Freeze-dried flash release tablet of GLP1 polypeptide drug and preparation method therefor

Abstract

Provided are a freeze-dried flash release tablet of a GLP1 polypeptide drug and a preparation method therefor. The provided freeze-dried orally disintegrating tablet of a GLP1 polypeptide drug comprises the following raw materials in percentage by mass: 30%-50% of a GLP1 polypeptide-mEV stock solution, 0.1%-1.0% of a surfactant, 3%-5% of a framework agent, 1%-3% of an adhesive, 0.01%-1% of a suspending agent and the balance of water. The GLP1 polypeptide is one or more of liraglutide and semaglutide. Further provided is a method for preparing the freeze-dried flash release tablet of a GLP1 polypeptide drug. The flash release tablet is prepared by using a freeze-drying technique, and the obtained product has a large loading capacity and a good dissolution effect, which is beneficial to the oral absorption of the GLP1 polypeptide. In addition, the preparation method for the flash release tablet is simple and convenient, and is easy to implement.

Description

GLP1 polypeptide drug freeze-dried flash-release tablet and preparation method thereof

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on August 28, 2023, with application number 202311090980.4 and invention name “GLP1 polypeptide drug freeze-dried flash-release tablets and preparation method thereof”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention relates to the field of pharmaceutical preparations, and in particular to a GLP1 polypeptide drug freeze-dried flash-release tablet and a preparation method thereof. Background Art

[0003] Glucagon-like peptide (GLP1) is widely used in the treatment of type 2 diabetes and severe obesity, and patients receive the drug through long-term injections. Despite the continuous iteration of innovative GLP1 drugs, it is still difficult to reverse a painful reality: patients need to continue to receive the drug through injections throughout their lives. After Novo Nordisk's oral semaglutide / semaglutide product was launched on the market, although it achieved the oral administration of GLP1 drugs for the first time, its oral bioavailability is still low (only 1%). In addition, the cost of the drug is high, and patients need to take it daily, which puts a heavy economic burden. Therefore, in the field of type 2 diabetes and severe obesity treatment, there is a lack of a technology that can deliver GLP1 drugs orally at low cost and high efficiency.

[0004] Milk extracellular vesicles (mEVs) are biological nanoparticles composed of a phospholipid bilayer membrane that can be extracted in large quantities from milk. Their natural function includes delivering bioactive molecules (proteins, small nucleic acids, etc.) from the mother (cow) to the offspring (calf) through the gastrointestinal tract. Studies have shown that mEVs can withstand damage from environmental factors such as digestive enzymes and low pH in the digestive tract and are absorbed into the bloodstream through the digestive tract. Due to the widespread consumption of milk, the long-term exposure of most people to mEVs (through drinking milk) determines that the oral application of mEVs in humans is safe and tolerable. Therefore, mEVs have received considerable attention in recent years as a potential oral drug delivery vehicle.

[0005] There are many precedents for loading small molecule drugs with mEV in previous technologies. In previous studies, although mEV loaded with GLP1 (Liraglutide) can be absorbed into the blood circulation by the sublingual venous plexus after oral administration. But so far, there is no mature oral solid dosage form for mEV loaded with GLP1 or using lipid nanoparticles (LNP) to load GLP1 for oral administration and sublingual absorption. In some existing technologies, solution preparations of mEV loaded with liraglutide are prepared. However, although the solution stability of mEV is significantly improved compared to other types of extracellular vesicles (EVs), how to keep mEV stable in high concentration solutions is still a technical problem to be solved. The solution concentration of mEV nanoparticles is more than 1×10 12 After the mEV concentration reaches 100 p / mL, problems such as aggregation, fusion, and adsorption to the container wall become prominent. In other approaches, although freeze-dried sublingual tablets were prepared, large amounts of mannitol or microcrystalline cellulose were required to maintain the activity of mEVs during the freeze-drying process. If compressed, the tablets would be too compact, resulting in difficulty in oral disintegration and release. Furthermore, the excessive increase in the volume of the freeze-dried powder makes it difficult to administer orally at the intended clinical dose.

[0006] The volume of a single tablet solution of a suitable freeze-dried solid preparation is usually small, less than 1 ml, while the clinically intended dose for oral absorption of mEV is 10 13 The particles are even higher. Therefore, a method that can reach 1×10 13 p / mL or even higher concentration of mEV stock solution concentration process and the corresponding stabilizer formula.

[0007] Summary of the Invention

[0008] In view of this, the technical problem to be solved by the present invention is to provide a GLP1 polypeptide drug freeze-dried flash-release tablet with high drug concentration, high dissolution efficiency and good oral sublingual absorption, and a preparation method thereof.

[0009] The present invention provides a GLP1 polypeptide drug freeze-dried orally disintegrating tablet, which comprises the following raw materials in percentage by weight: 30% to 50% of a GLP1 polypeptide-mEV stock solution or a GLP1 polypeptide-lipid nanoparticle (LNP) stock solution, 0.1% to 1.0% of a surfactant, 3% to 5% of a skeleton agent, 1% to 3% of a binder, 0.01% to 1% of a suspending agent, and the balance being water;

[0010] The surfactant is selected from Poloxamer 188.

[0011] The present invention uses GLP1 polypeptide-mEV stock solution to prepare GLP1 polypeptide drug freeze-dried orally disintegrating tablets. In addition, GLP1 polypeptide drug freeze-dried orally disintegrating tablets can also be prepared using SM-102, DLin-MC3-DMA and LNP formed by phospholipids as carriers.

[0012] Furthermore, the skeleton agent is selected from one of mannitol and trehalose; preferably, the skeleton agent is trehalose.

[0013] Furthermore, the adhesive is selected from one of pullulan and gelatin; preferably, the adhesive is pullulan.

[0014] Furthermore, the suspending agent is selected from one of pullulan and xanthan gum; preferably, the suspending agent is xanthan gum.

[0015] Furthermore, the raw materials for preparing the GLP1 polypeptide-mEV stock solution include: adding 0-1.2 mg of excipients and 1×10 10 ~1×10 13 mEV particles.

[0016] The raw materials for preparing the GLP1 polypeptide-LNP stock solution include: adding 0-3 mg of auxiliary materials and phospholipids, SM-102 and DLin-MC3-DMA lipid molecules to each mg of GLP1 polypeptide.

[0017] The auxiliary material is selected from one of polylysine, protamine, polyarginine, fucose or mannitol. Preferably, the auxiliary material is polyarginine.

[0018] In some embodiments, the freeze-dried orally disintegrating tablets of the GLP1 polypeptide provided herein contain the following raw materials in the following weight percentages: 50% GLP1 polypeptide-mEV stock solution or polypeptide-LNP stock solution, 0.2% surfactant, 3% backbone agent, 2% binder, 0.1% suspending agent, and the balance water. In these embodiments, the weight percentages of the raw materials are calculated based on the weight before lyophilization.

[0019] In the sublingual tablets provided by the present invention, the selection of excipients including surfactants, such as skeleton agents, adhesives or suspending agents, is reasonable, so that the prepared sublingual tablets have better disintegration effect and sublingual absorption efficiency. Preliminary experiments have shown that compared with the selection of other excipients, the combination of poloxamer 188, trehalose, pullulan and xanthan gum is more conducive to assisting GLP1-mEV to better maintain stability, improve release performance in the oral cavity and sublingual absorption efficiency. The GLP1 polypeptide is one or more of liraglutide and semaglutide.

[0020] In some embodiments, the GLP1 polypeptide is liraglutide, and the liraglutide freeze-dried orally disintegrating tablet contains the following raw materials in percentage by weight: 30% to 50% liraglutide-mEV stock solution or liraglutide-LNP stock solution, 0.1% to 1.0% poloxamer 188, 3% to 5% trehalose, 1% to 3% pullulan, 0.01% to 1% xanthan gum, and the balance is water.

[0021] In a specific embodiment, the liraglutide freeze-dried orally disintegrating tablets contain the following raw materials in percentage by weight: 50% liraglutide-mEV stock solution or liraglutide-LNP stock solution, 0.2% poloxamer 188, 3% trehalose, 2% pullulan, 0.1% xanthan gum, and the balance is water.

[0022] In some embodiments, the GLP1 polypeptide is semaglutide, and the semaglutide freeze-dried orally disintegrating tablet contains the following raw materials in percentage by weight: 30% to 50% semaglutide-mEV stock solution or semaglutide-LNP stock solution, 0.1% to 1.0% poloxamer 188, 3% to 5% trehalose, 1% to 3% pullulan, 0.01 to 1% xanthan gum, and the balance is water.

[0023] In a specific embodiment, the semaglutide freeze-dried orally disintegrating tablets contain the following raw materials in percentage by weight: 50% semaglutide-mEV stock solution or semaglutide-LNP stock solution, 0.2% poloxamer 188, 3% trehalose, 2% pullulan, 0.1% xanthan gum, and the balance is water.

[0024] The present invention also provides a method for preparing the aforementioned GLP1 polypeptide drug freeze-dried orally disintegrating tablets, comprising the following steps:

[0025] (1) Loading the GLP1 polypeptide into mEVs to obtain a GLP1 polypeptide-mEV stock solution, or loading the GLP polypeptide into LNPs to obtain a GLP1 polypeptide-LNP stock solution;

[0026] (2) The GLP1 polypeptide-mEV stock solution or the GLP1 polypeptide-LNP stock solution is concentrated by ultrafiltration to obtain a concentrated solution.

[0027] (3) mixing a surfactant, a skeleton agent, a binder, a suspending agent, a sweetener, and a flavoring agent, and adding the concentrate to obtain a drug solution;

[0028] (4) shearing the liquid medicine in step (3);

[0029] (5) degassing the liquid medicine after shearing in step (4);

[0030] (6) filling the degassed liquid medicine in step (5) into the cavity;

[0031] (7) Pre-freeze the filled liquid medicine;

[0032] (8) Freeze-dry the pre-frozen drug, setting the initial temperature to -30°C. The specific freeze-drying process is as follows:

[0033] Cold soaking temperature: ≤-40℃; Vacuum control: ≤200ubar

[0034] Plate temperature: from -30°C to -25°C, 5 minutes; maintain at -25°C for 60 minutes; from -25°C to -15°C, 10 minutes; maintain at 15°C for 60 minutes; from -15°C to -5°C, 10 minutes; maintain at -5°C for 60 minutes; from -5°C to 10°C, 25 minutes; maintain at 10°C for 100 minutes; from 10°C to 25°C, 15 minutes; maintain at 25°C for 60 minutes.

[0035] Furthermore, the loading of the GLP1 polypeptide into mEVs comprises:

[0036] The GLP1 peptide solution was mixed with the milk exosome solution and then ultrasonically treated.

[0037] In the GLP1 polypeptide solution, the solvent is 20 mM Tris buffer containing 100 mM NaCl, with a pH value of 8.0;

[0038] In the milk exosome solution, the solvent is 20 mM Tris buffer containing 100 mM NaCl, and the pH value is adjusted to 7.0-10.0 with Na2CO3 / NaHCO3 buffer containing glycine;

[0039] The ultrasonic treatment conditions include: 99% power, 5° C., and ultrasonic treatment for 12 h.

[0040] Furthermore, the loading of the GLP1 polypeptide into the LNP comprises:

[0041] The aqueous solution of GLP1 is mixed with the organic solution of lipid molecules to self-assemble into an aqueous dispersion of LNPs encapsulating GLP1.

[0042] Furthermore, the ultrafiltration concentration of the GLP1 polypeptide-mEV stock solution or the GLP1 polypeptide-LNP stock solution includes:

[0043] After dissolving the GLP1 polypeptide-mEV stock solution or the GLP1 polypeptide-LNP stock solution in PBS buffer containing PEG3350, the volume was concentrated to 1 / 5 of the stock solution using a hollow fiber column with a molecular weight cut-off of 750kDa (flow rate of 405mL / min). Experiments have shown that ultrafiltration and concentration in this step have important positive significance for increasing the loading capacity of the drug. The present invention optimizes the parameters of ultrafiltration and concentration to obtain the best effect, thereby further improving the loading effect.

[0044] Furthermore, the shearing in step (2) is carried out in an emulsifier;

[0045] Furthermore, the degassing in step (3) is carried out in an emulsifier or a degassing bottle.

[0046] Furthermore, the shearing speed is 100-3000 rpm, and the shearing time is 3-20 minutes; preferably, the emulsifying machine has a speed of 300 rpm, and the shearing time is 10 minutes.

[0047] Furthermore, the pre-freezing temperature is -60 to -80°C; preferably, the pre-freezing temperature is -60°C.

[0048] Furthermore, the pre-freezing time is 10-100 minutes; preferably, the pre-freezing time is 30 minutes.

[0049] The present invention provides a freeze-dried flash-release tablet of a GLP1 polypeptide drug. The tablet is prepared using freeze-drying technology, resulting in a large loading capacity, good dissolution effect, and high sublingual absorption efficiency. Furthermore, the preparation method of the flash-release tablet is simple and easy to implement. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 shows the electron microscopy results of mEV without additives;

[0051] Figure 2 shows the electron microscopy results of mEVs in the presence of 0.5% PEG3350 additive;

[0052] Figure 3 shows the electron microscopy results of mEVs in the presence of 5% PEG3350 additive;

[0053] FIG4 shows the blood glucose concentration test results in animal efficacy tests;

[0054] Figure 5 shows the results of animal blood drug concentrations;

[0055] FIG6 shows the blood glucose concentration test results of each group after drug administration;

[0056] FIG7 shows the blood glucose concentration test results after administration of LRT-mEV and SMT-mEV. DETAILED DESCRIPTION

[0057] In order to more clearly understand the technical content of the present invention, the following examples are given in detail, which are only for the purpose of better understanding the content of the present invention and not to limit the scope of protection of the present invention. The reagents used in the examples of the present invention are all commercially available.

[0058] Example 1 Preparation of high-concentration mEV stock solution loaded with liraglutide

[0059] Objective: After loading liraglutide (LRT) into mEVs, the mEV solution was concentrated by tangential flow ultrafiltration technology while maintaining the stability of mEV particle number and structure.

[0060] Materials: Milk exosomes (mEVs) were prepared according to patent 202110097550.X.

[0061] method:

[0062] 1. Loading:

[0063] (1) Chemical loading method

[0064] a. Take 7.5E+10 mEVs from each of four 15 mL tubes and dilute the EVs to a final volume of 2 mL using 20 mM Tris 8.0 + 100 mM NaCl buffer. Adjust the pH of the diluted mEV solution to 7.0, 8.0, 9.0, and 10.0 using 0.1 M Glycine (pH 7.0), 0.1 M Na2CO3 / NaHCO3 (pH 9.04), and 0.1 M Na2CO3 / NaHCO3 (pH 10.08), respectively. Bring all EV samples to a final volume of 6 mL using the corresponding pH buffer. Divide the mEVs at different pH values ​​into 15 equal portions until the total number of EV particles in each portion is 5.0E+09. These are referred to as mEV solutions.

[0065] b. Add 100 μL of the prepared 0.5-2 mg / mL liraglutide solution (Liraglutide, or LRT, dissolved in 20 mM Tris 8.0 + 100 mM NaCl buffer) to each of the mEV solutions with a total particle count of 5.0E+09 and different pH values. Invert and mix thoroughly to obtain a set of LRT-mEV solutions.

[0066] c. Add one of the following additives to the LRT-mEV solution: polylysine, protamine, polyarginine, fucose, or mannitol (ensure the final concentration of LRT:additive = 1:1).

[0067] d. Each sample was placed in a large ultrasonic cleaning machine, set at 99% power, 5°C temperature, and ultrasonic treatment time for 12 h.

[0068] e. The sample was processed through a Capto Core 700 column to remove free LRT and obtain a purified LRT-mEV stock solution sample.

[0069] (2) Osmotic pressure stimulation loading method

[0070] a. Prepare a 50% m / v sucrose solution by dissolving 25g of sucrose granules in 50mL of ultrapure water. Prepare a 0.1x PBS solution of LRT by dissolving 14.10mg of powder in 1.41mL of 0.1x PBS to a final concentration of 10mg / mL.

[0071] b. Prepare mEV solutions (4E+10 p / mL) containing different sucrose concentrations: 0%, 1%, 3%, 5%, 10%, 15%, 20%, and 30% (m / v).

[0072] c. Add 0.1*PBS solution of LRT to the mEV sucrose solution until the sucrose concentration in the solution drops to 0.2%.

[0073] d. Each sample was placed in a large ultrasonic cleaning machine, set at 99% power, 5°C temperature, and ultrasonic treatment time for 12 h.

[0074] e. The sample was processed through a Capto Core 700 column to remove free LRT and obtain a purified LRT-mEV stock solution sample.

[0075] 2. Hollow fiber column tangential flow ultrafiltration concentration

[0076] (1) Pre-experimental treatment:

[0077] Preparation of 10% m / v PEG3350: Take 60 mL of 50% PEG3350 stock solution and dilute to a final volume of 300 mL with water.

[0078] The LRT-mEV stock solution sample (450 mL, particle concentration 1.6E+13 p / mL) loaded and purified using the aforementioned chemical method was divided into three equal parts:

[0079] a. Name one portion 0% PEG3350-EV. Take 150 mL of LRT-mEV stock solution and add 1% PBS to a final volume of 400 mL.

[0080] b. In one portion, designated 0.5% PEG3350-EV, add 20 mL of 10% m / v PEG3350 to 150 mL of LRT-mEV stock solution and dilute to a final volume of 400 mL with PBS. This gives a final PEG3350 concentration of 0.5%.

[0081] c. In one aliquot, designated 5% PEG3350-EV, add 200 mL of 10% m / v PEG3350 to 150 mL of the LRT-mEV stock solution and dilute to a final volume of 400 mL with PBS. This gives a final PEG3350 concentration of 5%.

[0082] (2) Hollow fiber column tangential flow ultrafiltration concentration:

[0083] a. Cleaning Cytiva AKTAFLUX: Wash the hollow fiber column (MWCO = 750 kDa) with purified water at a flow rate of 405 mL / min until the pH of the filtrate is around 7.4.

[0084] b. Start concentration at a flow rate of 405 mL / min.

[0085] c. Concentrate to 20 mL.

[0086] d. Add 100 mL of PBS buffer to wash the filter.

[0087] e. Concentrate to 20 mL and collect the concentrate.

[0088] 3. Particle detection:

[0089] The obtained mEV-LRT product samples were tested for particle number using the Xiamen Fuliu nanoFCM nanoflow cytometer.

[0090] 4. LRT detection:

[0091] The LRT polypeptide content in the mEV-LRT product samples was detected using a GLP-1 polypeptide monoclonal antibody (ANTIBODYSHOP ABS 046-03 / ABS033-108). The specific ELISA detection method was referred to the antibody manufacturer's instructions.

[0092] result:

[0093] 1. Chemical loading method:

[0094] Comparing the single-particle loading capacity of mEV onto LRT after low-power sonication under different pH, additive, and LRT stock solution concentrations, the highest mEV loading capacity was observed at pH 10, with a single mEV particle capable of loading approximately 3,000 to 20,000 LRT peptide molecules. Overall, the loading capacity of mEV onto LRT was best with either polyarginine or no additive. Furthermore, when loading equal volumes of LRT stock solutions of varying concentrations, the higher the LRT addition, the higher the mEV loading capacity.

[0095] Table 1 Comparison of mEV loading capacity on LRT under different LRT stock solution concentrations, additives, and pH conditions

[0096] 2. Osmotic pressure stimulation loading results:

[0097] Comparing the effects of osmotic pressure stimulation of different intensities on the single-particle loading capacity of LRT in mEV, at a sucrose concentration of 20% w / v, the single mEV particle had the highest LRT loading capacity, which could load 2,600 LRT polypeptide molecules; the LRT loading effect of mEV under this method was not as good as that of the chemical loading method.

[0098] Table 2 Comparison of the effect of different osmotic pressure stimulation on the single particle loading of LRT in mEV

[0099] 3. Results of hollow fiber column tangential flow ultrafiltration concentration

[0100] The LRT-mEV stock solution had a concentration of 6.0E+12p / mL and a total particle count of 2.4E+15p before concentration. After concentration by tangential flow ultrafiltration on a hollow fiber column in the presence of 0.5% and 5% PEG3350, the total particle count was higher than that in the presence of 0% PEG3350. The maximum particle count reached 1.5E+15 at a concentration of 7.5E+13p / mL in the presence of 5% PEG3350. The total particle count accounted for 62.5% of the total before concentration, representing a 12.5-fold increase in concentration. Electron microscopy revealed that mEVs exhibited improved dispersion in the presence of 5% PEG3350.

[0101] Table 3 Concentration effect of mEV at different concentrations of PEG3350

[0102] Example 2 Preparation of high-concentration mEV stock solution loaded with semaglutide

[0103] Objective: After loading semaglutide (SMT) into mEVs, the mEV solution was concentrated by tangential flow ultrafiltration technology while maintaining the stability of mEV particle number and structure.

[0104] Materials: Milk exosomes (mEVs) were prepared according to patent 202110097550.X.

[0105] method:

[0106] 1. Loading: For details, see "Example 1"

[0107] 2. Hollow fiber column tangential flow ultrafiltration concentration (see "Example 1" for details)

[0108] 3. Particle detection: Use Xiamen Fuliu nanoFCM nano-flow cytometer to detect the number of particles in the mEV-SMT product samples.

[0109] 4. SMT detection: Use LC-MS / MS to detect the SMT peptide content in the obtained mEV-SMT product samples. The specific detection method can be found in the literature (DOI: 10.1016 / j.jchromb.2023.123688).

[0110] result:

[0111] 1. Loading:

[0112] Using a method similar to LRT loading conditions (pH 10.0, no additives), a single mEV particle can be loaded with 20,000-40,000 SMT peptide molecules.

[0113] Table 4 Single particle loading of SMT in mEVs

[0114] During loading, the ratio of SMT to mEV increases, leading to higher single-particle loading capacity. However, once the SMT loading rate reaches saturation, further increases in loading capacity will not increase the loading capacity and will reduce SMT utilization, resulting in waste (see table below). Both economic efficiency and loading efficiency should be considered in actual formulation production.

[0115] Table 5 Effect of the starting ratio of SMT to mEV on loading capacity

[0116] 2. Results of hollow fiber column tangential flow ultrafiltration concentration:

[0117] Similar to LRT, after mEV-SMT was concentrated using 5% PEG3350, the concentration increased by at least 10 times compared with before concentration, and the final concentration could reach up to 8E+13p / mL.

[0118] Table 6 Single particle loading of SMT in mEVs

[0119] Example 3: Preparation of GLP1 polypeptide-mEV freeze-dried orally disintegrating tablets

[0120] 1. Preparation of LRT-mEV Freeze-dried Orally Disintegrating Tablets

[0121] Liraglutide (LRT) loaded into mEV was used as the raw material and LRT-mEV freeze-dried orally disintegrating tablets were prepared according to the following formula.

[0122] Table 7 LRT-mEV freeze-dried orally disintegrating tablets formulation (calculated based on pre-freeze-drying mass)

[0123] (1) Weigh the prescribed amount of LRT-mEV, pullulan, trehalose, and poloxamer 188, dry mix them in a 100 ml shear cup, add approximately 50% of the prescribed amount of purified water, and stir to dissolve. Weigh the prescribed amount of xanthan gum, add the remaining prescribed amount of purified water, and stir to dissolve. Mix the two.

[0124] (2) Shearing: Use an emulsifier for shearing, speed: 300 rpm, time: 10 minutes.

[0125] (3) Degassing: Transfer the drug solution to a 125 ml degassing bottle and vacuum degas until there are no bubbles.

[0126] (4) Filling: Use a pipette to fill into a 0.4 ml aluminum container, 0.4 g / tablet.

[0127] (5) Prefreezing: Place the filled aluminum nests in a -60°C low-temperature refrigerator for prefreezing for 20 minutes. After prefreezing, store them in the refrigerator until they are placed in the freeze dryer.

[0128] (6) Freeze-drying process: The pre-frozen sample was transferred to a 1.7 m2 freeze dryer for freeze-drying. The freeze-drying curve is as follows:

[0129] Inlet temperature: -30℃; Cold trap temperature: ≤-40℃

[0130] Table 8 Freeze-drying procedure

[0131] Table 9 Evaluation results

[0132] 2. Preparation of SMT-mEV Freeze-dried Orally Disintegrating Tablets

[0133] SMT-mEV freeze-dried orally disintegrating tablets were prepared using semaglutide (SMT) loaded into mEV as the raw material according to the method and formulation described in (1).

[0134] Table 10 Evaluation results

[0135] Example 4: Animal efficacy test of GLP1 polypeptide-mEV freeze-dried flash-release tablets

[0136] Objective: To evaluate the hypoglycemic effect of GLP1 peptide-mEV freeze-dried orally disintegrating tablets and to measure the blood concentration of liraglutide using a randomized blood glucose test in db / db mice.

[0137] 1. Efficacy test method of LRT-mEV freeze-dried flash-release tablets:

[0138] 1. Animal efficacy test:

[0139] a. Experimental Grouping

[0140] Table 11

[0141] b. Dosage frequency: once at 0h and once at 7h

[0142] c. Detecting blood glucose at zero point in db / db mice, and administering the drug sublingually after isoflurane anesthesia:

[0143] a) Mice in group AC: Cut the freeze-dried flash-release tablet into 3-6 small pieces. Carefully insert one small piece under the tongue of the mouse. Then add 20 μL of water to dissolve the tablet. The tablet is administered in 3 or 6 doses, with a 10-min interval between each addition of water for absorption.

[0144] b) Positive control group: LRT-mEV solution was dripped sublingually into db / db mice according to the literature method (doi:10.1002 / cmdc.202100758), with a total of 90 μL administered in three doses, and each dose was absorbed after 10 minutes.

[0145] c) Negative control group: no drug administration.

[0146] d. Blood glucose levels in each group of mice were measured at 4, 7, 22, 24, 26, 28, and 33 hours after the first administration, with two replicates for each test (during the experiment, mice were not restricted in eating or drinking—random blood glucose testing was performed).

[0147] 2. Blood drug concentration detection

[0148] (1) Blood collection

[0149] Blood was collected from 9 mice in groups AC and the positive control group at different time points after administration (4 hours, 22 hours, and 33 hours after the first administration). 3 mice in each group were sacrificed at each time point for blood collection.

[0150] (2) Liraglutide content in blood samples was detected by LC-MS / MS method

[0151] The quantitative detection method of liraglutide in blood samples was carried out according to the operating procedures reported in the literature (Shiqi Dong, Yuan Gu, Guangli Wei, Duanyun Si, Changxiao Liu, Determination of liraglutide in rat plasma by a selective liquid chromatography tandem mass spectrometry method: Application to a pharmacokinetics study. Chromb (2017), doi:10.1016 / j.jchromb.2018.05.020)

[0152] result:

[0153] 1. Animal efficacy random blood glucose results

[0154] The results of random blood glucose tests showed that the blood glucose of mice in the positive control group decreased significantly within 4 hours after administration and rebounded significantly within 7 hours. After the second administration, the blood glucose level remained below the baseline level until it returned to the baseline level 33 hours later. The blood glucose level of the flash-release tablet group (AC) did not decrease significantly within 7 hours after the first administration, but decreased significantly after the second administration. The magnitude and duration of the decrease were positively correlated with the number of mEV particles per tablet. In addition, the blood glucose level of mice in group A decreased more than that of mice in the positive control group.

[0155] 2. Blood drug concentration results

[0156] The results of animal blood drug concentration showed that the LRT blood concentration of mice in the positive control group peaked 4 hours after the first administration and then gradually decreased. The LRT blood concentration of mice in the AC group peaked after the second administration (within 7-22 hours).

[0157] The above results show that the oral freeze-dried flash-release tablets made of LRT-mEV can dissolve rapidly in the mouse mouth, and LRT can be efficiently absorbed into the blood circulation under the mouse tongue, achieving a hypoglycemic effect.

[0158] 2. Efficacy test method of SMT-mEV freeze-dried flash-release tablets

[0159] Objective: To evaluate the hypoglycemic effect of SMT-mEV freeze-dried orally disintegrating tablets using a randomized blood glucose test in db / db mice and to compare the dosing frequency between LRT-mEV and SMT-mEV.

[0160] method:

[0161] 1. Animal efficacy test 1:

[0162] a. Experiment 1 Grouping

[0163] Table 12

[0164] b. Dosing frequency: oral administration group, once at 0h and 7h, subcutaneous injection group, once at 0h

[0165] c. The blood glucose test and drug administration methods of db / db mice were the same as before.

[0166] 2. Animal efficacy test 2

[0167] a. Experiment 2 Grouping

[0168] Table 13

[0169] b. Dosage frequency: The LRT-mEV group received the drug twice daily from Day 1 to Day 7, while the SMT-mEV group received the drug twice on Day 1 and no further dosing thereafter.

[0170] c. The blood glucose test and drug administration methods of db / db mice were the same as before.

[0171] result:

[0172] 1. Animal efficacy test 1 Random blood glucose results

[0173] Results from Animal Study 1 showed that oral administration of SMT-mEV flash-release tablets to db / db mice significantly decreased blood glucose levels, suggesting that SMT is orally absorbed through the mEVs. Furthermore, the formulation with a high mEV content (Group A) exhibited a more rapid onset of action than the formulation with a low mEV content (Group B), suggesting better absorption.

[0174] 2. Animal efficacy test 2 random blood glucose results

[0175] The results of Animal Experiment 2 showed that SMT, as a second-generation long-acting GLP1 drug, has a sustained efficacy of 5-6 days in db / db mice after the first day of oral administration of SMT-mEV, and its blood sugar-lowering effect is similar to that of the LRT-mEV group administered orally daily.

Claims

1. A freeze-dried orally disintegrating tablet of a GLP1 polypeptide drug, characterized in that: It contains the following raw materials in percentage by weight: 30% to 50% GLP1 polypeptide-mEV stock solution or GLP1 polypeptide-LNP stock solution, 0.1% to 1.0% surfactant, 3% to 5% skeleton agent, 1% to 3% binder, 0.01% to 1% suspending agent, and the balance is water; The surfactant is selected from Poloxamer 188.

2. The GLP1 polypeptide drug freeze-dried orally disintegrating tablet according to claim 1, characterized in that: The skeleton agent is selected from one of mannitol and trehalose; preferably, the skeleton agent is trehalose.

3. The detection method according to claim 1, characterized in that: The adhesive is selected from gelatin and pullulan; preferably, the adhesive is pullulan.

4. The GLP1 polypeptide drug freeze-dried orally disintegrating tablet according to claim 1, characterized in that: The suspending agent is selected from one of xanthan gum and pullulan; preferably, the suspending agent is xanthan gum.

5. The GLP1 polypeptide drug freeze-dried orally disintegrating tablet according to claim 1, characterized in that: The raw materials for preparing the GLP1 polypeptide-mEV stock solution include: adding 0-1.2 mg of auxiliary materials and 1×10 10 ~1×10 13 mEV particles; The raw materials for preparing the GLP1 polypeptide-LNP stock solution include: adding 0 to 3 mg of auxiliary materials and phospholipids, SM-102 and DLin-MC3-DMA per mg of GLP1 polypeptide; The auxiliary material is selected from one of poly-lysine, protamine, poly-arginine, fucose or mannitol. Preferably, the auxiliary material is poly-arginine.

6. The GLP1 polypeptide drug freeze-dried orally disintegrating tablet according to claim 1, characterized in that: The GLP1 polypeptide is liraglutide.

7. The GLP1 polypeptide drug freeze-dried orally disintegrating tablet according to claims 1 to 6, characterized in that: The invention comprises the following raw materials in percentage by mass: 30% to 50% liraglutide-mEV stock solution or liraglutide-LNP stock solution, 0.1% to 1.0% poloxamer 188, 3% to 5% trehalose, 1% to 3% pullulan, 0.01% to 1% xanthan gum, and the balance is water.

8. The GLP1 polypeptide drug freeze-dried orally disintegrating tablet according to claim 1, characterized in that: The GLP1 polypeptide is semaglutide.

9. The GLP1 polypeptide drug freeze-dried orally disintegrating tablet according to claim 1 to 5 or 8, characterized in that: The invention comprises the following raw materials in percentage by mass: 30% to 50% semaglutide-mEV stock solution or semaglutide-LNP stock solution, 0.1% to 1.0% poloxamer 188, 3% to 5% trehalose, 1% to 3% pullulan, 0.01% to 1% xanthan gum, and the balance is water.

10. The method for preparing the freeze-dried orally disintegrating tablet of GLP1 polypeptide drug according to any one of claims 1 to 9, characterized in that: The steps include: (1) Loading the GLP1 polypeptide into mEV to obtain a GLP1 polypeptide-mEV stock solution, or loading the GLP polypeptide into LNP to obtain a GLP1 polypeptide-LNP stock solution; (2) The GLP1 polypeptide-mEV stock solution or the GLP1 polypeptide-LNP stock solution is concentrated by ultrafiltration to obtain a concentrated solution. (3) mixing a surfactant, a skeleton agent, a binder, a suspending agent, a sweetener and a flavoring agent, and adding the concentrate to obtain a drug solution; (4) shearing the liquid medicine in step (3); (5) degassing the liquid medicine after shearing in step (4); (6) filling the degassed liquid medicine in step (5) into the cavity; (7) Pre-freeze the filled liquid medicine; (8) Freeze-dry the pre-frozen drug, setting the initial temperature to -30°C. The specific freeze-drying process is as follows: Cold soaking temperature: ≤-40℃; Vacuum control: ≤200ubar Plate temperature: increased from -30℃ to -25℃, 5 minutes; maintained at -25℃, 60 minutes; increased from -25℃ to -15℃, 10 minutes; maintained at 15℃, 60 minutes; increased from -15℃ to -5℃, 10 minutes; maintained at -5℃, 60 minutes; increased from -5℃ to 10℃, 25 minutes; maintained at 10℃, 100 minutes; increased from 10℃ to 25℃, 15 minutes; maintained at 25℃, 60 minutes.

11. The preparation method according to claim 10, characterized in that: The loading of the GLP1 polypeptide into mEV comprises: The GLP1 peptide solution was mixed with the milk exosome solution and then subjected to ultrasonic treatment. In the GLP1 polypeptide solution, the solvent is 20 mM Tris buffer containing 100 mM NaCl, and the pH value is 8.0; In the milk exosome solution, the solvent is a 20 mM Tris buffer containing 100 mM NaCl, and the pH value is adjusted to 7.0-10.0 with a Na2CO3 / NaHCO3 buffer containing glycine; The ultrasonic treatment conditions include: 99% power, 5° C., and ultrasonic treatment for 12 h.

12. The preparation method according to claim 10, characterized in that: The loading of the GLP1 polypeptide into the LNP comprises: mixing an aqueous solution of GLP1 with an organic solvent of lipid molecules, and self-packing to form an LNP aqueous dispersion containing GLP1.

13. The preparation method according to claim 10, characterized in that: The ultrafiltration concentration of the GLP1 polypeptide-mEV stock solution or the GLP1 polypeptide-LNP stock solution comprises: The GLP1 polypeptide-mEV stock solution or GLP1 polypeptide-LNP stock solution was dissolved in PBS buffer containing PEG3350, and then concentrated on a hollow fiber column with a molecular weight cutoff of 750 kDa at a flow rate of 405 mL / min to a volume of 1 / 20 of the stock solution.

14. The preparation method according to claim 10, characterized in that: The shearing speed is 100-3000 rpm, and the shearing time is 3-20 minutes; preferably, the emulsifier speed is 300 rpm, and the shearing time is 10 minutes.

15. The preparation method according to claim 10, characterized in that: The pre-freezing temperature is -60 to -80°C; preferably, the pre-freezing temperature is -60°C.

16. The preparation method according to claim 10, characterized in that: The pre-freezing time is 10-100 minutes; preferably, the pre-freezing time is 30 minutes.

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