Cannabidiol-type cannabinoid compound freeze-dried flash release tablet and preparation method therefor

Abstract

Provided are a cannabidiol-type cannabinoid compound freeze-dried flash release tablet and a preparation method therefor. The cannabidiol-type cannabinoid compound freeze-dried flash release tablet comprises the following raw materials: 10%-90% by mass of cannabidiol-type cannabinoid compound-milk exosome stock solution, 1%-10% by mass of a skeleton agent, 1%-10% by mass of an adhesive, 0.1%-1% by mass of a surfactant, 1%-10% by mass of a flavoring agent, and the balance of water. The cannabidiol-type cannabinoid compound is one or more of cannabidiol and a cannabidiol in-vivo metabolite. Further provided is a preparation method for the cannabidiol-type cannabinoid compound freeze-dried flash release tablet. The flash release tablet is prepared using a freeze drying technology, and the obtained product has good stability and a good dissolution effect, and is beneficial to oral absorption of cannabidiol. In addition, the preparation method for the flash release tablet is simple, convenient, and easy to implement.

Description

A cannabidiol-type cannabinoid compound freeze-dried flash-release tablet and preparation method thereof Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and in particular relates to a freeze-dried flash-release tablet of a cannabidiol-type cannabinoid compound and a preparation method thereof. Background Art

[0002] Cannabidiol (CBD) is a component extracted from hemp. Hemp is an annual herbaceous plant belonging to the Urticales order, Cannabinaceae family, Cannabis genus, and Cannabis sativa L., class Magnoliopsida. It has important medicinal properties. Hemp is composed of over 130 phytocannabinoids, as well as various terpenoids and flavonoids. The most well-known cannabinoids are cannabidiol and tetrahydrocannabinol (THC).

[0003] Cannabidiol (CBD) is the second most common cannabinoid after tetrahydrocannabinol (THC). It possesses numerous benefits, including significant effects in treating epilepsy and anxiety, with minimal side effects and no addictive potential. Furthermore, in cannabis-infused cosmetics, CBD is a new favorite in the cosmetics market, demonstrating its effectiveness in alleviating skin inflammation, soothing sensitive skin and redness, and building a protective barrier for the skin's surface.

[0004] Cannabidiol is a white to pale yellow crystalline powder that is highly lipophilic and hydrophobic, with extremely poor water solubility and is almost insoluble in water. When cannabidiol is exposed to light, heat, oxygen and other conditions, it undergoes isomerization, aggregation and degradation processes. In addition, its oral bioavailability is low, only 6% to 9%, and it is easily cleared during first-pass metabolism. Due to the polymorphism, poor water solubility, large individual differences in oral administration and stability issues of cannabidiol, it is difficult to maintain its proper pharmacological activity for a long time. To make it into an oral preparation, the key issues that need to be solved include: solidifying the liquid drug to facilitate the design of solid oral preparations; improving drug stability to facilitate long-term storage of preparations; and improving oral bioavailability to reduce the dosage.

[0005] Currently, the FDA-approved cannabidiol-containing pharmaceutical products on the market include Epidiolex oral solution for the treatment of intractable childhood epilepsy and Sativex oral mucosal spray for the treatment of pain associated with multiple sclerosis. Both products have poor bioavailability and require high dosages. Furthermore, the formulation processes used in these products are not conducive to maintaining the structure and content stability of the active ingredient, resulting in poor storage stability of the cannabidiol in existing formulations, which is susceptible to oxidation when exposed to light or air.

[0006] Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide a freeze-dried flash-release tablet of a cannabidiol-type cannabinoid compound with high drug stability, easy to disperse and dissolve evenly in the aqueous phase, high dissolution efficiency, and conducive to oral sublingual absorption, and a preparation method thereof.

[0008] The present invention provides a cannabidiol-type cannabinoid compound freeze-dried flash-release tablet, which comprises the following raw materials in percentage by mass: 10% to 90% cannabidiol-type cannabinoid compound-milk exosome stock solution, 1% to 10% skeleton agent, 1% to 10% binder, 0.1% to 1% surfactant, 1% to 10% flavoring agent, and the balance is water;

[0009] The surfactant is selected from Poloxamer 188.

[0010] Compared with existing dosage forms, it is more advantageous to encapsulate cannabidiol-type cannabinoid compounds in milk extracellular vesicles (mEVs) and make them into freeze-dried flash-release tablets. Cannabidiol is a poorly soluble drug that is almost insoluble in water, soluble in acetonitrile, and highly soluble in organic solvents such as benzene, chloroform, ether, methanol and ethanol. Since the solubility of this type of drug has a great influence on its bioavailability in the body, the solubility of cannabidiol is the limiting factor for its absorption. Therefore, in order to increase the drug loading of cannabidiol-type cannabinoid compound preparations or improve their bioavailability, it is a good choice to modify them into freeze-dried flash-release tablets after mEV encapsulation.

[0011] Freeze-dried flash-release tablets do not need to be taken with water, and saliva can cause them to quickly disintegrate and dissolve in the mouth; cannabidiol absorbed through the gastrointestinal tract is quickly metabolized and inactivated by the liver in the body, but after taking freeze-dried flash-release tablets, a considerable portion of it is absorbed through the oral mucosa, reducing the first-pass effect of the liver, which can effectively prolong the drug's action time and improve bioavailability.

[0012] 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 demonstrated that mEVs can withstand damage from digestive enzymes, low pH, and other environmental factors within the digestive tract, allowing them to be absorbed into the bloodstream. Due to the widespread consumption of milk, long-term exposure to mEVs (through milk consumption) in most populations suggests that oral administration of mEVs is safe and tolerable. Because mEVs are formed by a lipid bilayer membrane, their structure is conducive to the encapsulation of lipophilic small molecule drugs. Cannabidiol encapsulated in mEVs is isolated from air, effectively preventing oxidation of the active ingredient and eliminating the need for antioxidants. Furthermore, upon dissolution in the mouth, mEVs emit a natural milky aroma that effectively masks the bitter taste of the active drug. Therefore, mEV-encapsulated cannabidiol freeze-dried flash-release tablets offer advantages over traditional solid dosage forms in terms of bioavailability, safety, and clinical compliance.

[0013] Furthermore, the skeleton agent is selected from one of mannitol, trehalose, and glycine; preferably, the binder is trehalose.

[0014] Furthermore, the adhesive is selected from one of pullulan and gelatin; preferably, the adhesive is pullulan.

[0015] Furthermore, the weight content of the flavoring agent is preferably 1% to 8%, more preferably 2% to 5%.

[0016] Furthermore, the flavoring agent is selected from one or more of sweet orange flavor, strawberry flavor, mEV, neotame, milk flavor, cherry flavor and orange flavor; preferably, the flavoring agent is one of sweet orange flavor, strawberry flavor, neotame, milk flavor, cherry flavor and orange flavor and mEV; the mass ratio of the flavoring agent to one of sweet orange flavor, strawberry flavor, neotame, milk flavor, cherry flavor and orange flavor and mEV is preferably (0.05-0.2):10, more preferably (0.05-0.15):10, and even more preferably 0.1:10; preferably, the flavoring agent is one of neotame and milk flavor and mEV.

[0017] Furthermore, the raw materials for preparing the cannabidiol type cannabinoid compound-milk exosome stock solution are: 1×10 10 ~1×10 12 Milk exosome particles.

[0018] Since cannabidiol-type cannabinoid compounds will bind to and precipitate with the residual casein in the milk exosome particles during loading, it is necessary to further purify the milk exosomes in a targeted manner to remove the residual casein and ensure the loading efficiency of the cannabidiol-type cannabinoid compounds. Therefore, further, the milk exosome particles are milk exosome particles that have been purified by an anion exchange chromatography column; preferably, the anion exchange chromatography column is Nanogel-50Q.

[0019] Furthermore, the cannabidiol-type cannabinoid compound-milk exosome stock solution is a composition in which the cannabidiol-type cannabinoid compound is encapsulated in milk exosomes; preferably, the cannabidiol-type cannabinoid compound is selected from cannabidiol.

[0020] In some embodiments, the freeze-dried flash-release tablets of the cannabidiol-type cannabinoid compound provided by the present invention contain the following raw materials in percentage by weight: 30% to 70% cannabidiol-type cannabinoid compound-milk exosome stock solution, 3% to 7% skeleton agent, 1% to 5% binder, 0.1% to 0.5% surfactant, 1% to 10% flavoring agent, and the balance is water.

[0021] In some embodiments, the freeze-dried flash-release tablets of the cannabidiol-type cannabinoid compound provided by the present invention contain the following raw materials in percentage by weight: 10% to 90% cannabidiol-type cannabinoid compound-milk exosome stock solution, 1% to 10% trehalose, 1% to 10% pullulan, 0.1% to 1% poloxamer 188, 1% to 10% flavoring agent, and the balance is water.

[0022] In some embodiments, the cannabidiol-type cannabinoid compound freeze-dried flash-release tablets provided by the present invention contain the following raw materials in percentage by weight: 30% to 70% cannabidiol-type cannabinoid compound-milk exosome stock solution, 3% to 7% trehalose, 1% to 5% pullulan, 0.1% to 0.5% poloxamer 188, 1% to 10% flavoring agent, and the balance is water.

[0023] In some embodiments, the cannabidiol-type cannabinoid compound freeze-dried flash-release tablets provided by the present invention contain the following raw materials in percentage by weight: 30% to 70% cannabidiol-type cannabinoid compound-milk exosome stock solution, 3% to 7% trehalose, 1% to 5% pullulan, 0.1% to 0.5% poloxamer 188, 1% to 5% flavoring agent, and the balance is water.

[0024] In some embodiments, the freeze-dried flash-release tablets of cannabidiol-type cannabinoid compounds provided by the present invention contain the following raw materials in percentage by mass: 50% CBD-mEV stock solution, 5% skeleton agent, 2.5% binder, 0.2% surfactant, and the balance is water.

[0025] In some embodiments, the cannabidiol-type cannabinoid compound freeze-dried flash-release tablets provided by the present invention contain the following raw materials in percentage by weight: 50% CBD-mEV stock solution, 5% skeleton agent, 2.5% binder, 0.2% surfactant, 2.5% to 2.55% flavoring agent, and the balance is water.

[0026] In some embodiments, the cannabidiol-type cannabinoid compound freeze-dried flash-release tablets provided by the present invention contain the following raw materials in percentage by mass: 50% CBD-mEV stock solution, 5% trehalose, 2.5% pullulan, 0.2% poloxamer 188, and the balance is water.

[0027] In the present invention, unless otherwise specified, the mass fraction of the raw materials is calculated based on the mass before freeze-drying.

[0028] In the sublingual tablets provided by the present invention, the selection of excipients including surfactants, such as skeleton agents and adhesives, 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 pullulan, trehalose, and poloxamer 188 is more conducive to assisting exosomes to better maintain stability, improve release performance in the oral cavity, and sublingual absorption efficiency. The CBD-mEV stock solution is one or more combinations of active substances (i.e., cannabidiol) encapsulated in exosomes.

[0029] The present invention also provides a method for preparing the aforementioned freeze-dried flash-release tablets of cannabidiol-type cannabinoid compounds, comprising the following steps:

[0030] (1) loading a cannabidiol-type cannabinoid compound into milk exosomes to obtain a cannabidiol-type cannabinoid compound-milk exosome dilution solution;

[0031] (2) The cannabidiol-type cannabinoid compound-milk exosome dilution solution was concentrated by ultrafiltration to obtain a stock solution;

[0032] (3) Mixing the skeleton agent, binder, surfactant and flavoring agent, and adding the stock solution to obtain a drug solution;

[0033] (4) shearing the liquid medicine in step (3);

[0034] (5) Filling the liquid medicine cut in step (4) into the groove;

[0035] (6) Pre-freeze the filled liquid medicine;

[0036] (7) Freeze-dry the pre-frozen drug, setting the initial temperature to -30°C. The specific freeze-drying process is as follows:

[0037] Cold soaking temperature: ≤-60℃; Vacuum control: ≤200ubar;

[0038] Plate temperature: from -30°C to -25°C, 5 minutes; maintain at -25°C, 60 minutes; from -25°C to -15°C, 10 minutes; maintain at -15°C, 120 minutes; from -15°C to -5°C, 10 minutes; maintain at -5°C, 60 minutes; from -5°C to -1°C, 4 minutes; maintain at -1°C, 90 minutes; from -1°C to 10°C, 11 minutes; maintain at 10°C, 60 minutes; from 10°C to 25°C, 15 minutes; maintain at 25°C, 60 minutes.

[0039] Furthermore, the loading of cannabidiol-type cannabinoid compounds into milk exosomes (mEVs) includes:

[0040] mixing the cannabidiol-type cannabinoid compound solution with the milk exosome solution, stirring, and ultrasonicating the mixture;

[0041] In the cannabidiol-type cannabinoid compound solution, the solvent is anhydrous ethanol;

[0042] In the milk exosome solution, the solvent is 1×PBS buffer;

[0043] After the milk exosome solution is mixed with the cannabidiol-type cannabinoid compound solution, the final ethanol concentration does not exceed 50% v / v;

[0044] After the milk exosome solution is mixed with the cannabidiol-type cannabinoid compound solution, it needs to be diluted 10 times with 1×PBS;

[0045] The stirring conditions include: 25° C., 100-200 rpm, stirring for 1 hour;

[0046] The ultrasonic treatment conditions include: 100W to 800W power, 4°C, and ultrasonic treatment for 10 to 120 minutes.

[0047] Furthermore, the loading of cannabidiol-type cannabinoid compounds into milk exosomes (mEVs) includes:

[0048] mixing the cannabidiol-type cannabinoid compound solution with the milk exosome solution, stirring, and ultrasonicating the mixture;

[0049] In the cannabidiol-type cannabinoid compound solution, the solvent is anhydrous ethanol;

[0050] In the milk exosome solution, the solvent is 1×PBS buffer, and further comprises a non-ionic surfactant; preferably, the non-ionic surfactant is Tween 80;

[0051] After the milk exosome solution is mixed with the cannabidiol-type cannabinoid compound solution, the final ethanol concentration does not exceed 50% v / v;

[0052] After the milk exosome solution is mixed with the cannabidiol-type cannabinoid compound solution, it needs to be diluted 10 times with 1×PBS;

[0053] The stirring conditions include: 25° C., 100-200 rpm, stirring for 1 hour;

[0054] The ultrasonic treatment conditions include: 100W to 800W power, 4°C, and ultrasonic treatment for 10 to 120 minutes.

[0055] Furthermore, the milk exosome solution is prepared according to the following method:

[0056] Step 1, pre-treatment;

[0057] Step 2, preliminary purification and concentration;

[0058] Step 3, purification;

[0059] Step 4, purification treatment by anion exchange chromatography column;

[0060] The pre-treatment includes removing fat and / or removing protein;

[0061] The preliminary purification and concentration are carried out by tangential flow ultrafiltration;

[0062] The purification includes one or a combination of two or more of the following: CIMmultus QA column purification, Capto Core 700 column purification, and S400 molecular sieve chromatography purification.

[0063] Furthermore, steps 1, 2, and 3 in the preparation of the milk exosome solution are performed according to Chinese patent publication number CN114790439A.

[0064] Furthermore, in the preparation of the milk exosome solution, the eluate of the anion exchange chromatography column purification treatment in step 4 is a buffer solution containing sodium chloride; the concentration of sodium chloride in the buffer solution containing sodium chloride is 1.5 to 2.5 mol / L; preferably, the eluate of the anion exchange chromatography column purification treatment is a PBS buffer solution containing 2 mol / L sodium chloride.

[0065] Furthermore, the equilibrium solution purified by the anion exchange chromatography column is a buffer solution; preferably, the equilibrium solution purified by the anion exchange chromatography column is a PBS buffer solution.

[0066] Furthermore, the CIP cleaning solution for the anion exchange chromatography column purification treatment is preferably a 0.5-2 mol / L NaOH aqueous solution, more preferably a 0.8-1.5 mol / L NaOH aqueous solution, and even more preferably a 1 mol / L NaOH aqueous solution.

[0067] Furthermore, the operation of removing free cannabidiol-type cannabinoid compounds after the cannabidiol-type cannabinoid compound-milk exosome loading includes:

[0068] The loaded sample was filtered or centrifuged at 16,000 g to remove milk exosome (mEV) debris and undissolved cannabidiol-type cannabinoid compounds, and the supernatant was retained.

[0069] Furthermore, the ultrafiltration concentration of the cannabidiol-type cannabinoid compound-milk exosome dilution comprises:

[0070] After diluting the cannabidiol-type cannabinoid compound-milk exosome dilution with 1×PBS buffer containing PEG3350, the dilution was concentrated to a volume of 1 / 5 to 1 / 10 of the dilution using a hollow fiber column with a molecular weight cutoff of 750kDa (flow rate of 405mL / min). Experiments have shown that ultrafiltration concentration in this step has an important positive significance for increasing the drug loading capacity. The present invention optimizes the ultrafiltration concentration parameters to obtain the optimal effect, thereby further improving the loading effect.

[0071] Further, the skeleton agent, the binder, the surfactant and the flavoring agent are mixed and added to the stock solution to obtain a drug solution;

[0072] Furthermore, the skeleton agent, the binder and the surfactant are mixed, and the stock solution and the flavoring agent are added to obtain a liquid medicine;

[0073] Furthermore, the skeleton agent, binder and surfactant are mixed, part of the water is added and stirred until dissolved, the stock solution and the flavoring agent are added, and the remaining water is added to make up the volume to obtain the medicinal solution; preferably, the volume of the part of water is preferably 80% to 90% of the volume of water in the raw materials.

[0074] Furthermore, the liquid is sheared in the emulsifier;

[0075] Furthermore, the sheared liquid is degassed in an emulsifier or a degassing bottle.

[0076] Furthermore, the shearing speed is 100-3000 rpm, and the shearing time is 3-20 minutes; preferably, the shearing speed is 1000-3000 rpm, and the shearing time is 5-20 minutes; preferably, the shearing speed is 2000-3000 rpm, and the shearing time is 5-15 minutes; preferably, the emulsifier speed is 2500 rpm, and the shearing time is 10 minutes.

[0077] Furthermore, the pre-freezing temperature is -60°C to -80°C; preferably, the pre-freezing temperature is -60°C.

[0078] Furthermore, the pre-freezing time is 10 to 100 minutes; preferably, the pre-freezing time is 15 minutes.

[0079] The present invention provides a freeze-dried, flash-release tablet containing a cannabidiol-type cannabinoid compound encapsulated in milk exosomes (mEVs). The tablet is prepared using freeze-drying technology, resulting in excellent product stability, dissolution, and sublingual absorption efficiency. The preparation method for the tablet is simple and easy to implement. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 is a Nanogel-50Q purification chromatogram;

[0081] Figure 2 shows SDS-PAGE before and after Nanogel-50Q purification;

[0082] Figure 3 shows the precipitation phenomenon of mEVs loaded with CBD prepared by two methods;

[0083] Figure 4 shows the electron microscopy results of mEV without additives;

[0084] Figure 5 shows the electron microscopy results of mEVs in the presence of 0.5% PEG3350 additive;

[0085] Figure 6 shows the electron microscopy results of mEVs in the presence of 5% PEG3350 additive;

[0086] FIG7 shows the protective effect of CBD-mEV on glutamate-induced apoptosis in primary rat neurons;

[0087] Figure 8 is a full view of the CBD-mEV oral flash-release tablet;

[0088] Figure 9 is a cross-sectional view of a CBD-mEV oral flash-release tablet;

[0089] Figure 10 shows the blood concentration-time curve of CBD-mEV oral flash-release tablets for beagle dogs. DETAILED DESCRIPTION

[0090] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0091] Example 1 Preparation of mEV High-Concentration Stock Solution Loaded with Cannabidiol

[0092] Objective: After loading cannabidiol (CBD) into mEVs, the mEV solution was concentrated using tangential flow ultrafiltration technology while maintaining the stability of mEV particle number and structure.

[0093] Materials: Milk exosomes (mEV) and CBD powder

[0094] method:

[0095] 1. mEV preparation (two preparation methods)

[0096] (1) Prepared with reference to Example 12 in Patent ZL202110097550.X (non-purified).

[0097] (2) The mEVs prepared by method (1) were further purified by Nanogel-50Q, using PBS as the equilibration solution, PBS + 2M NaCl as the eluent, and 1M NaOH as the CIP cleaning solution.

[0098] Figure 1 is a chromatogram of Nanogel-50Q purification.

[0099] Figure 2 shows SDS-PAGE before and after Nanogel-50Q purification.

[0100] Results: Most of the casein was adsorbed on the Nanogel-50Q column and then eluted by high salt. mEVs could not be adsorbed by Nanogel-50Q. The flow-through sample finally collected during the flow-through was the purified mEVs.

[0101] 2. CBD loading:

[0102] 2.1 Comparison of the loading efficiency of two mEVs

[0103] a. Dissolution of CBD powder: Weigh 3 portions of CBD powder (20 mg each) and dissolve them in 6.0 mL of anhydrous ethanol.

[0104] b. CBD solution was added to mEVs prepared by the two methods described above (mEV solutions containing 6.0E+12p particles were diluted to a final volume of 14 mL with 1× PBS) to a final ethanol concentration of 30%. An equal volume of PBS was added with CBD solution as a control group. After mixing, the mixture was sonicated at 300W for 20 minutes and centrifuged at 16,000 g for 40 minutes. The supernatant and precipitate (the precipitate was reconstituted with anhydrous ethanol) were collected and quantified for CBD. The results are shown in Table 1.

[0105] Figure 3 shows the precipitation phenomenon of mEVs prepared by the two methods after loading with CBD.

[0106] Table 1: CBD quantitative results

[0107] Results: The impure mEV group produced a large amount of precipitate after loading, with most of the CBD in the precipitate, while the 50Q purified mEV group produced very little precipitate. Therefore, impure mEVs are not suitable for loading CBD.

[0108] 2.2 Nanogel-50Q pure mEV loaded with CBD

[0109] (1) Additive-free chemical loading method

[0110] A) CBD powder dissolution: 20 mg of CBD powder was weighed for each sample and dissolved in 6.0 mL of anhydrous ethanol. The CBD concentration was 3.33 mg / mL.

[0111] B) Dilute the anhydrous ethanol solution with 1× PBS to a 20% v / v ethanol concentration. Add the 20% v / v ethanol solution to the prepared CBD solution and mix thoroughly. Add the ethanol-CBD mixture to the mEV solution containing 6.0E+12 particles to a final volume of 62 mL. This results in a final mEV concentration of 9.7E+10 p / mL, a final ethanol concentration of 23.2% v / v, and a final CBD concentration of 0.32 mg / mL. This yields a set of CBD-mEV solution samples, labeled A.

[0112] Alternatively, take the mEV solution containing 6.0E+12p particles and dilute it to a final volume of 14 mL with 1× PBS. Add the mEV dilution to the prepared CBD solution to a final volume of 20 mL. At this time, the final mEV concentration is 3.0E+11p / mL, the final ethanol concentration is 30% v / v, and the final CBD concentration is 1.0 mg / mL. Mix well to obtain a group of CBD-mEV solution samples marked as B.

[0113] C) Simultaneously, prepare a mEV blank control without CBD and a CBD blank control without mEV using the same procedure.

[0114] D) After the sample preparation is completed, the sample is placed in an ultrasonic dissolution device, diluted 10-fold with 1× PBS, set at 25°C, 200 rpm, stirred for 1 hour, and then ultrasonicated at 4°C according to the conditions in Table 2.

[0115] Table 1 Ultrasonic power and time

[0116] D) After sonication, the sample was filtered or centrifuged at 16,000 g for 40 min to remove mEV fragments and undissolved CBD, and the supernatant was retained.

[0117] (2) Additive-assisted chemical loading method

[0118] A) Dissolution of Cannabidiol (CBD) Powder: 20 mg of CBD powder was weighed and completely dissolved in 6.0 mL of anhydrous ethanol to a CBD concentration of 3.33 mg / mL.

[0119] B) Preparation of additive-mEV dilution solution: Take the mEV solution containing 6.0E+12 particles, add Tween 80 solution and dilute to 14 mL with 1× PBS.

[0120] C) The additive-mEV dilution was added to the prepared CBD solution (solvent: anhydrous ethanol) to a final volume of 20 mL and mixed. The final concentration of the mEV in the sample was 3.0E+11 pM / mL, the final concentration of CBD was 1.0 mg / mL, the final concentration of ethanol was 30% v / v, and the final concentration of Tween 80 was 5% w / v. This yielded one set of CBD-mEV solution samples, labeled C.

[0121] D) Simultaneously, prepare a mEV blank control without CBD and a CBD blank control without mEV using the same procedure.

[0122] E) After the above sample preparation is completed, the sample is placed in an ultrasonic dissolution device, diluted 10 times with 1× PBS, set at 25°C, 200 rpm, stirred for 1 hour, and then continuously ultrasonicated at a power of 300W and 4°C for 20 minutes.

[0123] F) After sonication, the sample was filtered or centrifuged at 16,000 g for 40 min to remove mEV fragments and undissolved CBD, and the supernatant was retained.

[0124] 3. Hollow fiber column tangential flow ultrafiltration concentration:

[0125] (1) Pre-experimental treatment:

[0126] Preparation of 10% m / v PEG3350: Take 60 mL of 50% PEG3350 stock solution and dilute to a final volume of 30 mL with water.

[0127] The CBD-mEV dilution sample (sample A prepared by the additive-free chemical loading method) (200 mL, particle concentration 3.0E+12 p / mL) loaded above was divided into three portions:

[0128] One portion, designated as 0% PEG3350-EV, was prepared by taking 65 mL of the CBD-mEV dilution and adding 1× PBS to a final volume of 200 mL.

[0129] One portion, designated 0.5% PEG3350-EV, was prepared by adding 10 mL of 10% m / v PEG3350 to 65 mL of CBD-mEV dilution and then adjusting the volume to a final volume of 200 mL with 1× PBS. The final concentration of PEG3350 was 0.5%.

[0130] One portion was named 5% PEG3350-EV. 65 mL of CBD-mEV dilution was added to 100 mL of 10% m / v PEG3350 and then fixed to a final volume of 200 mL with 1× PBS. The final concentration of PEG3350 was 5%.

[0131] (2) Hollow fiber column tangential flow ultrafiltration concentration:

[0132] A) Cleaning of Cytiva AKTA FLUX: The hollow fiber column (MWCO = 750 kDa) was cleaned with purified water at a flow rate of 405 mL / min until the pH of the filtrate was approximately 7.4.

[0133] B) Start concentration at a flow rate of 405 mL / min.

[0134] C) Concentrate to 20 mL.

[0135] D) Add 100 mL of 1× PBS buffer containing PEG3350 each time and concentrate and filter 10 times.

[0136] E) Concentrate to 20 mL and collect the concentrate.

[0137] 4. Particle detection:

[0138] The particle number of the obtained CBD-mEV sample was detected using Xiamen Fuliu nanoFCM nanoflow cytometer.

[0139] 5. CBD testing:

[0140] The CBD-mEV stock solution was added to a final concentration of 80% (v / v) ethanol, mixed at room temperature for 30 minutes, and centrifuged at 12,000 g for 10 minutes. The supernatant was collected and filtered through a 0.22 μm filter. The supernatant was analyzed by high-performance liquid chromatography (HPLC). The column was an Agilent ZORBAX Extend-C18; the mobile phase A was 0.1% formic acid in water; the mobile phase B was 0.1% formic acid in acetonitrile. The detection wavelength was 273 nm. The CBD concentration in the sample was calculated using the area normalization algorithm and reference to a standard.

[0141] result:

[0142] 1. Loading:

[0143] The CBD loading capacity of mEVs using the same loading method is shown in Table 3. Comparison of the CBD loading capacity per mEV particle using different loading methods reveals that the single-particle loading efficiencies of samples in groups A and B (chemical loading without additives) are similar. When the ultrasonic power is 300 W and the ultrasonic time is 20 min, a single mEV particle can be loaded with a maximum of approximately 6,800,000 CBD molecules.

[0144] The single-particle loading capacity of mEVs on CBD in group C samples (additive-assisted chemical loading method) was higher than that in group A / B samples (chemical loading method without additives). The single-particle loading capacity of mEVs on CBD in group A. / B loaded samples was one order of magnitude lower than that in group C method, and the loading effect was relatively poor.

[0145] Table 3 Comparison of mEV loading capacity of CBD under different loading methods

[0146] 2. Results of tangential flow ultrafiltration using hollow fiber columns

[0147] Figure 4 is an electron micrograph of mEV without additive; Figure 5 is an electron micrograph of mEV in the presence of 0.5% PEG3350 additive; Figure 6 is an electron micrograph of mEV in the presence of 5% PEG3350 additive.

[0148] Table 4 shows the results of concentration of CBD-mEV dilutions at different PEG3350 concentrations. As shown in Table 4, the concentration of the CBD-mEV dilution before concentration was 3.0E+10p / mL, and the total particle count was 6.0E+12p. During tangential flow ultrafiltration concentration on a hollow fiber column, the total particle count after concentration was more than two-fold higher in the presence of 0.5% and 5% PEG3350 compared to 0% PEG3350. The highest particle count reached 3.54E+12 at a concentration of 1.77E+12p / mL in the presence of 5% PEG3350. The total particle count accounted for 59.1% of the total particle count before concentration, representing an approximately six-fold increase in concentration. Electron microscopy revealed that mEV dispersion was improved in the presence of 5% PEG3350.

[0149] Table 4 Concentration effect of CBD-mEV at different concentrations of PEG3350

[0150] Example 2: Investigating the protective effect of CBD-mEV on primary rat cortical neurons

[0151] Objective: To evaluate the protective effect of cannabidiol (CBD)-mEV against glutamate-induced apoptosis in rat primary cortical neurons.

[0152] Test method:

[0153] Rat primary cortical neurons (RPCN), pre-coated with PDL and cultured to day 10, were seeded into 96-well plates at 15,000 cells per well and incubated at 37°C in 5% CO2 for approximately 36 hours. The following stimuli were added:

[0154] a. The test groups were grouped according to Table 5 (sample C prepared by the additive-assisted chemical loading method in Example 1).

[0155] Table 5 Grouping criteria

[0156] b. After 20 hours of treatment, the supernatant was collected and the amount of LDH released was detected. The results are shown in Tables 6 and 7. The protective effect of CBD-mEV on primary rat neuronal apoptosis induced by glutamate was obtained. The results are shown in Figure 7, where the left figure shows the signal detection of LDH release by CBD-mEV in different stimulation groups, and the right figure shows the toxicity detection of CBD-mEV in different stimulation groups.

[0157] result:

[0158] The results of LDH release showed that compared with blank mEVs, CBD-loaded mEVs could significantly reduce the release of LDH from RPCN induced by glutamate, and the protective effect showed a significant dose-dependent effect. 8 The protective effect of CBD-loaded mEVs was similar to that of 10 μM CBD.

[0159] Table 6 Signal detection of LDH release by CBD-mEV in different stimulation groups

[0160] Table 7 Toxicity test of CBD-mEV in different stimulation groups

[0161] Example 3: Preparation of CBD-mEV freeze-dried flash-release tablets

[0162] 1. Preparation of CBD-mEV freeze-dried flash-release tablets

[0163] Objective: To prepare CBD-mEV freeze-dried flash-release tablets using cannabidiol (CBD) loaded into mEVs according to the formulation in Table 8. The CBD-mEV stock solution was prepared by concentrating sample A with 5% PEG3350 according to the non-additive loading method in Example 1.

[0164] Table 8 CBD-mEV freeze-dried flash-release tablets prescription (calculated based on the mass before freeze-drying)

[0165] (1) Pullulan, trehalose, and poloxamer 188 were weighed and dry-mixed for 5 min. 2-8°C pure water (approximately 90% of the total water volume) was weighed and stirred until completely dissolved to obtain an excipient solution. CBD-mEV stock solution (two concentrations, containing 3E+12 p / mL mEV and 10 mg / mL CBD or 1.5E+13 p / mL mEV and 50 mg / mL CBD) was weighed according to the prescription and added to the excipient solution. The prescribed amount of mEV was further weighed and added to the inclusion solution, and the remaining water was added to the volume. The mixture was stirred at 400 rpm for 5 min until uniformly mixed.

[0166] (2) Shearing: Use an emulsifier for shearing, speed: 280 rpm, time: 10 min.

[0167] (3) Filling: Use a pipette to fill into a 0.4 ml aluminum container, 0.4 g / tablet.

[0168] (4) 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.

[0169] (6) Freeze-drying process: transfer the pre-frozen sample to 1.7m 2 Freeze-drying was performed in a freeze dryer. The freeze-drying procedure is shown in Table 9:

[0170] Inlet temperature: -30℃; Cold trap temperature: ≤-40℃

[0171] Table 9 Freeze-drying procedure

[0172] Table 10 Evaluation results

[0173] The full view and cross-sectional view of the film are shown in Figures 8 and 9.

[0174] 2. Screening of flavoring agents for CBD-mEV freeze-dried flash-release tablets

[0175] Purpose: To add flavoring agents to the basic formula to optimize the taste. The formula is shown in Table 11, where the CBD-mEV stock solution was prepared by the non-additive loading method in Example 1 and then concentrated with 5% PEG3350.

[0176] Table 11 CBD-mEV freeze-dried flash-release tablets prescription (calculated based on the mass before freeze-drying)

[0177] (1) Pullulan, trehalose, poloxamer 188, neotame or milk flavor were weighed and dry mixed for 5 min; 2-8°C pure water (about 90% of the total water volume) was weighed and stirred until all dissolved to obtain an excipient solution; CBD-mEV stock solution (two concentrations, containing 3E+12p / mL mEV and 10mg / mL CBD or 1.5E+13p / mL mEV and 50mg / mL CBD) was weighed according to the prescription and added to the excipient solution, and the prescribed amount of mEV was further weighed and added to the inclusion solution, and the remaining water was added to the volume. Stirring was continued at 400 rpm for 5 min until the mixture was uniform.

[0178] (2) Shearing: Use an emulsifier for shearing, speed: 280 rpm, time: 10 min.

[0179] (3) Filling: Use a pipette to fill into a 0.4 ml aluminum container, 0.4 g / tablet.

[0180] (4) 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.

[0181] (6) Freeze-drying process: transfer the pre-frozen sample to 1.7m 2 Freeze-drying was performed in a freeze dryer. The freeze-drying procedure is shown in Table 12:

[0182] Inlet temperature: -30℃; Cold trap temperature: ≤-40℃

[0183] Table 12 Freeze-drying procedure

[0184] Table 13 Evaluation results

[0185] 3. Screening of flavoring agent dosage for CBD-mEV freeze-dried flash-release tablets

[0186] Objective: To optimize the dosage of the flavoring agent neotame. The prescription is shown in Table 14.

[0187] Table 14. Prescription of CBD-mEV freeze-dried flash-release tablets (calculated based on the mass before freeze-drying)

[0188] (1) Pullulan, trehalose, poloxamer 188, and neotame were weighed and dry-mixed for 5 min. 2-8°C pure water (approximately 90% of the total water volume) was weighed and stirred until all dissolved to obtain an excipient solution. CBD-mEV stock solution (two concentrations, containing 3E+12 p / mL mEV and 10 mg / mL CBD or 1.5E+13 p / mL mEV and 50 mg / mL CBD) was weighed according to the prescription and added to the excipient solution. The prescribed amount of mEV was further weighed and added to the inclusion solution. The remaining water was added to the volume and the mixture was stirred at 400 rpm for 5 min until the mixture was uniformly mixed.

[0189] (2) Shearing: Use an emulsifier for shearing, speed: 280 rpm, time: 10 min.

[0190] (3) Filling: Use a pipette to fill into a 0.4 ml aluminum container, 0.4 g / tablet.

[0191] (4) 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.

[0192] (6) Freeze-drying process: transfer the pre-frozen sample to 1.7m 2 Freeze-drying was performed in a freeze dryer. The freeze-drying procedure is shown in Table 15:

[0193] Inlet temperature: -30℃; Cold trap temperature: ≤-40℃

[0194] Table 15 Freeze-drying procedure

[0195] Table 16 Evaluation results

[0196] IV. 6-month stability evaluation of the active ingredient, as shown in Table 17.

[0197] Table 17 Stability evaluation results

[0198] Store the flash-release tablets at 25°C away from light.

[0199] Example 4: Animal Pharmacokinetic Study of CBD-mEV Freeze-Dried Flash-Release Tablets

[0200] Objective: To evaluate the changes in plasma concentration and exposure of CBD-mEV freeze-dried flash-release tablets after oral administration in beagle dogs.

[0201] Test method:

[0202] a. Experimental groups. The grouping criteria are shown in Table 12.

[0203] Table 11 Test grouping criteria

[0204] b. Administration method: Sublingual administration

[0205] c. Blood collection time: Blood was collected at 0.5h, 1h, 2h, 4h, 8h, 12h, and 24h after administration.

[0206] d. Detection: Blood samples were tested for CBD concentration using LC-MS / MS.

[0207] result:

[0208] The blood drug concentration curve after beagle dogs orally absorbed CBD-mEV freeze-dried flash-release tablets was shown in Figure 10, and the blood drug concentration change results after beagle dogs orally absorbed CBD-mEV freeze-dried flash-release tablets were shown in Table 12.

[0209] Table 12 Changes in blood drug concentrations after oral administration of CBD-mEV freeze-dried flash-release tablets in beagle dogs

[0210] BLQ: below the detection limit 1ng / ml

[0211] The results of blood drug concentration after beagle dogs orally absorbed CBD-mEV freeze-dried flash-release tablets showed that compared with the negative control Group B without mEV, the oral bioavailability of CBD-mEV flash-release tablets was significantly improved, the time to peak blood drug concentration was significantly delayed, and the low-dose flash-release tablets had better pharmacokinetic properties, which could significantly reach or even exceed the blood drug concentration that could be achieved by the original traditional oral preparation.

[0212] Example 5: Taste Evaluation of CBD-mEV Freeze-Dried Flash-Release Tablets

[0213] Objective: To evaluate the effect of free cannabidiol on the taste and mouthfeel of CBD-mEV flash-release tablets.

[0214] Test method:

[0215] Ten volunteers were recruited and administered oral doses of freeze-dried flash-release tablets on the first and fifth days. Feedback was recorded regarding the tablet's mouthfeel, flavor, and disintegration and absorption rate. Two types of CBD-mEV freeze-dried flash-release tablets were tested: the first was prepared directly after loading, and the second was prepared after loading and then subjected to chromatography to remove free CBD. Each volunteer was unaware of the type of freeze-dried flash-release tablets dispensed, and the order of tablet administration was randomized: one of the two freeze-dried flash-release tablets was administered on the first day, and the other on the fifth day.

[0216] result:

[0217] Nine out of ten volunteers thought the first freeze-dried flash-release tablet tasted significantly more bitter and caused stronger irritation to the throat mucosa, while the second freeze-dried flash-release tablet had a significantly less bitter taste and caused only a brief and weak irritation to the throat mucosa.

[0218] The above results show that the CBD-mEV freeze-dried flash-release tablets with free CBD removed are superior to the CBD-mEV freeze-dried flash-release tablets with no free CBD removed in terms of taste.

[0219] The preferred embodiments of the present invention disclosed above are merely intended to help illustrate the present invention, but the present invention is not limited thereto. Those skilled in the art will appreciate that, within the scope of the technical concept of the present invention, the technical solutions of the present invention may be modified, or some of the technical features may be combined in any other manner. Such modifications or combinations do not deviate from the essence of the corresponding technical solutions from the spirit and scope of the various technical solutions of the present invention, and should be regarded as the contents disclosed by the present invention and fall within the scope of protection of the present invention.

Claims

1. A freeze-dried flash-release tablet of a cannabidiol-type cannabinoid compound, characterized in that: It includes the following raw materials in percentage by mass: 10% to 90% cannabidiol-type cannabinoid compound-milk exosome stock solution, 1% to 10% skeleton agent, 1% to 10% binder, 0.1% to 1% surfactant, 1% to 10% flavoring agent, and the balance is water; The surfactant is selected from Poloxamer 188.

2. The freeze-dried flash-release tablet of cannabidiol type cannabinoid compound according to claim 1, characterized in that The skeleton agent is selected from one of mannitol, trehalose and glycine; preferably, the skeleton agent is trehalose.

3. The freeze-dried flash-release tablet of cannabidiol type cannabinoid compound according to claim 1, characterized in that The adhesive is selected from one of pullulan and gelatin; preferably, the adhesive is pullulan.

4. The freeze-dried flash-release tablet of cannabidiol type cannabinoid compound according to claim 1, characterized in that The flavoring agent is selected from one of sweet orange flavor, strawberry flavor, milk exosomes, cherry flavor and orange flavor; preferably, the flavoring agent is milk exosomes.

5. The freeze-dried flash-release tablet of cannabidiol type cannabinoid compound according to claim 1, characterized in that The raw materials for preparing the cannabidiol type cannabinoid compound-milk exosome stock solution include: adding 1×10 10 ~1×10 12 Milk exosome particles.

6. The freeze-dried flash-release tablet of cannabidiol type cannabinoid compound according to claim 5, characterized in that The milk exosome particles are milk exosome particles that have been purified by an anion exchange chromatography column; preferably, the anion exchange chromatography column is Nanogel-50Q.

7. The freeze-dried flash-release tablet of cannabidiol type cannabinoid compound according to claim 1, characterized in that The cannabidiol-type cannabinoid compound-milk exosome stock solution is a composition in which the cannabidiol-type cannabinoid compound is encapsulated in milk exosomes; preferably, the cannabidiol-type cannabinoid compound is selected from one or more of cannabidiol and cannabidiol's in vivo metabolites.

8. The freeze-dried flash-release tablet of cannabidiol type cannabinoid compound according to claim 1, characterized in that It includes the following raw materials in percentage by mass: 10% to 90% cannabidiol type cannabinoid compound-milk exosome stock solution, 1% to 10% trehalose, 1% to 10% pullulan, 0.1% to 1% poloxamer 188, 1% to 10% flavoring agent, and the balance is water.

9. The method for preparing the freeze-dried flash-release tablet of cannabidiol type cannabinoid compound according to any one of claims 1 to 8, characterized in that: The steps include: (1) loading a cannabidiol-type cannabinoid compound into milk exosomes to obtain a cannabidiol-type cannabinoid compound-milk exosome dilution solution; (2) The cannabidiol-type cannabinoid compound-milk exosome dilution solution was concentrated by ultrafiltration to obtain a stock solution; (3) Mixing the skeleton agent, binder, surfactant and flavoring agent, and adding the stock solution to obtain a drug solution; (4) shearing the liquid medicine in step (3); (5) Filling the liquid medicine cut in step (4) into the groove; (6) Pre-freeze the filled liquid medicine; (7) Freeze-dry the pre-frozen drug, setting the initial temperature to -30°C. The specific freeze-drying process is as follows: Cold soaking temperature: ≤-60℃; Vacuum control: ≤200ubar; Plate temperature: increased from -30°C to -25°C for 5 minutes; maintained at -25°C for 60 minutes; increased from -25°C to -15°C for 10 minutes; maintained at -15°C for 120 minutes; increased from -15°C to -5°C for 10 minutes; maintained at -5°C for 60 minutes; increased from -5°C to -1°C for 4 minutes; maintained at -1°C for 90 minutes; increased from -1°C to 10°C for 11 minutes; maintained at 10°C for 60 minutes; increased from 10°C to 25°C for 15 minutes; maintained at 25°C for 60 minutes.

10. The preparation method according to claim 9, characterized in that The loading of the cannabidiol-type cannabinoid compound into milk exosomes comprises: mixing the cannabidiol-type cannabinoid compound solution with the milk exosome solution, stirring, and ultrasonicating the mixture; In the cannabidiol-type cannabinoid compound solution, the solvent is anhydrous ethanol; In the milk exosome solution, the solvent is 1×PBS buffer; After the milk exosome solution is mixed with the cannabidiol-type cannabinoid compound solution, the final ethanol concentration does not exceed 50% v / v; After the milk exosome solution is mixed with the cannabidiol-type cannabinoid compound solution, it needs to be diluted 10 times with 1×PBS; The stirring conditions include: 25° C., 100-200 rpm, stirring for 1 hour; The ultrasonic treatment conditions include: 100W to 800W power, 4°C, and ultrasonic treatment for 10 to 120 minutes.

11. The preparation method according to claim 10, characterized in that: The milk exosome solution was prepared according to the following method: Step 1, pre-treatment; Step 2, preliminary purification and concentration; Step 3, purification; Step 4, purification treatment by anion exchange chromatography column; The pre-treatment includes removing fat and / or removing protein; The preliminary purification and concentration are carried out by tangential flow ultrafiltration; The purification in step 3 includes one or a combination of two or more of the following: CIMmultus QA column purification, Capto Core 700 column purification, and S400 molecular sieve chromatography purification.

12. The preparation method according to claim 11, characterized in that The eluate of the anion exchange chromatography column purification treatment in step 4 is a buffer solution containing sodium chloride; the concentration of sodium chloride in the buffer solution containing sodium chloride is 1.5 to 2.5 mol / L; preferably, the eluate of the anion exchange chromatography column purification treatment is a PBS buffer solution containing 2 mol / L sodium chloride.

13. The preparation method according to claim 9, characterized in that The ultrafiltration concentration of the cannabidiol type cannabinoid compound-milk exosome dilution comprises: The cannabidiol-type cannabinoid compound-milk exosome dilution was dissolved in 1×PBS buffer containing PEG3350, and then applied to a hollow fiber column with a molecular weight cutoff of 750 kDa at a flow rate of 405 mL / min and concentrated to a volume of 1 / 5 to 1 / 10 of the dilution.

14. The preparation method according to claim 9, characterized in that The shearing speed is 100 to 3000 rpm, and the shearing time is 3 to 20 minutes; preferably, the emulsifier speed is 2500 rpm, and the shearing time is 10 minutes.

15. The preparation method according to claim 9, characterized in that The pre-freezing temperature is -60°C to -80°C; preferably, the pre-freezing temperature is -60°C.

16. The preparation method according to claim 9, characterized in that The pre-freezing time is 10 to 100 minutes; preferably, the pre-freezing time is 15 minutes.

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