WO2024060997A1

WO2024060997A1 - Novel fullerene-lipid complex and enteric capsule thereof

Info

Publication number: WO2024060997A1
Application number: PCT/CN2023/117222
Authority: WO
Inventors: 陈伟光; 朱常锋; 刘雅玲; 朱晟; 陈标奇; 雷斌; 陈爱政
Original assignee: 厦门福纳新材料科技有限公司
Priority date: 2022-09-21
Filing date: 2023-09-06
Publication date: 2024-03-28
Also published as: CN117731617A

Abstract

The present invention relates to a novel fullerene-lipid complex and an enteric capsule thereof. Particularly, the present invention provides a fullerene-lipid complex, comprising: C60; lecithin; and cholesterol, wherein the weight ratio of lecithin to cholesterol is 4:1. The fullerene-lipid complex has a particle size of less than 200 nm and a high encapsulation efficiency of about 90% or even higher, and the lipid complex suspension system is stable and less prone to precipitation.

Description

A new type of fullerene lipid compound and its enteric-coated capsules

Technical Field

The invention belongs to the field of medicine and relates to a fullerene lipid compound and its capsule preparation, in particular to a fullerene lipid compound and its enteric capsule preparation.

Background technique

From the dissolution process of the compound, we can know that the dissolution of the compound needs to go through three steps: breaking the lattice energy-hole formation-hydration of the compound molecules. If the solubility of the compound molecules is low, we can generally judge that: ① It is difficult to improve the solubility of the compound by crushing or micro-powdering, unless the compound undergoes changes in physical stability, such as crystal transformation and amorphization during the crushing or micro-powdering process; ② Changing the solid form or the crystal habit of the compound under the microscope does not affect the solubility of the compound, but may affect the dissolution rate of the compound, or may affect the physical stability, chemical stability and mechanical properties of the compound, such as fluidity, bulk density and compressibility; ③ The compound is in an amorphous state, the compound molecules do not interact with each other, are in a long-range disordered state, have high-energy characteristics, the molecular lattice is no longer a limiting factor for the dissolution of the compound, and the compound particle size is in a small state, even at the molecular level. Adding a hydrophilic polymer will stabilize the amorphous form of the compound and protect the molecule in the solution to maintain a supersaturated state, which also improves the wettability of the compound molecules. For drug molecules with higher melting points, the lattice energy can limit the dissolution of the compound, and amorphous solid dispersion technology may be the best strategy to choose for solubilization; ④ For compound molecules with strong lipophilicity, lipophilicity affects their dissolution during the dissolution process, and if solid dispersion technology is used for solubilization, the effect is not ideal. Therefore, lipid components can be added to the prescription to encapsulate the compound molecules, and then the bioavailability of the compound can be improved by endocytosis, which is a good way to increase the solubility of compound molecules.

According to current research findings, fullerene C60 drugs are almost insoluble in water, only soluble in aromatic hydrocarbon organic solvents such as toluene, bixylene and slightly soluble in some organic solvents such as chloroform. When a drug enters the gastrointestinal tract, the factors affecting the absorption of the drug in the internal environment are very complex, including drug physical and chemical factors, physiological factors and factors related to dosage forms. It is more complicated for BCS Class IV drugs, because these drugs are affected by both the solubility of the drug molecules and the permeability of the gastrointestinal tract, which may lead to poor oral bioavailability of this type of drugs. Because BCS Class IV drugs have low solubility and permeability, both solubility and permeability become critical rate-limiting steps for their absorption. Some physiological factors, such as gastric emptying time and gastrointestinal transit time, can significantly affect the absorption of BCS Class IV drugs. Therefore, there are large individual differences in the absorption of BCS IV drugs, and these differences in absorption make the development and formulation design of BCS IV drugs extremely challenging. For BCS Class IV drugs, they are usually administered intravenously. Here, we creatively propose the use of lipid compound technology and enteric-coated capsule positioning drug release technology to improve the bioavailability of BCS Class IV drugs. The main advantages of improving the bioavailability of BCS IV drugs through lipid technology are: as a carrier, lipid compounds can absorb drugs with poor bioavailability through selective lymphatic absorption, enhancing the dissolution and absorption of drugs in the gastrointestinal tract. The lipoplex drug delivery system has the advantages of improving the solubility of poorly soluble drugs, improving lymphatic absorption, and protecting drugs from premature metabolism.

Liposome technology is an advanced drug delivery system. Since the main components of liposomes are equivalent to the main components of cells, liposomes have good biocompatibility, minimal toxicity to the human body, and no immune effect on the human body. Drugs are encapsulated in liposomes, which can solve the problem of bioavailability of poorly soluble drugs. At the same time, liposomes can make the encapsulated drugs have targeted characteristics of directional distribution in the body, thereby increasing the efficacy of drugs. More importantly, liposomes have the advantages of increasing the stability of encapsulated drugs, reducing the elimination rate of drugs in the body, and prolonging the duration of drug action. The main technologies currently include nanostructured lipid carrier technology (NLCs), solid lipid nanoparticle technology (SLN), self-emulsifying drug delivery system (SMEDDS), lipid nanocapsules, etc. At present, the use of self-emulsifying drug delivery technology for marketed drugs is more common than other technologies. The possible mechanisms of action of liposomes are as follows: nanostructured lipid carrier technology (NLCs) mainly promotes the entry of drugs into the systemic circulation by increasing paracellular absorption; solid lipid nanoparticle technology (SLN) mainly increases paracellular absorption and emulsifies lipids into micelles, thereby allowing them to be absorbed by the lymphatic system in the form of chylomicrons; self-emulsifying drug delivery system (SMEDDS) also emulsifies lipids into micelles, thereby allowing them to be absorbed by the lymphatic system in the form of chylomicrons, and can also inhibit P-gp protein; lipid nanocapsules mainly enter the systemic circulation through macropinocytosis.

However, fullerene lipid compounds require further research and development.

Contents of the invention

Through many years of experimental research, the inventor of the present application has obtained a new type of fullerene lipid compound, whose particle size is less than 200nm, and the encapsulation rate is high, about 90% or even higher, and the lipid compound The suspension system is stable and not prone to sedimentation.

In addition, most of the liposomes currently on the market are administered by injection. Due to damage by the blood environment, the structure of the liposomes is destroyed, and the contained drugs leak, resulting in high blood drug concentration, resulting in serious side effects and toxic effects. The absorption in the body is limited, which directly affects the efficacy of the drug, so the advantages of liposome drugs cannot be exerted. The inventor found that if C60 drugs are directly packed into enteric-coated hollow capsules, although the toxic side effects of the drugs on the stomach and the destruction of the drugs by gastric juice can be avoided, this method will directly affect the efficacy of the drugs due to the extremely poor water solubility of C60. Bioavailability and efficacy. In order to solve the above problems, the inventor first made C60 into a lipopolymer, which can not only solve its water solubility problem, but also improve the uniform dispersion of the drug. At the same time, the biofilm-like properties of the lipoplex can not only It promotes the absorption of drugs, increases blood drug concentration, and enhances drug efficacy. Moreover, the lipid compound also has targeted and long-acting effects, which can extend the clearance time of drugs, reduce the peak-to-trough ratio of blood drug concentrations, and reduce the toxic and side effects of drugs.

To this end, in a first aspect of the invention, the invention provides a prescription for a fullerene lipid compound, which includes: C60, lecithin and cholesterol; wherein the weight ratio of the lecithin and the cholesterol is 4 :1.

In some embodiments, the weight ratio of C60 to cholesterol is 3:2-1:1, preferably 6:5.

In a second aspect of the present invention, the present invention provides a method for preparing the fullerene lipid compound of the first aspect, which includes:

(1) Dissolve C60, lecithin, and cholesterol in chloroform, and disperse by ultrasonic until uniform to obtain the first dispersion system;

(2) Add water to the first dispersion system and continue ultrasonic to form a W/O emulsification system;

(3) Evaporate the W/O emulsified system under reduced pressure to remove the organic solvent chloroform, and then continue the constant temperature reaction to obtain a fullerene lipid compound suspension.

In some embodiments, in step (1), the weight ratio of the lecithin and the cholesterol is 4:1. For example, the weight of lecithin can be 40 mg, and the weight of cholesterol can be 10 mg.

In some embodiments, in step (1), the weight ratio of the C60 to the cholesterol is 3:2-1:1, preferably 6:5. For example, the weight of C60 may be 12 mg, and the weight of cholesterol may be 10 mg.

In some embodiments, in step (1), the ratio of the weight of the cholesterol to the volume of the chloroform is 1:6-1:10 mg/mL, preferably 1:8 mg/mL. For example, when the ratio of the weight of the cholesterol to the volume of the chloroform is 1:8 mg/mL, the weight of the cholesterol can be 10 mg and the volume of the chloroform can be 80 mL.

In some embodiments, the ratio of the weight of cholesterol in step (1) to the volume of water in step (2) is 1:8~1:16 mg/mL, preferably 1:12 mg/mL. For example, when the ratio of the weight of cholesterol described in step (1) to the volume of water described in step (2) is 1:12 mg/mL, then the weight of cholesterol described in step (1) can be 10 mg. The volume of water mentioned in (2) can be 120mL.

In some embodiments, in step (2), the water is purified water.

In some embodiments, in step (2), the ultrasonic time is 1-10 min, preferably 5 min.

In some embodiments, in step (3), the reduced pressure evaporation is reverse rotary evaporation.

In some embodiments, the rotation speed of the reverse rotary evaporation is 40-60 rpm, preferably 50 rpm.

In some embodiments, the temperature of the reverse rotary evaporation is 25-35°C, preferably 30°C.

In some embodiments, the reverse rotary evaporation is carried out for 4-6 hours, preferably 5 hours.

In some embodiments, in step (3), the isothermal reaction is carried out for 20-40 min, preferably 30 min. It can be understood that the term "constant temperature" means that the temperature of the isothermal reaction is consistent with the temperature of the previous evaporation under reduced pressure.

In some embodiments, after step (3), it further includes post-processing the fullerene lipid compound suspension.

In some embodiments, the post-treatment is filtration and extrusion granulation; preferably, the filtration is filtration with filter paper; preferably, the extrusion granulation is extrusion granulation with a water phase membrane.

In a third aspect of the invention, the invention provides a fullerene lipid compound enteric-coated capsule preparation, which includes: C60, lecithin, and cholesterol; wherein, the weight ratio of the lecithin and the cholesterol is 4: 1.

In some embodiments, the weight ratio of the C60 to the cholesterol is 3:2-1:1, preferably 6:5.

In some embodiments, the fullerene lipid compound enteric capsule preparation further includes: coating powder, and enteric capsule excipients.

In some embodiments, the enteric capsule excipient is a mixture of mannitol, lactose, micronized silica gel, and magnesium stearate.

In some embodiments, the weight ratio of the magnesium stearate to the mannitol is 1:4.

In some embodiments, the weight ratio of the magnesium stearate to the lactose is 1:8.

In some embodiments, the weight ratio of the magnesium stearate to the micronized silica gel is 1:2.

In some embodiments, the fullerene lipid compound enteric capsule formulation further includes: a pill core and a capsule.

In some embodiments, the pellet cores are microcrystalline fiber pellet cores.

In the fourth aspect of the present invention, the present invention provides a method for preparing the fullerene lipid compound enteric-coated capsule preparation of the third aspect, which includes:

(1) Perform tangential flow concentration on the fullerene lipid compound suspension, wherein the fullerene lipid compound suspension is prepared according to the method of the second aspect;

(2) Thoroughly mix the concentrated liquid obtained in step (1) and the coating liquid;

(3) Fluidize the mixed liquid obtained in step (2) until the weight of the pill core increases by 40%, and continue to wait for a period of time to obtain pellets;

(4) Thoroughly mix the pellets and enteric-coated capsule excipients;

(5) Fill the mixed powder obtained in step (4) into capsules to obtain a fullerene lipid compound enteric-coated capsule preparation.

In some embodiments, the coating liquid is obtained by uniformly mixing coating powder and water. In some embodiments, the coating powder is an enteric film coating powder. Among them, enteric film-coated powder is commercially available. In some embodiments, the enteric film-coated powder is purchased from Anhui Shanhe Pharmaceutical Excipients Co., Ltd.

In some embodiments, in step (1), in the concentrated fullerene lipid compound suspension, the C60 concentration is 1.0 mg/mL.

In some embodiments, in step (3), the pellet core is a microcrystalline fiber pellet core.

In some embodiments, in step (3), the continued standby time is 2 hours.

In some embodiments, in step (4), the enteric capsule excipient is a mixture of mannitol, lactose, micronized silica gel, and magnesium stearate.

In some embodiments, the weight ratio of the magnesium stearate to the pellets is 0.25:100.

In some embodiments, in step (4), before the pellets and enteric capsule excipients are fully mixed, the enteric capsule excipients are premixed.

In some embodiments, in step (5), when the capsule is filled, the weight difference of the capsule particles is controlled to be ±10%.

beneficial effects

1. The particle size of the fullerene lipid compound of the present invention is less than 200nm, the encapsulation rate is high, about 90% or even higher, and the lipid compound suspension system is stable and is not prone to sedimentation.

2. The fullerene lipid compound of the present invention has good dispersion and good sphericity. When the particle size is measured, it is mainly distributed between 100-200 nm. The particle size distribution is uniform and relatively small. narrow.

3. When preparing a fullerene lipid compound suspension, the dosage ratio of lecithin and cholesterol in the lipid compound affects the particle size, encapsulation rate, system stability and other properties of the lipid compound. Among them, the mass ratio of lecithin to cholesterol is 4:1, which is better than 2:1, 3:1 and 5:1. When the mass ratio of the two is 4:1, the particle size of the prepared lipid compounds is less than 200nm, including The sealing rate is also high, about 90% or even higher, and the lipid compound suspension system is stable and not prone to sedimentation.

4. When preparing enteric-coated capsules of fullerene lipid compounds, the lipid compounds are concentrated to increase the C60 concentration and encapsulation rate, and then sprayed on the microcrystalline fiber pellets through a fluidized bed to form a uniform film , it is better to solidify the lipid compound on the pill core. Compared with the lipid compound suspension system, C60 pellet capsules better solve the problems of long-term stability and uniform dispersion of the preparation, so as to improve the drug C60 in the body bioavailability, thereby exerting the therapeutic effect of the drug.

5. When preparing fullerene lipid compound enteric-coated capsules, if the C60 drug is directly put into the enteric-coated hollow capsule, although the toxic side effects of the drug on the stomach and the destruction of the drug by gastric juice are avoided, due to the water solubility of C60 The sex is extremely poor, and this method will directly affect the bioavailability and efficacy of the drug. In order to solve the above problems, the inventor first made C60 into a lipopolymer, which can not only solve its water solubility problem, but also improve the uniform dispersion of the drug. At the same time, the biofilm-like properties of the lipopolymer can not only It promotes the absorption of drugs, increases blood drug concentration, and enhances drug efficacy. Moreover, the lipid compound also has targeted and long-acting effects, which can extend the clearance time of drugs, reduce the peak-to-trough ratio of blood drug concentrations, and reduce the toxic and side effects of drugs.

6. The present invention combines the drug delivery technology of lipid compounds and the positioning drug release technology of enteric-coated capsules, and has the following five major advantages: (1) The present invention can encapsulate poorly soluble and fat-soluble drugs in lipid compounds. The use of co-solvents with toxic side effects is avoided in the film; (2) the problems of water solubility and drug uniform dispersion of C60 drugs are solved; (3) the drug is encapsulated in the lipid compound to increase drug absorption and improve drug efficacy ; (4) Filling the enteric-coated capsules with lipid compounds can position the drug in the small intestine to release the drug, avoiding the destruction of the lipid drug by gastric juice and the irritation and toxicity of the drug to the gastric mucosa; (5) For patients with gastric diseases Or drugs that are not easily absorbed in the stomach, this technology can make the drugs better absorbed in the small intestine.

Description of the drawings

Figure 1: Physical picture of C60 lipid compound;

Figure 2: Comparative physical picture of prescription 1;

Figure 3: Comparative physical picture of prescription two;

Figure 4: Comparing the actual picture of prescription three;

Figure 5: Actual picture of the pill core after applying the lipid compound;

Figure 6: Lipid compound particle size diagram;

Figure 7: Surface potential diagram of lipoplex;

Figure 8: Microscopic TEM image of the lipid compound;

Figure 9: Microscopic SEM image of lipoplex;

Figure 10: HPLC chart of C60 in the lipid compound before concentration;

Figure 11: C60 HPLC profile of concentrated 5X lipoplex.

Detailed ways

Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments.

The invention will now be described with reference to the following examples which are intended to illustrate the invention rather than to limit the invention.

Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents used in the examples of the present invention are all commercially available.

Some of the reagents and instruments used in the present invention are detailed in the table below.

Summary table of experimental instruments and equipment

Summary table of experimental materials and reagents

Example 1

1. Fullerene lipid compound prescription

Prescription one:

Prescription two:

Prescription three:

Prescription four:

Prescription five:

Comparative prescription one:

Comparative prescription two:

Comparative prescription three:

2. Preparation of fullerene lipid compounds

(1) Dissolve the prescribed amount of C60, lecithin, and cholesterol in the prescribed amount of chloroform, and use ultrasonic to disperse evenly;

(2) Add the prescribed amount of purified water to the system and continue ultrasonic for 5 minutes until a stable W/O emulsification system is formed;

(3) Place in an eggplant-shaped flask, use reverse rotary evaporation to remove the organic solvent, rotation speed: 50 rpm; reaction temperature: 30°C; reaction time: 5h; after the reaction is completed, continue the constant temperature reaction for 30 minutes.

(4) Collect the lipid compound suspension and filter it through filter paper and squeeze the aqueous membrane into granules, then place it in a brown bottle for later use.

Among them, the actual picture of the lipid complex suspension prepared by prescription 1 (the lipid complex suspension prepared by step (3) before filtering with filter paper) is shown in FIG1. From the appearance, it can be found that the system is uniform and stable. However, as shown in FIG2, the suspension prepared by step (3) of prescription 1 is not uniform (left picture), but a foamy mixture. The mixture is taken out and placed in a beaker (right picture), and it can be clearly found that solids are precipitated and the mixture is turbid; as shown in FIG3, the suspension prepared by step (3) of prescription 2 is not uniform (left picture), but there is obvious solid-liquid stratification. The liquid part is taken out and placed in a beaker (right picture), and it can be clearly found that solids are precipitated and the mixture is turbid; as shown in FIG4, the suspension prepared by step (3) of prescription 3 is not uniform (left picture), but there is also obvious solid-liquid stratification, and there is obvious foam. The liquid part is taken out and placed in a beaker (right picture), and it can be clearly found that solids are precipitated and the mixture is turbid. Therefore, from the physical pictures of Comparative Prescription 1, Comparative Prescription 2, and Comparative Prescription 3 shown in Figures 2 to 4, it can be found that the systems obtained by Comparative Prescription 1, Comparative Prescription 2, and Comparative Prescription 3 all showed obvious stratification and turbidity, and had poor stability. Therefore, Comparative Prescription 1, Comparative Prescription 2, and Comparative Prescription 3 were eliminated and no subsequent performance characterization research was conducted.

3. Preparation of fullerene lipid compound enteric-coated capsules

(1) Collect the lipid compound suspension obtained in the previous steps and conduct tangential flow concentration to control the C60 concentration at 1.0 mg/mL;

(2) Prepare a coating solution of appropriate concentration, and thoroughly mix the concentrated lipid compound suspension in step (1) and the coating solution evenly;

(3) Perform fluidized bed coating until the weight of the microcrystalline fiber pellet core increases by 40% (as shown in Figure 5), and continue to wait for 2 hours;

(4) Premixed excipients, the excipients are 1.0% (w/w) mannitol, 2.0% (w/w) lactose, 0.5% (w/w) micronized silica gel, 0.25% (w/w) magnesium stearate mixture;

(5) Collect the pellets obtained in step (3) and mix them thoroughly with the premixed excipients in step (4);

(6) Fill the mixed powder into capsules and control the weight difference of the capsules to ±10%;

(7) The resulting capsules are encapsulated by a blister machine into finished products, which are then packed into boxes and stored in warehouse.

Example 2

Perform sample characterization and analysis on the fullerene lipid compound prepared in Example 1.

(1) Lipid nanoparticle size and zeta potential

This instrument is used to measure the surface potential of fullerene nanolipid complexes and determine its hydrated particle size using the principle of dynamic light scattering. The main steps are: take 2 mL of water dispersion of appropriate concentration in a plastic sample cell, place it in the sample chamber, set the temperature, single measurement time, total number of measurements, number of single cycles and other parameters before testing. Based on phase analysis light scattering (PALS) technology, its Zeta potential is measured.

The fullerene lipid compound suspension prepared in Example 1 was formulated to a suitable concentration, and the hydrated particle size and surface charge of the fullerene nanolipid complex were further measured using a dynamic light scattering instrument (DLS). (Set the test parameters as: temperature 25°C, dispersion medium: purified water, total measurement times 100 times, cycle 6 times, etc.).

The test results are as follows:

The average hydrated particle size of the lipoplex obtained from prescription 1 was 169.2 nm, and the polydispersity coefficient PDI = 0.163 (Figure 6), indicating good dispersion. The surface charge test results showed that the surface of the nanoparticles was negatively charged, with a surface potential of -29.7mV (Figure 7).

(2) TEM characterization

Use TEM to observe the morphology of the fullerene nanolipid complex and take TEM images. The main steps are: select the fullerene nanolipid compound suspension (0.01%, W/V) obtained in Example 1, absorb a small amount and drop it on a 300-mesh copper mesh and let it stand for deposition, and then absorb the water with dust-free paper. Then negative stain with 2wt% phosphotungstic acid aqueous solution for 15~20 minutes. After the sample is fully dry, place it on a TEM for observation and photography.

The fullerene lipid compound suspension (0.01%, W/V) prepared in Example 1 was dropped on a copper grid. After negative staining with phosphotungstic acid and natural drying, a transmission electron microscope (TEM) was used to examine the fullerene lipids. The morphology of the compound was further observed.

The result is as follows:

A sample of the fullerene lipid compound suspension was prepared according to the prescription in Example 1, and was tested by TEM. The results are shown in Figure 8. Among them, the bar value in the figure is 500nm. It can be seen from the figure that the sphericity of the fullerene lipid compound is well distributed and evenly distributed.

(3) SEM characterization

The main steps of using SEM to observe the surface morphology of fullerene nanolipid complex are: select the fullerene nanolipid complex suspension (0.1%, W/V) prepared in Example 1, and drip a small amount onto the cut silicon wafer. After natural drying, the silicon wafer with the sample is attached to the sample stage, sprayed with gold for 30 s, and placed in the sample chamber. The morphology of the fullerene nanolipid complex is observed and photographed at different magnifications.

A scanning electron microscope (SEM) was used to observe the micromorphology of the fullerene nanolipid compound prepared in Example 1. The results are as follows.

A sample of the fullerene lipid compound suspension was prepared according to the prescription in Example 1 and was tested by SEM. The results are shown in Figure 9. In Figure 9, the bar value is 1.0µm. The results show that the fullerene nanolipid complex has good dispersion and good sphericity. The particle size is measured and it is mainly distributed between 100-200 nm. The particle size distribution is uniform and the particle size distribution is narrow.

(4) Fullerene content detection

2.1 Prepare reference solution

Reference solution: C60 toluene solution, 0.5mg/ml. Accurately weigh 10.0mg C60, dissolve it in 20.0ml toluene, and set aside.

2.2 Processing of samples to be tested

Accurately measure 2.0 ml of the solution to be tested (fullerene lipid compound suspension prepared in Example 1), add 1.0 ml of methanol, shake and mix thoroughly, and sonicate in a water bath for 3 minutes. Then centrifuge at 10,000 rpm for 15 minutes, discard the supernatant, add 1.0 ml of methanol to the precipitate, and sonicate to fully dissolve and disperse. Centrifuge again at 10,000 rpm for 15 min. Discard the supernatant. Add 2.0 ml of toluene to the precipitate and sonicate to ensure complete dissolution. Set aside.

2.3 Detection methods

HPLC chromatographic detection conditions: flow rate: 0.5mL/min; mobile phase: toluene; column specification: Buckypre 4.6IDx10mm; column temperature: 30°C; detection wavelength: 335nm; detector: UV detector; relative retention time: 15min, running time :45min.

2.4 Experimental results

The experimental results of Prescription 1 in Example 1 are shown in Figures 10 and 11. It has been calculated that the encapsulation rate of C60 in the lipid compound reaches more than 90%.

Example 3

Conduct long-term storage stability analysis on the fullerene lipid compound prepared in Example 1.

3.1 Experimental methods

The samples prepared in Example 1 were placed in a 4°C environment, and the particle size and encapsulation rate of the samples were detected according to the method described in Example 2 at 0, 1, 3, and 6 months respectively.

3.2 Experimental results

The experimental results show that the particle size and encapsulation efficiency of the samples prepared by Prescription 1 in Example 1 after 1, 3, and 6 months were close to those at 0 month, and the CV values were all less than 5.0%, which is within the controllable range of quality and has excellent long-term storage stability.

Example 4

The dissolution rate of the fullerene lipid compound enteric-coated capsules prepared in Example 1 was measured.

The dissolution rate of enteric-coated preparations was measured according to the Chinese Pharmacopoeia 2020 edition.

(1) Measure 750mL of 0.1 mol/L hydrochloric acid solution into each dissolution cup. The deviation between the actual measured volume and the specified volume should be within ±1%. When the temperature of the dissolution medium is constant at 37℃±0.5℃, Take the fullerene lipid compound enteric-coated capsules prepared in Example 1 and put them into the dissolution cups respectively; immediately start the instrument according to the rotation speed specified under each variety, and after 2 hours, absorb an appropriate amount of the dissolution liquid at the specified sampling point, and filter , from sampling to filtration should be completed within 30 seconds. Measure according to the method specified under each variety and calculate the amount of acid dissolved in each grain.

(2) Amount of dissolution in buffer: Add 250ml of 0.2mol/L sodium phosphate solution at a temperature of 37℃±0.5℃ to the above acid solution (if necessary, adjust the pH value with 2mol/L hydrochloric acid solution or 2mol/L sodium hydroxide solution) to 6.8), continue running for 45 minutes, or according to the time of 0.5h, 1.0h, 2h, 4h, absorb an appropriate amount of the eluate at the specified sampling point, and filter it. The time from sampling to filtration should be completed within 30 seconds. Measure according to the method specified under each variety, and calculate the dissolution amount of each particle in the buffer solution.

The experimental results are as follows:

The fullerene lipid compound enteric-coated capsule sample prepared according to prescription 1 in Example 1 was tested. The experimental results showed that the C60 enteric-coated preparation remained in 0.1 mol/L hydrochloric acid solution for 2 hours without dissolving the C60 drug. status, in line with the pharmacopoeia's regulations that the amount of dissolution in acid is not greater than 10% of the labeled amount; after testing in 0.1 mol/L hydrochloric acid solution and 750mL buffer for 45 minutes, the dissolution rate of C60 drugs is greater than 90%, which is in line with the Chinese Pharmacopoeia for enteric-coated preparations. The relevant quality standards stipulate that it is higher than 70% of the labeled quantity.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and do not limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art will understand that Modifications or equivalent substitutions may be made to the technical solution of the present invention without departing from the essence and scope of the technical solution of the present invention.

Claims

A fullerene lipid compound containing: C60; lecithin; and cholesterol;

Wherein, the weight ratio of the lecithin to the cholesterol is 4:1.
The fullerene liposome as described in claim 1, wherein the weight ratio of the C60 to the cholesterol is 3:2-1:1, preferably 6:5.
A method for preparing the fullerene lipid compound described in any one of claims 1-2, which includes:

(1) Dissolve C60, lecithin, and cholesterol in chloroform, and disperse by ultrasonic until uniform to obtain the first dispersion system;

(2) Add water to the first dispersion system and continue ultrasonic to form a W/O emulsification system;

(3) The W/O emulsified system is evaporated under reduced pressure to remove the organic solvent chloroform, and then the reaction is continued at a constant temperature to obtain a fullerene lipid complex suspension.
The preparation method of claim 3, wherein the method has technical features selected from one or more of the following:

(i) In step (1), the weight ratio of the lecithin and the cholesterol is 4:1;

(ii) In step (1), the weight ratio of the C60 to the cholesterol is 3:2-1:1, preferably 6:5;

(iii) In step (1), the ratio of the weight of the cholesterol to the volume of the chloroform is 1:6~1:10 mg/mL, preferably 1:8 mg/mL;

(iv) The weight of cholesterol described in step (1) and the volume of water described in step (2)

The ratio is 1:8~1:16 mg/mL, preferably 1:12 mg/mL;

(v) In step (2), the water is purified water;

(vi) In step (2), the ultrasonic time is 1-10 min, preferably 5 min;

(vii) In step (3), the reduced pressure evaporation is reverse rotary evaporation;

Preferably, the rotation speed of the reverse rotary evaporation is 40-60rpm, preferably 50rpm;

Preferably, the temperature of the reverse rotary evaporation is 25-35°C, preferably 30°C;

Preferably, the reverse rotary evaporation time is 4-6h, preferably 5h;

(viii) In step (3), the isothermal reaction is carried out for 20-40 minutes, preferably 30 minutes;

(ix) After step (3), it further includes post-processing the fullerene lipid compound suspension;

Preferably, the post-treatment is filtration and extrusion granulation; preferably, the filtration is filtration using filter paper; preferably, the extrusion granulation is extrusion granulation using a water phase membrane.
Fullerene lipid compound enteric-coated capsule preparation, which contains: C60; lecithin; cholesterol;

Wherein, the weight ratio of the lecithin and the cholesterol is 4:1.
The fullerene lipid compound enteric-coated capsule preparation according to claim 5, wherein the

The weight ratio of C60 to the cholesterol is 3:2-1:1, preferably 6:5.
The fullerene lipid compound enteric-coated capsule preparation according to any one of claims 5-6, wherein the fullerene lipid compound enteric-coated capsule preparation further includes: coating powder, and enteric-coated capsule excipients ;

Preferably, the enteric capsule excipients are mannitol, lactose, micronized silica gel, and stearic acid.

magnesium mixture;

Preferably, the weight ratio of the magnesium stearate to the mannitol is 1:4;

Preferably, the weight ratio of the magnesium stearate to the lactose is 1:8;

Preferably, the weight ratio of the magnesium stearate to the micropowder silica gel is 1:2.
The fullerene lipid compound enteric-coated capsule preparation according to any one of claims 5-7, wherein the fullerene lipid compound enteric-coated capsule preparation further includes: a pill core and a capsule;

Preferably, the pellet core is a microcrystalline fiber pellet core.
The method for preparing the fullerene lipid compound enteric-coated capsule preparation according to any one of claims 5-8, which includes:

(1) The fullerene lipid compound suspension is subjected to tangential flow concentration, wherein the fullerene lipid compound suspension is prepared according to the method described in any one of claims 3-4 of;

(2) fully mixing the concentrated solution obtained in step (1) with the coating solution;

(3) The mixed solution obtained in step (2) is subjected to fluidized bed coating until the pellet cores gain weight.

40%, continue to wait for a period of time to obtain pellets;

(4) Thoroughly mix the pellets and enteric-coated capsule excipients;

(5) Fill the mixed powder obtained in step (4) into capsules to obtain a fullerene lipid compound enteric-coated capsule preparation.
The method of claim 9, wherein the method has technical features selected from one or more of the following:

(i) In step (1), in the concentrated fullerene lipid compound suspension, the C60 concentration is 1.0 mg/mL;

(ii) In step (2), the coating liquid is obtained by uniformly mixing the coating powder and water;

(iii) In step (3), the pellet core is a microcrystalline fiber pellet core;

(iv) In step (3), the continued standby time is 2 hours;

(v) In step (4), the enteric capsule excipients are mannitol, lactose, and micropowder silicon.

A mixture of gum and magnesium stearate;

Preferably, the weight ratio of the magnesium stearate to the mannitol is 1:4;

Preferably, the weight ratio of the magnesium stearate to the lactose is 1:8;

Preferably, the weight ratio of the magnesium stearate to the micropowder silica gel is 1:2;

Preferably, the weight ratio of the magnesium stearate to the pellets is 0.25:100;

(vi) In step (4), before the pellets and the enteric-coated capsule excipients are fully mixed, the enteric-coated capsule excipients are premixed;

(vii) In step (5), when the capsule is filled, the weight difference of the capsule particles is controlled to be ±10%.