Encedipine-loaded lipid nanoparticle as well as preparation method and application thereof
By using a mixture of β-sitosterol and phospholipids as carriers, encidipine is loaded with encidipine and polyethylene glycol is modified on the surface to prepare lipid nanoparticles loaded with encidipine, which solves the adverse reactions and low drug utilization problems of traditional encidipine dosage forms, and achieves efficient IDH2 inhibition and drug sustained release effects.
Patent Information
- Application Number
- CN202510256822.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-05
- Publication Date
- 2025-05-13
AI Technical Summary
The traditional chemical dosage forms of Encidipine have problems such as adverse reactions, lack of selectivity and low drug utilization, and no research reports on other dosage forms have been found.
The lipid nanoparticles carrying encidipine were prepared by ultrasonic thin film hydration method using a mixture of β-sitosterol and phospholipids as the backbone, and the core was loaded with encidipine and polyethylene glycol was modified on the surface.
The prepared lipid nanoparticles have a uniform particle size, an encapsulation rate of more than 75%, and have IDH2 inhibitory activity and drug sustained release effect. They simplify the preparation process, are suitable for large-scale production, and reduce the risk of side effects.
Smart Images
Figure CN119970681A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of medicines, and particularly relates to lipid nanoparticles carrying ensidedipine, and a preparation method and application thereof. Background Art
[0002] Acute myeloid leukemia (AML) is a rapidly developing blood cancer that originates in the bone marrow and causes an abnormal increase in the number of white blood cells. Approximately 8-19% of AML patients have a mutation in isocitrate dehydrogenase-2 (IDH2). ) is the first IDH2 inhibitor, which was approved by the US FDA on August 1, 2017 for the treatment of relapsed or refractory acute myeloid leukemia in adults with IDH2 gene mutations. In clinical practice, ensidipine often causes adverse reactions such as nausea, vomiting, diarrhea, increased bilirubin, and decreased appetite. Although no side effects caused by drug overdose were found in clinical trials of ensidipine, this traditional chemical drug treatment method, which works by widely distributing throughout the body, lacks selectivity, increases drug side effects, and has a low drug utilization rate, resulting in drug waste.
[0003] At present, Ensidipine is only available in the form of tablets in the market as a small molecule drug, and no research reports on other dosage forms have been seen. Therefore, developing new dosage forms of Ensidipine to improve its in vivo absorption and prolong its duration of action has become an effective way to overcome its clinical treatment drawbacks.
[0004] Lipid nanoparticles are a new type of nanoparticles that have the characteristics of both liposomes and polymers. Their nanoscale size enables them to be absorbed by tissues or cells, while also avoiding phagocytosis by the reticuloendothelial system. In particular, when their surface is modified with a polyethylene glycol layer, they are also stealthy and can prolong their circulation time in the body.
[0005] Although a variety of lipid nanoparticles and their preparation methods have been reported in the literature, the ones disclosed are mainly lipid nanoparticles for delivering nucleic acid drugs, anti-tumor drugs such as doxorubicin and paclitaxel, and there is no preparation of lipid nanoparticles loaded with ensidipine. If the dosage form of ensidipine is easily changed, there will be a risk of change or loss of efficacy.
[0006] Moreover, some preparation methods of lipid nanoparticles disclosed in the prior art also have the following drawbacks. For example, 30-40% cholesterol is often added as a structural lipid in the preparation process of traditional lipid nanoparticles. However, the use of high cholesterol ratio is easy to cause the rupture of the bilayer membrane of the liposome and drug leakage; secondly, it will increase the risk of atherosclerosis, fatty liver, coronary heart disease, cerebral infarction, etc., which is particularly unfriendly to patients with cardiovascular diseases. In addition, some lipid nanoparticles are also added with surfactants such as Tween during preparation. Although these additives can play a solubilizing role and make the particle size uniform, they may cause allergic reactions, gastrointestinal discomfort, increase the burden on the liver and kidneys, heart damage and other side effects. Summary of the invention
[0007] In order to solve the above technical problems, the present invention provides a lipid nanoparticle carrying ensididipine and a preparation method and application thereof. The lipid nanoparticle carrying ensididipine has a uniform particle size, an encapsulation rate of ensididipine of more than 75%, has IDH2 inhibitory activity and drug sustained release effect, and has a simple preparation process, which is suitable for large-scale production in pharmaceutical and fine chemical enterprises.
[0008] The technical solution adopted by the present invention is as follows:
[0009] The invention discloses an ensidipine-loaded lipid nanoparticle, which has a beta-sitosterol and phospholipid mixture as a skeleton and an inner core loaded with ensidipine.
[0010] The surfaces of the ensididipine-loaded lipid nanoparticles are also modified with polyethylene glycol.
[0011] The phospholipid mixture is selected from at least two of lecithin, polyethylene glycol-distearoylphosphatidylethanolamine, and dipalmitoylphosphatidylcholine.
[0012] When the surface of the lipid nanoparticles carrying ensidipine is modified with polyethylene glycol, the phospholipid mixture is selected from a mixture of at least one of phosphatidylcholine and dipalmitoylphosphatidylcholine and polyethylene glycol-distearoylphosphatidylethanolamine.
[0013] Furthermore, the mass ratio of ensidipine to the mixture of β-sitosterol and phospholipid is 1:0.5-2:4-9.
[0014] The average particle size of the lipid nanoparticles carrying ensidipine is 150-250nm, the polydispersity index is 0.2-0.4, and the Zeta potential is -22--32mV; the encapsulation rate of ensidipine is above 75%.
[0015] The present invention also provides a method for preparing the lipid nanoparticles carrying encedidipine, and the preparation method comprises the following steps:
[0016] (1) dissolving ensidipine, β-sitosterol and a phospholipid mixture in an organic solvent, and performing vacuum rotary evaporation in a water bath until a lipid film is formed;
[0017] (2) ultrasonically dispersing the lipid film in deionized water, and then performing probe ultrasonic dispersion to obtain a uniform drug-loaded emulsion;
[0018] (3) The obtained drug-loaded emulsion is centrifuged at low temperature using an ultrafiltration tube, and the upper precipitate is dispersed in deionized water to obtain lipid nanoparticles loaded with ensididipine.
[0019] In step (1), the temperature of the water bath is 35-45°C.
[0020] In step (1), the organic solvent is selected from any one or more of chloroform, dichloromethane and tetrahydrofuran.
[0021] In step (2), the lipid film is dispersed in deionized water at a concentration of 0.2 to 1.5 mg / mL.
[0022] In step (2), the power of water bath ultrasound is 100-150W, and the ultrasound time is 5-20min; the power of probe ultrasound is 100-150W, and the ultrasound is performed for 6s with a rest period of 10s, and the ultrasound time is 5-15min.
[0023] In step (3), the molecular weight cut-off of the ultrafiltration tube is 30-50KD; and the temperature of the low-temperature centrifugation is 2-6°C.
[0024] In step (3), the centrifugal speed is 5000-6000 rpm and the time is 20-60 min.
[0025] The present invention also provides the use of the lipid nanoparticles carrying ensididipine in the preparation of drugs for inhibiting IDH2.
[0026] The lipid nanoparticles carrying ensidedipine provided by the present invention have a β-sitosterol and phospholipid mixture as a skeleton, and the inner core is loaded with ensidedipine. β-sitosterol is a plant sterol that can inhibit the intestinal absorption of cholesterol, reduce serum total cholesterol levels, and reduce the risk of cardiovascular diseases; it can also regulate the immune system and enhance immune function; it can also inhibit the growth of tumor cells, serve as an adjuvant treatment for cancer, and enhance the effects of chemotherapy and radiotherapy. Compared with lipid nanoparticles containing cholesterol and surfactant components, the lipid nanoparticles carrying ensidedipine provided by the present invention have simple and safe components, high encapsulation rate, small particle size and narrow distribution, can effectively deliver drugs, have a sustained release effect, and better meet the needs of ensidedipine delivery in vivo.
[0027] The preparation method of lipid nanoparticles carrying encedipin provided by the present invention adopts an ultrasonic thin film hydration method. Compared with the traditional melting-homogenization method, cooling-homogenization method, microemulsion method, etc., it does not need to undergo a high temperature process and is more friendly to heat-sensitive drugs; it does not need to stir emulsification for a long time, takes a short time and has a uniform particle size; it does not need to add a surfactant and is not prone to adverse reactions. The entire preparation process is simple, has few operating steps, and has strong continuity, and is suitable for large-scale production in pharmaceutical and fine chemical companies.
[0028] Compared with the prior art, the present invention has the following beneficial effects:
[0029] The present invention discloses for the first time other dosage forms of ensidipine besides tablets, breaking the current situation that there are few dosage forms of ensidipine; the lipid nanoparticles loaded with ensidipine provided by the present invention have IDH2 inhibitory activity and drug sustained release effect, and can be used to treat related diseases caused by IDH2 mutation. At the same time, the preparation method of the lipid nanoparticles loaded with ensidipine provided by the present invention is simple and the preparation cost is low. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] Figure 1 The particle size distribution diagram of the lipid nanoparticles loaded with ensidipine prepared in Example 1 and Example 3;
[0031] Figure 2 TEM images of lipid nanoparticles loaded with ensidipine prepared in Example 1, Example 3 and Comparative Example 2;
[0032] Figure 3 The drug release curves of the lipid nanoparticles loaded with ensidipine prepared in Example 1 and Example 3 are shown. DETAILED DESCRIPTION
[0033] The present invention is described in detail below with reference to the embodiments.
[0034] Example 1
[0035] A method for preparing lipid nanoparticles carrying encedidipine, comprising the following steps:
[0036] (1) 1.2 mg of ensidipine, 1 mg of β-sitosterol, 4 mg of lecithin, and 2 mg of polyethylene glycol-distearoylphosphatidylethanolamine were dissolved in a flask containing 3.5 mL of chloroform. The mixture was subjected to vacuum rotary evaporation for 30 min in a 40°C water bath until a uniform lipid film was formed. The mixture was then pumped dry for 10 min using an oil pump.
[0037] (2) Add 10 mL of deionized water to the flask, perform water bath ultrasonic emulsification for 5 min, and then switch to probe ultrasonic emulsification for 10 min, wherein the power of the water bath ultrasonic is 100 W, the power of the probe ultrasonic is 110 W, and the probe ultrasonic is performed with a 10-s rest every 6 s;
[0038] (3) The suspension obtained in step (2) was centrifuged at 6000 rpm for 20 min at 4° C. using a 30 KD ultrafiltration tube, and the upper precipitate was dispersed in 0.5 mL of deionized water to obtain lipid nanoparticles loaded with ensidipine.
[0039] Example 2
[0040] A method for preparing lipid nanoparticles carrying ensididipine, comprising the following steps:
[0041] (1) 1.2 mg of ensidipine, 1.5 mg of β-sitosterol, 5 mg of lecithin, and 2.5 mg of polyethylene glycol-distearoylphosphatidylethanolamine were dissolved in a flask containing 5 mL of dichloromethane, and vacuum rotary evaporated for 25 min in a 35°C water bath until a uniform lipid film was formed, and then the oil pump was switched to continue to pump dry for 8 min;
[0042] (2) Add 10 mL of deionized water to the flask, perform water bath ultrasonic emulsification for 5 min, and then switch to probe ultrasonic emulsification for 12 min. The power of the water bath ultrasonic emulsification was 100 W, the power of the probe ultrasonic emulsification was 110 W, and the probe ultrasonic emulsification was performed for 6 s with a rest period of 10 s.
[0043] (3) The obtained suspension was centrifuged at 6000 rpm for 30 min at 4°C using a 30KD ultrafiltration tube, and the upper precipitate was dispersed in 0.5 mL of deionized water to obtain lipid nanoparticles loaded with ensidipine.
[0044] Example 3
[0045] A method for preparing lipid nanoparticles carrying ensididipine, comprising the following steps:
[0046] (1) 1.2 mg of ensidipine, 1 mg of β-sitosterol, 4 mg of lecithin, and 2 mg of dipalmitoylphosphatidylcholine were dissolved in a flask containing 4 mL of chloroform. The mixture was subjected to vacuum rotary evaporation for 30 min in a 40°C water bath until a uniform lipid film was formed. The mixture was then pumped dry for 10 min using an oil pump.
[0047] (2) Add 10 mL of deionized water to the flask, perform water bath ultrasonic emulsification for 5 min, and then switch to probe ultrasonic emulsification for 10 min, wherein the power of the water bath ultrasonic is 100 W, the power of the probe ultrasonic is 110 W, and the probe ultrasonic is performed with a 10-s rest every 6 s;
[0048] (3) The obtained suspension was centrifuged at 6000 rpm for 30 min at 4°C using a 50KD ultrafiltration tube, and the upper precipitate was dispersed in 0.5 mL of deionized water to obtain lipid nanoparticles loaded with ensidipine.
[0049] Example 4
[0050] A method for preparing lipid nanoparticles carrying ensididipine, comprising the following steps:
[0051] (1) 1.2 mg of ensidipine, 1 mg of β-sitosterol, 6 mg of lecithin, and 4 mg of dipalmitoylphosphatidylcholine were dissolved in a flask containing 3.5 mL of chloroform. The mixture was subjected to vacuum rotary evaporation for 30 min in a 40°C water bath until a uniform lipid film was formed. The mixture was then pumped dry for 14 min using an oil pump.
[0052] (2) Add 10 mL of deionized water to the flask, perform water bath ultrasonic emulsification for 5 min, and then switch to probe ultrasonic emulsification for 10 min, wherein the power of the water bath ultrasonic is 100 W, the power of the probe ultrasonic is 110 W, and the probe ultrasonic is performed with a 10-s rest every 6 s;
[0053] (3) The obtained suspension was centrifuged at 6000 rpm for 30 min at 4°C using a 30KD ultrafiltration tube, and the upper precipitate was dispersed in 0.5 mL of deionized water to obtain lipid nanoparticles loaded with ensidipine.
[0054] Comparative Example 1
[0055] The main difference between this comparative example and Example 1 is that the preparation process only uses water bath ultrasonic emulsification, and probe ultrasonic emulsification is not used. The preparation method is as follows: 1.2 mg of ensidedipine, 1 mg of β-sitosterol, 4 mg of lecithin, and 2 mg of polyethylene glycol-distearoyl phosphatidylethanolamine are dissolved in a flask containing 3 mL of chloroform, and vacuum rotary evaporation is carried out for 30 min under 40 ° C water bath conditions to form a uniform lipid film, and then the oil pump is replaced to continue to drain for 10 min. 10 mL of deionized water is added to the flask, and water bath ultrasonic emulsification is carried out for 20 min, and the power of water bath ultrasound is 100 w. The resulting suspension is centrifuged at 6000 rpm for 25 min at 4 ° C with a 30KD ultrafiltration tube, and the upper precipitate is dispersed in deionized water to obtain lipid nanoparticles loaded with ensidedipine.
[0056] Comparative Example 2
[0057] The main difference between this comparative example and Example 1 is that β-sitosterol was not used as a skeleton material during preparation. The preparation method is as follows: 1.2 mg of ensidipine, 4 mg of lecithin, and 2 mg of polyethylene glycol-distetraylphosphatidylethanolamine were dissolved in a flask containing 3.5 mL of chloroform, and vacuum rotary evaporated for 30 min under a 35 ° C water bath condition to form a uniform lipid film, and then replaced with an oil pump to continue to drain for 10 min. 10 mL of deionized water was added to the flask, and water bath ultrasonic emulsification was performed for 5 min, and then switched to probe ultrasonic emulsification for 10 min. The resulting suspension was centrifuged at 5000 rpm for 20 min at 4 ° C with a 30KD ultrafiltration tube, and the upper precipitate was dispersed in deionized water to obtain lipid nanoparticles loaded with ensidipine.
[0058] Comparative Example 3
[0059] The main difference between this comparative example and Example 1 is that step (1) is different. Step (1) in this comparative example is:
[0060] (1) Dissolve 1.2 mg of ensidipine, 6 mg of β-sitosterol, 0.5 mg of lecithin, and 0.5 mg of polyethylene glycol-distearoylphosphatidylethanolamine in a flask containing 3.5 mL of chloroform. Vacuum rotary evaporation was performed in a 40°C water bath for 30 min until a uniform lipid film was formed. Then, the mixture was pumped dry for 10 min using an oil pump.
[0061] Test Example 1
[0062] The particle size, polydispersity index PDI and Zeta potential were measured using a dynamic light scattering instrument, and the encapsulation efficiency was measured using a UV-visible spectrophotometer. The test results of Examples 1 to 4 and Comparative Examples 1 to 3 are shown in Table 1. The particle size and particle size distribution of the lipid nanoparticles prepared in Example 1 and Example 3 are shown in Table 1. Figure 1 shown.
[0063] Table 1
[0064]
[0065] From the comparison between Example 1 and Comparative Example 1, it can be seen that the use of the probe emulsification method can make up for the shortcomings of water bath ultrasonic energy dispersion and low emulsification efficiency, the encapsulation rate of the obtained lipid nanoparticles is higher, the drug waste is less, and the cost is effectively reduced.
[0066] The SEM images of the lipid nanoparticles loaded with ensidipine prepared in Example 1, Example 3 and Comparative Example 2 are as follows: Figure 2 From the comparison between Examples 1 and 2 and Comparative Example 2, it can be seen that the addition of β-sitosterol as a skeleton during preparation can make the particle size of the obtained lipid nanoparticles smaller, effectively control the number of large particles, and have a higher encapsulation rate, which is more in line with the delivery requirements of drugs in vivo.
[0067] From the comparison between Example 1 and Comparative Example 3, it can be seen that if the dosage of β-sitosterol is too large, the stability of the obtained lipid nanoparticle system is greatly reduced (the absolute value of the zeta potential is greatly reduced), agglomeration occurs, and there are many particles with a particle size of about 6000 nm, making the average particle size greater than 1000 nm, and the encapsulation efficiency is also reduced.
[0068] Test Example 2
[0069] In vitro drug release from lipid nanoparticles
[0070] Take 1mL of lipid nanoparticle aqueous solution with a concentration of 600ug / mL and put it into a dialysis bag with a molecular weight cutoff of 3000MW, place it in a centrifuge tube containing 24mL PBS buffer, and seal it in a shaker for in vitro release experiment. The shaker temperature is set at 37°C and the speed is 100rpm. Take out 1.5mL of dissolution solution from the centrifuge tube at a certain interval and add 1.5mL of new PBS to continue the dissolution experiment. Use an ultraviolet spectrophotometer to measure the absorbance of the dissolution solution at 280nm to determine the concentration of the drug therein. The drug release experiment of lipid nanoparticles lasts for 7 days. The cumulative release amount is calculated based on the drug concentration in the dissolution solution measured each time, and the drug release curve is drawn.
[0071] Figure 3 The effects of the lipid nanoparticles prepared in Example 1 and Example 3 show that the drug release is relatively stable, without obvious sudden release and secondary release. After 7 days of the experiment, the drug release is basically completed, and the cumulative release rates of ensidipine in Examples 1 and 3 are 89.8% and 92.59%, respectively. This sustained release effect of ensidipine can simplify medication, effectively prolong the efficacy, avoid sudden increase in drug concentration, reduce the incidence of side effects, maintain a stable blood drug concentration, and enhance the therapeutic effect.
[0072] Test Example 3
[0073] Lipid nanoparticles for IDH2 R14QQ Determination of inhibitory activity of mutants
[0074] IDH2 R14QQ The mutant can catalyze the conversion of α-KG to 2-HG and simultaneously oxidize NADPH to NADP + Therefore, the effect of the lipid nanoparticles of the present invention on IDH2 can be evaluated by detecting the consumption value of NADPH. R14QQ Inhibitory activity of the mutants.
[0075] Ensidipin was selected as the positive control drug. Lyophilized lipid nanoparticles were graded diluted with DMSO in a 384 polypropylene plate compound dilution plate, and 0.1 μL of the dilution was transferred to a 384 reaction microplate (Corning 4514) using Echo to ensure that the final DMSO content was 1%. 5 μL of IDH2 (R140Q) enzyme solution was added to each well of the 384 reaction microplate and incubated at 25°C for 10 min. Wells containing 1% DMSO and enzyme were used as high controls, and wells containing the same amount of DMSO and buffer were used as low controls. 5 μL of α-ketoglutarate and β-NADPH solution were added to each well and incubated at 25°C for 120 min. (Final concentrations: 2nM IDH2 (R140Q), 100 μM β-NADPH and 1mM α-ketoglutarate). The fluorescence intensity signal (Ex = 355 nm, Em = 520 nm) was read on a BMG (CLARIO Star Plusacu) microplate reader. The inhibition percentage of the compound-treated wells was normalized between the high control group and the low control group, and finally the four-parameter IC was fitted by XLfit 5.5.0. 50 Curve and analyze.
[0076] Table 2 shows the effects of Ensidipine API, Example 1 and Example 3 on IDH2 R14QQ It can be seen that the lipid nanoparticles loaded with ensidipine prepared in Example 1 and Example 3 basically retain the IDH2 R14QQ The inhibitory activity of the mutants. Figure 3 The results show that the obtained lipid nanoparticles loaded with ensidipine can significantly improve the utilization rate of ensidipine and have better therapeutic effects.
[0077] Table 2
[0078] Group <![CDATA[IC 50 (ng / mL)]]> Ensidipine 1124 Example 1 1780 Example 3 1604
[0079] The detailed description of a lipid nanoparticle carrying encedipine and its preparation method and application with reference to the above-mentioned examples is illustrative rather than restrictive, and several embodiments can be listed according to the limited scope. Therefore, changes and modifications without departing from the overall concept of the present invention should fall within the scope of protection of the present invention.
Claims
1. A lipid nanoparticle carrying encedipine, characterized in that: The lipid nanoparticles carrying ensidipine have a mixture of beta-sitosterol and phospholipids as a skeleton, and the inner core is loaded with ensidipine.
2. The lipid nanoparticles carrying encedidipine according to claim 1, characterized in that: The surfaces of the ensididipine-loaded lipid nanoparticles are also modified with polyethylene glycol.
3. The lipid nanoparticles carrying encedidipine according to claim 1, characterized in that: The phospholipid mixture is selected from at least two of lecithin, polyethylene glycol-distearoylphosphatidylethanolamine, and dipalmitoylphosphatidylcholine.
4. The lipid nanoparticles carrying encedidipine according to claim 2, characterized in that: The phospholipid mixture is selected from a mixture of at least one of lecithin and dipalmitoylphosphatidylcholine and polyethylene glycol-distearoylphosphatidylethanolamine.
5. The lipid nanoparticles carrying encedidipine according to any one of claims 1 to 4, characterized in that: The mass ratio of ensidipine to the mixture of β-sitosterol and phospholipid is 1:0.5-2:4-9.
6. The lipid nanoparticles carrying encedidipine according to any one of claims 1 to 4, characterized in that: The average particle size of the lipid nanoparticles carrying ensidipine is 150-250nm, the polydispersity index is 0.2-0.4, and the Zeta potential is -22--32mV; the encapsulation rate of ensidipine is above 75%.
7. The method for preparing lipid nanoparticles carrying encedidipine according to any one of claims 1 to 6, characterized in that: The preparation method comprises the following steps: (1) dissolving ensidipine, β-sitosterol and a phospholipid mixture in an organic solvent, and performing vacuum rotary evaporation in a water bath until a lipid film is formed; (2) ultrasonically dispersing the lipid film in deionized water, and then performing probe ultrasonic dispersion to obtain a uniform drug-loaded emulsion; (3) The obtained drug-loaded emulsion is centrifuged at low temperature using an ultrafiltration tube, and the upper precipitate is dispersed in deionized water to obtain lipid nanoparticles loaded with ensididipine.
8. The preparation method according to claim 7, characterized in that: In step (1), the temperature of the water bath is 35-45° C.; in step (2), the lipid film is dispersed in deionized water at a concentration of 0.2-1.5 mg / mL.
9. The preparation method according to claim 7, characterized in that: In step (3), the molecular weight cut-off of the ultrafiltration tube is 30-50KD; and the temperature of the low-temperature centrifugation is 2-6°C.
10. Use of the lipid nanoparticles loaded with ensidipin as claimed in any one of claims 1 to 6 in the preparation of drugs for inhibiting IDH2.