A stiripentol liposome, its preparation method and application

Abstract

The present invention provides a stiripentol liposome, a preparation method thereof and an application thereof. The stiripentol liposome comprises stiripentol, phospholipid and cholesterol with a mass ratio of (5-15):(50-150):(1-3), and is prepared by a combination of the thin film hydration method and the ultrasonic method. Stiripentol, phospholipid and cholesterol are dissolved in an appropriate amount of ethanol solution, and evaporated under reduced pressure until the ethanol is completely volatilized. An appropriate amount of water is taken for rotary hydration to uniformly disperse the liposome in water, and ultrasonic treatment is carried out in an ice bath, followed by filtration to obtain the product. The stiripentol liposome prepared by the present invention has a particle size of 50 nm to 250 nm and an encapsulation efficiency of 50% to 90.0%. It can remain stable in gastric juice and pancreatic juice, can be rapidly dissolved in an aqueous medium, and significantly improves the in vivo bioavailability. The preparation method provided by the present invention has the advantages of simple process, controllable process, better stability, etc., is easy to scale up and industrialize, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a stiripentol liposome, a preparation method thereof and an application thereof. Background Art

[0002] Stiripentol (STP) is a drug that is poorly soluble in water (solubility is about 49.2 μg / ml), and is used for the treatment of severe myoclonic epilepsy in infants and young children with Dravet syndrome. Currently, capsule and dry suspension (two specifications of 250 mg and 500 mg) are available on the foreign market, and are administered 2 - 3 times a day. The recommended maximum total dose is 3000 mg / day. The blood drug concentration reaches the peak (4 - 22 μg / mL) 2 - 3 hours after a single dose, and generally needs to be taken with meals to achieve the best effect. Stiripentol (STP) belongs to BCS class II drugs with low water solubility and high permeability, has a slow and incomplete dissolution in the gastrointestinal tract, and poor stability in an acidic environment. Due to the above limitations of oral absorption, the daily oral dose of this drug is relatively high, severely restricting its further clinical application.

[0003] Therefore, it is of great clinical value to improve the solubility of stiripentol while retaining its therapeutic activity, and to design a new preparation of stiripentol that is safe, effective and convenient to use. Summary of the Invention

[0004] The purpose of the present invention is to provide a stiripentol liposome, a preparation method thereof and an application thereof in view of the above deficiencies of the prior art.

[0005] To achieve the above purpose, the present invention adopts the following technical solutions:

[0006] The first object of the present invention is to provide a stiripentol liposome, which comprises stiripentol, phospholipid and cholesterol, and the mass ratio of stiripentol, phospholipid and cholesterol is (5 - 15):(50 - 150):(1 - 3).

[0007] Further, the mass ratio of stiripentol, phospholipid and cholesterol is (5 - 9):(50 - 75):(1 - 2).

[0008] Further, the phospholipid is selected from soybean phospholipid, egg yolk phospholipid, hydrogenated phospholipid and synthetic phospholipid.

[0009] Further, the phospholipid is selected from phospholipids S45, S75, S100, SPC, E80, EPCS, EPG, SPC - 3, DMPC, DEPC, DPPA, DSPA of Lipoid Company in Germany; phospholipids PC98 - T, PL - 100M, HSPC, PGE, PGSH of Kewpie Corporation in Japan.

[0010] Further, the phospholipid is selected from any one of phospholipid S100, PC98-T, HSPC, and E80.

[0011] Further, the particle size of the stiripentol liposome is 50 nm to 250 nm.

[0012] The second object of the present invention is to provide a method for preparing a stiripentol liposome. The preparation method is to weigh stiripentol, phospholipid, and cholesterol according to a stoichiometric ratio, dissolve them in an appropriate amount of ethanol solution, evaporate under reduced pressure until the ethanol is completely evaporated, take an appropriate amount of water for rotary hydration to uniformly disperse the liposome in water, perform ice bath sonication, and filter to obtain the stiripentol liposome.

[0013] Further, the mass-volume ratio of the stiripentol raw material drug to the ethanol solution is (5 - 15) mg: 100 mL.

[0014] Further, the hydration temperature is 20°C to 60°C, the hydration time is 25 min to 45 min, and the hydration volume is 5 to 25 mL.

[0015] Further, the dispersion temperature is 20°C to 50°C.

[0016] Further, the ice bath sonication power is 65 w to 100 w; the sonication time is 3 min to 10 min.

[0017] The third object of the present invention is to provide an anti-epileptic drug composition prepared from the above-mentioned stiripentol liposome.

[0018] Antiepileptic drugs refer to compositions comprising stiripentol nanocrystals and at least one pharmaceutically acceptable and pharmacologically compatible component selected from the following: fillers, solvents, diluents, carriers, excipients, dispersants and acceptors, delivery agents such as preservatives, stabilizers, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, thickening agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants and extended delivery control agents, the selection and proportion of which depend on the nature and route of administration and the dose. Examples of suitable suspending agents are ethoxylated isostearyl alcohol, polyoxyethylene, sorbitol and sorbitol ethers, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth gum and mixtures thereof. Various antibacterial and antifungal agents such as parabens, chlorobutanol, sorbic acid, etc. can be used to provide protection against microorganisms. Antiepileptic drugs may also include isotonic agents such as sugars, sodium chloride, etc. The sustained action of the composition can be achieved by using reagents that slow down the absorption of the active ingredient, such as aluminum monostearate and gelatin. Examples of suitable carriers, solvents, diluents and delivery agents include water, ethanol, polyols and mixtures thereof, natural oils (such as olive oil) and organic esters for injection (such as ethyl oleate). Examples of fillers are lactose, milk sugar, sodium citrate, calcium carbonate, calcium phosphate, etc. Examples of disintegrants and dispersing agents are starch, alginic acid and its salts and silicates. Examples of lubricants are magnesium stearate, sodium lauryl sulfate, talc and high molecular weight polyethylene glycol. Pharmaceutical compositions for oral, sublingual, transdermal, intramuscular, intravenous, subcutaneous and topical or rectal administration of the active ingredient alone or in combination with another active compound can be administered to animals and humans as a mixture with conventional pharmaceutical carriers in standard dosage forms. Suitable standard dosage forms include oral forms such as tablets, capsules, pills, powders, granules, chewing gums and oral solutions or suspensions; sublingual and buccal administration forms; aerosols; implants; topical, transdermal, subcutaneous, intramuscular, intravenous, intranasal or intraocular forms; and rectal administration forms.

[0019] Compared with the prior art, the beneficial effects brought by the technical solution provided by the present invention are:

[0020] (1) The present invention provides a stiripentol liposome, a preparation method and an application thereof. The stiripentol liposome comprises stiripentol, phospholipid and cholesterol in a mass ratio of (5-15):(50-150):(1-3), and is prepared by a thin film hydration method combined with an ultrasonic method. Stiripentol, phospholipid and cholesterol are dissolved in an appropriate amount of ethanol solution, and evaporated under reduced pressure until the ethanol is completely volatilized. An appropriate amount of water is taken for rotary hydration to uniformly disperse the liposome in water, and ultrasonic treatment is carried out in an ice bath, followed by filtration to obtain the product. The stiripentol liposome prepared by the present invention has a particle size of 50 nm to 250 nm, an encapsulation efficiency of 50% to 90%, can remain stable in gastric juice and pancreatic juice, can be rapidly dissolved in an aqueous medium, and significantly improves the in vivo bioavailability;

[0021] (2) The preparation method provided by the present invention has a simple process, is controllable during the process, and is easy to scale up for industrial production. Description of the Drawings

[0022] Figure 1 It is the TEM image of stiripentol liposome;

[0023] Figure 2 It is the data analysis chart of the hydration layer thickness of stiripentol liposome;

[0024] Figure 3 It is the result chart of the stability study of stiripentol liposome in the stomach and pancreas;

[0025] Figure 4 It is the in vitro dissolution curve of stiripentol nanocrystals, stiripentol physical mixture, and stiripentol raw material drug in water. Detailed Description of the Invention

[0026] For those not specified in the present invention in terms of specific technologies or conditions, they shall be carried out according to the technologies or conditions described in the literature in this field or according to the product specifications. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0027] The reagent information used in this example is as follows:

[0028] The phospholipids S45, S75, S100, SPC, E80, EPCS, EPG, SPC-3, DMPC, DEPC, DPPA, DSPA used in the present invention are provided by Lipoid GmbH, Germany; the phospholipids PC98-T, PL-100M, HSPC, PGE, PGSH are provided by Kabushiki Kaisha Kyubey, Japan, and they are all commonly used commercially available phospholipid products.

[0029] The stiripentol dry suspension used in the present invention is provided by Biocodex and can be obtained commercially.

[0030] The method for characterizing the performance of stiripentol liposome is as follows:

[0031] 1) Determination of Size and PDI

[0032] Using a Malvern particle size analyzer, measure the particle size (Size), dispersion coefficient (PDI), and Zeta potential value of STP liposome. Experimental conditions: The solute is STP, the refractive index is 1.578, the solvent is water, the refractive index is 1.330, the temperature is 25 °C, the equilibrium time is 60 s, and measure 3 times in parallel and calculate the average value to obtain.

[0033] 2) Transmission electron microscopy (TEM)

[0034] The morphology of the STP liposome solution was observed by transmission electron microscopy. A drop of the STP liposome solution was adsorbed on a 400-mesh carbon-coated copper grid, and the excess solution was removed with filter paper. The sample was dried at room temperature and observed and photographed using a transmission electron microscope.

[0035] 3) Encapsulation efficiency

[0036] It was determined by the dialysis method. Treatment method of the dialysis bag: Cut the dialysis bag into equal-length segments, boil the dialysis bag in 500 mL of 2% sodium bicarbonate and 1 mmol / L EDTA·2Na (pH = 8.0) for 10 min, rinse with deionized water, then boil the dialysis bag in 1 mmol / L EDTA·2Na (pH = 8.0) for 10 min and then cool. Store in a 4°C refrigerator. Wear gloves when taking it. After rinsing it clean with deionized water, check whether there is liquid leakage before use. Take an appropriate amount of the liposome mixture and place it in a 12KD - 14KD dialysis bag, clamp both ends with a dialysis clip to prevent liquid leakage. Place the dialysis bag in 200 mL of water, stir at 200 r / min at room temperature on a magnetic stirrer for 12 h. Take 0.5 mL of the dialysis bag sample solution, dilute it 4 times with methanol, ultrasonicate for 30 min to break the emulsion, measure the concentration of the sample solution inside the dialysis bag, and thus calculate the amount of drug Qt encapsulated by the liposome. Take another 0.5 mL of STP liposome and calculate the total amount of drug Q of the liposome in the same way. 0 。

[0037] Encapsulation efficiency: W = Qt / Q0 * 100%.

[0038] 4) Investigation of the hydration layer thickness

[0039] The change in the Zeta potential of STP liposomes in NaCl solutions with different concentrations (0.1, 0.5, 1, 2, 5, 10, and 20 mM) was measured by a Malvern particle size analyzer to determine the hydration layer thickness in the liposomes. According to the Gouy-Chapman theory, the calculation formula for the hydration layer thickness is as follows:

[0040]

[0041] where ξ is the absolute value of the Zeta potential of each sample (V), A is a constant, K is the Debye-Huckel parameter, c is the concentration of the NaCl solution (M), and l is the hydration layer thickness (nm).

[0042] To make the objectives, technical solutions, and advantages of the present invention clearer, the following further describes in detail the specific implementation manners of the present invention in combination with specific embodiments and the accompanying drawings.

[0043] STP: Stiripentol.

[0044] PM: Physical mixture of stiripentol.

[0045] Example 1

[0046] Preparation of stiripentol liposomes

[0047] Weigh 5 mg of stiripentol, 50 mg of phospholipid S100 and 1 mg of cholesterol, dissolve them in 100 mL of ethanol solution, evaporate under reduced pressure until the ethanol is completely evaporated, take 5 mL of water for rotary hydration, the hydration temperature is 30 °C, and the hydration time is 30 min, so that the liposomes are evenly dispersed in water at 30 °C. The ice bath ultrasonic power is 90 w, and the ultrasonic time is 5 min. Filter through a 0.22 μm filter membrane to obtain stiripentol liposomes. The prepared stiripentol liposomes have a particle size of 76.8 nm, a PDI of 0.237, and an encapsulation efficiency of 85.7%.

[0048] Example 2

[0049] Preparation of stiripentol liposomes

[0050] Weigh 6 mg of stiripentol, 75 mg of phospholipid S100 and 1.5 mg of cholesterol, dissolve them in 100 mL of ethanol solution, evaporate under reduced pressure until the ethanol is completely evaporated, take 10 mL of water for rotary hydration, the hydration temperature is 30 °C, and the hydration time is 30 min, so that the liposomes are evenly dispersed in water at 30 °C. The ice bath ultrasonic power is 90 w, and the ultrasonic time is 5 min. Filter through a 0.22 μm filter membrane to obtain stiripentol liposomes. The prepared stiripentol liposomes have a particle size of 90.3 nm, a PDI of 0.265, and an encapsulation efficiency of 88.3%.

[0051] Example 3

[0052] Preparation of stiripentol liposomes

[0053] Weigh 9 mg of stiripentol, 100 mg of phospholipid S100 and 2 mg of cholesterol, dissolve them in 100 mL of ethanol solution, evaporate under reduced pressure until the ethanol is completely evaporated, take 10 mL of water for rotary hydration, the hydration temperature is 30 °C, and the hydration time is 30 min, so that the liposomes are evenly dispersed in water at 30 °C. The ice bath ultrasonic power is 90 w, and the ultrasonic time is 5 min. Filter through a 0.22 μm filter membrane to obtain stiripentol liposomes. The prepared stiripentol liposomes have a particle size of 85.9 nm, a PDI of 0.262, and an encapsulation efficiency of 82.8%.

[0054] Example 4

[0055] Preparation of stiripentol liposomes

[0056] Weigh 10 mg of stiripentol, 125 mg of phospholipid S100, and 2.5 mg of cholesterol, dissolve them in 100 mL of ethanol solution, evaporate under reduced pressure until the ethanol is completely evaporated, take 20 mL of water for rotary hydration, the hydration temperature is 30 °C, and the hydration time is 30 min to uniformly disperse the liposomes in water at 30 °C. The ice bath ultrasonic power is 90 w, and the ultrasonic time is 5 min. Filter through a 0.22 μm filter membrane to obtain stiripentol liposomes. The prepared stiripentol liposomes have a particle size of 116.1 nm, a PDI of 0.519, and an encapsulation efficiency of 81.5%.

[0057] Example 5

[0058] Preparation of Stiripentol Liposomes

[0059] Weigh 15 mg of stiripentol, 150 mg of phospholipid S100, and 3 mg of cholesterol, dissolve them in 100 mL of ethanol solution, evaporate under reduced pressure until the ethanol is completely evaporated, take 25 mL of water for rotary hydration, the hydration temperature is 30 °C, and the hydration time is 30 min to uniformly disperse the liposomes in water at 30 °C. The ice bath ultrasonic power is 90 w, and the ultrasonic time is 5 min. Filter through a 0.22 μm filter membrane to obtain stiripentol liposomes. The prepared stiripentol liposomes have a particle size of 122.4 nm, a PDI of 0.473, and an encapsulation efficiency of 76.9%.

[0060] Example 6

[0061] Preparation of Stiripentol Liposomes

[0062] Weigh 7 mg of stiripentol, 100 mg of phospholipid S100, and 1 mg of cholesterol, dissolve them in 100 mL of ethanol solution, evaporate under reduced pressure until the ethanol is completely evaporated, take 15 mL of water for rotary hydration, the hydration temperature is 45 °C, and the hydration time is 45 min to uniformly disperse the liposomes in water at 30 °C. The ice bath ultrasonic power is 65 w, and the ultrasonic time is 3 min. Filter through a 0.22 μm filter membrane to obtain stiripentol liposomes. The prepared stiripentol liposomes have a particle size of 88.4 nm, a PDI of 0.275, and an encapsulation efficiency of 84.2%.

[0063] Example 7

[0064] Preparation of Stiripentol Liposomes

[0065] Weigh 7 mg of stiripentol, 100 mg of phospholipid S100, and 1 mg of cholesterol, dissolve them in 100 mL of ethanol solution, evaporate under reduced pressure until the ethanol is completely evaporated, take 15 mL of water for rotary hydration, the hydration temperature is 45 °C, and the hydration time is 25 min. Then disperse the liposomes evenly in water at 50 °C, the ice bath ultrasonic power is 100 w, and the ultrasonic time is 10 min. Filter through a 0.22-μm filter membrane to obtain stiripentol liposomes. The prepared stiripentol liposomes have a particle size of 94.7 nm, a PDI of 0.247, and an encapsulation efficiency of 81.3%.

[0066] Example 8

[0067] Preparation of Stiripentol Liposomes

[0068] Weigh 7 mg of stiripentol, 100 mg of phospholipid S100, and 1 mg of cholesterol, dissolve them in 100 mL of ethanol solution, evaporate under reduced pressure until the ethanol is completely evaporated, take 15 mL of water for rotary hydration, the hydration temperature is 20 °C, and the hydration time is 30 min. Then disperse the liposomes evenly in water at 20 °C, the ice bath ultrasonic power is 90 w, and the ultrasonic time is 5 min. Filter through a 0.22-μm filter membrane to obtain stiripentol liposomes. The prepared stiripentol liposomes have a particle size of 97.0 nm, a PDI of 0.232, and an encapsulation efficiency of 70.9%.

[0069] Example 9

[0070] Weigh 7 mg of stiripentol, 50 mg of phospholipid S100, and 1.5 mg of cholesterol, dissolve them in 100 mL of ethanol solution, evaporate under reduced pressure until the ethanol is completely evaporated, take 15 mL of water for rotary hydration, the hydration temperature is 30 °C, and the hydration time is 30 min. Then disperse the liposomes evenly in water at 20 °C, the ice bath ultrasonic power is 90 w, and the ultrasonic time is 5 min. Filter through a 0.22-μm filter membrane to obtain stiripentol liposomes. The prepared stiripentol liposomes have a particle size of 83.3 nm, a PDI of 0.258, and an encapsulation efficiency of 73.2%.

[0071] Example 10

[0072] It is basically the same as Example 6, except that phospholipid PC98-T is selected. The prepared stiripentol liposomes have a particle size of 85.4 nm, a PDI of 0.261, and an encapsulation efficiency of 77.4%.

[0073] Example 11

[0074] It is basically the same as Example 6, except that phospholipid HSPC is selected. The prepared stiripentol liposomes have a particle size of 87.8 nm, a PDI of 0.277, and an encapsulation efficiency of 79.0%.

[0075] Example 12

[0076] It is basically the same as Example 6, except that phospholipid E80 is selected. The prepared stiripentol liposome has a particle size of 90.6 nm, a PDI of 0.259, and an encapsulation efficiency of 84.7%.

[0077] Example 13

[0078] It is basically the same as Example 6, except that phospholipid DMPC is selected. The prepared stiripentol liposome has a particle size of 54.6 nm, a PDI of 0.559, and an encapsulation efficiency of 67.3%.

[0079] Example 14

[0080] It is basically the same as Example 6, except that phospholipid DSPA is selected. The prepared stiripentol liposome has a particle size of 232.6 nm, a PDI of 0.417, and an encapsulation efficiency of 87.3%.

[0081] Comparative Example 1

[0082] It is basically the same as Example 6, except that 200 mg of phospholipid S100 is selected. The prepared stiripentol liposome has a particle size of 336.5 nm, a PDI of 0.737, and an encapsulation efficiency of 78.1%.

[0083] Comparative Example 2

[0084] It is basically the same as Example 6, except that 20 mg of stiripentol is selected. The prepared stiripentol liposome has a particle size of 267.3 nm, a PDI of 0.629, and an encapsulation efficiency of 65.8%.

[0085] Comparative Example 3

[0086] It is basically the same as Example 6, except that the ultrasonic power is 110 w and the ultrasonic time is 2 min. The prepared stiripentol liposome has a particle size of 261.2 nm, a PDI of 0.569, and an encapsulation efficiency of 60.7%.

[0087] Comparative Example 4

[0088] It is basically the same as Example 6, except that the hydration time is 50 min and the hydration volume is 30 mL. The prepared stiripentol liposome has a particle size of 252.0 nm, a PDI of 0.653, and an encapsulation efficiency of 59.8%.

[0089] The characterization results of the stiripentol liposome prepared in Example 6 are as follows:

[0090] As Figure 1 shown, after the stiripentol liposome solution is diluted five times and dried, the TEM image obtained shows that the stiripentol liposome is spherical and evenly dispersed.

[0091] AsFigure 2 As shown, according to the formula, the slope is the film thickness. The film thickness of the liposome is about 3.5611 nm, and the particle size is about 83 nm. It can be judged that it should be a monolayer vesicle with a medium particle size.

[0092] As Figure 3 shown, the stability results of stiripentol liposomes in artificial gastric juice and artificial pancreatic juice are that the particle size of stiripentol liposomes in gastric juice and pancreatic juice does not change significantly within 4 h, indicating that the overall structure of STP liposomes can exist stably in the stomach and intestine. The absolute value of the potential of stiripentol liposomes is very small, 6.96 mV, but it can maintain good stability, indicating that the structure of phospholipids on the liposome surface endows the system with steric stability.

[0093] In order to better explore the bioavailability characteristics of stiripentol liposomes, in vitro dissolution investigation and bioavailability evaluation were carried out. The specific contents are as follows:

[0094] (1) In vitro dissolution investigation

[0095] The dissolution curve of stiripentol liposomes was determined by the small cup method. The determination conditions were as follows: the dissolution medium was water, the volume of the dissolution medium was 250 mL, the rotation speed was 100 rpm / min, and the temperature was 37.0 °C. Three portions (equivalent to 10 mg of STP) of STP, physical mixture, and the stiripentol liposome sample prepared in Example 1 were accurately weighed and simultaneously put into the dissolution cup. Timing started when the raw drug contacted the dissolution medium. Samples were taken at 2, 5, 10, 20, 30, 45, 60, 90, 120, 240, 480, and 720 min respectively. Each time, 5 mL was drawn and 5 mL of the dissolution medium was added in time. The filtrate was filtered through a 0.45 μm pore size filter membrane, and the filtrate was used as the test sample. The absorbance was measured by ultraviolet spectrophotometry at a wavelength of 270 nm.

[0096] The preparation process of the STP physical mixture was as follows: 5 mg of stiripentol, 50 mg of phospholipid S100, and 1 mg of cholesterol were mixed evenly.

[0097] The results are as Figure 4 shown. Compared with the stiripentol raw drug, stiripentol liposomes have more dissolution in water. This indicates that preparing stiripentol in the form of liposomes not only enhances the dissolution rate of the drug, but also is expected to improve its bioavailability due to the high stability of stiripentol liposomes in gastric juice and pancreatic juice for up to 4 h.

[0098] Without conflict, the above-mentioned embodiments and the features in the embodiments in this article can be combined with each other.

[0099] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Stiripentol liposome, characterized in that, the stiripentol liposome comprises stiripentol, phospholipid S100 and cholesterol. The preparation process of the stiripentol liposome is as follows: weigh 7 mg of stiripentol, 100 mg of phospholipid S100 and 1 mg of cholesterol, dissolve them in 100 mL of ethanol solution, evaporate under reduced pressure until the ethanol is completely evaporated, take 15 mL of water for rotary hydration, the hydration temperature is 45 °C, the hydration time is 45 min, so that the liposome is uniformly dispersed in water at 30 °C, the ice bath ultrasonic power is 65 w, the ultrasonic time is 3 min, and filter through a 0.22 μm filter membrane to obtain the stiripentol liposome.

2. The stiripentol liposome according to claim 1, characterized in that, the particle size of the stiripentol liposome does not change significantly within 4 h in artificial gastric juice and artificial pancreatic juice.

3. An anti-epileptic drug composition, characterized in that, it is prepared by using the stiripentol liposome according to any one of claims 1-2.

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