wherein the ultrasonication is carried out with an input power level between 450 W and 600 W and a sonication time between 20 min and 1 h.
18. The method according to claim 16, wherein the electroporation is carried out with a voltage of 400V-500V and a capacitance of 300 F-400 F.
19. The method according to claim 16, wherein the co-incubation is carried out with the temperature of 42 C.-60 C. and the time of 0.5 h-4 h.
20. A method in preparation of sublingual tablets, wherein drug-loaded milk exosome is mixed with excipients to prepare sublingual tablets.
21. The method according to claim 20, wherein the drug includes liraglutide, insulin degludec, insulin detemir, semaglutide or tirzepatide.
22. The method according to claim 20, wherein for ultrasonication, electroporation or co-incubation approach is used for loading the drug into milk exosomes.
23. The method according to claim 22, wherein the ultrasonication is carried out with an input power level between 450 W and 600 W and a sonication time between 20 min and 1 h.
24. The method according to claim 22, wherein the electroporation is carried out with a voltage of 400V-500V and a capacitance of 300 F-400 F.
25. The method according to claim 22, wherein the co-incubation is carried out with the temperature of 42 C.-60 C. and the time of 0.5 h-4 h.
26. The method according to claim 20, wherein the milk exosome is prepared by: step 1, pre-treatment, wherein the pre-treatment comprises fat removal and/or protein removal; step 2, preliminary purification and concentration, wherein the preliminarily purification and concentration is performed by tangential flow ultrafiltration; step 3, downstream purification, wherein the downstream purification is performed by a method selected from the group consisting of CIMmultus QA column purification, Capto core 700 column purification, S400 size exclusion chromatography purification, and a combination thereof.
27. A sublingual tablet, wherein the sublingual tablet includes drug-loaded milk exosomes.
28. The sublingual tablet according to claim 27, wherein the sublingual tablet includes milk exosomes loaded with Liraglutide.
29. The sublingual tablet according to claim 28, wherein the sublingual tablet includes cross-linked sodium carboxymethyl cellulose, mannitol, purified water and magnesium stearate.
30. The sublingual tablet according to claim 28, wherein the sublingual tablet includes sodium carboxymethyl cellulose, mannitol, microcrystalline cellulose and magnesium stearate.
31. The sublingual tablet according to claim 27, wherein the sublingual tablet includes milk exosomes loaded with insulin degludec, insulin detemir, semaglutide or tirzepatide.
Description
BRIEF DESCRIPTION OF DRAWINGS
[0058] For more clearly illustrating embodiments of the present disclosure or the technical solutions in the prior art, drawings for describing the embodiments or the prior art will be briefly described hereinafter.
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DETAILED DESCRIPTION
[0082] The present disclosure discloses use of a milk exosome in the preparation of a drug carrier. Those skilled in the art can learn from the contents of the disclosure and appropriately adapt the process parameters to achieve the present disclosure. Particularly, it should be noted that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included within the present disclosure. The method and the application of the present disclosure have been described through the preferred embodiments, and it is obvious that the method and application described herein may be changed or appropriately modified and combined to realize and apply the technology of the present disclosure by those skilled in the art without departing from the content, spirit and scope of the present disclosure.
[0083] All materials and reagents used in the use of a milk exosome in the preparation of a drug carrier provided by the present disclosure are commercially available.
[0084] The present disclosure is further illustrated below in combination with the embodiments.
Example 1 Industrially Scalable Purification Process for Milk Exosomes
1) Pre-Treatment of Milk Samples by Different Protein Removal Methods
[0085] a. EDTA precipitation method: Skimmed milk was mixed with EDTA of the same volume, different concentrations and different pHs, and incubated at room temperature for 15 min to precipitate casein. The EDTA used had a concentration of 0.15-0.35 mol.Math.L.sup.1 and a pH of 6.0-8.0. Centrifugation was carried out at 12,000 r.Math.min.sup.1 for 40 min, and the supernatant was filtered through 0.80, 0.45 and 0.20 m membrane filters sequentially.
[0086] b. Sodium phosphate precipitation method: An equal amount of skimmed milk was added to a sodium phosphate solution. Several pre-tests were carried out on the concentration and pH range of sodium phosphate, such as using 0.5 mol.Math.L.sup.1 (pH 5.3, pH 6.5 and pH 7.6), 1.0 mol.Math.L.sup.1 (pH 5.2, pH 6.3 and pH 7.6) and 2 mol.Math.L.sup.1 (pH 6.0, pH 7.0 and pH 8.0) sodium phosphate. This mixture was placed at 4 C. overnight to precipitate protein, and the supernatant after precipitation was sequentially filtered through 0.80, 0.45, and 0.20 m membrane filters.
[0087] c. Sodium citrate dissolution method: skimmed milk was mixed with equal amounts of 2%, 4%, 6%, 8% and 16% sodium citrate, incubated on ice for 2 h to dissolve casein, and filtered through a 0.20 m membrane filter.
2) Preliminary Purification and Concentration of Milk Exosomes by TFF Method
[0088] The milk supernatant after pre-treatment was processed using a hollow fiber column with a relative molecular mass of 750,000. The system parameters were set as follows: TMP maximum pressure at 2 atmospheres, alarm pressure at 2 atmospheres; 100 mL minimum volume, and 100 mL alarm volume. The sample was poured into a reusable beaker, and exchanged with 1PBS until the concentration of permeate proteins was 0.00 g. L.sup.1. Then the liquid in the beaker was further concentrated to 1/20 of the original solution, and the sample was collected.
3) Downstream Purification of Milk Exosomes by BE-SEC and UC
[0089] The KTA Pure 25 purification system was rinsed with ultrapure water, connected to a HiScreen Capto Core 700 column, and then rinsed with 3 column volumes of ultrapure water. After rinsing, the equilibration was carried out with 3 column volumes of 1PBS, and the preliminarily purified sample was purified using a HiScreen Capto Core 700 column at a flow rate of 2 mL min.sup.1. The sample was collected at the position of the flow-through peak, i.e. milk exosomes.
Experimental Results
[0090] As shown in
[0091] As shown in
[0092] The particle size, concentration and purity of exosomes detected by NanoFCM are shown in
[0093] As shown in FIG. 4, the amount of free proteins (particle size smaller than that of exosomes) in the exosome sample was detected by HPLC. The retention time of milk exosomes extracted by the three methods on a TSKgel G6000 PWXL column was all 8 min. However, the proportion of impurity peaks (RT>8 min) of the exosome samples extracted by TFF and BE-SEC was smaller than that of the samples obtained by UC, and the final product obtained by TFF+BE-SEC showed more of a single peak.
[0094] The use of protein precipitation pre-treatment in combination with purification by TFF and BE-SEC of milk can obtain exosome samples with a higher yield and purity. More importantly, TFF could achieve the preliminary purification and concentration of samples from a few liters to hundreds of liters, and the large-scale preparation of milk exosomes could finally be achieved by combining TFF with BE-SEC.
Example 2 Drug Loading Process for Milk Exosomes-Ultrasonication Method
[0095] Principle: Ultrasonication can disrupt the arrangement of phospholipid bilayer in the membrane and create temporary pores. After exosomes are subjected to low-frequency continuous ultrasonication, the sound wave causes the encapsulated air to expand and the pressure inside the vesicle to rise to the elastic limit of the bilayer, resulting in pores in the phospholipid layer. Due to the high drug concentration outside the membrane, the difference in osmotic pressure causes the drug to diffuse into the liposome through the pores.
Operation Steps
[0096] The optimal conditions for loading by ultrasonication were explored using PKH67. Specifically, after co-incubation of PKH67 with milk exosomes at room temperature for 30 min, ultrasonication was carried out using a cell crusher at the power of 150 w, 300 w, 450 w, 600 w, 800 w, 1000 w and 1200 w for 0 s, 50 s, 250 s, 500 s, 750 s, 20 min, 30 min, 1 h, 2 h, 4 h and 6 h. The particle number and PKH67 positive rate of the ultrasonicated samples were determined by using NanoFCM. Based on the optimal conditions for loading by ultrasonication, the volume for loading by ultrasonication was explored. The particle number, particle size and PKH67 positive rate of the ultrasonicated samples were determined by using NanoFCM.
Experimental Results
TABLE-US-00001 TABLE 1 Effects of power and duration of ultrasonication on positive rate of exosome loading Time Power 0 s 50 s 250 s 500 s 750 s 20 min 30 min 1 h 2 h 4 h 6 h 0 w 5.4% 6.1% 7.2% 8.3% 8.9% 10.3% 10.0% 15.2% 15.4% 27.5% 31.0% 150 w 5.4% 8.6% 23.5% 31.4% 31.9% 40.3% 47.6% 45.5% 48.3% 50.1% 50.6% 300 w 5.4% 9.7% 12.1% 19.3% 35.0% 33.6% 50.6% 45.1% 48.3% 58.0% 52.7% 450 w 5.4% 15.8% 31.2% 60.2% 54.6% 44.5% 63.3% 74.3% 76.1% 75.9% 79.1% 600 w 5.4% 15.4% 67.6% 77.6% 76.1% 78.1% 76.5% 78.6% 85.6% 74.1% 78.7% 800 w 5.4% 30.0% 65.5% 78.1% 78.4% 75.4% 80.4% 79.3% 78.0% 80.1% 80.0% 1000 w 5.4% 42.2% 64.8% 77.3% 77.2% 80.4% 87.8% 80.0% 85.7% 81.2% 88.1% 1200 w 5.4% 41.0% 66.3% 77.5% 78.9% 75.5% 75.4% 88.7% 88.3% 85.0% 87.5%
TABLE-US-00002 TABLE 2 Effects of power and duration of ultrasonication on particle number (10.sup.10 particles/mL) of exosomes Time Power 0 s 50 s 250 s 500 s 750 s 20 min 30 min 1 h 2 h 4 h 6 h 0 w 7.39 7.90 7.68 7.21 6.56 6.43 5.88 6.31 5.21 5.10 4.52 150 w 7.39 6.77 7.33 6.88 6.21 6.07 6.22 5.77 5.48 5.09 4.71 300 w 7.39 6.53 7.08 7.01 6.38 6.37 6.11 6.41 5.88 4.87 5.00 450 w 7.39 7.01 6.81 6.55 4.56 5.21 5.43 6.01 4.78 4.45 4.03 600 w 7.39 5.91 5.99 5.76 3.80 3.90 4.01 3.54 3.48 3.61 3.67 800 w 7.39 5.90 6.42 5.87 4.22 5.11 4.63 4.37 3.71 3.31 3.44 1000 w 7.39 6.44 6.53 6.11 5.89 5.33 5.41 3.51 2.69 2.24 2.30 1200 w 7.39 6.54 7.11 6.38 6.67 7.31 6.06 2.67 3.08 2.87 2.01
TABLE-US-00003 TABLE 3 Effects of different volumes for ultrasonication (600 w, 500 s) on exosome loading Particle concentration Volume (ml) Positive rate (%) (10.sup.10 particles/mL) 3 77.8 5.23 10 75.3 5.60 50 80.1 4.85 100 78.1 5.08 200 75.6 4.66 500 74.7 4.97
[0097] There was a large difference in exosome loading efficiency after ultrasonication at different power, and the positive rate after ultrasound was between 5% and 90%. For ultrasonication at 450 W-1200 W power for more than 20 min, the difference in loading efficiency (as gauged by the positive rates, see table 1) was not obvious and was mostly around 80%. With longer ultrasonication time, the particle number decreased. The loading scale of 3 ml-500 ml system could be achieved, and the particle number and loading positive rate after loading were relatively stable.
Loading Liraglutide by Ultrasonication:
[0098] After mixing according to the ratio of 110.sup.11 exosome particles to 5 mg liraglutide, ultrasonication was performed followed by purification using Capto Core 700 or QA columns. There was an obvious change in the state of the solution before and after ultrasonication.
[0099] 25 mg of liraglutide, and a mixture of 25 mg liraglutide and 510.sup.11 exosome particles were ultrasonicated and then passed through a Capto Core 700 column to collect flow-through samples. The results are shown in
[0100] 250 mg liraglutide, and a mixture of 250 mg liraglutide and 510.sup.12 exosome particles were ultrasonicated and then passed through a QA column for purification, and flow-through and eluted samples were collected. The results are shown in
[0101] GLP-1R-CHO cells were seeded into a 96-well plate and cultured for 24 h, and then the medium was replaced with blank milk exosomes, milk exosome-liraglutide (10.sup.5/mL, 10.sup.6/mL, 10.sup.7/mL, 10.sup.8/mL, 10.sup.9/mL and 10.sup.10/mL) or liraglutide solutions with the corresponding particle concentration for 5 h of culture. The samples were collected according to the instructions of luciferase kit, and 100 l of cell lysate was added to each well to lyse cells for 5 min. The cell lysate was collected and centrifuged at 1200 rpm for 5 min. Then the substrate was added and incubated for 5 min, and the fluorescence was detected using an enzyme reader. The results are shown in
TABLE-US-00004 TABLE 4 Exosome-liraglutide_Capto Core 700 Liraglutide (ultrasonication)_Capto Core 700 Blank 20617 19685 22845 19513 20557 21774 Positive control 160126 161819 164485 166442 168105 182139 Blank exosomes 21244 24053 24830 22927 25572 26806 1 10.sup.10 117775 129093 137324 23620 23463 23987 1 10.sup.9 49581 52138 56060 22659 23782 25679 1 10.sup.8 24063 25156 24889 24413 23676 23367 1 10.sup.7 20066 20339 22143 23953 23706 24012 1 10.sup.6 17023 18083 20867 22732 22735 22400 1 10.sup.5 18999 17390 18551 17634 17430 21039
[0102] Experimental conclusion: From the comparison of the liquid appearance of liraglutide with that of the milk exosome-liraglutide mixture after ultrasonication, it can be seen that liraglutide did not precipitate when containing exosomes, but was loaded on the exosomes. After ultrasonication and QA purification of the milk exosome-liraglutide mixture, the chromatogram showed that the A260/A280 absorption ratio of the samples after loading by ultrasonication in high salt elution (conductivity of 70.44 mS/cm) was increased to 1.88, which represented the absorption peak of nucleic acid contents released from exosomes of which the bilayer membrane was damaged by ultrasonication. The particle number of exosomes decreased after prolonged ultrasound time. After purification through Capto Core 700 or QA columns, the drug-treated cells were proportionally diluted, and it was shown that the expression of luciferase was dose-dependent. The treatment of 110.sup.10 particles of milk exosome-exosome-liraglutide on cells significant increased luciferase expression, indicating that the loading of liraglutide in milk exosomes was achieved with biological activity.
Example 3 Drug Loading Process for Milk Exosomes-Electroporation Method
[0103] Principle: Electroporation is a microbiological technique, where an electric field is applied to a cell to increase the permeability of the cell membrane, thereby allowing chemicals, drugs or DNA to be introduced into the cell.
Operation Steps:
[0104] The optimal conditions for drug loading by electroporation were explored using PKH67. Specifically, after co-incubation of PKH67 with milk exosomes at room temperature for 30 min, electroporation was carried out using an electroporator at a voltage of 100-500 V and a capacitance of 100-500 F. The particle number, particle size and PKH67 positive rate of the electroporated samples were determined by using NanoFCM.
[0105] 25 mg of liraglutide, and a mixture of 25 mg liraglutide and 510.sup.11 exosome particles were electroporated and then passed through a Capto Core 700 column to remove free liraglutide. The flow-through samples were collected.
[0106] GLP-IR-CHO cells were seeded into a 96-well plate and cultured for 24 h, and then the medium was replaced with blank milk exosomes, milk exosome-liraglutide (10.sup.5/mL, 10.sup.6/mL, 10.sup.7/mL, 10.sup.8/mL, 10.sup.9/mL and 10.sup.10/mL) or liraglutide solutions with the corresponding particle concentration for 5 h of culture. The samples were collected according to the instructions of luciferase kit, and 100 l of cell lysate was added to each well to lyse cells for 5 min. The cell lysate was collected and centrifuged at 1200 rpm for 5 min. Then the substrate was added and incubated for 5 min, and the fluorescence was detected using an enzyme reader.
[0107] The results are shown in
TABLE-US-00005 TABLE 5 Effect of voltage on exosomes loading Voltage V Positive rate % Concentration particles 10.sup.10/mL 0 7.12 8.55 100 8.2 2.8 200 28.5 2.52 300 39 2.26 400 62.9 1.56 500 59.5 2.14
TABLE-US-00006 TABLE 6 Effect of capacitance on exosomes loading Capacitance F Positive rate % Concentration particles 10.sup.10/mL 0 6.35 8.55 100 46.8 3.2 200 51.8 3.78 300 71.2 2.78 400 69.8 2.96 500 57.1 2.11
TABLE-US-00007 TABLE 7 Exosome-liraglutide_Capto Core 700 Liraglutide (electroporation)_Capto Core 700 Blank 16617 15685 21845 17513 18557 19774 Positive control 170126 131819 144485 196442 128105 122139 Blank exosomes 18244 20053 24830 22927 18572 20806 1 10.sup.10 97775 92093 87324 20620 20463 20987 1 10.sup.9 50581 42138 46060 21659 22782 18679 1 10.sup.8 22063 22156 22889 19413 18676 20367 1 10.sup.7 20066 20339 22143 23953 23706 24012 1 10.sup.6 17023 18083 20867 22732 22735 22400 1 10.sup.5 18999 17390 18551 17634 17430 21039
Example 4 Drug Loading Process for Milk Exosomes-Co-Incubation Method
[0108] Principle: The drug will be adsorbed on the drug carrier by the interaction between substances.
Operation Steps
[0109] The optimal conditions for loading by co-incubation were explored using PKH67. Specifically, after co-incubation of PKH67 with milk exosomes at room temperature for 30 min, the samples were incubated at 4 C., 37 C. and 50 C. with shaking at 500 rpm for 30 min, 1 h, and 2 h. The particle number, particle size and PKH67 positive rate of the co-incubated samples were determined by using NanoFCM.
[0110] 25 mg of liraglutide, and a mixture of 25 mg liraglutide and 510.sup.11 exosome particles were co-incubated and then passed through a Capto Core 700 column to remove free liraglutide. The flow-through samples were collected.
[0111] GLP-1R-CHO cells were seeded into a 96-well plate and cultured for 24 h, and then the medium was replaced with blank milk exosomes, milk exosome-liraglutide (10.sub.5/mL, 10.sup.6/mL, 1.sup.07/mL, 10.sup.8/mL, 10.sup.9/mL and 10.sup.10/mL) or liraglutide solutions with the corresponding particle concentration for 5 h of culture. The samples were collected according to the instructions of luciferase kit, and 100 l of cell lysate was added to each well to lyse cells for 5 min. The cell lysate was collected and centrifuged at 1200 rpm for 5 min. Then the substrate was added and incubated for 5 min, and the fluorescence was detected using an enzyme reader.
[0112] The results are shown in
TABLE-US-00008 TABLE 8 Effect of incubation temperature on exosome loading Temperature Positive rate % Concentration particles 10.sup.10/mL 4 C. 7.12 8.55 37 C. 8.2 3.18 42 C. 28.5 3.32 50 C. 49 3.26 60 C. 22.9 1.56
TABLE-US-00009 TABLE 9 Effect of incubation time on exosome loading Time Positive rate % Concentration particles 10.sup.10/mL 0.5 h 32.1 3.6 1 h 46.8 3.78 2 h 29.8 3.78 4 h 21.2 2.78 8 h 9.8 1.96
TABLE-US-00010 TABLE 10 Cellular efficacy by co-incubation method Exosome-liraglutide_Capto Core 700 Liraglutide (incubation)_Capto Core 700 Blank 16617 15685 21845 17513 18557 19774 Positive control 190126 131819 144485 156442 128105 122139 Blank exosomes 18244 20053 24830 22927 18572 20806 1 10.sup.10 77775 72093 87324 14620 15463 13987 1 10.sup.9 40581 32138 46060 11659 12782 11679 1 10.sup.8 12063 12156 12889 9413 8676 80367 1 10.sup.7 10066 10339 12143 7953 7306 74012 1 10.sup.6 7023 8083 7867 5732 6735 6400 1 10.sup.5 6999 7390 6551 4634 5430 4039
[0113] Experimental conclusion: After purification through Capto Core 700, the drug-treated cells were proportionally diluted, and it was shown that the expression of luciferase was dose-dependent. The treatment of 110.sup.10 particles of milk exosome-liraglutide on cells significant increased luciferase expression, indicating that the loading of liraglutide in milk exosomes was achieved with biological activity.
Example 5 Loading Different T2DM Drugs in Milk Exosomes
[0114] At present, there are many drugs for treating type 2 diabetes mellitus (T2DM) on the market, such as insulin degludec, insulin detemir, liraglutide, semaglutide, and tirzepatide which has been listed abroad. The present disclosure realized loading different T2DM drugs in milk exosomes, so that these drugs can be administered orally instead of by injection, solving the dilemma of no oral diabetes drugs on the market.
Operation
[0115] 1) According to the drug loading method of milk exosomes introduced in 4.2, the electroporation method was selected. The milk exosomes were loaded with insulin degludec, insulin detemir, liraglutide, semaglutide and tirzepatide separately, and purified through a Capto Core 700 column.
[0116] 2) GLP-1R-CHO cells were seeded into a 96-well plate and cultured for 24 h, and then the medium was replaced with blank milk exosomes, or milk exosome loaded with different drugs (10.sup.5/mL, 10.sup.6/mL, 10.sup.7/mL, 10.sup.8/mL, 10.sup.9/mL and 10.sup.10/mL) for 5 h of culture. The samples were collected according to the instructions of luciferase kit, and 100 l of cell lysate was added to each well to lyse cells for 5 min. The cell lysate was collected and centrifuged at 1200 rpm for 5 min. Then the substrate was added and incubated for 5 min, and the fluorescence was detected using an enzyme reader.
[0117] The results are shown in
TABLE-US-00011 TABLE 11 Cellular efficacy of exosomes loaded with different drugs Exosmome-liraglutide Exosome-tirzepatide Exosome-semaglutide Blank 16617 15685 21845 17513 18557 19774 20617 19685 22845 Positive control 170126 171819 144485 206442 208105 212139 160126 161819 164485 Blank exosomes 23244 24053 24830 22927 26572 20806 21244 24053 24830 1 10.sup.10 97775 92093 87324 110620 110463 120987 97775 99093 87324 1 10.sup.9 50581 42138 46060 51659 52782 58679 49581 52138 56060 1 10.sup.8 22063 22156 22889 19413 18676 20367 24063 25156 24889 1 10.sup.7 20066 20339 22143 23953 23706 24012 20066 20339 22143 1 10.sup.6 17023 18083 20867 22732 22735 22400 17023 18083 20867 1 10.sup.5 18999 17390 18551 17634 17430 21039 18999 17390 18551
Experimental Conclusion:
[0118] From the results, milk exosomes loaded with different drugs enable cells to release GLP in a dose-dependent manner, indicating that the present disclosure can realize the loading of different types of protein and polypeptide drugs.
Example 6 Sublingual Administration of Fluorescently Labeled Milk Exosomes
[0119] Principle: Utilizing the characteristics of the sublingual mucosa that is not keratinized, has abundant capillaries, and has fast blood flow speed, and aiming to solve problems that milk exosomes are easily destroyed by gastric acid after passing through the gastrointestinal tract, resulting in low bioavailability of the loaded drug, the present disclosure prepares liraglutide-loaded milk exosomes into sublingual tablets to achieve oral administration of liraglutide, which can be absorbed through the sublingual mucosa and directly enter the blood circulation through the jugular vein and superior vena cava, with a rapid onset of action. In addition, the drug can be taken without water by placing it under the tongue to dissolve, which is convenient to take and has a good taste.
Experimental Operation:
[0120] A. PKH67-labelled milk exosomes were administered separately via 1) gavage, 2) sublingually, and 3) as enteric capsules. [0121] B. After administration, blood was collected from mice at different time points, and the number of positive particles in the blood was detected by NanoFCM to determine the absorption efficiency of exosomes into the blood.
[0122] For PKH67-labelled milk exosomes administered to mice by gavage, the blood was collected at different time points to detect PKH67-positive particles and 410.sup.11 particles were administered per mouse. After 5 min, the maximum number of PKH67-posivie particles were detected in the blood of the mice (210.sup.9), corresponding to 0.5% exosomes entering the blood. For PKH67-labelled milk exosomes administered sublingually, the maximum number of PHK67-positive particles detected in the blood (also at 5 min) was 210.sup.10, corresponding to 5% exosomes entering the blood. For administration by enteric capsules, the number of PKH67-positive particles in the blood of mice was not detected. Therefore, the bioavailability by sublingual administration is significantly higher compared to administration by gavage.
[0123] The experimental results are shown in
TABLE-US-00012 TABLE 12 Data from administration by gavage Mode of administration PKH67 Positive particles PBS 8.70 10.sup.7 1.74 10.sup.7 6.46 10.sup.7 3.57 10.sup.7 4.29 10.sup.7 6.83 10.sup.7 PKH67-15 min 3.30 10.sup.8 2.44 10.sup.8 2.01 10.sup.8 2.09 10.sup.8 3.04 10.sup.8 9.87 10.sup.7 Tail vein .sup.1.68 10.sup.11 .sup.2.34 10.sup.11 .sup.2.12 10.sup.11 .sup.1.98 10.sup.11 .sup.2.01 10.sup.11 .sup.2.05 10.sup.11 5 min 1.85 10.sup.9 2.32 10.sup.9 1.94 10.sup.9 2.05 10.sup.9 1.77 10.sup.9 2.11 10.sup.9 15 min 7.48 10.sup.8 3.66 10.sup.8 6.96 10.sup.8 4.87 10.sup.8 6.12 10.sup.8 5.34 10.sup.8 30 min 4.00 10.sup.8 2.62 10.sup.8 1.57 10.sup.8 3.56 10.sup.8 1.22 10.sup.8 3.19 10.sup.8 45 min 3.30 10.sup.8 2.26 10.sup.8 1.22 10.sup.8 2.98 10.sup.8 3.09 10.sup.8 1.78 10.sup.8 1 h 2.44 10.sup.8 2.08 10.sup.8 1.74 10.sup.8 1.35 10.sup.8 9.98 10.sup.7 2.04 10.sup.8 2 h 1.57 10.sup.8 2.78 10.sup.8 1.39 10.sup.8 1.34 10.sup.8 1.24 10.sup.8 8.99 10.sup.7 4 h 1.39 10.sup.8 1.04 10.sup.8 1.57 10.sup.8 1.02 10.sup.8 1.11 10.sup.8 8.34 10.sup.7
TABLE-US-00013 TABLE 13 Data from sublingual administration Mode of administration PKH67 Positive particles PBS 6.60 10.sup.7 6.60 10.sup.7 5.89 10.sup.7 6.01 10.sup.7 5.46 10.sup.7 5.28 10.sup.7 Tail vein .sup.2.60 10.sup.11 .sup.2.30 10.sup.11 .sup.2.45 10.sup.11 .sup.1.98 10.sup.11 .sup.2.45 10.sup.11 .sup.2.19 10.sup.11 30 min 7.88 10.sup.9 5.78 10.sup.9 6.56 10.sup.9 .sup.2.89 10.sup.10 .sup.4.02 10.sup.10 .sup.3.27 10.sup.10 1 h 3.50 10.sup.9 5.08 10.sup.9 1.04 10.sup.9 7.54 10.sup.8 4.78 10.sup.9 1.67 10.sup.9 2 h 1.11 10.sup.9 5.36 10.sup.8 8.97 10.sup.8 7.29 10.sup.8 1.02 10.sup.9 6.92 10.sup.8 4 h 4.26 10.sup.8 4.72 10.sup.8 4.72 10.sup.8 6.30 10.sup.8 2.78 10.sup.9 3.10 10.sup.9
TABLE-US-00014 TABLE 14 Data of enteric capsules mean SD 0 min 4.58 10.sup.7 2.67 10.sup.7 30 min 6.10 10.sup.7 1.89 10.sup.7 1 h 1.53 10.sup.7 9.87 10.sup.6 2 h 1.53 10.sup.7 8.22 10.sup.6 3 h 3.05 10.sup.7 1.24 10.sup.7 4 h 6.86 10.sup.7 2.78 10.sup.7 5 h 1.30 10.sup.8 9.18 10.sup.6 8 h 1.53 10.sup.7 7.35 10.sup.6 22 h 9.92 10.sup.7 3.22 10.sup.7
Example 7 Sublingual Tablets of Milk Exosomes Loaded with Liraglutide
[0124] Experimental operation: Lyophilized powder of milk exosomes loaded with liraglutide was mixed with a disintegrant, filler, binder, lubricant and flavoring agent to prepare into sublingual tablets. In each 1,000 tablets of sublingual tablets of liraglutide milk exosomes, the drug-loaded exosome has a content of 105-1017 particles, the disintegrating agent 1-50 g, the filler 20-400 g, the flavoring agent 0-50 g, the binder 5-100 g, and the lubricant 1 g-10 g;
[0125] The disintegrating agent used was selected from the group consisting of microcrystalline cellulose, cross-linked sodium carboxymethyl cellulose, cross-linked polyvinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, starch, Tween, sodium dodecyl sulfate, and a mixture thereof.
[0126] The filler used was selected from the group consisting of microcrystalline cellulose, microcrystalline cellulose-mannitol, microcrystalline cellulose-micronized silica gel, lactose, starch, modified starch, mannitol, sorbitol, xylitol, erythrose, trehalose, pregelatinized starch, icing sugar, glucose, dextrin, calcium sulfate, and a mixture thereof.
[0127] The flavoring agent used was selected from the group consisting of stevioside, icing sugar, glycyrrhizin, aspartame, sucralose, cyclamate, thaumatin, saccharin, and a mixture thereof.
[0128] The binder used was selected from the group consisting of purified water, starch slurry, hydroxypropyl methylcellulose, polyvinylpyrrolidone, carbomer, dextrin, gelatin slurry, gum arabic slurry, sodium alginate, syrup, and a mixture thereof.
[0129] The lubricant used was selected from the group consisting of stearic acid, magnesium stearate, calcium stearate, micronized silica gel, talc, hydrogenated vegetable oil, polyethylene glycol 4000, polyethylene glycol 6000, sodium dodecyl sulfate, magnesium dodecyl sulfate, sodium stearyl fumarate, and a mixture thereof.
[0130] The optimal formula is as follows: 110.sup.15 particles of exosome-liraglutide, 4 g cross-linked sodium carboxymethyl cellulose, 80 g mannitol, 25 g purified water and 3.5 g magnesium stearate. The preparation was performed according to the following preparation steps.
Preparation steps:
[0131] Step 1: The drug-loaded exosome was lyophilized and passed through 80-120 mesh sieves.
[0132] Step 2: The filler, disintegrating agent, flavoring agent and lubricant were dried, crushed and passed through 80-120 mesh sieves for pre-treatment.
[0133] Step 3: The pre-treated drug-loaded exosome, filler, flavoring agent and disintegrating agent were mixed well.
[0134] Step 4: The binder was added to the above well-mixed materials to prepare soft materials, which were then granulated and dried.
[0135] Step 5: The lubricant was added to the dry granules, mixed well and then pressed to obtain the final product.
[0136] Example 8 An improved formulation for the sublingual tablets of milk exosomes loaded with Liraglutide
[0137] 110.sup.13 particles of the drug-loaded exosome was added to 0.6 g mannitol, lyophilized, dried with 55 g microcrystalline cellulose the filler, 1 g sodium carboxymethyl cellulose the disintegrant, and 1 g magnesium stearate the lubricant, crushed, sieved, mixed, prepared into a soft material, granulated with a 20 mesh sieve, air dried to moisture3%, then added with lubricant for final blending, and finally pressed to obtain sublingual tablets of liraglutide-milk exosomes.
Example 9 Sublingual tablets of milk exosomes, loaded with T2DM treatment drugs Experimental operation
[0138] By electroporation method, insulin degludec, insulin detemir, liraglutide, semaglutide, and tirzepatide were loaded into milk exosomes and purified to obtain drug-loaded exosomes, which were prepared into sublingual tablets according to the formula in Example 8.
[0139] Each mouse was sublingually administered with sublingual tablets containing 110.sup.11 particles of drug-loaded milk exosomes. The animals were evaluated for their tolerance of elevated blood glucose level (induced via injection of glucose).
[0140] The results are shown in
TABLE-US-00015 TABLE 15 Time (min) 60 0 15 30 60 120 Vehicle 80 81 431 398 298 199 83 85 452 382 301 203 87 85 403 401 265 217 Exosome-liraglutide 87 88 135 121 110 100 82 86 146 135 109 102 85 81 132 129 101 90 Exosome-tirzepatide 91 93 101 99 95 93 87 81 110 97 94 90 89 84 109 98 92 88 Exosome-semaglutide 92 89 129 112 109 98 91 93 137 130 110 95 86 83 140 129 103 100 Exosome-insulin detemir 86 88 139 120 106 90 83 89 125 111 103 92 82 80 131 109 101 95 Exosome-insulin degludec 88 81 135 121 107 95 89 84 126 114 102 93 81 89 139 117 109 90 Exosome-insulin 83 89 155 141 120 116 81 88 189 139 126 120 89 85 167 129 118 111
Result Analysis
[0141] The use of milk exosomes can achieve loading of multiple T2DM treatment drugs. The drug-loaded exosomes can achieve significant efficacy within 15 min after sublingual administration. And it can maintain the stable blood glucose and improve the glucose tolerance of mice.
Example 10 In Vivo Efficacy (Random Blood Glucose Level) of Sublingual Tablets Containing Liraglutide-Loaded Milk Exosome
[0142] By ultrasonication method, liraglutide (not limited to liraglutide, but also including insulin degludec, insulin detemir, semaglutide and tirzepatide) was loaded into milk exosomes and purified to obtain drug-loaded exosomes, which were prepared into sublingual tablets according to the optimal preparation formula in Example 8. Here, the results of milk exosomes loaded with liraglutide are illustrated as an example.
[0143] Diabetic mice were administered sublingually with different dosages of milk exosomes loaded with liraglutide, and random blood glucose levels of the mice were detected.
[0144] The results are shown in
TABLE-US-00016 TABLE 16 min Blank (Unit: mg/dL of glucose) 0 277 245 288 266 329 312 289 287 290 321 30 302 282 340 299 340 340 323 319 307 285 60 215 234 265 221 298 284 290 290 279 320 120 194 194 238 213 259 251 283 283 261 266 150 210 219 253 220 223 225 275 274 281 272 180 225 211 327 340 298 291 284 284 273 297 210 235 267 262 273 320 322 296 291 286 279 240 306 259 243 249 299 316 314 316 307 309 300 169 150 296 255 320 303 340 333 320 286 min 1.5 10.sup.8 particles per mouse (Unit: mg/dL of glucose) 0 212 213 251 252 277 277 281 284 332 330 30 303 340 238 269 209 192 239 233 302 282 60 245 263 282 252 232 217 250 224 290 279 120 208 229 259 257 210 225 251 307 259 274 150 167 160 276 276 215 198 236 225 261 270 180 190 220 291 289 225 258 183 233 269 269 210 214 192 287 301 212 201 182 174 274 284 240 246 204 269 300 276 249 166 155 279 290 300 239 235 279 286 212 273 193 197 291 286 min 1.5 10.sup.10 particles per mouse (Unit: mg/dL of glucose) 0 298 254 271 253 292 281 299 274 310 302 30 340 333 166 156 258 272 198 176 192 192 60 207 205 75 78 197 169 89 82 179 189 120 209 199 95 91 212 210 187 285 192 196 150 188 190 166 186 220 240 219 211 227 219 180 191 194 151 165 218 232 229 221 231 228 210 237 219 162 156 221 229 235 230 239 239 240 226 175 211 199 229 231 239 231 228 240 300 221 202 209 206 237 230 238 229 242 241 min 1.5 10.sup.12 particles per mouse (Unit: mg/dL of glucose) 0 284 271 283 294 277 245 238 226 231 233 30 115 118 151 158 246 282 105 85 94 83 60 125 113 116 126 215 234 85 104 90 71 120 124 138 137 138 194 194 115 118 56 58 150 133 130 149 121 210 219 129 135 39 44 180 151 161 181 143 155 151 176 169 162 156 210 216 218 194 197 195 207 194 194 176 197 240 222 226 174 154 176 199 194 196 188 197 300 229 207 209 204 179 210 195 193 202 204
[0145] Conclusion: From the results of random blood glucose levels, it could be seen that the exosomes loaded with liraglutide can significantly lower blood glucose levels in diabetic mice.
Example 11 In Vivo Efficacy (Fasting Blood Glucose Level) of Sublingual Tablets Containing Liraglutide-Loaded Milk Exosome
[0146] By electroporation, liraglutide (not limited to liraglutide, but also including insulin degludec, insulin detemir, semaglutide and tirzepatide) was loaded into milk exosomes and purified to obtain drug-loaded exosomes, which were prepared into sublingual tablets. Here, the results of milk exosomes loaded with liraglutide are illustrated as an example.
[0147] Diabetic mice were fasted for 6 h, and then orally administered with different concentrations of milk exosomes loaded with liraglutide. The fasting blood glucose of the mice was detected.
[0148] The results are shown in
TABLE-US-00017 TABLE 17 h Blank (Unit: mg/dL of glucose) 0 448.2 390.6 342 284.4 433.8 435.6 462.6 441 342 351 0.25 457.2 439.2 423 473.4 468 522 525.6 570.6 466.2 487.8 0.5 460.8 392.4 399.6 361.8 459 451.8 599.4 585 473.4 495 1 612 612 374.4 421.2 493.2 486 612 612 486 495 2 612 612 365.4 342 480.6 473.4 428.4 403.2 451.8 466.2 3 430.2 376.2 448.2 478.8 612 586.8 493.2 451.8 388.8 361.8 4 504 444.6 376.2 381.6 522 568.8 363.6 401.4 322.2 313.2 5 338.4 329.4 493.2 459 531 495 376.2 340.2 259.2 246.6 6 612 545.4 329.4 309.6 388.8 322.2 379.8 354.6 327.6 298.8 i.v. h 1.5 10.sup.10 particles per mouse (Unit: mg/dL of glucose) 0 594 525.6 331.2 342 448.2 466.2 518.4 502.2 421.2 392.4 0.25 450 424.8 241.2 282.6 385.2 424.8 480.6 466.2 277.2 277.2 0.5 361.8 419.4 259.2 194.4 338.4 334.8 428.4 446.4 160.2 190.8 1 196.2 235.8 111.6 109.8 230.4 264.6 450 457.2 127.8 117 2 122.4 108 82.8 82.8 253.8 257.4 417.6 415.8 142.2 126 3 64.8 73.8 66.6 55.8 88.2 90 419.4 417.6 72 86.4 4 55.8 37.8 50.4 45 45 48.6 399.6 370.8 52.2 52.2 5 50.4 45 50.4 55.8 36 36 279 271.8 54 55.8 6 54 43.2 59.4 57.6 34.2 32.4 212.4 230.4 41.4 36 s.l. (sublingual) h 1.5 10.sup.12 particles per mouse (Unit: mg/dL of glucose) 0 502.2 428.4 352.8 351 414 451.8 435.6 471.6 351 423 0.25 516.6 599.4 558 504 532.8 612 612 576 612 612 0.5 511.2 487.8 379.8 437.4 496.8 453.6 457.2 466.2 428.4 365.4 1 612 594 180 216 329.4 300.6 257.4 244.8 228.6 223.2 2 457.2 462.6 151.2 135 210.6 271.8 262.8 210.6 257.4 226.8 3 417.6 498.6 120.6 115.2 241.2 235.8 196.2 153 185.4 199.8 4 462.6 493.2 95.4 88.2 196.2 136.8 192.6 169.2 163.8 169.2 5 336.6 347.4 73.8 66.6 118.8 108 95.4 106.2 154.8 160.2 6 286.2 316.8 77.4 64.8 70.2 73.8 100.8 90 169.2 156.6 s.l. (sublingual) h 1.5 10.sup.10 particles per mouse (Unit: mg/dL of glucose) 0 462.6 428.4 469.8 428.4 415.8 369 426.6 482.4 324 273.6 0.25 612 612 450 451.8 612 576 540 532.8 318.6 356.4 0.5 318.6 284.4 417.6 426.6 612 612 500.4 477 315 396 1 552.6 592.2 379.8 372.6 426.6 469.8 612 581.4 388.8 385.2 2 570.6 581.4 354.6 336.6 612 590.4 552.6 500.4 266.4 250.2 3 487.8 415.8 475.2 480.6 592.2 554.4 367.2 383.4 158.4 145.8 4 577.8 583.2 221.4 266.4 612 612 273.6 288 154.8 149.4 5 455.4 432 358.2 295.2 532.8 538.2 304.2 279 135 133.2 6 594 612 612 612 349.2 410.4 468 482.4 153 151.2
[0149] Conclusion: From the results of the fasting blood glucose, it could be seen that the exosomes loaded with liraglutide can significantly reduce blood glucose in diabetic mice.
[0150] The use of a milk exosome in the preparation of drug-loaded complex and subsequent production of sublingual tablets provided by the present disclosure is described in detail above. The principle and implementation of the present disclosure are illustrated by using specific embodiments herein. The above descriptions of the embodiments are only used to facilitate understanding of the method and the core idea of the present disclosure. It should be noted that, several improvements and modifications may be made by those skilled in the art to the present disclosure without departing from the principle of the present disclosure, and these improvements and modifications also fall within the protection scope of the claims of the present disclosure.